Allotopic expression of mtDNA genes

Abstract

Human mitochondrial DNA (mtDNA) is a 16,569-base pair, double-stranded, circular DNA that encodes 13 key proteins of the electron transport chain. Embodiments of the present invention include allotopic expression of mtDNA genes to compensate for functional deficiencies resulting from underlying mtDNA damage or mutations. In allotopic expression, genes normally expressed only from the mitochondrial genome are modified for expression using nuclear-cytosolic transcription and machinery. The method can be used therapeutically to treat diseases caused by mitochondrial DNA mutations. The method can also be used to slow down the aging process and / or reduce the effects of aging.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 414,406, filed October 7, 2022. The contents of the foregoing application are incorporated herein by reference.

[0002] The present invention relates to the field of molecular genetics, and more specifically to the expression of mitochondrial genes in human cells to treat mitochondrial disorders and combat aging processes and phenotypes. [Background technology]

[0003] Mitochondria are double-membrane-bound organelles found in most eukaryotes. Although most of a cell's DNA is contained in the nucleus, mitochondria have their own genome (the "mitogenome"), similar to bacterial genomes. Mitochondrial proteins (i.e., proteins transcribed from mitochondrial DNA) vary between tissues and species. In humans, 615 different proteins have been identified in cardiac mitochondria, while 940 proteins have been reported in rats. The mitochondrial proteome is thought to be dynamically regulated.

[0004] Mitochondria use aerobic respiration to generate the majority of the cellular adenosine triphosphate (ATP) supply, which is then used throughout the cell as an energy source. Oxidative phosphorylation (OXPHOS), the Krebs cycle, the urea cycle, heme biosynthesis, and fatty acid oxidation occur within mitochondria. In addition to providing cellular energy, mitochondria are involved in signal transduction, cell differentiation and death, and maintaining control of the cell cycle and cell proliferation. Mitochondrial biogenesis is temporally coordinated with these cellular processes. Mitochondria are implicated in several human disorders and conditions, including mitochondrial disease, cardiac dysfunction, heart failure, and autism.

[0005] The majority of mitochondrial proteins are encoded by the nuclear genome, translated in the cytosol, and imported into mitochondria. However, the 13 core subunits of respiratory complexes are encoded by the reduced mitochondrial genome and synthesized within the mitochondrial matrix. Mutations in these 13 genes (or their associated non-protein-coding genes) tend to be particularly severe because all 13 proteins are core subunits of the oxidative phosphorylation pathway. Disruption of subunit structure, stability, or function can have significant biochemical and physiological consequences. For example, mtDNA mutations that cause structural changes in OXPHOS subunits disrupt the electron transport relay, resulting in inefficient energy production. Inefficient transport can generate superoxide byproducts, resulting in increased reactive oxygen species (ROS) and reactive nitrogen species (RNS), causing a state of chronic cellular stress.

[0006] There are several known mitochondrial diseases and disorders caused by pathogenic point mutations in mitochondrial DNA (mtDNA), one-third of which are located in coding genes. Primary defects in mitochondrial function commonly present clinical problems in tissues with high energy demands, such as the retina, heart, muscle, kidney, pancreas, and liver. Their incidence is estimated at 1 in 5,000 live births. Therefore, mitochondrial pathology is considered one of the most common genetically determined diseases and represents a major health problem, as both curative and palliative therapies remain inaccessible.

[0007] Mitochondrial dysfunction is also a hallmark of aging and cellular senescence. Age-related changes in mitochondrial metabolism are associated with altered mitochondrial composition. The dynamic equilibrium between fusion and fission is essential for healthy mitochondrial function. In aging cells, fusion exceeds fission, resulting in the formation of larger mitochondria that contribute to cellular senescence. The fission process is coupled to ER-microtubule function; therefore, mt-ER-lysosome interactions influence the fission process. There is evidence of exercise-induced attenuation of senescence, and findings suggest that mechanical forces alter the MT-ER junction protein complex through unknown processes in fission and fusion function. Furthermore, there is evidence of a decline in mitochondrial quality and copy number, as well as an increased incidence of mtDNA mutations with age, which contribute to cellular senescence and age-related organismal decline.

[0008] With aging, the quantity, integrity, and functionality of mitochondrial DNA decline due to the accumulation of mutations and oxidative damage induced by reactive oxygen species (ROS). In aging subjects, mitochondria are characterized by dysfunction, including reduced oxidative capacity, decreased oxidative phosphorylation, decreased ATP production, significantly increased ROS generation, and reduced antioxidant defenses. Mitochondrial biogenesis declines with age due to altered mitochondrial dynamics and inhibition of mitophagy, an autophagy process that removes dysfunctional mitochondria. Age-dependent abnormalities in mitochondrial quality control further weaken and impair mitochondrial function. In aged tissues, enhanced mitochondrial-mediated apoptosis contributes to an increased proportion of apoptotic cells.

[0009] Currently, there is no cure for mitochondrial disorders. The few available treatment options are generally limited to symptomatic management. Traditional treatments include nutritional supplements and lifestyle interventions such as diet and exercise.

[0010] Allotopic expression has been proposed to treat these mitochondrial disorders. In allotopic expression, a wild-type (i.e., healthy) copy of the mutated gene is introduced into the genome. A normal copy of the gene product is imported from the cytosol into the mitochondria. Currently, several mitochondrial genes have been successfully recoded and expressed in the nucleus in yeast. However, efforts at allotopic expression in mammalian cells have been generally unsuccessful.

[0011] Therefore, methods for allotopic expression must be improved before they can be used therapeutically in humans. Allotopic expression of mitochondrial genes presents several difficulties, including the following: a) The different codon dictionaries used by the mitochondrial and nuclear genomes, b) different codon preferences between mitochondrial and nuclear cytosolic translation systems, and c) Providing import signals to ensure that newly translated proteins in the cytosol are successfully imported into mitochondria. These and other issues require further research to elucidate the steps of nuclear gene expression, mitochondrial import, processing, and functional integration of allotopically expressed polypeptides into mitochondrial protein complexes.

[0012] There is a need for reliable methods of allotopic expression in mammalian cells. The present invention overcomes the problems of previous approaches. The methods described herein can be used therapeutically to treat mitochondrial diseases and age-related conditions. Summary of the Invention

[0013] The invention described and claimed herein has many attributes and embodiments, including but not limited to those described or illustrated or referenced in this brief summary. The invention described and claimed herein is not limited to, and is not limited by, the features or embodiments identified in this summary, which are included for purposes of example only and not limitation.

[0014] The present invention provides means, including compositions and methods, that enable mitochondrial import with improved efficiency and stability compared to conventional techniques. The means of the present invention enable targeted localization of mRNA to the mitochondrial surface.

[0015] Thus, embodiments include methods of introducing modified gene products into cells, e.g., by plasmid or viral vectors, as RNA or cDNA therapeutics, or as therapeutic proteins. In aspects, the gene products are expressed from the nucleus (i.e., genomically integrated). In other aspects, the gene products are provided as non-integrated vectors or as cDNA or mRNA, with expression / regulatory elements appropriate for the type of nucleic acid. In yet other aspects, the therapeutic proteins are administered to the subject (i.e., directly to the tissue).

[0016]

[0003] Embodiments include methods for allotopic expression of a gene in a cell. The method may include (a) identifying a mitochondrial gene in the cell that has one or more mutations, (b) expressing a wild-type version of the mitochondrial gene in the nucleus of the cell, and (c) importing a gene product of the wild-type version of the mitochondrial gene into the mitochondria of the cell. In embodiments, the cell is a mammalian cell, such as a human cell.

[0017] In embodiments, the gene product is an oxidative phosphorylation (OXPHOS) complex subunit that is translated from mitochondrial DNA.

[0018] In embodiments, one or more codon sequences are incorporated into the wild-type version of a mitochondrial gene. The methods described herein may employ codon optimization, translation slowing, and / or ribosome stalling.

[0019] In embodiments, an N-terminal matrix export signal is incorporated into a wild-type version of a mitochondrial gene. In embodiments, one or more codon sequences are introduced into a wild-type version of a mitochondrial gene to restore the natural orientation of the transmembrane helices of the gene product to the inner mitochondrial membrane.

[0020] In embodiments, the methods of allotopic expression described herein are used therapeutically to improve mitochondrial function, e.g., to treat mitochondrial dysfunction, aging-related diseases or disorders, or to prevent or slow the aging process.

[0021] Embodiments also include methods for reducing the hydrophobicity of coding sequences to improve the import and function of allotopic mtDNA proteins, which may include (a) identifying non-deleterious mutations and evaluating the resulting hydrophobicity changes, and (b) evaluating the three-dimensional (3D) structural impact of the mutations.

[0022] Embodiments include methods of administering a nucleic acid construct encoding a therapeutic mitochondrial protein. In aspects, the construct is integrated into the genome. In aspects, the construct is not integrated into the genome (e.g., expressed from an episome). In aspects, the gene product is imported into mitochondria.

[0023] Embodiments include the administration of a nucleic acid encoding a therapeutic mitochondrial protein. In aspects, the nucleic acid is RNA.

[0024] Embodiments include allotopic administration of therapeutic mitochondrial proteins.

[0025] In embodiments, the mitochondrial targeting sequences (MTS) identified in Table 1 (ie, SEQ ID NOs: 1-29) are paired with nuclear-encoded mitochondrial proteins of respiratory complex I.

[0026] In embodiments, the mitochondrial targeting sequences (MTS) identified in Table 2 (ie, SEQ ID NOs: 30-48) are paired with the nuclear-encoded mitochondrial protein of respiratory complex V.

[0027] In embodiments, the mitochondrial targeting sequences (MTS) identified in Table 3 (ie, SEQ ID NOs: 49-56) are paired with the nuclear-encoded mitochondrial protein of respiratory complex IV.

[0028] In embodiments, the mitochondrial targeting sequences (MTS) identified in Table 4 (ie, SEQ ID NOs: 57-63) are paired with nuclear-encoded mitochondrial proteins of respiratory complex III.

[0029] In embodiments, the mitochondrial targeting sequences (MTS) identified in Table 5 (i.e., SEQ ID NOs: 64-118) are paired with nuclear-encoded inner membrane proteins that contain transmembrane domains.

[0030] In embodiments, the mitochondrial targeting sequences (MTS) identified in Table 6 (i.e., SEQ ID NOs: 119-280) are paired with nuclear-encoded inner membrane proteins that lack transmembrane (TM) domains.

[0031] In embodiments, the therapeutic nucleic acid or protein is encapsulated in a liposome, nanoparticle or other pharmaceutically acceptable carrier.

[0032] In embodiments, a viral vector is used in the process of expressing a nucleic acid construct encoding a therapeutic mitochondrial protein. In aspects, the viral vector is a lentiviral vector, a herpes simplex virus (HSV) vector, an adenoviral vector, or an adeno-associated virus (AAV) vector.

[0033] The accompanying drawings illustrate embodiments of the present invention. In such drawings: [Brief explanation of the drawings]

[0034] [Figure 1A] FIG. 1A is a diagram of the steps in allotopic expression.

[0035] [Figure 1B] FIG. 1B is a diagram of a pCMV construct according to an embodiment of the present invention.

[0036] [Figure 1C] FIG. 1C is a bar graph showing the expression levels of oND1 mRNA in ND1 null cell lines (relative to GAPDH) for each construct.

[0037] [Figure 1D] FIG. 1D is a bar graph showing the expression levels of oND1 mRNA in ND1 null cell lines for each construct (relative to COX10).

[0038] [Figure 2A] FIG. 2A is a diagram of pCMV and pCAG constructs according to an embodiment of the invention.

[0039] [Figure 2B] Figure 2B is a bar graph showing the expression levels of oND1 mRNA in ND1 null cell lines (relative to GAPDH) for each construct.

[0040] [Figure 2C] FIG. 2C is a bar graph showing the expression levels of oND1 mRNA in ND1 null cell lines for each construct (relative to COX10).

[0041] [Figure 3A] FIG. 3A is a diagram of a construct having a hexapeptide and an OXA1L sequence according to an embodiment of the present invention.

[0042] [Figure 3B] FIG. 3B is a bar graph showing the expression levels of oND1 mRNA in ND1 null cell lines (relative to GAPDH) for each construct.

[0043] [Figure 3C] FIG. 3C is a bar graph showing the expression levels of oND1 mRNA in ND1 null cell lines for each construct (relative to COX10).

[0044] [Figure 4A] FIG. 4A is a diagram of a construct having an MPCP sequence according to an embodiment of the present invention.

[0045] [Figure 4B] FIG. 4B is a bar graph showing the expression levels of oND1 mRNA in ND1 null cell lines (relative to GAPDH) for each construct.

[0046] [Figure 4C] FIG. 4C is a bar graph showing the expression levels of oND1 mRNA in ND1 null cell lines for each construct (relative to COX10).

[0047] [Figure 5A] FIG. 5A is a diagram of a construct having an ABCBA sequence according to an embodiment of the present invention.

[0048] [Figure 5B] FIG. 5B is a bar graph showing the expression levels of oND1 mRNA in ND1 null cell lines (relative to GAPDH) for each construct.

[0049] [Figure 5C] FIG. 5C is a bar graph showing the expression levels of oND1 mRNA in ND1 null cell lines for each construct (relative to COX10).

[0050] [Figure 6A] FIG. 6A is a diagram of a construct with a triple protein tag according to an embodiment of the invention.

[0051] [Figure 6B] FIG. 6B is a bar graph showing the expression levels of oND1 mRNA in ND1 null cell lines (relative to GAPDH) for each construct.

[0052] [Figure 6C] FIG. 6C is a bar graph showing the expression levels of oND1 mRNA in ND1 null cell lines for each construct (relative to COX10).

[0053] [Figure 7A] FIG. 7A is a graphical depiction of mesohydrophobicity versus local hydrophobicity of transmembrane domain 1 of ATP6.

[0054] [Figure 7B] FIG. 7B is a graphical depiction of mesohydrophobicity versus local hydrophobicity of transmembrane domain 1 of ATP6.

[0055] [Figure 8A] FIG. 8A is a bar graph showing the expression levels of oATP6 mRNA in ATP6 mutant cell lines for different constructs relative to COX10.

[0056] [Figure 8B] FIG. 8B is a bar graph showing the expression levels of oATP6 mRNA in ATP6 mutant cell lines for different constructs relative to GAPDH.

[0057] [Figure 9] FIG. 9 is an image of an SDS PAGE of mitochondrial fractions showing a comparison of anti-FLAG and anti-aconitase expression.

[0058] [Figure 10A] FIG. 10A shows the cleaved and uncleaved COX2 proteins.

[0059] [Figure 10B] FIG. 10B is an SDS PAGE showing stable expression of COX2 site-directed mutagenesis constructs.

[0060] [Figure 11A] FIG. 11A shows mRNA levels for different versions of the codon-optimized allotopic COX2 gene compared to GAPDH.

[0061] [Figure 11B] FIG. 11B shows the SDS PAGE profiles for different versions of codon-optimized allotopic COX2 in a COX2 null cell line upon stable selection.

