WO2000053773A2 - Methods for mitochondrial gene therapy - Google Patents

Abstract

The present invention provides methods for functionally complementing one or more defects, mutations, or deletions in a mitochondrial genome. The method includes the steps of selecting a mitochondrial gene; determining a nucleic acid sequence of the mitochondrial gene; linking the nucleic acid sequence of the mitochondrial gene to one or more targeting sequences to generate a target construct; linking one or more control sequences to the target construct to generate a nucleic acid construct; and inserting the nucleic acid construct into the nuclear genome of the cell for expression of the nucleic acid in the cell. The method is used to treat a disease or disorder that arises from a paucity or lack of mitochondrial DNA in a cell. The invention further provides methods for performing total functional complementation of a mitochondrial genome. Corresponding methods for detecting functional complementation of one or more defects, mutations or deletions in a mitochondrial genome, as well as a selectable marker for detecting functional complementation of one or more mitochondrial genes, are also provided.

Description

METHODS FOR MITOCHONDRIAL GENE THERAPY

FIELD OF THE INVENTION

The present invention relates to the field of gene therapy. More particularly, the present invention relates to methods of providing functional complementation for mutated, missing, or nonfunctional mitochondrial genes.

BACKGROUND OF THE INVENTION

Although the role of the mitochondrion in providing energy for the cell by the process of oxidative phosphorylation has been known for a long time, the role of the mitochondrial genome and the consequences of defects in the mitochondrial genome are just being understood. This has led to the understanding that such defects can cause or contribute to a broad range of diseases and syndromes in humans and other eukaryotic organisms. These diseases and syndromes often present unusual or rare combinations of symptoms and can be difficult to diagnose. Moreover, they are frequently resistant to treatment by standard methods.

Although rigorous epidemiologic data for mitochondrial disease are not yet available, specialists at referral centers for mitochondrial and metabolic disease have developed estimates of the incidence and prevalence of mitochondrial diseases by reference to genetic disorders for which good epidemiologic data are available. One such example is medium chain acyl CoA dehydrogenase deficiency (MCAD). One Dutch study that looked at a common mutation in newborn screening found a carrier frequency of 1 in 55 subjects, which led to predictions of homozygote frequency

(disease frequency) of 1 in 12,100. de Vries HG, et al, Human Genetics 1996;98:1-2. Another study in Spain found the carrier frequency for the common mutant allele to be 1 in 17, which led to an estimated homozygote frequency of 1 in 1,156. Martinez, G., et al, Ped. Res. 1998;44:83-84. In a prospective surveillance report from the UK, the disease frequency was 1 in 22,222 (Pollitt et al., Arch. Dis. Childhood 1998; 79: 1 lol l 9).

Experience at the Mitochondrial and Metabolic Disease Center (MMCD) at the University of California, San Diego, is consistent with an MCAD incidence on the order of 1 in 20,000 to 1 in 40,000 live births. Since approximately 1995, studies at the MMCD have found the ratio of local children presenting with one of the 20 most common mitochondrial disease to MCAD was 10 to 20 to 1. This leads to a rough estimate of the incidence of mitochondrial disease of 1 in 4,000 to 1 in 1,000. According to these estimates, as many as 4,000 children are born each year in the

United States with mitochondrial disease. This is about half the frequency of childhood cancer, which is 8,000 new cases each year.

Mitochondrial disease is not always present in the first ten years of life. Adults with mitochondrial encephalmyopathy with lactic acidosis and stroke-like episodes (MELAS) or myoclonic epilepsy with ragged red fibers (MERRF) may not become symptomatic until their thirties. Kearns-Sayre may not present until the teenage years, and many adults are not diagnosed until the twenties or thirties. The first patient diagnosed with neurogenic muscular weakness, atoxia, retinitis pigmentosa (NARP) was in her fifties. Treatment of mitochondrial genetic disease has met with limited success and simply treats the symptoms rather than the cause of the genetic disorder. See Bresolin et al., Neurol. 1989, 39(Suppl. 1):259; Desnuelle et al, Neuro I. 1988, 38(Suppl. 1):102.; Ihara et al., J Neurol. Sci. 1989, 90:263-271; Nishikawa et al., Neurol. 1989, 39:399-403; Ogasahara et al., Neurol. 1985, 35:372-377; Ogasahara et al, Neurol. 1986, 36:45-53; Shoffneret al, Neurol. 1989, 39(Suppl. 1):404; Shoffner et al., Neurol. 1989, 39(Suppl. 1):256.

The emerging field of gene therapy endeavors to treat the cause of genetic diseases; i.e., replace the defective gene or supply the missing gene. Generally, gene therapy relies on the presence of recombination machinery at the site of the genome, i.e., in the nucleus for mendelian-inherited and sex-linked-inherited disease, and in the mitochondria for mitochondrial genome-inherited diseases.

The entire mitochondrial genome has been cloned and sequenced for a number of different organisms, including humans. Of the hundreds of proteins required for mitochondrial structure and function, the mammalian mitochondrial genome encodes only thirteen (13). The thirteen polypeptides are all subunits of the electron transport chain, and are essential to the energetic health of mitochondria and the individual. Aside from the thirteen protein-encoding mitochondrial genes, the remaining mitochondrial proteins are encoded in the nucleus, translated in the cytoplasm and imported into mitochondria. Amino-terminal "leader" peptides direct many of the nuclear-encoded, cytoplasmically translated mitochondrial proteins to the mitochondrial membrane, where the leader peptides are removed in one or two steps as the protein is transported into the mitochondrion.

Each mitochondrion contains numerous copies of mitochondrial DNA

(mtDNA), constituting the mitochondrial genome. Somatic mutations in a copy or copies of mtDNA can create the phenomenon of heteroplasmy (literally, multiple "alleles" in "plasmic" (i.e., mitochondrial) DNA of the same cytoplasm). Linnane, et al, (1989. Lancet. i:642-645) suggested that the accumulation of mtDNA mutations and deletions, and the heteroplasmy resulting from the stochastic distribution of the mutant molecules, may contribute to the aging process and several diseases. Mutations, duplications, and deletions of mtDNA-encoded genes have indeed been found in human diseases, such as Leber's hereditary optic neuropathy (LHON); mitochondrial myopathy; chronic progressive external ophthalmoplegia (CPEO); and Kearns-Sayre (KS) syndrome. Organ systems that rely heavily on mitochondrial function are primarily affected, in spite of the fact that each cell contains hundreds of mitochondria and each mitochondrion contains numerous mitochondrial DNA copies. Further, somatic mtDNA mutations may be among the more important contributors to various disorders of the central nervous system (CNS).

Many cells affected by mitochondrial maladies are post-mitotic, e.g., central nervous system (CNS) cells, and turnover of mtDNA may lead to a damaged DNA molecule becoming the predominant type in the cell. Ozawa, et al. (1990. Biochem. Biophys. Res. Comm. 172:483-489) observed that the ratio of mutated mtDNA, containing a 4977 base pair (bp) deletion, to normal mtDNA (heteroplasmy) increased over ten-fold, from 0.3% in normal striatum to 5% in parkinsonian striatum. Merril, et al, (1996. Arch. Biochem. Biophys. 326:172-177) used the polymerase chain reaction (PCR) to detect an increase of the 4977 bp mtDNA deletion in autopsy tissue of subjects with conditions associated with chronic hypoxia, and with no known mitochondrial disease. The deletion is flanked by a 13 bp direct repeat, indicating a possible recombination mechanism. These increases of deleted mtDNA molecules may be important in mitochondrial energy metabolism and the disease processes. SUMMARY

The present invention provides a method for providing functional complementation of one or more defects, mutations, or deletions in the mitochondrial genome. Generally, the method involves placing one or more functional copies of the defective mitochondrial genetic material in the nuclear genome, with alterations of the nucleic acid base sequence as appropriate to take into account the differences between the mitochondrial genetic code and the universal genetic code. These functional copies are then expressed and targeted to the mitochondria to provide the gene product that the mitochondrion is unable to make. The method is used to functionally complement one or more defects, mutations or deletions in a mitochondrial genome of a cell.

