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WO2024077283A2 - Expression allotopique de gènes d'adn mitochondrial - Google Patents

Expression allotopique de gènes d'adn mitochondrial Download PDF

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WO2024077283A2
WO2024077283A2 PCT/US2023/076302 US2023076302W WO2024077283A2 WO 2024077283 A2 WO2024077283 A2 WO 2024077283A2 US 2023076302 W US2023076302 W US 2023076302W WO 2024077283 A2 WO2024077283 A2 WO 2024077283A2
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mitochondrial
wild
modified
gene
type version
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WO2024077283A3 (fr
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Amutha BOOMINATHAN
Matthew S. O' CONNOR
Caitlin J. LEWIS
Bhavna DIXIT
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Sens Research Foundation
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Sens Research Foundation
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/05—Oxidoreductases acting on the CH-CH group of donors (1.3) with a quinone or related compound as acceptor (1.3.5)
    • C12Y103/05001—Succinate dehydrogenase (ubiquinone) (1.3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y109/00—Oxidoreductases acting on a heme group of donors (1.9)
    • C12Y109/03—Oxidoreductases acting on a heme group of donors (1.9) with oxygen as acceptor (1.9.3)
    • C12Y109/03001—Cytochrome-c oxidase (1.9.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
    • C12Y110/02—Oxidoreductases acting on diphenols and related substances as donors (1.10) with a cytochrome as acceptor (1.10.2)
    • C12Y110/02002—Ubiquinol-cytochrome-c reductase (1.10.2.2), i.e. electron-transport-complex-III
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/07—Fusion polypeptide containing a localisation/targetting motif containing a mitochondrial localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/095—Fusion polypeptide containing a localisation/targetting motif containing a nuclear export signal
    • C—CHEMISTRY; METALLURGY
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the 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 counteract aging processes and phenotypes.
  • a mitochondrion is a double-membrane-bound organelle found in most eukaryotic organisms. Although most of a cell's DNA is contained in the cell nucleus, the mitochondrion has its own genome ("mitogenome”) that is similar to bacterial genomes. Mitochondrial proteins (i.e. , proteins transcribed from mitochondrial DNA) vary depending on the tissue and the species. In humans, 615 distinct types of proteins have been identified from cardiac mitochondria, whereas in rats, 940 proteins have been reported. The mitochondrial proteome is thought to be dynamically regulated.
  • Mitochondria use aerobic respiration to generate most of the cell's supply of adenosine triphosphate (ATP), which is subsequently used throughout the cell as a source of energy.
  • Oxidative phosphorylation (OXPHOS) the Krebs's cycle, the urea cycle, heme biosynthesis and fatty acid oxidation take place within the mitochondria.
  • mitochondria are involved in signaling, cellular differentiation, and cell death, as well as maintaining control of the cell cycle and cell growth.
  • Mitochondrial biogenesis is in turn temporally coordinated with these cellular processes. Mitochondria have been implicated in several human disorders and conditions, such as mitochondrial diseases, cardiac dysfunction, heart failure and autism.
  • mitochondrial diseases and disorders that are caused by pathogenic point mutations of mitochondrial DNA (mtDNA), one-third of which are located in coding genes.
  • Primary defects in mitochondrial function generally present clinical problems in tissues that have high energy requirements, such as retina, heart, muscle, kidney, pancreas and liver. Their incidence is estimated at 1 in 5,000 live births.
  • mitochondrial pathologies are considered among the most common genetically determined diseases and are a major health issue since they remain inaccessible to both curative and palliative therapies.
  • Mitochondrial dysfunction is also a hallmark of aging and cellular senescence.
  • mitochondrial metabolism There are changes in mitochondrial metabolism with aging that are linked to changes in mitochondrial organization.
  • the dynamic equilibrium between fusion and fission is essential for healthy mitochondrial function.
  • fusion exceeds fission, and large mitochondria form that contribute to cell senescence.
  • the fission process is coupled with ER-microtubule function; therefore, interaction of mt-ER- Lysosome affects the fission process.
  • There is evidence of decreased senescence with exercise and findings indicate that mechanical forces alter MT-ER junction protein complexes through unknown processes in the fission and fusion functions.
  • mitochondrial quality and copy number and an increasing incidence of mtDNA mutations with age, which are consequently implicated in cellular senescence and age-related organismal decline.
  • ROS reactive oxygen species
  • mitochondria are characterized by impaired function such as lowered oxidative capacity, reduced oxidative phosphorylation, decreased ATP production, significant increase in ROS generation, and diminished antioxidant defense.
  • Mitochondrial biogenesis declines with age due to alterations in 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.
  • enhanced mitochondria-mediated apoptosis contributes to an increase in the percentage of apoptotic cells.
  • Allotopic expression has been proposed to treat these mitochondrial disorders.
  • a wild-type (i.e. , healthy) copy of a mutated gene is introduced in the genome. Normal copies of the gene product are imported into mitochondria from the cytosol.
  • a number of mitochondrial genes have been successfully recoded and nuclearly expressed in yeast.
  • efforts at allotopic expression in mammalian cells have been generally unsuccessful.
  • the invention provides means, including compositions and methods, which enable mitochondrial importation at enhanced efficiency and stability compared to conventional techniques.
  • the means of the invention enable a targeted localization of the mRNA to the mitochondrial surface.
  • embodiments include methods of introducing a modified gene product into a cell by, for example, plasmid or viral vector, as an RNA or cDNA therapeutic or as a therapeutic protein.
  • the gene product is expressed from the nucleus (i.e. , genome integration).
  • the gene product is provided as a non-integrating vector, or as cDNA or mRNA, with expression/regulatory elements appropriate for the nucleic acid type.
  • a therapeutic protein is administered to a subject (i.e. , direct to tissue).
  • Embodiments include methods of al lotopic expression of a gene in a cell.
  • the methods can include steps of (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 mitochondria of the cell.
  • the cell is a mammalian cell such as a human cell.
  • the gene product is an oxidative phosphorylation (OXPHOS) complex subunit translated on the mitochondrial DNA.
  • OXPHOS oxidative phosphorylation
  • one or more codon sequences are incorporated into the wild-type version of the mitochondrial gene.
  • the methods described herein can use codon optimization, translational slow-down and/or ribosome stalling.
  • an N-terminal matrix export signal is incorporated into the wild-type version of the mitochondrial gene.
  • one or more codon sequences are introduced into the wild-type version of the mitochondrial gene to restore natural orientation of transmembrane helices of the gene product into the mitochondrial inner membrane.
  • methods of allotopic expression described herein are used therapeutically to improve mitochondrial function, for example, to treat a mitochondrial dysfunction, a senescence-associated disease or disorder or to prevent or slow the aging process.
  • Embodiments also include methods of reducing the hydrophobicity of the coding sequences to improve import and function of allotopic mtDNA proteins.
  • the methods can include steps of (a) identifying non-deleterious mutations and to assess the resulting hydrophobicity alterations and (b) evaluating the three-dimensional (3D) structural impact of the mutations.
  • Embodiments include methods of administering a nucleic acid construct that encodes a therapeutic mitochondrial protein.
  • the construct is integrated into the genome.
  • the construct does not integrate into the genome (e.g., expressed from an episome).
  • the gene product is imported into mitochondria.
  • Embodiments include administration of nucleic acids encoding therapeutic mitochondrial protein.
  • the nucleic acids are RNA.
  • Embodiments include allotopic administration of a therapeutic mitochondrial protein.
  • a mitochondrial targeting sequence (MTS) identified in Table 1 is paired with a nuclear-encoded mitochondrial protein of respiratory complex I.
  • a mitochondrial targeting sequence (MTS) identified in Table 2 i.e., SEQ ID NO: 30 - 48
  • MTS mitochondrial targeting sequence
  • a mitochondrial targeting sequence (MTS) identified in Table 3 i.e., SEQ ID NO: 49 - 56
  • MTS mitochondrial targeting sequence
  • a mitochondrial targeting sequence (MTS) identified in Table 4 i.e., SEQ ID NO: 57 - 63
  • MTS mitochondrial targeting sequence
  • a mitochondrial targeting sequence (MTS) identified in Table 5 i.e., SEQ ID NO: 64 - 118
  • a mitochondrial targeting sequence (MTS) identified in Table 6 is paired with a nuclear encoded protein of the inner membrane lacking transmembrane (TM) domains.
  • therapeutic nucleic acids or protein are encapsulated in a liposome, nanoparticle or other pharmaceutically acceptable carrier.
  • a viral vector is used in the step of expressing a nucleic acid construct that encodes a therapeutic mitochondrial protein.
