US20200054682A1 - Methods and compositions for treating mitochondrial disease or disorders and heteroplasmy - Google Patents

Methods and compositions for treating mitochondrial disease or disorders and heteroplasmy Download PDF

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US20200054682A1
US20200054682A1 US16/539,993 US201916539993A US2020054682A1 US 20200054682 A1 US20200054682 A1 US 20200054682A1 US 201916539993 A US201916539993 A US 201916539993A US 2020054682 A1 US2020054682 A1 US 2020054682A1
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mitochondria
mtdna
cells
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Satoshi Gojo
Daisuke KAMI
Hideki Maeda
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Imel Biotherapeutics Inc
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Definitions

  • the present invention provides a composition of cells with reduced mitochondrial DNA and/or replacement of mitochondrial DNA, methods for their production, and methods for treating various diseases associated with genetic or age-related mitochondrial dysfunctions.
  • Mitochondria play a major and critical role in cellular homeostasis, and are involved in a diverse range of disease processes. They participate in intracellular signaling, apoptosis and perform numerous biochemical tasks, such as pyruvate oxidation, the Krebs cycle, and metabolism of amino acids, fatty acids, nucleotides and steroids. One crucial task is their role in cellular energy metabolism. This includes ⁇ -oxidation of fatty acids and production of ATP by means of the electron-transport chain and the oxidative-phosphorylation system.
  • the mitochondrial respiratory chain consists of five multi-subunit protein complexes embedded in the inner membrane, comprising: complex I (NADH-ubiquinone oxidoreductase), complex II (succinate-ubiquinone oxidoreductase), complex III (ubiquinol-ferricytochrome c oxidoreductase), complex IV (cytochrome c oxidoreductase), and complex V (FIFO ATPase).
  • complex I NADH-ubiquinone oxidoreductase
  • complex II succinate-ubiquinone oxidoreductase
  • complex III ubiquinol-ferricytochrome c oxidoreductase
  • complex IV cytochrome c oxidoreductase
  • V FIFO ATPase
  • the mammalian mitochondrial genome is a small, circular, double-stranded molecule containing 37 genes, including 13 protein-encoding genes, 22 transfer RNA (tRNA) genes and two ribosomal RNA (rRNA) genes. Of these, 24 (22 tRNAs and two rRNAs) are needed for mitochondrial DNA translation, and 13 encode subunits of the respiratory chain complexes.
  • nuclear DNA nDNA encodes most of the approximately 900 gene products in the mitochondria.
  • Mitochondrial disease or disorders are a clinically heterogeneous group of disorders that are characterized by dysfunctional mitochondria. Disease onset can occur at any age and can manifest with a wide range of clinical symptoms. Mitochondrial disease or disorders can involve any organ or tissue, characteristically involve multiple systems, typically affecting organs that are highly dependent on aerobic metabolism, and are often relentlessly progressive with high morbidity and mortality. Mitochondrial disease or disorders are the most common group of inherited metabolic disorders and are among the most common forms of inherited neurological disorders.
  • Mitochondrial disease or disorders can be caused by mutations in genes in the nuclear DNA (nDNA) and/or mitochondrial DNA (mtDNA) that encode structural mitochondrial proteins or proteins involved in mitochondrial function. While some mitochondrial disorders only affect a single organ (e.g., the eye in Leber hereditary optic neuropathy [LHON]), many involve multiple organ systems and often present with prominent neurologic and myopathic features. Even though tissues with high energy demand, such as brain, muscle, and eye, are more frequently involved, patients' phenotype can be extremely varied and heterogeneous. This variation is due in part because of several factors, such as, the dual genetic control (nDNA and mtDNA), level of heteroplasmy (percentage of mutated DNA in single cells and tissues), tissue energy demand, maternal inheritance, and mitotic segregation.
  • heteroplasmy Many patients with a mitochondrial disease or disorder have a mixture of mutated and wild-type mtDNA (known as heteroplasmy); the proportion of mutated and wild-type mtDNA is a key factor that determines whether a cell expresses a biochemical defect.
  • the majority of pathogenetic mtDNA mutations are heteroplasmic, with a mixture of mutated and wild-type mtDNA inside an individual cell.
  • High levels of heteroplasmy refer to cells with high levels of mutant mtDNA and low levels of wild-type mtDNA
  • low levels of heteroplasmy refer to cells with low levels of mutant mtDNA and high levels of wild-type mtDNA.
  • mutated and wild-type mtDNA are very important for determining the cellular phenotype. For example, cells become respiratory deficient if they contain high levels of mutated mtDNA and low levels of wild-type mtDNA (that is, high levels of heteroplasmy). The threshold at which this deficiency occurs depends on the precise mutation and the cell type.
  • mutated mtDNA typically, high percentage levels are required to result in cellular defects, but some mtDNA mutations only generate a deficiency if present at very high levels (typically mt tRNA mutations) and others (such as single, large-scale mtDNA deletions) produce a deficiency when there is ⁇ 60% deleted mtDNA.
  • mt tRNA mutations typically mt tRNA mutations
  • others such as single, large-scale mtDNA deletions
  • NARP neurogenic weakness
  • clinical phenotypes in MELAS and MERRF correlate with heteroplasmy (see, e.g., Chinnery, P. F., et al., Brain 120 (Pt 10), 1713-1721 (1997)).
  • mitochondrial transfer protocols have attempted to add mitochondria without depletion of endogenous mtDNA, but this approach has been found to be inefficient or harmful to a cell.
  • mitochondrial transfer using simple coincubation has been reported to be ineffective and not equally efficient among different cell types.
  • Additional techniques to transfer have involved injection using invasive instruments, which caused harm to the recipient cell, or other invasive instruments, such as nanoblades, but all were less efficient than coincubation (Caicedo et al, Stem Cells International, (2017), vol. 2017, Article ID 7610414, 23 pages).
  • a method of generating a mitochondria replaced cell comprising: (a) contacting a recipient cell with an agent that reduces endogenous mtDNA copy number; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mtDNA copy number in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mtDNA has been partially reduced, and (2) exogenous mitochondria from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell.
  • a method of treating a subject in need of mitochondrial replacement comprising (a) generating a mitochondria replaced cell ex vivo or in vitro, comprising the steps of (i) contacting a recipient cell with an agent that reduces mtDNA copy number (ii) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the mtDNA copy number in the recipient cell; and (iii) co-incubating (1) the recipient cell from step (ii) in which the endogenous mtDNA has been partially reduced, and (2) exogenous mitochondria from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell; and (b) administering a therapeutically effective amount of the mitochondria replaced recipient cell from step (a) to the subject in need of mitochondrial replacement.
  • a method of treating a subject having or suspected of having an age-related disease comprising: (a) generating a mitochondria replaced cell ex vivo or in vitro, comprising the steps of: (i) contacting a recipient cell with an agent that reduces mtDNA copy number; (ii) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the mtDNA copy number in the recipient cell; and (iii) co-incubating (1) the recipient cell from step (ii) in which the endogenous mtDNA has been partially reduced, and (2) exogenous mitochondria from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell; and (b) administering a therapeutically effective amount of the mitochondria replaced recipient cell from step (a) to the subject having or suspected of having an age-related disease.
  • a method of treating a subject having or suspected of having a mitochondrial disease or disorder comprising: (a) generating a mitochondria replaced recipient cell ex vivo or in vitro, comprising the steps of: (i) contacting a recipient cell with an agent that reduces mtDNA copy number; (ii) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the mtDNA copy number in the recipient cell; and (iii) co-incubating (1) the recipient cell from step (ii) in which the endogenous mtDNA has been partially reduced, and (2) exogenous mitochondria from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell; and (b) administering a therapeutically effective amount of the mitochondria replaced recipient cell from step (a) to the subject having or suspected of having a mitochondrial disease or disorder.
  • the exogenous mitochondria is a functional mitochondria.
  • the exogenous mitochondria comprises wild-type mtDNA.
  • the exogenous mitochondria is isolated mitochondria.
