Importation Of Mitochondrial Protein By An Enhanced Allotopic Approach

Abstract

An expression vector containing appropriate mitochondrion-targeting sequences (MTS) and appropriate 3'UTR sequences provides efficient and stable delivery of a mRNA encoding a protein (CDS) to the mitochondrion of a mammalian cell. The MTS and 3'UTR sequences guide the CDS mRNA from the nuclear compartment of the cell to mitochondrion-bound polysomes, where the CDS is translated. This provides an efficient translocation of a mature functional protein into the mitochondria. A method of targeting mRNA expressed in the nuclear compartment of a mammalian cell to the mitochondrion is also provided. The vector and methods can be used to treat defects in mitochondrial function.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to the field of cell biology, molecular genetics, and medicine. It more particularly relates to the importation of proteins into the mitochondrion of animal and human cells.

BACKGROUND OF THE INVENTION

[0002] Mitochondria occupy a central position in the overall metabolism of eukaryotic cells; hence the oxidative phosphorylation (OXPHOS), the Krebs's cycle, the urea cycle, the heme biosynthesis and the fatty acid oxidation take place within the organelle. Recently, another major role for mitochondria in determining the cellular life span was established, as they are recognized to be a major early mediator in the apoptotic cascade. Mitochondria are also a major producer of reactive oxygen species (ROS) causing oxidative stress and therefore inducers of cell death.

[0003] Primary defects in mitochondrial function are implicated in over 120 diseases and the list continues to grow, they encompass an extraordinary assemblage of clinical problems, commonly involving tissues that have high energy requirements, such as retina, heart, muscle, kidney, pancreas and liver. Their incidence is estimated of 1 in 5,000 live births. Indeed, combining epidemiological data on childhood and adult mitochondrial diseases suggests this prevalence as minimum, and could be much higher. Therefore, mitochondrial pathologies are considered among the most common genetically determined diseases, and are a major health issue since they remain inaccessible to both curative and palliative therapies.

[0004] Mitochondrion is assembled with proteins encoded by genes distributed between mitochondrial and nuclear genomes. These genes include those encoding the structural proteins of the respiratory chain complexes I-V, their associated substrates and products, the proteins necessary for mitochondrial biogenesis, the apparatus to import cytoplasmically synthesized precursors and the proteins necessary for mitochondrial assembly and turnover. Studies leading to the identification of genes involved in mitochondrial disorders have made considerable progress in the last decade. Indeed, numerous mutations in both mitochondrial DNA and a number of nuclear genes have been reported in association with a striking diversity of clinical presentations.

[0005] Approximately half of human mitochondrial disorders are caused by pathogenic point mutations of mtDNA, one-third of which are located in coding genes. There is currently no treatment for any of these disorders, a possible therapeutic approach is to introduce in the nucleus a wild-type copy of the gene mutated in the mitochondrial genome and import normal copies of the gene product into mitochondria from the cytosol. This approach has been termed "allotopic expression".

[0006] There have already some reports describing that engineered nucleus-localized version of some mtDNA genes could be expressed in mammalian cells. For example, in a Leigh's disease case, a plasmid was constructed in which the mitochondrial targeting signal of the nuclear encoded COX8 gene was appended to a recoded mitochondrial ATP6 gene, mutated in patients. Stably transfected cells from patients present an improvement of growth in galactose medium and a mild increase in ATP synthesis, however the amount of Atp6 protein imported into mitochondria was relatively low (18.5%), implying that the precursor was not imported efficiently (Manfredi, G., et al., Rescue of a deficiency in ATP synthesis by transfer of MTATP6, a mitochondrial DNA-encoded gene to the nucleus. Nature Genet., 2002. 30: p. 394-399).

[0007] Oco-Cassio and co-workers have demonstrated that allotopic expression of apocytochrome b and ND4 into Cos-7 and HeLa cells, did not lead to an efficient mitochondrial import of these proteins (Oca-Cossio, J., et al., Limitations of allotopic expression of mitochondrial genes in mammalian cells. Genetics, 2003. 165: p. 707-720).

[0008] Hence, up today important limitations are found to the allotopic expression as a therapeutic approach and require optimization to overcome the significant hurdles before it can be applied in genetic therapy.

[0009] One hypothesis that can explain the poor import ability of the mitochondrial protein is its high hydrophobicity. Thus, the precursor synthesized in the cytoplasm remains stuck on the outer mitochondrial membrane.

[0010] Mitochondria assembly depends on balanced synthesis of 13 proteins encoded by mtDNA with more than a thousand others encoded by nuclear DNA. As the vast majority of mitochondrial polypeptides are synthesized in the cytoplasm, there is the requirement for an efficient and specific protein targeting system. This process involves the transport of mRNAs from the nucleus to the surface of mitochondria.

[0011] The inventors examined the possibility that allotopic expression of DNA such as mtDNA could be optimized by a targeted localization of the mRNA to the mitochondrial surface.

SUMMARY OF THE INVENTION

[0012] Mitochondrial proteins are encoded by nucleic acids which are located in the mitochondrion, i.e. mitochondrial nucleic acids (mtDNA, mtRNA), as well as by nucleic acids which originate from the nucleus, i.e. nuclear nucleic acids (nDNA, nRNA).

[0013] The inventors describe an enhanced allotopic approach for importation of proteins into the mitochondrion. The present invention provides means, including compositions and methods, which enable mitochondrial importation at enhanced efficiency and stability compared to prior art techniques. The means of the invention enable a targeted localization of the mRNA to the mitochondrial surface.

[0014] Compared to prior art techniques, the means of the invention enable the efficient and stable importation of protein into the mitochondrion of an animal of human in need thereof, such as an animal or human having a cellular dysfunction caused by one or several mutations in a gene encoding a mitochondrial protein.

[0015] The inventors demonstrate that mRNA sorting to the mitochondrial surface is an efficient way to proceed to such an allotopic expression, and that this mRNA sorting can be controlled by selecting appropriate mitochondrion-targeting sequence (MTS) and appropriate 3'UTR sequences. The CDS sequence which codes for the protein to be delivered into the mitochondrion is guided by these appropriate MTS and 3'UTR sequences from the nuclear compartment to the mitochondrion-bound polysomes (where the CDS is translated), and aids in an efficient translocation of a mature functional protein into the mitochondria.

[0016] The inventors demonstrate that, to obtain a stable therapeutically-effective importation, both an appropriate MTS and an appropriate 3'UTR should preferably be used.

[0017] Appropriate MTS and 3'UTR sequences correspond to those of nuclearly-transcribed mitochondrially-targeted mRNAs. If a vector is used, it is preferred that it does not contain any 3'UTR which would correspond to the 3'UTR of a nuclearly-transcribed but not-mitochondrially-targeted mRNAs. To the best of the inventors' knowledge, all commercially-available vectors contain such a not-mitochondrially-targeted mRNA; it is then preferred to delete this inappropriate 3'UTR from the vector before use as mitochondrial importer.

[0018] The means of the invention are especially adapted to animal and human cells, and more particularly to mammalian cells. They give access to therapeutically-effective means for such cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGS. 1A and 1B: Map and sequence of COX10 MTS-nATP6, SOD2MTS-nATP6, COX10 MTS-nATP6-COX10 3'UTR, SOD2MTS-nATP6-SOD2 3'UTR obtained in the pCMV-Tag 4A vector.

[0020] FIG. 1A. The four constructs are schematically represented.

[0021] FIG. 1B. The COX10 MTS-nATP6 and SOD2MTS-nATP6 are introduced at the EcoRI restriction site of the pCMV-tag4A vector. EcoRI restriction sites are framed. The ATG of SOD2 MTS, COX10 MTS and nATP6, are bold and underlined. The COX10 3'UTR and the SOD2 3'UTR are inserted at the PvuI and MluI restriction sites, represented in bold. FLAG tag epitope is in italics.

[0022] FIG. 2: RT-PCR analyses of RNAs purified from HeLa transfected cells

[0023] 100 ng of total RNAs were reversed transcribed and subjected to 30 cycles of PCR amplification, 10% of amplified product were subjected to agarose electrophoresis. [0024] 1: Transiently transfected cells with the COX10 MTS-nATP6 vector (SV40 3'UTR). [0025] 2: Transiently transfected cells with COX10 MTS-nATP6-COX10 3'UTR vector. [0026] In 3 and 4 we examined RNAs from the same transfection experiment but in this case it represents the stably transfected cells. [0027] 5: HeLa transfected cells with the empty pCMV-tag4A vector. [0028] 6: Hela cells.

[0029] Specific oligonucleotide primers were used to detect hybrid ATP6 mRNA in transfected cells, for the MTS COX10-ATP6 product MTS COX10 and ATP6 ORF 3' were used (cf. Table 2 below, in example 1). For the amplification of the complete ATP6 ORF and the entire COX10 3'UTR the ATP6 ORF 5' primer and the 3' UTR COX10 3' Primer were used (cf. Table 2 below, in example 1). As internal control, the steady-state levels of COX6c mRNA were examined in all the RNA preparations using both COX6 primers shown in said Table 2.

[0030] FIG. 3: Subcellular localization of the recoded Atp6 protein in HeLa cells

[0031] Stably transfected cells with either COX10 MTS-nATP6 (SV40 3'UTR) or COX10 MTS-nATP6-COX10 3'UTR vector were visualized by indirect immunofluorescence using antibodies to Flag and ATP synthase subunit beta. The punctuate pattern of Flag antibody staining indicates that the fusion Atp6 protein is efficiently transported to mitochondria in vivo, since the same pattern of mitochondria labeling was observed with the beta subunit of ATP synthase.

[0032] FIGS. 4A and 4B: nATP6 proteins are efficiently imported into mitochondria in vivo.

[0033] FIG. 4A: Proteins were extracted from HeLa cells and HeLa transfected cells (COX10 MTS-nATP6-SV40 3'UTR or COX10 MTS-nATP6-COX10 3'UTR vectors) and assayed for import in the absence and presence of proteinase K (PK). Proteins were treated with 200 .mu.g/ml of proteinase K at 0.degree. C. for 30 min. Then, they were separated on 4-12% polyacrylamide SDS gel and transferred into a nitrocellulose membrane. The resulting blot was probed with mouse monoclonal anti-ATP synthase subunit alpha or mouse monoclonal anti-Flag M2 antibodies.

[0034] FIG. 4B: Histograms of the amount of COX10 MTS-nATP6-Flag and Atp synthase subunit alpha with or without proteinase K. Signals from immunoblots were scanned and quantified by the MultiAnalyst System (Bio-Rad). The amount of the mature ATP6 protein insensitive to proteinase K proteolysis is approximately 185% higher in cells transfected with COX10 MTS-nATP6-COX10 3'UTR vector compared to cells expressing the COX10 MTS-nATP6 without COX10 3'UTR but with the SV40 Poly A signal. Besides, the amount of the mature form of the recoded ATP6 protein inside mitochondria is very similar to the one measured for the naturally imported ATP synthase subunit alpha, confirming that recoded ATP6 proteins are efficiently translocated into the organelle.

[0035] FIGS. 5A and 5B: Map and sequence of COX10 MTS-nND1, COX10MTS-nND4, COX10 MTS-nND1-COX10 3'UTR, COX10MTS-nND4-COX10 3'UTR obtained in the pCMV-Tag 4A vector.

[0036] FIG. 5A. The four constructs are schematically represented.

[0037] FIG. 5B. The COX10 MTS-nND1 and COX10MTS-nND4 are introduced at the XhoI/SalI restriction sites of the pCMV-tag4A vector. XhoI and SalI restriction sites are framed. The ATG of COX10 MTS, nND1 and nND4, are bold and underlined. The COX10 3'UTR is inserted at the PvuI and MluI restriction sites, represented in bold. FLAG tag is in italics.

[0038] FIG. 6: Immunocytochemistry of G3460A LHON fibroblats

[0039] The fusion protein was visualized by indirect immunofluorescence using antibodies to Flag. Indicative of mitochondrial import, cells transfected with either COX10 MTS-nND1-SV40 3'UTR or COX10 MTS-nND1-COX10 3'UTR vectors exhibited a typically punctuate staining pattern, also observed with the beta subunit of ATP synthase, which localize in vivo to the inner mitochondrial membrane. In contrast, cells transfected with the empty pCMV-Tag 4A vector exhibited a very low intensity and diffuse cytoplasmic staining when antibodies to Flag were used.

[0040] FIG. 7: Immunocytochemistry of G11778A LHON fibroblats

[0041] The fusion protein was visualized by indirect immunofluorescence using antibodies to Flag. Cells transfected with either COX10 MTS-nND4-SV40 3'UTR or COX10 MTS-nND4-COX10 3'UTR vectors exhibited a typically punctate staining pattern, also observed with the beta subunit of ATP synthase, which localize in vivo to the inner mitochondrial membrane. This data indicates that ND4 is efficiently imported into mitochondria. In contrast, cells transfected with the empty pCMV-Tag 4A vector exhibited a very low intensity and diffuse cytoplasmic staining when antibodies to Flag were used.

[0042] FIG. 8: Growth in glucose-free medium of non-transfected fibroblasts with the G3460A mutation and transfected fibroblasts with the MTS COX10-nND1-COX10 3'UTR vector Fibroblasts from LHON patients presenting the G3460A mutation were stably transfected with the MTS COX10-nND1-COX10 3'UTR vector and examined for their ability to growth on DMEM medium supplemented with 10 mM galactose. Non-transfected fibroblasts (LHON G3460A ND1) show a severe growth defect on galactose medium, the ability to grow on galactose was significantly improved when the recoded nND1 protein is expressed in stably transfected cells (LHON G3460A ND1 MTSCOX10-nND1). Cells were photographed after 6 day culture.

[0043] FIG. 9: recoded CDS of mtDNA (SEQ ID NO: 27-29)

[0044] FIG. 9 shows the human nucleic acid coding sequence of the mitochondrial ATP6, ND1, ND4, recoded according to the universal genetic code (nATP6, nND1, nND4 of SEQ ID NO:27, 28 and 29, respectively). The recoded ND1 and ND4 which are shown in FIG. 9 also take into account the preferred human codon usage (see example 2 below).

[0045] FIG. 10: illustrative human co-translational MTS and 3'UTR (SEQ ID NOS: 30, 47, 31, 60)

[0046] FIG. 10 shows the sequence of human COX10 MTS (SEQ ID NO: 30), human COX10 3'UTR (SEQ ID NO: 47), human SOD2 MTS (SEQ ID NO: 31), and human SOD2 3'NTR (SEQ ID NO: 60).

TABLE-US-00001 TABLE 5 Nucleic acid MTS Nucleic acid 3'UTR COX10 SEQ ID NO: 30 SEQ ID NO: 47 SOD2 SEQ ID NO: 31 SEQ ID NO: 60

[0047] FIGS. 11A-11I show the protein sequence coded by illustrative human mitochondrially-targeted mRNA, as well as their respective MTS peptide sequences and their respective 3'UTR sequences. ATCC accession number is indicated for each of these protein sequences.

ACO2=Aconitase;

[0048] SOD2 Mitochondrial Superoxide dismutase; ATP5b=P synthase subunit beta; UQCRFS1=Ubiquinol-cytochrome c reductase. Rieske iron-sulfur polypeptide 1; NDUFV1 NADH-ubiquinone oxidoreductase 51 kDa subunit, mitochondrial precursor (Complex I-51KD) (CI-51KD) (NADH dehydrogenase flavoprotein 1); NDUFV2=NADH dehydrogenase (ubiquinone) flavoprotein 2, 24 kDa; ALDH2=Mitochondrial aldehyde dehydrogenase 2 precursor; COX10 Heme A:farnesyltransferase;

AK2=Adenylate Kinase 2.

[0049] SEQ ID NO are as follows:

TABLE-US-00002 TABLE 6 MTS peptide 3'UTR Protein ACO2 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 48 SOD2 SEQ ID NO: 34 SEQ ID NO: 35 SEQ ID NO: 49 ATP5b SEQ ID NO: 36 SEQ ID NO: 37 SEQ ID NO: 50 UQCRFS1 SEQ ID NO: 38 SEQ ID NO: 39 SEQ ID NO: 51 NDUFV1 SEQ ID NO: 40 SEQ ID NO: 41 SEQ ID NO: 52 NDUFV2 SEQ ID NO: 42 SEQ ID NO: 43 SEQ ID NO: 53 ALDH2 SEQ ID NO: 44 SEQ ID NO: 45 SEQ ID NO: 54 COX10 SEQ ID NO: 46 SEQ ID NO: 59 SEQ ID NO: 55 AK2 SEQ ID NO: 57 SEQ ID NO: 56

[0050] FIGS. 12A, 12B, 12C: Subcellular distribution of hybrid ATP6 mRNAs in HeLa cells

[0051] A. Total RNAs extracted from cells expressing the SOD2.sup.MTS ATP6-3'UTR.sup.SV40 (S.T 1 and S.T 2) or the SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 (S.T 3 and S.T 4) vectors were subjected to RT-PCR analysis to reveal amounts of hybrid ATP6 (SOD2.sup.MTS ATP6) mRNAs and endogenous SOD2, ATP6 and COX6c mRNAs. The amount of RNAs used for the reverse transcription, FOR conditions and specific oligonucleotides used for each gene are summarized in Table 9.

[0052] B. RNAs were purified from mitochondrion-bound polysomes (M-P) and free-cytoplasmic polysomes (F-P) of stably transfected cell lines with either SOD2.sup.MTS ATP6-3'UTR.sup.SV40 (S.T 1 and S.T 2) or SOD2'.sup.MTS ATP6-3'UTR.sup.SOD2 (S.T 3 and S.T 4) vectors and subjected to RT-PCR analysis. The abundance of endogenous ATP6, SOD2 and COX6c mRNAs was determined in each polysomal population using the conditions shown in Table 9.

[0053] C. Densitometric Analyses were Performed Using the Quantity One Biorad Software System.

[0054] The difference between the amounts of hybrid ATP6 mRNAs in cells expressing respectively SOD2.sup.MTS ATP6-3'UTR.sup.SV40 or SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 constructions was significant according to the paired Student's t-test (P<0.0034, n=6).

[0055] FIG. 13: Subcellular localization of the recoded Atp6 protein in vivo

[0056] Stably transfected cells with either the empty pCMV-tag4A vector, SOD2.sup.MTS ATP6-3'UTR.sup.SV40 or SOD2.sup.MTS ATP6-3' UTR.sup.SOD2 plasmids were visualized by indirect immunofluorescence using antibodies to Flag and ATP synthase subunit .alpha.. For each cell type visualized, a merged image in association with DAPI staining is shown at the right panel. Indicative of the mitochondrial localization of recoded ATP6 proteins, cells transfected with either SOD2.sup.MTS ATP6-3'UTR.sup.SV40 or SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 plasmids showed a significant colocalization of both Flag and ATP synthase .alpha. signals. In contrast, cells transfected with the empty vector exhibited a low diffuse cytoplasmic staining.

[0057] FIGS. 14A, 14B, 14C: Recoded ATP6 proteins are efficiently imported into mitochondria in vivo.

[0058] A. Six independent mitochondria purifications were performed with cells stably transfected with either SOD2.sup.MTS ATP6-3'UTR.sup.SV40 SOD2.sup.MTS ATP6-3'.sup.SOD2 plasmids and subjected to Western blot analysis. Signals for the ATP6 precursors and mature forms were scanned and quantified by the Quantity One System (Bio-Rad). No significant differences between the amounts of the precursor and the mature form of the recoded ATP6 proteins were observed in each cell line examined. B. Upper panel: Schematic representation of mitochondrial import intermediates. The hydrophobic passenger protein can be trapped en route to the matrix. In this step, the protein can be blocked or represented an intermediate of translocation. This doesn't prevent the cleavage of the MTS by a mitochondrial processing peptidase, the rest of the protein remains accessible to PK digestion and therefore if digested it becomes undetectable in the Western blot assay. The fraction of the protein completely translocated turns into a mature protein insensitive to PK located in the inner mitochondrial membrane. MM: mitochondrial matrix; OM: outer membrane; MIS: mitochondrial intermembrane space, TOM: Translocase of the Outer Membrane, TIM: Translocase of the Inner membrane.

[0059] Middle panel: Mitochondria extracted from transfected cells with either the empty pCMV-Tag 4A vector, SOD2.sup.MTS ATP6-3'UTR.sup.SV40 or SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 plasmids were subjected to Western blot essays. 20 .mu.g of proteins were treated with 150 .mu.g/ml of PK at 0.degree. C. for 30 minutes and subjected to immunoblotting analysis using anti-ATP synthase subunit .alpha. and anti-Flag M2 antibodies. Densitometric analyses of experiments performed with six independent mitochondrial purifications were represented at the lower panel. We normalized values measured for the signal of the mature form of ATP6 resistant to PK with ATP.alpha. signal revealed after PK digestion. We then compared the value obtained for cells expressing either the SOD2.sup.MTS ATP6-3'UTR.sup.SV40 or the SOD2.sup.MTS ATP6-3'UTR3.sup.SOD2 plasmids. Signals from Western blots were scanned and quantified by the Quantity One System (Bio-Rad). The difference between the amounts of fully mitochondrial translocated ATP6 protein in cells expressing respectively SOD2.sup.MTS ATP6-3'UTR.sup.SV40 or SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 constructions was significant according to the paired Student's t-test (P<0.0022, n=6).

[0060] C. 20 .mu.g of mitochondria isolated from cells stably transfected with SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 vector treated with 150 .mu.g/ml of PK and 1% Triton X100 at 0.degree. C. for 30 min and subsequently subjected to Western analysis

[0061] FIG. 15: Mitochondrial import ability of ATP6 proteins based on the mesohydrophobicity index

[0062] A plot developed by Claros and Vincens was used to measure mitochondrial import ability of fusion ATP6 proteins. By this approach, the fusion SOD2.sup.MTS ATP6 protein would not be importable. Mesohydrophobicity, which is the average regional hydrophobicity over a 69 amino acid region, was calculated using Mito-ProtII. Values obtained are the following: ATP6: 1.41; SOD2.sup.MTS ATP6: 1.41; COX8.sup.MTS ATP6: 1.41; SOD2: -1.26; COX8: -1.63.

[0063] FIG. 16: rescue of NARP cells; survival rate on galactose medium of NARP cells (mutated ATP6), and of NARP cells transfected by a SOD2 MTS-ATP vector (SOD2 MTS-ATP6-SV40 3'UTR), or by a vector of the invention (SOD2 MTS-ATP6 SOD2 3'UTR); see also example 3, table 11.

[0064] FIG. 17: rescue of LHON fibroblasts; survival rate on galactose medium of LHON fibroblasts (mutated ND1), and of LHON fibroblasts transfected by a COX10 MTS-ND1-SV40 3'UTR vector, or by a vector of the invention (COX10 MTS-ND1-COX10 3'UTR); see also example 4, table 12.

[0065] FIG. 18: mitochondrial distribution in retinal gangion cells (RGC) transfected with the mutated version of ND1.

[0066] FIG. 19: rescue of NARP cells; anti-Flag, Mito-tracker and merged+DAPI staining of NARP cells transfected either with SOD2 MTS ATP6-SV40 3' UTR, or with SOD2 MTS-ATP6-SOD2 3'UTR.

DETAILED DESCRIPTION OF THE INVENTION

[0067] The present invention relates to the use and control of mRNA sorting at the surface of mitochondria.

[0068] Schematically, the present invention relates to the use of nucleic acid sequences corresponding to a co-translational MTS and of a co-translational 3'UTR, for guiding a desired mRNA (which codes a desired mitochondrial protein) from the nucleus to the mitochondria-bound polysomes, and for inducing the effective translocation of the translated protein into the mitochondrion.

[0069] By "co-translational", it is herein referred to a nuclearly-encoded mitochondrially-targeted pathway.

Mitochondrion-Targeting Sequences (MTS):

[0070] Sequences known as mitochondrion-targeting signal or mitochondrial targeting signal are referred to as MTS by the skilled person.

[0071] A MTS sequence can be identified within a protein or nucleic acid sequence by a person of ordinary skill in the art.

[0072] Most mitochondrion-targeting peptides consist of a N-terminal pre-sequence of about 15 to 100 residues, preferably of about 20 to 80 residues. They are enriched in arginine, leucine, serine and alanine. Mitochondrial pre-sequences show a statistical bias of positively charged amino acid residues, provided mostly through arginine residues; very few sequences contain negatively charged amino acids. Mitochondrion-targeting peptides also share an ability to form an amphilic alpha-helix.

[0073] A complete description of a method to identify a MTS is available in: M. G. Claros, P. Vincens, 1996 (Eur. J. Biochem. 241, 779-786 (1996), "Computational method to predict mitochondrially imported proteins and their targeting sequences"), the content of which is herein incorporated by reference.

[0074] Software is available to the skilled person to identify the MIS of a given sequence. Illustrative software notably comprises the MitoProt.RTM. software, which is available e.g. on the web site of the Institut fur Humangenetik; Technische Universitat Munchen, Germany. The MitoProt.RTM. software calculates the N-terminal protein region that can support a Mitochondrial Targeting Sequence and the cleavage site. The identification of the N-terminal mitochondrial targeting peptide that is present within a protein gives a direct access to the nucleic acid sequence, i.e. to the MTS (e.g. by reading the corresponding positions in the nucleic acid sequence coding for said protein).