  • the viral vector is a lentiviral vector, a herpes simplex virus (HSV) vector, an adenoviral vector or an adeno-associated viral (AAV) vector.
  • HSV herpes simplex virus
  • AAV adeno-associated viral
  • FIG. 1 A is a depiction of steps in allotopic expression.
  • FIG. 1 B is a depiction of pCMV constructs according to embodiments of the invention.
  • FIG. 1 C is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to GAPDH).
  • FIG. 1 D is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to COX10).
  • FIG. 2A is depiction of pCMV and pCAG constructs according to embodiments of the invention.
  • FIG. 2B is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to GAPDH).
  • FIG. 2C is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to COX10).
  • FIG. 3A is depiction of constructs with Hexapeptide and 0XA1 L sequences according to embodiments of the invention.
  • FIG. 3B is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to GAPDH).
  • FIG. 3C is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to COX10).
  • FIG. 4A is a depiction of constructs with MPCP sequences according to embodiments of the invention.
  • FIG. 4B is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to GAPDH).
  • FIG. 4C is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to 00X10).
  • FIG. 5A is a depiction of constructs with ABCBA sequences according to embodiments of the invention.
  • FIG. 5B is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to GAPDH).
  • FIG. 50 is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to COX10).
  • FIG. 6A is a depiction of constructs with triple protein tags according to embodiments of the invention.
  • FIG. 6B is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to GAPDH).
  • FIG. 6C is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to COX10).
  • FIG. 7A is graphical depiction of mesohydrophobicity versus local hydrophobicity of transmembrane region 1 of ATP6.
  • FIG. 7B is graphical depiction of mesohydrophobicity versus local hydrophobicity of transmembrane region 1 of ATP6.
  • FIG. 8A is a bar graph showing the levels of 0ATP6 mRNA expression in ATP6 mutant cell lines for different constructs to COX10.
  • FIG. 8B is a bar graph showing the levels of 0ATP6 mRNA expression in ATP6 mutant cell lines for different constructs to GAPDH.
  • FIG. 9 is an image of SDS PAGE of mitochondrial fractions with a comparison of anti-FLAG and anti-ACONITASE expression.
  • FIG. 10A depicts the cleaved and uncleaved C0X2 protein.
  • FIG. 10B is an SDS PAGE showing stable expression in C0X2 site-directed mutagenesis constructs.
  • FIG. 11 A shows mRNA levels for different versions of codon-optimized allotopic C0X2 gene in comparison to GAPDH.
  • FIG. 11 B shows SDS PAGE profiles for different versions of codon- optimized allotopic COX2 in a COX2 null cell line upon stable selection.
  • FIG. 12 shows a plasmid sequence of a C0X2 variant according to embodiments.
  • references in this specification to "one embodiment/aspect” or “an embodiment/aspect” means that a particular feature, structure, or characteristic described in connection with the embodiment/aspect is included in at least one embodiment/aspect of the disclosure.
  • the use of the phrase “in one embodiment/aspect” or “in another embodiment/aspect” in various places in the specification are not necessarily all referring to the same embodiment/aspect, nor are separate or alternative embodiments/aspects mutually exclusive of other embodiments/aspects.
  • various features are described which may be exhibited by some embodiments/aspects and not by others.
  • various requirements are described which may be requirements for some embodiments/aspects but not other embodiments/aspects.
  • Embodiment and aspect can be in certain instances be used interchangeably.
  • AE allotopic expression
  • genes normally expressed only from the mitochondrial genome using nucleo-cytosolic machinery Biomedically engineered AE has been suggested as a possible tool in gene therapy to treat certain mitochondria-related diseases.
  • Mitochondrial diseases refers to chronic, genetic, often inherited disorders that occur when mitochondria fail to produce enough energy for the body to function properly. Mitochondrial diseases can affect almost any part of the body, including the cells of the brain, nerves, muscles, kidneys, heart, liver, eyes, ears or pancreas. Mitochondrial diseases can be caused by mitochondrial DNA (mtDNA) disorders or nuclear DNA (nDNA) disorders.
  • mtDNA mitochondrial DNA
  • nDNA nuclear DNA
  • Mitochondrial DNA (mtDNA) disorders include, for example, Leigh syndrome, leukodystrophy w/complex II deficiency, cardiomyopathy & encephalopathy (complex I deficiency), optic atrophy and ataxia (complex II deficiency), hypokalemia and lactic acidosis, hepatopathy & ketoacidosis, hypertrophic cardiomyopathy, liver failure, renal tubulopathy (w/complex III deficiency) and encephalopathy (w/complex V deficiency), autosomal progressive external ophthalmoplegia, mitochondrial neurogastrointestinal encephalomyopathy, Alpers-Huttenlocher syndrome, ataxia neuropathy syndromes, infantile myopathy/spinal muscular atrophy and hypotonia.
  • mtDNA Mitochondrial DNA
  • primary mitochondrial disorders refers to a clinically heterogeneous group of disorders that arise as a result of dysfunction of the mitochondrial respiratory chain.
  • the mitochondrial respiratory chain is the essential final common pathway for aerobic metabolism. Tissues and organs that are highly dependent on aerobic metabolism are usually most affected by mitochondrial disorders. Many genetic and non-genetic disorders involve mitochondrial mechanisms as a secondary feature.
  • primary mitochondrial disorders are considered to be known or presumed genetic disorders caused by pathogenic variants in genes coding for the mitochondrial respiratory chain and related proteins.
  • Nuclear DNA (nDNA) disorders include, for example, Leigh syndrome, leukodystrophy w/complex II deficiency, cardiomyopathy & encephalopathy (complex I deficiency), optic atrophy & ataxia (complex II deficiency), hypokalemia & lactic acidosis (complex III deficiency), hepatopathy and ketoacidosis, cardiomyopathy and encephalopathy, leukodystrophy & renal tubulopathy, hypertrophic cardiomyopathy, liver failure, renal tubulopathy (w/complex III deficiency), encephalopathy (w/complex V deficiency), coenzyme Q10 deficiency, Barth syndrome, autosomal progressive external ophthalmoplegia, mitochondrial neurogastrointestinal encephalomyopathy, Alpers- Huttenlocher syndrome, ataxia neuropathy syndromes, infantile myopathy/spinal muscular atrophy, hypotonia, reversible hepatopathy, myopathy with cataract and combined
  • Secondary mitochondrial dysfunction refers to any abnormal mitochondrial function other than a primary mitochondrial disorder.
  • Secondary mitochondrial dysfunction can be caused by genes encoding neither function nor production of the oxphos proteins and accompanies many hereditary non-mitochondrial diseases. Secondary mitochondrial dysfunction can also be due to nongenetic causes such as environmental factors. Secondary mitochondrial dysfunction is seen in many different genetic disorders, including ethylmalonic aciduria (caused by mutation of ETHE1 ), Friedreich ataxia (FXN), hereditary spastic paraplegia 7 (SPG7), and Wilson disease (ATP7B), and is also seen as part of the aging process.
  • ETHE1 ethylmalonic aciduria
  • FXN Friedreich ataxia
  • SPG7 hereditary spastic paraplegia 7
  • Wilson disease ATP7B
  • mutation refers to a change that occurs in our DNA sequence as the result of chemical mutagens such as reactive byproducts of cellular metabolism, environmental factors such as UV light and cigarette smoke, or due to DNA replication errors arising from inherent or acquired deficiency of replication machinery.
  • a mitochondrial mutation may be described as a non-silent point mutation in one or more mtDNA genes encoding subunits of the respiratory chain.
  • a mutation may also consist of base insertions or deletions, or deletion or duplication of mtDNA genomic regions.
  • Mitochondrial mutation in this context, also refers to a deficiency in mtDNA copy number which inhibits compensation of a deleterious phenotype.
  • mitochondrial deficiency refers to a deficiency characterized by inadequate mtDNA copy number, reduced or absent expression of mtDNA subunits, or expression of faulty mtDNA subunits, and subsequent state of inadequate energy metabolism of a cell (or tissue or organ system or individual).
  • a mitochondrial deficiency can also be caused by a deficiency in mtDNA copy number which leads to a phenotypic deficiency.
  • inadequate energy metabolism refers to a condition of reduced mitochondrial function such as inefficient electron transport, ATP synthesis, or an alteration of mitochondrial membrane potential.
  • wild type refers to the phenotype of the typical form of a species as it occurs in nature.