In one embodiment, the invention provides a method for functionally complementing one or more defects, mutations, or deletions in a mitochondrial genome of a cell having a nuclear genome, the method comprising steps of: (a) selecting a mitochondrial gene;

(b) determining a nucleic acid sequence of the mitochondrial gene;

(c) linking the nucleic acid sequence of the mitochondrial gene to one or more targeting sequences to generate a target construct;

(d) linking one or more control sequences to the target construct to generate a nucleic acid construct; and

(e) inserting the nucleic acid construct into the nuclear genome of the cell for expression of the nucleic acid in the cell.

The method is used to treat diseases or disorders that arise from one or more defects, mutations or deletions in the mitochondrial genome. In one embodiment, the method is used to treat a disease or disorder that is caused by one or more mutated or defective mitochondrial genes, the method comprising functionally complementing the mutated mitochondrial-encoded gene with a nucleus-located and expressed functional copy of the mutated mitochondrial gene. The method is also used to counteract the deleterious effects of a reduction in oxidative phosphorylation capacity during a disease process, or during the aging process.

In another aspect, the invention provides a method for total complementation of the function of the mitochondrial genome. According to this embodiment, the thirteen mitochondrial genes are selected and inserted into the nuclear genome of the cell for expression, and the resulting proteins are targeted to the mitochondrion. In this embodiment, there is no need for functioning mitochondrial tRNA or ribosomal RNA, since transcription and translation occur outside the mitochondrion.

In general, this method comprises:

(a) selecting all mitochondrial genes of the cell that encode proteins; (b) determining a nucleic acid sequence of each of the mitochondrial genes selected in step (a);

(c) linking the nucleic acid sequence of each of the mitochondrial genes to one or more target sequences to generate target constructs;

(d) linking one or more control sequences to the target constructs to generate nucleic acid constructs; and

(e) inserting the nucleic acid constructs into the nuclear genome of the cell for expression of the nucleic acid sequences in the cell.

The method is used, in one embodiment, to treat a disease or disorder that arises from deletion of the protein-encoding genes of the mitochondrial genome. Alternatively, the method is used to treat a disease or disorder that arises from one or more defects, deletions or mutations in mitochondrial genes encoding ribosomes or tRNA for transcription and translation in the mitochondria. The method is used, in another embodiment, to treat a disease or disorder that arises from a paucity or lack of mitochondrial DNA in the cell, such as mitochondrial depletion syndrome. In yet another embodiment, the method is used for the recovery of adequate oxidative phosphorylation capacity.

Preferably, the control element comprises a promoter. In one embodiment, the promoter is a constitutive promoter, such as SV40. Alternatively, the promoter is an inducible promoter, such as a tetracycline promoter. The nucleic acid construct or constructs are inserted into the nuclear genome of the cell using a suitable vector. Suitable vectors include vectors that are plasmid or viral in origin, to introduce the desired genetic material into the cells. Other vectors can be chosen to achieve the same purpose. In one embodiment, when the invention is used to functionally complement a large portion of, or the entire mitochondrial genome, a preferred vector is a lentiviral vector.

The nucleic acid construct is introduced into the cell by a method such as, for example, a polycation, calcium phosphate coprecipitation, electroporation, transfection, lipofection, micro injection, or the use of viral or retroviral vectors. Other methods allowing the introduction of foreign DNA into a cell can be utilized.

A preferred method is electroporation.

The cell can be an animal cell, such as a mammalian cell, including a human cell. Alternatively, the cell can be a plant cell. The method is used to introduce the mitochondrial gene into non-dividing cells, or dividing cells, such as stem cells.

In one embodiment, the eukaryotic nucleus having the nuclear genome into which the thirteen nucleic acid constructs are inserted is then transplanted into an enucleated egg to form a transgenic animal.

In yet another aspect, the present invention provides a selectable marker for detecting functional complementation of a mitochondrial gene comprising the coding region of the nucleotide sequence of pUOATP2.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description, appended claims, and accompanying drawings where:

Figure 1 is a map of the human mitochondrial genome;

Figure 2 is a diagram of the structure of the plasmid pUOATP2 that includes a gene for oligomycin-resistant mitochondrial ATPaseό;

Figure 3 depicts the complete nucleotide sequence of the plasmid pUOATP2;

Figure 4 is a diagram of the vector pZeoSV2(+) that incorporates a constitutive SV40 promoter; Figure 4a is a photograph of the results of an in vitro translation of the pUOATP2 vector;

Figure 5 is a photomicrograph of results obtained by fluorescent in situ hybridization (FISH), showing that the nucleic acid construct is present in metaphase chromosomes of transformed Chinese hamster ovary (CHO) cells; Figure 6 is a graph of the results obtained by growing the transformed and nontransformed CHO cells in the presence and absence of oligomycin;

Figure 7 is a graph of the results obtained by measuring the oxygen consumption of the transformed and nontransformed human cybrid cells in the presence or absence of oligomycin; and Figure 8 is a diagram of the structure of the HSV/AAV vector.

DETAILED DESCRIPTION

To date, dietary supplements are the only method used to ameliorate the effects of mitochondrial DNA (mtDNA) mutations or deletions. The phenomenon of heteroplasmy and the sheer number of mtDNA molecules per cell make direct gene therapy of mtDNA defects impractical.

The present invention provides a novel method for mitochondrial gene therapy involving introduction of a mtDNA gene to the nuclear genome. The invention provides a combination of advantages. For example, in one embodiment, the method provides a selectable marker for determining incorporation of the nucleic acid construct into the mitochondria, for example, by the oli^r phenotype conferred by mutation of the ATPaseό gene. Additionally, the number of copies of the replacement protein in the mitochondria can be controlled using the control elements. Further, the invention is not limited to functional complementation of a gene that is missing from the mitochondrial genome, but rather can be used to functionally complement any defect, deletion, mutation or deficit of a mitochondrial gene. In one preferred embodiment, a CHO mutant ATPaseό confers oligomycin resistance (oli¹) to the wild type CHO cell. The transformed CHO cell lines grow in 1000 ng/ml oligomycin while the untransformed sensitive CHO cells are eliminated in 1 ng/ml oligomycin. Additionally, human cybrids (gift of Doug Wallace-Emory) containing the NARP mutation in 100% of the mitochondrial genomes were transformed with the CHO mutant. The transformed cybrids grow in up to 100 ng/ml oligomycin, while the untransformed are eliminated in 1 ng/ml oligomycin.

In another embodiment, the invention provides a method for performing total functional complementation of the mitochondrial genome of a cell. In yet another embodiment, the invention provides a selectable marker for detecting functional complementation of a mitochondrial gene.

Most of the enzyme subunits of the mitochondrial electron transport and oxidative phosphorylation systems are nuclear-encoded and are normally incorporated in a functional state. This present method does not correct the genetic defect but rather ameliorates the clinical pathology resulting from the defect by supplying the wild type protein from an exogenous source, in effect treating the cause by replacing the defective protein. This exogenously supplied protein can be supplied in a constitutive fashion or an inducible fashion. In a preferred embodiment, the mitochondrial gene is attached to a control sequence, such as a promoter, to control expression of the protein.