[0075] Illustrative human MIS peptide sequences and human 3'UTR originating from human nuclearly-encoded mitochondrially-targeted mRNA are given in FIGS. 11A, 11B, 11C, 11D, 11E, 11F, 11G, 11H and 11I.

[0076] SEQ ID NOs are as follows:

TABLE-US-00003 TABLE 7 Illustrative human mRNAs which are nuclearly-encoded but mitochondrially-targeted MTS peptide FIG. ACO2 SEQ ID NO: 32 11A SOD2 SEQ ID NO: 34 11B ATP5b SEQ ID NO: 36 11C UQCRFS1 SEQ ID NO: 48 11D NDUFV1 SEQ ID NO: 40 11E NDUFV2 SEQ ID NO: 42 11F ALDH2 SEQ ID NO: 44 11G COX10 SEQ ID NO: 46 11H

3'UTR:

[0077] The 3'UTR of a RNA molecule is defined as the fragment of this RNA molecule that extends from the STOP codon to the end of the molecule. According to the universal genetic code, there are three possible STOP codons: TGA, TAA, TAG.

[0078] An online data base gives direct access to 3'UTR sequences.

[0079] Illustrative 3'UTR sequences which can used in accordance with the invention are shown in FIGS. 11A, 11B, 110, 11D, 11E, 11F, 11G, 11H and 11I (Accession numbers of these sequences are also indicated).

[0080] SEQ ID NOs are as follows:

TABLE-US-00004 TABLE 8 Illustrative human mRNAs which are nuclearly-encoded but mitochondrially-targeted 3'UTR FIG. ACO2 SEQ ID NO: 33 11A SOD2 SEQ ID NO: 35 11B ATP5b SEQ ID NO: 37 11C UQCRFS1 SEQ ID NO: 39 11D NDUFV1 SEQ ID NO: 41 11E NDUFV2 SEQ ID NO: 43 11F ALDH2 SEQ ID NO: 45 11G COX10 SEQ ID NO: 47 10 AK2 SEQ ID NO: 57 11I

Vectors of the Invention:

[0081] The present invention relates to a vector which is adapted to the efficient and stable delivery of a protein into the mitochondrion of an animal or human cell, preferably a mammalian cell, most preferably a human cell.

[0082] The vector of the invention can be produced in the form of a recombinant vector. Advantageously, the vector of the invention is an expression vector.

[0083] A vector of the invention comprises: [0084] at least one nucleic acid sequence encoding a mitochondrion-targeting signal (also referred to as: MTS nucleic acid sequence), [0085] at least one nucleic acid sequence which encodes said protein to be delivered, in accordance with the universal genetic code (also referred to as: CDS), and [0086] at least one 3' nucleic acid sequence.

[0087] Said at least one MTS nucleic acid sequence is a co-translational MTS nucleic acid sequence, or a conservative fragment or variant thereof.

[0088] Said at least one 3' nucleic acid sequence is a co-translational 3'UTR nucleic acid sequence or the DNA sequence of such a co-translational 3'UTR, or a conservative fragment or variant thereof.

[0089] Preferably, said vector does not comprise a non-co-translational 3'UTR.

[0090] Said vector does not use a post-translation importation pathway, but uses a co-translation importation pathway from nucleus to said mitochondrion.

[0091] The delivery of protein according to the invention not only comprises the translocation of the protein-encoding nucleic acid from nucleus towards mitochondrion, but also comprises the translation of the encoded protein in the cytosol but at proximity of the mitochondrion (on mitochondrion-bound polysomes), and the effective importation of the translated protein into said mitochondrion. The invention provides a very advantageous importation mechanism compared to prior art techniques, which provided the mitochondrion with mature proteins at an unsatisfactory level of efficiency.

[0092] The present invention further provides a stable importation of said protein into the mitochondrion. It means that the protein rescue obtained by the invention is a rescue that is stable over time: the fibroblasts of a LHON patient transfected by a vector of the invention (expressing N.COPYRGT.1) has grown in vitro for at least 20 days on a galactose culture medium. To the best of the inventors' knowledge, this is the first time that a culture of LHON patient fibroblasts can be kept growing for such a long period on a galactose medium.

[0093] The invention thus relates to a vector adapted to the efficient and stable delivery of a protein into the mitochondrion of an animal or human cell, preferably a mammalian cell, most preferably a human cell, which comprises: [0094] at least one mitochondrion-targeting nucleic acid sequence (MTS nucleic acid sequence), [0095] at least one nucleic acid sequence which encodes said protein in accordance with the universal genetic code (CDS), and [0096] at least one 3' nucleic acid sequence, which is located in 3' of said at least one MTS nucleic acid sequence and said at least one CDS.

[0097] Preferably, said at least MTS nucleic acid sequence is in 5' position compared to said at least one CDS sequence, whereby the vector has the following structure (from 5' to 3'): at least one MTS nucleic acid sequence--at least one CDS--at least one 3'UTR.

[0098] Said at least one MTS nucleic acid sequence is: [0099] the MTS RNA sequence of a nuclearly-encoded mitochondrially-targeted mRNA, preferably the MTS RNA sequence of a naturally-occurring nuclearly-encoded mitochondrially-targeted m RNA, or [0100] the cDNA sequence of such a MTS RNA sequence, or [0101] a DNA sequence coding for such a MTS RNA sequence in accordance with the universal genetic code, or [0102] a conservative variant or fragment of such a RNA or cDNA or DNA MTS sequence, which derives therefrom by deletion and/or substitution and/or addition of one or several nucleotides, but has retained a mitochondrion-targeting function.

[0103] In other words, said at least one MTS nucleic acid sequence is: [0104] the RNA sequence of a MTS which targets a (preferably naturally-occurring) nuclearly-transcribed mRNA to the surface of a mitochondrion in a cell collected from a healthy animal or human being, or in a normal animal or human cell, or [0105] the cDNA sequence of such a MTS RNA sequence, or [0106] a DNA sequence coding for such a MTS RNA sequence in accordance with the universal genetic code, or [0107] a conservative variant or fragment of such a RNA or cDNA or DNA MTS sequence, which derives therefrom by deletion and/or substitution and/or addition of one or several nucleotides, but has retained a mitochondrion-targeting function.

[0108] Preferably, said at least one MTS nucleic acid sequence is: [0109] the cDNA sequence of a MTS of a nuclearly-encoded mitochondrially-targeted mRNA, or [0110] a conservative variant or fragment of such a cDNA sequence, which derives therefrom by deletion and/or substitution and/or addition of one or several nucleotides, but has retained a mitochondrion-targeting function.

[0111] Said at least one 3' nucleic acid sequence is: [0112] the 3'UTR sequence of a nuclearly-encoded mitochondrially-targeted mRNA, preferably the 3'UTR sequence of a naturally-occurring nuclearly-encoded mitochondrially-targeted mRNA, or [0113] the cDNA sequence of such a 3'UTR sequence, or [0114] a DNA sequence coding for such a 3'UTR sequence in accordance with the universal genetic code, or [0115] a conservative variant or fragment of such a RNA or cDNA or DNA 3'UTR sequence, which derives therefrom by deletion and/or substitution and/or addition of one or several nucleotides, and which, when replacing the wild-type 3'UTR of said nuclearly-encoded mitochondrially-targeted mRNA, still allows for a mitochondrial targeting of the resulting mRNA.

[0116] In other words, said at least one 3' nucleic acid sequence is: [0117] the RNA sequence of the 3'UTR of a nuclearly-transcribed mitochondrially-targeted mRNA, i.e. the RNA sequence of the 3'UTR of a (preferably naturally-occurring) nuclearly-transcribed RNA which is targeted to the surface of a mitochondrion in a cell collected from a healthy animal or human being, or in a normal animal or human cell, or [0118] the cDNA sequence of such a 3'UTR sequence, or [0119] a DNA sequence coding for such a 3'UTR sequence in accordance with the universal genetic code, or [0120] a conservative variant or fragment of such a RNA or cDNA or DNA 3'UTR sequence, which derives therefrom by deletion and/or substitution and/or addition of one or several nucleotides, and which, when replacing the wild-type 3'UTR of said nuclearly-encoded mitochondrially-targeted mRNA, still allows for a mitochondrial targeting of the resulting mRNA.

[0121] Preferably, said at least one 3' nucleic acid sequence is: [0122] the cDNA sequence of the 3'UTR sequence of a nuclearly-encoded mitochondrially-targeted mRNA, or [0123] a conservative variant or fragment of such a cDNA sequence, which derives therefrom by deletion and/or substitution and/or addition of one or several nucleotides, and which, when replacing the wild-type 3'UTR of said nuclearly-encoded mitochondrially-targeted mRNA, still allows for a mitochondrial targeting of the resulting mRNA.

[0124] The resulting vector does not use a post-translation importation pathway, but uses a co-translation importation pathway from nucleus to said mitochondrion.

[0125] Preferably, said vector (inserted nucleic acid construct included) does not comprise any sequence which would be identical to: [0126] the 3'UTR of a naturally-occurring mRNA which is a (preferably naturally-occurring) nuclearly-transcribed but non-mitochondrially-targeted mRNA, or [0127] the cDNA sequence of such a 3'UTR sequence, or [0128] a DNA sequence coding such a 3'UTR in accordance with the universal genetic code.

[0129] Preferably, said vector (inserted nucleic acid construct included) does not comprise any sequence which would be identical to: [0130] the 3'UTR of a mRNA which is not targeted to the surface of a mitochondrion, and preferably the 3'UTR of a naturally-occurring mRNA which is not targeted to the surface of a mitochondrion, or [0131] the cDNA sequence of such a 3'UTR sequence, or [0132] a DNA sequence coding such a naturally-occurring mRNA 3'UTR in accordance with the universal genetic code.

[0133] The present invention more particularly relates to a vector adapted to the efficient and stable delivery of a protein into the mitochondrion of a mammalian cell, which comprises: [0134] at least one mitochondrion-targeting nucleic acid sequence (referred to as MTS nucleic acid sequence), [0135] at least one nucleic acid sequence which encodes said protein in accordance with the universal genetic code (referred to as CDS sequence), and [0136] at least one 3' nucleic acid sequence, which is located in 3' of said at least one MTS nucleic acid sequence and of said at least one CDS, wherein said at least one MTS nucleic acid sequence is: [0137] the cDNA sequence of a MTS of a nuclearly-encoded mitochondrially-targeted mRNA, or [0138] a conservative variant or fragment of such a cDNA sequence, which derives therefrom by deletion and/or substitution and/or addition of one or several nucleotides, but has retained a mitochondrion-targeting function, wherein said at least one 3' nucleic acid sequence is: [0139] the cDNA sequence of the 3'UTR sequence of a nuclearly-encoded mitochondrially-targeted mRNA, or [0140] a conservative variant or fragment of such a cDNA sequence, which derives therefrom by deletion and/or substitution and; or addition of one or several nucleotides, and which, when replacing the wild-type 3'UTR of said naturally-occurring mRNA, still allows for a mitochondrial targeting of the resulting mRNA, wherein said vector does not comprise any sequence which would be identical to: [0141] the 3'UTR of a naturally-occurring mRNA which is a nuclearly-transcribed but not-mitochondrially-targeted mRNA, or [0142] the cDNA sequence of such a 3'UTR sequence, or [0143] a DNA sequence coding for such a naturally-occurring mRNA 3'UTR in accordance with the universal genetic code, whereby said vector does not use a post-translation importation pathway, but uses a co-translation importation pathway from nucleus to said mitochondrion.

[0144] Said at least one MTS nucleic acid sequence can e.g. be the MTS nucleic acid sequence of ACO2, or of SOD2, or of ATP5b, or of UQCRFS1, or of NDUFV1, or of NDUFV2, or of ALDH2, or of COX10.

[0145] Said at least one MTS nucleic acid sequence may thus code for a sequence of SEQ ID NO:32, or SEQ ID NO:34, or SEQ ID NO:36, or SEQ ID NO:38, or SEQ ID NO:40, or SEQ ID NO:42, or SEQ ID NO:44, or SEQ ID NO:46 the MTS peptidic or polypeptidic sequence of human ACO2, SOD2, ATP5b, UQCRFS1, NDUFV1, NDUFV2, ALDH2, COX10, respectively; see FIGS. 11A-11H).

[0146] Preferably, said at least one MTS nucleic acid sequence is the MTS nucleic acid sequence of ACO2, or of SOD2, or of ATP5b, or of COX10.

[0147] Said at least one MTS nucleic acid sequence may thus code for a sequence of SEQ ID NO:32, or SEQ ID NO:34, or SEQ ID NO:36, or SEQ ID NO:46 (=the MTS peptidic or polypeptidic sequence of human ACO2, SOD2, ATP5b, COX10, respectively).

[0148] Preferably, said at least one MTS nucleic acid sequence is SEQ ID NO: 30, or SEQ ID NO: 31 (MTS nucleic acid sequence of human COX10 and SOD2, respectively; see FIG. 10).

[0149] Said at least one 3' nucleic acid sequence can be e.g. be: [0150] the 3'UTR sequence of ACO2, or of SOD2, or of ATP5b, or of UQCRFS1, or of NDUFV1, or of NDUFV2, or of ALDH2, or of COX10, or of AK2, or [0151] the cDNA sequence of such a 3'UTR sequence, or [0152] a DNA sequence coding for such a 3'UTR sequence in accordance with the universal genetic code.

[0153] Said at least one 3' nucleic acid sequence may thus comprise or consist of those of SEQ ID NO: 33, or SEQ ID NO: 35, or SEQ ID NO: 37, or SEQ ID NO: 39, or SEQ ID NO: 41, or SEQ ID NO: 43, or SEQ ID NO: 45, or SEQ ID NO: 47, or SEQ ID NO: 57 (=the sequences corresponding to the human 3'UTR of ACO2, SOD2, ATP5b, UQCRFS1, NDUFV1, NDUFV2, ALDH2, COX10, AK2, respectively; see FIGS. 10 and 11A-11I).

[0154] Preferably, said at least one 3' nucleic acid sequence is [0155] the 3'UTR sequence of ACO2, or of SOD2, or of ATP5b, or of COX10, or of AK2, or [0156] the cDNA sequence of such a 3'UTR, or [0157] a DNA sequence coding for such a 3'UTR sequence.

[0158] Said at least one 3' nucleic acid sequence may thus comprise or consist of SEQ ID NO: 33, or SEQ ID NO: 35, or SEQ ID NO: 37, or SEQ ID NO: 47, or SEQ ID NO: 57 (=the sequences corresponding to the human 3'UTR of ACO2, SOD2, ATP5b, COX10, AK2, respectively; see FIGS. 10 and 11A-11I).

[0159] Preferably, said at least one 3' nucleic acid sequence is SEQ ID NO: 35 (human SOD2 3'UTR), or SEQ ID NO: 47 (human COX10 3'UTR).

[0160] Said at least one CDS nucleic acid sequence can be a RNA, a cDNA or a DNA sequence. Preferably, said at least one CDS sequence is a cDNA sequence.

[0161] According to a very advantageous aspect of the invention, said at least one CDS may be any nucleic acid which codes for a protein that may be found useful for a mitochondrion. Contrary to prior art techniques, the technology of the invention is indeed not limited by the level of hydrophobicity of the encoded protein.

[0162] Said at least one CDS may thus be any nucleic acid coding for a mitochondrial protein. This nucleic acid may be a mitochondrial nucleic acid, or a nuclear nucleic acid coding for a mitochondrial protein.

[0163] Most preferably said at least one CDS sequence codes for a naturally-occurring functional mitochondrial protein, such as Cox1, Cox2, Cox3, Atp6, Atp8, Cytb, Nd1, Nd2, Nd3, Nd4, Nd41, Nd5, Nd6.

[0164] Preferably, said at least one CDS sequence is the sequence of a naturally-occurring mitochondrial nucleic acid, recoded in accordance with the universal genetic code.

[0165] The mitochondrial nucleic acids use a mitochondrial genetic code which is slightly different from the universal genetic code that is used by nuclear nucleic acids.

[0166] When the protein to be imported into said mitochondrion corresponds to a naturally-occurring mitochondrial protein, the naturally-occurring form of its nucleic acid sequence follows the mitochondrial genetic code.

[0167] When such a mitochondrial nucleic acid has to be inserted in the vector of the invention, the mitochondrial nucleic acid sequence has to be recoded in accordance with the universal genetic code, as the vector directs a co-translational importation process from nucleus to mitochondrion. Hence, a nuclear-encoded version of the mitochondrial nucleic acid sequence has to be created. This nuclear-encoded version can be produced by codon substitution in the mitochondrial nucleic acid, so as to replace those codons which are read by the mitochondrial genetic system with codons of the universal genetic code. For example, the mammalian UGA codon directs insertion of a tryptophan in mitochondria, but is a stop codon in the nuclear genetic code. Therefore, the UGA codon of a mitochondrial nucleic acid has to be replaced with UGG which codes for tryptophan in the universal genetic code.

TABLE-US-00005 TABLE 4 universal vs. mitochondrial genetic code codon Universal code Human mitochondrial code UGA Stop Trp AGA Arg Stop AGG Arg Stop AUA Ile Met

[0168] Codon usage in mitochondria vs. the universal genetic code is described in Lewin, Genes V, Oxford University Press; New York 1994, the content of which being incorporated by reference.

[0169] Codon substitutions notably include: [0170] UGA to UGG, [0171] AGA to UAA, UAG or UGA, [0172] AGG to UAA, UAG or UGA, [0173] AUA to AUG, CUG or GUG, [0174] AUU to AUG, CUG or GUG.

[0175] Said at least one CDS sequence may e.g. a nucleic acid sequence coding for Atp6, or Nd1, or Nd4, such as a nucleic acid sequence of ATP6, or of ND1, or of ND4, recoded in accordance with the universal genetic code (e.g. a sequence of SEQ ID NO:27, NO:28 or NO:29, see FIG. 9).

[0176] Said at least one CDS sequence may e.g. a nucleic acid sequence a nucleic acid sequence coding for Cox1, Cox2, Cox3, Atp8, Cytb, Nd2, Nd3, Nd41, Nd5, Nd6, such as a nucleic acid sequence of COX1, COX2, COX3, ATP8, Cytb, ND2, ND3, ND41, ND5, ND6.

[0177] The description of the thirteen naturally-occurring mitochondrial nucleic acids can be found in Andrew et al. 1999 (Nat Genet. 1999 October; 23(2): 147).

[0178] Preferably, said recoding is made taking into account the preferred usage codon of said mammalian cell, and most preferably taking into account the human preferred usage codon.

[0179] When recoding mitochondrial nucleic acid according to the universal genetic code, it is according to the present invention very advantageous to take into account the preferred codon usage of the subject or patient, to which the vector or nucleic acid of the invention is to be administered.

[0180] Preferred codon usage principles, as well as examples of preferred codon usage for various organisms can e.g. be found in Klump and Maeder, 1991 (Pure & Appl. Chem., vol. 63, No. 10, pp. 1357-1366 "the thermodynamic basis of the genetic code"), the content of which is herein incorporated by reference. An illustrative preferred codon usage for human beings is shown in Table 3 below (see example 2).

[0181] Said at least one CDS sequence may e.g. the nucleic acid sequence of SEQ ID NO:28 or of SEQ ID NO:29 (i.e. a nucleic acid sequence of ND1 or of ND4, recoded in accordance with the universal genetic code, and taking into account the human preferred usage codon).

[0182] The vector of the invention may e.g. comprise: [0183] at least one SOD2 MTS nucleic acid sequence and at least one SOD2 3'UTR, or [0184] at least one COX10 MTS nucleic acid sequence and at least one COX10 3'UTR, or [0185] any combination of these MTS nucleic acid sequences and 3'UTR that the skilled person may find appropriate.

[0186] Such a vector may e.g. comprise a recoded ATP6, ND1 or ND4 as CDS.

[0187] The vector of the invention may e.g. comprise at least one sequence of SEQ ID NO:21 (COX10 MTS-re-coded ATP6-COX10 3'UTR), SEQ ID NO:22 (SOD2 MTS-re-coded ATP6-SOD2 3'UTR), SEQ ID NO:25 (COX10 MTS-re-coded ND1-COX10 3'UTR), SEQ ID NO:25 (COX10 MTS-re-coded ND4 COX10 3'UTR).

[0188] Alternatively, said at least one CDS sequence may be the nucleic acid sequence of a nuclear nucleic acid which encodes a functional mitochondrial protein, e.g., a naturally-occurring nuclear nucleic acid which encodes a functional mitochondrial protein.

[0189] More particularly, said at least one nuclear nucleic acid can be a nuclearly-transcribed mitochondrially-targeted mRNA, or the cDNA sequence of such a mRNA, or the DNA sequence coding for such a mRNA.

[0190] More particularly, said at least one nuclear nucleic acid can be a nuclearly-transcribed mRNA which is not mitochondrially-targeted, or the cDNA sequence of such a mRNA, or the DNA sequence coding for such a mRNA.

[0191] Said vector may further comprise one or several expression control sequences. The selection of suitable expression control sequences, such as promoters is well known in the art, as is the selection of appropriate expression vectors (see e.g. Sambrook et al. "Molecular Cloning: A laboratory Manual", 2.sup.nd ed., vols. 1-3, Cold Spring Harbor Laboratory, 1989, the content of which is herein incorporated by reference).

[0192] Said vector may thus further comprise at least one promoter operably linked to said at least one MTS sequence, said at least one CDS sequence, said at least one 3' sequence.

[0193] Said promoter may e.g. be a constitutive promoter, such as e.g. a CMV promoter.

[0194] Said vector may further comprise a termination site.

[0195] Said vector may further comprise one of several of the following expression control sequences: insulators, silencers, IRES, enhancers, initiation sites, termination signals.

[0196] Said vector may further comprise an origin of replication.

[0197] Preferably, said promoter and said origin of replication are adapted to the transduction or infection of animal or human cells, preferably to the transduction or infection of human cells.

[0198] Said vector can e.g. a plasmid, or a virus, such as an integrating viral vector, e.g. a retrovirus, an adeno-associated virus (AAV), or a lentivirus, or is a non-integrating viral vector, such as an adenovirus, an alphavirus, a Herpes Simplex Virus (HSV).

[0199] Said vector may further comprise a nucleic acid coding for a detectable marker, such as a FLAG epitope or green fluorescent protein (GFP)

[0200] The present invention also relates to a process for the production of a vector of the invention, which comprises: [0201] providing a vector, and depleting from its original 3'UTR, if any, [0202] inserting in this vector at least one MTS nucleic acid sequence, at least one CDS sequence, and at least one 3' sequence as above-described.

[0203] As already-mentioned, said vector should preferably not comprise any sequence corresponding to the 3'UTR of a nuclearly-encoded but non-mitochondrially-targeted mRNA. To the best of the inventors' knowledge, all commercially-available vectors contain such an inappropriate 3'UTR; according to the present invention, such a 3'UTR should hence be removed from the vector. It may e.g. be replaced by an appropriate 3' sequence corresponding to a nuclearly-encoded and mitochondrially-targeted mRNA.

Nucleic Acid Construct of the Invention:

[0204] The nucleic acid construct which is carried by the vector of the invention is also encompassed by the present invention. The present invention more particularly relates to a non-naturally occurring nucleic acid construct.

[0205] A non-naturally occurring nucleic acid construct of the invention comprises: [0206] at least one mitochondrion-targeting nucleic acid sequence (referred to as MTS nucleic acid sequence), [0207] at least one nucleic acid sequence which encodes said protein in accordance with the universal genetic code (referred to as CDS sequence), and [0208] at least one 3' nucleic acid sequence, which is located in 3' of said at least one MTS nucleic acid sequence and of said at least one CDS sequence.

[0209] Said at least one MTS nucleic acid sequence is: [0210] the MTS RNA sequence of a nuclearly-encoded mitochondrially-targeted mRNA, such as the MTS RNA sequence of a naturally-occurring nuclearly-encoded mitochondrially-targeted mRNA, or [0211] the cDNA sequence of such a RNA, or [0212] a DNA sequence coding for such a MTS RNA sequence, or [0213] a conservative variant or fragment of such a RNA or DNA MTS sequence, which derives therefrom by deletion and/or substitution and/or addition of one or several nucleotides, but has retained a mitochondrion-targeting function.

[0214] Said at least one 3' nucleic acid sequence is: [0215] the 3'UTR sequence of a nuclearly-encoded mitochondrially-targeted mRNA, such as the 3'UTR sequence of a naturally-occurring nuclearly-encoded mitochondrially-targeted mRNA, or [0216] the cDNA sequence of such a RNA, or [0217] a DNA sequence coding for such a 3'UTR sequence, or [0218] a conservative variant or fragment of such a RNA or DNA 3'UTR sequence, which derives therefrom by deletion and/or substitution and/or addition of one or several nucleotides, and which, when replacing the wild-type 3'UTR of said nuclearly-encoded mitochondrially-targeted mRNA, still allows for a mitochondrial targeting of the resulting mRNA.

[0219] It may be provided that, when said at least one MTS nucleic acid sequence is the MTS RNA sequence of a naturally-occurring nuclearly-encoded mitochondrially-targeted mRNA, or the cDNA sequence of such a mRNA, or a DNA sequence coding for such a MTS RNA sequence in accordance with the universal genetic code, said at least one nucleic acid CDS sequence is not the CDS of this naturally-occurring nuclearly-encoded mitochondrially-targeted mRNA.