  • mutations in regulatory elements can be deleterious if the regulatory function is impaired. These mutations can arise from single base changes or more extensive insertions, deletions or frame shifts. Mutations in protein coding genes and regulatory genes can also be neutral. A base change in a protein coding gene that does not alter the amino acid sequence of the protein is termed a synonymous change. These are commonly neutral. A base change that alters the amino acid sequence is likely to be deleterious but can be neutral.
  • conservative substitution refers to the replacement of an amino acid residue by another, biologically similar residue.
  • conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine for another, or the substitution of one polar or charged residue for another residue with similar polarity or charge, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like.
  • Neutral hydrophilic amino acids that can be substituted for one another include asparagine, glutamine, serine and threonine.
  • the term "conservative substitution” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.
  • senescence refers to gradual deterioration of functional characteristics in living organisms.
  • Cellular senescence is often defined as a stress- induced, durable cell cycle arrest of previously replication-competent cells.
  • the effects of senescent cells can be thought of as beneficial or detrimental with regard to host physiology and disease, although in some contexts, senescent cells affect a disease state in a complex manner both promoting and opposing certain conditions.
  • the term “senescence-associated disease or disorder” refers to an ailment that is associated with age and can include, for example, atherosclerosis, osteoarthritis, osteoporosis, hypertension, arthritis, cataracts, cancer, Alzheimer’s disease, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis.
  • Other ailments (including age-related conditions) associated with age or senescence include hair graying, sarcopenia, adiposity, neurogenesis, fibrosis and glaucoma.
  • cardiovascular disease e.g., atherosclerosis, angina, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, coronary thrombosis, myocardial infarction, hypertension, aortic aneurysm, cardiac diastolic dysfunction, hypercholesterolemia, hyperlipidemia, mitral valve prolapsed, peripheral vascular disease, cardiac stress resistance, cardiac fibrosis, brain aneurysm, and stroke).
  • cardiovascular disease e.g., atherosclerosis, angina, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, coronary thrombosis, myocardial infarction, hypertension, aortic aneurysm, cardiac diastolic dysfunction, hypercholesterolemia, hyperlipidemia, mitral valve prolapsed, peripheral vascular disease, cardiac stress resistance, cardiac fibrosis, brain an
  • a senescence-associated disease or disorder can also be an inflammatory or autoimmune disease or disorder (e.g., osteoarthritis, osteoporosis, oral mucositis, inflammatory bowel disease or kyphosis).
  • a senescence-associated disease or disorder can also be a neurodegenerative disease (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, mild cognitive impairment or motor neuron dysfunction).
  • a senescence-associated disease or disorder can also be a metabolic disease (e.g., diabetes, diabetic ulcer, metabolic syndrome or obesity).
  • a senescence-associated disease or disorder can also be a pulmonary disease (e.g., pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, cystic fibrosis, emphysema, bronchiectasis or age-related loss of pulmonary function).
  • a senescence- associated disease or disorder can also be an eye disease or disorder (e.g., macular degeneration, glaucoma, cataracts, presbyopia or vision loss).
  • a senescence- associated disease or disorder can also be renal disease, renal failure, frailty, hearing loss, muscle fatigue, skin conditions, skin wound healing, liver fibrosis, pancreatic fibrosis, oral submucosa fibrosis or sarcopenia.
  • a senescence-associated disease or disorder can also be a dermatological disease or disorder (e.g., eczema, psoriasis, hyperpigmentation, nevi, rashes, atopic dermatitis, urticaria, diseases or disorders related to photosensitivity or photoaging).
  • Oxidative phosphorylation or “OXPHOS” refers to the process by which ATP synthesis is coupled to the movement of electrons through the mitochondrial electron transport chain and the associated consumption of oxygen. In eukaryotes, this takes place inside mitochondria. In eukaryotes, redox reactions are catalyzed by a series of protein complexes within the inner membrane of the cell's mitochondria.
  • mtDNA mitochondrial DNA
  • nDNA nuclear genome
  • the term “codon optimization” refers to experimental approaches designed to improve the codon composition of a recombinant gene 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 term “promoter” refers to a sequence of DNA to which proteins bind to initiate transcription of a single RNA transcript from the DNA downstream of the promoter.
  • the RNA transcript may encode a protein (mRNA), or can have a function in and of itself, such as tRNA or rRNA. Promoters are located near the transcription start sites of genes, upstream on the DNA (towards the 5' region of the sense strand) and contain usually about 100-1000 base pairs.
  • the CMV promoter is a strong synthetic promoter frequently used to drive high levels of gene expression in mammalian expression vectors.
  • pCMV or “pCMV-Script vector” refers to a promoter sequence that is derived from a high-copy-number pUC-based plasmid and is designed to allow protein expression in mammalian systems. Mammalian expression is driven by the human cytomegalovirus (CMV) immediate early promoter to promote constitutive expression of cloned inserts in a wide variety of cell lines.
  • CMV cytomegalovirus
  • 5' untranslated region refers to the region of a messenger RNA (mRNA) that is directly upstream from the initiation codon. This region is important for the regulation of translation of a transcript by differing mechanisms in viruses, prokaryotes and eukaryotes. While called untranslated, the 5' UTR or a portion of it is sometimes translated into a protein product. This product can then regulate the translation of the main coding sequence of the mRNA. In many organisms, however, the 5' UTR is completely untranslated, instead forming complex secondary structure to regulate translation.
  • mRNA messenger RNA
  • MLS canonical mitochondrial localization signal
  • mitochondrial targeting sequence refers to a short peptide, about 15-70 amino acids long, bearing positively charged basic residues, that directs the transport of a protein to the mitochondria.
  • N-terminal mitochondrial targeting sequence refers to an MTS on the amino terminal portion of a protein which may interact with mitochondrial import machinery to aid in translocation of soluble proteins to the matrix.
  • translational stalling or “ribosome stalling” refers to a situation in ribosomes moving along the mRNAs slow down or stall. Stalling can happen when, for example, the mRNA sequence utilizes codons which rely on low-abundance tRNA species.
  • OXA1 L or “mitochondrial inner membrane protein OXA1 L” refers to a protein that in humans is encoded by the OXA1 L gene located on 14q 11 .2. The C- terminus of this protein interacts with mitochondrial ribosomes and helps insert both mitochondrial and nuclear produced protein termini into or translocate across the inner membrane of the mitochondria from the matrix.
  • PUMILIO refers to sequence specific RNA-binding proteins that regulate protein expression.
  • Pumilio family of proteins regulate translation and mRNA stability in a variety of eukaryotic organisms.
  • Pumilio family members are characterized by the presence of eight tandem copies of an imperfectly repeated 36 amino acids sequence motif (i.e. , the Pumilio repeat) surrounded by a short N- and C-terminal conserved region.
  • Pumilio interaction motifs are defined in several species.
  • treating refers to one or more of (1 ) inhibiting the disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology); and (2) ameliorating the disease condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology) such as decreasing the severity of disease.
  • compositions include a therapeutic peptide encoded by a DNA or RNA sequence and an acceptable carrier.
  • the therapeutic peptide can be contained within a delivery vehicle.
  • the two main approaches used to deliver the therapeutic genetic material are (a) synthetic and (b) viral delivery vehicles.
  • Synthetic vectors include, for example, encapsulation in liposomes, nanoparticles, cyclodextrins, and microvesicles.
  • Viruses include, for example, AAV and retroviral vectors.
  • liposome refers to a spherical vesicle having at least one lipid bilayer (i.e. , an aqueous solution 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. Upon dispersion in water the phospholipids form closed vesicles called “liposomes,” which are characterized by lipid bilayers encapsulating an aqueous core. Liposomes have therapeutic applications including delivering drugs to target cells after systemic administration.
  • Liposomes can be modified by the incorporation of polyethylene glycol or other hydrophilic polymers (e.g., a PEG liposome where one or more of the constituent lipids is modified by attachment of PEG). Liposomes can also be modified to target particular cell types by incorporating targeting factors (e.g., “targeting ligands”) for particular cell types. Examples include asialoglycoprotein, folate, transferrin, antibodies, etc.
  • stable formulation or “stable pharmaceutical formulation” refers to a formulation which preserves its physical stability/identity/integrity and/or chemical stability/identity/integrity and/or biological activity/identity/integrity during manufacturing, storage, transportation, and application.
  • Various analytical techniques for evaluating virus stability are available in the art and reviewed in Felix A. Rey and Shee-Mei Lok (2016) “Common Features of Enveloped Viruses and Implications for Immunogen Design for Next-Generation Vaccines,” Cell 172, pp 1319-1334 and Guy Unsmellings et al.