[0062] [Figure 12] FIG. 12 shows the plasmid sequence of a COX2 variant according to an embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0063] definition References herein to "one embodiment / aspect" or "embodiment / aspect" mean that a particular feature, structure, or characteristic described in connection with the embodiment / aspect is included in at least one embodiment / aspect of the present disclosure. Use of the phrase "in one embodiment / aspect" or "in another embodiment / aspect" in various places herein does not necessarily all refer to the same embodiment / aspect, nor to separate or alternative embodiments / aspects that are mutually exclusive of other embodiments / aspects. Furthermore, various features are described that may be exhibited by some embodiments / aspects but not other embodiments / aspects. Similarly, various requirements are described that may be requirements of some embodiments / aspects but are not requirements of other embodiments / aspects. Embodiment and aspect may be used interchangeably in certain instances.

[0064] The terms used in this specification generally have their usual meaning in the art within the context of this disclosure and in the specific context in which each term is used.Certain terms used to describe this disclosure will be discussed below or elsewhere in this specification to provide practitioners with additional guidance regarding the description of this disclosure.It will be understood that the same thing can be said in more than one way.

[0065] Therefore, alternative language and synonyms may be used for any one or more of the terms discussed herein. Also, no particular importance is attached to whether a term is recited or discussed herein. Synonyms for certain terms are provided. The listing of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any term discussed herein, is merely illustrative and is not intended to further limit the scope and meaning of the disclosure or any exemplified term. Similarly, the present disclosure is not limited to the various embodiments provided herein.

[0066] Without intending to further limit the scope of the present disclosure, examples of devices, apparatuses, methods and their related results according to embodiments of the present disclosure are provided below. Please note that for the convenience of the reader, titles or subtitles may be used in the examples, but should not limit the scope of the present disclosure in any way. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, the present document, including definitions, shall prevail.

[0067] The term "allotopic expression" or "AE" refers to the expression of genes normally expressed exclusively from the mitochondrial genome using nuclear-cytosolic machinery. Biomedically engineered AE has been proposed as a possible tool in gene therapy for treating certain mitochondrial-related diseases.

[0068] The term "mitochondrial disease" refers to a chronic, genetic, often inherited disorder that occurs when mitochondria are unable to produce enough energy for the body to function properly. Mitochondrial disease can affect almost any part of the body, including cells of the brain, nerves, muscles, kidneys, heart, liver, eyes, ears, or pancreas. Mitochondrial disease can be caused by mitochondrial DNA (mtDNA) damage or nuclear DNA (nDNA) damage.

[0069] Mitochondrial DNA (mtDNA) disorders include, for example, Leigh syndrome, leukodystrophy with complex II deficiency, cardiomyopathy and encephalopathy (complex I deficiency), optic atrophy and ataxia (complex II deficiency), hypokalemia and lactic acidosis, hepatopathy and ketoacidosis, hypertrophic cardiomyopathy, liver failure, renal tubulopathy (with complex III deficiency) and encephalopathy (with complex V deficiency), autosomal progressive external ophthalmoplegia, mitochondrial neurogastrointestinal encephalomyopathy, Alpers-Huttenlocher syndrome, ataxic neuropathy syndrome, infantile myopathy / spinal muscular atrophy, and hypotonia.

[0070] The term "primary mitochondrial disorders" refers to a clinically heterogeneous group of disorders resulting from dysfunction of the mitochondrial respiratory chain. The mitochondrial respiratory chain is the final common pathway essential for aerobic metabolism. Tissues and organs highly dependent on aerobic metabolism are usually most affected by mitochondrial disorders. Many genetic and non-genetic disorders involve the mitochondrial machinery as a secondary feature. However, "primary mitochondrial disorders" are considered to be known or presumed genetic disorders caused by pathogenic variants in genes encoding the mitochondrial respiratory chain and associated proteins.

[0071] Nuclear DNA (nDNA) disorders include, for example, Leigh syndrome, leukodystrophy with complex II deficiency, cardiomyopathy and encephalopathy (complex I deficiency), optic atrophy and ataxia (complex II deficiency), hypokalemia and lactic acidosis (complex III deficiency), liver damage and ketoacidosis, cardiomyopathy and encephalopathy, leukodystrophy and renal tubulopathy, hypertrophic cardiomyopathy, liver failure, renal tubulopathy (with complex III deficiency), encephalopathy (with complex V deficiency), coenzyme Q10 deficiency, Barth syndrome, autosomal progressive external ophthalmoplegia, mitochondrial neurogastrointestinal encephalomyopathy, Alpers-Huttenlocher syndrome, ataxic neuropathy syndrome, infantile myopathy / spinal muscular atrophy, hypotonia, reversible liver damage, myopathy with cataracts, and combined RC deficiency.

[0072] The term "secondary mitochondrial dysfunction" refers to any abnormal mitochondrial function other than a primary mitochondrial disorder. Secondary mitochondrial dysfunction (SMD) can be caused by genes that do not encode the function or production of oxphos proteins and is associated with many inherited non-mitochondrial diseases. Secondary mitochondrial dysfunction can also result from non-genetic causes, such as environmental factors. Secondary mitochondrial dysfunction is seen in many different genetic disorders, including ethylmalonic aciduria (caused by mutations in ETHE1), Friedreich's ataxia (FXN), hereditary spastic paraplegia 7 (SPG7), and Wilson's disease (ATP7B), and is also seen as part of the aging process.

[0073] The term "mutation" refers to changes that occur in our DNA sequence as a result of chemical mutagens such as reactive by-products of cellular metabolism, environmental factors such as UV light and cigarette smoke, or due to DNA replication errors resulting from congenital or acquired defects in the replication machinery.

[0074] Mitochondrial mutations can be described as non-silent point mutations in one or more mtDNA genes encoding subunits of the respiratory chain. Mutations can also consist of base insertions or deletions, or deletions or duplications of mtDNA genomic regions. In this context, mitochondrial mutations also refer to mtDNA copy number defects that prevent compensation for deleterious phenotypes.

[0075] The term "mitochondrial deficiency" refers to a deficiency characterized by inadequate mtDNA copy number, reduced or absent expression of mtDNA subunits, or expression of defective mtDNA subunits, and the subsequent inadequate energy metabolic state of a cell (or tissue or organ system or individual). Mitochondrial deficiencies can also be caused by defects in mtDNA copy number, resulting in phenotypic defects.

[0076] The term "improper energy metabolism" refers to a state of impaired mitochondrial function, such as inefficient electron transport, ATP synthesis, or altered mitochondrial membrane potential.

[0077] The term "wild type" refers to the phenotype that occurs in nature and is typical of a species.

[0078] The term "deleterious mutation" refers to a mutation in which the protein product of a gene is not produced, is produced but is non-functional, or is produced but interferes with normal function. Similarly, mutations in regulatory elements can be deleterious if regulatory function is impaired. These mutations can result from single base changes or more extensive insertions, deletions, or frameshifts. Mutations in protein-coding and regulatory genes can also be neutral. Base changes in protein-coding genes that do not change the amino acid sequence of the protein are called synonymous changes. These are generally neutral. Base changes that change the amino acid sequence are more likely to be deleterious, but can also be neutral.

[0079] The term "conservative substitution" refers to the replacement of an amino acid residue with another biologically similar residue.Examples of conservative substitution include the replacement of one hydrophobic residue, such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine, with another, or the replacement of a residue with a certain polarity or charge with another residue with a similar polarity or charge, such as the replacement of arginine with lysine, the replacement of glutamic acid with aspartic acid, or the replacement of glutamine with asparagine.Neutral hydrophilic amino acids that can be substituted for each other include asparagine, glutamine, serine and threonine.The term "conservative substitution" also includes the use of a substituted amino acid instead of the unsubstituted parent amino acid.

[0080] The term "senescence" refers to the gradual deterioration of functional properties in living organisms. Cellular senescence is often defined as the stress-induced permanent cell cycle arrest of previously replicative cells. The effects of senescent cells can be considered beneficial or detrimental with respect to host physiology and disease, but in some contexts, senescent cells influence disease states in complex ways that both promote and oppose certain conditions.

[0081] The term "aging-related disease or disorder" refers to a disease associated with aging, and may include, for example, atherosclerosis, osteoarthritis, osteoporosis, hypertension, arthritis, cataracts, cancer, Alzheimer's disease, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis. Other diseases associated with aging or senescence (including symptoms associated with aging) include hair graying, sarcopenia, adiposity, neurogenesis, fibrosis, and glaucoma.

[0082] Other diseases associated with aging or senescence include cardiovascular diseases (e.g., atherosclerosis, angina pectoris, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, coronary artery thrombosis, myocardial infarction, hypertension, aortic aneurysm, cardiac diastolic dysfunction, hypercholesterolemia, hyperlipidemia, mitral valve prolapse, peripheral vascular disease, cardiac stress resistance, cardiac fibrosis, cerebral aneurysm, and stroke). Aging-related diseases or disorders can also be inflammatory or autoimmune diseases or disorders (e.g., osteoarthritis, osteoporosis, oral mucositis, inflammatory bowel disease, or kyphosis). Aging-related diseases or disorders can also be neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, mild cognitive impairment, or motor neuron dysfunction). Aging-related diseases or disorders can also be metabolic diseases (e.g., diabetes, diabetic ulcer, metabolic syndrome, or obesity). The aging-related disease or disorder may also be a pulmonary disease (e.g., pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, cystic fibrosis, emphysema, bronchiectasis, or age-related loss of lung function). The aging-related disease or disorder may also be an ocular disease or disorder (e.g., macular degeneration, glaucoma, cataracts, presbyopia, or vision loss). The aging-related disease or disorder may also be a kidney disease, renal failure, weakness, hearing loss, muscle fatigue, skin conditions, skin wound healing, liver fibrosis, pancreatic fibrosis, oral submucous fibrosis, or sarcopenia. The aging-related disease or disorder may also be a dermatological disease or disorder (e.g., eczema, psoriasis, hyperpigmentation, nevus, rash, atopic dermatitis, urticaria, photosensitivity, or a disease or disorder associated with photoaging).

[0083] The term "oxidative phosphorylation" or "OXPHOS" refers to the process by which ATP synthesis is coupled with the movement of electrons through the mitochondrial electron transport chain and the associated consumption of oxygen. In eukaryotes, this occurs within the mitochondria. In eukaryotes, redox reactions are catalyzed by a series of protein complexes within the inner membrane of the cell's mitochondria. This set of linked proteins is called the electron transport chain. In eukaryotes, it involves five major protein complexes that use a variety of electron donors and acceptors.

[0084] Over 70 different polypeptides interact on the inner mitochondrial membrane to form the electron transport chain, also known as the respiratory chain. Thirteen essential subunits, along with ribosomal and transfer RNAs required for intramitochondrial protein synthesis, are encoded by mitochondrial DNA (mtDNA), located within the mitochondria. The remaining respiratory chain polypeptides, as well as proteins essential for the respiratory chain, mitochondrial structural assembly, and mtDNA maintenance and expression, are encoded by the nuclear genome (nDNA).

[0085] The term "codon optimization" refers to an experimental approach designed to improve the codon composition of a recombinant gene based on various criteria without changing the amino acid sequence. This is possible because most amino acids are coded for by more than one codon.

[0086] The term "promoter" refers to a sequence of DNA to which proteins bind and initiate transcription of a single RNA transcript from the DNA downstream of the promoter. The RNA transcript may encode a protein (mRNA) or may have a function in itself, such as tRNA or rRNA. The promoter is located upstream of the DNA (toward the 5' region of the sense strand) near the transcription start site of the gene and usually contains about 100 to 1000 base pairs. The CMV promoter is a strong synthetic promoter frequently used to drive high levels of gene expression in mammalian expression vectors.

[0087] The term "pCMV" or "pCMV-Script vector" refers to a promoter sequence derived from a high-copy-number pUC-based plasmid and designed to enable protein expression in mammalian systems. Mammalian expression is driven by the human cytomegalovirus (CMV) immediate-early promoter, which promotes constitutive expression of cloned inserts in a wide variety of cell lines.

[0088] The terms "5' untranslated region," "5' UTR," "leader sequence," "transcript leader," or "leader RNA" refer to the region of a messenger RNA (mRNA) immediately upstream of the start codon. This region is important for regulating the translation of the transcript by different mechanisms in viruses, prokaryotes, and eukaryotes. Although called untranslated, the 5' UTR, or portions thereof, may be translated into a protein product. This product can then regulate the translation of the primary coding sequence of the mRNA. However, in many organisms, the 5' UTR is completely untranslated and instead forms complex secondary structures that regulate translation.

[0089] The terms "canonical mitochondrial localization signal," "MLS," "mitochondrial targeting sequence," or "MTS" refer to a short peptide, approximately 15-70 amino acids in length, containing positively charged basic residues that direct the transport of proteins into mitochondria. The term "N-terminal mitochondrial targeting sequence" refers to the MTS in the amino-terminal portion of a protein, which can interact with the mitochondrial import machinery to aid in the translocation of soluble proteins into the matrix.

[0090] The terms "translation stall" or "ribosome stall" refer to a situation in which a ribosome moving along an mRNA slows down or stalls. For example, stalling can occur if the mRNA sequence utilizes codons that depend on low-abundance tRNA species.

[0091] The term "OXA1L" or "mitochondrial inner membrane protein OXA1L" refers to the protein encoded by the OXA1L gene, which in humans is located at 14q11.2. The C-terminus of this protein interacts with the mitochondrial ribosome and serves to insert or translocate both mitochondrially and nuclear-produced protein ends from the matrix into or across the inner mitochondrial membrane.

[0092] The terms "Pumilio," "PUM," or "human pumilio protein" refer to sequence-specific RNA-binding proteins that regulate protein expression. Members of the Pumilio family of proteins (Puf) regulate translation and mRNA stability in various eukaryotic organisms. Members of the Pumilio family are characterized by the presence of eight tandem copies of an imperfectly repeated 36-amino acid sequence motif (i.e., pumilio repeats) surrounded by short N- and C-terminal conserved regions. Pumilio interaction motifs have been defined in several species.

[0093] The term "treat" or "treatment" refers to one or more of: (1) inhibiting a disease, condition, or disorder (i.e., arresting further progression of the pathology and / or symptomology) in an individual experiencing or exhibiting the pathology or symptomology of the disease, condition, or disorder; (2) ameliorating a disease symptom or disorder (i.e., reversing the pathology and / or symptomology), e.g., reducing the severity of the disease, in an individual experiencing or exhibiting the pathology or symptomology of the disease, condition, or disorder.

[0094] The term "administration" refers to the introduction of a certain amount of a predetermined substance into a patient by a certain appropriate method. The compositions disclosed herein can be administered via any of the common routes, such as inhalation, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary, or rectal administration, so long as they can reach the desired tissue.

[0095] The composition comprises a therapeutic peptide encoded by DNA or RNA sequence and an acceptable carrier.The therapeutic peptide can be contained in a delivery vehicle.The two main approaches used to deliver therapeutic genetic material are (a) synthetic and (b) viral delivery vehicles.Synthetic vectors include, for example, liposomes, nanoparticles, cyclodextrins, and microvesicles.Viruses include, for example, AAV and retroviral vectors.