[0220] It may be provided that, when said at least one 3' nucleic acid sequence is the 3'UTR sequence of a naturally-occurring nuclearly-encoded mitochondrially-targeted mRNA, or the cDNA sequence of such a mRNA, or a DNA sequence coding for such a 3'UTR sequence, said at least one CDS sequence is not the CDS of this naturally-occurring nuclearly-encoded mitochondrially-targeted m RNA.

[0221] It may be provided that, when said at least one MTS nucleic acid sequence and said 3' nucleic acid sequence, respectively, are the MTS and 3'UTR sequences of a naturally-occurring nuclearly-encoded mitochondrially-targeted mRNA, or the cDNA sequences of such a mRNA, or a DNA sequence coding for such a mRNA sequence, then said at least one CDS sequence is not the CDS of this naturally-occurring nuclearly-encoded mitochondrially-targeted mRNA.

[0222] Preferably, said nucleic acid construct does not comprise any sequence which would be identical to: [0223] the 3'UTR of a naturally-occurring mRNA which is a nuclearly-transcribed but not-mitochondrially-targeted mRNA, or [0224] the cDNA sequence of such a 3'UTR sequence, or [0225] a DNA sequence coding for such a naturally-occurring mRNA 3'UTR in accordance with the universal genetic code.

[0226] The resulting nucleic acid construct does not use a post-translation importation pathway, but uses a co-translation importation pathway from nucleus to said mitochondrion.

[0227] The present invention more particularly relates to a non-naturally occurring nucleic acid construct which comprises: [0228] at least one mitochondrion-targeting nucleic acid sequence (referred to as MTS nucleic acid sequence), [0229] at least one nucleic acid sequence which encodes said protein in accordance with the universal genetic code (referred to as CDS sequence), and [0230] at least one 3' nucleic acid sequence, which is located in 3' of said at least one MTS nucleic acid sequence and of said at least one CDS sequence, wherein said at least one MTS nucleic acid sequence is: [0231] the cDNA sequence of the MTS RNA sequence of a nuclearly-encoded mitochondrially-targeted mRNA, or [0232] a conservative variant or fragment of such a cDNA sequence, which derives therefrom by deletion and/or substitution and/or addition of one or several nucleotides, but has retained a mitochondrion-targeting function, wherein said at least one 3' nucleic acid sequence is: [0233] the cDNA sequence of the 3'UTR sequence of a nuclearly-encoded mitochondrially-targeted mRNA, or [0234] a conservative variant or fragment of such a cDNA 3'UTR sequence, which derives therefrom by deletion and/or substitution and/or addition of one or several nucleotides, and which, when replacing the wild-type 3'UTR of said nuclearly-encoded mitochondrially-targeted mRNA, still allows for a mitochondrial targeting of the resulting mRNA, provided that, when said at least one MTS nucleic acid sequence and said at least one 3' nucleic acid sequence, respectively, are the MTS and 3'UTR sequences of a naturally-occurring nuclearly-encoded mitochondrially-targeted mRNA, or the cDNA sequences of such a mRNA, or a DNA sequence coding for such a mRNA sequence, then said at least one CDS sequence is not the CDS of this naturally-occurring nuclearly-encoded mitochondrially-targeted mRNA, and wherein said nucleic acid construct does not comprise any sequence which would be identical to: [0235] the 3'UTR of a naturally-occurring mRNA which is a nuclearly-transcribed but not-mitochondrially-targeted mRNA, or [0236] the cDNA sequence of such a 3'UTR sequence, or [0237] a DNA sequence coding for such a naturally-occurring mRNA 3'UTR in accordance with the universal genetic code.

[0238] Each and every feature, herein and above described for the MTS, CDS, 3' nucleic acid sequences in relation with the vector of the invention, and notably those features relating to the MTS, CDS, 3' nucleic acid sequences, of course applies mutatis mutandis to the nucleic acid contruct of the invention, and more particularly to the non-naturally occurring nucleic acid contruct of the invention.

[0239] Hence, it notably follows that: [0240] a MTS nucleic acid sequence of said nucleic acid construct can be the MTS nucleic acid sequence of ACO2, or of SOD2, or of ATP5b, or of UQCRFS1, or of NDUFV1, or of NDUFV2, or of ALDH2, or of COX10; [0241] a 3' nucleic acid sequence of said nucleic acid construct can be: [0242] the 3'UTR sequence of ACO2, or of SOD2, or of ATP5b, or of UQCRFS1, or of NDUFV1, or of NDUFV2, or of ALDH2, or of COX10, or of AK2, or [0243] the cDNA sequence of such a 3'UTR sequence, or [0244] a DNA sequence coding for such a 3'UTR sequence in accordance with the universal genetic code; and that [0245] illustrative nucleic acid constructs of the invention comprise or consist of a sequence of SEQ ID NO:21 (COX10 MTS-re-coded ATP6-COX10 3'UTR), and/or of SEQ ID NO:22 (SOD2 MTS-re-coded ATP6-SOD2 3'UTR), and/or of SEQ ID NO:25 (COX10 MIS-re-coded ND1-COX10 3'UTR), and/or of SEQ ID NO:26 (COX10 MTS-re-coded ND4-COX10 3'UTR).

[0246] Said non-naturally occurring nucleic acid construct may be transfected in a cell in the form of naked DNA, or in the form of a plasmid. Any transfection technology which is found convenient by the skilled person is convenient. The skilled person may e.g. proceed by electroporation, DEAE Dextran transfection, calcium phosphate transfection, cationic liposome fusion, creation of an in vivo electrical field, DNA-coated microprojectile bombardment, ex vivo gene therapy, and the like.

[0247] Said non-naturally occurring nucleic acid construct may of course alternatively and/or complementarily be inserted into a vector, such as a viral vector.

[0248] The vector and nucleic acid construct of the invention are useful for nucleic acid therapy, e.g. to reverse a cellular dysfunction caused by a mutation in nucleic acid coding for a mitochondrial protein. They enable to restore a protein function in a cell.

Engineered Cell:

[0249] The present invention also relates to an engineered cell which has been transduced or infected by a vector according to the invention, and/or transfected by a nucleic acid construct according to the invention.

[0250] Preferably, said engineered cell is an engineered animal or human cell, most preferably to a mammalian engineered cell, still more preferably to an engineered human cell.

[0251] Said engineered cell may e.g. be a bone-marrow cell, a clonal cell, a germ-line cell, a post-mitotic cell, such as a cell of the central nervous system; a neuronal cell, a retinal ganglion cell, a progenitor cell; or a stem cell, a hematopoietic stem cell, a mesenchymal stem cell. Preferably, said engineered cell is a neuronal cell, a retinal ganglion cell.

[0252] Said transduction, infection or transfection may be impleted by any means available to the skilled person, e.g. by electroporation, DEAE Dextran transfection, calcium phosphate transfection, cationic liposome fusion, creation of an in vivo electrical field, DNA-coated microprojectile bombardment, injection with a recombinant replicative-defective virus, homologous recombination, ex vivo gene therapy, a viral vector, naked DNA transfer, and the like.

[0253] Said engineered cell may e.g. be a cell, such as a neuronal cell, collected from a patient suffering from a disease related to a mitochondrial dysfunction. The vector and nucleic acid construct of the invention can indeed be used for ex vivo cell therapy.

Pharmaceutical Compositions and Applications:

[0254] The present invention also relates to a pharmaceutical composition comprising at least one vector according to the invention, or at least one nucleic acid construct according to the invention, or at least one engineered mammalian cell according to the invention.

[0255] The present invention also relates to a drug comprising at least one vector according to the invention, or at least one nucleic acid construct according to the invention, or at least one engineered mammalian cell according to the invention.

[0256] The compositions of the present invention may further comprise at least one pharmaceutically and/or physiologically acceptable vehicle (diluent, excipient, additive, pH adjuster, emulsifier or dispersing agent, preservative, surfactant, gelling agent, as well as buffering and other stabilizing and solubilizing agent, etc.).

[0257] Appropriate pharmaceutically acceptable vehicles and formulations include all known pharmaceutically acceptable vehicles and formulations, such as those described in "Remington: The Science and Practice of Pharmacy", 20.sup.th edition, Mack Publishing Co.; and "Pharmaceutical Dosage Forms and Drug Delivery Systems", Ansel, Popovich and Allen Jr., Lippincott Williams and Wilkins.

[0258] In general, the nature of the vehicle will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise, in addition to the one or more contrast agents, injectable fluids that include pharmaceutically and physiologically acceptable fluids, including water, physiological saline, balanced salt solutions, buffers, aqueous dextrose, glycerol, ethanol, sesame oil, combinations thereof, or the like as a vehicle. The medium also may contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like. The carrier and composition can be sterile, and the formulation suits the mode of administration.

[0259] The composition can be e.g., be in the form of a liquid solution, suspension, emulsion, capsule, sustained release formulation, or powder.

[0260] The pharmaceutical composition and drug of the invention are useful for the therapeutic and/or palliative and/or preventive treatment of a disease, condition, or disorder related to a defect in activity or function of mitochondria.

[0261] A mitomap is available on the MITOMAP website; this site notably provides with a list of mitochondrial disease-associated mutations.

[0262] Scientific publication review relating to mitochondrial disease, condition, or disorder notably comprise Carelli et al. 2004 (Progress in Retinal and Eye Research 23: 53-89), DiMauro 2004 (Biochimica et Biophysica Acta 1659:107-114), Zeviani and Carelli 2003 (Curr Opin Neurol 16:585-594), and Schaefer et al. 2004 (Biochimica et Biophysica Acta 1659: 115-120), the contents of which being herein incorporated by reference.

[0263] Diseases, conditions or disorders related to a defect in mitochondria activity or function notably comprise myopathies and neuropathies, such as optic neuropathies. Examples of mitochondrial diseases, conditions or disorders comprise: aging, aminoglycoside-induced deafness, cardiomyopathy, CPEO (chronic progressive external ophtalmoplegia), encephalomyopathy, FBSN (familial bilateral stritial necrosis), KS (Kearns-Sayre) syndrome, LHON (Leber's hereditary optic neuropathy), MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), MERRF (myoclonic epilepsy with stroke-like episodes), MILS (maternally-inherited Leigh syndrome), mitochondrial myopathy, NARP (neropathy, ataxia, and retinis pigmentosa), PEO, SNE (subacute necrotizing encephalopathy).

[0264] Optic neuropathies notably comprise: [0265] Leber's hereditary optic neuropathy (LHON), which involves one or several point mutation(s) in mitochondrial DNA, more particularly point mutation(s) in the ND1 and/or ND4 and/or ND6 gene(s), such as G3460A (ND1 mutation), G11778A (ND4 mutation), T144840 (ND6 mutation), [0266] the dominant optic atrophy (DOA), also known as Kjer's optic neuropathy, which involves a defect in nuclear gene OPA1, [0267] the FBSN, MILS and NARP, which are the result of a mutation in the MTATP6 gene (defective ATP synthesis), which can be corrected by restoring the activity of function of ATP6.

[0268] Leber's hereditary optic neuropathy (LHON) was the first maternally inherited disease to be associated with point mutations in mitochondrial DNA and is now considered the most prevalent mitochondrial disorder. The pathology is characterized by selective loss of retinal ganglion cells leading to central vision loss and optic atrophy, prevalently in young males. It is a devastating disorder with the majority of patients showing no functional improvement and remaining within the legal requirement for blind registration. Other clinical abnormalities have also been reported in LHON patients. These include postural tremor, peripheral neuropathy, non-specific myopathy, movement disorders and cardiac arrhythmias [8]. The three most common pathogenic mutations from LHON affect complex I ND1 and/or ND4 and/or ND6 genes with the double effect of lowering ATP synthesis and increasing oxidative stress chronically.

[0269] Each of said disease, condition or disorder could be corrected by restoring activity or function of the mutated DNA.

[0270] Example 1 below illustrates the rescue of an ATP6 activity or function with a vector and nucleic acid of the invention. Example 2 below illustrates the rescue of a ND1 and ND4 activity or function with a vector and nucleic acid of the invention (fibroblasts collected from LHON patients).

[0271] The pharmaceutical compositions or drugs of the invention are more particularly intended for the therapeutic and/or palliative and/or preventive treatment of a myopathy or of an optic neuropathy, such as LHON, DOA, FBSN, MILS or NARP.

[0272] The present invention relates to the use of at least one vector or nucleic acid construct for in vivo or ex vivo therapy of a subject or patient in need of a therapeutic, palliative or preventive treatment of a disease, condition, or disorder related to a defect in activity or function of mitochondria.

[0273] The present invention also relates to the use of at least one vector or nucleic acid construct of the invention, or of at least one engineered cell of for the treatment of a disease, condition, or disorder related to a defect in activity or function of mitochondria, and more particularly for the production of a composition, pharmaceutical composition or drug intended for the treatment of such a disease condition, or disorder.

[0274] The present invention more particularly relates to a method for the therapeutic and/or palliative and/or preventive treatment of a disease, condition, or disorder related to a defect in activity or function of mitochondria, which comprises: [0275] administering to a subject or patient in need thereof a vector and/or a nucleic acid construct and/or an engineered cell of the invention, in a quantity effective for the therapeutic and/or palliative and/or preventive treatment of said subject or patient, [0276] ex vivo treating cells collected from a subject or patient in need thereof, and returning the treated cells to the subject or patient.

[0277] The term "comprising", which is synonymous with "including" or "containing", is open-ended, and does not exclude additional, unrecited element(s), ingredient(s) or method step(s), whereas the term "consisting of" is a closed term, which excludes any additional element, step, or ingredient which is not explicitly recited.

[0278] The term "essentially consisting of" is a partially open term, which does not exclude additional, unrecited element(s), step(s), or ingredient(s), as long as these additional element(s), step(s) or ingredient(s) do not materially affect the basic and novel properties of the invention.

[0279] The term "comprising" (or "comprise(s)") hence includes the term "consisting of" ("consist(s) of"), as well as the term "essentially consisting of" ("essentially consist(s) of"). Accordingly, the term "comprising" (or "comprise(s)") is, in the present application, meant as more particularly encompassing the term "consisting of" ("consist(s) of"), and the term "essentially consisting of" ("essentially consist(s) of").

[0280] Each of the relevant disclosures of all references cited herein is specifically incorporated by reference.

[0281] The present invention is illustrated by the following examples, which are given for illustrative purposes only.

EXAMPLES

Example 1: Allotopic Expression of the ATP6 Mitochondrial Gene is Significantly Improved by the Localization of its mRNAs to the Surface of Mitochondria Leading to an Efficient Import of the Precursor

Abstract

[0282] It is clear that impairment of mitochondrial energy metabolism is the key pathogenic factor in a growing number of neurodegenerative disorders. With the discovery of mtDNA mutations, the replacement of defective genes became an important goal for mitochondrial geneticists worldwide. Unfortunately, before the present invention, it was still not possible to introduce foreign genes into the mitochondria of mammalian cells.

[0283] To circumvent this problem, allotopic expression in the nucleus of genes encoded by mitochondrial DNA (mtDNA), became an attractive idea. However, for most is mitochondrial genes tested, there were important limitations related to the high hydrophobicity of the corresponding proteins, which impedes their mitochondrial translocation.

[0284] We herein elucidate the mechanisms that enable the delivery of mRNAs encoding mitochondrial proteins to the organelle surface, and demonstrate that this delivery depends on two sequences: the region coding for the mitochondrial targeting sequence (MTS) and the 3'UTR. mRNA sorting to mitochondrial surface permits to optimize allotopic approach, by enhancing the mitochondrial import efficiency of the precursor synthesized in the cytosol. As an illustration of this mechanism, we have chosen to utilize the sequence coding for the MTS and the 3'UTR of two nuclear genes encoding mitochondrial proteins: COX10 and SOD2 associated to a recoded mitochondrial ATP6 gene. Indeed, COX10 and SOD2 mRNAs localize to the mitochondrial surface in HeLa cells. HeLa cells transfected with these constructions express an Atp6 protein which is successfully delivered to the mitochondria. Hence, we have been able to optimize the allotopic approach for Atp6, and our procedure will next be tried to rescue mitochondrial dysfunction in patients presenting ATP6 mutations.

Introduction

[0285] To examine the possibility that allotopic expression of mtDNA genes could be optimized by a targeted localization of the mRNA to the mitochondrial surface, we have chosen to utilize the sequences coding for the MTS and the 3'UTR of two nuclear genes encoding mitochondrial proteins: COX10 and SOD2 associated to an reengineered nucleus-localized ATP6 gene. COX10 encodes a highly hydrophobic protein of the inner mitochondrial membrane, its mRNAs localizes to the mitochondrial surface [5]. SOD2 encodes a mitochondrial protein involved in detoxification, its mRNA, as COX10 mRNA, localizes to the mitochondrial surface [5] and a recent report described that in HeLa cells, its 3'UTR is associated to the mitochondrial surface via the Akap121 protein [6]. The ability to synthesize and direct the Atp6 protein to mitochondria was examined in Hela cells for 4 plasmids: two of them only contain the mts of COX10 or SOD2, and the two other possess both the MTS and the 3'UTR of each gene. Hybrid mRNAs were detected for each construction in both transiently and stably transfected cells. Further, Atp6 protein was also visualized by indirect immunofluorescence associated to the surface of mitochondria. Mitochondria isolated from transfected cells were examined for the presence of Atp6 protein. Remarkably, hybrids mRNAs possessing both the MTS and the 3'UTR of either COX10 and SOD2 allow the synthesis of a polypeptide which is imported in a highly efficient way from the cytosol into the mitochondria. Thus, the strategy of directing a hybrid mRNA to the mitochondrial surface significantly improves the feasibility of the allotopic approach for mitochondrial genes.

Material and Methods:

[0286] Plasmid Construction:

[0287] The full-length ATP6 mitochondrial gene was reengineered after the production of the 677 pb product by RT-PCR (Superscript III one step RT-PCR Platinium Taq HiFi, Invitrogen), using total RNA from HeLa cells. The KR product obtained was cloned in the PCR 2.1-Topo vector (Invitrogen, Life technologies). In this vector, we recoded 11 non-universal codons in the ATP6 gene by four rounds of in vitro mutagenesis (Quik change Multi site-directed mutagenesis kit; Stratagene, La Jolla, Calif.). Six oligonucleotide primers were designed to alter AUA codons to AUG and UGA to UGG (Table 1).

TABLE-US-00006 TABLE 1 In vitro mutagenesis of the ATP6 mitochondrial gene Length Name sequence (bp) ATP6.1 CAATGGCTAATCAAACTAACCTCAAAACAAATGA 78 TGACCATGCACAACACTAAAGGACGAACCTGGTC TCTTATGCTA (SEQ ID NO: 1) ATP6.2 TCTATGAACCTAGCCATGGCCATCCCCTTATGGG 54 CGGGCACAGTGATTATGGGC (SEQ ID NO: 2) ATP6.3 CCCATGCTAGTTATTATCGAAACCATCAGCCTAC 51 TCATTCAACCAATGGCC (SEQ ID NO: 3) ATP6.4 ACCCTAGCAATGTCAACCATTAAC (SEQ ID 24 NO: 4) ATP6.5 ACTAAAGGACGAACCTGGTCTCTTATGCTAGTAT 42 CCTTAATC (SEQ ID NO: 5) ATP6.6 ACACCAACCACCCAACTATCTATGAACCTAGCCA 42 TGGCCATC (SEQ ID NO: 6)

[0288] The intermediate construct was sequenced for accuracy. To this recoded ATP6, we appended in frame either the MTS of COX10 or 5002, obtained by RT-PCR using total RNA from HeLa cells (Superscript III one step RT-PCR Platinium Taq HiFi; Invitrogen, Life technologies). For COX10 we amplified the sequence corresponding to the first 28 amino acids, for SOD2 the sequence coding for the first 30 amino acids. Oligonucleotide primers used for the amplification include at its 3' extremity a Sal1 restriction site for the subsequent cloning in frame with the reengineered ATP6 gene which possesses a Sal1 restriction site at its 5' extremity (Table 2).

TABLE-US-00007 TABLE 2 Oligonucleotides primers for RT-PCR analysis RT-PCR product length Name 5' Primer (5'-3') 3'Primer (5'-3') (bp) ATP6 GTCGACCGCATGA CCGGGCGGCCGCTGT 677 ORF ACGAAAATCTGTTC GTTGTCGTGCAGGTA GCTTCATTCATT GAGGCTTAC (SEQ ID NO: 7) (SEQ ID NO: 8) MTS CGCTCTAGAATGG GCGGTCGACTTCAAG 84 COX10 CCGCATCTCCGCA ATACCAGACAGAGCC CACTCTC TCC (SEQ ID NO: 9) (SEQ ID NO: 10) 'UTR CCCGATCGGAGCA CGCACGCGTAAAGCT 1429 COX10 CTGGGACGCCCAC TCTACAAATGTGAAGG CGCCCCTTTCCC CTGTAACA (SEQ ID NO: 11) (SEQ ID NO: 12) MTS CGCTCTAGAATGTT GTCGACCGCGTCGGG 90 SOD2 GAGCCGGGCAGTG GAGGCTGTGCTTCTG TGCGGC CCT (SEQ ID NO: 13) (SEQ ID NO: 14) 3'UTR ACCACGATCGTTAT CGCACGCGTCAATCA 215 SOD2 GCTGAGTATGTTAA CACAAAGCATTTACTA GCTCTTTA TTTTC (SEQ ID NO: 15) (SEQ ID NO: 16) COX6c ATGGCTCCCGAAG CTGAAAGATACCAGC 250 TTTTGCCAAAACCT CTTCCTCATCTC (SEQ ID NO: 17) (SEQ ID NO: 18) SOD2 CGACTACGGCGCC CTGGAACCTCACA TCAACGC (SEQ ID NO: 58)

[0289] The final sequences of the fusion ATP6 genes were checked for accuracy, and inserted in the pCMV-Tag 4A vector (Stratagene, La Jolla Calif.), which will direct the synthesis of the protein via the CMV promoter and its detection by the presence of a FLAG epitope tag appended to the C-terminal region of Atp6. To obtain hybrid mRNAs which will also contain the 3'UTR of COX10 or SOD2 genes we replaced the SV40 polyA signal present in the pCMV-Tag 4A vector (positions 1373-1679) using Pvu1 and Mlu1 restriction enzymes, by the 1429 bp of the full-length COX10 3'UTR or the 215 pb of the SOD2 3'UTR. Both 3'UTR were first obtained by RT-PCR using RNAs purified from HeLa cells and specific oligonucleotide primers containing Pvu1 and Mlu1 restriction sites at each end (cf. Table 2 above). PCR fragments were first cloned in the PCR 2.1-Topp vector (Invitrogen, Life technologies) and sequenced to verify that no mistakes were generated before subcloning in the pCMV-Tag 4A vector. The four final constructs were entirely sequenced for accuracy using specific oligonucleotide primers to verify the full-length sequences of either the fusion ATP6 genes or the 3'UTR regions appended to them. Final sequences inserted in the pCMV-Tag 4A vectors are shown in FIG. 1B.

[0290] The sequence of recoded ATP6 (SEQ ID NO: 27) is shown on FIG. 9. The MTS and 3'UTR sequences of COX10 and SOD2 are shown on FIG. 10 (SEQ ID NO: 30, 47, 31 and 60).

Cell Culture and Transfection:

[0291] We cultured HeLa cells with RPMI medium complemented with 10% of foetal bovine serum (Gibco, Invitrogen), gentamicin (0.01%), 2 mM glutamine, optionally with pyruvate (e.g., 2.5 mM), optionally with antibiotics (such as 100 u/mL penicillin, 100 .mu.g/mL streptomycin). They were transfected with FuGENE 6 transfection reagent as recommended by the manufacturer (Roche Biochemicals, Indianapolis). Briefly, monolayer Hela cells were seeded a day before transfection at 50% confluence, so the next day they will be at approximately 80% confluence, the cells were plated in a medium without antibiotics. 2 microgrammes of different plasmids purified with Quiagen plasmid midi kit (Quiagen; Valencia, Calif.) were used. Between 48 to 60 hr later, 80% of the transfected cells were used either for immunochemistry analyses or RNA and mitochondria extractions. The remaining 20% of cells were selected for neomycine, G418, resistance (selectable marker present in the pCMV-Tag 4A vector) at a final concentration of 1 mg/ml. Stable clones were expanded for several weeks, immunochemistry analyses were performed. Mitochondria were also isolated to determine the import ability of the Atp6 protein.