  • Stability can be evaluated by, for example, without limitation, storage at selected climate conditions for a selected time period, by applying mechanical stress such as shaking at a selected shaking frequency for a selected time period, by irradiation with a selected light intensity for a selected period of time, or by repetitive freezing and thawing at selected temperatures.
  • a “virus’” may encompass any chemical or biochemical component portion of a virus, including viral component preparations, virus-like particles, viral vectors, nanoparticles that are enveloped in lipid, a related particle (e.g. prion), or the like and it need not be infective or capable of self-replication.
  • a viral particle can comprise nanoparticles that are enveloped with lipid.
  • viral vector refers to a viral genome that has been adapted into a plasmid-based technology and modified for safety through the removal of many essential genes and the separation of the viral components.
  • the use of viral vectors is a means of gene transfer to modify a specific cell type or tissue and can be manipulated to express therapeutic genes.
  • AAV adeno-associated virus
  • ssDNA linear single-stranded DNA genome of approximately 4.8 kilobases
  • Plasmid refers to a genetic structure in a cell that can replicate independently of the chromosomes, typically a small circular DNA strand. Plasmids are often used in the laboratory manipulation of genes. Plasmids represent the simplest form of vector for transport of DNA into the cell nucleus. The generally include a circular, double-stranded DNA molecule varying in size from ⁇ 1000 to >200 000 bp. Compared with recombinant viruses, plasmids are simple to construct and easily propagated in large quantities. They also possess an excellent safety profile, with virtually no risk of oncogenesis (as genomic integration is very inefficient) and relatively little immunogenicity. Plasmids can be administered by, for example, infusion or injection and can be administered using techniques (e.g., carrier vehicles) to improve their update by targeted cells.
  • techniques e.g., carrier vehicles
  • a carrier vehicle for a plasmid can reduce susceptibility to circulating nucleases and increase cellular uptake. They may also target plasmids to a specific tissue. Like carrier microbubbles, most vehicles are cationic. Their positive charge enables electrostatic complex formation with negatively charged pDNA. Complexes are prepared with a residual positive charge which enhances cellular uptake via electrostatic interaction with the negatively charged cell membrane. These carrier vehicles can substantially increase transfection; non-viral systems can be as effective as viruses at delivering DNA to cell nuclei.
  • the term “episome” or “plasmid” refers to a length of DNA that exists either in the cytoplasm or attached to the chromosome of a mammalian cell. They replicate in synchrony with the host chromosome and are thus perpetuated as long as the parent strain exists.
  • the main disadvantage of integrating vector systems is their potential risk of causing insertional mutagenesis. Episomal vector systems have the potential to avoid these undesired side effects because they behave as separate extrachromosomal elements in the nucleus of a target cell.
  • subject refers to those who are susceptible to an ailment (e.g., a disease related to senescence) or who are suspected of having or diagnosed with the ailment.
  • ailment e.g., a disease related to senescence
  • any subject to be treated with the therapeutic methods described herein is included without limitation.
  • formulation(s) refers to a combination of at least one active ingredient with one or more other ingredient, also commonly referred to as excipients, which may be independently active or inactive.
  • excipients also commonly referred to as excipients, which may be independently active or inactive.
  • formulation may or may not refer to a pharmaceutically acceptable composition for administration to humans or animals and may include compositions that are useful intermediates for storage or research purposes.
  • mtDNA mitochondrial DNA
  • mtDNA mitochondrial DNA
  • mtDNA is particularly vulnerable to damage and accumulation of mutations. Its location adjacent to the oxidative phosphorylation machinery exposes the genetic material to higher risk of mutagenic events.
  • the mtDNA is susceptible to mutations from reactive oxygen species generated via oxidative phosphorylation as well as inadequate DNA repair mechanisms in the organelle.
  • Allotopic expression is a promising therapeutic tool to genetically remedy deleterious mtDNA mutations through nuclear complementation of the affected genes.
  • a mitochondrial gene is deliberately recoded and relocated into the nucleus and the encoded polypeptide is imported back into the mitochondrion.
  • FIG. 1 is a schematic for allotopic expression that shows the various steps involved in the successful implementation of the strategy.
  • the first step (1 ) is the design of the optimal DNA expression construct. As described herein, a modified or wild-type version of the mitochondrial gene can be used in the construct.
  • the plasmid is integrated into an allotopic gene (2). This step is followed by transcription (3).
  • sufficient amounts of the mRNA must be exported into the cytosol (4) and translated into a peptide (5).
  • efforts must be taken to avoid aggregation (6).
  • the peptide is targeted to mitochondria (7) and imported across the outer and inner membrane of the mitochondria (8).
  • the peptide is delivered to correct a component of the respiratory chain (RC) complex (8). If these steps are successful, the peptide is delivered to correct a deficiency in a mitochondrial deficiency (9) and functional recovery can be achieved (10). [00113] Conventional methods of allotopic expression have been unsuccessful for numerous reasons. The present invention provides methods that overcome hurdles of past efforts of allotopic expression.
  • translational slowdown or ribosome stalling is induced during the synthesis of allotopically expressed (AE) proteins. This is especially relevant in conjunction with codon optimized constructs.
  • the systematic reduction in the hydrophobicity of specific domains in the allotopic protein is addressed to improve import while preserving function.
  • a combination of synergistic elements is addressed to improve allotopic expression and targeting to mitochondria.
  • the methods described herein account for expression of several genes at the same time.
  • Codon optimization refers to experimental approaches designed to improve the codon composition of a recombinant gene 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 genetic code of mitochondria furthermore often differs from the standard genetic code. Mitochondrial genomes have a relatively conserved gene content and small size. Moreover, the relative use of particular codons for each amino acid is vastly different between the two genomes. This is a facet that has not been utilized in conventional methods of allotopic expression.
  • Codon optimization of transgenes can lead to an increased transcription (mRNA levels) by synchronizing transgene sequences to the codon preferences of the nuclear genome. Because the mitochondrial genome uses codons differently than the nuclear genome, all allotopically expressed genes require several changes to maintain the amino acid sequence of the encoded protein. The codon-optimized transgenes described herein results in greater transcription of mtDNA genes.
  • the import mechanisms by which soluble proteins are translocated across the membranes of peroxisomes, mitochondria, chloroplasts or of the ER are remarkably different.
  • the targeting signals for mitochondria, chloroplasts, or the ER appear structurally similar, because they all involve an a-helical domain in proximity to the N-terminus.
  • the targeting signals for mitochondria, chloroplasts, and the ER are encoded within N-terminal sequences with different denominations (presequence, transit sequence, and signal peptide).
  • the 13 mitochondrially-encoded proteins are all hydrophobic subunits of electron transport chain (ETC) enzyme complexes, and are necessary for oxidative phosphorylation (OXPHOS), which occurs across the mitochondrial inner membrane (MIM).
  • ETC electron transport chain
  • OXPHOS oxidative phosphorylation
  • MIM mitochondrial inner membrane
  • Each of these subunits contain one or more transmembrane domains spanning the MIM region, and adopt a final topology characterized by the position of the N- and C-termini of the protein with respect to the inter membrane space (IMS) and the matrix.
  • IMS inter membrane space
  • N-terminal export signal in the construct, to stimulate translocation of the N-terminus back towards the IMS following initial import to the matrix.
  • Mitochondria are separated from the cytoplasm by the outer and inner mitochondrial membrane.
  • the outer membrane is porous and freely traversed by ions and small, uncharged molecules through pore-forming membrane proteins (i.e., porins), such as the voltage-dependent anion channel (VDAC). Any larger molecules, especially proteins, must be imported by special translocases. Because of its porosity, there is no membrane potential across the outer membrane.
  • the inner membrane is a tight diffusion barrier to all ions and molecules. These can only get across with the aid of specific membrane transport proteins, each of which is selective for a particular ion or molecule.
  • an electrochemical membrane potential of about 180 mV builds up across the inner mitochondrial membrane.
  • the inner membrane is where oxidative phosphorylation takes place in a suite of membrane protein complexes that create the electrochemical gradient across the inner membrane or use it for ATP synthesis.
  • loop regions connecting extra helices to the AE protein can include sites for cleavage by specific local proteases. Different cleavage signatures (e.g., for MMP vs 0CT1 vs IMP vs IMS proteases) can be used depending on where the loop is located.
  • the transmembrane domain can also be used to reduce hydrophobicity.
  • specific sequences in the transmembrane domain can code for hydrophilic amino acids.
  • surface charge can determine protein localization.
  • the surface charge can affect electrostatic interactions between anionic lipids and cationic amino acids which work in combination with other processes to direct protein localization.
  • the surface charge of the AE protein is modulated or reduced.