[0096] The term "liposome" refers to a spherical vesicle having at least one lipid bilayer (ie, an aqueous core surrounded by a hydrophobic membrane). Liposomes can be prepared by disrupting biological membranes (such as by sonication). Liposomes are formed when phospholipids and their derivatives are dispersed in water. When dispersed in water, phospholipids form closed vesicles called "liposomes," characterized by a lipid bilayer enclosing an aqueous core. Liposomes have therapeutic uses, including delivering drugs to target cells after systemic administration. Liposomes can be modified by incorporating polyethylene glycol or other hydrophilic polymers (e.g., PEG-liposomes, in which one or more constituent lipids are modified by PEG attachment). Liposomes can also be modified to target specific cell types by incorporating targeting factors (e.g., "targeting ligands") for those cell types. Examples include asialoglycoprotein, folate, transferrin, antibodies, etc.

[0097] The term "stable formulation" or "stable pharmaceutical formulation" refers to a formulation that preserves its physical stability / identity / integrity and / or chemical stability / identity / integrity and / or biological activity / identity / integrity during manufacture, storage, transportation, and application. Various analytical techniques for assessing virus stability are available in the art and are reviewed in Felix A. Rey and Shee-Mei Lok (2018) "Common Features of Enveloped Viruses and Implications for Immunogen Design for Next-Generation Vaccines," Cell 172, pp. 1319-1334 and Guy Ungerechts et al. (2016) "Moving oncolytic viruses into the clinic: clinical-grade production, purification, and characterization of diverse oncolytic viruses," Nature: Molecular Therapy - Methods & Clinical Development 3, 16018. Stability can be assessed, for example, but not limited to, by storage for a selected period of time in selected climatic conditions, by application of mechanical stress such as rocking at a selected rocking frequency for a selected period of time, by irradiation at a selected light intensity for a selected period of time, or by repeated freezing and thawing at a selected temperature.

[0098] As used herein, "virus" can encompass any chemical or biochemical component part of a virus, including viral component preparations, virus-like particles, viral vectors, lipid-enveloped nanoparticles, related particles (e.g., prions), etc., and need not be infectious or capable of self-replicating. Viral particles can include lipid-enveloped nanoparticles.

[0099] The term "viral vector" refers to a viral genome that has been adapted to plasmid-based technology and modified for safety through the removal of many essential genes and the separation of viral components. The use of viral vectors is a means of gene transfer to modify specific cell types or tissues, and can be engineered to express therapeutic genes.

[0100] The term "adeno-associated virus" or "AAV" refers to a small virus that infects humans and some other primate species. They are small (20 nm), replication-deficient, non-enveloped viruses with a linear, single-stranded DNA (ssDNA) genome of approximately 4.8 kilobases (kb). Several characteristics make AAV an attractive candidate for generating viral vectors for gene therapy and the generation of isogenic human disease models. AAV-based vectors have a good safety record in clinical trials and have emerged as the preferred vector system for neurological gene therapy.

[0101] The term "plasmid" refers to a genetic structure within a cell, typically a small, circular DNA strand, that can replicate independently of chromosomes. Plasmids are often used in laboratory manipulation of genes. Plasmids represent the simplest form of vector for transporting DNA into the cell nucleus. They generally comprise circular, double-stranded DNA molecules that vary in size from less than 1,000 to more than 200,000 base pairs. Compared to recombinant viruses, plasmids are simple to construct and easy to amplify in large quantities. They also have an excellent safety profile, with virtually no risk of tumorigenesis (because genome integration is highly inefficient) and relatively low immunogenicity. Plasmids can be administered, for example, by infusion or injection, and can be administered using techniques (e.g., carrier vehicles) to improve their uptake by target cells.

[0102] Carrier vehicles for plasmids (pDNA) can reduce susceptibility to circulating nucleases and increase cellular uptake. They can also target plasmids to specific tissues. Like carrier microbubbles, most vehicles are cationic. Their positive charge allows electrostatic complex formation with negatively charged pDNA. Complexes are prepared with a residual positive charge that enhances cellular uptake through electrostatic interactions with negatively charged cell membranes. These carrier vehicles can substantially increase transfection; nonviral systems can be as effective as viruses in delivering DNA to the cell nucleus.

[0103] The terms "episome" or "plasmid" refer to a length of DNA present in the cytoplasm or chromosome of a mammalian cell. They replicate synchronously with the host chromosome and therefore persist as long as the parent strain exists. The main drawback of integrating vector systems is the potential risk of insertional mutagenesis. Episomal vector systems have the potential to avoid these undesirable side effects because they behave as separate extrachromosomal elements in the nucleus of the target cell.

[0104] The term "subject" refers to a person who is susceptible to, suspected of having, or diagnosed with a disease (e.g., an age-related disease), including, but not limited to, any subject treated with the therapeutic methods described herein.

[0105] All numerical designations (e.g., pH, temperature, time, concentration, and molecular weight), including ranges, should be understood as approximations, in accordance with common practice in the art. As used herein, the term "about" can mean a variation of (+) or (-) 1%, 5%, or 10% of the stated amount, depending on the context. It should be understood that, although not always explicitly stated, the reagents described herein are merely exemplary, and that equivalents of such reagents are known in the art.

[0106] Many known useful compounds are listed in Remington's Pharmaceutical Sciences (13 th "These formulations are generally considered to be pharmaceutically acceptable compositions for human or animal administration, and may be found in a variety of standard reference works on various types of administration," Ed., Mack Publishing Company, Easton, PA. As used herein, the term "formulation" refers to a combination of at least one active ingredient with one or more other ingredients (also commonly referred to as excipients), which may be independently active or inactive. The term "formulation" may or may not refer to a pharmaceutically acceptable composition for administration to humans or animals, and may also include compositions that are useful intermediates for storage or research purposes.

[0107] Other technical terms used herein have their ordinary meaning in the art in which they are used, as exemplified by various technical dictionaries. The specific values ​​and configurations discussed in these non-limiting examples may vary and are cited only to illustrate at least one embodiment and are not intended to limit its scope.

[0108] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the claimed subject technology. Additional features and advantages of the subject technology will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the subject technology. The advantages of the subject technology will be realized and attained by the structure particularly pointed out in the written description and claims.

[0109] De novo synthesis of human mitochondria requires coordinated translation of nuclear and mitochondrial genes. The mitochondrial proteome contains over 1,500 proteins, only 13 of which are encoded by mitochondrial DNA (mtDNA). However, mtDNA is particularly vulnerable to damage and the accumulation of mutations. Its location adjacent to the oxidative phosphorylation machinery places genetic material at a higher risk of mutational events. mtDNA is susceptible to mutations from reactive oxygen species generated through oxidative phosphorylation as well as inadequate DNA repair mechanisms in the organelle.

[0110] The quality and quantity of mtDNA are related to mitochondrial function. Inappropriate function can lead to mitochondrial disease. Aging is also related to mitochondrial function. Age-related changes in mitochondrial metabolism can result in, for example, a decrease in the number of mitochondria, a decrease in the rate of ATP synthesis, or a decrease in oxidative capacity. Other mitochondrial changes observed with aging include a depletion of mtDNA copy number and large deletions in the mtDNA coding region.

[0111] Allotopic expression is a promising therapeutic tool for genetically treating deleterious mtDNA mutations through nuclear complementation of affected genes, in which mitochondrial genes are deliberately recoded and relocated to the nucleus, and the encoded polypeptides are then retroported into mitochondria.

[0112] Figure 1 is a schematic diagram of allotopic expression, showing the various steps involved in implementing a successful strategy. The first step (1) is the design of an optimal DNA expression construct. As described herein, modified or wild-type versions of mitochondrial genes can be used in the construct. Next, a plasmid is integrated with the allotopic gene (2). This step is followed by transcription (3). Next, sufficient amounts of mRNA must be exported to the cytosol (4) and translated into a peptide (5). As described herein, efforts must be made to avoid aggregation (6). The peptide is targeted to mitochondria (7) and imported across the outer and inner mitochondrial membranes (8). The peptide is then delivered to normalize components of the respiratory chain (RC) complex (8). If these steps are successful, the peptide can be delivered to normalize the mitochondrial deficiency (9) and achieve functional recovery (10).

[0113] Previous methods of allotopic expression have been unsuccessful for a number of reasons. The present invention provides a method that overcomes the difficulties in past efforts at allotopic expression. In embodiments of the present invention, the methods described herein address differences in codon usage preferences between genomes. In embodiments, translational slowing or ribosome stalling is induced during synthesis of allotopically expressed (AE) proteins. This is particularly important in conjunction with codon-optimized constructs. In embodiments, the influence of mitochondrial import direction on the final functional assembly of AE subunits is considered. Topological changes that can result from post-translational import versus matrix synthesis are also addressed. In embodiments, components utilized by specific mitochondrial import machinery or multi-pass inner membrane proteins are targeted. In embodiments, a systematic reduction in the hydrophobicity of specific domains in allotopic proteins is addressed to improve import while preserving function. In embodiments, feedback regulation of AE gene expression is also addressed. In embodiments, combinations of synergistic elements are addressed to improve allotopic expression and mitochondrial targeting. In embodiments, the methods described herein involve the expression of several genes simultaneously.

[0114] Codon optimization Codon optimization refers to experimental approaches designed to improve the codon composition of recombinant genes based on various criteria without altering the amino acid sequence. This is possible because most amino acids are encoded by more than one codon. The mitochondrial genetic code also often differs from the standard genetic code. The mitochondrial genome has a relatively conserved gene content and small size. Furthermore, the relative usage of specific codons for each amino acid differs significantly between the two genomes, an aspect not exploited by traditional methods of allotopic expression.

[0115] Codon optimization of transgenes can result in increased transcription (mRNA levels) by matching the transgene sequence to the codon preferences of the nuclear genome. Because the mitochondrial genome uses different codons than the nuclear genome, all allotopically expressed genes require some modification to maintain the amino acid sequence of the encoded protein. The codon-optimized transgenes described herein result in greater transcription of mtDNA genes.

[0116] N-terminal export The import mechanisms that translocate soluble proteins across peroxisome, mitochondrial, chloroplast, or ER membranes are significantly different. However, mitochondrial, chloroplast, and ER targeting signals appear to be structurally similar, as they all contain an α-helical domain near the N-terminus. Mitochondrial, chloroplast, and ER targeting signals are encoded within N-terminal sequences with different names (presequence, transit sequence, and signal peptide).

[0117] All 13 mitochondrially encoded proteins are hydrophobic subunits of electron transport chain (ETC) enzyme complexes and are required for oxidative phosphorylation (OXPHOS) across the mitochondrial inner membrane (MIM). When mitochondrial genes are normally expressed, their transcripts are translated within the mitochondrial matrix, the innermost mitochondrial compartment, and then inserted into the inner membrane. Each of these subunits contains one or more transmembrane domains that span the MIM region and adopt a final topology characterized by the positioning of the protein's N- and C-termini relative to the intermembrane space (IMS) and matrix. Because mtDNA-encoded subunits are translated from within the matrix, proteins whose N-termini face the IMS can be "exported" across the inner membrane. However, when these subunits are allotopically expressed, they are imported into mitochondria from the opposite face of the inner membrane, with their N-termini delivered first to the matrix.

[0118] Previous studies have identified a six-residue sequence in the nuclear-encoded inner membrane protein OXA1L that is responsible for translocating (exporting) its N-terminus across the MIM after import, resulting in a final N-out (facing the IMS) topology. Therefore, for allotopic expression of OXPHOS subunits, which should adopt a final N-terminus-facing IMS topology, Applicants included this six-residue "N-terminal export signal" in their constructs to direct the N-terminus back toward the IMS after initial import into the matrix.

[0119] Transmembrane architecture Mitochondria are separated from the cytoplasm by the outer and inner mitochondrial membranes. The outer membrane is porous, allowing the free passage of ions and small, uncharged molecules through pore-forming membrane proteins (i.e., porins) such as the voltage-dependent anion channel (VDAC). Larger molecules, especially proteins, must be imported by specialized translocases. Due to its porosity, there is no membrane potential across the outer membrane. In contrast, the inner membrane is a severe diffusion barrier to all ions and molecules. These can only pass through with the assistance of specific membrane transport proteins, each selective for a particular ion or molecule. As a result of its ion selectivity, an electrochemical membrane potential of approximately 180 mV is established across the inner mitochondrial membrane. The inner membrane is where oxidative phosphorylation occurs in a series of membrane protein complexes that either create an electrochemical gradient across the inner membrane or use it for ATP synthesis.

[0120] Many multi-pass inner membrane proteins are thought to translocate into the matrix as helical pairs, with the helices stabilizing each other. Paired translocation across the membrane / lateral insertion alters the subsequent insertion and exit of the transmembrane domain.

[0121] Instead of (or in conjunction with) an N-terminal export signal, the addition of an "inactive" (non-interfering) transmembrane domain region at either the amino or carboxy terminus of the AE protein can be included to restore the natural orientation of the transmembrane helix relative to the inner mitochondrial membrane. The loop region connecting the extra helix to the AE protein may contain sites for cleavage by specific local proteases. Depending on where the loop is located, different cleavage signatures (e.g., for MMPs vs. OCT1 vs. IMPs vs. IMS proteases) can be used.

[0122] Transmembrane domains can also be used to reduce hydrophobicity, for example, specific sequences in transmembrane domains can encode hydrophilic amino acids.

[0123] localized charge Research has demonstrated that surface charge can determine protein localization.Surface charge can affect the electrostatic interaction between anionic lipids and cationic amino acids, which works in combination with other processes to direct protein localization.In embodiments, the surface charge of AE protein is adjusted or reduced.

[0124] For example, in the case of ND1, the net negative charge of the reporter tag may also promote association of the carboxy terminus of ND1 with localized positive charges on the intermembrane side of the inner membrane, preventing full translocation and helping to maintain that terminus on the correct side of the MIM. Similarly, in the case of A6, localized charges near the N-terminus may help promote interaction with the MIM-IMS interface to allow proper topology (or by avoiding affecting potential endogenous signals apparently present in the C-terminal region).

[0125] Promoter selection In embodiments, the cellular state is considered in relation to the promoter. Long-term adaptive expression of AE transgenes often responds to cellular states (e.g., energy status). The inclusion of transcription factor binding sites can improve transcriptional regulation in response to cellular states (e.g., Pgcla / errla, HIF, NRF1 / 2, or other effectors). Regulatory elements are also included to allow tissue- and condition-specific gene expression levels. For example, pcgla / errla target genes are highly expressed in tissues with high oxygen demand. Metabolic feedback loops / gene circuits can also be considered.

[0126] pCMV is recognized as a strong promoter and is often used in allotopic research.However, due to its strong constitutive expression, pCMV may cause aggregation and therefore proteotoxicity.In an embodiment, the pCMV promoter is replaced with the pCAG promoter to affect the endogenous chimeric promoter.As described herein, pCAG does not cause the overexpression of downstream genes to the same extent as pCMV.

[0127] There are additional advantages to using an alternative promoter, such as pCAG. Existing pCMV constructs may have translation initiation sites within the promoter, which, when recognized by cells, could result in the generation of an incorrect protein product. The pCAG promoter also has an upstream ORF, but the observed upstream stop site would prevent the formation of aberrant promoter fusion proteins. uSTOP can be included to normalize translation initiation. In an embodiment, the chimeric promoter contains a CMV enhancer sequence but is otherwise derived from chicken beta globulin, which is known to have a better, more stable expression profile in the mouse safe harbor nuclear expression region than CMV, and which can be silenced over time.