  • the isolated mitochondria is an intact mitochondria.
  • the exogenous mitochondria is allogeneic.
  • Also provided herein is a method of generating a mitochondria replaced cell, comprising (a) contacting a recipient cell with an agent that reduces endogenous mtDNA copy number; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mtDNA copy number in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mtDNA has been partially reduced, and (2) exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mtDNA into the recipient cell, thereby generating a mitochondria replaced cell.
  • the disclosure also provides a method of treating a subject in need of mitochondrial replacement, comprising (a) generating a mitochondria replaced cell ex vivo or in vitro, comprising the steps of: (i) contacting a recipient cell with an agent that reduces mtDNA copy number, (ii) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the mtDNA copy number in the recipient cell; and (iii) co-incubating (1) the recipient cell from step (ii) in which the endogenous mtDNA has been partially reduced, and (2) exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mtDNA into the recipient cell, thereby generating a mitochondria replaced cell; and (b) administering a therapeutically effective amount of the mitochondria replaced recipient cell from step (a) to the subject in need of mitochondrial replacement.
  • a method of treating a subject having or suspected of having an age-related disease comprising: (a) generating a mitochondria replaced cell ex vivo or in vitro, comprising the steps of: (i) contacting a recipient cell with an agent that reduces mtDNA copy number; (ii) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the mtDNA copy number in the recipient cell; and (iii) co-incubating (1) the recipient cell from step (ii) in which the endogenous mtDNA has been partially reduced, and (2) exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mtDNA into the recipient cell, thereby generating a mitochondria replaced cell; and (b) administering a therapeutically effective amount of the mitochondria replaced recipient cell from step (a) to the subject having or suspected of having an age-related disease.
  • a method of treating a subject having or suspected of having a mitochondrial disease or disorder comprising: (a) generating a mitochondria replaced recipient cell ex vivo or in vitro, comprising the steps of: (i) contacting a recipient cell with an agent that reduces mtDNA copy number; (ii) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the mtDNA copy number in the recipient cell; and (iii) co-incubating (1) the recipient cell from step (ii) in which the endogenous mtDNA has been partially reduced, and (2) exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mtDNA into the recipient cell, thereby generating a mitochondria replaced cell; and (b) administering a therapeutically effective amount of the mitochondria replaced recipient cell from step (a) to the subject having or suspected of having a mitochondrial disease or disorder.
  • the agent that reduces endogenous mtDNA copy number is selected from the group consisting of a polynucleotide encoding a fusion protein comprising a mitochondrial-targeted sequence (MTS) and an endonuclease, a polynucleotide encoding an endonuclease, and a small molecule.
  • the small molecule is a nucleoside reverse transcriptase inhibitor (NRTI).
  • the polynucleotide is comprised of messenger ribonucleic acid (mRNA) or deoxyribonucleic acid (DNA).
  • the recipient cell transiently expresses the fusion protein.
  • the endonuclease is selected from the group consisting of XbaI, EcoRI, BamHI, HindIII, PstI, Cas9, zinc finger nuclease (ZFN), and transcription activator-like effector nuclease (TALEN).
  • the MTS targets a mitochondrial matrix protein.
  • the mitochondrial matrix protein is selected from the group consisting of cytochrome c oxidase subunit IV, cytochrome c oxidase subunit VIII, and cytochrome c oxidase subunit X.
  • the agent that reduces endogenous mtDNA copy number reduces about 5% to about 99% of the endogenous mtDNA copy number. In certain embodiments, the agent that reduces endogenous mtDNA copy number reduces about 30% to about 70% of the endogenous mtDNA copy number. In further embodiments, the agent that reduces endogenous mtDNA copy number reduces about 50% to about 95% of the endogenous mtDNA copy number. In yet further embodiments, the agent that reduces endogenous mtDNA copy number reduces about 60% to about 90% of the endogenous mtDNA copy number. In some embodiments, the agent that reduces endogenous mtDNA copy number reduces mitochondrial mass.
  • Also provided herein is a method of generating a mitochondria replaced cell, comprising: (a) contacting a recipient cell with an agent that reduces mitochondrial function; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mitochondrial function in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mitochondrial function has been partially reduced, and (2) exogenous mitochondria from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell.
  • the present disclosure also provides a method of generating a mitochondria replaced cell, comprising: (a) contacting a recipient cell with an agent that reduces mitochondrial function; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mitochondrial function in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mitochondrial function has been partially reduced, and (2) exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mtDNA into the recipient cell, thereby generating a mitochondria replaced cell.
  • the agent that reduces mitochondrial function transiently reduces endogenous mitochondrial function. In other embodiments, the agent that reduces mitochondrial function permanently reduces endogenous mitochondrial function.
  • the subject in need of mitochondrial replacement has a dysfunctional mitochondria; a disease selected from the group consisting of an age-related disease, a mitochondrial disease or disorder, a neurodegenerative disease, a retinal disease, diabetes, a hearing disorder, a genetic disease; or a combination thereof.
  • the neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, Friedreich's ataxia, Charcot Marie Tooth disease and leukodystrophy.
  • the retinal disease is selected from the group consisting of age-related macular degeneration, macular edema and glaucoma.
  • the age-related disease is selected from the group consisting of an autoimmune disease, a metabolic disease, a genetic disease, cancer, a neurodegenerative disease, and immunosenescence.
  • the metabolic disease is diabetes.
  • the neurodegenerative disease is Alzheimer's disease, or Parkinson's disease.
  • the genetic disease is selected from the group consisting of Hutchinson-Gilford Progeria Syndrome, Werner Syndrome, and Huntington's disease.
  • the mitochondrial disease or disorder is caused by mitochondrial DNA abnormalities, nuclear DNA abnormalities, or both.
  • the mitochondrial disease or disorder caused by mitochondrial DNA abnormalities is selected from the group consisting of chronic progressive external ophthalmoplegia (CPEO), Pearson syndrome, Kearns-Sayre syndrome (KSS), diabetes and deafness (DAD), mitochondrial diabetes, Leber hereditary optic neuropathy (LHON), LHON-plus, neuropathy, ataxia, and retinitis pigmentosa syndrome (NARP), maternally-inherited Leigh syndrome (MILS), mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), myoclonic epilepsy and ragged-red fiber disease (MERRF), familial bilateral striatal necrosis/striatonigral degeneration (FBSN), Lucas disease, aminoglycoside-induced Deafness (AID), and multiple deletions of mitochondrial DNA syndrome.
  • CPEO chronic progressive external ophthalmoplegia
  • KSS
  • the mitochondrial disease or disorder caused by nuclear DNA abnormalities is selected from the group consisting of Mitochondrial DNA depletion syndrome-4A, mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase gamma (POLG)-related disorders, sensory ataxia neuropathy dysarthria ophthalmoplegia (SANDO), leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LB SL), co-enzyme Q10 deficiency, Leigh syndrome, mitochondrial complex abnormalities, fumarase deficiency, ⁇ -ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenase complex deficiency (PDHC), pyruvate carboxylase deficiency (PCD), carnitine
  • the endogenous mtDNA encodes for a dysfunctional mitochondria.
  • the endogenous mtDNA comprises mutant mtDNA.
  • the endogenous mtDNA in the recipient cell comprises wild-type mtDNA.
  • the endogenous mtDNA comprises mtDNA associated with a mitochondrial disease or disorder.
  • the endogenous mtDNA is heteroplasmic.
  • the recipient cell has endogenous mitochondria that is dysfunctional.
  • the mitochondria replaced cell has a total mtDNA copy number no greater than about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, or more, relative to the total mtDNA copy number of the recipient cell prior to contacting with the agent that reduces endogenous mtDNA copy number.