  • net-negative charge of a reporter tag may also help facilitate association of the carboxy terminus of ND1 with the localized positive charges on the intermembrane space side of the inner membrane, preventing complete translocation and maintaining that terminus on the correct side of the MIM.
  • localized charges near the N-terminus can aid in stimulating interaction with the MIM-IMS interface to allow for proper topology (or by avoiding affecting the cryptic, endogenous signals seemingly present in the c-terminal region).
  • cellular state is considered in regard to the promoter.
  • Long-term adaptive expression of AE transgenes is often in response to cellular state (e.g., energy status).
  • Inclusion of transcription factor binding sites can improve transcriptional regulation in response to cellular state (e.g., Pgc1 a/err1 a, HIF, NRF1/2 or other effectors).
  • regulatory elements permitting tissue and condition- specific gene expression levels.
  • Pcg1 a/err1 a target genes for example, are highly expressed in tissues with high oxygen demand. Metabolic feedback loop/gene circuit can also be considered.
  • pCMV is recognized as a strong promoter and is often used in allotopic studies. However, because of strong constitutive expression, pCMV can lead to aggregation and thus proteotoxicity.
  • the pCMV promoter is replaced with a pCAG promoter to influence an endogenous chimeric promoter. As described herein, pCAG does not cause the same degree of overexpression of downstream genes as pCMV.
  • pCAG pCAG
  • the existing pCMV constructs can have translation initiation sites within the promoter, which can generate an incorrect protein product if recognized by the cell.
  • the pCAG promoter also has an upstream ORF, however an observed upstream stop site would prevent the formation of an aberrant promoter-fusion protein.
  • uSTOPs can be included to correct translation initiation.
  • a chimeric promoter has CMV enhancer sequence, however otherwise derived from chicken beta-globulin, and is known to have a better stable expression profile in safe harbor nuclear expression regions in mice compared to CMV, which can be silenced over time.
  • promoters that can be used include, for example, PGK, PGC1 alpha, ER1 alpha, or common tissue-specific promoters such as alphaMyosin, creatinine kinase, or hybrid promoters such as C5-12, MHCK7. Still others include AAV serotypes 1 - 9, AAV2/6 or AAV2/8.
  • Upstream architecture and 5’ UTR [00130] The 5’ UTR regions from specific classes of mitochondrial proteins have been appended upstream of the expression construct. In addition to general transfactor recognition, 5’ UTR regions may form stable structures which can improve mRNA quality or may facilitate transcript localization for translation near the mitochondrial surface.
  • CMV is a constitutive promoter, it does not usually require transcription factor induction. However, under some instances of cellular stress (such as particular mitochondrial stress) certain transcriptional programs are up- or down-regulated. One of these pathways is responsive to NRF1 (nuclear respiratory factor 1 ) which functions like PGC1 a to activate OXPHOS genes in response to metabolic conditions and aerobic demand. Further, it is involved in the antioxidant response. Many mitochondrial genes are upregulated in response to NRF-1 , including ATP5A1 . A putative binding site is identified. ATP5A1 was chosen arbitrarily among NEMGs (nuclear-encoded mitochondrial genes). The 5’ UTR may also contain a Y-box promoter binding motif. Some examples include:
  • An upstream in-frame stop codon near the translation start site can aid in translational initiation rates and prevent upstream starts from interfering with downstream translation.
  • a 5’ UTR of nuclear encoded mitochondrial protein is used.
  • Examples include sequences of complex I, complex II, complex IV, complex V, inner membrane proteins or matrix proteins.
  • COX4 3’UTR was similarly selected (somewhat arbitrary of NEMGs with various RBP binding sites). Contains a site for binding of YBX1 , an RBP identified in stabilizing mRNA and helps regulate transcription by modulating interaction between EIFs and mRNA.
  • the 3’ UTR of ATP5A1 is also being studied, as it contains both a PUM (pumilio) binding site, as well as a sequence similar to the potential CLUH sequence found to be enriched in mitochondrial proteins.
  • CLUH was identified in drosophila as a protein which is implicated in the transport of OXPHOS mRNAs to the mitochondrial surface.
  • yeast some PUM proteins assist in the local enrichment of OXPHOS transcripts at the OMM.
  • Pum proteins influence translation dynamics by ribosome stalling, binding at the 3’ end of many mRNAs and forming secondary structures which inhibit (or delay) translation.
  • PUM binds the mRNAs of many mitochondria-targeted proteins, and is believed to be involved in both nutrient conditiondependent translation (when there is demand for OXPHOS) and in facilitating localized translation at the OMM.
  • This UTR also forms a highly stable secondary structure, which has been implicated in general mRNA stability. It is proposed, in some instances, to also play a role in RBP recognition and binding.
  • a 3’ UTR of nuclear encoded mitochondrial protein is used.
  • Examples include sequences of complex I, complex II, complex IV, complex V, inner membrane proteins or matrix proteins.
  • a target peptide is a short (3 - 70 amino acids long) peptide chain that directs the transport of a protein to a specific region in the cell (e.g., the mitochondria).
  • the mitochondrial targeting signal (“MTS”) is a 10 - 70 amino acid long peptide that directs a newly synthesized protein to the mitochondria. It is predominantly found at the N-terminus end and includes an alternating pattern of hydrophobic and positively charged amino acids to form an amphipathic helix. Mitochondrial targeting signals can contain additional signals that subsequently target the protein to different regions of the mitochondria, such as the mitochondrial matrix or inner membrane.
  • Embodiments include an MTS sequence that overcomes limitations of MTS sequences used in conventional methods of allotopic expression.
  • the MTS sequences described herein can:
  • MTSs from protein classes known to interact with specific chaperones
  • applicants take advantage of additional signals they may encode, such as those for chaperone recognition/recruitment (e.g., Hsp90), and outer membrane receptor recognition, as well as native context for codon composition, which may play a role in modulating translation dynamics (which, in turn, influence folding/m isfolding, chaperone recognition, targeting, etc.).
  • 0XA1 L is an inner-membrane insertase conserved from bacteria and utilizes the hexapeptide export signal in adopting its final topology. Because of the protein’s structural similarity to many OXPHOS proteins embedded in the MIM, this MTS may encode additional information which aid in its targeting and insertion into the inner membrane.
  • Other embodiments could include sequences derived from other non-carrier, multipass, nuclear-encoded mitochondrial inner membrane proteins, such as: TMM70, TMM65, TMM11 , TMM177, TI17B, T123B, TI17A, T126A, SURF1 , SPNS1 , SPG7, SFXN4, SFXN1 , PARL, 0MA1 , NNTM, NDUAB, NCLX, MFTEP1 , MRS2, MVP17, MIC27, MCUR1 , MCUB, MCU, M17L2, IFI6, HIG2A, HIG1A, GHITM, DMAC1 , DHSD, CRLS1 , COX20* C0X18*, COQ2, CDS2, C560, AGK, AFG32 *COX-derived sequences found in several existing patents.
  • sequences derived from other non-carrier, multipass, nuclear-encoded mitochondrial inner membrane proteins such as: TMM70,
  • MPCP a mitochondrial carrier family protein
  • MPCP a mitochondrial carrier family protein
  • MPCP a mitochondrial carrier family protein
  • the structure and biochemical properties of ND1 are similar to carrier family proteins, and thus cooperative import via the TIM23 and TIM22 pathways may be achieved through inclusion of this targeting region, (see: e.g., V Zara, F Palmieri, K Mahlke, 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).
  • Metabolite/carrier family proteins (which constitute the bulk of multipass MIM proteins) are specifically targeted to TOM70 on the outer membrane prior to being passed to TOM20/22 for import.
  • TOM70 substrates go on to be inserted in the MIM via TIM22.
  • TOM70 is also a known interaction partner of Hsp90, a ubiquitous chaperone involved in facilitating and correcting protein folding. This suggests that Hsp90 specifically (unlike the even more ubiquitous Hsp70) may recognize and bind to sequences in nascent carrier proteins and aid in their delivery to MOM receptors in an import-competent state. Because HSPs bind promiscuously, no specific signal or motif is identified that can be appended to its constructs; thus, sequences instead derived from its client proteins are candidates for recognition. In this case, the presequence of MPCP may have dual benefit if it is both recognized as a misfolding-prone, mitochondria-destined client by Hsp90 chaperones, and if it further can direct inner membrane assembly via TIM22.
  • OXA1 L the insertase responsible for the (co-translational) inner membrane insertion and assembly of most mitochondrially- encoded OXPHOS subunits
  • the presequence of MPCP may constitute a signal which can be recognized by the import and assembly machinery as being destined for the MIM, and potentially assist in targeting allotopically-expressed ND1 and other multi-pass subunits to the machinery responsible for correct inner membrane organization and final topology.