For example, it may be desirable to constitutively express the protein because the mitochondrial electron transport enzymes are undoubtedly replaced regularly.

Alternatively, the protein can be supplied in inducible fashion when it is desirable to allow selective activation of the transgenes to augment oxidative phosphorylation at selected times or for selected durations. In one embodiment, a selectable marker is incorporated into the nucleic acid construct to enable the feasibility and efficacy of the method to be evaluated.

I. Methods of Providing Functional Complementation

According to the present invention, a method of providing functional complementation for defects, mutations, or deletions in the mitochondrial genome is based on the addition of the required mitochondrial genetic material to the nuclear genome. The mitochondrial genetic material to be added is modified to reflect the difference between the mitochondrial and nuclear genetic codes, so that the added genetic material is capable of being transcribed and translated outside the mitochondria, and result in a functional protein once it is imported into the mitochondria.

In general, this method comprises: (a) selecting a mitochondrial gene;

(b) determining a nucleic acid sequence of the mitochondrial gene;

(c) linking the nucleic acid sequence of the mitochondrial gene to one or more target sequences to generate a target construct;

(d) linking one or more control sequences to the target construct to generate a nucleic acid construct; and

(e) inserting the nucleic acid construct into a nuclear genome of the cell for expression of the nucleic acid in the cell.

As used herein the term "nucleic acid sequence" includes both DNA and RNA unless otherwise specified, and, unless otherwise specified, includes both double-stranded and single-stranded nucleic acids. Also included are hybrids such as DNA-RNA hybrids. In particular, a reference to DNA includes RNA that has either the equivalent base sequence except for the substitution of uracil in RNA for thymine in DNA, or has a complementary base sequence except for the substitution of uracil for thymine, complementarity being determined according to the Watson- Crick base pairing rules. Reference to nucleic acid sequences can also include modified bases as long as the modifications do not significantly interfere either with binding of a ligand such as a protein by the nucleic acid or with Watson-Crick base pairing. Terms such as "oligonucleotide" and "polynucleotide" are subject to the same qualifications as "nucleic acid sequence" unless otherwise limited.

As there is no reliable mechanism for recombination known in mammalian mitochondria at this time, replacement of a missing or mutant mitochondrial gene directly into the mitochondrial genome seems unlikely. Additionally, heteroplasmy makes direct gene replacement unlikely. However, insertion of a mitochondrial gene that has been modified to reflect the nuclear genetic code, into the nuclear genome with a mitochondrial targeting sequence attached results in a functional protein being delivered to mitochondria.

These steps are discussed in detail below.

A. Selecting a Mitochondrial Gene

The sequence of the mitochondrial genome in humans is shown in Figure 1. The standard sequence has been designated the "Cambridge Sequence." The mitochondrial genome of the Cambridge Sequence is 16,569 bases in length and is predominantly circular, although it can exist in a linear form of the same length. This genome encodes 13 proteins, 22 transfer RNAs (tRNAs) and 2 ribosomal RNAs (rRNAs). Any of these genes can be used in the method of the present invention. However, it is generally preferred to use a gene encoding a mitochondrial protein. The desired gene is selected on the basis of the disease, condition, or defect to be ameliorated. Thus, a physician will often select the gene to be functionally complemented, based upon the condition of the patient.

Typically, the mitochondrial gene to be selected and inserted into the nuclear genome in accordance with the invention will have the same species origin as the mitochondrial DNA to be complemented. For example, a human mtATPaseό gene can be used to functionally complement a defect, mutation or deletion in the human wild type ATPaseό gene.

However, the method of the present invention is not limited to applications in which the nucleic acid sequence to be incorporated has the same species origin as the mitochondrial DNA; these can be of different species origins. Generally, the more similar a nucleic acid sequence is to the mtDNA sequence to be complemented, the more likely the nucleic acid sequence will be suitable for the present invention. For example, the Chinese hamster ovary (CHO) mtATPaseό protein is 77% identical and

86%) similar to the human mtATPaseό protein, and 83% identical and 91% similar to the mouse mtATPaseό protein. A nucleic acid sequence encoding the CHO mtATPaseό protein can complement mitochondrial genes encoding the analogues of that protein in mouse or human mitochondria. In general, the present method can be used whenever there is at least about 70% identity or about 80% similarity, preferably when there is at least about 75% identity or about 85% similarity between the protein encoded by the mitochondrial genome and by the nucleic acid sequence. Sequence identity or similarity is judged in terms of the amino acid sequence of the resulting protein. Sequence identity or similarity is determined by methods known in the art, such as those described in M.S. Waterman, "Introduction to Computational Biology" (Chapman & Hall, London, 1995), pp. 183-186. The CHO mtATPase 6 protein is 85% identical to both the mouse and human mtATPaseό protein. These proteins are highly conserved in the region of the oligomycin resistance mutation. They are also well conserved in the regions of the oligomycin resistance mutation of the mouse, as well as in the region of a mutation found in Leigh's syndrome. Thus, in one preferred embodiment, the oli^r mtATPase 6 could impart oligomycin-resistance on both human and mouse cells, as well as replace the mutant protein in Leigh's syndrome cell line.

B. Sequencing the Mitochondrial Gene

Once a mitochondrial gene is selected for functional complementation, the nucleic acid sequence of the selected gene is determined. As mentioned supra, the human mitochondrial genome, as well as other eukaryotic systems, has been sequenced in its entirety. The known nucleic acid sequence of the mitochondrial gene can be used, or the gene can be sequenced. In most individuals, a number of variations from the Cambridge Sequence are noted, and in most cases, these variations are polymorphisms. However, in some cases, the differences in sequences may lead to differences in function. In most cases, it will be preferable to use a nucleic acid segment from the Cambridge Sequence, which has already been sequenced. In some cases, however, where tissue-specific variation exists, it may be desirable to use the sequence of mitochondrial genes found in a specific tissue.

In other cases, it may be necessary to sequence the nucleic acid segment to be added. This can be done by standard techniques well known in the art, such as the

Maxam-Gilbert chemical degradation method or the Sanger dideoxyribonucleotide chain termination method. In some cases, the nucleic acid segment to be sequenced can be amplified by methods such as the polymerase chain reaction (PCR) method.

These methods are well known in the art and need not be detailed further here.

C. Modification of the Nucleic Acid Sequence According to the present invention, the nucleic acid sequence of the mitochondrial gene of interest is inserted into the nuclear genome for expression. The gene is transcribed and translated outside the mitochondrion, and the resulting protein is then transported to the mitochondrion. In some embodiments, the nucleic acid sequence of the mitochondrial gene will need to be modified to reflect the differences in codon utilization between the mitochondrial protein translation apparatus and the protein translation apparatus employing cellular ribosomes and utilizing mRNA produced as the result of transcription of nuclear DNA. These genetic codes are referred to for convenience herein as the "mitochondrial genetic code" and the "universal genetic code," respectively. This step is required if the nucleic acid sequence includes at least one codon whose meaning is different in the mitochondrial genetic code than in the universal genetic code. For example, in vertebrates, UGA, a termination codon in the universal genetic code, specifies tryptophan in the mitochondrial genetic code. AGG and AGA, both of which code for arginine in the universal genetic code, are used as termination codons in the mitochondrial genetic code. AUA, which codes for isoleucine in the universal genetic code, codes for methionine in the mitochondrial genetic code. Further, AUU, which codes for isoleucine in the universal genetic code, codes for methionine in the mitochondrial genetic code of mammals and yeasts. When the nucleic acid sequence of the mitochondrial gene contains a codon that has a different meaning for cytoplasmic ribosomal machinery, versus mitochondrial ribosomal machinery, such modification will be desirable to result in a functional protein in the mitochondria.