[0128] Other promoters that can be used include, for example, PGK, PGC1 alpha, ER1 alpha, or general tissue-specific promoters such as alpha myosin, creatine kinase, or hybrid promoters such as C5-12, MHCK7, etc. Still others include AAV serotypes 1-9, AAV2 / 6, or AAV2 / 8.

[0129] Although there is no evidence that our CMV construct is silenced (high mRNA levels are maintained), expression from this promoter is predicted to be lower overall, which may be beneficial if constitutively high expression levels (functional or non-functional) are causing excessive intracellular proteostatic (or other) stress.

[0130] Upstream architecture and 5'UTR 5'UTR regions from specific classes of mitochondrial proteins have been added upstream of expression constructs. In addition to general transactivation factor recognition, the 5'UTR regions may form stable structures that can improve mRNA quality or promote transcript localization near the mitochondrial surface for translation.

[0131] Various UTR regions can improve mRNA / nascent strand targeting. Because CMV is a constitutive promoter, it does not usually require transcription factor induction. However, under some cellular stresses (e.g., certain mitochondrial stresses), certain transcriptional programs are up- or down-regulated. One of these pathways responds to NRF1 (nuclear respiratory factor 1), which functions like PGC1a to activate OXPHOS genes in response to metabolic conditions and aerobic demands. Furthermore, it is involved in antioxidant responses. Many mitochondrial genes, including ATP5A1, are up-regulated in response to NRF-1. Putative binding sites have been identified. ATP5A1 was arbitrarily selected from the NEMG (nuclear-encoded mitochondrial genes). The 5'UTR may also contain a Y-box promoter-binding motif. Some examples include: -YYI join -ABCBA -ATP5A1 NRF1 / NRF2 -ATP5A1 -uSTOP -MPCP -ABCBA An in-frame stop codon located near the upstream translation start site can aid in the rate of translation initiation and prevent upstream initiation from interfering with downstream translation.

[0132] In embodiments, the 5'UTR of a nuclear-encoded mitochondrial protein (NUMP) is used, including sequences of complex I, complex II, complex IV, complex V, inner membrane proteins, or matrix proteins.

[0133] 3'UTR(COXIV) The 3'UTR of COX4 was similarly chosen (a somewhat arbitrary NEMG with various RBP binding sites) to contain a binding site for YBX1, an RBP identified as stabilizing mRNA and helping to regulate transcription by modulating the interaction between EIF and mRNA.

[0134] 3'UTR(ATP5A1) The 3'UTR of ATP5A1 has also been studied because it contains both a PUM (pumilio) binding site and a sequence similar to a potential CLUH sequence found to be enriched in mitochondrial proteins. CLUH was identified in Drosophila as a protein involved in the transport of OXPHOS mRNAs to the mitochondrial surface. In yeast, several PUM proteins aid in the localization of OXPHOS transcripts at the OMM. PUM proteins affect translation dynamics by ribosome stalling, binding to the 3' ends of many mRNAs, and forming secondary structures that inhibit (or slow) translation. PUM (pufp3 in yeast) binds to the mRNAs of many mitochondrially targeted proteins and is thought to be involved in both nutrient-dependent translation (when there is a demand for OXPHOS) and promoting localized translation at the OMM. This UTR also forms a highly stable secondary structure that has been implicated in general mRNA stability. In some cases, it has also been proposed to play a role in RBP recognition and binding.

[0135] In embodiments, the 3'UTR of a nuclear-encoded mitochondrial protein (NUMP) is used, including sequences of complex I, complex II, complex IV, complex V, inner membrane proteins, or matrix proteins.

[0136] Mitochondrial targeting / MTS selection A targeting peptide is a short (3-70 amino acid long) peptide chain that directs the transport of a protein to a specific region within the cell (e.g., mitochondria). A mitochondrial targeting signal ("MTS") is a 10-70 amino acid long peptide that directs newly synthesized proteins to mitochondria. It is found primarily at the N-terminus and contains an alternating pattern of hydrophobic and positively charged amino acids, forming an amphipathic helix. The mitochondrial targeting signal may then contain additional signals that target the protein to different regions of the mitochondria, such as the mitochondrial matrix or inner membrane.

[0137] Previous efforts to target precursor proteins to mitochondria have utilized either ATP5G1 mts or COX VIII mts. Embodiments include MTS sequences that overcome the limitations of MTS sequences used in conventional methods of allotopic expression. The MTS sequences described herein enable: · Guides AE proteins to the specific import machinery of nuclear mitochondrial proteins (NuMPs), ·Distinguish the targeting of AE proteins to the import machinery utilized by multipath, endomembrane NUMPs, and Facilitating recognition by outer membrane receptors as having a destination embedded in the MIM. Provides complex specificity Furthermore, by utilizing MTSs from protein classes known to interact with specific chaperones, Applicants take advantage of additional signals that can be encoded, such as those for chaperone recognition / recruitment (e.g., Hsp90), and outer membrane receptor recognition, as well as the native context of codon composition that can play a role in regulating translational kinetics (which in turn affects folding / misfolding, chaperone recognition, targeting, etc.).

[0138] OXA1L Endogenous context of the HEX signal after the OXA1L MTS. OXA1L is an inner membrane insertase conserved from bacteria that utilizes a hexapeptide export signal to adopt its final topology. Due to the protein's structural similarity to many OXPHOS proteins embedded in the MIM, this MTS may encode additional information that aids its targeting and insertion into the inner membrane.

[0139] Other embodiments may include sequences derived from other non-carrier, multi-pass, nuclear-encoded mitochondrial inner membrane proteins such as TMM70, TMM65, TMM11, TMM177, TI17B, T123B, TI17A, T126A, SURF1, SPNS1, SPG7, SFXN4, SFXN1, PARL, OMA1, NNTM, NDUAB, NCLX, MFTEP1, MRS2, MVP17, MIC27, MCUR1, MCUB, MCU, M17L2, IFI6, HIG2A, HIG1A, GHITM, DMAC1, DHSD, CRLS1, COX20*, COX18*, COQ2, CDS2, C560, AGK, AFG32*COX-derived sequences found in several existing patents.

[0140] MTS(MPCP) The mitochondrial carrier family protein MPCP is unique in that it has a truncated N-terminal MTS domain, which is typically absent in carrier proteins imported via the TIM22 pathway rather than the TIM23 pathway. The structural and biochemical properties of ND1 are similar to those of carrier family proteins, and therefore, cooperative import via the TIM23 and TIM22 pathways may be achieved by including this targeting domain (see, e.g., V. Zara, F. Palmieri, K. Mahlke, and N. Pfanner, "The cleavable presequence is not essential for import and assembly of the phosphate carrier of mammalian mitochondria but enhances the specificity and efficiency of import," Journal of Biological Chemistry, Volume 267, Issue 17, 1992).

[0141] Metabolite / carrier family proteins (which constitute the majority of multi-pass MIM proteins) are specifically targeted to TOM70 on the outer membrane before being routed to TOM20 / 22 for import. TOM70 substrates are inserted into the MIM via TIM22.

[0142] TOM70 is also a known interacting partner of Hsp90, a ubiquitous chaperone involved in promoting and normalizing protein folding. This suggests that Hsp90 (unlike the even more ubiquitous Hsp70) may specifically recognize and bind to sequences in nascent carrier proteins and assist in their delivery to MOM receptors in an import-competent state. Because HSPs bind promiscuously, specific signals and motifs that may be added to their constructs have not been identified. Therefore, sequences derived from their client proteins are instead candidates for recognition. In this case, the presequence of MPCP may have a dual advantage if it can both be recognized by Hsp90 chaperones as clients destined for misfolding-prone mitochondria and, further, direct inner membrane assembly via TIM22.

[0143] Furthermore, OXA1L, the insertase responsible for the (co-translational) inner membrane insertion and assembly of most mitochondrially encoded OXPHOS subunits, has been shown to be required for the assembly of other multi-pass inner membrane proteins, including many metabolite carrier families (as well as many TIMs, SDH subunits, and ABC transporters). Thus, the presequence of MPCP may constitute a signal that can be recognized by the import and assembly machinery as destined for the MIM, potentially helping to target allotopically expressed ND1 and other multi-pass subunits to the machinery responsible for correct inner membrane organization and final topology.

[0144] Other embodiments may include sequences from any of the mitochondrial carrier family proteins (gene designation SLC25A), including UCP5, UCP4, UCP3, UCP2, UCP1, TXTP, TPC, SCMC3 / 2 / 1, SAMC, S2553, S2552, S2551, S2548, S2547, S2545, S2544, S2543, S2542, S25441, S2540, S2539, S2538, S2536, S2535, S2534, S2533, ORNT2*, ORNT1*, ODC, MTCH2, MTCH1, MPC2, MPC1, MFTC, MFRN2, MFRN1, MCATL, MCAT, M2OM, KMCP1, GHC1, GDC, DIC, CMC2, and ADT4. *ORNT1 / 2 are ornithine transporters and are distinct from the OTC enzymes that are the therapeutic targets of several patents.

[0145] MTS(ABCBA) ABCBA is a mitochondrial ABC transporter of the inner membrane and is unique in that it possesses an N-terminal MTS, a feature not characteristic of this family. It has also been demonstrated that the long MTS region and subsequent first transmembrane domain can promote the correct folding and topology of associated inner membrane proteins, but not matrix proteins. Therefore, we tested the ability of the MTS alone and the MTS plus the first TM region of ABCBA proteins to direct the topology of allotopically expressed ND1. The subsequent MTS and TM regions also contain relatively abundant slowly reading codons, particularly proline and serine (which are rare), that may play a role in regulating translation kinetics and / or promoting trans-factor specification. For example, Ethan R. Graf, Xue Zhao Zhang, Shan-Xue Jin, Michael W. Linhoff, Ann Marie Craig, Neurexins Induce Differentiation of GABA and Glutamate Postsynaptic Specializations via Neuroligins, Cell, Volume 119, Issue 7, 2004, pp. 1013-1026, and Mitsunori Miyazaki and Karyn. See A. Esser, "Cellular mechanisms regulating protein synthesis and skeletal muscle hypertrophy in animals," Journal of Applied Physiology, Vol. 106, No. 4, 2005.

[0146] Allotopic proteins ("AE proteins") can pair with the MTS of a nuclear-encoded mitochondrial protein (NUMP) from the same respiratory complex to facilitate complex assembly. This includes other members of the mature respiratory complex, as well as assembly factors. In some cases, the exact location of the MTS is unknown. Estimates can be made based on literature knowledge to incorporate the appropriate length of the protein N-terminus. In embodiments, allotopic proteins pair with the MTS of proteins identified in Table 1 (i.e., SEQ ID NOS: 1-63). [Table 1] JPEG2025533896000003.jpg188159JPEG2025533896000004.jpg57159 [Table 2] JPEG2025533896000006.jpg25159 [Table 3] [Table 4] JPEG2025533896000009.jpg43159

[0147] In embodiments, MTSs from nuclear-encoded proteins containing transmembrane domains (TM) are added to allotopically expressed proteins based on structural and biochemical properties, such as length and hydrophobicity. All mitochondrially encoded proteins are integral components of membrane complexes and therefore contain hydrophobic TM domains. Therefore, a region of a similar TM domain-containing protein that localizes the allotopically expressed mitochondrial protein to the inner mitochondrial membrane can be added. This can include the MTS of the nuclear-encoded protein. Alternatively, many inner membrane proteins with hydrophobic TM domains lack a canonical N-terminal MTS and are instead targeted via a cryptic internal sequence. In such cases, a region corresponding to the N-terminus of the protein by the first TM domain (e.g., 1-30 AA) can be added instead. Table 5 lists examples of MTS sequences for nuclear-encoded inner membrane proteins containing transmembrane domains. In embodiments, allotopic proteins are paired with the MTSs of proteins identified in Table 5 or Table 6 (i.e., SEQ ID NOS: 64-280). [Table 5] JPEG2025533896000011.jpg200159JPEG2025533896000012.jpg193159JPEG2025533896000013.jpg122159 [Table 6] JPEG2025533896000015.jpg201159JPEG2025533896000016.jpg203159JPEG2025533896000017.jpg197159JPEG20255338960 00018.jpg202159JPEG2025533896000019.jpg197159JPEG2025533896000020.jpg197159JPEG2025533896000021.jpg118159

[0148] N-terminal export We propose that a significant proportion of allotopically expressed ND1 does not fold / assemble correctly in the inner membrane, with both the short N- and C-termini facing the intermembrane space. A previously identified "matrix export signal" derived from OXA1L has been shown to promote export of the appropriate transmembrane domain of mature inner membrane proteins from the matrix to the IMS after MTS cleavage. Adding this sequence after the MTS signal, but upstream of the first transmembrane domain of ND1, may promote orientation of the N-terminus toward the IMS.

[0149] Additional TM1 (ABCBA1~165) The region following the MTS (approximately up to the second TM helix) has been shown to specifically target multipass membrane proteins to the inner mitochondrial membrane and is required for ABCBA. In the absence of this region, proteins accumulate within the matrix. This additional sequence has also been shown to promote similar inner membrane targeting of proteins that would otherwise be recruited to the ER and assemble into other organelle membranes.

[0150] Translation stall The allotopically expressed genes described are codon-optimized, utilizing the most preferred codons at nearly every position, which can promote robust and rapid translation of polypeptides. However, in nuclear-encoded proteins, including those targeted to mitochondria, codon usage is more commonly distributed. Many of these proteins, particularly membrane proteins, have an overrepresentation of "rare" codons near the N-terminus and in regions surrounding highly hydrophobic membrane-spanning segments. The presence of rare codons at these positions is often conserved and is thought to be involved in protein folding and chaperone recruitment through regulating translation rate. Introducing "rare" codons into otherwise rapidly translated regions can help slow translation kinetics and thus increase the yield of high-quality mitochondrially targeted proteins. Rare codons can also be placed downstream of hydrophobic sequence regions (i.e., at the C-terminus) so that nascent peptides can electrostatically interact with the ribosomal surface. Such interactions can promote the formation of transient, stable secondary structures and prevent aggregation of highly hydrophobic proteins upon emergence from the ribosomal exit tunnel. Slowing translation upon the occurrence of hydrophobic sequence regions can also facilitate recognition by and interaction with protein chaperones such as Hsp70 and Hsp90.

[0151] PUMILIO Because PUM binding is thought to cause ribosome stalling, applicants proposed that translation elongation could be prevented or inhibited by including an upstream PUM binding site between the RBS and the TSS / start of the MTS coding sequence (i.e., as a negative control and evidence of PUM binding).

[0152] However, previous preliminary studies suggest that upstream PUM binding, and therefore translation stalling, may be beneficial for allotopic expression, potentially delaying translation of transgenic mRNA until it reaches the mitochondrial vicinity. Furthermore, studies suggest that rapid translation may be detrimental to the expression of high-quality heterologous protein products, particularly due to the relationship between translation kinetics and nascent chain folding.

[0153] Because the allotopic constructs described herein primarily utilize the most frequent codons for each amino acid (which correlates with translation rate), PUM domains, if interspersed throughout the CDS (as is also common), may also be beneficial for slowing translation rate. Instead, previous preliminary studies revealed improved expression likely due to ribosome stalling (which may promote localized translation) or a slowing of the translational tempo of mRNAs that would otherwise translate very rapidly due to frequent codon optimization.