Immunocytochemistry:

[0292] Coverslips were placed on the bottom of 24-well dishes and HeLa cells seeded at approximately 50% confluence (80000 cells). 60 hours after transfection, cells were fixed with 2% paraformaldehyde in PBS for 15 min and processed for indirect immunofluorescence. After permeabilization of the cells for 5 min with Triton 1% in PBS, cells were incubated for one hour in PBS with 1% BSA before the addition of the primary antibodies: mouse monoclonal anti-Flag M2 antibodies (Stratagene, La Jolla Calif.) or mouse monoclonal anti-ATP synthase subunit beta (Molecular Probes, Invitrogen). Both antibodies were used at a final concentration of 1 microgramme/ml. The incubation with primary antibodies was performed for either 2 hr at room temperature or overnight at 4.degree. C. After washing the primary antibody three times five min with PBS, cells were incubated with the secondary antibody: labeled goat-anti-mouse.quadrature.IgG Alexa Fluor 488 (Molecular Probes, Invitrogen). This antibody was used in 1% BSA-PBS at 1:600 dilution and placed on the top of the coverslips for two hr. The cells were, subsequently, washed once in PBS for 5 min. For DNA and mitochondria staining, a second wash was performed with 0.3 microgrammes/ml of DAPI (Sigma, Saint Louis, Mich.) and 100 nM of MitoTracker Deep Red 633 (Molecular probes, Invitrogen) for 20 min. A last 10 min wash was performed in PBS and the coverslips were mounted using Biomeda Gel/Mount. Immunofluorescence was visualized in a Leica DM 5000 B Digital Microscope. Digital images were acquired and processed with the MetaVue imaging system software.

[0293] Mitochondria Isolation and Western Blot Analysis:

[0294] Between 20 to 40 millions or 100 millions of transiently or stably transfected HeLa cells were treated with trypsin (Gifco, Invitrogen) for 5 min and spin down. One wash in PBS was performed. The pellets were resuspended in 10 ml of homogenization buffer: 0.6 Mannitol, 30 mM Tris-HCl PH 7.6, 5 mM MgAc and 100 mM KCl, 0.1% fatty acid-free bovine serum albumin (BSA), 5 mM beta-mercaptoethanol and 1 mM PMFS. To the resuspended cells 0.01% of digitonin was added. After a 4 min incubation on ice the homogenization was performed with 15 strokes in a Dounce glass homogenenizer with a manually driven glass pestle type B. Homogenates are centrifuged for 8 min at 1000 g at 4.degree. C. to pellet unbroken cells and nuclei. Since many mitochondria remain trapped in this pellet, it was resuspended and rehomogenized again with 5 ml of homogenization buffer and 25 additional strokes. Then a second round of centrifugation under the same conditions was performed. Both supernatants were assembled and centrifuged again to discard any nuclear or cell contaminant. The supernatant obtained was centrifuged at 12000 g at 4.degree. C. for 30 min to pellet mitochondria. Four washes in homogenization buffer were performed to free the mitochondrial fraction of particles containing membranes, reticulum endoplasmic and proteases. The last two washes were performed in a homogenization buffer devoid of BSA and PMFS to allow a better estimation of the protein concentration in the final mitochondrial fraction and its subsequent analysis by proteinase K digestion. Protein concentrations in the extracts were measured using the dye-binding assay Bradford. To determine whether Atp6 was translocated into the organelle, 15 microgrammes of mitochondrial proteins were treated with 200 microgrammes/ml of proteinase K (PK) at 0.degree. C. for 30 min. Samples were then resolved in 4-12 gradient or 12% polyacrylamide SDS-PAGE, and transferred to nitrocellulose. Filters were probed with the following antibodies: mouse monoclonal anti-Flag M2 antibodies (Stratagene, La Jolla Calif.) which recognizes the nuclear recoded Atp6 protein in which a flag epitope was appended at its C-terminus or mouse monoclonal anti-ATP synthase subunit alpha (Molecular Probes, Invitrogen), which recognizes the 65 kDa nuclear encoded alpha-subunit of the ATP synthase, Complex V. Immunoreactive bands were visualized with anti-mouse coupled to horseradish peroxidase (1:10000) followed by ECL Plus detection (Amersham International) according to the manufacturer's instructions.

[0295] Five independent mitochondria purifications from cells stably transfected with either SOD2.sup.MTS ATP6-3'UTR.sup.SV40 or SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 vectors were performed. The amount of precursor and mature forms of ATP6 in mitochondria, as well as the quantities of both the mature form of ATP6 and ATP .quadrature. resistant to PK proteolysis were compared by densitometric analyses (Quantity One, Biorad software system). The significance of the differences observed was validated with a paired Student's t-test.

RNA Extraction and RT-PCR Analyses:

[0296] Mitochondria extractions were performed as described in the precedent section, with the following modifications: 400 millions of cells were treated with 250 .mu.g/ml cycloheximide for 20 minutes at 37.degree. C. To HB was added 200 Mg/ml cycloheximide, 500 .mu.g/ml heparine and 1/1000 RNase inhibitor (rRNasin, Promega). The last pellet of crude mitochondria associated with polysomes (M-P) was stored at -80.degree. C. until RNA extraction. Free-cytoplasmic polysomes (F-P) were obtained from the post-mitochondrial supernatant fraction by sedimentation through a step gradient of 2 M and 0.5 M sucrose. RNAs from these two fractions, as well as total RNAs from each stably transfected cell line, were obtained using RNeasy Protect Mini kit (Qiagen). Generally, 10 millions of cells are sufficient to obtain approximately 30 microgrammes of total RNA. The presence of the hybrid ATP6 mRNA was examined using primers which recognize the first 27 nt of either COX10 or SOD2 MTS and a primer which recognize the last 27 nt of the ATP6 ORF. For the pCMV-Tag 4A vector containing both the MTS and the 3'UTR of COX10 or SOD2, we used a primer recognizing the last 27 nt of each 3'UTR. 100 ng of RNA was used for reverse transcription (cf. Table 1 above). The products were then subjected to 25 cycles of PCR using Superscript III one step RT-PCR Platinium Taq kit (Invitrogen). As an internal control a 250 nt fragment within the ORF of COX6c gene, encoding a mitochondrial protein, was also amplified. Ten percent of the amplified products were run in agarose gels, and the quantities of amplified products reflecting hybrid ATP6 mRNA amount in each preparation was estimated using the Photocap software (Vilber Lourmat; Torcy, France).

[0297] Tables 1 and 2 above, and table 9 below, show primer sequences, the expected sizes of the FOR products, the quantity of RNA used for reverse-transcription and the number of FOR cycles performed.

[0298] Densitometric analyses (Quantity One, Bio-Rad software) were performed the amount of both hybrid ATP6 and SOD2 transcripts in either mitochondrion-bound polysomes or free-cytoplasmic polysomes. Three independent RNA preparations from M-P and F-P fractions were subjected three times to RT-PCR analyses.

TABLE-US-00008 TABLE 9 RT-PCR Polysomal RNAs product Total RNAs (M-P/F-P) length Primers Quantity Cycle Quantity Cycle mRNA (bp) 5' Primer 3'Primer (ng) numbers (ng) numbers SOD2.sup.MTS 780 MTS ATP6 200 28 250 28 ATP6 SOD2 5' ORF3' ATP6 677 ATP6 ATP6 50 28 150 20 ORF 5' ORF 3' SOD2 785 SOD2 5' 3'UTR 100 28 20 20 SOD2 3' COX6c 250 COX6c 5' COX6c 3' 200 28 250 20

Results:

Construction of Reengineered Mitochondrial ATP6 Gene for Allotopic Expression

[0299] To accomplish allotopic expression we synthesized the full-length version of nuclear-encoded ATP6 mitochondrial gene converting codons AUA to AUG and codons UGA to UGG. Indeed, AUA in the mitochondrial genetic system leads to the insertion of a methionine, but according to the universal code it is an isoleucine. Additionally, UGA into mitochondria codes for a tryptophan, whereas in the cytosol it represents a stop codon. We, therefore, recoded all 11 mitochondrial codons present in ATP6 ensuring the accurate translation of the transcript by cytoplasmic ribosomes. These alterations were performed by four rounds of in vitro mutagenesis using six independent oligonucleotide primers (Table 1) and the Quik change Multi site-directed mutagenesis kit (Stratagene, La Jolla, Calif.).

[0300] The concept of allotopic approach has important implications for the development of therapies to patients with mitochondrial DNA mutations. However, up today a major obstacle remains to be overcome and is the targeting of the recoded protein to mitochondria. We then decided to force the localization of the recoded ATP6 mRNA to the mitochondrial surface. The rationale behind this specific mRNA targeting is to allow a co-translational import mechanism which will maintain the precursor in an import competent conformation impeding its aggregation before or during translocation through the TOM (Translocase of the outer membrane) and TIM (Translocases of the inner membrane) import complexes. Two sequences within mRNAs are believed to be involved in their localization to the mitochondrial membrane: the sequence coding for the MTS and the 3'UTR. We have chosen two nuclearly-encoded mitochondrial genes, which mRNAs are preferentially localized to the surface of mitochondria in HeLa cells: COX10 and SOD2 [5]. Interestingly, the SOD2 mRNA has been shown to be associated to the mitochondrial surface via its 3'UTR and the Akap121 protein.

[0301] We therefore, obtained four different plasmids.

[0302] Two contain either the MTS of COX10 or the sequence encoding the first 30 amino acids of SOD2 (=the 20 amino acids of the MTS sequence of SOD2, and the ten consecutive amino acids that follows within the SOD2 sequence, i.e., fragment 1-30 of SEQ ID NO:49), in frame with the AUG codon of the recoded ATP6 gene (COX10 MTS-recoded ATP6-SV40 3' UTR; SOD2 MTS-recoded ATP6-SV40 3' UTR). In these plasmids, the SV40 polyA signal functions as the 3'UTR.

[0303] The other two combine both the MTS and the 3'UTR of COX10 and SOD2 respectively, and do not comprise the cytosolic 3'UTR of SV40 (COX10 MTS-recoded ATP6-COX10 3' UTR; SOD2 MTS-recoded ATP6-SOD2 3' UTR).

[0304] FIGS. 1A and 1B illustrate the constructs obtained and the full-length sequences inserted in the pCMV-Tag 4A vector than we named respectively: COX10 MTS-nATP6, SOD2 MTS-nATP6 and COX10 MTS-nATP6-COX10 3'UTR and SOD2 MTS-nATP6-SOD2 3'UTR.

Detection of Hybrid ATP6 mRNAs in Transiently and Stably HeLa Transfected Cells

[0305] To determine whether transfected cells express hybrid ATP6 mRNAs, steady-state levels of the transcripts were measured in both transiently and stably transfected cells after the isolation of total RNAs. 100 ng of total RNAs were subjected to RT-KR analyses using specific primer oligonucleotides for hybrid ATP6 mRNA. COX6c gene encoding a mitochondrial protein was used as an internal control, with specific primers allowed the amplification of a 250 bp fragment (FIG. 2). RNAs from non-transfected HeLa cells as well as HeLa cells transfected with the empty pCMV-Tag 4A vector were also tested as negative controls. FIG. 2 shows a 780 bp PCR product corresponding to the amplification of the first 27 nt of COX10 ORF and the last 27 nt of the ATP6 ORF in cells transfected with both COX10 MTS-nATP6 and COX10 MTS-nATP6-COX10 3'UTR vectors. Additionally, RNAs isolated from cells transfected with the COX10 MTS-nATP6-COX10 3'UTR vector amplified a 2374 bp product corresponding to the entire ATP6 ORF and the full-length COX10 3'UTR.

[0306] The results obtained with RNAs purified from transfected cells with SOD2 MTS-nATP6 and SOD2 MTS-nATP6-SOD2 3'UTR vectors show that hybrid ATP6 transcript was detected as a 780 nt amplified product. Further, the SOD2 MTS-nATP6-SOD2 3'UTR region amplified a 1060 bp fragment, corresponding to the entire ATP6 ORF and the full-length SOD2 3'UTR. These results indicate that HeLa cells express the reengineered ATP6 gene. Moreover, no significant differences in the steady-state levels of hybrid mRNAs were found by the addition of either COX10 3'UTR or SOD2 3'UTR.

[0307] To examine the ability of SOD2 signals associated to the recoded ATP6 gene to direct hybrid mRNAs to the mitochondrial surface, we determined their subcellular localization in the four stably cell lines obtained. In this purpose, we isolated RNAs from mitochondrion-bound polysomes (M-P) and free-cytoplasmic polysomes (F-P) and we determined by AT-FOR the steady-state levels of hybrid mRNAs in both polysomal populations (FIG. 12B). As internal controls, the subcellular distribution of endogenous mitochondrial ATP6. SOD2 and COX6c mRNAs were determined. Endogenous ATP6 mRNA exclusively localized to the mitochondrial compartment as expected. Besides, endogenous SOD2 mRNA is enriched in mitochondrion-bound polysomes (M-P), whereas COX6c mRNA is preferentially detected in free-cytoplasmic polysomes (F-P) as we have previously observed. The SOD2MTSATP6-3'UTRSOD2 vector directed the synthesis of a hybrid mRNA that was almost undetectable in free-cytoplasmic polysomes. Hybrid mRNA produced from the SOD2MTSATP6-3'UTRSV40 plasmid was also detected preferentially in mitochondrion-bound polysomes. However, it was also present in free-cytoplasmic polysomes (FIG. 12B). Densitometric analyses were performed to determine the amount of both endogenous SOD2 and hybrid ATP6 mRNAs in each polysomal population examined. SOD2 mRNA signal in mitochondrion-bound polysomes was 85.6%.+-.6.15 in cell lines expressing the SOD2MTSATP6-3'UTRSV40 plasmid and 82.5%.+-.4.87 in cells expressing the SOD2MTSATP6-3'UTRSOD2 vector. Interestingly, it was found for the ATP6 hybrid mRNA that only 72.4%.+-.5.2 localized to the mitochondrial surface in cells expressing the SOD2MTSATP6-3'UTRSV40 vector. Instead, in cells expressing the SOD2MTSATP6-3'UTRSOD2 vector 84.6%.+-.4.7 of the hybrid mRNA localized to the mitochondrial surface (FIG. 120). These values were significantly different according to the paired Student's t-test (P<0.0034, n=6). Thus, the combination of both the MTS and 3'UTR of SOD2 to the reengineered ATP6 gene leads to the synthesis in the nucleus of a transcript which was almost exclusively sorted to the mitochondrial surface. Indeed, its subcellular distribution is not significantly different to the one of the endogenous SOD2 mRNA.

TABLE-US-00009 TABLE 10 ATP6 mRNA signal localized at the ATP6 mRNA within Hela cells mitochondrial surface the mitochondria With a cytosolic 3'UTR 72.4% .+-. 5 0.71 .+-. 0.12 (SV40 3' UTR) With a mitochondrial .sup. 82.5 .+-. 4.8 1.28 .+-. 0.24 3'UTR (SOD2 3'UTR), and without any cytosolic 3'UTR

Detection of ATP6 Allotopic Expression in HeLa Cells by Indirect Immunofluorescence

[0308] We analyzed the ability of the reengineered ATP6 product to localize to mitochondria in vivo.

[0309] For this, we appended a Flag epitope in frame to the C-terminus of the ATP6 ORF and we examined stably transfected cells by indirect immunofluorescence (FIG. 13). HeLa cells transfected with the empty pCMV-Tag 4A vector were used as negative controls and showed a low diffused signal in cytoplasm when antibodies to Flag were used (FIG. 13, left panel). Stably transfected cells with either SOD2.sup.MTS ATP6-3'UTR.sup.SV40 or SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 vectors were visualized by indirect immunofluorescence using antibodies to Flag (FIG. 14, left panel) and to ATP synthase subunit .alpha. (FIG. 13 middle panel). For each cell type visualized, a merged image in association with DAPI staining is shown in the right panel. A typical punctuate mitochondrial pattern was observed in cells expressing the recoiled ATP6 polypeptides, when the Flag antibody was used. This indicates that fusion ATP6 proteins localized to mitochondria.

[0310] Immunocytochemistry to detect the flag epitope in HeLa cells transiently or stably transfected with the four pCMV-Tag 4A vectors showed a typical punctuate mitochondrial pattern, suggesting that the fusion Atp6 protein had been localized within the mitochondria (FIG. 3). Indeed, this typical punctuate mitochondrial pattern was also observed using either the mitochondrion-specific dye Mito Tracker Red or specific antibodies anti-ATP synthase subunit beta. HeLa cells transfected with the empty pCMV-Tag 4A vector were used as negative controls and showed a diffuse cytoplasmic distribution but with a low intensity (FIG. 3). The localization patterns of the different Atp6 peptides which synthesis were directed by the four pCMV-Tag 4A vectors were essentially identical confirming that both COX10 and SOD2 sequences successfully allowed the reengineered Atp6 protein to localize to the mitochondria in vivo.

Translocation of the Fusion Atp6 Protein into Mitochondria of HeLa Cells

[0311] To determine whether reengineered ATP6 gene products are efficiently imported into mitochondria in vivo, mitochondria isolated from stably transfected HeLa cells were subjected to western blot analysis (FIG. 4). We visualized two forms with anti-flag antibodies of approximately 30 and 20 kDa, representing the precursor and mature forms of the recoded ATP6 protein.

[0312] The predicted molecular weights of both proteins are respectively 34 and 30 kDa, larger than the ones implied by the molecular weight markers. This discrepancy has often been observed when extremely hydrophobic proteins were migrated in SDS-PAGE. In general, the electrophoretic mobility on SDS-PAGE of proteins encoded by mtDNA is higher than the one expected for their theoretical molecular weights.

[0313] The steady-state levels of both polypeptides are similar in the two cell lines examined: cells transfected with MTS COX10-nATP6 vector (MTS COX10-nATP6), and cells transfected with MTS COX10-nATP6-COX10 3'UTR (MTS COX10-nATP6-3'UTR).

[0314] To determine the amounts of the recoded ATP6 polypeptides produced in HeLa cells expressing either SOD2.sup.MTS ATP6-3'UTR.sup.SV40 or SOD2.sup.MTS ATP6-3'UTR'.sup.SOD2 vectors, we compared six independent mitochondrial extractions (FIG. 14A). Both precursor and mature polypeptides were equally abundant in mitochondria from each cell line, indeed the expression of SOD2.sup.MTS ATP6-3'YTR.sup.SV40 vector leads to an accumulation of 61.4%.+-.6 of the precursor form. Instead, SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 vector directed the synthesis of 64.4%.+-.6.5 of the precursor. These values were not significantly different according to the paired Student's t-test. Similar results were obtained when total extracts from each cell line were examined by Western blotting. This data is in agreement with the overall amounts of hybrid ATP6 mRNAs detected when total RNAs from cell lines expressing either SOD2.sup.MTS ATP6-3'UTR.sup.SV40 or SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 vectors were subjected to RT-PCR analyses (FIG. 12A). Therefore, the steady-state levels of the ATP6 precursor and its ability to recognize the TOM complex in the outer mitochondrial membrane do not depend on the presence of the SOD2 3'UTR. Notably, the relative proportions of ATP6 precursor and mature forms were analogous to the ones shown in cells for highly hydrophobic proteins en route to the mitochondria.

[0315] When mitochondria were treated with 150 or 200 microgrammes/ml of proteinase K (PK) the precursor forms of the fusion ATP6 protein were sensitive to proteolysis in both cells lines. In contrast the mature form of ATP6 is resistant to PK digestion, especially in cells expressing the MTS COX10-nATP6-COX10 3'UTR transcript. Indeed, in these cells the amount of the mature ATP6 protein is approximately 185% higher than in cells expressing the MTS COX10-nATP6 mRNA. These data strongly indicates that not only the precursor polypeptide is correctly addressed to the surface of mitochondria, as we observed by indirect immunofluorescence (FIG. 3), but also that it was efficiently translocated into the organelle and correctly processed. Moreover, FIG. 4 shows that the quantity of the mature form of the Atp6 protein and the 65 kDa ATPalpha protein inside the mitochondria were quite similar after proteinase K digestion. Therefore, the use of COX10 MTS allows an efficient mitochondrial translocation of the recoded ATP6 protein, and when COX10 MTS is combined to the 3'UTR of COX10 a significant more efficient in vivo translation/import of the allotopically expressed ATP6 gene is obtained.

[0316] FIG. 14B upper panel shows a schematic representation of the theoretically expected ATP6 import intermediate. The hydrophobic passenger ATP6 precursor can be trapped en route to the matrix and a mitochondrial processing peptidase can cleave the MTS. Nevertheless, the rest of the protein remained accessible to PK action and therefore becoming undetectable on Western blotting. Instead, the fraction of the ATP6 protein which can be completely translocated is insensitive to PK-induced proteolysis and can therefore be integrated into the inner mitochondrial membrane, hence, remaining detectable on immunoblotting.

[0317] FIG. 14B shows that precursor forms of the fusion proteins were sensitive to proteolysis in both cell line examined. Nearly all the ATP6 precursor signal disappeared after PK digestion, so precursors that were engaged in the process of translocation or loosely attached to the outer mitochondrial membrane but not fully translocated, were entirely digested (FIG. 14B, middle panel). In contrast, a significant amount of the mature form of ATP6 is resistant to PK digestion, indicating its location inside the organelle. To examine the levels of another complex V protein in these cells, immunoblots were performed using anti-ATP synthase .alpha. antibody. This naturally imported mitochondrial protein was present at similar extents in all cells tested. Only one band of approximately 65 kDa was visualized suggesting that either we were unable to discriminate the precursor and mature forms of this protein under the electrophoretic conditions used or precursor polypeptides were very rapidly and efficiently translocated. Additionally, no major differences of the ATP synthase .alpha. signals were detected after PK treatment, confirming the integrity of the mitochondrial isolations (FIG. 14B, middle panel). To compare the import efficiency of the recoded ATP6 proteins in cells transfected with either SOD2.sup.MTS ATP6-3'UTR.sup.SV40 or SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 vectors, we measured the amount of the mature form of ATP6 insensitive to PK digestion in each cell line, after normalization with the amount of ATP synthase .alpha. resistant to PK proteolysis. Results for six independent mitochondrial extractions subjected to immunoblotting analyses were shown in FIG. 15B, lower panel. Overall results show that both SOD2 signals lead to a high efficient import of the recoded ATP6 precursor. Remarkably, the level of the mature form insensitive to PK proteolysis in cells transfected with SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 (1.28.+-.0.24) was 1.8 fold higher than in cells expressing the SOD2.sup.MTS ATP6-3'UTR.sup.SV40 (0.71.+-.0.12). The difference measured was significant according to the paired Student's t-test (P<0.0022, n=6). This observation could be related to the higher enrichment in the mitochondrion-bound polysomes of the corresponding mRNA (FIG. 12B).

[0318] The question arises whether imported reengineered ATP6 proteins were assembled into the ATP synthase complex. The complex is organized in F0-F1 domains, F1 sector is a water-soluble unit located in the matrix and having the ability to hydrolyse ATP. The F0 domain is embedded in the inner membrane and is composed by hydrophobic subunits forming a proton pathway. ATP6 is an intrinsic protein of F0, composed of five putative transmembranous .alpha.-helices. In contrast, ATP synthase .alpha. is a located in the matrix F1 domain. Studies performed with bovine heart mitochondria demonstrated that ATP6 was degraded at a very low rate when F0 subunits were subjected to trypsine treatment. Therefore, we treated mitochondria with both PK and Triton X-100 (1%). The detergent disrupts both mitochondrial membranes and theoretically leads to the entire proteolysis of mitochondrial proteins, demonstrating their localization somewhere inside the organelle in a protease-sensitive form. FIG. 3C shows that indeed ATP synthase .alpha. was fully digested by PK; instead a significant amount of ATP6 remained insensitive to PK proteolysis. This result suggests that the recoded ATP6 was assembled into complex V.

Discussion:

[0319] Recent, epidemiological studies demonstrated that as a group, disorders of the mitochondrial function affect at least 1 in 5000 of the population, making them among the most common genetically determined disorders. In spite of the fact that over the last decade, the underlying genetic bases of several mitochondrial diseases involving central nervous system degeneration, no effective therapy is available for mitochondrial disorders. Pathogenic point mutations of genes encoded by the mitochondrial genome have been described as the cause of many mitochondrial disorders. A possible therapeutic approach is therefore to exploit the natural mitochondrial protein import pathway. The basic concept is to introduce a wild-type copy of the mutated mitochondrial gene into the nucleus and import normal copies of the gene product into mitochondria from cytosol. This concept has been termed allotopic expression and several reports in yeast described that a number of non mitochondrial polypeptides can be relocated to the mitochondrial matrix simply by conjugating a targeting sequence to their N-terminus. However, when this approach has been tried in mammalian cells using different MTS and genes encoded by mtDNA, precursors were not imported efficiently into mitochondria. By consequence, the rescue of mitochondrial defect in patient cells was not only partial but also temporary [7]. Therefore, up today the spectrum of mtDNA encoded polypeptides than can be successfully expressed and integrated into mitochondrial respiratory chain complexes is very limited. This limitation is thought to be the consequence of the high hydrophobicity nature of mtDNA encoded proteins, which possess transmembrane domains refractive to mitochondrial import. The precursor synthesized in the cytosol could lack the import-competent structure required for an efficient mitochondrial membrane translocation.