Suitable methods for mutating the nucleic acid sequence include point mutations, such as base substitution mutations. As used herein, "point mutations" are mutations that involve a single nucleotide, or a few adjacent nucleotides, within the gene. "Base substitutions" are mutations in which a particular nucleotide is replaced by a different nucleotide. Generally, these mutations do not result in addition or deletion of a nucleotide from the gene, and therefore the reading frame of the gene is not changed by the mutation. Suitable methods include site-directed mutagenesis, are described, for example, in J. Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989), vol. 2, ch. 15, "Site-Directed Mutagenesis of Cloned DNA," pp. 15.1-15.113. Typically, such methods employ oligonucleotides containing one or more mismatches to introduce the mutation. Other methods for modifying the nucleic acid sequence are known in the art and can be utilized in the invention.

Alternatively, once the sequence of the selected mitochondrial gene is known, the gene can be synthesized by standard nucleotide synthesis methods. The synthesis can take into account any required mutations in the nucleic acid sequence. Examples of suitable nucleic acid synthesis methods include solid-phase oligodeoxyribonucleotide synthesis methods, such as the phosphotriester or phosphite triester methods. These methods are well known in the art and need not be discussed in detail here. If the nucleic acid segment encodes a protein, but contains no codons whose assignments are different in the universal and mitochondrial genetic codes, the mutation step can be omitted.

Although, as indicated above, it is generally preferred that the nucleic acid segment encode a protein, if the nucleic acid segment encodes a tRNA or a rRNA, there is no need to modify the sequence to reflect codon utilization. D. Linking a Mitochondrial Targeting Sequence to the Nucleic Acid

Sequence

In a preferred embodiment, a mitochondrial targeting sequence is linked to the nucleic acid sequence so that the cytoplasmically transcribed and translated protein is transported to the mitochondria. As used herein, a "targeting sequence" is a sequence on the translated protein that directs the protein from the cytosol to the correct organelle of the cell, such as the mitochondria. "Target Construct" means a nucleic acid sequence having linked thereto a targeting sequence. Preferably, the targeting sequences is linked to the N-terminus of the nucleic acid sequence. Typically, the targeting sequence is located at the amino terminus of the protein. This N-terminal sequence is usually removed once the protein is transported into the mitochondria. Generally, the targeting sequence is rich in positively charged amino acids and hydroxylated amino acids, yet is devoid of acidic residues. Typically, the targeting sequence includes approximately 3 to 5 nonconsecutive arginine (Arg) or lysine (Lys) residues and often contains serine (Ser) and threonine (Thr) residues. The targeting sequence does not generally include glutamic acid (Glu) or aspartic acid (Asp) residues. The targeting sequence contains all the information required to target the protein from the cytosol to the mitochondrial matrix. The targeting sequence directs the protein to the mitochondrial matrix, and then to the correct mitochondrial subcompartment, when necessary. Proteins targeted to the matrix or inner membrane will preferably have a single targeting sequence. Following translocation of the protein to the matrix, and cleavage of the targeting sequence, the protein either remains in the matrix or inserts into the inner membrane by binding to the other subunits of the multiprotein oxidase complex. Proteins that are destined for the intermembrane space will preferably carry two or more sequences: a matrix targeting sequence that directs the N-terminus of the protein to the matrix and is cleaved, and an intermembrane-space-targeting sequence of hydrophobic amino acids that redirects the protein to the intermembrane space, where it is cleaved.

A matrix protease removes all N-terminal matrix-targeting sequences once they arrive in the mitochondrial matrix. One or more targeting sequences can be used, depending upon the application. Several targeting sequences are known, and one of skill in the art, following the principles of the present invention, can choose an appropriate targeting sequence.

In this embodiment, the protein that is generated by the transcription and translation processes is a fusion protein containing an amino-terminal mitochondrial targeting sequence. One preferred functional mitochondrial targeting sequence is the ornithine transcarbamylase targeting sequence. Alternatively, one could utilize a targeting sequence from a nuclear-encoded subunit of the NADH complex

(Complex I) for the seven NADH subunits encoded by the mitochondrial genome.

Other suitable targeting sequences are known in the art and can be substituted without departing from the spirit of the invention.

The attachment of the functional mitochondrial targeting sequence to the nucleic acid segment is performed by standard genetic engineering techniques so that the fusion protein is transcribed and translated correctly. This is typically accomplished through ligation, such as using Escherichia coli or bacteriophage T4 ligase. Conditions for the use of these enzymes are well known in the art.

According to the invention, the ligation is done in such a way so that the nucleic acid segment and the targeting sequence are joined in a single contiguous reading frame so that a single protein is produced. This may, in some cases, involve addition or deletion of bases of the cloned DNA segment to maintain a single reading frame. This can be done by using standard techniques.

It has long been hypothesized that the proteins still encoded by mitochondrial DNA, rather than being transferred to the nucleus, in eukaryotic organisms are too hydrophobic to be imported into the mitochondrion from the cytoplasm. This does not appear to be the case in the present method. In a preferred embodiment, import of the transgene can be enhanced by cotransfection with the yeast karyopherin Pselp/Kapl21p, or its mammalian homologue (or human homologue in human cells, mouse homologue in mouse cells, and the like). Overexpression of yeast karyopherin Pselp/Kapl21p stimulates the mitochondrial import of hydrophobic proteins in vivo. Optionally, import of the relatively hydrophobic, normally mtDNA-encoded, ATPase 6 protein into the mitochondria can be enhanced through the overexpression of a mammalian homologue of the yeast Pselp/Kapl21p or Kapl23p, which belong to the superfamily of karyopherin beta proteins. Overexpression of yeast karyopherin Pselp/Kapl21p stimulates the mitochondrial import of hydrophobic proteins in vivo.

Suitable recombinant plasmids can be chosen to achieve this import enhancement.

E. Linking One or More Control Elements to the Target Construct

Linkage of the target sequence(s) to the nucleic acid sequence generates a target construct, which is, in turn, linked to at least one control element to generate a nucleic acid construct. This step is performed by standard gene cloning techniques, as described above, and involves incorporating the protein targeting sequence and the nucleic acid sequence into a vector possessing the desired control element or elements.

The step of linking the control element to the target construct can be performed in any suitable manner, e.g., simultaneously or sequentially, with the step of linking the target sequence to the nucleic acid sequence.

As used herein, a "control element" is a DNA sequence that determines the site of transcription initiation for an RNA polymerase. The control element also includes promoter proximal regulatory sequences, the binding of which either stimulates or decreases the rate of transcription of the associated gene. Such promotion-proximal elements are typically located within approximately 200bp of a promoter. Control elements provide sufficient level of expression of the gene product in the mitochondria to allow complementation of a defect in the mitochondrial genome.