  • the recipient cell is an animal cell or a plant cell. In certain embodiments, the animal cell is a mammalian cell. In specific embodiments, the recipient cell is a somatic cell. In other embodiments, the recipient cell is a bone marrow cell. In some embodiments, the bone marrow cell is a hematopoietic stem cell (HSC), or a mesenchymal stem cell (MSC). In other embodiments, the recipient cell is a cancer cell. In further embodiments, the recipient cell is a primary cell. In yet further embodiments, the recipient cell is an immune cell. In specific embodiments, the immune cells is selected from the group consisting of a T cell, a phagocyte, a microglial cell, and a macrophage. In further embodiments, the T cell is a CD4+ T cells. In other embodiments, the T cell is a CD8+ T cells. In certain embodiments, the T cell is a chimeric antigen receptor (CAR) T cell.
  • CAR chimeric antigen receptor
  • the exogenous mitochondria and/or exogenous mtDNA is stable.
  • the exogenous mtDNA alters heteroplasmy in the recipient cell.
  • the method further comprises delivering a small molecule, a peptide, or a protein.
  • the disclosure also provides methods provided herein, further comprising contacting the recipient cell with a second active agent prior to co-incubating the recipient cell with exogenous mitochondria and/or exogenous mtDNA.
  • the second active agent is selected from the group consisting of large molecules, small molecules, or cell therapies, and the second active agent is optionally selected from the group consisting of rapamycin, NR (Nicotinamide Riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxolone (RTA408), KH176, Vatiquinone (Epi743), thioctic acid, A0001 (alpha-tocopherolquinone), mitochondrial CoQ10 (MitoQ), SkQ1 (Visomitin), resveratrol, curcumin, ketogenic treatment, hypoxia, and an activator of endocytosis.
  • the activator of endocytosis is a modulator of cellular metabolism.
  • the modulator of cellular metabolism comprises nutrient starvation, a chemical inhibitor, or a small molecule.
  • the chemical inhibitor or the small molecule is an mTOR inhibitor.
  • the mTOR inhibitor comprises rapamycin or a derivative thereof.
  • the disclosure also provides a composition comprising one or more mitochondria replaced cells obtained by the method of: (a) contacting a recipient cell with an agent that reduces endogenous mtDNA copy number; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mtDNA copy number in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mtDNA has been partially reduced, and (2) exogenous mitochondria from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell, wherein said mitochondria replaced cell comprises greater than 5% of exogenous mtDNA.
  • the disclosure further provides a composition of one or more mitochondria replaced cells obtained by the method of (a) contacting a recipient cell with an agent that reduces endogenous mtDNA copy number (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mtDNA copy number in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mtDNA has been partially reduced, and (2) exogenous mtDNA from healthy donor, for a sufficient period of time to non-invasively transfer exogenous mtDNA into the recipient cell, thereby generating a mitochondria replaced cell, wherein said mitochondria replaced cell comprises greater than 5% of exogenous mtDNA.
  • the one or more mitochondria replaced cells comprise a total mtDNA copy number no greater than about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, or more, relative to the total mtDNA copy number of the recipient cell prior to contacting with the agent that reduces endogenous mtDNA copy number.
  • compositions for use in a method of generating one or more mitochondria replaced cells comprising an agent that reduces endogenous mtDNA copy number, and a second active agent.
  • the composition further comprising one or more recipient cells, or a combination thereof.
  • the composition further comprising exogenous mtDNA exogenous mtDNA and/or exogenous mitochondria.
  • the agent that reduces endogenous mtDNA copy number is a small molecule or a fusion protein.
  • the small molecule is a nucleoside reverse transcriptase inhibitor (NRTI).
  • the fusion protein comprises an endonuclease that cleaves mtDNA and a mitochondrial target sequence (MTS).
  • the endonuclease cleaves wild-type mtDNA.
  • the endonuclease is selected from the group consisting of XbaI, EcoRI, BamHI, HindIII, PstI, Cas9, zinc finger nuclease (ZFN), and transcription activator-like effector nuclease (TALEN).
  • the MTS targets a mitochondrial matrix protein.
  • the mitochondrial matrix protein is cytochrome c oxidase subunit IV, cytochrome c oxidase subunit VIII, and cytochrome c oxidase subunit X.
  • the fusion protein is transiently expressed.
  • the reduction of endogenous mtDNA copy number is a partial reduction.
  • the partial reduction is a reduction of about 5% to about 99% of endogenous mtDNA.
  • the partial reduction is a reduction of about 50% to about 95% of the endogenous mtDNA copy number.
  • the partial reduction is a reduction of about 60% to about 90% of the endogenous mtDNA copy number.
  • the disclosure also provides a composition comprising one or more mitochondria replaced cells obtained by the method of: (a) contacting a recipient cell with an agent that reduces mitochondrial function; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce endogenous mitochondrial function in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mitochondrial function has been partially reduced, and (2) exogenous mitochondria from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell, wherein said mitochondria replaced cell comprises greater than 5% of exogenous mtDNA.
  • composition of one or more mitochondria replaced cells obtained by the method of: (a) contacting a recipient cell with an agent that reduces mitochondrial function; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce endogenous mitochondrial function in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mitochondrial function has been partially reduced, and (2) exogenous mtDNA from healthy donor, for a sufficient period of time to non-invasively transfer exogenous mtDNA into the recipient cell, thereby generating a mitochondria replaced cell, wherein said mitochondria replaced cell comprises greater than 5% of exogenous mtDNA.
  • the one or more mitochondria replaced cells comprise a total mtDNA copy number no greater than about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, or more, relative to the total mtDNA copy number of the recipient cell prior to contacting with the agent that reduces endogenous mtDNA copy number.
  • the disclosure also provides a composition for use in a method of generating one or more mitochondria replaced cells comprising an agent that reduces mitochondrial function, and a second active agent.
  • the composition further comprises an exogenous mitochondria, one or more recipient cells, or a combination thereof.
  • the composition further comprises exogenous mtDNA.
  • the one or more mitochondria replaced cells comprise wild-type exogenous mtDNA.
  • compositions further comprising a second active agent.
  • the second active agent is selected from the group consisting of large molecules, small molecules, or cell therapies, and the second active agent is optionally selected from the group consisting of rapamycin, NR (Nicotinamide Riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxolone (RTA408), KH176, Vatiquinone (Epi743), thioctic acid, A0001 (alpha-tocopherolquinone), mitochondrial CoQ10 (MitoQ), SkQ1 (Visomitin), resveratrol, curcumin, ketogenic treatment, hypoxia, and an activator of endocytosis.
  • the activator of endocytosis is an activator of a clathrin-independent endocytosis pathway. In some embodiments, the activator of endocytosis is an activator of a clathrin-independent endocytosis pathway. In further embodiments, the clathrin-independent endocytosis pathway is selected from the group consisting of a CLIC/GEEC endocytic pathway, Arf6-dependent endocytosis, flotillin-dependent endocytosis, macropinocytosis, circular doral ruffles, phagocytosis, and trans-endocytosis.
  • the clathrin-independent endocytosis pathway is macropinocytosis.
  • the activator of endocytosis comprises nutrient stress, and/or an mTOR inhibitor.
  • the mTOR inhibitor comprises rapamycin or a derivative thereof.
  • the disclosure further provides a composition where the total mtDNA copy number of the one or more mitochondria replaced cells comprises greater than 5% of exogenous mtDNA. In some embodiments, the total mtDNA copy number of the one or more mitochondria replaced cells comprises greater than 30% of exogenous mtDNA. In specific embodiments, the total mtDNA copy number of the one or more mitochondria replaced cells comprises greater than 50% of exogenous mtDNA. In further embodiments, the total mtDNA copy number of the one or more mitochondria replaced cells comprises greater than 75% of exogenous mtDNA.
  • the exogenous mitochondria is isolated mitochondria. In specific embodiments, the isolated mitochondria is intact. In some embodiments, the exogenous mitochondria and/or exogenous mtDNA is allogeneic. In specific embodiments, the exogenous mitochondria further comprises exogenous mtDNA.
  • the one or more cells are animal cells or plant cells.
  • the animal cells are mammalian cells.
  • the cells are somatic cells.
  • the somatic cells are epithelial cells.