[0320] The concept of mesohydrophobicity is likely to be an important factor for mitochondrial import competency. Mesohydrophobicity describes the average hydrophobicity in a window of 60-80 amino acids, together with the calculation of the most hydrophobic 17-amino acid segment. This calculation could predict importability of hydrophobic peptides. Using their algorithm, we analyzed this correlation to assess the mitochondrial importability of SOD2.sup.MTS ATP6 gene product and compared to ATP6, COX8 and SOD2 polypeptides as well as the previously tested fusion protein COX8.sup.MTS ATP6: as the wild-type ATP6, both fusion proteins examined cannot be translocated into mitochondria, mainly due to the high hydrophobicity of ATP6. Hence, a possibility that can allow the import of a recoded ATP6 protein into the organelle is that the precursor is engaged in a co-translational pathway of import. Thereby, the precursor would be maintained in a loosely folded nonaggregated conformation required for translocation through the mitochondrial import apparatus. To overcome this limitation and try to develop a more long-term and definitive rescue of mtDNA mutations by allotopic expression leading to its application in gene therapy, we decided to construct nuclear versions of the mtDNA encoded ATP6 gene in which we appended the signals intended for forcing the hybrid mRNA to localize to the mitochondrial surface. We have chosen COX10 and SOD2 genes, which transcripts are enriched in the mitochondrial surface.

[0321] We were able to demonstrate that the association to a recoded ATP6 gene of both the MTS and 3'UTR signals leading to a mRNA delivery to the mitochondrial surface unambiguously improves the feasibility of the allotopic approach for mitochondrial genes. Indeed, not only we were able to visualize the protein in the mitochondria by indirect immunofluorescence, but most important the amounts of the processed Atp6 polypeptide inside the organelle were quite similar to the naturally imported ATPalpha protein. This result strongly indicates that the recoded Atp6 precursor was efficiently imported, the improvement we were able to produce as compared to other recent reports [1], [2], [3], [4] is certainly due to the fact that the hybrid mRNA was addressed to the mitochondrial surface, therefore enhancing the coupling between translation and mitochondrial import processes.

[0322] Most interestingly, we obtained a gradual improvement, indeed the use of either COX10 or SOD2 MTS alone, gave a good result in which approximately 50% of the mature ATP6 protein is translocated inside the mitochondria. When each MTS was combined to the corresponding 3'UTR, at least 85% of the mature ATP6 protein is insensitive to proteinase K digestion indicating that almost all the protein synthesized in the cytoplasm is successfully translocated inside the mitochondria.

[0323] To accomplish allotopic expression, the localization of an mRNA to the mitochondrial surface has never been tried before. In the allotopic approaches reported, even though different MTS were appended to recoded mitochondrial genes, all the constructs examined contained at their 3' extremities the SV40 polyA signal that does not lead to any specific subcellular localization of the transcript. Our data clearly demonstrate that the association to a recoded ATP6 gene of both the MTS and 3'UTR signals of the SOD2 gene leads to a high efficient delivery of the hybrid mRNA to the mitochondrial surface. This improves unambiguously the feasibility of the allotopic approach for this mitochondrial gene. Indeed, not only were we able to visualize ATP6 protein in the mitochondria by indirect immunofluorescence but definitely the amount of the processed ATP6 polypeptide inside the organelle was quite similar to the naturally imported ATP synthase subunit .alpha., a Complex V component, such as is ATP6. These data strongly indicate that recoded ATP6 precursors were successfully imported. The improvement we obtained compared to a recent report, which measured 18.5% of the precursor translocated, is certainly due to the localization of hybrid mRNAs to the mitochondrial surface. This specific localization obviously enhances the coupling between translation and import processes, therefore, diminishing the block of the precursor during its translocation through the TOM and TIM complexes. It is worth mentioning that we obtained a gradual improvement on mitochondrial import of the ATP6 precursor. When both the MTS and the 3'UTR of SOD2 were combined, the amount of fully translocated ATP6 protein was 1.8-fold higher than when just the MTS was present. This is likely related to the improvement of mRNA sorting to the mitochondrial surface when both cis-acting elements of SOD2 were associated to the recoded ATP6 gene. Remarkably, proteolysis insensitivity of the translocated ATP6 protein in the presence of both PK and Triton X-100 suggested that the protein could be correctly assembled in the F0 domain of the respiratory chain Complex V. Notably, combining the cis-acting elements of the COX10 gene to the recoded ATP6 gene, we obtained a very efficient mitochondrial import ability of the fusion protein. Indeed, COX10 mRNA codes for a highly hydrophobic protein involved in Complex IV biogenesis, and SOD2 mRNA is enriched at the mitochondrial surface in human cells.

[0324] We clearly demonstrated that the association to a recoded ATP6 gene of both the MTS and 3'UTR signals of either SOD2 or COX10 genes leads to a high efficient delivery of hybrid ATP6 mRNAs to the mitochondrial surface, especially when both the MTS and the 3'UTR of SOD2 or COX10 were associated to the reengineered ATP6 gene. This specific subcellular localization of hybrid mRNAs leads to a high efficiency in the mitochondrial translocation of the recoded ATP6 proteins. Remarkably, when both the MTS and the 3'UTR of either SOD2 or COX10 were combined, the amount of fully translocated ATP6 protein was 1.8-fold higher than when the MTS was associated to the cytosolic SV40 3'UTR. Therefore, the improvement of mRNA sorting to the mitochondrial surface when both cis-acting elements of SOD2 or COX10 were associated to the recoded ATP6 gene definitely increase the amount of the processed ATP6 polypeptide inside the organelle which became quite similar to the naturally imported ATP synthase subunit .alpha., a Complex V component, such as is ATP6. Thus, by directing a hybrid mRNA to the mitochondrial surface we significantly improve the feasibility of the allotopic approach for the ATP6 mitochondrial gene.

[0325] In conclusion, we optimize the allotopic expression approach for ATP6, by the use of mRNA targeting signals without any amino acid change in the protein that could affect biologic activity.

[0326] This approach becomes henceforth available to rescue mitochondrial deficiencies caused by mutations in mtDNA genes.

Example 2: Correct Mitochondrial Localization of the Recoded Mitochondrial ND1 and ND4 Genes in Fibroblastes from LHON Patients

[0327] The three most common pathogenic mutations from LHON affect complex I ND1, ND4 and ND6 genes with the double effect of lowering ATP synthesis and increasing oxidative stress chronically.

[0328] Since we have demonstrated that reengineered mitochondrial Atp6 proteins were successfully translocated inside the mitochondria in HeLa cells (see example 1 above), we decided to synthesize recoded mitochondrial genes ND1 and ND4. To ensure the efficient import of the allotopically expressed proteins we appended to them signals which will direct the corresponding mRNAs to the mitochondrial surface. We have chosen to use the MTS of COX10 gene alone or in combination with its entire 3'UTR. FIGS. 5A and 5B illustrate the constructs obtained and the full-length sequences inserted in the pCMV-Tag 4A vector.

Material and Methods:

[0329] Cell culture and transfection: Fibroblasts were obtained from LHON patients of the Hopital Necker Enfants Malades, Paris, France (Departement de Genetique). We cultured these cells with D-MEM medium complemented with 10% of foetal bovine serum, pyruvate, gentamicin (0.01%), and 2 mM glutamine. When indicated cells were grown in glucose-free medium supplemented with 10 mM galactose.

[0330] Fibroblasts were transfected with EUGENE 6 transfection reagent as recommended by the manufacturer (Roche Biochemicals, Indianapolis). Briefly, monolayer fibroblast cells were seeded a day before transfection at 50% confluence, so the next day they will be at approximately 80% confluence, the cells were plated in a medium without antibiotics. 2 microgrammes of different plasmids purified with Quiagen plasmid midi kit (Quiagen; Valencia, Calif.) were used. Between 48 to 60 hr later, 80% of the transfected cells were used for immunochemistry analyses. The remaining 20% of cells were selected for neomycine, G418, resistance (selectable marker present in the pCMV-Tag 4A vector) at a final concentration of 0.25 mg/ml. Stable clones were expanded for several weeks.

Optimized Recoding into Human Genetic Code

[0331] mtDNA has been recoded according to human genetic code, taking into account the preferred codon usage in human:

TABLE-US-00010 TABLE 3 preferred codon usage in human Human preferred Source codon usage ARG CGA -- CGC -- CGG -- CGU -- AGA -- AGG AGG LEU CUA -- CUC -- CUG CUG CUU -- UUA -- UUG -- SER UCA -- UCC UCC UCG -- UCU -- AGC -- AGU -- THR ACA -- ACC ACC ACG -- ACU -- PRO CCA -- CCC CCC CCG -- CCU -- ALA GCA -- GCC GCC GCG -- GCU -- GLY GGA -- GGC GGC GGG -- GGU -- VAL GUA -- GUC -- GUG GUG GUU -- LYS AAA -- AAG AAG ASN AAC AAC AAU -- GLN CAA -- CAG CAG HIS CAC CAC CAU -- GLU GAA -- GAG GAG ASP GAC GAC GAU -- TYR UAC UAC UAU -- CYS UGC UGC UGU -- PHE UUC UUC UUU -- ILE AUA -- AUC AUC AUU -- MET AUG AUG TRP UGG UGG % GC 63/42

ND1 and ND4 Constructs:

[0332] COX10 MTS-nND1-SV40 3' UTR, COX10 MTS-nND4-SV40 3' UTR, COX10 MTS-nND1-COX10 3'UTR, and COX10 MTS-nND4-COX10 3'UTR were produced as described above in example 1 for ATP6. The resulting sequences are shown on FIG. 5B (SEQ ID NO:23, 24, 25, 26, respectively).

Results:

[0333] Detection of ND1 Ailotopic Expression in Fibroblasts from LHON Patients Presenting the G3460A ND1 Mutation

[0334] We analyzed the ability of the reengineered ND1 product to localize to mitochondria in viva. Immunocytochemistry analyses were performed to detect the flag epitope in fibroblasts, from a patient presenting the ND1 gene mutated, transiently transfected with either COX10 MTS-nND1-SV40 3' UTR or COX10 MTS-nND1-COX10 3'UTR showed a typical punctuate mitochondrial pattern, suggesting that the fusion Nd1 protein had been localized within the mitochondria (FIG. 6). Indeed, this typical punctuate mitochondrial pattern was also observed using specific antibodies anti-ATP synthase subunit beta. Cells transfected with the empty pCMV-Tag 4A vector were used as negative controls and showed a diffuse cytoplasmic distribution but with a low intensity (FIG. 6). The localization patterns of Nd1 peptides which synthesis were directed by the two vectors examined were essentially identical confirming that COX10 sequences successfully allowed the reengineered Nd1 protein to be addressed inside the mitochondria.

Detection of ND4 Allotopic Expression in Fibroblasts from LHON Patients Presenting the G11778A ND4 Mutation

[0335] Two plasmids directing the synthesis in the cytosol of a recoded wild-type ND4 gene were obtained. One of them, COX10 MTS-nND4-SV40 3' UTR, possesses appended to the N-terminus of the protein the sequence corresponding to the first 28 amino acids of Cox10. The second one, COX10 MTS-nND4-COX10 3'UTR, has in addition at the end of the ORF the full-length 3'UTR of COX10. Fibroblasts from a patient presenting 100% of mtDNA molecules with the G11778A ND4 mutation were transiently transfected with either one of these plasmids. 60 h later cells were fixed and visualized to determine the ability of the COX10 sequences to target the recoded protein to the mitochondria. FIG. 7 shows that in both cases the fusion MTS Cox10ND4Flag protein did have a punctuate staining pattern, which is very similar to the one observed for the same cells with the naturally imported mitochondrial protein ATP synthase subunit beta. Thus, implying that the recoded Nd4 fusion protein was imported into mitochondria.

[0336] In conclusion, as for the mitochondrial ATP6 gene, we were able to optimize the allotopic expression approach for ND1 and ND4 genes, by the simply use of mRNA targeting signals without any amino acid change in the protein that could affect biologic activity.

Growth Ability of LHON Fibroblasts in Galactose Medium

[0337] Fibroblasts presenting the G3460A ND1 mutation were grown with galactose, which slowly enters glycolysis as compared to glucose. FIG. 8 shows major differences in cell growth after six day culture: fibroblasts presented a severe growth defect, less that 10% of the cells survived in medium containing galactose as compared to cells seeded in glucose-rich medium. Stably transfected fibroblasts with the MTS COX10-nND1-COX10 3'UTR vector had a markedly improved rate of growth in galactose compared with that of non-transfected cells. This result implies that the mitochondrially imported recoded ND1 protein had assembled successfully into functional complex I allowing, therefore, a rescue of mitochondrial dysfunction in these cells.

Example 3: Rescue of Mitochondrial Deficiency Causing Human Diseases (Transfection of Fibroblasts from a NARP Patient)

[0338] We also determined whether the reengineered ATP6 protein would be able to rescue mitochondrial deficiency in cells having a mutated ATP6 gene.

[0339] We obtained fibroblasts from a patient presenting NARP disease caused by the T8993G mutation in the ATP6 gene.

[0340] Fibroblasts were cultured on media containing sodium pyruvate and relatively high amounts of FBS, more particularly: [0341] on a medium containing glucose (D-MEM with L-glutamine, 4500 mg/L D-glucose, 110 mg/L sodium pyruvate 2.5 mM, FBS 15%, uridine 28 microM), or [0342] on a medium, which does not contain glucose, but contain galactose (liquid D-MEM (1.times.), with L-Glutamine without Glucose, sodium pyruvate 2.5 mM, galactose 10 mM, FBS 15%, uridine 28 microM).

[0343] Stably transfected cells expressing the nuclear version of ATP6 associated with either SOD2 MTS alone, or in combination with SOD2 3'UTR, were obtained. Respiratory chain activity has been examined by the ability of these cells to grow in a medium in which glucose has been replaced by galactose for either 10 or 20 days. NARP cells expressing the empty vector had a low survival rate (30%). Cells expressing ATP6 with either the MTS of SOD2 or both the MTS and the 3'UTR of SOD2 present a growth survival of approximately 60%. If the selection was maintained for 20 days, the survival growth rate of cells expressing our optimized vectors was superior to 80% (FIG. 16). Only subtle differences of survival rate were observed for fibroblasts expressing either the vector with both the MTS and 3'UTR of the SOD2 gene or the vector with the SOD2 MTS associated to the SV40 3'UTR. This, is certainly due to the fact that these cells are heteroplasmic for the T8993G mutation, indeed they possess approximately 10% of the wild-type gene. Therefore, in their mitochondria probably 10% of a functional ATP6 protein could be assembled in Complex V. We can envision that the expression of the vector with SOD2 MTS associated to the SV40 3'UTR, will lead to the mitochondrial import of enough ATP6 protein to allow the cells to growth a good rate in galactose medium.

[0344] Preliminary measures of the real amount of ATP produced in vitro by fibroblasts expressing either ones of our vectors clearly show a difference in the activity of Complex V related to the presence of either SOD2 3'UTR or SV40 3'UTR. Hence, when compared to control fibroblasts (100% of ATP synthesis in galactose medium) NARP fibroblasts expressing the vector with SOD2 MTS associated to the SV40 3'UTR had 50%, representing an increase compared to non transfected NARP cells (30%) but was less important when compared to the amount found in cells expressing the vector which combines to the recoded ATP6 gene both the MTS and the 3'UTR of the SOD2 gene (approximately 85%); cf. FIG. 19. By consequence, a more complete and efficient rescue of mitochondrial dysfunction is obtained when allotopic approach implies the presence of both the MTS and 3'UTR targeting signals.

TABLE-US-00011 TABLE 11 Rate of ATP Survival rate on synthesis on Fibroblasts galactose galactose Control 100% 100% NARP (mutated ATP6) 30% 30% NARP + cytosolic 3'UTR 60% 50% (SV40 3'UTR) NARP + mitochondrial 60% 85% 3'UTR (SOD2 3'UTR)

Example 4: Rescue of Mitochondrial Deficiency Causing Human Diseases (Transfection of Fibroblasts from LHON Patients)

[0345] The applicability potential of the improved allotopic expression approach of the inventors has been further confirmed by examining two other mtDNA genes involved in LHON. The fibroblasts obtained presented a total homoplasmy of the mutation; indeed all the molecules of mitochondrial DNA are mutated.

[0346] Fibroblasts were cultured on media containing sodium pyruvate and relatively high amounts of FBS, more particularly: [0347] on a medium containing glucose (D-MEM with L-glutamine, 4500 mg/L D-glucose, 110 mg/L sodium pyruvate 2.5 mM, FBS 15%, uridine 28 microM), or [0348] on a medium, which does not contain glucose, but contain galactose (liquid D-MEM (1.lamda.), with L-Glutamine without Glucose, sodium pyruvate 2.5 mM, galactose 10 mM, FBS 15%, uridine 28 microM).

[0349] The engineered nucleus-localized versions of ND1 and ND4 were obtained; ND1 and ND4 transcripts possess both at their 5' and 3' extremities COX10 mRNA targeting sequences. Stable transfections of these constructions in fibroblasts from LHON's patients with either ND1 or ND4 mutations were performed. Indirect immunofluorescence showed that both proteins localize to the surface of mitochondria in vivo. The OXPHOS activity of these cells has been also examined by growing in a galactose rich medium. Interestingly, fibroblast cells allotopically expressing the wild-type ND4 protein showed a markedly improved rate of growth on galactose medium. This improvement is higher when both the MTS and 3'UTR of COX10 were associated to the ND4 gene (54.3%) as compared to that of mock transfected cells (8%) or to the cells transfected with the ND4 gene associated to the MTS of COX10 and the cytosolic SV40 3'UTR (12.7%) (FIG. 17, MTS: ND4 associated to COX10 MTS and the SV40 3'UTR, 3'UTR: ND4 associated to both the MTS and 3'UTR of COX10). This data imply that in spite of the presence in these cells of the ND4 mutated polypeptide, the wild-type protein was successfully imported into the organelle and assembled in Complex I. Preliminary experiments, of in vitro measurements of ATP synthesis confirm these results, indeed in untransfected cells very little ATP was synthesized in galactose medium (14% of the control level measured in healthy fibroblasts), when cells express the ND4 gene associated to the MTS of COX10 and the cytosolic SV40 3'UTR an increased is observed (56%). Remarkably, this increase is more important when cells express the ND4 gene associated to both the MTS and 3'UTR of COX10 (84%).

[0350] Hence, our results undeniably confirm that we have optimized the allotopic approach for three mtDNA encoded genes by the use of mRNA targeting signals, without any amino acid change in the proteins. This is particularly the case when our vectors presented both the MTS and the 3'UTR targeting signals of a mRNA which exclusively localized to the mitochondrial surface.

TABLE-US-00012 TABLE 12 Rate of ATP Survival rate on synthesis on Fibroblasts galactose galactose Control 100% 100% LHON (mutated ND4) 8% 14% LHON + cytosolic 3'UTR 12.7% 56% (SV40 3'UTR) LHON + mitochondrial 54.3% 84% 3'UTR (COX10 3'UTR)

Example 5: Transduction of Retinal Ganglion Cells

[0351] The inventors obtained, by in vitro mutagenesis, reengineered ND1, ND4, ND6 and ATP6 genes, which possess the most common mutations found in LHON's and NARP's patients: G3460A, G11778A, T144840 and T8993G respectively. Both wild-type and mutated genes have been integrated in the p-AAV-IRES-hrGFP vector, which will allow the production of infectious recombinant human Adeno-Associated Virus Type 2 (AAV2) virions. For all constructions, each nuclear version of mtDNA genes is associated to the two mRNA targeting sequences of the COX10 gene, which allow the enrichment of corresponding mRNAs at the surface of mitochondria. In accordance with the present invention, this will ensure the efficient delivery of the polypeptides inside the organelle.

[0352] Retinal ganglion cells (RGC) represent the primary cellular target of the pathogenic process of LHON disease.

[0353] The inventors purified RGCs from adult rat retina, thereby obtaining enriched RGC populations, and maintained them in culture for two weeks. Mitochondria are distributed along actin filaments and they specifically concentrated at the extremities of neuron extensions. The inventors transfected these cells with the mutated version of the ND1 gene. Preliminary results showed that the expression at high levels of the mutated protein during 8 days leads to an abnormal distribution of mitochondria along the neurite and cone extensions (FIG. 18).

BIBLIOGRAPHIC REFERENCES CITED IN THE EXAMPLES

[0354] 1. Owen, R., et al., Recombinant Adeno-associated virus vector-based gene transfer for defects in oxidative metabolism. Hum. Gene Ther., 2000. 11: p. 2067-2078. [0355] 2. Guy, J., et al., Rescue of a mitochondrial deficiency causing Leber Hereditary Optic Neuropathy. Ann. Neurol., 2002. 52: p. 534-542. [0356] 3. Manfredi, G., et al., Rescue of a Deficiency in ATP Synthesis by Transfer of MTATP6, a mitochondrial DNA-encoded gene to the nucleus. Nature Genet., 2002. 30: p. 394-399. [0357] 4. Oca-Cossio, J., et al., Limitations of allotopic expression of mitochondrial genes in mammalian cells. Genetics, 2003. 165: p. 707-720. [0358] 5. Sylvestre, J., et al., The role of the 3'UTR in mRNA sorting to the vicinity of mitochondria is conserved from yeast to human cells. Md. Biol. Cell, 2003. 14: p. 3848-3856. [0359] 6. Ginsberg, M. D., et al., PKA-dependent binding of mRNA to the mitochondrial AKAP121 protein. J. Mol. Biol., 2003. 327(4): p. 885-897. [0360] 7. Smith, P. M., et al., Strategies for treating disorders of the mitochondrial genome. Biochem. Biophys. Acta, 2004. 1659: p. 232-239. [0361] 8. Carelli et al., Progress in Retinal and Eye Research, 2004. 23: p. 53-89