Because the mitochondria contains several copies of mtDNA, any complementation of a defective gene must be present in sufficient copies to overcome the defect. In this aspect, the present invention provides a method of functionally complementing one or more defective mitochondrial genes, wherein the amount of nuclear-encoded protein introduced into the mitochondria can be controlled. This control is accomplished by using a suitable control sequence. In one embodiment, the control sequence comprises a constitutive promoter, so that expression of the gene is independent of any internal or external stimuli. In an alternative embodiment, the control sequence comprises an inducible promoter, which allows regulation of transcription of the gene, thereby controlling the amount of protein introduced into the mitochondria. The invention thus provides the ability to effectively increase the transgene product during periods of stress or other periods when increased energy output is needed, or during episodes when a previously recognized defect in the mtDNA of the subject is affecting a particular activity known to be affected by the defect. Thus, an inducible promoter allows production of the transgene product primarily when it is needed, or in anticipation of its need. In one embodiment, the control element is a promoter that functions constitutively. As used herein, a "constitutive promoter" is any promoter that operates at a constant rate, which is not regulated by internal or external stimuli. A particularly suitable constitutive promoter is the SV40 promoter, which is found in the vector pZeoSN2 (+) (Example 1). Other promoters, such as the Rous sarcoma virus promoter, the adeno virus major late promoter, and the human cytomegalovirus (CMV) immediate early promoter, can be used. Still other promoters can be used, such as those found in herpes simplex virus (HSV) vectors (P.G.E. Kennedy, "Potential Use of Herpes Simplex Virus (HSV) Vectors for Gene Therapy of Neurological Disorders," Brain: 120: 1245-1259 (1997)). Tissue-specific promoters can be used, incorporated in appropriate vectors.

In another embodiment, the control element is an inducible promoter. As used herein, an "inducible promoter" is any promoter that is mediated by a molecule, e.g., an inducer, to regulate the rate of transcription. A preferred inducible promoter is a tetracycline promoter. Other suitable inducible promoters include estrogen- inducible promoters, OXBOX/REBOX promoters, and the like. Preferably, the inducible promoter is capable of being regulated without affecting other systems of the cell. One of skill in the art, given the guidance herein, can select other suitable inducible promoters known in the art.

F. Inserting the Construct into the Eukaryotic Nuclear Genome for Expression

The nucleic acid construct is inserted into the eukaryotic cell for expression so that functional complementation of at least one defect, deletion, or mutation in the mitochondrial genome is provided. Insertion of the construct into the eukaryotic cell is typically done by methods well known in the art. These methods include the use of poly cations such as DEAE-dextran, calcium phosphate coprecipitation, electroporation, transfection, lipofection or liposome fusion, micro injection, and the use of viral or retroviral vectors. A particularly preferred method for insertion is electroporation, but the particular route to be chosen can be selected by one skilled in the art by consideration of factors such as the size of the construct, the concentration of construct available, and the nature of the target cell.

The method of the present invention can be used in any eukaryotic cells, including animal or plant, and can be used in any animal cells, including mammalian or non-mammalian. If the method is used in mammalian cells, it can be used in human or non-human cells. Additionally, the method is to introduce the nucleic acid construct into non-dividing cells or dividing cells (such as stem cells).

Suitable vectors are used in accordance with the invention to provide insertion of the nucleic acid construct into the nuclear genome of the cell. Factors influencing the choice of vector include: size of the nucleic acid construct to be introduced into the nuclear genome, the presence of control elements and/or targeting sequences, the number of nucleic acid constructs to be incorporated, the type of cell to receive the nucleic acid construct, and any other additional selectable markers desired to be included in the vector. Suitable vectors include vectors that are plasmid or viral in origin, allowing expression of the desired genetic material. Preferred vectors include lentiviral vectors, (HSV-l)-based amplicon vectors, and the like.

Preferred vectors according to the invention provide a combination of such advantages as sufficient transgene capacity to deliver the gene or genes of interest into the nuclear genome, ability to transduce either dividing or non-dividing cells, high transduction efficiency, stability of gene expression, and lack of toxicity or inflammatory response.

When the present invention is used to functionally complement more than one defective mitochondrial gene, more than one vector can be used, depending upon such factors as those identified above. Alternatively, all of the nucleic acid constructs to be inserted into the nuclear genome can be included in a single vector.

In one embodiment, gene targeting via homologous recombination (Mansour, et al. 1988. Nature 336:348-352) can be used to inactivate the UOATPase 6 gene present in the nucleus.

The present method provides the desired gene product, typically a protein, that is lacking as the result of the mutation, deletion, or defect in the mitochondrial genome. Among the diseases or conditions that can be ameliorated by such functional complementation are: 1 ) mitochondrial encephalmyopathy with lactic acidosis and stroke- ike episodes (MELAS);

2) Leber hereditary optic neuropathy;

3) myoclonic epilepsy with ragged-red fibers (MERRF);

4) neurogenic muscular weakness, ataxia, retinitis pigmentosa

(NARP);

5) Kearns-Sayre syndrome;

6) Leigh syndrome

7) Pearson Marrow pancreas syndrome (PMPS)

8) aminoglycoside-associated deafness;

9) diabetes with deafness;

10 epilepsy with abrupt onset at 1-8 years with nonfocal EEG; 11 leukodystrophy with hypotonia; 12 autism with seizures; 13 sudden infant death syndrome with hypoglycemia; 14 leukemia with maternally inherited thrombocytopenia; 15 migraines associated with hearing loss, strokes, or diabetes; 16 early hearing loss; 17 refractory infantile reflux with carnitine deficiency; 18 multiple sclerosis with seizures; 19 blindness with optic atrophy and dystonia; 20 renal tubular acidosis with elevated lactic acid and hypotonia; 21 nonvalvular hypertrophic cardiomyopathy before age 50; and 22 chronic pancreatitis with stroke-like episodes.

This list is illustrative but not exclusive. Although some of these conditions and syndromes are rare, it is likely that the mitochondrial defects and conditions are underdiagnosed and are responsible for many conditions that present atypically or with an unusual combination of symptoms. Additionally, reduced oxidative phosphorylation capacity generally declines with age, and the present method can be used to assist in ameliorating this decline. II. Total Functional Complementation of the Mitochondrial Genome

The methods of Section (I), above, can be used in a method for total functional complementation of the mitochondrial genome. As indicated above, the mitochondrial genome includes 13 genes that encode proteins, 22 genes that encode tRNAs, and 2 genes that encode rRNAs. The tRNAs and rRNAs transcribed within the mitochondrion are used solely for production of proteins within the mitochondrion and have no other function. Thus, if the methods of Section (I), above, are used to incorporate 13 nucleic acid segments, one for each of the proteins encoded by the mitochondrion, the total function of the mitochondrial genome can be complemented. In this embodiment there is no need to complement the genes that encode tRNAs or rRNAs.

In general, this method comprises:

(a) selecting all mitochondrial genes of the cell that encode proteins; (b) determining a nucleic acid sequence of each of the mitochondrial genes selected in step (a);

(c) linking the nucleic acid sequence of each of the mitochondrial genes to one or more targeting sequences to generate target constructs;

(d) linking one or more control sequences to the target constructs to generate nucleic acid constructs; and

(e) inserting the nucleic acid constructs into the nuclear genome of the cell for expression of the nucleic acid sequences in the cell.

Optionally, the method further comprises mutating the nucleic acid sequence to account for differences between codon usage in the nucleus versus the mitochondria, as discussed above.