  • the epithelial cells are thymic epithelial cells (TECs).
  • the somatic cells are immune cells.
  • the immune cells are T cells.
  • the T cells are CD4+ T cells.
  • the T cells are CD8+ T cells.
  • the T cells are chimeric antigen receptor (CAR) T cells.
  • the immune cells are phagocytic cells.
  • the one or more mitochondria replaced cells are bone marrow cells.
  • the bone marrow cells are a hematopoietic stem cell (HSC), or a mesenchymal stem cell (MSC).
  • the one or more mitochondria replaced cells are more viable than an isogenic cell having homoplasmic endogenous mtDNA. In other embodiments, the one or more mitochondria replaced cells are efficacious in killing a cancer cell, treating an age-related disease, treating a mitochondrial disease or disorder, treating a neurodegenerative disease, treating diabetes, or a genetic disease.
  • the composition further comprises a small molecule, a peptide, or a protein.
  • compositions for use in delaying senescence and/or extending lifespan in a cell comprising: (a) a senescent or near senescent cell having endogenous mitochondria; (b) isolated exogenous mitochondria from a non-senescent cell; and (c) an agent that reduces endogenous mtDNA copy number.
  • the agent is a fusion protein.
  • the fusion protein comprises an endonuclease that cleaves mtDNA and a mitochondrial target sequence (MTS).
  • MTS mitochondrial target sequence
  • the endonuclease cleaves wild-type mtDNA.
  • the endonuclease is selected from the group consisting of XbaI, EcoRI, BamHI, HindIII, PstI, Cas9, zinc finger nuclease (ZFN), and transcription activator-like effector nuclease (TALEN).
  • the MTS targets a mitochondrial matrix protein.
  • the mitochondrial matrix protein is selected from the group consisting of cytochrome c oxidase subunit IV, cytochrome c oxidase subunit VIII, and cytochrome c oxidase subunit X.
  • the fusion protein is transiently expressed in said senescent or near senescent cell.
  • the disclosure further provides a composition for use in delaying senescence and/or extending lifespan in a cell comprising: (a) a senescent or near senescent cell having endogenous mitochondria; (b) isolated exogenous mitochondria from a non-senescent cell; and (c) an agent that reduces mitochondrial function.
  • the agent that reduces mitochondrial function transiently reduces endogenous mitochondrial function.
  • the agent that reduces mitochondrial function permanently reduces endogenous mitochondrial function.
  • the exogenous mitochondria from the non-senescent cell has enhanced function relative to the endogenous mitochondria.
  • the composition for use in delaying senescence and/or extending lifespan in a cell further comprises a second active agent.
  • the second active agent is selected from the group consisting of large molecules, small molecules, or cell therapies, and the second active agent is optionally selected from the group consisting of rapamycin, NR (Nicotinamide Riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxolone (RTA408), KH176, Vatiquinone (Epi743), thioctic acid, A0001 (alpha-tocopherolquinone), mitochondrial CoQ10 (MitoQ), SkQ1 (Visomitin), resveratrol, curcumin, ketogenic treatment, hypoxia, and an activator of endocytosis.
  • the activator of endocytosis is an activator of a clathrin-independent endocytosis pathway.
  • the clathrin-independent endocytosis pathway is selected from the group consisting of a CLIC/GEEC endocytic pathway, Arf6-dependent endocytosis, flotillin-dependent endocytosis, macropinocytosis, circular doral ruffles, phagocytosis, and trans-endocytosis.
  • the clathrin-independent endocytosis pathway is macropinocytosis.
  • said activator of endocytosis comprises nutrient stress, and/or an mTOR inhibitor.
  • said mTOR inhibitor comprises rapamycin or a derivative thereof.
  • the disclosure also provides a pharmaceutical composition comprising an isolated population of mitochondria replaced cells having an exogenous mitochondria from a healthy donor, wherein the cells are obtained by any of the methods provided herein for obtaining a mitochondrial replaced cell.
  • the disclosure provides a pharmaceutical composition comprising an isolated population of mitochondria replaced cells having an exogenous mtDNA from a healthy donor, wherein the cells are obtained by any of the methods provided herein for obtaining a mitochondrial replaced cell.
  • the pharmaceutical composition comprising an isolated population of mitochondria replaced cells having an exogenous mtDNA from a healthy donor further comprises exogenous mitochondria.
  • a pharmaceutical composition comprising an exogenous mitochondria from a healthy donor are obtained by a method of generating a mitochondrial replaced cell that includes (a) contacting a recipient cell with an agent that reduces endogenous mtDNA copy number; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mtDNA copy number in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mtDNA has been partially reduced, and (2) exogenous mitochondria from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell.
  • the cells are obtained by a method comprising (a) contacting a recipient cell with an agent that reduces mitochondrial function; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mitochondrial function in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mitochondrial function has been partially reduced, and (2) exogenous mitochondria from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell.
  • the cells are obtained by a method comprising (a) contacting a recipient cell with an agent that reduces endogenous mtDNA copy number; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mtDNA copy number in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mtDNA has been partially reduced, and (2) exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mtDNA into the recipient cell, thereby generating a mitochondria replaced cell.
  • the cells are obtained by a method comprising: (a) contacting a recipient cell with an agent that reduces mitochondrial function; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mitochondrial function in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mitochondrial function has been partially reduced, and (2) exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mtDNA into the recipient cell, thereby generating a mitochondria replaced cell.
  • the cells are obtained by a method further comprising further comprising contacting the recipient cell with a second active agent prior to co-incubating the recipient cell with exogenous mitochondria and/or exogenous mtDNA.
  • the second active agent is selected from the group consisting of large molecules, small molecules, or cell therapies, and the second active agent is optionally selected from the group consisting of rapamycin, NR (Nicotinamide Riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxolone (RTA408), KH176, Vatiquinone (Epi743), thioctic acid, A0001 (alpha-tocopherolquinone), mitochondrial CoQ10 (MitoQ), SkQ1 (Visomitin), resveratrol, curcumin, ketogenic treatment, hypoxia, and an activator of endocytosis.
  • rapamycin NR (Nicotinamide Riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxo
  • the activator of endocytosis is a modulator of cellular metabolism.
  • the modulator of cellular metabolism comprises nutrient starvation, a chemical inhibitor, or a small molecule.
  • the chemical inhibitor or the small molecule is an mTOR inhibitor.
  • said mTOR inhibitor comprises rapamycin or a derivative thereof.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the cells are T cells. In other embodiments, the cells are hematopoietic stem cells.
  • FIG. 1A depicts a scheme for generation of a Mitochondria replaced cell (MirC).
  • FIG. 1B depicts a plasmid construct for the Mitochondrial Targeting Sequence (MTS)-XbaI restriction enzyme (XbaIR) plasmid.
  • FIG. 1C depicts that isolated mitochondrial DNA is digested at multiple sites by the XbaI restriction enzyme, whereas NotI digestion of mitochondrial DNA resulted in a single fragment, as predicted by Cambridge Reference Sequence (CRS) of mitochondrial DNA.
  • CRS Cambridge Reference Sequence
  • FIG. 1D depicts five XbaIR endonuclease sites (1193, 2953, 7440, 8286, 10256) on human mitochondrial DNA as predicted by Cambridge Reference Sequence (CRS).
  • FIG. 1E depicts microscopy of human dermal fibroblasts under phase contrast (left), immunofluorescence of green fluorescence protein (middle), and merged fields (right) after uptake of a fusion MTS-green fluorescence protein (GFP) plasmid using electroporator (Nucleofector). Top, low magnification. Bottom, high magnification.
  • GFP green fluorescence protein
  • FIG. 1F depicts the construct for the pCAGGS-MTS-EGFP-PuroR and pCAGGS-MTS-XbaIR-PuroR plasmids.
  • FIG. 1G depicts the localization of exogenous transgene products MTS-EGFP in mitochondria by mitochondria-specific staining with tetramethylrhodamine, methyl ester (TMRM).