Sequence CWU 1

1

60178DNAArtificial SequenceSynthetic Primer 1caatggctaa tcaaactaac ctcaaaacaa atgatgacca tgcacaacac taaaggacga 60acctggtctc ttatgcta 78254DNAArtificial SequenceSynthetic Primer 2tctatgaacc tagccatggc catcccctta tgggcgggca cagtgattat gggc 54351DNAArtificial SequenceSynthetic Primer 3cccatgctag ttattatcga aaccatcagc ctactcattc aaccaatggc c 51424DNAArtificial SequenceSynthetic Primer 4accctagcaa tgtcaaccat taac 24542DNAArtificial SequenceSynthetic Primer 5actaaaggac gaacctggtc tcttatgcta gtatccttaa tc 42642DNAArtificial SequenceSynthetic Primer 6acaccaacca cccaactatc tatgaaccta gccatggcca tc 42739DNAArtificial SequenceSynthetic Primer 7gtcgaccgca tgaacgaaaa tctgttcgct tcattcatt 39839DNAArtificial SequenceSynthetic Primer 8ccgggcggcc gctgtgttgt cgtgcaggta gaggcttac 39933DNAArtificial SequenceSynthetic Primer 9cgctctagaa tggccgcatc tccgcacact ctc 331033DNAArtificial SequenceSynthetic Primer 10gcggtcgact tcaagatacc agacagagcc tcc 331138DNAArtificial SequenceSynthetic Primer 11cccgatcgga gcactgggac gcccaccgcc cctttccc 381239DNAArtificial SequenceSynthetic Primer 12cgcacgcgta aagcttctac aaatgtgaag gctgtaaca 391333DNAArtificial SequenceSynthetic Primer 13cgctctagaa tgttgagccg ggcagtgtgc ggc 331433DNAArtificial SequenceSynthetic Primer 14gtcgaccgcg tcggggaggc tgtgcttctg cct 331536DNAArtificial SequenceSynthetic Primer 15accacgatcg ttatgctgag tatgttaagc tcttta 361636DNAArtificial SequenceSynthetic Primer 16cgcacgcgtc aatcacacaa agcatttact attttc 361727DNAArtificial SequenceSynthetic Primer 17atggctcccg aagttttgcc aaaacct 271827DNAArtificial SequenceSynthetic Primer 18ctgaaagata ccagccttcc tcatctc 27191337DNAArtificial SequenceSynthetic COX10 MTS-nATP6 19gaattcgccc ttcgctctag aatggccgca tctccgcaca ctctctcctc acgcctcctg 60acaggttgcg taggaggctc tgtctggtat cttgaagtcg accgcatgaa cgaaaatctg 120ttcgcttcat tcattgcccc cacaatccta ggcctacccg ccgcagtact gatcattcta 180tttccccctc tattgatccc cacctccaaa tatctcatca acaaccgact aatcaccacc 240caacaatggc taatcaaact aacctcaaaa caaatgatga ccatgcacaa cactaaagga 300cgaacctggt ctcttatgct agtatcctta atcattttta ttgccacaac taacctcctc 360ggactcctgc ctcactcatt tacaccaacc acccaactat ctatgaacct agccatggcc 420atccccttat gggcgggcac agtgattatg ggctttcgct ctaagattaa aaatgcccta 480gcccacttct taccacaagg cacacctaca ccccttatcc ccatgctagt tattatcgaa 540accatcagcc tactcattca accaatggcc ctggccgtac gcctaaccgc taacattact 600gcaggccacc tactcatgca cctaattgga agcgccaccc tagcaatgtc aaccattaac 660cttccctcta cacttatcat cttcacaatt ctaattctac tgactatcct agaaatcgct 720gtcgccttaa tccaagccta cgttttcaca cttctagtaa gcctctacct gcacgacaac 780acagcggccg cccggaaggg cgaattcgat atcaagctta tcgataccgt cgacctcgag 840gattacaagg atgacgacga taagtagggc ccggtacctt aattaattaa ggtaccaggt 900aagtgtaccc aattcgccct atagtgagtc gtattacaat tcactcgatc gcccttccca 960acagttgcgc agcctgaatg gcgaatggag atccaatttt taagtgtata atgtgttaaa 1020ctactgattc taattgtttg tgtattttag attcacagtc ccaaggctca tttcaggccc 1080ctcagtcctc acagtctgtt catgatcata atcagccata ccacatttgt agaggtttta 1140cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt 1200gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca 1260aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc 1320aatgtatctt aacgcgt 1337201356DNAArtificial SequenceSynthetic SOD2MTS-nATP6 20gaattcgccc ttcgctctag aatgttgagc cgggcagtgt gcggcaccag caggcagctg 60gctccggttt tggggtatct gggctccagg cagaagcaca gcctccccga cgcggtcgac 120cgcatgaacg aaaatctgtt cgcttcattc attgccccca caatcctagg cctacccgcc 180gcagtactga tcattctatt tccccctcta ttgatcccca cctccaaata tctcatcaac 240aaccgactaa tcaccaccca acaatggcta atcaaactaa cctcaaaaca aatgatgacc 300atgcacaaca ctaaaggacg aacctggtct cttatgctag tatccttaat catttttatt 360gccacaacta acctcctcgg actcctgcct cactcattta caccaaccac ccaactatct 420atgaacctag ccatggccat ccccttatgg gcgggcacag tgattatggg ctttcgctct 480aagattaaaa atgccctagc ccacttctta ccacaaggca cacctacacc ccttatcccc 540atgctagtta ttatcgaaac catcagccta ctcattcaac caatggccct ggccgtacgc 600ctaaccgcta acattactgc aggccaccta ctcatgcacc taattggaag cgccacccta 660gcaatgtcaa ccattaacct tccctctaca cttatcatct tcacaattct aattctactg 720actatcctag aaatcgctgt cgccttaatc caagcctacg ttttcacact tctagtaagc 780ctctacctgc acgacaacac agcggccgcc cggtaagggc gaattcgata tcaagcttat 840cgataccgtc gacctcgagg attacaagga tgacgacgat aagtagggcc cggtacctta 900attaattaag gtaccaggta agtgtaccca attcgcccta tagtgagtcg tattacaatt 960cactcgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggaga tccaattttt 1020aagtgtataa tgtgttaaac tactgattct aattgtttgt gtattttaga ttcacagtcc 1080caaggctcat ttcaggcccc tcagtcctca cagtctgttc atgatcataa tcagccatac 1140cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa 1200acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa 1260ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 1320tggtttgtcc aaactcatca atgtatctta acgcgt 1356212381DNAArtificial SequenceSynthetic COX10MTS-nATP6-COX10 3'UTR 21gaattcgccc ttcgctctag aatggccgca tctccgcaca ctctctcctc acgcctcctg 60acaggttgcg taggaggctc tgtctggtat cttgaagtcg accgcatgaa cgaaaatctg 120ttcgcttcat tcattgcccc cacaatccta ggcctacccg ccgcagtact gatcattcta 180tttccccctc tattgatccc cacctccaaa tatctcatca acaaccgact aatcaccacc 240caacaatggc taatcaaact aacctcaaaa caaatgatga ccatgcacaa cactaaagga 300cgaacctggt ctcttatgct agtatcctta atcattttta ttgccacaac taacctcctc 360ggactcctgc ctcactcatt tacaccaacc acccaactat ctatgaacct agccatggcc 420atccccttat gggcgggcac agtgattatg ggctttcgct ctaagattaa aaatgcccta 480gcccacttct taccacaagg cacacctaca ccccttatcc ccatgctagt tattatcgaa 540accatcagcc tactcattca accaatggcc ctggccgtac gcctaaccgc taacattact 600gcaggccacc tactcatgca cctaattgga agcgccaccc tagcaatgtc aaccattaac 660cttccctcta cacttatcat cttcacaatt ctaattctac tgactatcct agaaatcgct 720gtcgccttaa tccaagccta cgttttcaca cttctagtaa gcctctacct gcacgacaac 780acagcggccg cccggaaggg cgaattcgat atcaagctta tcgataccgt cgacctcgag 840gattacaagg atgacgacga taagtagggc ccggtacctt aattaattaa ggtaccaggt 900aagtgtaccc aattcgccct atagtgagtc gtattacaat tcactcgatc ggagcactgg 960gacgcccacc gcccctttcc ctccgctgcc aggcgagcat gttgtggtaa ttctggaaca 1020caagaagaga aattgctggg tttagaacaa gattataaac gaattcggtg cccagtgatc 1080acttgacagt tttttttttt tttaaatatt acccaaaatg ctccccaaat aagaaatgca 1140tcagctcagt cagtgaatac aaaaaaggaa ttatttttcc ctttgagggt ctttatacat 1200ctctcctcca accccaccct ctattctgtt tcttcctcct cacatggggg tacacataca 1260cagcttcctc ttttggttcc atccttacca ccacaccaca cgcacactcc acatgcccag 1320cagagtggca cttggtggcc agaaagtgtg agcctcatga tctgctgtct gtagttctgt 1380gagctcaggt ccctcaaagg cctcggagca cccccttcct ggtgactgag ccagggcctg 1440catttttggt tttccccacc ccacacattc tcaaccatag tccttctaac aataccaata 1500gctaggaccc ggctgctgtg cactgggact ggggattcca catgtttgcc ttgggagtct 1560caagctggac tgccagcccc tgtcctccct tcacccccat tgcgtatgag catttcagaa 1620ctccaaggag tcacaggcat ctttatagtt cacgttaaca tatagacact gttggaagca 1680gttccttcta aaagggtagc cctggactta ataccagccg gatacctctg gcccccaccc 1740cattactgta cctctggagt cactactgtg ggtcgccact cctctgctac acagcacggc 1800tttttcaagg ctgtattgag aagggaagtt aggaagaagg gtgtgctggg ctaaccagcc 1860cacagagctc acattcctgt cccttgggtg aaaaatacat gtccatcctg atatctcctg 1920aattcagaaa ttagcctcca catgtgcaat ggctttaaga gccagaagca gggttctggg 1980aattttgcaa gttatcctgt ggccaggtgt ggtctcggtt accaaatacg gttacctgca 2040gctttttagt cctttgtgct cccacgggtc tgcagagtcc catctgccca aaggtcttga 2100agcttgacag gatgttttca ttactcagtc tcccagggca ctgctggtcc gtagggattc 2160attggtcggg gtgggagagt taaacaacat ttaaacagag ttctctcaaa aatgtctaaa 2220gggattgtag gtagataaca tccaatcact gtttgcactt atctgaaatc ttccctcttg 2280gctgccccca ggtatttact gtggagaaca ttgcatagga atgtctggaa aaagctccta 2340caacttgtta cagccttcac atttgtagaa gctttacgcg t 2381221179DNAArtificial SequenceSynthetic SOD2MTS-nATP6-SOD2 3'UTR 22gaattcgccc ttcgctctag aatgttgagc cgggcagtgt gcggcaccag caggcagctg 60gctccggttt tggggtatct gggctccagg cagaagcaca gcctccccga cgcggtcgac 120cgcatgaacg aaaatctgtt cgcttcattc attgccccca caatcctagg cctacccgcc 180gcagtactga tcattctatt tccccctcta ttgatcccca cctccaaata tctcatcaac 240aaccgactaa tcaccaccca acaatggcta atcaaactaa cctcaaaaca aatgatgacc 300atgcacaaca ctaaaggacg aacctggtct cttatgctag tatccttaat catttttatt 360gccacaacta acctcctcgg actcctgcct cactcattta caccaaccac ccaactatct 420atgaacctag ccatggccat ccccttatgg gcgggcacag tgattatggg ctttcgctct 480aagattaaaa atgccctagc ccacttctta ccacaaggca cacctacacc ccttatcccc 540atgctagtta ttatcgaaac catcagccta ctcattcaac caatggccct ggccgtacgc 600ctaaccgcta acattactgc aggccaccta ctcatgcacc taattggaag cgccacccta 660gcaatgtcaa ccattaacct tccctctaca cttatcatct tcacaattct aattctactg 720actatcctag aaatcgctgt cgccttaatc caagcctacg ttttcacact tctagtaagc 780ctctacctgc acgacaacac agcggccgcc cggtaagggc gaattcgata tcaagcttat 840cgataccgtc gacctcgagg attacaagga tgacgacgat aagtagggcc cggtacctta 900attaattaag gtaccaggta agtgtaccca attcgcccta tagtgagtcg tattacaatt 960cactcgatcg ttatgctgag tatgttaagc tctttatgac tgtttttgta gtggtataga 1020gtactgcaga atacagtaag ctgctctatt gtagcatttc ctgatgttgc ttagtcactt 1080atttcataaa caacttaatg ttctgaataa tttcttacta aacattttgt tattgggcaa 1140gtgattgaaa atagtaaatg ctttgtgtga ttgacgcgt 1179231552DNAArtificial SequenceSynthetic COX10MTS-nND1 23gtcgacatgg ctgctagccc ccacactctg agcagccgcc tgctgaccgg ttgcgtgggc 60ggctctgtgt ggtatctgga gaggagaacc atgccaatgg caaatctgct gctcctcatc 120gtgccaatcc tgatcgccat ggccttcctc atgctgactg aaagaaaaat tctgggatac 180atgcagctca ggaaggggcc taacgtggtg ggaccttatg gactgctcca gccctttgct 240gatgctatga agctgttcac aaaagagccc ctgaaaccag ccacctctac aatcaccctg 300tacattaccg ctcctaccct ggctctgaca attgccctgc tgctgtggac ccctctccct 360atgccaaatc ctctggtgaa cctgaatctg ggcctcctct ttatcctggc caccagcagc 420ctggccgtgt actccatcct gtggagcgga tgggcttcta acagcaatta cgccctgatc 480ggtgccctga gggccgtggc ccagaccatt tcttacgagg tgaccctcgc cattatcctg 540ctctcaaccc tgctgatgag cggctctttc aacctctcaa ccctgattac aacccaggag 600cacctctggc tgctcctccc cagctggcca ctggccatga tgtggtttat cagcaccctg 660gctgagacaa accggacccc ctttgatctg gctgagggcg agtctgagct ggtctccgga 720ttcaatattg agtacgcagc agggccattc gctctgttct tcatggccga gtatacaaat 780attattatga tgaacacact gactactact atcttcctgg gtactacata cgatgctctg 840agtcccgaac tctacaccac ttacttcgtg accaaaaccc tgctgctgac tagcctgttc 900ctgtggatca ggaccgccta tccacgattc cgatacgacc agctgatgca tctgctgtgg 960aagaacttcc tgccactcac cctggctctg ctcatgtggt acgtgagtat gccaatcact 1020atcagctcta tccctccaca gacctactcg aggaggatta caaggatgac gacgataagt 1080agggcccggt accttaatta attaaggtac caggtaagtg tacccaattc gccctatagt 1140gagtcgtatt acaattcact cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa 1200tggagatcca atttttaagt gtataatgtg ttaaactact gattctaatt gtttgtgtat 1260tttagattca cagtcccaag gctcatttca ggcccctcag tcctcacagt ctgttcatga 1320tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc 1380tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag 1440cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt 1500cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc gt 1552241973DNAArtificial SequenceSynthetic COX10MTS-nND4 24gtcgacatgg ccgcctcacc ccacaccctg agtagcaggc tgctgaccgg ctgtgtggga 60ggaagcgtgt ggtatctgga gcggagaacc atgctgaagc tgatcgtgcc caccattatg 120ctgctgcctc tgacatggct gtctaagaag cacatgatct ggattaacac aaccacccac 180agcctgatta tctccatcat tcccctcctg ttcttcaacc agatcaacaa caacctgttc 240tcctgctcac ctacttttag cagcgatcca ctgacaaccc cactgctgat gctgacaacc 300tggctcctcc ccctgacaat catggcttcc cagaggcacc tgagcagcga gccactgtcc 360cgcaaaaagc tgtacctgtc catgctgatt tctctccaga tctcactcat catgaccttc 420actgccaccg agctgattat gttctatatc ttcttcgaga ctactctgat ccctacactc 480gccattatca cccggtgggg caaccagcct gagagactga atgccgggac ttattttctg 540ttctacaccc tggtggggtc actgcccctg ctgattgccc tgatctacac ccataacaca 600ctgggctctc tcaatatcct gctgctcaca ctgacagccc aggagctgtc caattcttgg 660gctaacaatc tgatgtggct cgcatacact atggccttca tggtgaagat gccactctat 720gggctccacc tctggctccc taaggcccac gtcgaagccc caattgcagg gtccatggtg 780ctggcagctg tgctcctgaa gctgggtggc tatgggatga tgcgcctgac cctgatcctg 840aatcctctca caaagcatat ggcttaccct tttctggtgc tgtccctgtg gggaatgatt 900atgacaagct ctatttgcct gcgccagaca gacctgaaaa gcctgattgc ctacagcagt 960atcagtcata tggccctggt ggtgaccgct attctgattc agacaccatg gtcttttaca 1020ggggccgtca ttctgatgat cgcccacgga ctgacctcat cactcctctt ctgtctggcc 1080aactcaaact acgaaaggac acactcaaga attatgattc tgagccaggg actccagact 1140ctgctccccc tcatggcctt ctggtggctg ctcgcctctc tcgccaacct ggccctccct 1200cccacaatca atctgctggg cgagctcagc gtgctggtga ccacttttag ttggtccaac 1260atcacactgc tgctcaccgg actcaatatg ctggtcaccg ccctgtacag tctgtacatg 1320ttcaccacaa cacagtgggg tagcctcact catcacatta ataacatgaa gccttctttt 1380actagggaaa atactctgat gtttatgcat ctctccccaa tcctcctcct gagtctgaac 1440cccgacatca tcaccggctt tagctctctc gaggaggatt acaaggatga cgacgataag 1500tagggcccgg taccttaatt aattaaggta ccaggtaagt gtacccaatt cgccctatag 1560tgagtcgtat tacaattcac tcgatcgccc ttcccaacag ttgcgcagcc tgaatggcga 1620atggagatcc aatttttaag tgtataatgt gttaaactac tgattctaat tgtttgtgta 1680ttttagattc acagtcccaa ggctcatttc aggcccctca gtcctcacag tctgttcatg 1740atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac 1800ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca 1860gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 1920tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg cgt 1973252595DNAArtificial SequenceSynthetic COX10MTS-nND1-COX10 3'UTR 25gtcgacatgg ctgctagccc ccacactctg agcagccgcc tgctgaccgg ttgcgtgggc 60ggctctgtgt ggtatctgga gaggagaacc atgccaatgg caaatctgct gctcctcatc 120gtgccaatcc tgatcgccat ggccttcctc atgctgactg aaagaaaaat tctgggatac 180atgcagctca ggaaggggcc taacgtggtg ggaccttatg gactgctcca gccctttgct 240gatgctatga agctgttcac aaaagagccc ctgaaaccag ccacctctac aatcaccctg 300tacattaccg ctcctaccct ggctctgaca attgccctgc tgctgtggac ccctctccct 360atgccaaatc ctctggtgaa cctgaatctg ggcctcctct ttatcctggc caccagcagc 420ctggccgtgt actccatcct gtggagcgga tgggcttcta acagcaatta cgccctgatc 480ggtgccctga gggccgtggc ccagaccatt tcttacgagg tgaccctcgc cattatcctg 540ctctcaaccc tgctgatgag cggctctttc aacctctcaa ccctgattac aacccaggag 600cacctctggc tgctcctccc cagctggcca ctggccatga tgtggtttat cagcaccctg 660gctgagacaa accggacccc ctttgatctg gctgagggcg agtctgagct ggtctccgga 720ttcaatattg agtacgcagc agggccattc gctctgttct tcatggccga gtatacaaat 780attattatga tgaacacact gactactact atcttcctgg gtactacata cgatgctctg 840agtcccgaac tctacaccac ttacttcgtg accaaaaccc tgctgctgac tagcctgttc 900ctgtggatca ggaccgccta tccacgattc cgatacgacc agctgatgca tctgctgtgg 960aagaacttcc tgccactcac cctggctctg ctcatgtggt acgtgagtat gccaatcact 1020atcagctcta tccctccaca gacctactcg aggaggatta caaggatgac gacgataagt 1080agggcccggt accttaatta attaaggtac caggtaagtg tacccaattc gccctatagt 1140gagtcgtatt acaattcact cgatcggagc actgggacgc ccaccgcccc tttccctccg 1200ctgccaggcg agcatgttgt ggtaattctg gaacacaaga agagaaattg ctgggtttag 1260aacaagatta taaacgaatt cggtgctcag tgatcacttg acagtttttt ttttttttaa 1320atattaccca aaatgctccc caaataagaa atgcatcagc tcagtcagtg aatacaaaaa 1380aggaattatt tttccctttg agggtcttta tacatctctc ctccaacccc accctctatt 1440ctgtttcttc ctcctcacat gggggtacac atacacagct tcctcttttg gttccatcct 1500taccaccaca ccacacgcac actccacatg cccagcagag tggcacttgg tggccagaaa 1560gtgtgagcct catgatctgc tgtctgtagt tctgtgagct caggtccctc aaaggcctcg 1620gagcaccccc ttcctggtga ctgagccagg gcctgcattt ttggttttcc ccaccccaca 1680cattctcaac catagtcctt ctaacaatac caatagctag gacccggctg ctgtgcactg 1740ggactgggga ttccacatgt ttgccttggg agtctcaagc tggactgcca gcccctgtcc 1800tcccttcacc cccattgcgt atgagcattt cagaactcca aggagtcaca ggcatcttta 1860tagttcacgt taacatatag acactgttgg aagcagttcc ttctaaaagg gtagccctgg 1920acttaatacc agccggatac ctctggcccc caccccatta ctgtacctct ggagtcacta 1980ctgtgggtcg ccactcctct gctacacagc acggcttttt caaggctgta ttgagaaggg 2040aagttaggaa gaagggtgtg ctgggctaac cagcccacag agctcacatt cctgtccctt 2100gggtgaaaaa tacatgtcca tcctgatatc tcctgaattc agaaattagc ctccacatgt 2160gcaatggctt taagagccag aagcagggtt ctgggaattt tgcaagttat cctgtggcca 2220ggtgtggtct cggttaccaa atacggttac ctgcagcttt ttagtccttt gtgctcccac 2280gggtctgcag agtcccatct gcccaaaggt cttgaagctt gacaggatgt tttcattact 2340cagtctccca gggcactgct ggtccgtagg gattcattgg tcggggtggg agagttaaac 2400aacatttaaa cagagttctc tcaaaaatgt ctaaagggat tgtaggtaga taacatccaa 2460tcactgtttg cacttatctg aaatcttccc tcttggctgc ccccaggtat ttactgtgga 2520gaacattgca taggaatgtc tgaaaaagct tctacaactt gttacagcct tcacatttgt 2580agaagcttta cgcgt 2595263016DNAArtificial SequenceSynthetic COX10MTS-nND4-COX10 3'UTR 26gtcgacatgg ccgcctcacc ccacaccctg agtagcaggc tgctgaccgg ctgtgtggga 60ggaagcgtgt ggtatctgga gcggagaacc atgctgaagc tgatcgtgcc caccattatg 120ctgctgcctc tgacatggct gtctaagaag cacatgatct ggattaacac aaccacccac