[0154] In embodiments, compositional similarity to endogenous genes is used to identify Avoiding transcriptional repression, Avoiding immune response / foreign nucleic acid responses, and Mimic the natural periodicity of translation rate.

[0155] Reduced hydrophobicity The highly hydrophobic nature of mitochondrially encoded subunits is thought to hinder mitochondrial import of cytosolically translated subunits, thus promoting the retention of these genes in the mitochondrial genome.

[0156] For example, the average maximum hydrophobicity across the first X amino acids (or first TM domain or MesoH, etc.) of a sample of mitochondrial proteins (or proteins spanning the MIM, mitochondrial proteins containing a TM domain, etc.) is known to be a numerical value using a hydrophobicity scale and any corresponding window size. However, mtDNA-encoded subunits exhibit both high maximum local hydrophobicity across the same region (as measured in controls) and higher overall hydrophobicity than many nuclear-encoded subunits (see, e.g., Figure 7A, which shows the clustering of mitochondria subunits MesoH and H17 compared to their nuclear counterparts).

[0157] To facilitate the import of exogenously expressed subunits, the present invention describes targeted amino acid substitutions within the hydrophobic transmembrane regions of allotopic proteins, the effect of which is a reduction in local hydrophobicity without functional impairment of the protein product. Thus, in embodiments, annotated transmembrane regions of mitochondrial protein subunits are evaluated for overall hydrophobicity (MesoH) and maximum local hydrophobicity (H17), a measure of local hydrophobicity over a 17-residue peptide window that approximates the peptide length spanning a biological membrane. The hydrophobicity of amino acid residues is ranked using the Goldman-Engelman-Seitz (GES) hydrophobicity scale.

[0158] In embodiments, the hydrophobic transmembrane region of a mitochondrial subunit is computationally evaluated by substituting each amino acid position within the region with each of the other 19 standard amino acids. Amino acid substitutions can be evaluated for suitability based on the local (H17) and global (MesoH) hydrophobicity-reducing effect of a given residue substitution. Examples include: · Substitutions within the transmembrane region of the protein with a maximum H17 greater than 2.5 as measured by the GES scale; Substitutions that reduce the maximum H17 of the transmembrane region to less than 2.5 as measured by the GES scale.

[0159] Substitutions can be further characterized using software to predict the deleterious effects of position-specific substitutions on protein function. Such predictions can use local (e.g., block substitution matrix, BLOSUM) or global (e.g., point-allowed mutation, PAM) evolutionary conservation matrices (or derivatives such as the Dayhoff mutation data matrix) to show positional and functional residue conservation across species homologs of a protein or domain, in addition to the frequency of substitutions with each of the other 19 amino acids. Such matrices can thus be used to filter out substitutions predicted to have deleterious effects, for example, if the residues show a high degree of conservation.

[0160] Thus, a functional impact prediction threshold can be established to eliminate hydrophobicity-reducing substitutions predicted to adversely affect protein function, for example, by eliminating highly conserved residues with mutation frequencies below an arbitrary threshold. Other methods for eliminating potentially deleterious residue substitutions include evaluation of annotated and predicted active sites and ligand-binding regions.

[0161] The method may also consider physicochemical properties such as hydrophobicity, aromaticity, and charge, which often contribute to protein-protein or protein-ligand interactions. Substitutions annotated as associated with human disease or having adverse functional implications are excluded. Residue substitutions within a defined functional impact threshold that also effectively reduce H17 and / or MesoH are candidates for selection.

[0162] Furthermore, through computational modeling of residue substitutions and measuring the resulting deviations from the established wild-type crystal structure, candidate substitutions can be evaluated for risk of structural protein perturbation. Evaluation metrics can include phi / psi angles within transmembrane helices, distances between residue side chains, proximity of atoms to adjacent side chains, local hydrophobicity trends, interactions with membrane lipids, ligand interactions, and changes to residues in intermolecular bonds and interfaces.

[0163] Thus, in embodiments, the transmembrane region may have one or more amino acid substitutions introduced that are predicted to reduce hydrophobicity without any adverse effect on the function of the protein.

[0164]

[0003] Embodiments include allotopic expression of one or more mtDNA genes to compensate for functional deficiencies resulting from mtDNA damage and / or mutations in mitochondrial genes. In aspects, the mtDNA genes are MT-ND1, MT-ND2, MT-ND3, MT-ND4L, MT-ND4, MT-ND5, MT-ND6, NDUFA6, NDUFA3, NUBPL, NDUFS3, NDUFV3, NDUFV2, DMAC1, AIFM1, NDUFS4, OXA1L, NDUFA13, NDUFAF5, NDUFAF4, NDUFA4, TIMM21, NDUFA1, NDUFB4, NDUFA8, NDUFAF8, NDUFB1, NDUFS6, NDUFA2, NDUFB6, NDUFB9, NDUFB11, WDR93, NDUFC1, DMAC2, NDUFA7, NDUFAF1, and NDUFB8. , NDUFAF3, NDUFAF2, NDUFAF7, TAFAZZIN, NDUFB7, NDUFAB1, NDUFB2, TMEM126B, TMEM126A, NDUFC2-KCTD14, NDUFA5, NDUFC2, NDUFA10, NDUFAF6, NDUFV1, TMEM186, BCS1L, NDUFB5, ACAD9, NDUFA11, NDUFS5, NDUFS2, NDUFB10, NDUFA12, NDUFS7, NDUFB3, ECSIT, COA1, FOXRED1, NDUFA9, NDUFS8 and NDUFS1.

[0165] In embodiments, the mtDNA gene is a subunit and / or assembly factor of Complex III selected from MT-CYB, UQCC2, TTC19, UQCRB, SLC25A33, UQCRH, UQCR10, UQCRFS1, CYC1, LYRM7, UQCC1, UQCRC2, UQCRQ, UQCC3, BCS1L, C12orf73, UQCRC1, UQCR11, and UQCRHL.

[0166] In embodiments, the mtDNA gene is selected from the group consisting of MT-CO1, MT-CO2, MT-CO3, SURF1, COX8C, FASTKD3, COX4I2, PET117, COX7C, COX5A, COX7B, OXA1L, COX14, COX7A2L, COA3, NDUFA4, COX19, COX7A1, TIMM21, COA5, SCO2, UQCRFS1, COX16, COX8A, SURF1, SC and a subunit and / or assembly factor of Complex IV selected from O1, UQCRC2, COX18, COX6C, COA8, C15orf48, COX6A2, COA4, COX6A1, COX8C, COX17, BCS1L, SMIM20, COX7A2, TACO1, COA1, COX4I1, COX6B1, PET100, NDUFA4L2, COX5B, and COX20.

[0167] In embodiments, the mtDNA gene is a subunit and / or assembly factor of Complex V selected from MT-ATP8, MT-ATP6, ATP23, ATP5PO, ATP5F1C, ATP5F1E, ATPAF2, ATP5PF, OXA1L, ATP5MJ, ATP23, TMEM242, ATP5MK, ATP5MC1, ATP5MGL, ATP5F1A, FMC1, PPIF, ATP5MF, TMEM70, ATP5F1B, ATP5F1D, ATP5PD, ATP5MC3, ATP5ME, ATP5MC2, ATP5MG, ATP5PB, and ATPAF1.

[0168] In one aspect, the mtDNA genes are MT-ND1, MT-ND2, MT-CO1, MT-CO2, MT-ATP8, MT-ATP6, MT-CO3, MT-ND3, MT-ND4L, MT-ND4, MT-ND5, MT-ND6, MT-CYB, NDUFA6, SMDT1, TIMM22, SMDT1, RDH13, BDH1, RDH13, AGK, NDUFA3, SMDT1, RDH13, RDH13, ATP23, RDH13, MRPS18B, MRPS18B, MRPS18B, TAMM41, CHCHD10, RDH13, NEU4, TIMM22, RDH13, MRPS36, MRPL45, PAM16, RDH13, MRPS18B, RDH13, SURF1, COX8C, MRPS18B, SLC25A15, DNAJC15, MRPS18B, SLC25A26, MRPS6, NDUFS1, MRPS31, SLC25A6, SLC25A24, HCCS, MRPS12, OTC, SAMM50, SLC25A17, PLSCR3, ATP5PO, MRPL39, ATP5F1C, MRPL36, ATP5F1E, SLC25A37, SLC25A30, NDUFS3, PTPMT1, NDUFV3, NDUFV2, SLC25A16, ATPSCKMT, MRPS30, LYN, MRPS26, SLC25A29, COX4I2, BCL2L1, SLC, MRPL23, SLC25A52, SLC25A47, UQCC2, CYP11A1, CDS2, TTC19, CKMT2, MRPL57, MICU2, GRPEL1, ROMO1, ATP5PF, DMAC1, COX7C, COX5A, MRPL54, AIFM1, NDUFS4, MRPS28, COX7B, OXA1L, ATAD3B, HADHA, UQCRB, CYP11A1, IMMP1L, TIMM8A, SFXN2, MTG1, DUSP21, MRPL16, SLC25A53, ADCK1, NDUFA13, MRPL13, NDUFAF5, NNT, MRPL15, HADHB, MICOS10-NBL1, ATP5MJ, SFXN4, CABS1, MTG2, COX7B2, FPGS, COX7A2L, SFXN5, ALAS2, SLC25A33, COQ4, SHMT2, SLC27A1, COA3, SLC9B2, UQCRH, UQCR10, MRPL1, MRPL19, MRPL41, MTFP1, MRPL,NDUFAF4, PDE2A, ATP23, CYP11B2, MICOS10, MRPS9, TMEM242, NDUFA4, CPOX, SLC25A1, ABCB7, MRPS11, COX7A1, GCAT, DELE1, COX11, CPT2, ATP5MK, PTPN1 、SRC、MRPL40、PRODH、TIMM21、OMA1、GRPEL2、AIFM3、MRPL46、ATP5MC1、NDUF A1、SCO2、MCUB、FECH、CYP11B1、TIMMDC1、TIMM9、MRPL22、CKMT1B、CKMT1A、TM EM177, RAB5IF, RPS3, UQCRFS1, SFXN1, PMPCA, APOO, MRPS23, TYMS, SLC25A3 1、AFG3L2、COQ7、SLC25A14、MRPS17、CYCS、ATP5MGL、SMDT1、CYP24A1、SLC25 A42, TRMT10B, SLC25A22, SLC25A51, MPC1L, NDUFB4, TIMM50, COQ9, LETM2, P ISD、CHCHD6、NDUFA8、SLC25A24、TMEM14C、NDUFB1、TIMM8B、SDHD、TMEM11、GA TM、L2HGDH、ATP5MF-PTCD1、CYC1、SQOR、COX16、MRPL18、HSD3B2、MRPL23、AT P5F1A, MTX3, CHCHD10, MRPL2, LGALS3, DUSP18, PLSCR3, TOML2, NDUFS6, MRP S10、TRAP1、SLC25A4、SLC25A35、COX8A、MPC1、MRS2、PAM16、SURF1、IMMP2L、 BOK、SLC25A12、SCO1、NDUFA2、GADD45GIP1、COX6B2、MPC2、APOOL、NDUFB6、ST MP1、NDUFB9、UQCC1、MRPL52、COX10、CHCHD1、TIMM23、UQCRC2、COX18、MRPL4 SPNS1, CRLS1, NDUFB11, WDR93, MGARP, COX6C, NDUFC1, LETM1, ENDOG, MRPL 14、MPV17L2、CRAT、NOA1、TIMM23B、MRPL12、SLC25A10、TMEM65、MTX2、DMAC2 、CLPX、AGK、MRPS18B、MRPL38、BDH1、MRPL21、NDUFA7、CHCHD3、PPIF、MRPS14、<h2 style=";text-align:left;direction:ltr">SLC25A25、ALDH18A1、COA8、DMAC2L、SPHK2、HSPA9、NDUFAF1、NDUFB8、TIMM17B、GPD2、MRPS18A、SLC25A43、SLC25A5、SLC25A20、NDUFAF3、NDUFAF2、C15or f48、PLA2G4A、SLC25A13、MRPL53、TAFAZZIN、COX6A2、TIMM13、PARL、PTCD3、 MRPS34、MRPL35、MRPL48、AMBP、NDUFB7、UCP3、NDUFAB1、TIMM44、FLVCR1、PLA 2G4B、MRPL10、NDUFB2、MRPS33、CIBAR1、ATP5MF、COX6A1、SPG7、COQ5、COQ3、 MRPL11、TIMM17A、MRPS7、SLC25A19、PTPMT1、COQ8B、SLC25A27、TIMM29、PINK 1、TMEM70、LDHD、TIMM10、MRPL47、UCP2、DMD、DNAJC30、DAP3、COQ2、MRPS18C 、CYP2E1、MICU1、MRPL32、MRPL34、MRPS12、UCP1、COQ6、PMPCB、MRPS35、MICU3 、SLC25A32、YME1L1、ETFDH、TMEM126B、TMEM126A、UQCRQ、CYP2U1、LDHB、NDU FC2-KCTD14、NDUFA5、NDUFC2、MRPL17、SDHA、MCU、CYP1A1、GUF1、MRPS2、COX8 C、SLC25A21、SLC25A2、SLC25A41、SLC25A23、MRPL30、PPOX、CLU、ABCB8、MRP S16、HSPD1、ATP5F1B、GHITM、NDUFA3、NDUFA10、UQCC3、ACAD11、MRPL43、TIMM 22、MRPL51、NDUFAF6、TOMM40、SDHB、PSEN1、HSD3B1、HIGD2A、MRPL27、SLC41 A3、OPA1、SLC25A40、SLC25A39、NDUFV1、PHB2、TMEM186、MRPL28、NME4、TIMM1 0B、BCS1L、ERAL1、DHRS13、ATP5F1D、CYP27A1、FDXR、MRPL58、ATP5PD、MRPS3 6、ATP5MC3、RCC1L、ACADVL、NDUFB5、MRPS27、MRPL42、DHODH、ACAD9、MRPL20、ABCB10, DHFR2, LETMD1, SFXN3, SLC25A48, SMIM20, COX15, HIGD1A, MRPS24, MCUR1, C12orf73, LRRK2, M ICOS13, ATP5ME, CCDC51, SLC25A28, NDUFA11, UQCRC1, MRPL55, MTHFD2L, RSAD2, SLC8B1, COQ10A, COQ8 A, NDUFS5, CNP, SDHC, NDUFS2, NDUFB10, SIRT4, NDUFA12, CHDH, SLC25A3, NDUFS7, COX7A2, SLC22A14, M TLN, NDUFB3, ECSIT, FGR, IFI6, COA1, MRPL37, ATP5MC2, PSEN2, SLC25A11, COX4I1, MRPL24, COX6B1, PH B1, MRPS22, NEU4, MRPL3, RDH13, UQCR11, PGS1, PRODH2, PET100, FOXRED1, SLC25A26, IMMT, NDUFA4L2, MTX1, MRPL9, THEM4, NDUFA9, CPS1, ATP5MG, TAMM41, EFHD1, MYOC, ATAD3A, MRPL49, EXOG, COQ10B, SLC25A45, SLC25A38, SLC25A36, MPV17, NDUFS8, MRPS15, COX5B, MRPS25, DNAJC19, MAIP1, MRPL33, DNAJC11, MRPS5, ATP5PB, MRPL44, SLC25A34, UQCRHL, COX20, MRPS21 and AURKAIP1.