The nucleic acid constructs are introduced into the cell using procedures described above. In this embodiment, introduction of the nucleic acid constructs into the nuclear genome of the cell is accomplished using one or more of the suitable vectors described above. As discussed above, when multiple nucleic acid constructs are inserted into the nuclear genome of the cell for functional complementation, multiple vectors can be used. Alternatively, all nucleic acid constructs are included in a single vector for insertion into the nuclear genome. A preferred vector for complementation of total mitochondrial genome function is a recombinant (HSV- l)-based amplicon vector. Suitable (HSV-l)-based amplicons contain three types of genetic elements: (i) sequences that allow propagation of the amplicon as a bacterial plasmid; (ii) a transgene cassette with the genes of interest; and (iii) approximately 1% of the 152-kb HSV-1 genome, in particular an origin of DNA replication (orϊ) and a DNA cleavage/packaging signal (pac), to support replication of amplicon DNA and subsequent packaging into HSV-1 virions in the presence of helper functions, respectively.

Preferred (HSV-l)-based amplicons have large transgene capacity, for example, from 15-kb to approximately 150-kb, high transduction efficiency, ability to infect both dividing and nondividing cells, and stability of the virion. In one preferred embodiment, the vector comprises a hybrid amplicon that contains, in addition to HSV-1 ori and pac, the adeno-associated virus (AAV) inverted terminal repeats (ITRs), and the AAV rep gene. An example of this hybrid vector is shown in Figure 8. In AAV infections, the rep gene encodes proteins that mediate the amplification of the ITR-flanked genome and subsequent integration into a specific site on chromosome 19 of human cells. The HSV/ AAV hybrid vector utilizes HSV- 1 properties for entry into the cell and nuclear localization, and amplifies and integrates AAV ITR-flanked transgenes into a specific locus on the human genome. Suitable HSV/ AAV vectors are described, for example, in Fraefel, C. et al, Adv. Virol. Research (in press), and Constantini, L.C. et al, Human Gene Therapy, 10:2481-2494 (1999).

This invention provides a mechanism to ameliorate the effects of not only those maladies resulting from defects in the 13 mtDNA genes encoding proteins, but also those maladies resulting from defects in the 22 mitochondrial DNA genes encoding tRNAs, and the 2 mitochondrial DNA genes encoding ribosomal RNAs. Additionally, since this invention could eliminate the need for mitochondrial DNA, it can be utilized to treat mitochondrial DNA (mtDNA) depletion syndromes.

Additionally, transgenic animals could be made from these cell lines (e.g., if done in mouse cells, to take advantage of the genetic information in mouse) by transplanting the nuclei into enucleated eggs, with consequent procedures as used currently and known in the art. The use of transgenic animal procedures is described, for example, in CA. Pinkert et al., "Transgenic Animal Modeling," in Molecular Biology and Biotechnology A Comprehensive Desk Reference (R.A. Meyers, ed., VCH Publishers, Inc., 1995), pp. 901-907, and is known in the art. Further, stem cells can be transformed with the entire complement of mtDNA encoded genes to produce a stem cell culture that can be utilized to produce any type of cell through appropriate selection. In one exemplified embodiment, a mitochondrial gene is introduced into embryonic stem (ES) cells. Introduction of one or more mitochondria-encoded genes into stem cells provides a useful model system for studying mitochondrial diseases.

III. Selectable Marker

In yet another aspect, the present invention provides a selectable marker for identifying cells in which functional complementation of one or more mitochondria- encoded genes has taken place. Utilization of the known mutation in ATPaseό that confers oligomycin resistance on the Complex V ATPase, can effectively allow for identification of cells that have likely been effectively transformed with nuclear genome expressible mitochondrial genes for which a mutation allowing selection by a pressure (such as oligomycin resistance) is known, and thus not present in the transgene.

The present invention provides a combination of the following advantages. The method provides an efficient method of overcoming defects in mitochondrial metabolism due to defects, mutations, or deletions in the mitochondrial genome. The method can be used to provide functional complementation for any of the mitochondrial proteins, and can be used to provide total complementation of the functions of the mitochondrial genome, obviating the necessity for functioning mitochondrial DNA. The method can be used to treat a wide variety of diseases, syndromes and conditions that are due to such defects, mutations, or deletions in the mitochondrial genome. Many of these diseases, syndromes, or conditions are refractory to treatment and are life threatening. Further, the method can be used in conjunction with transgenic techniques.

The invention is illustrated by the following Examples. These Examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any manner. EXAMPLE 1

Functional Complementation of ATPase 6 in Chinese Hamster Ovary Cells

Functional complementation of ATPase 6 in Chinese hamster ovary (CHO) cells was accomplished as follows.

An oligomycin resistant Chinese hamster ovary (CHO) cell line was obtained which contains a single nucleotide change in the mitochondrially-encoded ATPase 6 gene (mtATPase 6). (Kindly provided by Dr. Gail Breen) This nucleotide change renders the ATPase subunit and thus the cell line oligomycin resistant (oli^r; Breen, et al, 1986).

The plasmid pUOATP2 contains the mutant oli^r ATPase 6 gene linked to ornithine transcarbamylase DNA sequences utilized for targeting the protein to the mitochondria. The construct contains Kozak sequences (Kozak, M., Nucl Acids Res. 1987; 15:8125-8148) that were inserted between the Hindlll site and Kpnl sites 5' to the UO ATPaseό sequence. The construct was digested with Hindlll and Kpnl. Sense and antisense oligomers encoding the Kozak sequence, with Hindlll and Kpnl overhangs appropriately situated were annealed and ligated into the digested plasmid. The new construct was thus cloned. Figure 1 shows the map of pUOATP2, including promoters, origin of replication, and other functional regions. The complete nucleotide sequence of pUOATP2 is shown in Figure 2. Figure 2 shows the amino acid sequence of the UOATPaseό gene product. The coding region for UO ATPase 6 was inserted between the SV40 constitutive promoter and polyadenylation signal for constitutive expression. The coding region of UOATPaseό is located approximately between base pair numbers 481 and 1260.

In order for the nuclear-encoded ATPase 6 gene to produce a protein product identical to the mitochondrial-encoded ATPase 6, specific codons were altered to accommodate for the cytoplasmic ribosomal machinery. Specifically, codons were altered so that tryptophan could be synthesized at amino acid positions 48, 68 and 109 rather than reading as stop codons. In addition, 12 other codons were modified to correctly incorporate methionine instead of isoleucine in the ATPase 6 protein. These modifications were propagated by synthesizing the entire coding sequence (sequence obtained from Oligos, Etc., Inc., Wilsonville, Oregon). The sequence was then cloned into the pZeoSV2(+) vector (obtained from Invitrogen, Inc., Carlsbad, California).

The structure of the pZeoSV2(+) vector, showing the origin of replication, promoters, and other functional regions is shown in Figure 3. This vector contains genes for ampicillin, zeocin and gentamycin resistance and for replication in bacteria.

The pUOATP2 gene was transcribed and translated in a rabbit reticulocyte lysate system (Sambrook, et al. 1989. Molecular Cloning. 2^nd ed. Cold Spring Harbor.

NY). The expected 28.8 kD gene product of the pUOATP2 insert was produced (data not shown). The CHO cells into which the UO ATPase 6 gene would be electroporated contain the normal mtATPase 6 gene product. The processed UO ATPase 6 gene product was nearly identical to the mtATPase 6 gene product. Both proteins were precipitated by an antibody against the carboxy terminus of the human mtATPase 6 gene (kindly supplied by Dr. R. Doolittle, UCSD) (Data not shown). To demonstrate import of the UO ATPase 6 gene product into the mitochondria, a radiolabeled UO ATPase 6 protein was generated by in vitro translation in the presence of [³⁵S]-methionine (Sambrook et al. 1989). The translation mixture contained only the 28.8 kD gene product of UO ATPase 6.