180agcctgatta tctccatcat tcccctcctg ttcttcaacc agatcaacaa caacctgttc 240tcctgctcac ctacttttag cagcgatcca ctgacaaccc cactgctgat gctgacaacc 300tggctcctcc ccctgacaat catggcttcc cagaggcacc tgagcagcga gccactgtcc 360cgcaaaaagc tgtacctgtc catgctgatt tctctccaga tctcactcat catgaccttc 420actgccaccg agctgattat gttctatatc ttcttcgaga ctactctgat ccctacactc 480gccattatca cccggtgggg caaccagcct gagagactga atgccgggac ttattttctg 540ttctacaccc tggtggggtc actgcccctg ctgattgccc tgatctacac ccataacaca 600ctgggctctc tcaatatcct gctgctcaca ctgacagccc aggagctgtc caattcttgg 660gctaacaatc tgatgtggct cgcatacact atggccttca tggtgaagat gccactctat 720gggctccacc tctggctccc taaggcccac gtcgaagccc caattgcagg gtccatggtg 780ctggcagctg tgctcctgaa gctgggtggc tatgggatga tgcgcctgac cctgatcctg 840aatcctctca caaagcatat ggcttaccct tttctggtgc tgtccctgtg gggaatgatt 900atgacaagct ctatttgcct gcgccagaca gacctgaaaa gcctgattgc ctacagcagt 960atcagtcata tggccctggt ggtgaccgct attctgattc agacaccatg gtcttttaca 1020ggggccgtca ttctgatgat cgcccacgga ctgacctcat cactcctctt ctgtctggcc 1080aactcaaact acgaaaggac acactcaaga attatgattc tgagccaggg actccagact 1140ctgctccccc tcatggcctt ctggtggctg ctcgcctctc tcgccaacct ggccctccct 1200cccacaatca atctgctggg cgagctcagc gtgctggtga ccacttttag ttggtccaac 1260atcacactgc tgctcaccgg actcaatatg ctggtcaccg ccctgtacag tctgtacatg 1320ttcaccacaa cacagtgggg tagcctcact catcacatta ataacatgaa gccttctttt 1380actagggaaa atactctgat gtttatgcat ctctccccaa tcctcctcct gagtctgaac 1440cccgacatca tcaccggctt tagctctctc gaggaggatt acaaggatga cgacgataag 1500tagggcccgg taccttaatt aattaaggta ccaggtaagt gtacccaatt cgccctatag 1560tgagtcgtat tacaattcac tcgatcggag cactgggacg cccaccgccc ctttccctcc 1620gctgccaggc gagcatgttg tggtaattct ggaacacaag aagagaaatt gctgggttta 1680gaacaagatt ataaacgaat tcggtgctca gtgatcactt gacagttttt ttttttttta 1740aatattaccc aaaatgctcc ccaaataaga aatgcatcag ctcagtcagt gaatacaaaa 1800aaggaattat ttttcccttt gagggtcttt atacatctct cctccaaccc caccctctat 1860tctgtttctt cctcctcaca tgggggtaca catacacagc ttcctctttt ggttccatcc 1920ttaccaccac accacacgca cactccacat gcccagcaga gtggcacttg gtggccagaa 1980agtgtgagcc tcatgatctg ctgtctgtag ttctgtgagc tcaggtccct caaaggcctc 2040ggagcacccc cttcctggtg actgagccag ggcctgcatt tttggttttc cccaccccac 2100acattctcaa ccatagtcct tctaacaata ccaatagcta ggacccggct gctgtgcact 2160gggactgggg attccacatg tttgccttgg gagtctcaag ctggactgcc agcccctgtc 2220ctcccttcac ccccattgcg tatgagcatt tcagaactcc aaggagtcac aggcatcttt 2280atagttcacg ttaacatata gacactgttg gaagcagttc cttctaaaag ggtagccctg 2340gacttaatac cagccggata cctctggccc ccaccccatt actgtacctc tggagtcact 2400actgtgggtc gccactcctc tgctacacag cacggctttt tcaaggctgt attgagaagg 2460gaagttagga agaagggtgt gctgggctaa ccagcccaca gagctcacat tcctgtccct 2520tgggtgaaaa atacatgtcc atcctgatat ctcctgaatt cagaaattag cctccacatg 2580tgcaatggct ttaagagcca gaagcagggt tctgggaatt ttgcaagtta tcctgtggcc 2640aggtgtggtc tcggttacca aatacggtta cctgcagctt tttagtcctt tgtgctccca 2700cgggtctgca gagtcccatc tgcccaaagg tcttgaagct tgacaggatg ttttcattac 2760tcagtctccc agggcactgc tggtccgtag ggattcattg gtcggggtgg gagagttaaa 2820caacatttaa acagagttct ctcaaaaatg tctaaaggga ttgtaggtag ataacatcca 2880atcactgttt gcacttatct gaaatcttcc ctcttggctg cccccaggta tttactgtgg 2940agaacattgc ataggaatgt ctgaaaaagc ttctacaact tgttacagcc ttcacatttg 3000tagaagcttt acgcgt 301627696DNAArtificial SequenceSynthetic nATP6 (recoded ATP6) 27atgaacgaaa atctgttcgc ttcattcatt gcccccacaa tcctaggcct acccgccgca 60gtactgatca ttctatttcc ccctctattg atccccacct ccaaatatct catcaacaac 120cgactaatca ccacccaaca atggctaatc aaactaacct caaaacaaat gatgaccatg 180cacaacacta aaggacgaac ctggtctctt atgctagtat ccttaatcat ttttattgcc 240acaactaacc tcctcggact cctgcctcac tcatttacac caaccaccca actatctatg 300aacctagcca tggccatccc cttatgggcg ggcacagtga ttatgggctt tcgctctaag 360attaaaaatg ccctagccca cttcttacca caaggcacac ctacacccct tatccccatg 420ctagttatta tcgaaaccat cagcctactc attcaaccaa tggccctggc cgtacgccta 480accgctaaca ttactgcagg ccacctactc atgcacctaa ttggaagcgc caccctagca 540atgtcaacca ttaaccttcc ctctacactt atcatcttca caattctaat tctactgact 600atcctagaaa tcgctgtcgc cttaatccaa gcctacgttt tcacacttct agtaagcctc 660tacctgcacg acaacacagc ggccgcccgg aagggc 69628956DNAArtificial SequenceSynthetic nND1 (recoded ND1) 28atgccaatgg caaatctgct gctcctcatc gtgccaatcc tgatcgccat ggccttcctc 60atgctgactg aaagaaaaat tctgggatac atgcagctca ggaaggggcc taacgtggtg 120ggaccttatg gactgctcca gccctttgct gatgctatga agctgttcac aaaagagccc 180ctgaaaccag ccacctctac aatcaccctg tacattaccg ctcctaccct ggctctgaca 240attgccctgc tgctgtggac ccctctccct atgccaaatc ctctggtgaa cctgaatctg 300ggcctcctct ttatcctggc caccagcagc ctggccgtgt actccatcct gtggagcgga 360tgggcttcta acagcaatta cgccctgatc ggtgccctga gggccgtggc ccagaccatt 420tcttacgagg tgaccctcgc cattatcctg ctctcaaccc tgctgatgag cggctctttc 480aacctctcaa ccctgattac aacccaggag cacctctggc tgctcctccc cagctggcca 540ctggccatga tgtggtttat cagcaccctg gctgagacaa accggacccc ctttgatctg 600gctgagggcg agtctgagct ggtctccgga ttcaatattg agtacgcagc agggccattc 660gctctgttct tcatggccga gtatacaaat attattatga tgaacacact gactactact 720atcttcctgg gtactacata cgatgctctg agtcccgaac tctacaccac ttacttcgtg 780accaaaaccc tgctgctgac tagcctgttc ctgtggatca ggaccgccta tccacgattc 840cgatacgacc agctgatgca tctgctgtgg aagaacttcc tgccactcac cctggctctg 900ctcatgtggt acgtgagtat gccaatcact atcagctcta tccctccaca gaccta 956291377DNAArtificial SequenceSynthetic nND4 (recoded ND4) 29atgctgaagc tgatcgtgcc caccattatg ctgctgcctc tgacatggct gtctaagaag 60cacatgatct ggattaacac aaccacccac agcctgatta tctccatcat tcccctcctg 120ttcttcaacc agatcaacaa caacctgttc tcctgctcac ctacttttag cagcgatcca 180ctgacaaccc cactgctgat gctgacaacc tggctcctcc ccctgacaat catggcttcc 240cagaggcacc tgagcagcga gccactgtcc cgcaaaaagc tgtacctgtc catgctgatt 300tctctccaga tctcactcat catgaccttc actgccaccg agctgattat gttctatatc 360ttcttcgaga ctactctgat ccctacactc gccattatca cccggtgggg caaccagcct 420gagagactga atgccgggac ttattttctg ttctacaccc tggtggggtc actgcccctg 480ctgattgccc tgatctacac ccataacaca ctgggctctc tcaatatcct gctgctcaca 540ctgacagccc aggagctgtc caattcttgg gctaacaatc tgatgtggct cgcatacact 600atggccttca tggtgaagat gccactctat gggctccacc tctggctccc taaggcccac 660gtcgaagccc caattgcagg gtccatggtg ctggcagctg tgctcctgaa gctgggtggc 720tatgggatga tgcgcctgac cctgatcctg aatcctctca caaagcatat ggcttaccct 780tttctggtgc tgtccctgtg gggaatgatt atgacaagct ctatttgcct gcgccagaca 840gacctgaaaa gcctgattgc ctacagcagt atcagtcata tggccctggt ggtgaccgct 900attctgattc agacaccatg gtcttttaca ggggccgtca ttctgatgat cgcccacgga 960ctgacctcat cactcctctt ctgtctggcc aactcaaact acgaaaggac acactcaaga 1020attatgattc tgagccaggg actccagact ctgctccccc tcatggcctt ctggtggctg 1080ctcgcctctc tcgccaacct ggccctccct cccacaatca atctgctggg cgagctcagc 1140gtgctggtga ccacttttag ttggtccaac atcacactgc tgctcaccgg actcaatatg 1200ctggtcaccg ccctgtacag tctgtacatg ttcaccacaa cacagtgggg tagcctcact 1260catcacatta ataacatgaa gccttctttt actagggaaa atactctgat gtttatgcat 1320ctctccccaa tcctcctcct gagtctgaac cccgacatca tcaccggctt tagctct 13773084DNAHomo sapiens 30atggccgcat ctccgcacac tctctcctca cgcctcctga caggttgcgt aggaggctct 60gtctggtatc ttgaagtcga ccgc 8431102DNAHomo sapiens 31atgttgagcc gggcagtgtg cggcaccagc aggcagctgg ctccggtttt ggggtatctg 60ggctccaggc agaagcacag cctccccgac gcggtcgacc gc 1023230PRTHomo sapiens 32Met Ala Pro Tyr Ser Leu Leu Val Thr Arg Leu Gln Lys Ala Leu Gly 1 5 10 15 Val Arg Gln Tyr His Val Ala Ser Val Leu Cys Gln Arg Ala 20 25 30 33382DNAHomo sapiens 33gggcagtgcc tccccgcccc gccgctggcg tcaagttcag ctccacgtgt gccatcagtg 60gatccgatcc gtccagccat ggcttcctat tccaagatgg tgtgaccaga catgcttcct 120gctccccgct tagcccacgg agtgactgtg gttgtggtgg gggggttctt aaaataactt 180tttagccccc gtcttcctat tttgagtttg gttcagatct taagcagctc catgcaactg 240tatttatttt tgatgacaag actcccatct aaagtttttc tcctgcctga tcatttcatt 300ggtggctgaa ggattctaga gaaccttttg ttcttgcaag gaaaacaaga atccaaaacc 360agaaaaaaaa aaaaaaaaaa aa 3823420PRTHomo sapiens 34Met Leu Ser Arg Ala Val Cys Gly Thr Ser Arg Gln Leu Ala Pro Ala 1 5 10 15 Leu Gly Tyr Leu 20 35214DNAHomo sapiens 35accacgatcg ttatgctgat cataccctaa tgatcccagc aagataacgt cctgtcttct 60aagatgtgca tcaagcctgg tacatactga aaaccctata aggtcctgga taatttttgt 120ttgattattc attgaagaaa catttatttt ccaattgtgt gaagtttttg actgttaata 180aaagaatctg tcaaccatca aaaaaaaaaa aaaa 2143663PRTHomo sapiens 36Met Leu Gly Phe Val Gly Arg Val Ala Ala Ala Pro Ala Ser Gly Ala 1 5 10 15 Leu Arg Arg Leu Thr Pro Ser Ala Ser Leu Pro Pro Ala Gln Leu Leu 20 25 30 Leu Arg Ala Ala Pro Thr Ala Val His Pro Val Arg Asp Tyr Ala Ala 35 40 45 Gln Thr Ser Pro Ser Pro Lys Ala Gly Ala Ala Thr Gly Arg Ile 50 55 60 37162DNAHomo sapiens 37ggggtctttg tcctctgtac tgtctctctc cttgccccta acccaaaaag cttcattttt 60ctgtgtaggc tgcacaagag ccttgattga agatatattc tttctgaaca gtatttaagg 120tttccaataa aatgtacacc cctcagaaaa aaaaaaaaaa aa 1623836PRTHomo sapiens 38Met Leu Ser Val Ala Ala Arg Ser Gly Pro Phe Ala Pro Val Leu Ser 1 5 10 15 Ala Thr Ser Arg Gly Val Ala Gly Ala Leu Arg Pro Leu Val Gln Ala 20 25 30 Thr Val Pro Ala 35 39233DNAHomo sapiens 39gagacttgga ctcaagtcat aggcttcttt cagtctttat gtcacctcag gagacttatt 60tgagaggaag ccttctgtac ttgaagttga tttgaaatat gtaagaattg atgatgtatt 120tgcaaacatt aatgtgaaat aaattgaatt taatgttgaa tactttcagg cattcactta 180ataaagacac tgttaagcac tgttatgctc agtcaaaaaa aaaaaaaaaa aaa 2334020PRTHomo sapiens 40Met Leu Ala Thr Arg Arg Leu Leu Gly Trp Ser Leu Pro Ala Arg Val 1 5 10 15 Ser Val Arg Phe 20 41102DNAHomo sapiens 41cccaccaccc tggcctgctg tcctgcgtct atccatgtgg aatgctggac aataaagcga 60gtgctgccca ccctccaaaa aaaaaaaaaa aaaaaaaaaa aa 1024243PRTHomo sapiens 42Met Phe Phe Ser Ala Ala Leu Arg Ala Arg Ala Ala Gly Leu Thr Ala 1 5 10 15 His Trp Gly Arg His Val Arg Asn Leu His Lys Thr Ala Met Gln Asn 20 25 30 Gly Ala Gly Gly Ala Leu Phe Val His Arg Asp 35 40 4397DNAHomo sapiens 43tttatattga actgtaaata tgtcactaga gaaataaaat atggacttcc aatctacgta 60aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 974424PRTHomo sapiens 44Met Leu Arg Ala Ala Ala Arg Phe Gly Pro Arg Leu Gly Arg Arg Leu 1 5 10 15 Leu Ser Ala Ala Ala Thr Gln Ala 20 45450DNAHomo sapiens 45gaatcatgca agcttcctcc ctcagccatt gatggaaagt tcagcaagat cagcaacaaa 60accaagaaaa atgatccttg cgtgctgaat atctgaaaag agaaattttt cctacaaaat 120ctcttgggtc aagaaagttc tagaatttga attgataaac atggtgggtt ggctgagggt 180aagagtatat gaggaacctt ttaaacgaca acaatactgc tagctttcag gatgattttt 240aaaaaataga ttcaaatgtg ttatcctctc tctgaaacgc ttcctataac tcgagtttat 300aggggaagaa aaagctattg tttacaatta tatccccatt aaggcaactg ctacaccctg 360ctttgtattc tgggctaaga ttcattaaaa actagctgct cttaaaaaaa aaaaaaaaaa 420aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4504620PRTHomo sapiens 46Met Ala Ala Ser Pro His Thr Leu Ser Ser Arg Leu Leu Thr Gly Cys 1 5 10 15 Val Gly Gly Ser 20 471424DNAHomo sapiens 47gagcactggg acgcccaccg cccctttccc tccgctgcca ggcgagcatg ttgtggtaat 60tctggaacac aagaagagaa attgctgggt ttagaacaag attataaacg aattcggtgc 120ccagtgatca cttgacagtt tttttttttt ttaaatatta cccaaaatgc tccccaaata 180agaaatgcat cagctcagtc agtgaataca aaaaaggaat tatttttccc tttgagggtc 240tttatacatc tctcctccaa ccccaccctc tattctgttt cttcctcctc acatgggggt 300acacatacac agcttcctct tttggttcca tccttaccac cacaccacac gcacactcca 360catgcccagc agagtggcac ttggtggcca gaaagtgtga gcctcatgat ctgctgtctg 420tagttctgtg agctcaggtc cctcaaaggc ctcggagcac ccccttcctg gtgactgagc 480cagggcctgc atttttggtt ttccccaccc cacacattct caaccatagt ccttctaaca 540ataccaatag ctaggacccg gctgctgtgc actgggactg gggattccac atgtttgcct 600tgggagtctc aagctggact gccagcccct gtcctccctt cacccccatt gcgtatgagc 660atttcagaac tccaaggagt cacaggcatc tttatagttc acgttaacat atagacactg 720ttggaagcag ttccttctaa aagggtagcc ctggacttaa taccagccgg atacctctgg 780cccccacccc attactgtac ctctggagtc actactgtgg gtcgccactc ctctgctaca 840cagcacggct ttttcaaggc tgtattgaga agggaagtta ggaagaaggg tgtgctgggc 900taaccagccc acagagctca cattcctgtc ccttgggtga aaaatacatg tccatcctga 960tatctcctga attcagaaat tagcctccac atgtgcaatg gctttaagag ccagaagcag 1020ggttctggga attttgcaag ttatcctgtg gccaggtgtg gtctcggtta ccaaatacgg 1080ttacctgcag ctttttagtc ctttgtgctc ccacgggtct gcagagtccc atctgcccaa 1140aggtcttgaa gcttgacagg atgttttcat tactcagtct cccagggcac tgctggtccg 1200tagggattca ttggtcgggg tgggagagtt aaacaacatt taaacagagt tctctcaaaa 1260atgtctaaag ggattgtagg tagataacat ccaatcactg tttgcactta tctgaaatct 1320tccctcttgg ctgcccccag gtatttactg tggagaacat tgcataggaa tgtctggaaa 1380aagctcctac aacttgttac agccttcaca tttgtagaag cttt 142448780PRTHomo sapiens 48Met Ala Pro Tyr Ser Leu Leu Val Thr Arg Leu Gln Lys Ala Leu Gly 1 5 10 15 Val Arg Gln Tyr His Val Ala Ser Val Leu Cys Gln Arg Ala Lys Val 20 25 30 Ala Met Ser His Phe Glu Pro Asn Glu Tyr Ile His Tyr Asp Leu Leu 35 40 45 Glu Lys Asn Ile Asn Ile Val Arg Lys Arg Leu Asn Arg Pro Leu Thr 50 55 60 Leu Ser Glu Lys Ile Val Tyr Gly His Leu Asp Asp Pro Ala Ser Gln 65 70 75 80 Glu Ile Glu Arg Gly Lys Ser Tyr Leu Arg Leu Arg Pro Asp Arg Val 85 90 95 Ala Met Gln Asp Ala Thr Ala Gln Met Ala Met Leu Gln Phe Ile Ser 100 105 110 Ser Gly Leu Ser Lys Val Ala Val Pro Ser Thr Ile His Cys Asp His 115 120 125 Leu Ile Glu Ala Gln Val Gly Gly Glu Lys Asp Leu Arg Arg Ala Lys 130 135 140 Asp Ile Asn Gln Glu Val Tyr Asn Phe Leu Ala Thr Ala Gly Ala Lys 145 150 155 160 Tyr Gly Val Gly Phe Trp Lys Pro Gly Ser Gly Ile Ile His Gln Ile 165 170 175 Ile Leu Glu Asn Tyr Ala Tyr Pro Gly Val Leu Leu Ile Gly Thr Asp 180 185 190 Ser His Thr Pro Asn Gly Gly Gly Leu Gly Gly Ile Cys Ile Gly Val 195 200 205 Gly Gly Ala Asp Ala Val Asp Val Met Ala Gly Ile Pro Trp Glu Leu 210 215 220 Lys Cys Pro Lys Val Ile Gly Val Lys Leu Thr Gly Ser Leu Ser Gly 225 230 235 240 Trp Ser Ser Pro Lys Asp Val Ile Leu Lys Val Ala Gly Ile Leu Thr 245 250 255 Val Lys Gly Gly Thr Gly Ala Ile Val Glu Tyr His Gly Pro Gly Val 260 265 270 Asp Ser Ile Ser Cys Thr Gly Met Ala Thr Ile Cys Asn Met Gly Ala 275 280 285 Glu Ile Gly Ala Thr Thr Ser Val Phe Pro Tyr Asn His Arg Met Lys 290 295 300 Lys Tyr Leu Ser Lys Thr Gly Arg Glu Asp Ile Ala Asn Leu Ala Asp 305 310 315 320 Glu Phe Lys Asp His Leu Val Pro Asp Pro Gly Cys His Tyr Asp Gln 325 330 335 Leu Ile Glu Ile Asn Leu Ser Glu Leu Lys Pro His Ile Asn Gly Pro 340 345 350 Phe Thr Pro Asp Leu Ala His Pro Val Ala Glu Val Gly Lys Val Ala 355 360 365 Glu Lys Glu Gly Trp Pro Leu Asp Ile Arg Val Gly Leu Ile Gly Ser 370 375 380 Cys Thr Asn Ser Ser Tyr Glu Asp Met Gly Arg Ser Ala Ala Val Ala 385 390 395 400 Lys Gln Ala Leu Ala His Gly Leu Lys Cys Lys Ser Gln Phe Thr Ile 405 410 415 Thr Pro Gly Ser Glu Gln Ile Arg Ala Thr Ile Glu Arg Asp Gly Tyr 420 425 430 Ala Gln Ile Leu Arg Asp Leu Gly Gly Ile Val Leu Ala Asn Ala Cys 435 440 445 Gly Pro Cys Ile Gly Gln Trp Asp Arg Lys Asp Ile Lys Lys Gly Glu 450 455 460 Lys Asn Thr Ile Val Thr Ser Tyr Asn Arg Asn Phe Thr Gly Arg Asn 465 470 475 480 Asp Ala Asn Pro Glu Thr His Ala Phe Val Thr Ser Pro Glu Ile Val 485 490 495 Thr Ala Leu Ala Ile Ala Gly Thr Leu Lys Phe Asn Pro Glu Thr Asp 500 505 510 Tyr Leu Thr Gly Thr Asp Gly Lys Lys Phe Arg Leu Glu Ala Pro Asp