  • SLC25A mitochondrial carrier family proteins
  • SLC25A mitochondrial carrier family proteins
  • SLC25A mitochondrial carrier family proteins
  • SLC25A genes derived from any of the mitochondrial carrier family proteins which include: 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, M20M, KMCP1 , GHC1 , GDC, DIC, CMC2 and ADT4.
  • *ORNT1/2 are ornithine transporters, different from the OTC enzyme
  • ABCBA is a mitochondrial ABC transporter of the inner membrane and is also unique in that it possesses an N-terminal MTS, uncharacteristic of this family. It is also demonstrated that the long MTS region and subsequent first transmembrane domain can facilitate correct folding and topology of attached inner membrane proteins, but not for matrix proteins. For this reason we will test the ability of the MTS alone and the MTS plus first TM region of the ABCBA protein to direct topology of al lotopically expressed ND1. MTS region and TM region afterward also contain a relative abundance of slow-reading codons, specifically for proline and serine (rare), which may play a role in modulating translation kinetics and/or facilitating trans-factor identification. See, e.g., Ethan R.
  • An allotopic protein (“AE protein”) can be paired with the MTS of a nuclear encoded mitochondrial protein (NUMP) from the same respiratory complex to facilitate complex integration. This includes other members of the mature respiratory complex, as well as assembly factors. In some cases, the precise location of the MTS is not known. An estimate may be made based on literature findings to incorporate an appropriate length of the protein N-terminus.
  • an allotopic protein is paired with an MTS of a protein identified in Table 1 (i.e., SEQ ID NO: 1 - 63).
  • MTS transmembrane domain
  • TM transmembrane domain
  • All mitochondrially-encoded proteins are integral components of membrane complexes, and as such, contain hydrophobic TM domains. Allotopically expressed mitochondrial proteins may therefore be appended with regions of similar TM domain containing proteins, which localize to the mitochondrial inner membrane. This can include the MTS of a nuclear-encoded protein.
  • many inner membrane proteins with hydrophobic TM domains lack a canonical N-terminal MTS, and instead are targeted via cryptic internal sequences.
  • a region corresponding to the N-terminus of the protein through the first TM domain, plus (e.g., 1 - 30 AAs) can instead be appended.
  • Table 5 lists examples of MTS sequences of transmembrane domaincontaining nuclear encoded proteins of the inner membrane.
  • an allotopic protein is paired with an MTS of a protein identified in Table 5 or Table 6 (i.e. , SEQ ID NO: 64 - 280).
  • the identified "matrix export signal" derived from 0XA1 L has been shown to facilitate the export of appropriate transmembrane regions of a mature inner membrane protein from the matrix to the IMS, following MTS cleavage. Appending this sequence after the MTS signal, but upstream of the first transmembrane region of ND1 may facilitate orientation of the N-terminus toward the IMS.
  • the allotopically-expressed genes described are codon-optimized, and utilize the most preferred codons at nearly every position, which can facilitate robust and rapid translation of the polypeptide.
  • codon use is more normally distributed.
  • membrane proteins especially, have an overrepresentation of "rare" codons near the N-terminus and in regions surrounding highly hydrophobic membrane-spanning segments. The presence of rare codons in these positions is often conserved and believed to participate in protein folding and chaperone recruitment through modulation of translational speed.
  • the introduction of "rare" codons into otherwise rapidly translated regions may help to slow translation dynamics and thus increase the yield of high-quality mitochondrial ly-targeted proteins.
  • Rare codons can also be placed downstream of (i.e. , c-terminal to) hydrophobic sequence regions, such that the nascent peptide may electrostatically interact with the ribosome surface. Such interactions can facilitate formation of transient, stable secondary structures and to prevent aggregation of highly hydrophobic proteins upon emergence from the ribosome exit tunnel. Translational slowing upon emergence of hydrophobic sequence regions can also facilitate recognition by and interaction with protein chaperones such as Hsp70 and Hsp90.
  • PUM domains may also be beneficial for slowing translation speed if interspersed throughout the CDS (as is also common). Instead, previous preliminary testing revealed improved expression, possibly due to ribosome stalling (which may facilitate localized translation) or slowing the translational tempo of mRNAs which may otherwise translate very rapidly due to high-frequency codon optimization.
  • compositional similarity to endogenous genes is used to:
  • the average maximum hydrophobicity of a sample of mitochondrial proteins (or proteins spanning the MIM, TM domain containing mitochondrial proteins, etc.) across the first X amino acids (or first TM domain, or MesoH, etc.) is found to be a numerical value using the hydrophobicity scale and any corresponding window size.
  • mtDNA-encoded subunits demonstrate both high maximum local hydrophobicities across (same region measured in controls) and higher overall hydrophobicities than many nuclear-encoded subunits, (see, e.g., Fig. 7A showing clustering of MesoH and H17 of mito subunits as compared to nuclear comparables).
  • annotated transmembrane regions of mitochondrial protein subunits are evaluated for overall hydrophobicity (MesoH) as well as maximal local hydrophobicity (H17), a measure of local hydrophobicity across a 17-residue peptide window, which approximates the peptide length spanning a biological membrane.
  • the hydrophobicity of amino acid residues is ranked using the Goldman-Engelman-Seitz (GES) hydrophobicity scale.
  • hydrophobic transmembrane regions of mitochondrial subunits are evaluated computationally by substitution of each amino acid position within the region with each of the other standard 19 amino acids.
  • Amino acid substitutions may be evaluated for suitability on the basis of the local (H17) and global (MesoH) hydrophobicity-reducing effects of a given residue substitution. Examples include:
  • Substitutions can be further be characterized using software to predict the deleterious effect of a position-specific substitution on protein function. Such predictions can use local (e.g., BLocks Substitution Matrix, BLOSUM) or global (e.g., Point Accepted Mutation, PAM) evolutionary conservation matrices (or derivatives such as Dayhoff mutation data matrix) to demonstrate positional and functional residue conservation across species homologs of a protein or domain, in addition to the frequency of substitution with each of the other 19 amino acids. Such matrices may be used in this way to filter out substitutions which are predicted to have deleterious impact, for example, if a residue shows a high degree of conservation.
  • BLOSUM BLocks Substitution Matrix
  • PAM Point Accepted Mutation
  • Such matrices may be used in this way to filter out substitutions which are predicted to have deleterious impact, for example, if a residue shows a high degree of conservation.
  • a functional impact prediction threshold may thus be established to exclude hydrophobicity-reducing substitutions which are predicted to adversely affect protein function, for example, through exclusion of highly conserved residues with a mutation frequency below an arbitrary threshold.
  • Other methods to exclude potentially harmful residue substitutions include evaluation of annotated and predicted active sites and ligand binding regions.
  • Methods can also include consideration of physiochemical properties such as hydrophobicity, aromaticity and charge, which often contribute to protein-protein or protein-ligand interactions. Substitutions annotated to be associated with human disease, or those annotated with adverse functional implications are excluded. Residue substitutions within a defined functional impact threshold which also demonstrate effective reduction of H17 and/or MesoH are selected candidates.
  • candidate substitutions may be evaluated for risk of structural protein perturbation through computational modeling of residue substitutions and measurement of the resulting deviation from the established wild-type crystal structure.
  • Evaluation metrics may include changes to: phi/psi angles within transmembrane helices; distance between residue side chains; proximity of atoms to neighboring sidechains; regional hydrophobic tendency; interaction with membrane lipids; ligand interactions; intermolecular bonding and interfacial residues.
  • a transmembrane region may have one or more amino acid substitutions introduced which are predicted to reduce hydrophobicity without any deleterious effect on protein function.
  • Embodiments include allotopic expression of one or more mtDNA genes to compensate for a functional deficiency arising from mtDNA damage and/or mutations in mitochondrial genes.
  • the mtDNA gene is a complex I subunit and/or assembly factor selected from MT-ND1, MT-ND2, MT-ND3, MT-ND4L, MT-ND4, MT- ND5, MT-ND6, NDUFA6, NDUFA3, NUBPL, NDUFS3, NDUFV3, NDUFV2, DMAC1, AIFM1, NDUFS4, 0XA1L, NDUFA13, NDUFAF5, NDUFAF4, NDUFA4, TIMM21 , NDUFA1, NDUFB4, NDUFA8, NDUFAF8, NDUFB1, NDUFS6, NDUFA2, NDUFB6, NDUFB9, NDUFB11, WDR93, NDUFC1, DMAC2, NDUFA7, NDUFAF1, NDUFB8, NDUFAF3, NDUF
  • the mtDNA gene is a complex III subunit and/or assembly factor selected from MT-CYB, UQCC2, TTC19, UQCRB, SLC25A33, UQCRH, UQCR10, UQCRFS1, CYC1, LYRM7, UQCC1, UQCRC2, UQCRQ, UQCC3, BCS1L, C12orf73, UQCRC1, UQCR11 and UQCRHL.