  • TMRM tetramethylrhodamine
  • FIG. 2A depicts a scheme of the time schedule to compare the MTS-XbaIR endonuclease method (top) with the traditional method using ethidium bromide (EtBr) (middle), relative to non-contacted cells.
  • FIG. 2B depicts quantification of human (3-actin (Actb), left columns, and mitochondria DNA (mtDNA), right columns, following contact with either the MTS-XbaIR endonuclease method or the ethidium bromide treatment, relative to non-contacted cells.
  • Actb was used as a housekeeping gene.
  • FIG. 2C depicts a greater reduction in mitochondria following exposure to the gene transfer of MTS-XbaIR, relative to EtBr treatment, based on DsRed fluorescence that had been expressed in mitochondria.
  • FIG. 2D depicts semi-quantification of mitochondrial membrane potentials (surrogate marker for mitochondrial content) in cells contacted with the gene transfer of MTS-XbaIR or EtBr using FACS analyses by using TMRM, and shows that MTS-XbaIR resulted in a greater reduction in mitochondria.
  • FIG. 2E depicts a time course quantification of transgene expression in the gene transfer system over fourteen days.
  • FIG. 2F and FIG. 2G depicts fluorescent images ( FIG. 2F ) following the transfer of the plasmid carrying GFP prior to (“pre”) and after (“post”) puromycin section, and quantification of the GFP/Mitochondria ratio ( FIG. 2G ) demonstrates enrichment of the GFP plasmid post-puromycin selection.
  • FIG. 3A depicts a scheme of the time schedule for mitochondria replacement.
  • TF Gene transfection of XbaIR or Mock; Puro: Puromycin for enrichment of gene transferred cells; U+: Addition of uridine to rescue ⁇ ( ⁇ ) cells devoid of mitochondrial ATP production; Mt Tx: Mitochondria transfer; NHDF: Normal Human Dermal Fibroblasts, EPC100: Placental venous endothelium-derived cell lines.
  • FIG. 3B depicts reduction in mitochondria on Day 6 following the gene transfer of XbaIR (top), but not following the transfer of the negative control vector expression GFP (bottom), as measured by TMRM staining.
  • FIG. 3D depicts Photographs from time lapse movie: Upper left: Cocultivation of ⁇ ( ⁇ ) cells with isolated and DsRed-marked mitochondria; Upper right: ⁇ ( ⁇ ) cells as a control; Lower left: Cocultivation of NHDF with mitochondria; Lower right: Cocultivation of mock transfectant of NHDF with mitochondria;
  • FIG. 3E depicts a series of 10 still images from time lapse movie depicted in FIG. 3D , arranged chronologically, vertically;
  • FIG. 3F depicts measurement of DsRed labeled mitochondria by FACS analysis and revealed that the present invention (“DsRed-Mt EPC100”) resulted in increased uptake of exogenous mitochondria compared to previously described methods.
  • FIG. 3G and FIG. 311 depict microscopy images of DsRed labeled mitochondria ( FIG. 3G ) and phase contrast ( FIG. 3H ) after mitochondria transfer in ⁇ (0) cells treated with, or without, antimycin, and demonstrates that no engulfment of exogenous mitochondria occurred in cells with complete destruction of mitochondria;
  • FIG. 3I depicts a series of 5 still images from time lapse movie depicted in FIG. 3G , arranged chronologically, vertically.
  • FIG. 3J depicts quantification of fluorescent intensities of DsRed-labeled isolated exogenous mitochondria, measured every 24 hours in ⁇ ( ⁇ ) cells, or in ⁇ ( ⁇ ), mock transfected cells, or untreated cells (add on Mt) co-incubated with the Ds-Red mitochondria.
  • FIG. 4A depicts a scheme for measuring the fate of donor mitochondria following the engulfment by the recipient cells using DsRed marked mitochondria as donors and EGFP marked cells as recipients.
  • FIG. 4B depicts representative images from movies to observe engulfed exogenous mitochondria (indicated as red) in recipient cells with GFP marked mitochondria. Movies were recorded by using superfine microscopy, and few fusion images were recognized, and a major of the donor mitochondria separately exist to the pre-existing mitochondria.
  • FIG. 4C depicts three dimensional reconstitutional photograph of the fusion.
  • FIG. 4D depicts photos of NHDF transferred of gene coding DsRed fused with mitochondria transfer signal.
  • FIG. 4E depicts photos of EPC100 transferred of gene coding EGFP fused with TFAM.
  • FIG. 4F depicts time course of mitochondria transfer using DsRed marked cells as recipients and TFAM targeted EGFP as donor mitochondria.
  • FIG. 4G depicts exogenous TFAMs were stably engrafted in the pre-existing mitochondria, after the exogenous mitochondria were transiently contacted with the recipient cell, suggesting that mitochondrial nucleoids including TFAMs were transferred to the pre-existing mitochondria via the transient contact, analogous to mouth-to-mouth feeding.
  • FIG. 5A depicts the whole circular mitochondrial DNA with the Cambridge reference sequence (CRS) of human mitochondrial DNA indicating hypervariable (“HV”) regions 1/2, and 5 primers to identify the difference between NHDF and EPC100;
  • CRS Cambridge reference sequence
  • HV hypervariable
  • FIG. 5B depicts DNA sequencing data for the nucleotides surrounding hmt16362 in NHDF ctrl recipient cells (SEQ ID NO: 1), EPC100 ctrl donor cells (SEQ ID NO: 2), NHDF derived ⁇ ( ⁇ ) cells without mitochondria replacement (SEQ ID NO: 3), and NHDF derived ⁇ ( ⁇ ) cells with mitochondria replacement (SEQ ID NO: 4) and demonstrated that NHDF derived ⁇ ( ⁇ ) cells with mitochondria replacement (SEQ ID NO: 4) changed from A in the original recipient cells to G in the donor mtDNA at hmt16362.
  • FIG. 5C depicts the hmt16318-F (SEQ ID NO: 6) and hmt16414-R (SEQ ID NO: 9) set of primers used for amplification of the HV1 region of human mitochondrial DNA D-Loop (SEQ ID NO: 8) surrounding hmt16362 and the NHDF specific probe (SEQ ID NO: 5) and the EPC100 specific probe (SEQ ID NO: 7) that were designed for the TaqMan SNP genotyping assay.
  • FIG. 5D depicts quantification of the NHDF specific hmtDNA (left) and EPC100 specific hmtDNA (right) in the parental NHDF and EPC100 cell lines, or in NHDF cells treated with XbaIR, with (XbaIR Mt+) or without mitochondria (XbaIR Mt ⁇ ) from EPC100 cells, and revealed that EPC100 mitochondria was successfully transferred in XbaIR Mt+ cells, as evaluated by using single nucleotide polymorphism assay (SNP).
  • SNP single nucleotide polymorphism assay
  • FIG. 6A depicts representative oxygraphies from a mitochondria functioning assay performed using Oroboros Oxygraph-2k, and demonstrated that NHDF cells with mitochondria replaced ( ⁇ ( ⁇ ) Mt) (bottom) regained the mitochondrial function, relative to control NHDF cells (top), and ⁇ ( ⁇ ) NHDF cells without mitochondrial replacement (middle).
  • the machine depicts respiratory flow in red line (pmol/sec/1 ⁇ 10 6 cells, right axis) and oxygen concentrations in blue line ( ⁇ M, left axis).
  • FIG. 6B depicts that the respiratory flows (routine, Electron Transfer System (ETS), ROX), free routine activities (mitochondrial ATP production), proton leakage, and coupling efficiency in each stage demonstrated that mitochondrial replacement in NHDF cells ( ⁇ ( ⁇ ) Mt) regained the mitochondrial function, relative to NHDF control cells, and NHDF without mtDNA replacement ( ⁇ ( ⁇ )).