[0169] In embodiments, the mtDNA gene is selected from the group consisting of MT-CYB, SMDT1, SMDT1, AGK, SMDT1, CHCHD10, SAMM50, OXA1L, SFXN2, MICOS10-NBL1, SFXN4, SFXN5, COA3, MICOS10, COX11, SCO2, MCUB, TMEM177, SFXN1, APOO, AFG3L2, SMDT1, MPC1L, PISD, CHCHD6, TMEM11, L2HGDH, COX16, MTX3, CHCHD10, MPC1, SCO1, M are essential components of the inner mitochondrial membrane selected from PC2, APOOL, TIMM23, COX18, TIMM23B, MTX2, AGK, CHCHD3, HSPA9, TIMM17B, SPG7, TIMM17A, SLC25A19, COQ2, ETFDH, MCU, GHITM, UQCC3, SFXN3, MCUR1, MICOS13, CCDC51, SLC25A3, SLC22A14, MTLN, COA1, PET100, IMMT, MTX1 and DNAJC11.

[0170] In the embodiment, mtDNA gene is、SMDT1、SMDT1、BDH1、SSBP1、SMDT1、MRPS18B、MRPS18B、MRPS18B、MRPS36、MRPL45、PAM16、DNAJA3、MRPS18B、MRM1、HSD17B8、HSD17B8、 HSD17B8、MRPS18B、MIPEP、DNAJC15、MRPS18B、HSD17B8、ACSS2、HSD17B8、MRPS6、NDUFS1、MCAT、MRPS31、SARS2、MRPS12、OTC、PCCA、IDH3B、ACSS1、MRPL3 9、AK3、NUBPL、ATP5F1C、MRPL36、FASTKD3、TOP3A、PABPC5、PIN4、ATP5F1E、SUCLA2、NDUFS3、ISCA2、PCK2、CARS2、MRPS30、RAD51、NAXD、MRPS26、ME2、LDH AL6B、PDSS1、BCL2L1、GLUD2、MRPL23、OXCT1、TFAM、GLRX5、UQCC2、MMUT、CYP11A1、DHTKD1、TMLHE、TEFM、GLUD2、MRPL57、GRPEL1、NFS1、SDHAF4、HOGA1、PD P1、MRPL54、GLYAT、MRPS28、SOD1、OXA1L、DMGDH、ISCA1、MTRES1、RIDA、TXNRD2、HADHA、DNA2、CYP11A1、OAT、MTG1、DUSP21、METTL15、MRPL16、TOP3A、ALD H4A1、DLST、ETFBKMT、MRPL13、MRPL15、HADHB、DHRS2、MTG2、MPST、PRDX3、FPGS、TTC5、ACADSB、CA5B、ALAS2、TRUB2、NUDT9、VDAC1、SHMT2、GLS2、PNPT1、P DE12、HYKK、MRPL1、LACTB2、MRPL19、MRPL41、MRPL50、TOP1MT、GLDC、PDE2A、MRPS9、PDSS2、PPA2、FDX1、PCK2、ETNPPL、ACSS3、AUH、MRPS11、ECI1、FOXO3、 PARS2、ACO2、PTPN1、MRPL40、PRODH、GRPEL2、SUCLG1、MALSU1、ABHD10、MRPL46、BCKDHB、FTMT、SC02、NR3C1、MTRF1L、AK4、FECH、ALDH1L2、MRPL22、TBRG4、<h2 style=";text-align:left;direction:ltr">ALDH7A1、PDHA2、METTL4、FXN、ERBB4、RPS3、IDH3A、METTL17、ACOT9、PMPCA、 MRPS23、PRDX5、TYMS、GPT2、SDHAF3、MTERF1、POLDIP2、ARG2、ARL2、MRPS17、 FARS2、ALDH2、SMDT1、CBR4、FASTKD5、TXN2、ALDH5A1、ACOT13、HSD17B10、DG LUCY、ACADL、MRRF、PPM1K、PDP2、NT5M、BCO2、SOD2、MTERF2、MRPL18、MRPL23、 ATP5F1A、POLG、MRPL2、AADAT、FASTKD2、ACAA2、MRPS10、SARS2、TFB1M、BCAT 2、TRAP1、LYRM7、PAM16、MTERF4、TST、DNAJA3、GADD45GIP1、HARS2、IDH3G、H IBADH、LYRM4、ELAC2、BCKDK、MRPL52、CASQ1、GSR、GSTZ1、CHCHD1、SUPV3L1、 MRPL4、TUFM、PITRM1、BCKDHA、SSBP1、MRPL14、ACAT1、REXO2、MPV17L2、TFB2M 、AKR1B15、PDK3、MRPL12、HSPA1L、MTHFS、D2HGDH、CLPX、NMNAT3、LIAS、MRPS 18B、MRPL38、BDH1、GCSH、MRPL21、NDUFA7、PPIF、MRPS14、PDPR、HSPA9、OGDH L、NUDT2、NDUFAF1、NDUFB8、TWNK、MTHFD1L、MRPS18A、KARS1、GLS、SLC25A5、 ECHS1、PDHA1、DDX28、NADK2、TSFM、PARG、NDUFAF7、RNASEL、MLYCD、SIRT5、PT CD1、MRPL53、ACAD10、PTCD3、MRPS34、MRPL35、MRPL48、HAGH、GARS1、EARS2、 NDUFAB1、TIMM44、SARDH、GLUD1、MRPL10、MRPS33、POLG2、COQ5、PRIMPOL、PD K1、DIMT1、COQ3、MRPL11、NAT8L、MRPS7、ATXN3、LIPT2、ALDH1B1、COASY、MRP L47、ATG4D、CCAR2、IDH2、DAP3、MRPS18C、ARL2BP、HSD17B8、MRPL32、MRPL34、<h2 style=";text-align:left;direction:ltr">MRPS12、RPUSD4、SUCLG2、ACSF3、NAGS、MMAA、MCCC2、PMPCB、ALDH6A1、MRPS35、ETFDH、SIRT3、PRORP、ISCU、IVD、AMT、PYCR1、EXD2、ACOT2、CDKN2A、MRPL1 7、DECR1、VDAC2、MRM3、HADH、GUF1、MRPS2、MMAB、CLPP、ALKBH7、LIPT1、MRPL30、ALAS1、C2orf69、NUDT13、HSPE1、MRPS16、HSPD1、QARS1、GFM2、SDHAF2、TK 2、ATP5F1B、ADHFE1、NDUFA10、AASS、MYG1、DLAT、MRPL43、MRM2、MRPL51、TP5 3AIP1、GSTK1、FASTKD1、TDRD7、ACSM5、ACSM2A、GCDH、NUDT1、CCNB1、GOT2、P DK2、HMGCS2、ARHGAP11B、MRPL27、TRMT5、LRRC59、PHYKPL、ACSM2B、ACSM3、ETFA、ACSM1、ABAT、MTHFD2、ACP6、MRPL28、NME4、ERAL1、ATP5F1D、CYP27A1、FD XR, WARS2, MRPL58, GLRX2, GPX1, MRPS36, PDHB, GRSF1, ACADVL, HTD2, ACSM6 、MRPS27、MRPL42、PYROXD2、MRPL20、DHFR2、NSUN3、AGXT2、ETFB、RARS2、PUS 1、STYXL1、MDH2、MRPS24、CDK1、LRRK2、FASTK、LONP1、PPTC7、MRPL55、MTHFD2L、IBA57、TERT、SUOX、PDHX、DLD、OGG1、OGDH、MECR、DARS2、CS、DHX30、YARS2 、NDUFS2、LARS2、SIRT4、NDUFS7、MPG、DGUOK、ACOT11、PDK4、POLRMT、SDHAF1 、ABCE1、LRPPRC、MRPL37、ETHE1、NAXE、MRPL24、ACADS、MRPS22、MRPL3、DBT、M CCC1、ACAD8、FAHD1、ACSM4、BLOC1S1、CA5A、MRM1、TP53、TRMT10C、FDX2、MRP L9、THEM5、THEM4、NDUFA9、CPS1、NARS2、ACSF2、RPUSD3、FH、ATAD3A、MRPL49、The matrix protein is selected from NSUN4, PC, ADPRS, NDUFS8, MRPS15, C1QBP, SHC1, ME3, FLAD1, GFM1, TRNT1, BTD, MRPS25, DNAJC19, CHPF, MAIP1, PYCR2, MCEE, TARS2, MARS2, PCCB, HMGCL, HIBCH, IARS2, MRPL33, TRMT61B, MRPS5, ATP5PB, MRPL44, OXCT2, TRIT1, MRPS21, AURKAIP1, ACADM, and PARK7. [Example]

[0171] The following non-limiting examples are provided for illustrative purposes only, to facilitate a more complete understanding of the currently contemplated representative embodiments.These examples are intended to be merely a subset of all possible situations in which the components of the formulation can be combined.Therefore, these examples should not be interpreted as limiting any of the embodiments described herein, including those related to the type and amount of the components of the formulation and / or the method and use thereof. [Example]

[0172] Re-engineering 13 mtDNA-encoded proteins for nuclear expression Figure 1B shows the gene constructs used in this study. Each construct utilized the same promoter (pCMV) with different elements. Expression levels of oND1 were compared as described below.

[0173] First, the mRNA expression level was determined for each construct. Figure 1C is a bar graph showing the expression level of the oxidative phosphorylation (OxPhos) protein oND1 mRNA in the ND1 null cell line (relative to GAPDH) for each construct. The highest expression level was found in the PUM construct. Similarly, Figure 1D is a bar graph showing the expression level of oND1 mRNA in the ND1 null cell line (relative to COX10) for each construct. The PUM construct also had the highest expression level.

[0174] Next, expression levels were compared using different promoters (i.e., pCMV and pCAG). Figure 2A shows the pCMV and pCAG constructs. Figure 2B is a bar graph showing the expression level of oND1 mRNA in ND1-null cell lines (relative to GAPDH) for each construct. Figure 2C is a bar graph showing the expression level of oND1 mRNA in ND1-null cell lines (relative to COX10) for each construct. In both studies, the pCMV promoter showed higher levels of oND1 expression.

[0175] In the next study, expression levels were compared using different N-terminal export signals (i.e., hexapeptide and OXA1L). Figure 3A shows the constructs compared.

[0176] Figure 3B is a bar graph showing the expression level of oND1 mRNA in ND1-null cell lines (relative to GAPDH) for each construct. Figure 3C is a bar graph showing the expression level of oND1 mRNA in ND1-null cell lines (relative to COX10) for each construct. The C2 (pCAG, OXA1L 5'UTR, OXA1L MTS, Hex) construct showed the highest expression.

[0177] The MPCP presequence may constitute a signal that can be recognized by the import and assembly machinery as being destined for the inner mitochondrial membrane (MIM). Figure 4A shows constructs carrying the MPCP sequence. Figure 4B is a bar graph showing the expression level of oND1 mRNA in ND1-null cell lines (relative to GAPDH) for each construct. Figure 4C is a bar graph showing the expression level of oND1 mRNA in ND1-null cell lines (relative to COX10) for each construct. The D1 and CAG constructs each showed the highest expression levels.

[0178] ABCBA is a mitochondrial ABC transporter of the inner membrane and has an N-terminal mitochondrial targeting sequence (MTS). Figure 5A shows constructs containing the ABCBA sequence. Figure 5B is a bar graph showing the expression level of oND1 mRNA in ND1-null cell lines (relative to GAPDH) for each construct. Figure 5C is a bar graph showing the expression level of oND1 mRNA in ND1-null cell lines (relative to COX10) for each construct. The E5 construct showed the highest expression level.

[0179] Figure 6A is a diagram of a triple protein tagged construct according to an embodiment of the present invention. Figure 6B is a bar graph showing the expression level of oND1 mRNA in an ND1 null cell line (relative to GAPDH) for each construct. Figure 6C is a bar graph showing the expression level of oND1 mRNA in an ND1 null cell line (relative to COX10) for each construct. The 3xF construct showed the highest expression level.

[0180] Figure 7A is a graphical representation of mesohydrophobicity versus local hydrophobicity of transmembrane domain 1 of ATP6. Similarly, Figure 7B is a graphical representation of mesohydrophobicity versus local hydrophobicity of transmembrane domain 1 of ATP6.

[0181] Figure 8A is a bar graph showing the expression level of oATP6 mRNA in ATP6 mutant cell lines for different COX10 constructs. Figure 8B is a bar graph showing the expression level of oATP6 mRNA in ATP6 mutant cell lines for different COX10 constructs relative to GAPDH. The TM2 mutant cell line showed the highest expression level.

[0182] Figure 9 is an image of an SDS PAGE of mitochondrial fractions showing a comparison of anti-FLAG and anti-aconitase expression. The TM2 mutant exhibited the highest Flag:aconitase density ratio (i.e., 17.88).

[0183] Figure 10A shows cleaved and uncleaved COX2 proteins. The predicted molecular weights and marker detection of cleaved and uncleaved COX2 proteins correspond to the molecular weights shown in kDa on an SDS PAGE gel.

[0184] Figure 10B is an SDS PAGE showing stable expression of COX2 site-directed mutagenesis constructs. This demonstrates the effect of reduced transmembrane hydrophobicity on COX2 import. Purified mitochondrial fractions were separated on a 4-12% SDS PAGE gel and probed for anti-FLAG and anti-TOMM20 (mitochondrial outer membrane protein markers). Lanes represent (1) the 293 mock-negative control, (2) the original COX2 construct, (3) the I31R mutagenesis construct, (4) the Y40R mutagenesis construct, (5) the I31R+Y40R mutagenesis construct, (6) the L74P mutagenesis construct, and (7) the V75M mutagenesis construct, respectively.

[0185] Figure 11A shows mRNA levels for different versions of the codon-optimized allotopic COX2 gene compared to GAPDH. V1 showed the highest expression of mRNA, while V4 had the lowest.

[0186] Similarly, Figure 11B shows the SDS PAGE profiles for different versions of codon-optimized allotopic COX2 in a COX2 null cell line upon stable selection. V1 and V4 showed the highest protein expression levels.

[0187] FIG. 12 shows the plasmid sequence of a COX2 variant according to an embodiment. [Example]

[0188] Treatment of Leigh syndrome with allotopic expression Human mitochondrial DNA (mtDNA) is a 16,569-base-pair double-stranded circular DNA that encodes 13 key proteins of the electron transport chain. Mitochondria occupy a central position in the overall metabolism of eukaryotic cells. Oxidative phosphorylation (OXPHOS), the Krebs cycle, the urea cycle, heme biosynthesis, and fatty acid oxidation occur within the organelle. Recently, mitochondria have been recognized as key early mediators of the apoptotic cascade, establishing another major role for mitochondria in determining cellular lifespan. Mitochondria are also a major source of reactive oxygen species (ROS), which cause oxidative stress and thus induce cell death.

[0189] Defects in mitochondrial function are implicated in over 120 diseases. Mitochondrial pathology is considered one of the most common genetically determined diseases and is a major health concern due to the lack of effective treatments or therapies. Mitochondria are assembled from proteins encoded by genes distributed between the mitochondrial and nuclear genomes. These genes include those encoding the structural proteins of respiratory chain complexes I-V, their associated substrates and products, proteins required for mitochondrial biogenesis, the machinery for importing precursors synthesized in the cytoplasm, and proteins required for mitochondrial assembly and turnover.