  • the mtDNA gene is a complex IV subunit and/or assembly factor selected from 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, SCO1, UQCRC2, COX18, COX6C, COA8, C15orf48, COX6A2, COA4, COX6A1, COX8C, COX17, BCS1L, SMIM20, COX7A2, TACO1, COA1, COX4I1, COX6B1, PET100, NDUFA4L2, COX5B and COX20.
  • the mtDNA gene is a complex V subunit and/or assembly factor 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.
  • MT-ATP8 MT-ATP6, ATP23, ATP5PO, ATP5F1C, ATP5F1E, ATPAF2, ATP5PF, OXA1L, ATP5MJ, ATP23, TMEM242, ATP5MK, ATP5MC1 , ATP5MG
  • the mtDNA gene is a component of mitochondrial inner membrane selected from 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, MRPS18B, TAMM41, CHCHD10, RDH13, NEU4, TIMM22, RDH13, MRPS36, MRPL45, PAM16, RDH13, MRPS18B, RDH13, SURF1, COX8C, MRPS18B, SLC25A15, DNAJC15, MRPS18B, SLC25A26
  • the mtDNA gene is an integral component of the mitochondrial inner membrane selected from 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, MPC2, APOOL, TIMM23, COX18, TIMM23B, MTX2, AGK, CHCHD3, HSPA9, TIMM17B, SPG7, TIMM17A, SLC25A19, COQ2, ETFDH, MCU, GHITM, UQCC3, SF
  • the mtDNA gene is a matrix protein selected from SMDT 1 , 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, MRPL39, AK3, NUBPL, ATP5F1C, MRPL36, FASTKD3, TOP3A, PABPC5, PIN4, ATP5F1E, SUCLA2, NDUFS3, ISCA2, PCK2, CARS2, MRPS30, RAD51, NAXD, MRPS26, ME2,
  • FIG. 1 B depicts gene constructs that were used in this study. Each construct utilized the same promoter (pCMV) with different elements. Levels of expression of oND1 were compared as described below.
  • FIG. 1 C is a bar graph showing the levels of oxidative phosphorylation (OxPhos) protein oND1 mRNA expression in ND1 null cell lines for each construct (relative to GAPDH). The highest level of expression was found in the PUM construct.
  • FIG. 1 D is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to COX10). The PUM construct also had the highest level of expression.
  • levels of expression were compared using different promoters (i.e. , pCMV and pCAG).
  • FIG. 1 C is a bar graph showing the levels of oxidative phosphorylation (OxPhos) protein oND1 mRNA expression in ND1 null cell lines for each construct (relative to GAPDH). The highest level of expression was found in the PUM construct.
  • FIG. 1 D is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines
  • FIG. 2A depicts pCMV and pCAG constructs.
  • FIG. 2B is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to GAPDH).
  • FIG. 20 is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to 00X10). In both studies, the pCMV promoter demonstrated higher levels of oND1 expression.
  • FIG. 3A depicts constructs that were compared.
  • FIG. 3B is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to GAPDH).
  • FIG. 3C is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to 00X10).
  • the C2 (pCAG, OXA1 L 5’UTR, OXA1 L MTS, Hex) construct demonstrated the highest expression.
  • the pre-sequence of MPCP can constitute a signal which can be recognized by the import and assembly machinery as being destined for the mitochondrial inner membrane (MIM).
  • FIG. 4A depicts constructs with MPCP sequences.
  • FIG. 4B is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to GAPDH).
  • FIG. 4C is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to COX10).
  • the D1 construct and CAG construct demonstrated the highest levels of expression respectively.
  • FIG. 5A depicts constructs that include ABCBA sequences.
  • FIG. 5B is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to GAPDH).
  • FIG. 5C is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to COX10). The E5 construct demonstrated the highest levels of expression.
  • FIG. 6A depicts constructs with triple protein tags according to embodiments of the invention.
  • FIG. 6A depicts constructs with triple protein tags according to embodiments of the invention.
  • FIG. 6B is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to GAPDH).
  • FIG. 6C is a bar graph showing the levels of oND1 mRNA expression in ND1 null cell lines for each construct (relative to COX10). The 3xF construct demonstrated the highest levels of expression.
  • FIG. 7A is graphical depiction of mesohydrophobicity versus local hydrophobicity of transmembrane region 1 of ATP6.
  • FIG. 7B is graphical depiction of mesohydrophobicity versus local hydrophobicity of transmembrane region 1 of ATP6.
  • FIG. 8A is a bar graph showing the levels of 0ATP6 mRNA expression in ATP6 mutant cell lines for different constructs of COX10.
  • FIG. 8B is a bar graph showing the levels of 0ATP6 mRNA expression in ATP6 mutant cell lines for different constructs to GAPDH. The TM2 mutant cell line demonstrated the highest levels of expression.
  • FIG. 9 is an image of SDS PAGE of mitochondrial fractions with a comparison of anti-FLAG and anti-ACONITASE expression.
  • the TM2 Mutant demonstrated the highest flag:aconitase density ratio (i.e. 17.88).
  • FIG. 10A depicts the cleaved and uncleaved C0X2 protein.
  • the predicted molecular weight and marker detection for the cleaved and uncleaved correspond to the molecular weights demonstrated as kDa in the SDS PAGE gel.
  • FIG. 10B is an SDS PAGE showing stable expression in C0X2 site-directed mutagenesis constructs. It shows the influence of reducing transmembrane hydrophobicity on the import of COX2.
  • the purified mitochondrial fractions were resolved on 4 - 12% SDS PAGE gels and probed for Anti FLAG and Anti TOMM 20 (an outer mitochondrial membrane protein marker).
  • the lanes represent the (1 ) 293 mock negative control (2) original COX2 construct (3) 131 R mutagenesis construct (4) Y40R mutagenesis construct (5) 131 R + Y40R mutagenesis construct (6) L74P mutagenesis construct and (7) V75M mutagenesis construct respectively.
  • FIG. 11 A shows mRNA levels for different versions of codon-optimized allotopic COX2 gene in comparison to GAPDH. V1 showed the highest expression of mRNA while V4 was lowest.
  • FIG. 11 B shows SDS PAGE profiles for different versions of codon-optimized allotopic COX2 in a COX2 null cell line upon stable selection.
  • V1 and V4 demonstrated the highest level of protein expression.
  • FIG. 12 shows a plasmid sequence of a COX2 variant according to embodiments.
  • Mitochondrial DNA in humans is a 16,569 base pair doublestranded circular DNA that encodes 13 vital proteins of the electron transport chain. Mitochondria occupy a central position in the overall metabolism of eukaryotic cells. Oxidative phosphorylation (OXPHOS), the Krebs's cycle, the urea cycle, the heme biosynthesis and the fatty acid oxidation take place within the organelle. Recently, another major role for mitochondria in determining the cellular life span was established, as they are recognized to be a major early mediator in the apoptotic cascade. Mitochondria are also a major producer of reactive oxygen species (ROS) causing oxidative stress and therefore inducers of cell death.
  • ROS reactive oxygen species
  • Mitochondrial pathologies are considered among the most common genetically determined diseases and are a health concern because they lack effective treatments or therapies.
  • Mitochondrion is assembled with proteins encoded by genes distributed between mitochondrial and nuclear genomes. These genes include those encoding the structural proteins of the respiratory chain complexes I - V, their associated substrates and products, the proteins necessary for mitochondrial biogenesis, the apparatus to import cytoplasmically synthesized precursors and the proteins necessary for mitochondrial assembly and turnover.
  • Mitochondrial DNA (mtDNA) disorders include Leigh syndrome, leukodystrophy w/complex II deficiency, cardiomyopathy & encephalopathy (complex I deficiency), optic atrophy and ataxia (complex II deficiency), hypokalemia and lactic acidosis, hepatopathy & ketoacidosis, hypertrophic cardiomyopathy, liver failure, renal tubulopathy (w/complex III deficiency) and encephalopathy (w/complex V deficiency), autosomal progressive external ophthalmoplegia, mitochondrial neurogastrointestinal encephalomyopathy, Alpers-Huttenlocher syndrome, ataxia neuropathy syndromes, infantile myopathy/spinal muscular atrophy and hypotonia.