  • ETS Electron Transfer System
  • ROX free routine activities
  • proton leakage proton leakage
  • FIG. 6C depicts a time-lapse microphotograph, which enabled to estimate continuous cell number based on the surface area of cells, and demonstrated that ⁇ ( ⁇ ) cells were quiescent state between days 3 to 12, whereas mitochondria replaced cells regained the growth capability after day 6.
  • FIG. 6D depicts a scheme of the protocol used to examine the molecular mechanism for macropinocytosis, which involved transfecting NHDF cells with the MTS-XbaIR-P2A-PuroR plasmid, selecting with puromycin, and then serum starving the cells for 60 min, or treating the cells with palmitic acid (PA) or rapamycin for 24 hours.
  • PA palmitic acid
  • FIG. 6E - FIG. 611 depict quantification of the WES' analysis for phosphorylation of S6 kinase ( FIG. 6E ) and phosphorylation of AMPK ( FIG. 6G ), and corresponding WESTM blots ( FIG. 6F ) and ( FIG. 6H ), respectively, which demonstrated that AMPK is activated and mTOR is completely suppressed in ⁇ ( ⁇ ) cells.
  • Rapa Rapamycin
  • PA Palmitic acid
  • EAA ⁇ Essential amino acid-deficient.
  • FIG. 6I depicts the protocol used to examine the effect of mTOR mediated macropinocytosis in the setting of MirC generation protocol.
  • FIG. 6J - FIG. 6L depict quantification ( FIG. 6J and FIG. 6K ) AND FACS analysis ( FIG. 6L ) and of DsRed labeled mitochondrial uptake in control (top), mock transfected cells (middle), and ⁇ ( ⁇ ) cells, with or without rapamycin treatment, or with or without palmitic acid (PA) treatment.
  • ⁇ ( ⁇ ) cells exhibited greater uptake of mitochondria, relative to control of mock TF cells, and the uptake of mitochondria was significantly increased after rapamycin treatment, whereas palmitic acid decreased mitochondria uptake in ⁇ ( ⁇ ) cells.
  • FIG. 7A depicts the whole mtDNA sequence showing the Leigh syndrome associated mutation of 10158T>C in the respiratory chain complex I (CI) subunit of the ND3 gene in mitochondrial DNA.
  • FIG. 7B depicts DNA sequencing data for the nucleotides surrounding hmt10158 within ND3 in EPC100 cells (top; SEQ ID NO: 10) and Leigh syndrome (7SP) fibroblasts (bottom; SEQ ID NO: 11), and revealed the mutation, 10158T>C, with a mosaic of C in the major wave and T in the minor wave, indicating the heteroplasmy.
  • FIG. 7C depicts photographs from time lapse movies that demonstrated similar behavior in both ⁇ ( ⁇ ) 7SP fibroblasts, with and without exogenous mitochondria, as in NHDF experiments.
  • FIG. 7E depicts DNA sequencing data for the nucleotides surrounding hmt10158 in 7SP ctrl recipient cells (SEQ ID NO: 14), EPC100 ctrl donor cells (SEQ ID NO: 12), 7SP derived ⁇ ( ⁇ ) cells without mitochondria replacement (SEQ ID NO: 13), and 7SP derived ⁇ ( ⁇ ) cells with mitochondria replacement (SEQ ID NO: 15), and revealed that 7SP ctrl cells are heteroplasmic (majority 10158C; SEQ ID NO: 14), whereas EPC100 has only T in the same site in mitochondrial DNA (SEQ ID NO: 12).
  • the ⁇ ( ⁇ ) cells stem from 7SP cells expressed the same wave as the original (SEQ ID NO: 13), whereas mitochondria replaced 7SP cells demonstrated T as major wave (SEQ ID NO: 15).
  • FIG. 7F depicts the hmt10085-F (SEQ ID NO: 17) and hmt10184-R (SEQ ID NO: 20) set of primers used for amplification of ND3 of human mitochondrial DNA (SEQ ID NO: 16) surrounding the Leigh syndrome associated SNP at hmt10158, and the EPC100 specific probe (SEQ ID NO: 18) and the 7SP specific probe (SEQ ID NO: 19) that were designed for the TaqMan SNP genotyping assay.
  • the ND3 peptide sequence is also depicted (SEQ ID NO: 46).
  • FIG. 7G depicts quantification of the percentage of hmt10158 heteroplasmy in each cell group evaluated by SNP assay, and revealed that exogenous normal sequence (“healthy”) dominated up to 80% in mitochondria replaced 7SP cells, in spite that the original heteroplasmy of mutant sequence was over 90%. In case of mock transfectant, the heteroplasmy did not significantly change, and maintained the almost same ratio.
  • FIG. 7H and FIG. 7I depict quantification of heteroplasmy level percentage ( FIG. 7H ) and absolute mtDNA copy number ( FIG. 7I ) in three independent experiments in 7SP cells treated with mock control and subjected to mitochondrial transfer.
  • FIG. 7J depicts a series of 10 still images from the time lapse movie depicted in FIG. 7C , arranged chronologically, vertically.
  • FIG. 8A depicts microscopic photos in ⁇ ( ⁇ ) mitochondria replaced 7SP fibroblasts with time, compared with the original 7SP fibroblasts and ⁇ ( ⁇ ) 7SP fibroblasts, and revealed that the growth of mitochondria replaced cells recovered to near control level.
  • FIG. 8B depicts time-lapse-estimated cellular growth in 7SP fibroblasts, ⁇ ( ⁇ ) 7SP fibroblast, and ⁇ ( ⁇ ) 7SP fibroblasts with mitochondria replacement, and revealed that ⁇ ( ⁇ ) 7SP fibroblasts were quiescent, whereas mitochondria replaced 7SP cells recovered cellular growth to levels equivalent to the original 7SP fibroblasts around day 12.
  • FIG. 8C depicts senescence in 7SP fibroblasts around population doubling levels (PDL) 25, which was extended to about PDL 63 in ⁇ ( ⁇ ) 7SP fibroblasts with healthy mitochondria replacement performed at PDL 8, indicating the lifespan extension of ⁇ ( ⁇ ) 7SP fibroblasts with healthy mitochondria replacement.
  • PDL population doubling levels
  • FIG. 8D depicts the increase in PDL produces an increase in cell size (left), which is reverted following mitochondria replacement, and is maintained even past PDL 50 (right).
  • FIG. 8E depicts short tandem repeat (STR) assay, which discriminates cells with different origins and identifies contamination of different type of cells.
  • STR short tandem repeat
  • FIG. 8F depicts RT-PCR quantification of telomerase in 7SP fibroblasts and mitochondria replaced cells for different PDLs, relative to HeLa and EPC100, indicating that the cells were not transformed into cancer cells.
  • FIG. 9A depicts oxygraphies in 7SP fibroblasts at different PDLs following mitochondria replacement using Oroboros 02k according to coupling-control protocol (CCP), and the kinetics demonstrated that mitochondria function dropped at early PDL followed by a gradual recovery that eventually surpassed the original capability, relative to the original 7SP fibroblasts as control.
  • CCP coupling-control protocol
  • FIG. 9B and FIG. 9C depict that the respiratory flows (routine, Electron Transfer System (ETS), ROX), free routine activities (mitochondrial ATP production), proton leakage, and coupling efficiency ( FIG. 9B ), as well as the flux control ratios (FCRs), ROVE, L/E, R/E, and (R-L)/E ( FIG. 9C ) regained to near control levels in mitochondrial replaced cells ( ⁇ ( ⁇ ) Mt) after approximately PDL30.
  • ETS Electron Transfer System
  • ROX free routine activities
  • mitochondrial ATP production proton leakage
  • FCRs flux control ratios
  • ROVE L/E
  • R/E R/E
  • R-L R-L/E
  • FIG. 10A depicts microscopy images of NHDF, 7SP, an 7SP MirC cells under basal conditions or following reperfusion using H 2 O 2 , and show that 7SP cells are highly sensitive to H 2 O 2 relative to NHDF cells, whereas the 7SP MirC are not.