The plasmid pUOATP2 was introduced into CHO oligomycin-sensitive (oli^s) cells by electroporation. After selection in zeocin (Invitrogen®, Carlsbad, California) to isolate transformants with acquired G418-resistance, transformants were subjected to 0.1 μg/mL oligomycin. Two transformants grew in the presence of oligomycin. These two transformants, the recipient CHO cell line, and the mtATPase 6 donor oli^r cells were plated in Dulbeco's Modified Eagles Medium (DMEM) growth medium containing increasing concentrations of oligomycin (0.00001, 0.0001, 0.001, 0.01, 0.1, 1.0) μg/mL.

The untransformed CHO cells were eliminated in 0.001 μg/ml oligomycin, while the transformed cells grew in up to 1.0 μg/mL oligomycin. Figure 5 illustrates the growth characteristics of the untransformed recipient cell line 11-11, and the transformed zrl 1 cell line in the presence and absence of 0.01 μg/mL oligomycin. The cell lines were plated in 12 position culture dishes. Selection occurred in DMEM, 10% FBS, with or without (Controls) 0.01 μg/mL oligomycin. At the end of the experiment, the experimental cell lines were returned to DMEM without oligomycin, the 11-11 did not grow, while the zrl 1 did, illustrating the successful gene therapy of the "oligomycin-sensitive" defect.

The results show that the recipient oli^s CHO cells were 1000 times more sensitive to oligomycin than the ATPase 6 oli^r-transformed zrl 1 cell line. The results thus showed that placement of mitochondrial genes into the nuclear genome yielded proteins that were incorporated into the mitochondria.

Figure 6 is a graph of the results obtained by measuring the oxygen consumption of the transformed and nontransformed CHO cells in the presence or absence of oligomycin. As shown in the graph, oxidative phosphorylation was restored.

The pUOATP2 insert was detected in the nuclear genome as follows. Fluorescence in situ hybridization (FISH) of metaphase chromosomes, essentially as described in SJ. Zullo et al., "Localization by Fluorescence in Situ Hybridization (FISH) of human Mitochondrial γ (POLG) to Human Chromosome Band 15q24→q26, and of Mouse Mitochondrial Polymerase γ (Polg) to Mouse Chromosome Band 7E, with Confirmation by Direct Sequence Analysis of Bacterial Artificial Chromosomes (BACs), Cytogenet. Cell Genetics 78: 281-284 (1997)) indicated that there was a single insertion of pUOATP2 on one homologue of chromosome 1 in CHO transformed line ZRl 1 (Fig. 4). This indicated that the cells were transformed by the construct and that the ATP 6 gene was found at a specific chromosomal location. The chromosome 1 homologue without an insertion verifies the mitotic nature of the CHO cell line.

The PCR products of the mtDNA have been sequenced to confirm that no mutation of the endogenous mtDNA-encoded ATPase 6 gene to the oligomycin- resistant form took place (Lark Sequencing, Houston, Texas).

This Example demonstrated that an oligomycin-resistant mitochondrial genome-encoded Chinese hamster ovary ATPase 6 gene can still confer oligomycin resistance after transfer to the nuclear genome.

Detection of Unprocessed Gene Product in Transformants and Activity Assay

Western blot of total proteins isolated from wild-type CHO cells, oli^r cells, stable CHO transformant line 1, and stable CHO transformant line 2 with antisera to the carboxyl-end of the human mtATPase 6 (kindly supplied by Dr. R. Doolittle

UCSD) is used to detect the endogenous mtATPase 6 gene product in all the cell lines, and an additional band representing the unprocessed cytoplasmically-translated

UO ATPase 6 gene product. Western blot shows that the unprocessed UOATPaseό gene product is indeed present only in the transformants, and in neither the untransformed wild-type CHO cells nor the oli^r cells.

The molecular weights and pi's calculated from the sequence data are as follows: for the endogenous mt-encoded ATPase 6, molecular weight 25039.21 daltons, pi 11.02; for the imported nuclear encoded ATPase 6, molecular weight 25038.27, pi 11.29; for the unprocessed nuclear encoded ATPase 6, molecular weight

28826.73, pi 11.88. Thus, the nuclear encoded ATPase 6 is imported as a molecule that is identical or virtually identical to the mt-encoded ATPase 6.

EXAMPLE 2

Functional Complementation of mtDNA ATPase in Human Cybrid Cells

Mutant CHO mtATPase was placed into the nucleus of human cybrid cells for expression to functionally complement mitochondrial ATPase 6. The plasmid pUOATP2 was constructed as described in Example 1. The plasmid contains the mutant oli^r ATPase 6 gene, flanked by transcarbamylase targeting sequence, as well as Kozak sequences.

The ATPase 6 gene is altered to accommodate for the cytoplasmic ribosomal machinery as described in Example 1. The pUOATP2 gene was transcribed and translated in a rabbit reticulocyte lysate system as described in Example 1 above. The expected 28.8 kD gene product of the pUOATP2 insert was produced. The processed UO ATPase 6 gene product was nearly identical to the mtATPase 6 gene product. Both proteins were precipitated by an antibody against the carboxyl terminus of the human mtATPase 6 gene (as described above; kindly supplied by Dr. R. Doolittle, UCSD).

The plasmid pUOATP2 was introduced into human cybrid cells (obtained from

D. Wallace, Emory) by electroporation. After selection in zeocin (Invitrogen®) to isolate transformants with acquired G418-resistance, transformants were subjected to

0.1 μg/mL oligomycin. Three transformants grew in the presence of oligomycin.

These transformants, and the recipient cybrid cell line were plated in DMEM growth medium as described above containing increasing concentrations of oligomycin (0.00001, 0.0001, 0.001, 0.01, 0.1, 1.0) μg/mL.

The results show that the recipient oli^s human rho-zero cybrids cells were 100 times more sensitive to oligomycin than either of the two transformants.

Fluorescence in situ hybridization (FISH) of metaphase chromosomes, essentially as described in S.J. Zullo et al, Cytogenet. Cell Genetics, 78: 281-284 (1997) indicated that there is a single insertion of the pUO ATP2 viral vector on one homologue of chromosome x in the transformed line y (Fig. 4). This indicates that the cells are indeed transformed by the construct and that the mtDNA gene cluster is localized to a specific chromosomal location.

The PCR products of the mtDNA were sequenced to ensure there was little if any mutation of the endogenous mtDNA-encoded ATPase 6 gene to the oligomycin- resistant form (Lark Sequencing, Houston, Texas).

Detection of Unprocessed Gene Product in Transformants and Activity Assay

To demonstrate direct evidence of import of the UO ATPase 6 gene product into the mitochondria, a radiolabeled UO ATPase 6 protein is generated by in vitro translation in the presence of [³⁵S]-methionine (Sambrook et al. 1989). The translation mixture contains only the 28.8 kD gene product of UO ATPase 6.

Western blot of total proteins isolated from untransformed cybrids, and stable transformant line with antisera to the mtDNA-encoded proteins (kindly supplied by Dr. R. Doolittle UCSD; C. Bernadier UGA) detects the gene products in the transformed cell lines, but not in the untransformed cell lines.