[0190] Approximately half of human mitochondrial disorders are caused by pathogenic point mutations in mtDNA, one-third of which are located in coding genes. Mitochondrial DNA (mtDNA) disorders include Leigh syndrome, leukodystrophy with complex II deficiency, cardiomyopathy and encephalopathy (complex I deficiency), optic atrophy and ataxia (complex II deficiency), hypokalemia and lactic acidosis, hepatopathy and ketoacidosis, hypertrophic cardiomyopathy, liver failure, renal tubulopathy (with complex III deficiency) and encephalopathy (with complex V deficiency), autosomal progressive external ophthalmoplegia, mitochondrial neurogastrointestinal encephalomyopathy, Alpers-Huttenlocher syndrome, ataxic neuropathy syndrome, infantile myopathy / spinal muscular atrophy, and hypotonia.

[0191] In this example, the pediatric patient had been diagnosed with Leigh syndrome (early onset), a genetic condition that affects the central nervous system. The infant appeared healthy at birth but gradually developed symptoms as cells in the nervous system began to break down or degenerate. Symptoms included feeding problems, seizures, and continuous crying.

[0192] A pediatrician performs a genetic analysis to identify the gene responsible for the patient's disease: the MT-ATP6 gene, which produces ATP. This is the most common mtDNA alteration in Leigh syndrome, preventing the MT-ATP6 gene from producing ATP. In this example, allotopic expression is used to introduce a wild-type (i.e., healthy) copy of the mutated gene in the mitochondrial genome into the nucleus, delivering a normal copy of the gene product from the cytosol into the mitochondria. The gene is introduced into the patient by allotopic expression using the methods described herein.

[0193] The expression vector is produced in the form of a recombinant vector. The vector contains a nucleic acid sequence encoding a mitochondrial targeting signal (i.e., MTS sequence), a sequence encoding the protein to be delivered, and a 3' nucleic acid sequence. Additional codon sequences are introduced into the vector to (a) reduce the hydrophobicity of the gene product, (b) reduce the localized charge of the gene product, and (c) cause ribosome stalling. The vector is then introduced into the patient. The vector can be, for example, a plasmid, a virus, such as an integrating viral vector (e.g., a retrovirus, an adeno-associated virus (AAV) or a lentivirus), or a non-integrating viral vector, such as an adenovirus, an alphavirus, or a herpes simplex virus (HSV).

[0194] Within 48 hours, the patient's condition gradually improves. After about one month of treatment, the patient has no signs or symptoms of the disease. Doctors perform periodic testing of the expression and activity of the MT-ATP6 gene. [Example]

[0195] Treatment of apical hypertrophic cardiomyopathy and neuropathy by allotopic expression In this example, a 17-year-old male patient presented to his healthcare provider with signs and symptoms of apical hypertrophic cardiomyopathy and neuropathy due to mitochondrial dysfunction. The provider measured the performance of the mitochondrial energy-generating system (MEGS) in muscle, along with enzyme analysis in muscle and fibroblasts. The relevant portion of the patient's mitochondrial DNA was then analyzed by sequencing. A homoplasmic nonsense mutation, m.8529G→A (p.Trp55X), was found in the mitochondrial ATP8 gene in the patient's fibroblasts and muscle tissue. Decreased complex V activity was measured in the patient's fibroblasts and muscle tissue and confirmed in a cybrid clone containing the patient's mitochondrial DNA.

[0196] Allotopic expression is used to introduce a wild-type (i.e., healthy) copy of a gene mutated in the mitochondrial genome into the nucleus and import a normal copy of the gene product from the cytosol into the mitochondria. The gene is introduced into the patient by allotopic expression using the methods described herein.

[0197] The expression vector is produced in the form of a recombinant vector. The vector contains a nucleic acid sequence encoding a mitochondrial targeting signal (i.e., MTS sequence), a sequence encoding the protein to be delivered, and a 3' nucleic acid sequence. Additional codon sequences are introduced into the vector to (a) reduce the hydrophobicity of the gene product, (b) reduce the localized charge of the gene product, and (c) cause ribosome stalling. The vector is then introduced into the patient.

[0198] The vector can be, for example, a plasmid, a virus, such as an integrating viral vector (e.g., a retrovirus, adeno-associated virus (AAV), or lentivirus), or a non-integrating viral vector, such as an adenovirus, alphavirus, or herpes simplex virus (HSV). In this example, the vector is introduced as a plasmid by electroporation.

[0199] Within 48 hours, the patient's condition gradually improves. After about one month of treatment, the patient has no signs or symptoms of the disease. Doctors perform periodic testing of the expression and activity of the MT-ATP6 gene. [Example]

[0200] Treatment of Leber's Hereditary Optic Neuropathy (LHON) Leber's hereditary optic neuropathy (LHON), or sudden vision loss, is an inherited form of vision loss. It begins with painless clouding or blurring in one or both eyes and then often worsens with loss of sharpness and loss of color vision. LHON affects central vision, which is necessary for fine tasks such as reading, driving, and facial recognition. LHON can result in legal blindness.

[0201] LHON is a mitochondrial disease caused by mutations in mitochondrial DNA. Because it is a mitochondrial disease, it can only be inherited through the mother. Individuals who have lost central vision are referred to as "affected." Individuals who carry one of the mitochondrial mutations but do not experience vision loss are referred to as "carriers." LHON is caused by mutations in the following genes: MT-NDI1, MT-ND4, MT-ND4L, and MT-ND6. Vision loss occurs because cells in the optic nerve die.

[0202] Most LHON-causing mutations affect the mitochondrial DNA (mtDNA) gene encoding the NADH dehydrogenase (ND) subunit of respiratory chain complex I, leading to subacute and devastating degeneration of retinal ganglion cells (RGCs) with the end result of optic nerve atrophy. The majority of LHON cases are caused by one of three mtDNA missense point mutations (positions m.3460G>A / MT-ND1, m.11778G>A / MT-ND4, and m.14484T>C / MT-ND6).

[0203] In this example, a 19-year-old male patient visits his healthcare provider with complaints of sudden vision loss, particularly central vision loss. After the diagnosis is confirmed, physicians perform genetic analysis to identify a missense point mutation (position m.3460G>A / MT-ND1) in the gene responsible for the disease.

[0204] In this example, allotopic expression is used to introduce a wild-type (i.e., healthy) copy of a gene mutated in the mitochondrial genome into the nucleus and import a normal copy of the gene product from the cytosol into the mitochondria. The gene is introduced into the patient via a modified adeno-associated virus gene therapy method, which complements the defective ND4 gene by allotopic expression of the wild-type ND4 subunit from the nucleus and subsequent mitochondrial import of the protein product.

[0205] The vector is introduced by intravitreal injection (IVT). Within six months of treatment, the patient's vision improved (i.e., to about 80% of normal). Doctors perform regular tests of the patient's vision.

[0206] How to use Embodiments include methods for treating pathologies or defects in the mitochondrial respiratory chain or oxidative phosphorylation system. The methods may include allotopic expression of a wild-type version of a mutated gene (e.g., a gene with a deleterious mutation). Embodiments also include methods for slowing or retarding the aging process in humans by allotopic expression of one or more genes in human cells. Embodiments also include methods for treating age-related diseases by allotopic expression of one or more genes in human cells. In embodiments, the genes are mitochondrial genes.

[0207] Also included is the use of the gene to manufacture a medicament for treating a disease affecting the mitochondrial respiratory chain or the oxidative phosphorylation system.

[0208] In one embodiment, the methods of allotopic expression disclosed herein express, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the wild-type gene product introduced into the cell nucleus (i.e., to replace a mutated gene from the mitochondrial genome). In other aspects of this embodiment, the methods disclosed herein can be used to achieve a cellular saturation rate of, for example, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, and about 50% to about 100%. %, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70% expression of the gene product.

[0209] In one embodiment, a therapeutic agent disclosed herein can reduce signs / symptoms of a mitochondrial disease or aging-related disease or disorder by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, as compared to a patient not receiving the same treatment. In other aspects of this embodiment, the therapeutic agent reduces the number of signs / symptoms of a mitochondrial disease or aging-related disease or disorder in an individual, for example, by about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 9 ...50% to about 100%, about 60% to about 100%, about The reduction can be about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

[0210] In one embodiment, a therapeutic agent disclosed herein can reduce signs / symptoms in an individual suffering from a mitochondrial disease or aging-related disease or disorder, e.g., by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, compared to a patient not receiving the same treatment. In other aspects of this embodiment, the therapeutic agent reduces signs / symptoms in an individual suffering from a mitochondrial disease or aging-related disease or disorder, for example, by about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about The decrease can be from 20% to about 90%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 10% to about 80%, from about 20% to about 80%, from about 30% to about 80%, from about 40% to about 80%, from about 50% to about 80%, or from about 60% to about 80%, from about 10% to about 70%, from about 20% to about 70%, from about 30% to about 70%, from about 40% to about 70%, or from about 50% to about 70%.

[0211] In one embodiment, a therapeutic agent disclosed herein can reduce signs / symptoms of aging in an individual by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% compared to a patient not receiving the same treatment. In other aspects of this embodiment, the therapeutic agent reduces signs / symptoms of aging in a subject, for example, by about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 9 ... The reduction can be about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

[0212] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that such variations will be employed by those skilled in the art as appropriate, and the inventors intend that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.Furthermore, this invention includes any combination of all possible variations of the above-described embodiments unless otherwise indicated herein or clearly contradicted by context.

[0213] The grouping of alternative embodiments, elements, or steps of the present invention should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When such inclusion or deletion occurs, the specification is deemed to include the group as modified and thus fulfill all Markush group descriptions used in the appended claims.

[0214] Unless otherwise indicated, all numbers expressing features, items, quantities, parameters, characteristics, periods, etc. used in the specification and claims are understood to be modified in all instances by the term "about." As used herein, the term "about" means that a given feature, item, quantity, parameter, characteristic, or period encompasses a range of 10% above and below the value of the stated feature, item, quantity, parameter, characteristic, or period. Thus, unless indicated to the contrary, the numerical parameters set forth in this specification and the appended claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical indication should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values ​​setting forth the broad scope of the invention are approximations, the numerical ranges and values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical range or value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of numerical ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise stated herein, each individual value of the numerical range is incorporated herein as if it were individually recited herein.

[0215] As used in the context of describing the present invention (particularly in the context of the claims below), the terms "a," "an," "the," and similar referents should be construed to encompass both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better illustrate the invention and does not limit the scope of the invention as claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0216] Certain embodiments disclosed herein may be further limited in the claims using the phrases "consisting of" or "consisting essentially of." When used in a claim, the transitional term "consisting of" excludes any element, step, or ingredient not specified in the claim, whether added at the time of filing or by amendment. The transitional term "consisting essentially of" limits the claim to certain materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0217] All patents, patent publications, and other publications referenced and identified herein are individually and expressly incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methodologies described in such publications that may be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0218] Finally, while aspects of the present specification have been emphasized by reference to specific embodiments, it should be understood that those skilled in the art will readily appreciate that these disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Accordingly, it should be understood that the disclosed subject matter is in no way limited to the particular methodology, protocols, and / or reagents, etc., described herein. Accordingly, various modifications or variations of the disclosed subject matter or alternative configurations can be made in accordance with the teachings herein without departing from the spirit of the specification. Finally, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Therefore, the present invention is not limited to that precisely as shown and described.

Claims

1. 1. A method for allotopic expression of a gene in a cell, said method comprising: a) identifying a mitochondrial defect in said cell; b) identifying a mitochondrial gene associated with said mitochondrial defect; c) expressing in the cell a modified or wild-type version of the mitochondrial gene; and d) importing the gene product of said modified or wild-type version of said mitochondrial gene into the mitochondria of said cell.

2. The method of claim 1 , wherein the cell is a mammalian cell.

3. The method of claim 1 , wherein the cell is a human cell.

4. 2. The method of claim 1, wherein the gene product is an oxidative phosphorylation (OXPHOS) complex subunit.

5. 2. The method of claim 1, wherein the gene product is a subunit of NADH ubiquinone oxidoreductase (complex I), succinate dehydrogenase (complex II), ubiquinol-cytochrome c oxidoreductase (complex III or cytochrome bc1 complex), cytochrome c oxidase (complex IV), or ATP synthase (complex V).

6. 2. The method of claim 1, wherein one or more mutations in the mitochondrial genes cause the mitochondrial defect.

7. 10. The method of claim 1, wherein the mitochondrial defect causes one or more of seizures, ataxia, cortical blindness, dystonia, exercise intolerance, ophthalmoplegia, optic atrophy, cataracts, diabetes mellitus, short stature, cardiomyopathy, sensorineural hearing loss, renal failure, blindness, hearing loss, movement disorder (ataxia), dementia, cardiovascular disease, muscle weakness, renal insufficiency, and endocrine disorders.

8. 2. The method of claim 1, wherein the step of expressing a modified or wild-type version of the mitochondrial gene further comprises incorporating sequences into the modified or wild-type gene for codon optimization.

9. 2. The method of claim 1, wherein the step of expressing an engineered or wild-type version of the mitochondrial gene further comprises incorporating into the engineered or wild-type gene a sequence that causes translation slowdown or ribosome stalling.

10. 2. The method of claim 1, wherein the step of expressing an engineered or wild-type version of the mitochondrial gene further comprises incorporating into the engineered or wild-type gene a sequence that causes translation slowdown or ribosome stalling.

11. 2. The method of claim 1, wherein the step of expressing a modified or wild-type version of the mitochondrial gene further comprises the step of incorporating a PUMILIO binding site into the modified or wild-type gene.

12. 2. The method of claim 1, wherein the step of expressing a modified or wild-type version of the mitochondrial gene further comprises incorporating into the modified or wild-type gene a sequence that reduces the hydrophobicity of a transmembrane domain within the gene product.

13. wherein the step of expressing the modified or wild-type version of the mitochondrial gene further comprises incorporating one or more codon sequences into the modified or wild-type version of the mitochondrial gene; 2. The method of claim 1, wherein the one or more codon sequences reduce the hydrophobicity of the gene product, reduce the local charge of the gene product, or cause ribosome stalling.

14. wherein the step of expressing the modified or wild-type version of the mitochondrial gene further comprises incorporating one or more codon sequences into the modified or wild-type version of the mitochondrial gene; 2. The method of claim 1, wherein the one or more codon sequences encode an N-terminal matrix export signal for the gene product.

15. The method of claim 14, wherein the N-terminal matrix export signal comprises a 6-residue sequence from OXA1L.

16. wherein the step of expressing the modified or wild-type version of the mitochondrial gene further comprises incorporating one or more codon sequences into the modified or wild-type version of the mitochondrial gene; 2. The method of claim 1, wherein the one or more codon sequences restore the natural orientation of the transmembrane helices of the gene product to the inner mitochondrial membrane.

17. wherein the step of expressing the modified or wild-type version of the mitochondrial gene further comprises incorporating one or more codon sequences into the modified or wild-type version of the mitochondrial gene; 2. The method of claim 1, wherein the one or more codon sequences reduce the localized charge of the gene product.

18. wherein the step of expressing the modified or wild-type version of the mitochondrial gene further comprises adding a promoter sequence to the modified or wild-type version of the mitochondrial gene; 2. The method of claim 1, wherein the promoter sequence is selected from pCAG, PGK, PGC1a, ER1a, myosin, and CK.