  • FIG. 10B - FIG. 10D depict FACS analysis ( FIG. 10B ) and quantification of Annexin V ( FIG. 10C ) and propidium iodine (PI; FIG. 10D ) positive cells following no treatment or treatment with H 2 O 2 , and demonstrate that 7SP cells are highly sensitive to H 2 O 2 relative to NHDF cells, whereas the 7SP MirC are not.
  • FIG. 10E depicts microscopy images of NHDF, 7SP, an 7SP MirC cells under basal conditions or following starvation conditions (EAA ⁇ ), and show that 7SP cells are highly sensitive to starvation conditions relative to NHDF cells whereas the 7SP MirC are not.
  • FIG. 10F - FIG. 1011 depict FACS analysis ( FIG. 10F ) and quantification of Annexin V ( FIG. 10G ) and PI ( FIG. 10H ) positive cells following no treatment or starvation, and demonstrate that 7SP cells are highly sensitive to starvation conditions relative to NHDF cells, whereas the 7SP MirC are not.
  • FIG. 11 depicts quantification of the expression levels of representative SASP cytokines, IL-6 and IL-8, chemokine, CXCL-1, and growth factor, ICAM1 for NHDF, 7SP fibroblast, and 7SP fibroblast-derived MirC cells, whose PDLs were almost the same, about 15 to 20, which demonstrated a significant reduction in IL-6, indicating a reversal of SASP in the MirC. GAPDH was used for normalization.
  • FIG. 12A depicts the scheme for generation of induced pluripotent stem cells (iPSCs) from mitochondria replaced 7SP fibroblasts.
  • iPSCs induced pluripotent stem cells
  • FIG. 12B - FIG. 12D depicts alkaline phosphatase (AP) staining and quantification as an indicator of iPSCs, which were generated from either 7SP fibroblasts, 7SP fibroblast-derived MirC, or mock transfectants originated from 7SP fibroblasts.
  • Microscopic FIG. 12B left panel; 7SP fibroblasts, middle panel; 7SP fibroblast-derived MirC, right panel: Mock transfectant of 7SP fibroblast
  • FIG. 12C left panel; 7SP fibroblasts, middle panel; 7SP fibroblast-derived MirC, right panel: Mock transfectant of 7SP fibroblast microscopy of AP stained cells, as well as quantification of the AP stained cells ( FIG. 12D ), revealed that mitochondrial replacement in either NHDF or 7SP fibroblasts following Xb
  • FIG. 12E depicts colony formation of iPSCs derived from mitochondria replaced 7SP fibroblasts. Photos of 3 representative colonies in 75 days and 170 days after gene transfer of reprogramming factors.
  • FIG. 12F depicts immunohistochemical staining for OCT3/4, NANOG, TRA1-80, and TRA-160 in iPSCs generated from 7SP fibroblasts following mitochondrial replacement, which are representative markers for pluripotent stem cells;
  • FIG. 12G depicts mitochondrial DNA copy number in iPSCs derived from 7SP fibroblast derived MirC, compared with the original 7SP fibroblasts and the standard human iPSCs (201B7) as references, and revealed that iPSCs had limited number of mitochondrial DNA that was similar that of the standard human iPSCs (201B7).
  • FIG. 12H and FIG. 12I depict the percentage of heteroplasmy ( FIG. 12H ) and absolute mtDNA copy number ( FIG. 12I ) in iPSCs derived from 7SP fibroblast-derived MirC in 170 days after the reprogramming procedure, and revealed that 7SP fibroblast-derived MirC that formed iPSC showed negligible levels of mutated genome sequence, reduced total mtDNA, and nearly 100% donor mtDNA in at least three clones, suggesting that the change of the heteroplasmy in MirC could be reverted into the original state, and different from the mitochondrial replacement therapy in IVF.
  • FIG. 13A depicts a scheme of the protocol for mitochondrial transfer from a donor cell to a recipient cell, where the donor cell and recipient cell are from different stages of a lifespan.
  • FIG. 13B depicts DNA sequencing data for the nucleotides surrounding hmt16145 in NHDF ctrl recipient cells (SEQ ID NO: 21) which have the genotype hmt16145 A, and TIG1 ctrl donor cells (SEQ ID NO: 22) which have the genotype hmt16145 G.
  • FIG. 13C depicts quantitation of hmt16145 heteroplasmy level (%) by SNP assay of the cells from mitochondria replaced cells (MirC) (“old” NHDF recipient cells with mitochondrial transfer of mitochondria from “young” TIG1 donor cells) and indicated that greater than 90% of the mtDNA in the NHDF derived MirC cells with mitochondria replacement from TIG1 derived mitochondria donor cells was hmt16145 G (i.e., from TIG1 mtDNA), whereas 100% of the NHDF ctrl cell's mtDNA was hmt16145 A.
  • FIG. 13D depicts quantification of the population doubling level (PDL) versus time (days) (left), and doubling time (hours) versus population doubling level (right) in recipient NHDF cells transfected with MTS-GFP (“mock”), or MTS-XbaIR (“MirC”) and coincubated with exogenous mitochondria from TIG1 donor cells, or untransfected (“Ctrl”).
  • MTS-GFP MTS-GFP
  • MirC MTS-XbaIR
  • MirC with “young” donor TIG1 embryonic lung cell (PDL 10) to an “old” normal human dermal fibroblasts (NHDF) recipient cell (PDL 41) showed an extension in lifespan, as indicated by the upward shift in PDL (left) and rightward shift in PDL (right).
  • FIG. 13E depicts quantification of the population doubling level (PDL) versus time (days) (left), and doubling time (hours) versus population doubling level (right) in normal human dermal fibroblasts transfected with MTS-GFP plus mitochondrial transfer (“mock”), MTS-XbaIR plus mitochondrial transfer (“MirC”), or untransfected (“Ctrl”).
  • Mitochondrial transfer from an “old” donor cell (PDL 49) to a “young” recipient cell (PDL ⁇ 21) showed reduction in lifespan, as indicated by the downward shift in PDL (left), and leftward shift in PFL (right).
  • FIG. 14A depicts quality assessment of mRNA generated by in vitro transcription, as measured by electrophoresis of mRNA for MTS-EGFP and MTS-XbaIR.
  • FIG. 14B depicts strong expression of the MTS-GFP transgene in mitochondria of T cells 24 hours following electroporation.
  • FIG. 14C depicts FACS analysis of GFP expression in T cells following transfection of the MTS-GFP mRNA by electroporation, and revealed that GFP expression is present in nearly all T cells.
  • FIG. 14D depicts FACS analysis of DsRed labeled mitochondria and demonstrates that the MTS-XbaIR construct robustly degraded the endogenous mitochondria, whereas the MTS-GFP did not.
  • FIG. 14E depicts a scheme of the protocol design for determining the optimal time period of mitochondrial co-incubation.
  • FIG. 14F depicts fluorescent images of control electroporated cells (upper panels) and MTS-GFP electroporated cells (lower panels) at 4 hr, 2 days, 4 days, 6 days, and 8 days after electroporation (EP), and indicated the MTS-GFP construct displayed high expression within 4 hours post-electroporation and was nearly absent by day 6.
  • FIG. 14G and FIG. 14 I 1 depict electrophoresis ( FIG. 14H ) and quantification ( FIG. 14G ) of GFP in cells receiving the MTS-GFP mRNA, relative to GAPDH. The peak expression occurred at day 4, and expression was lost by day 6.
  • FIG. 14I depicts quantification of XbaIR transcript levels, at 4 hr, day 2 (d2), day 4 (d4), day 6 (d6) and day 8 (d8), indicating that the transcript expressions of the endonuclease were quite highest at 4 hours post-gene transfer.
  • FIG. 14J depicts quantification of mitochondrial contents (12S rRNA) in cells subjected to MTS-XbaI, and demonstrated that mitochondria decreased to about 30% by day 2, and was maintained at less than 20% throughout the length of the experiment.
  • FIG. 15A depicts a scheme of the MirC protocol for human primary T cells, with electroporation at day 0, analysis at day 2, mitochondria (mt) transfer at day 7, SNP assays at day 9 and 14, and ddPCR heteroplasmy assay at day 14.