This Example demonstrated that an oligomycin-resistant mitochondrial genome-encoded Chinese hamster ovary ATPase 6 gene supplanted and functioned in place of a mutant human mtDNA-encoded ATPase 6 gene. The oli^r mtATPase 6 gene imparted oligomycin-resistance in the human cells, replacing the mutant protein in Leigh's syndrome cell line. EXAMPLE 3

MtDNA-less, Rho-zero, Mouse and Human Cells Regain Oxidative Phosphorylation after Transfer of the 13 mtDNA-encoded Proteins to the Nuclear Genome

All 13 mtDNA-encoded proteins in mouse and human cells are inserted into the nuclear genome of the cells and targeted to the mitochondria for expression as follows. The HS V/AAV viral vector vMdMTGEN is used to introduce all 13 mitochondria-encoded genes into murine cells (for human cybrid cells, the HSV/AAV viral vector vHsMTGEN is used to introduce the mitochondria-encoded genes). The structure of vMdMTGEN is shown in Figure 8, which shows the origin of replication (ori-S), AAV rep, AAV ITRs, green fluorescent protein (GFP), neomycin resistance gene (NeoR), the pac gene, and other functional regions of the vector. The AAV rep gene encodes a protein that mediates the amplification of the ITR-flanked genome, and facilitates site-specific integration into the human genome on chromosome 19. AAV inverted terminal repeats (ITRs) carry signals promoting extrachromosomal replication and integration of transgenes at multiple sites in the genome. The HSV/AAV viral vector vMdMTGEN is cloned to contain all 13 mtDNA protein-encoding genes in a cassette of 13 distinct transcriptional units. Each cassette includes a gene that has been modified to be encoded by the universal code, an ornithine transcarbamylase (OTC) targeting sequence, and a dedicated SV40 constitutive promoter for transcriptional control. Also included in the vMdMTGEN vector is a mutant oli^r mouse mtATPase 6 gene to select for the transgene cassette. Each cassette contains Kozak sequences 5' to each gene sequence, to ensure proper ATG codon selection by the ribosome.

A transgene cassette containing all thirteen mt-encoded genes is constructed as follows. Each mt-encoded gene is linked to its own dedicated control element(s), and targeting signal(s) without disrupting the reading frame of the gene. The genes are then linked together using techniques known in the art, maintaining the reading frame of each of the genes. This transgene cassette is then cloned into the vMdMTGEN vector using standard cloning techniques known in the art.

The vMdMTGEN insert is transcribed and translated in a rabbit reticulocyte lysate system as described in the Examples above (Sambrook, et al. 1989. Molecular Cloning. 2^nd ed. Cold Spring Harbor. NY). The expected gene products are identified with antibodies to each construct of the 13 mtDNA-encoded proteins (Data not shown).

The vMdMTGEN vector is introduced into rho-zero oligomycin-sensitive (oli^s) murine cells by electroporation. After selection in zeocin (Invitrogen®) to isolate transformants with acquired G418-resistance, transformants are subjected to 0.1 μ g/mL oligomycin. Two transformants grow in the presence of oligomycin. These two transformants, the recipient CHO cell line, and the mtATPase 6 donor oli^r cells were plated in DMEM growth medium (as described above) containing increasing concentrations of oligomycin (0.00001, 0.0001, 0.001, 0.01, 0.1, 1.0) μg/mL Although the present invention has been described in considerable detail with regard to certain preferred versions thereof, other versions are possible. Therefore, the spirit and scope of the appended claims should not be limited to the descriptions of the preferred versions contained herein.

Claims

WE CLAIM:

1. A method for functionally complementing one or more defects, mutations, or deletions in a mitochondrial genome of a cell having a nuclear genome, the method comprising steps of:

(a) selecting a mitochondrial gene;

(b) determining a nucleic acid sequence of the mitochondrial gene;

(c) linking the nucleic acid sequence of the mitochondrial gene to one or more targeting sequences to generate a target construct;

(d) linking one or more control sequences to the target construct to generate a nucleic acid construct; and

(e) inserting the nucleic acid construct into the nuclear genome of the cell for expression of the nucleic acid in the cell.

2. The method according to claim 1 , wherein the nucleic acid sequence comprises one or more codons that is different from one or more codons of a nuclear-encoded protein of the cell, the method further comprising the step of modifying the nucleic acid sequence of the mitochondrial gene to generate a modified sequence, so that a polypeptide that is expressed as a result of nuclear transcription of the modified sequence has an amino acid sequence similar to an amino acid sequence of a polypeptide originally expressed in the mitochondrion.

3. The method according to claim 1 wherein the step of selecting a mitochondrial gene comprises selecting a plurality of mitochondrial genes.

4. The method of claim 1 wherein the nucleic acid sequence encodes a mitochondrial protein.

5. The method of claim 3 wherein the mitochondrial protein targeting sequence is an ornithine transcarbamylase targeting sequence.

6. The method of claim 1 wherein one or more of the control elements comprises a constitutive promoter.

7. The method of claim 6 wherein the constitutive promoter is an SV40 constitutive promoter.

8. The method according to claim 1 wherein one or more of the control elements comprises an inducible promotor.

9. The method according to claim 8 wherein the inducible promotor is a tetracycline inducible promoter.

10. The method of claim 1 wherein the nucleic acid construct is inserted into the nuclear genome of the cell by a method selected from the group consisting of the use of a poly cation, calcium phosphate coprecipitation, electroporation, lipofection, transfection, microinjection, and the use of viral or retro viral vectors.

11. The method of claim 10 wherein the method comprises electroporation.

12. The method of claim 1 wherein the cell is an animal cell.

13. The method of claim 12 wherein the cell is a mammalian cell.

14. The method of claim 13 wherein the cell is a human cell.

15. A method for performing total functional complementation of a mitochondrial genome of a cell having a nuclear genome located in a nucleus, the method comprising steps of:

(a) selecting all mitochondrial genes of the cell that encode proteins;

(b) determining a nucleic acid sequence of each of the mitochondrial genes selected in step (a);

(c) linking the nucleic acid sequence of each of the mitochondrial genes to one or more targeting sequences to generate target constructs for each of the mitochondrial genes; (d) linking one or more control sequences to the target construct to generate nucleic acid constructs for each of the mitochondrial genes; and

(e) inserting the nucleic acid constructs into the nuclear genome of the cell for expression of the nucleic acid sequences in the cell.

16. The method according to claim 15, wherein the step of linking one or more control sequences to the target construct to generate nucleic acid constructs for each of the mitochondrial genes comprises linking one or more control sequences to each of the target constructs individually.

17. The method according to claim 15, wherein the nucleic acid sequence comprises one or more codons that is different from one or more codons of a nuclear-encoded protein of the cell, the method further comprising the step of modifying the nucleic acid sequence of the mitochondrial gene to generate a modified sequence, so that a polypeptide that is expressed as a result of nuclear transcription of the modified sequence has an amino acid sequence similar to an amino acid sequence of a polypeptide originally expressed in the mitochondrion.

18. The method of claim 15 further comprising transplanting the nucleus of the cell into an enucleated egg to form a transgenic animal.

19. A method for detecting functional complementation of one or more defects, mutations, or deletions in a mitochondrial genome of a cell having a nuclear genome, the method comprising steps of:

(a) selecting a mitochondrial gene;

(b) determining a nucleic acid sequence of the mitochondrial gene;

(c) linking the nucleic acid sequence of the mitochondrial gene to one or more targeting sequences to generate a target construct;

(d) linking one or more control sequences to the target construct to generate a nucleic acid construct;

(e) linking the nucleic acid construct to a marker comprising the coding region of the plasmid pUOATP2; (f) inserting the nucleic acid construct linked to a marker into the nuclear genome of the cell for expression of the nucleic acid in the cell; and

(g) detecting the presence of the marker in the cell.

20. A selectable marker for detecting functional complementation of one or more mitochondrial genes comprising the coding region of the nucleotide sequence of pUOATP2.