19. wherein the step of expressing the modified or wild-type version of the mitochondrial gene further comprises incorporating one or more codon sequences into the modified or wild-type version of the mitochondrial gene; 2. The method of claim 1, wherein the one or more codon sequences encode a mitochondrial targeting sequence to the amino-terminal portion of the gene product.

20. the modified or wild-type version of the mitochondrial gene a) expressed from the cell nucleus via genomic integration, or b) expressed as a non-integrated vector, cDNA or mRNA; The method of claim 1.

21. 2. The method of claim 1, wherein the modified or wild-type version of the mitochondrial gene is a nuclear-encoded mitochondrial protein of respiratory complex I, and the modified or wild-type version of the mitochondrial gene is paired with a mitochondrial targeting sequence (MTS) selected from SEQ ID NOs: 1-29.

22. 2. The method of claim 1, wherein the modified or wild-type version of the mitochondrial gene is a nuclear-encoded mitochondrial protein of respiratory complex V, and wherein the modified or wild-type version of the mitochondrial gene is paired with a mitochondrial targeting sequence (MTS) selected from SEQ ID NOs: 30-48.

23. 2. The method of claim 1, wherein the modified or wild-type version of the mitochondrial gene is a nuclear-encoded mitochondrial protein of respiratory complex IV, and the modified or wild-type version of the mitochondrial gene is paired with a mitochondrial targeting sequence (MTS) selected from SEQ ID NOs: 49-56.

24. 2. The method of claim 1, wherein the modified or wild-type version of the mitochondrial gene is a nuclear-encoded mitochondrial protein of respiratory complex III, and the modified or wild-type version of the mitochondrial gene is paired with a mitochondrial targeting sequence (MTS) selected from SEQ ID NOs: 57-63.

25. 2. The method of claim 1, wherein the modified or wild-type version of the mitochondrial gene is a nuclear-encoded inner membrane protein that contains a transmembrane domain, and the modified or wild-type version of the mitochondrial gene is paired with a mitochondrial targeting sequence (MTS) selected from SEQ ID NOs: 64-118.

26. 2. The method of claim 1, wherein the modified or wild-type version of the mitochondrial gene is a nuclear-encoded inner membrane protein lacking a transmembrane (TM) domain, and the modified or wild-type version of the mitochondrial gene is paired with a mitochondrial targeting sequence (MTS) selected from SEQ ID NOs: 119-280.

27. 1. A method of treating a mitochondrial defect in a subject, said method comprising: a) identifying said mitochondrial defect in a cell of a subject; b) identifying a mitochondrial gene associated with said mitochondrial defect; c) expressing an altered or wild-type version of a mitochondrial gene in the nucleus of a cell of said subject; and d) importing the gene product of said modified or wild-type version of said mitochondrial gene into mitochondria in cells of said subject.

28. 28. The method of claim 27, wherein the subject is a human.

29. 28. The method of claim 27, wherein the gene product is a subunit of the oxidative phosphorylation (OXPHOS) complex.

30. 28. The method of claim 27, wherein the gene product is a subunit of NADH ubiquinone oxidoreductase (complex I), succinate dehydrogenase (complex II), ubiquinol-cytochrome c oxidoreductase (complex III or cytochrome bc1 complex), cytochrome c oxidase (complex IV), or ATP synthase (complex V).

31. 28. The method of claim 27, wherein mitochondrial dysfunction causes one or more of seizures, ataxia, cortical blindness, dystonia, exercise intolerance, ophthalmoplegia, optic atrophy, cataracts, diabetes mellitus, short stature, cardiomyopathy, sensorineural hearing loss, renal failure, blindness, hearing loss, movement disorder (ataxia), dementia, cardiovascular disease, muscle weakness, renal insufficiency, and endocrine disorders in the subject.

32. 28. The method of claim 27, wherein the step of expressing a modified or wild-type version of the mitochondrial gene further comprises incorporating sequences into the modified or wild-type gene for codon optimization.

33. 28. The method of claim 27, wherein the step of expressing an engineered or wild-type version of the mitochondrial gene further comprises incorporating into the engineered or wild-type gene a sequence that causes translation slowdown or ribosome stalling.

34. 28. The method of claim 27, wherein the step of expressing an engineered or wild-type version of the mitochondrial gene further comprises the step of incorporating a PUMILIO binding site into the engineered or wild-type version of the mitochondrial gene.

35. 28. The method of claim 27, wherein the step of expressing a modified or wild-type version of the mitochondrial gene comprises incorporating into the modified or wild-type gene a sequence that reduces the hydrophobicity of the gene product.

36. wherein the step of expressing the modified or wild-type version of the mitochondrial gene comprises incorporating one or more codon sequences into the wild-type version of the mitochondrial gene; 28. The method of claim 27, wherein the one or more codon sequences reduce the hydrophobicity of the gene product, reduce the local charge of the gene product, or cause ribosome stalling.

37. wherein the step of expressing the modified or wild-type version of the mitochondrial gene further comprises incorporating one or more codon sequences into the modified or wild-type version of the mitochondrial gene; 28. The method of claim 27, wherein the one or more codon sequences encode an N-terminal matrix export signal for the gene product.

38. 38. The method of claim 37, wherein the N-terminal matrix export signal is a six-residue sequence from OXA1L.

39. wherein the step of expressing the modified or wild-type version of the mitochondrial gene further comprises incorporating one or more codon sequences into the wild-type version of the mitochondrial gene; 28. The method of claim 27, wherein the one or more codon sequences restore the natural orientation of the transmembrane helices of the gene product to the inner mitochondrial membrane.

40. wherein the step of expressing the modified or wild-type version of the mitochondrial gene further comprises incorporating one or more codon sequences into the wild-type version of the mitochondrial gene; 28. The method of claim 27, wherein the one or more codon sequences reduce or increase localized charge on the gene product.

41. wherein the step of expressing the modified or wild-type version of the mitochondrial gene further comprises adding a promoter sequence to the modified or wild-type version of the mitochondrial gene; 28. The method of claim 27, wherein the promoter sequence is selected from pCAG, PGK, PGC1a, ER1a, myosin, and CK.

42. wherein the step of expressing the modified or wild-type version of the mitochondrial gene further comprises incorporating one or more codon sequences into the modified or wild-type version of the mitochondrial gene; 28. The method of claim 27, wherein the one or more codon sequences encode a mitochondrial targeting sequence to the amino-terminal portion of the gene product.

43. 28. The method of claim 27, wherein the mitochondrial defect is manifested in an aging-related disease or disorder.

44. 44. The method of claim 43, wherein the aging-related disease or disorder is one or more of atherosclerosis, osteoarthritis, osteoporosis, hypertension, arthritis, cataracts, cancer, Alzheimer's disease, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis, hair greying, sarcopenia, adiposity, neurogenesis, fibrosis, and glaucoma.

45. 28. The method of claim 27, wherein the modified or wild-type version of the mitochondrial gene is a nuclear-encoded mitochondrial protein of respiratory complex I, and wherein the modified or wild-type version of the mitochondrial gene is paired with a mitochondrial targeting sequence (MTS) selected from SEQ ID NOs: 1-29.

46. 28. The method of claim 27, wherein the modified or wild-type version of the mitochondrial gene is a nuclear-encoded mitochondrial protein of respiratory complex V, and wherein the modified or wild-type version of the mitochondrial gene is paired with a mitochondrial targeting sequence (MTS) selected from SEQ ID NOs: 30-48.

47. 28. The method of claim 27, wherein the modified or wild-type version of the mitochondrial gene is a nuclear-encoded mitochondrial protein of respiratory complex IV, and wherein the modified or wild-type version of the mitochondrial gene is paired with a mitochondrial targeting sequence (MTS) selected from SEQ ID NOs: 49-56.

48. 28. The method of claim 27, wherein the modified or wild-type version of the mitochondrial gene is a nuclear-encoded mitochondrial protein of respiratory complex III, and wherein the modified or wild-type version of the mitochondrial gene is paired with a mitochondrial targeting sequence (MTS) selected from SEQ ID NOs: 57-63.

49. 28. The method of claim 27, wherein the modified or wild-type version of the mitochondrial gene is a nuclear-encoded inner membrane protein containing a transmembrane domain, and the modified or wild-type version of the mitochondrial gene is paired with a mitochondrial targeting sequence (MTS) selected from SEQ ID NOs: 64-118.

50. 28. The method of claim 27, wherein the modified or wild-type version of the mitochondrial gene is a nuclear-encoded inner membrane protein lacking a transmembrane (TM) domain, and the modified or wild-type version of the mitochondrial gene is paired with a mitochondrial targeting sequence (MTS) selected from SEQ ID NOs: 119-280.

51. the modified or wild-type version of the mitochondrial gene a) expressed from the nucleus via genomic integration, or b) expressed as a non-integrated vector, cDNA or mRNA; 22. The method of claim 21.

52. 22. The method of claim 21, wherein a viral vector is used in the step of expressing the modified or wild-type version of the mitochondrial gene in the nucleus of the subject's cells.

53. 53. The method of claim 52, wherein the viral vector is a lentiviral vector, a herpes simplex virus (HSV) vector, an adenoviral vector, or an adeno-associated virus (AAV) vector.

54. 1. A method of preventing or slowing the aging process in a subject, said method comprising: a) identifying a mitochondrial defect in the cells of said subject; b) identifying a mitochondrial gene associated with said mitochondrial defect; c) expressing an altered or wild-type version of the mitochondrial gene in the nucleus of a cell of the subject; and d) importing the gene product of said modified or wild-type version of said mitochondrial gene into mitochondria in cells of said subject.

55. 55. The method of claim 54, wherein the mitochondrial defect is one or more of: reduced oxidative capacity, decreased oxidative phosphorylation, reduced ATP production, increased ROS generation, and reduced antioxidant defense.

56. 55. The method of claim 54, wherein the gene product is a subunit of the oxidative phosphorylation (OXPHOS) complex.

57. 55. The method of claim 54, wherein the gene product is a subunit of NADH ubiquinone oxidoreductase (complex I), succinate dehydrogenase (complex II), ubiquinol-cytochrome c oxidoreductase (complex III or cytochrome bc1 complex), cytochrome c oxidase (complex IV), or ATP synthase (complex V).

58. 55. The method of claim 54, wherein the step of expressing a modified or wild-type version of the mitochondrial gene further comprises incorporating sequences into the modified or wild-type gene for codon optimization.

59. 55. The method of claim 54, wherein the step of expressing an engineered or wild-type version of the mitochondrial gene further comprises incorporating into the engineered or wild-type gene a sequence that causes translation slowdown or ribosome stalling.

60. 55. The method of claim 54, wherein said step of expressing an engineered or wild-type version of said mitochondrial gene further comprises the step of incorporating a PUMILIO binding site into said engineered or wild-type gene.

61. 55. The method of claim 54, wherein the step of expressing an engineered or wild-type version of the mitochondrial gene further comprises incorporating into the engineered or wild-type gene a sequence that reduces the hydrophobicity of a transmembrane domain within the gene product.

62. wherein the step of expressing the modified or wild-type version of the mitochondrial gene further comprises incorporating one or more codon sequences into the modified or wild-type version of the mitochondrial gene; 55. The method of claim 54, wherein the one or more codon sequences reduce the hydrophobicity of the gene product, reduce the local charge of the gene product, or cause ribosome stalling.

63. wherein the step of expressing the modified or wild-type version of the mitochondrial gene comprises incorporating one or more codon sequences into the modified or wild-type version of the mitochondrial gene; 55. The method of claim 54, wherein the one or more codon sequences encode an N-terminal matrix export signal for the gene product.

64. 64. The method of claim 63, wherein the N-terminal matrix export signal is a six-residue sequence from OXA1L.

65. wherein the step of expressing the modified or wild-type version of the mitochondrial gene further comprises incorporating one or more codon sequences into the wild-type version of the mitochondrial gene; 55. The method of claim 54, wherein said one or more codon sequences restore the natural orientation of the transmembrane helices of said gene product to the inner mitochondrial membrane.

66. wherein the step of expressing the modified or wild-type version of the mitochondrial gene comprises incorporating one or more codon sequences into the modified or wild-type version of the mitochondrial gene; 55. The method of claim 54, wherein the one or more codon sequences reduce the localized charge of the gene product.

67. wherein the step of expressing the modified or wild-type version of the mitochondrial gene further comprises adding a promoter sequence to the modified or wild-type version of the mitochondrial gene; 55. The method of claim 54, wherein the promoter sequence is selected from pCAG, PGK, PGC1a, ER1a, myosin, and CK.

68. wherein the step of expressing the modified or wild-type version of the mitochondrial gene further comprises incorporating one or more codon sequences into the modified or wild-type version of the mitochondrial gene; 55. The method of Claim 54, wherein said one or more codon sequences encode a mitochondrial targeting sequence to the amino terminal portion of said gene product.

69. the modified or wild-type version of the mitochondrial gene a) expressed from the nucleus via genomic integration, or b) expressed as a non-integrated vector, cDNA or mRNA; 55. The method of claim 54.

70. 55. The method of claim 54, wherein a viral vector is used in the process of expressing the modified or wild-type version of the mitochondrial gene in the nucleus of the subject's cells.

71. 71. The method of claim 70, wherein the viral vector is a lentiviral vector, a herpes simplex virus (HSV) vector, an adenoviral vector, or an adeno-associated virus (AAV) vector.

72. 55. The method of claim 54, wherein the modified or wild-type version of the mitochondrial gene is a nuclear-encoded mitochondrial protein of respiratory complex I, and wherein the modified or wild-type version of the mitochondrial gene is paired with a mitochondrial targeting sequence (MTS) selected from SEQ ID NOs: 1-29.

73. 55. The method of claim 54, wherein the modified or wild-type version of the mitochondrial gene is a nuclear-encoded mitochondrial protein of respiratory complex V, and wherein the modified or wild-type version of the mitochondrial gene is paired with a mitochondrial targeting sequence (MTS) selected from SEQ ID NOs: 30-48.

74. 55. The method of claim 54, wherein the modified or wild-type version of the mitochondrial gene is a nuclear-encoded mitochondrial protein of respiratory complex IV, and wherein the modified or wild-type version of the mitochondrial gene is paired with a mitochondrial targeting sequence (MTS) selected from SEQ ID NOs: 49-56.

75. 55. The method of claim 54, wherein the modified or wild-type version of the mitochondrial gene is a nuclear-encoded mitochondrial protein of respiratory complex III, and wherein the modified or wild-type version of the mitochondrial gene is paired with a mitochondrial targeting sequence (MTS) selected from SEQ ID NOs: 57-63.

76. 55. The method of claim 54, wherein the modified or wild-type version of the mitochondrial gene is a nuclear-encoded inner membrane protein containing a transmembrane domain, and wherein the modified or wild-type version of the mitochondrial gene is paired with a mitochondrial targeting sequence (MTS) selected from SEQ ID NOs: 64-118.

77. 55. The method of claim 54, wherein the modified or wild-type version of the mitochondrial gene is a nuclear-encoded inner membrane protein lacking a transmembrane (TM) domain, and wherein the modified or wild-type version of the mitochondrial gene is paired with a mitochondrial targeting sequence (MTS) selected from SEQ ID NOs: 119-280.

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