515 520 525 Ala Asp Glu Leu Pro Lys Gly Glu Phe Asp Pro Gly Gln Asp Thr Tyr 530 535 540 Gln His Pro Pro Lys Asp Ser Ser Gly Gln His Val Asp Val Ser Pro 545 550 555 560 Thr Ser Gln Arg Leu Gln Leu Leu Glu Pro Phe Asp Lys Trp Asp Gly 565 570 575 Lys Asp Leu Glu Asp Leu Gln Ile Leu Ile Lys Val Lys Gly Lys Cys 580 585 590 Thr Thr Asp His Ile Ser Ala Ala Gly Pro Trp Leu Lys Phe Arg Gly 595 600 605 His Leu Asp Asn Ile Ser Asn Asn Leu Leu Ile Gly Ala Ile Asn Ile 610 615 620 Glu Asn Gly Lys Ala Asn Ser Val Arg Asn Ala Val Thr Gln Glu Phe 625 630 635 640 Gly Pro Val Pro Asp Thr Ala Arg Tyr Tyr Lys Lys His Gly Ile Arg 645 650 655 Trp Val Val Ile Gly Asp Glu Asn Tyr Gly Glu Gly Ser Ser Arg Glu 660 665 670 His Ala Ala Leu Glu Pro Arg His Leu Gly Gly Arg Ala Ile Ile Thr 675 680 685 Lys Ser Phe Ala Arg Ile His Glu Thr Asn Leu Lys Lys Gln Gly Leu 690 695 700 Leu Pro Leu Thr Phe Ala Asp Pro Ala Asp Tyr Asn Lys Ile His Pro 705 710 715 720 Val Asp Lys Leu Thr Ile Gln Gly Leu Lys Asp Phe Thr Pro Gly Lys 725 730 735 Pro Leu Lys Cys Ile Ile Lys His Pro Asn Gly Thr Gln Glu Thr Ile 740 745 750 Leu Leu Asn His Thr Phe Asn Glu Thr Gln Ile Glu Trp Phe Arg Ala 755 760 765 Gly Ser Ala Leu Asn Arg Met Lys Glu Leu Gln Gln 770 775 780 49222PRTHomo sapiens 49Met Leu Ser Arg Ala Val Cys Gly Thr Ser Arg Gln Leu Ala Pro Ala 1 5 10 15 Leu Gly Tyr Leu Gly Ser Arg Gln Lys His Ser Leu Pro Asp Leu Pro 20 25 30 Tyr Asp Tyr Gly Ala Leu Glu Pro His Ile Asn Ala Gln Ile Met Gln 35 40 45 Leu His His Ser Lys His His Ala Ala Tyr Val Asn Asn Leu Asn Val 50 55 60 Thr Glu Glu Lys Tyr Gln Glu Ala Leu Ala Lys Gly Asp Val Thr Ala 65 70 75 80 Gln Ile Ala Leu Gln Pro Ala Leu Lys Phe Asn Gly Gly Gly His Ile 85 90 95 Asn His Ser Ile Phe Trp Thr Asn Leu Ser Pro Asn Gly Gly Gly Glu 100 105 110 Pro Lys Gly Glu Leu Leu Glu Ala Ile Lys Arg Asp Phe Gly Ser Phe 115 120 125 Asp Lys Phe Lys Glu Lys Leu Thr Ala Ala Ser Val Gly Val Gln Gly 130 135 140 Ser Gly Trp Gly Trp Leu Gly Phe Asn Lys Glu Arg Gly His Leu Gln 145 150 155 160 Ile Ala Ala Cys Pro Asn Gln Asp Pro Leu Gln Gly Thr Thr Gly Leu 165 170 175 Ile Pro Leu Leu Gly Ile Asp Val Trp Glu His Ala Tyr Tyr Leu Gln 180 185 190 Tyr Lys Asn Val Arg Pro Asp Tyr Leu Lys Ala Ile Trp Asn Val Ile 195 200 205 Asn Trp Glu Asn Val Thr Glu Arg Tyr Met Ala Cys Lys Lys 210 215 220 50529PRTHomo sapiens 50Met Leu Gly Phe Val Gly Arg Val Ala Ala Ala Pro Ala Ser Gly Ala 1 5 10 15 Leu Arg Arg Leu Thr Pro Ser Ala Ser Leu Pro Pro Ala Gln Leu Leu 20 25 30 Leu Arg Ala Ala Pro Thr Ala Val His Pro Val Arg Asp Tyr Ala Ala 35 40 45 Gln Thr Ser Pro Ser Pro Lys Ala Gly Ala Ala Thr Gly Arg Ile Val 50 55 60 Ala Val Ile Gly Ala Val Val Asp Val Gln Phe Asp Glu Gly Leu Pro 65 70 75 80 Pro Ile Leu Asn Ala Leu Glu Val Gln Gly Arg Glu Thr Arg Leu Val 85 90 95 Leu Glu Val Ala Gln His Leu Gly Glu Ser Thr Val Arg Thr Ile Ala 100 105 110 Met Asp Gly Thr Glu Gly Leu Val Arg Gly Gln Lys Val Leu Asp Ser 115 120 125 Gly Ala Pro Ile Lys Ile Pro Val Gly Pro Glu Thr Leu Gly Arg Ile 130 135 140 Met Asn Val Ile Gly Glu Pro Ile Asp Glu Arg Gly Pro Ile Lys Thr 145 150 155 160 Lys Gln Phe Ala Pro Ile His Ala Glu Ala Pro Glu Phe Met Glu Met 165 170 175 Ser Val Glu Gln Glu Ile Leu Val Thr Gly Ile Lys Val Val Asp Leu 180 185 190 Leu Ala Pro Tyr Ala Lys Gly Gly Lys Ile Gly Leu Phe Gly Gly Ala 195 200 205 Gly Val Gly Lys Thr Val Leu Ile Met Glu Leu Ile Asn Asn Val Ala 210 215 220 Lys Ala His Gly Gly Tyr Ser Val Phe Ala Gly Val Gly Glu Arg Thr 225 230 235 240 Arg Glu Gly Asn Asp Leu Tyr His Glu Met Ile Glu Ser Gly Val Ile 245 250 255 Asn Leu Lys Asp Ala Thr Ser Lys Val Ala Leu Val Tyr Gly Gln Met 260 265 270 Asn Glu Pro Pro Gly Ala Arg Ala Arg Val Ala Leu Thr Gly Leu Thr 275 280 285 Val Ala Glu Tyr Phe Arg Asp Gln Glu Gly Gln Asp Val Leu Leu Phe 290 295 300 Ile Asp Asn Ile Phe Arg Phe Thr Gln Ala Gly Ser Glu Val Ser Ala 305 310 315 320 Leu Leu Gly Arg Ile Pro Ser Ala Val Gly Tyr Gln Pro Thr Leu Ala 325 330 335 Thr Asp Met Gly Thr Met Gln Glu Arg Ile Thr Thr Thr Lys Lys Gly 340 345 350 Ser Ile Thr Ser Val Gln Ala Ile Tyr Val Pro Ala Asp Asp Leu Thr 355 360 365 Asp Pro Ala Pro Ala Thr Thr Phe Ala His Leu Asp Ala Thr Thr Val 370 375 380 Leu Ser Arg Ala Ile Ala Glu Leu Gly Ile Tyr Pro Ala Val Asp Pro 385 390 395 400 Leu Asp Ser Thr Ser Arg Ile Met Asp Pro Asn Ile Val Gly Ser Glu 405 410 415 His Tyr Asp Val Ala Arg Gly Val Gln Lys Ile Leu Gln Asp Tyr Lys 420 425 430 Ser Leu Gln Asp Ile Ile Ala Ile Leu Gly Met Asp Glu Leu Ser Glu 435 440 445 Glu Asp Lys Leu Thr Val Ser Arg Ala Arg Lys Ile Gln Arg Phe Leu 450 455 460 Ser Gln Pro Phe Gln Val Ala Glu Val Phe Thr Gly His Met Gly Lys 465 470 475 480 Leu Val Pro Leu Lys Glu Thr Ile Lys Gly Phe Gln Gln Ile Leu Ala 485 490 495 Gly Glu Tyr Asp His Leu Pro Glu Gln Ala Phe Tyr Met Val Gly Pro 500 505 510 Ile Glu Glu Ala Val Ala Lys Ala Asp Lys Leu Ala Glu Glu His Ser 515 520 525 Ser 51274PRTHomo sapiens 51Met Leu Ser Val Ala Ala Arg Ser Gly Pro Phe Ala Pro Val Leu Ser 1 5 10 15 Ala Thr Ser Arg Gly Val Ala Gly Ala Leu Arg Pro Leu Val Gln Ala 20 25 30 Thr Val Pro Ala Thr Pro Glu Gln Pro Val Leu Asp Leu Lys Arg Pro 35 40 45 Phe Leu Ser Arg Glu Ser Leu Ser Gly Gln Ala Val Arg Arg Pro Leu 50 55 60 Val Ala Ser Val Gly Leu Asn Val Pro Ala Ser Val Cys Tyr Ser His 65 70 75 80 Thr Asp Ile Lys Val Pro Asp Phe Ser Glu Tyr Arg Arg Leu Glu Val 85 90 95 Leu Asp Ser Thr Lys Ser Ser Arg Glu Ser Ser Glu Ala Arg Lys Gly 100 105 110 Phe Ser Tyr Leu Val Thr Gly Val Thr Thr Val Gly Val Ala Tyr Ala 115 120 125 Ala Lys Asn Ala Val Thr Gln Phe Val Ser Ser Met Ser Ala Ser Ala 130 135 140 Asp Val Leu Ala Leu Ala Lys Ile Glu Ile Lys Leu Ser Asp Ile Pro 145 150 155 160 Glu Gly Lys Asn Met Ala Phe Lys Trp Arg Gly Lys Pro Leu Phe Val 165 170 175 Arg His Arg Thr Gln Lys Glu Ile Glu Gln Glu Ala Ala Val Glu Leu 180 185 190 Ser Gln Leu Arg Asp Pro Gln His Asp Leu Asp Arg Val Lys Lys Pro 195 200 205 Glu Trp Val Ile Leu Ile Gly Val Cys Thr His Leu Gly Cys Val Pro 210 215 220 Ile Ala Asn Ala Gly Asp Phe Gly Gly Tyr Tyr Cys Pro Cys His Gly 225 230 235 240 Ser His Tyr Asp Ala Ser Gly Arg Ile Arg Leu Gly Pro Ala Pro Leu 245 250 255 Asn Leu Glu Val Pro Thr Tyr Glu Phe Thr Ser Asp Asp Met Val Ile 260 265 270 Val Gly 52464PRTHomo sapiens 52Met Leu Ala Thr Arg Arg Leu Leu Gly Trp Ser Leu Pro Ala Arg Val 1 5 10 15 Ser Val Arg Phe Ser Gly Asp Thr Thr Ala Pro Lys Lys Thr Ser Phe 20 25 30 Gly Ser Leu Lys Asp Glu Asp Arg Ile Phe Thr Asn Leu Tyr Gly Arg 35 40 45 His Asp Trp Arg Leu Lys Gly Ser Leu Ser Arg Gly Asp Trp Tyr Lys 50 55 60 Thr Lys Glu Ile Leu Leu Lys Gly Pro Asp Trp Ile Leu Gly Glu Ile 65 70 75 80 Lys Thr Ser Gly Leu Arg Gly Arg Gly Gly Ala Gly Phe Pro Thr Gly 85 90 95 Leu Lys Trp Ser Phe Met Asn Lys Pro Ser Asp Gly Arg Pro Lys Tyr 100 105 110 Leu Val Val Asn Ala Asp Glu Gly Glu Pro Gly Thr Cys Lys Asp Arg 115 120 125 Glu Ile Leu Arg His Asp Pro His Lys Leu Leu Glu Gly Cys Leu Val 130 135 140 Gly Gly Arg Ala Met Gly Ala Arg Ala Ala Tyr Ile Tyr Ile Arg Gly 145 150 155 160 Glu Phe Tyr Asn Glu Ala Ser Asn Leu Gln Val Ala Ile Arg Glu Ala 165 170 175 Tyr Glu Ala Gly Leu Ile Gly Lys Asn Ala Cys Gly Ser Gly Tyr Asp 180 185 190 Phe Asp Val Phe Val Val Arg Gly Ala Gly Ala Tyr Ile Cys Gly Glu 195 200 205 Glu Thr Ala Leu Ile Glu Ser Ile Glu Gly Lys Gln Gly Lys Pro Arg 210 215 220 Leu Lys Pro Pro Phe Pro Ala Asp Val Gly Val Phe Gly Cys Pro Thr 225 230 235 240 Thr Val Ala Asn Val Glu Thr Val Ala Val Ser Pro Thr Ile Cys Arg 245 250 255 Arg Gly Gly Thr Trp Phe Ala Gly Phe Gly Arg Glu Arg Asn Ser Gly 260 265 270 Thr Lys Leu Phe Asn Ile Ser Gly His Val Asn His Pro Cys Thr Val 275 280 285 Glu Glu Glu Met Ser Val Pro Leu Lys Glu Leu Ile Glu Lys His Ala 290 295 300 Gly Gly Val Thr Gly Gly Trp Asp Asn Leu Leu Ala Val Ile Pro Gly 305 310 315 320 Gly Ser Ser Thr Pro Leu Ile Pro Lys Ser Val Cys Glu Thr Val Leu 325 330 335 Met Asp Phe Asp Ala Leu Val Gln Ala Gln Thr Gly Leu Gly Thr Ala 340 345 350 Ala Val Ile Val Met Asp Arg Ser Thr Asp Ile Val Lys Ala Ile Ala 355 360 365 Arg Leu Ile Glu Phe Tyr Lys His Glu Ser Cys Gly Gln Cys Thr Pro 370 375 380 Cys Arg Glu Gly Val Asp Trp Met Asn Lys Val Met Ala Arg Phe Val 385 390 395 400 Arg Gly Asp Ala Arg Pro Ala Glu Ile Asp Ser Leu Trp Glu Ile Ser 405 410 415 Lys Gln Ile Glu Gly His Thr Ile Cys Ala Leu Gly Asp Gly Ala Ala 420 425 430 Trp Pro Val Gln Gly Leu Ile Arg His Phe Arg Pro Glu Leu Glu Glu 435 440 445 Arg Met Gln Arg Phe Ala Gln Gln His Gln Ala Arg Gln Ala Ala Ser 450 455 460 53249PRTHomo sapiens 53Met Phe Phe Ser Ala Ala Leu Arg Ala Arg Ala Ala Gly Leu Thr Ala 1 5 10 15 His Trp Gly Arg His Val Arg Asn Leu His Lys Thr Ala Met Gln Asn 20 25 30 Gly Ala Gly Gly Ala Leu Phe Val His Arg Asp Thr Pro Glu Asn Asn 35 40 45 Pro Asp Thr Pro Phe Asp Phe Thr Pro Glu Asn Tyr Lys Arg Ile Glu 50 55 60 Ala Ile Val Lys Asn Tyr Pro Glu Gly His Lys Ala Ala Ala Val Leu 65 70 75 80 Pro Val Leu Asp Leu Ala Gln Arg Gln Asn Gly Trp Leu Pro Ile Ser 85 90 95 Ala Met Asn Lys Val Ala Glu Val Leu Gln Val Pro Pro Met Arg Val 100 105 110 Tyr Glu Val Ala Thr Phe Tyr Thr Met Tyr Asn Arg Lys Pro Val Gly 115 120 125 Lys Tyr His Ile Gln Val Cys Thr Thr Thr Pro Cys Met Leu Arg Asn 130 135 140 Ser Asp Ser Ile Leu Glu Ala Ile Gln Lys Lys Leu Gly Ile Lys Val 145 150 155 160 Gly Glu Thr Thr Pro Asp Lys Leu Phe Thr Leu Ile Glu Val Glu Cys 165 170 175 Leu Gly Ala Cys Val Asn Ala Pro Met Val Gln Ile Asn Asp Asn Tyr 180 185 190 Tyr Glu Asp Leu Thr Ala Lys Asp Ile Glu Glu Ile Ile Asp Glu Leu 195 200 205 Lys Ala Gly Lys Ile Pro Lys Pro Gly Pro Arg Ser Gly Arg Phe Ser 210 215 220 Cys Glu Pro Ala Gly Gly Leu Thr Ser Leu Thr Glu Pro Pro Lys Gly 225 230 235 240 Pro Gly Phe Gly Val Gln Ala Gly Leu 245 54517PRTHomo sapiens 54Met Leu Arg Ala Ala Ala Arg Phe Gly Pro Arg Leu Gly Arg Arg Leu 1 5 10 15 Leu Ser Ala Ala Ala Thr Gln Ala Val Pro Ala Pro Asn Gln Gln Pro 20 25 30 Glu Val Phe Cys Asn Gln Ile Phe Ile Asn Asn Glu Trp His Asp Ala 35 40 45 Val Ser Arg Lys Thr Phe Pro Thr Val Asn Pro Ser Thr Gly Glu Val 50 55 60 Ile Cys Gln Val Ala Glu Gly Asp Lys Glu Asp Val Asp Lys Ala Val 65 70 75 80 Lys Ala Ala Arg Ala Ala Phe Gln Leu Gly Ser Pro Trp Arg Arg Met 85 90 95 Asp Ala Ser His Arg Gly Arg Leu Leu Asn Arg Leu Ala Asp Leu Ile 100 105 110 Glu Arg Asp Arg Thr Tyr Leu Ala Ala Leu Glu Thr Leu Asp Asn Gly 115 120 125 Lys Pro Tyr Val Ile Ser Tyr Leu Val Asp Leu Asp Met Val Leu Lys 130 135 140 Cys Leu Arg Tyr Tyr Ala Gly Trp Ala Asp Lys Tyr His Gly Lys Thr 145 150 155 160 Ile Pro Ile Asp Gly Asp Phe Phe Ser Tyr Thr Arg His Glu Pro Val 165 170 175 Gly Val Cys Gly Gln Ile Ile Pro Trp Asn Phe Pro Leu Leu Met Gln 180 185 190 Ala Trp Lys Leu Gly Pro Ala Leu Ala Thr Gly Asn Val Val Val Met 195 200 205 Lys Val Ala Glu Gln Thr Pro Leu Thr Ala Leu Tyr Val Ala Asn Leu 210 215 220 Ile Lys Glu Ala Gly Phe Pro Pro Gly Val Val Asn Ile Val Pro Gly 225 230 235 240 Phe Gly Pro Thr Ala Gly Ala Ala Ile Ala Ser His Glu Asp Val Asp 245 250 255 Lys Val Ala Phe Thr Gly Ser Thr Glu Ile Gly Arg Val Ile Gln Val 260 265 270 Ala Ala Gly Ser Ser Asn Leu Lys Arg Val Thr Leu Glu Leu Gly Gly 275 280 285 Lys Ser Pro Asn Ile Ile Met Ser

Asp Ala Asp Met Asp Trp Ala Val 290 295 300 Glu Gln Ala His Phe Ala Leu Phe Phe Asn Gln Gly Gln Cys Cys Cys 305 310 315 320 Ala Gly Ser Arg Thr Phe Val Gln Glu Asp Ile Tyr Asp Glu Phe Val 325 330 335 Glu Arg Ser Val Ala Arg Ala Lys Ser Arg Val Val Gly Asn Pro Phe 340 345 350 Asp Ser Lys Thr Glu Gln Gly Pro Gln Val Asp Glu Thr Gln Phe Lys 355 360 365 Lys Ile Leu Gly Tyr Ile Asn Thr Gly Lys Gln Glu Gly Ala Lys Leu 370 375 380 Leu Cys Gly Gly Gly Ile Ala Ala Asp Arg Gly Tyr Phe Ile Gln Pro 385 390 395 400 Thr Val Phe Gly Asp Val Gln Asp Gly Met Thr Ile Ala Lys Glu Glu 405 410 415 Ile Phe Gly Pro Val Met Gln Ile Leu Lys Phe Lys Thr Ile Glu Glu 420 425 430 Val Val Gly Arg Ala Asn Asn Ser Thr Tyr Gly Leu Ala Ala Ala Val 435 440 445 Phe Thr Lys Asp Leu Asp Lys Ala Asn Tyr Leu Ser Gln Ala Leu Gln 450 455 460 Ala Gly Thr Val Trp Val Asn Cys Tyr Asp Val Phe Gly Ala Gln Ser 465 470 475 480 Pro Phe Gly Gly Tyr Lys Met Ser Gly Ser Gly Arg Glu Leu Gly Glu 485 490 495 Tyr Gly Leu Gln Ala Tyr Thr Glu Val Lys Thr Val Thr Val Lys Val 500 505 510 Pro Gln Lys Asn Ser 515 55443PRTHomo sapiens 55Met Ala Ala Ser Pro His Thr Leu Ser Ser Arg Leu Leu Thr Gly Cys 1 5 10 15 Val Gly Gly Ser Val Trp Tyr Leu Glu Arg Arg Thr Ile Gln Asp Ser 20 25 30 Pro His Lys Phe Leu His Leu Leu Arg Asn Val Asn Lys Gln Trp Ile 35 40 45 Thr Phe Gln His Phe Ser Phe Leu Lys Arg Met Tyr Val Thr Gln Leu 50 55 60 Asn Arg Ser His Asn Gln Gln Val Arg Pro Lys Pro Glu Pro Val Ala 65 70 75 80 Ser Pro Phe Leu Glu Lys Thr Ser Ser Gly Gln Ala Lys Ala Glu Ile 85 90 95 Tyr Glu Met Arg Pro Leu Ser Pro Pro Ser Leu Ser Leu Ser Arg Lys 100 105 110 Pro Asn Glu Lys Glu Leu Ile Glu Leu Glu Pro Asp Ser Val Ile Glu 115 120 125 Asp Ser Ile Asp Val Gly Lys Glu Thr Lys Glu Glu Lys Arg Trp Lys 130 135 140 Glu Met Lys Leu Gln Val Tyr Asp Leu Pro Gly Ile Leu Ala Arg Leu 145 150 155 160 Ser Lys Ile Lys Leu Thr Ala Leu Val Val Ser Thr Thr Ala Ala Gly 165 170 175 Phe Ala Leu Ala Pro Gly Pro Phe Asp Trp Pro Cys Phe Leu Leu Thr 180 185 190 Ser Val Gly Thr Gly Leu Ala Ser Cys Ala Ala Asn Ser Ile Asn Gln 195 200 205 Phe Phe Glu Val Pro Phe Asp Ser Asn Met Asn Arg Thr Lys Asn Arg 210 215 220 Pro Leu Val Arg Gly Gln Ile Ser Pro Leu Leu Ala Val Ser Phe Ala 225 230 235 240 Thr Cys Cys Ala Val Pro Gly Val Ala Ile Leu Thr Leu Gly Val Asn 245 250 255 Pro Leu Thr Gly Ala Leu Gly Leu Phe Asn Ile Phe Leu Tyr Thr Cys 260 265 270 Cys Tyr Thr Pro Leu Lys Arg Ile Ser Ile Ala Asn Thr Trp Val Gly 275 280 285 Ala Val Val Gly Ala Ile Pro Pro Val Met Gly Trp Thr Ala Ala Thr 290 295 300 Gly Ser Leu Asp Ala Gly Ala Phe Leu Leu Gly Gly Ile Leu Tyr Ser 305 310 315 320 Trp Gln Phe Pro His Phe Asn Ala Leu Ser Trp Gly Leu Arg Glu Asp 325 330 335 Tyr Ser Arg Gly Gly Tyr Cys Met Met Ser Val Thr His Pro Gly Leu 340 345 350 Cys Arg Arg Val Ala Leu Arg His Cys Leu Ala Leu Leu Val Leu Ser 355 360 365 Ala Ala Ala Pro Val Leu Asp Ile Thr Thr Trp Thr Phe Pro Ile Met 370 375 380 Ala Leu Pro Ile Asn Ala Tyr Ile Ser Tyr Leu Gly Phe Arg Phe Tyr 385 390 395 400 Val Asp Ala Asp Arg Arg Ser Ser Arg Arg Leu Phe Phe Cys Ser Leu 405 410 415 Trp His Leu Pro Leu Leu Leu Leu Leu Met Leu Thr Cys Lys Arg Pro 420 425 430 Ser Gly Gly Gly Asp Ala Gly Pro Pro Pro Ser 435 440 56239PRTHomo sapiens 56Met Ala Pro Ser Val Pro Ala Ala Glu Pro Glu Tyr Pro Lys Gly Ile 1 5 10 15 Arg Ala Val Leu Leu Gly Pro Pro Gly Ala Gly Lys Gly Thr Gln Ala 20 25 30 Pro Arg Leu Ala Glu Asn Phe Cys Val Cys His Leu Ala Thr Gly Asp 35 40 45 Met Leu Arg Ala Met Val Ala Ser Gly Ser Glu Leu Gly Lys Lys Leu 50 55 60 Lys Ala Thr Met Asp Ala Gly Lys Leu Val Ser Asp Glu Met Val Val 65 70 75 80 Glu Leu Ile Glu Lys Asn Leu Glu Thr Pro Leu Cys Lys Asn Gly Phe 85 90 95 Leu Leu Asp Gly Phe Pro Arg Thr Val Arg Gln Ala Glu Met Leu Asp 100 105 110 Asp Leu Met Glu Lys Arg Lys Glu Lys Leu Asp Ser Val Ile Glu Phe 115 120 125 Ser Ile Pro Asp Ser Leu Leu Ile Arg Arg Ile Thr Gly Arg Leu Ile 130 135 140 His Pro Lys Ser Gly Arg Ser Tyr His Glu Glu Phe Asn Pro Pro Lys 145 150 155 160 Glu Pro Met Lys Asp Asp Ile Thr Gly Glu Pro Leu Ile Arg Arg Ser 165 170 175 Asp Asp Asn Glu Lys Ala Leu Lys Ile Arg Leu Gln Ala Tyr His Thr 180 185 190 Gln Thr Thr Pro Leu Ile Glu Tyr Tyr Arg Lys Arg Gly Ile His Ser 195 200 205 Ala Ile Asp Ala Ser Gln Thr Pro Asp Val Val Phe Ala Ser Ile Leu 210 215 220 Ala Ala Phe Ser Lys Ala Thr Cys Lys Asp Leu Val Met Phe Ile 225 230 235 57922DNAHomo sapiens 57tatcagaagg ccaggcgaga ctgcaacact gctcatcacc ccgcggcgtg atccctgctc 60ttaggtgctg ggcagagggg aagggtggtc agggtgagga tggtgaggga gggctggtga 120ggggctcaga ggaatacttg gaacaatagc agtgttattg tagtgtggca gtttctttta 180tacataggtg agagttttta aagtgtaagg gaaaaattaa ttttttaaaa aacaccatgc 240ttggagggtg ggggtagaaa tagacacaat attatttcta aggaatcggg ttttcattta 300ctctggactg gtgaaaatat tttttaaagc cagtgctcta agacctcagc ttttatctca 360gaaccccatg ggttccagac caagagtaca ggaaatcaaa ttgttgtcct gtctgtctat 420agcttggaac agggagcttt gattactgac tccggttcca cacactgtaa gatcaaaaac 480catctccaca tttgaaagag atgtaaggtg tattcatagg gatggtggct caacaaatca 540agcaaactgg aatcaagggg agggggaagg gaatgaaatg gaaagggagg ctgattccct 600tcccctgact taccactaat ttactaggct acctactttc atgagtaacc tctcacagct 660acccagcaca tgccacaatc ctatgctctt gccttctttt atctgcactg tgtgaaggga 720ctcttttaaa taaatgagca agtgtcctaa gctatgtcat ccaaagattg tcctttccat 780tctcaaatcc tgtgactggg atcactcaac agcactgtga tgtattattt tcaatgaggt 840gccttctaaa actgaccaaa tgctgccttg tttggcccct aaatcaataa aatatgttaa 900aatttgaaaa aaaaaaaaaa aa 9225833DNAArtificial SequenceDescription of Artificial Sequence syntheticPrimer 58cgactacggc gccctggaac ctcacatcaa cgc 33591425DNAHomo sapiens 59gtgttgatgt atcacctccc caaaactgtt ggtaaatgtc agattttttc ctccaagagt 60tgtgcttttg tgttatttgt tttcactcaa atattttgcc tcattattct tgttttaaaa 120gaaagaaaac aggccgggca cagtggctca tgcctgtaat cccagcactt tgggaggtcg 180aggtgggtgg atcacttggg gtcagggttt gagaccagcc tggccaacat ggcggaaccc 240tgtctctacc aaaattacaa aaattagccg agcatggtgg cgcatgcctg tagtcgcagc 300tactcgcgag gttgaggcag gagaattgct tgaacccagg aagtggcagt tgcagtgagc 360cgagacgaca ccactgcact ccagcctggg tgacagaggg agactctgtc tcgaaagaaa 420gaaagaaaaa aaggaaggaa ggagaaggaa ggaaggagaa gaaaaggtac ctgttctacg 480tagaacacct ttggtggagt tccatcaact cgcaaagtag aatccttacc tactactctt 540ctgataataa ttttaatatt ttttatgttt ggttgatgcg agcagctgca ctgctcatgc 600agttagctag catgtgacat catgtgacaa agttcatgta attagatgga agaaacctca 660ctgattaatt ttaagaacct tttagggatg caggaacaat gaagtggcca cagtatgtgc 720tgtttttgaa gcatttttaa aaacgaattg tagttgtttt tcttcattta aaatggatct 780gttggaggtt atgtgtgtat gttgtagttt tattgcagcc acaataattt taccaaagtt 840ttcacatagg cagttagcct ttacttaata tcaagacaag tgaaaaaata ttggcatcga 900tgaaaccgat aacattggcc tcattggatt tctttaccca ttcacagtgt aaagaagtta 960ccttcatgct ttcattgtac ctgcaggcct gtgggcttgt acagtagata attaatttct 1020aaaaagaaca gctgcctatt ttcttcctag gttaggttat atcttcataa tcacaagaat 1080tagtgatggc aaaataaaat tttgcttatg aatcttttac attgtttata tatgattaat 1140atcatcatat atattttctg tattaagctc atttggcttc atttaagctg tatacttagt 1200catatatctt tcattagttc tatggatatg agcagatccc tttactggag cccagtatgt 1260gctgtgtgag ttagaagtca ttcttgctga gaaggtgaat aggtagggat ttgccttgtt 1320ttgtaagtct acaatttgcc aagagtaaat aacactggac cagctgtaaa agtaaacagt 1380gtgtttatgc attgagatac taaagcattt aagaaaaaat taaaa 142560202DNAHomo sapiens 60ttatgctgag tatgttaagc tctttatgac tgtttttgta gtggtataga gtactgcaga 60atacagtaag ctgctctatt gtagcatttc ctgatgttgc ttagtcactt atttcataaa 120caacttaatg ttctgaataa tttcttacta aacattttgt tattgggcaa gtgattgaaa 180atagtaaatg ctttgtgtga tt 202

Claims

1. An expression vector configured for the efficient and stable delivery of a protein into the mitochondrion of a mammalian cell, the vector comprising: a mitochondrion-targeting nucleic acid sequence (MTS); a nucleic acid sequence encoding said protein in accordance with a universal genetic code (CDS); and a 3'UTR nucleic acid sequence, located 3' of said CDS, configured to target mRNA expressed by the expression vector in the nucleus to the mitochondrion-bound polysomes of the mammalian cell, wherein, said MTS consists of a nucleic acid sequence encoding the peptide SEQ ID NO: 46, and said 3'UTR nucleic acid sequence consists of a conservative variant or fragment of the nucleic acid sequence SEQ ID NO: 47, said conservative variant or fragment still allowing for the mitochondrial targeting of the mRNA.

2. The expression vector of claim 1, wherein the CDS nucleic acid sequence encodes the NADH-ubiquinone oxireductase subunit 4 protein (ND4).

3. The expression vector of claim 1, wherein the nucleic acid sequence encoding the peptide SEQ ID NO: 46 is SEQ ID NO: 30.

4. The expression vector of claim 2, wherein the CDS nucleic acid sequence encoding ND4 is SEQ ID NO: 29.

5. The expression vector of claim 1, wherein said mammalian cell is a human cell.

6. The expression vector of claim 1, wherein the nucleic acid sequence encoding said protein has been optimized with preferred human codon usage.

7. The expression vector of claim 2, wherein the nucleic acid sequence encoding the ND4 protein has been optimized with preferred human codon usage.

8. The expression vector of claim 1, wherein the CDS nucleic acid sequence encodes a protein selected from the group consisting of mitochondrially encoded ATP synthase 6 (ATP6), mitochondrially encoded NADH dehydrogenase subunit 1 (ND1), and NADH-ubiquinone oxidoreductase subunit 4 (ND4).

9. The expression vector of claim 8, wherein the CDS nucleic acid sequence encoding said protein has been optimized with preferred human codon usage.

10. The expression vector of claim 8, wherein the CDS nucleic acid sequence is selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 28, and SEQ ID NO: 27.

11. The expression vector of claim 1, wherein said 3'UTR nucleic acid sequence consists of the nucleic acid sequence SEQ ID NO: 47.

12. The expression vector of claim 1, wherein said 3'UTR nucleic acid sequence consists of a conservative variant of the nucleic acid sequence SEQ ID NO: 47, said conservative variant still allowing for the mitochondrial targeting of the mRNA.

13. The expression vector of claim 1, wherein said 3'UTR nucleic acid sequence consists of a fragment of the nucleic acid sequence SEQ ID NO: 47, said fragment still allowing for the mitochondrial targeting of the mRNA.