  • mtDNA Mitochondrial DNA
  • a pediatric patient is diagnosed with Leigh syndrome (early onset), a genetic condition that affects the central nervous system.
  • the infant appeared healthy at birth but gradually presented symptoms as cells in the nervous system began to break down or degenerate. Symptoms included feeding problems, seizures and continuous crying.
  • a pediatrician conducts a genetic analysis to identify the gene responsible for the disease in the patient: MT-ATP6 gene from making ATP. This is the most common mtDNA change in Leigh syndrome and prevents the MT-ATP6 gene from making ATP.
  • allotopic expression is used to introduce in the nucleus a wild-type (i.e. , healthy) copy of the gene mutated in the mitochondrial genome and import normal copies of the gene product into mitochondria from the cytosol.
  • the gene is introduced into the patient via allotopic expression using the methods described herein.
  • An expression vector is produced in the form of a recombinant vector.
  • the vector includes: a nucleic acid sequence encoding a mitochondrion-targeting signal (i.e., an MTS sequence), a sequence that encodes the protein to be delivered and a 3' nucleic acid sequence. Additional codon sequences are introduced into the vector to (a) reduce hydrophobicity of the gene product, (b) reduce a localized charge of the gene product and (c) cause ribosome stalling.
  • the vector is introduced into the patient.
  • the vector can be, for example, a plasmid, or 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, a Herpes Simplex Virus (HSV).
  • an integrating viral vector e.g., a retrovirus, an adeno-associated virus (AAV), or a lentivirus
  • a non-integrating viral vector such as an adenovirus, an alphavirus, a Herpes Simplex Virus (HSV).
  • HSV Herpes Simplex Virus
  • a 17-year-old male patient visits a healthcare provider and presents signs/symptoms of apical hypertrophic cardiomyopathy and neuropathy due to a mitochondrial disorder.
  • the provider measures mitochondrial energy-generating system (MEGS) capacity in the muscle along with enzyme analysis in muscle and fibrobasts.
  • MEGS mitochondrial energy-generating system
  • relevant portions of the patient’s mitochondrial DNA were 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.
  • Reduced complex V activity was measured in the patient’s fibroblasts and muscle tissue and was confirmed in cybrid clones containing patient-derived mitochondrial DNA.
  • Allotopic expression is used to introduce in the nucleus a wild-type (i.e. , healthy) copy of the gene mutated in the mitochondrial genome and import normal copies of the gene product into mitochondria from the cytosol.
  • the gene is introduced into the patient via allotopic expression using the methods described herein.
  • An expression vector is produced in the form of a recombinant vector.
  • the vector includes: a nucleic acid sequence encoding a mitochondrion-targeting signal (i.e. , an MTS sequence), a sequence that encodes the protein to be delivered and a 3' nucleic acid sequence. Additional codon sequences are introduced into the vector to (a) reduce hydrophobicity of the gene product, (b) reduce a localized charge of the gene product and (c) cause ribosome stalling.
  • the vector is introduced into the patient.
  • the vector can be, for example, a plasmid, or a virus, such as an integrating viral vector (e.g., a retrovirus, an adeno-associated virus (AAV), or a lentivirus) or a nonintegrating viral vector, such as an adenovirus, an alphavirus, a Herpes Simplex Virus (HSV).
  • an integrating viral vector e.g., a retrovirus, an adeno-associated virus (AAV), or a lentivirus
  • a nonintegrating viral vector such as an adenovirus, an alphavirus, a Herpes Simplex Virus (HSV).
  • HSV Herpes Simplex Virus
  • the patient’s condition gradually improves. Approximately one month after treatment, the patient has no signs/symptoms of the disease. The physician conducts regular testing of MT-ATP6 gene expression and activity.
  • LHON Leber hereditary optic neuropathy
  • sudden vision loss is an inherited form of vision loss. It often starts with a painless clouding or blurring in one or both eyes, and then worsens with a loss of sharpness and loss of color vision. LHON affects the central vision needed for detailed tasks such as reading, driving and recognizing faces. LHON can lead to one becoming legally blind.
  • LHON is a mitochondrial disease caused by a mutation in the mitochondrial DNA. Because it is a mitochondrial disease, it is only inherited through the mother. Individuals who have lost their 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 the cells in the optic nerve die.
  • LHON causative mutations affect mitochondrial DNA (mtDNA) genes encoding for NADH dehydrogenase (ND) subunits of the respiratory chain complex I, leading to a subacute and catastrophic degeneration of retinal ganglion cells (RGCs) (with the final outcome of optic nerve atrophy).
  • mtDNA mitochondrial DNA
  • ND NADH dehydrogenase
  • RRCs retinal ganglion cells
  • the majority of LHON cases are caused by one of the three mtDNA missense point mutations at positions m.3460G>A/MT-ND1 , m.11778G>A/MT-ND4 and m.14484T>C/MT-ND6.
  • a 19-year-old male patient visits a healthcare provider and presents complaints of sudden vision loss, particularly to his central vision.
  • a physician conducts a genetic analysis to identify the gene responsible for the disease: a missense point mutations at positions m.3460G>A/MT- ND1.
  • allotopic expression is used to introduce in the nucleus a wild-type (i.e. , healthy) copy of the gene mutated in the mitochondrial genome and import normal copies of the gene product into mitochondria from the cytosol.
  • the gene is introduced into the patient via a modified adeno-associated virus gene therapy to complement the defective ND4 gene by the allotopic expression of the wild-type ND4 subunit from the nucleus followed by mitochondrial import of the protein product.
  • the vector is introduced by intravitreal injection (IVT). Within six months of treatment, the patient’s vision has improved (i.e., to about 80% of normal). The physician conducts regular testing of the patient’s vision.
  • IVT intravitreal injection
  • Embodiments include methods of treating pathogenesis or defects in the mitochondrial respiratory chain or the oxidative phosphorylation system.
  • the methods can include allotopic expression of a wild-type version of a mutated gene (e.g., a gene with a deleterious mutation).
  • Embodiments also include a method for slowing or retarding aging processes in humans by allotopic expression of one or more genes in human cells.
  • Embodiments also include methods of treating aging-associated diseases by allotopic expression of one or more genes in human cells.
  • the gene is a mitochondrial gene.
  • a method of allotopic expression disclosed herein expresses, e.g., 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 a wild-type gene product that is introduced in the cell nucleus (i.e. , to replace a mutated gene from the mitochondrial genome).
  • a method disclosed herein leads to expression of a gene product by, e.g., 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%, 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%.
  • a therapeutic disclosed herein is capable of reducing the signs/symptoms of a mitochondrial disease or senescence-associated disease or disorder by, e.g., 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.
  • a therapeutic is capable of reducing the number of signs/symptoms of a mitochondrial disease or a senescence-associated disease or disorder in an individual by, e.g., 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%, 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% as compared to a patient not receiving the same treatment.
  • a therapeutic disclosed herein is capable of reducing signs/symptoms in an individual suffering from a mitochondrial disease or a senescence-associated disease or disorder by, e.g., 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.
  • a therapeutic is capable of reducing signs/symptoms in an individual suffering from a mitochondrial disease or a senescence-associated disease or disorder by, e.g., 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%, 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% as compared to a patient not receiving the same treatment.
  • a therapeutic disclosed herein is capable of reducing signs/symptoms of aging in an individual by, e.g., 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.
  • a therapeutic is capable of reducing signs/symptoms of aging in a subject by, e.g., 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%, 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% as compared to a patient not receiving the same treatment.

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Abstract

L'ADN mitochondrial (ADNmt) chez l'homme est un ADN circulaire double brin à 16 569 paires de bases qui code 13 protéines vitales de la chaîne de transport d'électrons. Des modes de réalisation de l'invention comprennent l'expression allotopique de gènes d'ADNmt pour compenser une déficience fonctionnelle résultant d'une lésion ou de mutations sous-jacentes d'ADNmt. Dans une expression allotopique, des gènes qui sont normalement exprimés uniquement à partir du génome mitochondrial sont modifiés à des fins d'expression à l'aide d'une transcription nucléo-cytosolique et d'une machinerie. Les procédés peuvent être utilisés thérapeutiquement afin de traiter des maladies provoquées par des mutations d'ADN mitochondrial. Les procédés peuvent également être utilisés afin de ralentir le processus de vieillissement et/ou de réduire les effets du vieillissement.
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WO2025064906A1 (fr) * 2023-09-21 2025-03-27 The Board Of Regents Of The University Of Texas System Peptides de signal ciblant les mitochondries endogènes et leurs utilisations

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