  • FIG. 15B depicts DNA sequencing data for the nucleotides surrounding hmtDNA 218 and hmtDNA 224 of the HV1 region of the human mitochondrial DNA D-loop in human primary NH T cell control recipient cells (top; SEQ ID NO: 23) and EPC100 control donor cells (bottom; SEQ ID NO: 24).
  • hmtDNA 218 and hmtDNA 224 were C/C (SEQ ID NO: 23) and T/T (SEQ ID NO: 24) for T cells and EPC100 cells, respectively.
  • FIG. 15C depicts the hmtHV1-F (SEQ ID NO: 26) and hmtHV1-R (SEQ ID NO: 27) set of primers used for amplification of the HV1 region of human mitochondrial DNA D-loop (SEQ ID NO: 25) surrounding the SNPs at hmtDNA 218 and hmtDNA 224, as well as the SNP assay Primer1-F (SEQ ID NO: 40), the SNP assay-Primer1-R (SEQ ID NO: 41), the N-terminal VIC labeled EPC100 specific probe (SEQ ID NO: 38), and the N-terminal FAM labeled T cell specific probe (SEQ ID NO: 39) that were designed for the TaqMan SNP genotyping assay.
  • FIG. 15D depicts quantification of the amount of exogenous mtDNA present in the recipient cells at day 7 and day 12 for mock (MTS-GFP) or MTS-XbaIR (XbaIR) treated cells following coincubation with exogenous mitochondria from donor EPC100 cells. Quantification of recipient and donor cells was performed as positive controls.
  • FIG. 15E depicts quantification of respirometry experiments performed using Oroboros 02k, and demonstrated a recovery of ATP production and coupling efficiency in human T cell-derived MirC, whereas ⁇ ( ⁇ ) human T cells that were generated by XbaIR mRNA transfer with electroporation maintained the loss of ATP production throughout the experiment.
  • FIG. 15F and FIG. 15G depict representative raw data using coupling-control protocol (CCP), and show that MirC T cells are able to restore mitochondrial respiration.
  • CCP coupling-control protocol
  • FIG. 16A depicts comparison viability (left panel) of mouse primary T cells cultured in RPMI1640 (top) or TexMACS (bottom) at day 2 (left side of left panel), day 4 (middle of left panel), and day 6 (right side of left panel), or CD3 expression (right panel), and demonstrated that RPMI1640 produced greater viability and higher cell count, as well as a slight increase in CD3 expression, relative to TexMACS culture medium.
  • FIG. 16B depicts qualitative analysis of GFP expression in T cells following electroporation (EP) with pmax GFP (middle), or MTS-GFP (right), or without electroporation (left), at 6 hours after EP (top left panel), day 2 after EP (top right panel), day 4 after EP (bottom left panel), and day 6 after EP (bottom right panel). Viability was not significantly affected following EP with MTS-GFP at day 2 or day 4.
  • FIG. 16C depicts qPCR quantification of XbaIR levels in T cells electroporated with the MTS-XbaIR vector at 4 hr, day 2, day 4, and day 6 following electroporation and indicated that the XbaIR expression slowly decreased.
  • FIG. 16D depicts quantification of 12S rRNA levels in T cells electroporated with MTS-XbaIR and indicated that the murine mtDNA was decreased by approximately 60% by day 4.
  • FIG. 16E depicts a scheme of the protocol used for MirC generation in T cells using mitochondrial coincubation on day 5.
  • FIG. 16F depicts FACS analysis of engulfed DsRed-labeled mitochondria 48 hours in the recipient T cells, following the co-incubation with isolated DsRed-labeled mitochondria and revealed a significant positive fraction (9.73%) of T cells expressing exogenous mitochondria in MTS-XbaIR (right), compared with 0.43% in control cells without electroporation (i.e., “add-on”).
  • FIG. 17A depicts DNA sequencing data for the nucleotides surrounding ND1 in mouse mtDNA C57BL6 recipient cells (“BL6”; top; SEQ ID NO: 34) which have the genotype mmt2766-A and mmt2767-T, and NZB donor cells (bottom; SEQ ID NO: 35), which have the genotype mmt2766-G and mmt2767-C.
  • FIG. 17B depicts the 2716-F (SEQ ID NO: 28) and 2883-R (SEQ ID NO: 33) set of primers used for amplification of ND1 of mouse mitochondrial DNA (SEQ ID NO: 32) surrounding the polymorphic nucleotides mmt2766 and mmt2767, and the BL6 specific probe (SEQ ID NO: 29) and the NZB specific probe (SEQ ID NO: 31), that were designed for the TaqMan SNP genotyping assay, as well as the BamH1-mND1-F primer (SEQ ID NO: 30) used to clone the nucleotide sequence in a plasmid for generation of a standard curve to enable absolute quantification.
  • the ND1 peptide sequence is also depicted (SEQ ID NO: 47).
  • FIG. 17C depicts quantification of mouse mtND1 heteroplasmy levels in BL6 recipient cells at day 7 and day 12 following control electroporation (columns 1 and 2, respectively) or MTS-XbaI electroporation and coincubation with isolated mitochondria from NZB cells (columns 3 and 4, respectively). Basal levels of BL6 (column 5) and NZB (column 6) cells were measured as controls.
  • FIG. 17D depicts measurement of telomere length following the treatment of old murine cells with the MTS-XbaIR mRNA and co-incubation with exogenous mitochondria from the young donor cells to generate the MirC (Young to Old: YtoO) and revealed an increase in the length of telomeres in MirC compared to the parental “Old” cells.
  • FIG. 17E depicts measurement of SASP associated cytokines CXCL1, ICAM1, IL-6, and IL-8 in the parental old T cell, or the MirC-derived T cell, and indicated that CXCL1 and IL6 were lower in the MirC-derived T cells.
  • FIG. 17F depicts measurement of DNA damage response in the MirC and the original T cells using the histone 2 A (H2A) phosphorylation antibody, which indicated that the positive fraction for DDR was lower in the MirC (1.53%), compared with the original T cells (4.75%).
  • H2A histone 2 A
  • FIG. 18A depicts a scheme of the in vivo ACT experiment using old mice with ACT of T cells from young mouse (Group 1), old mice with ACT (Group 2) or old mice with ACT of MirC derived from a T cell of an old mouse transferred with exogenous mitochondria from a young mouse (Group 3).
  • FIG. 18B depicts a representative image of tumor growth imaging performed during the experimental protocol.
  • FIG. 18C depicts the body weight of the mock, young T cell, or MirC groups, and reveals that no significant difference between the three groups was observed during the 25 days experiment.
  • FIG. 18D and FIG. 18E depict quantification of the individual ( FIG. 18D ) and mean ( FIG. 18E ) cancer mass size, and revealed that the MirC group reduced cancer mass size to levels equivalent to the Young T cell group (lower lines), whereas the mock group increased in cancer mass throughout the length of the experiment (top lines).
  • FIG. 18F depicts a scheme of the protocol used to analyze the present of infused T cells in the animals.
  • FIG. 18G depicts FACS analysis of peripheral blood (left panels) or spleen (right panels). Negative controls using C57BL/6 mice (left upper panel), and positive controls using GFP transgenic mice (left lower panel) were generated for both the peripheral blood and the spleen. Positive fractions of T cells expressing GFP fluorescence were recognized in both the peripheral blood and spleen, 0.057% and 0.9%, respectively.
  • FIG. 18H depicts immunofluorescence images of the transferred T cells detected in the mice on day 6 following transplantation.
  • FIG. 18I depicts the percentage of chimerism following infusion of the exogenous T cells in peripheral blood (PB) or the spleen after injection of 1 ⁇ 10 7 or 2 ⁇ 10 7 cells.
  • FIG. 19A and FIG. 19B depict evaluation of MTS-GFP transfection into hematopoietic cells (HSCs) using the X-001, Y-001, and T-030 programs (MTS-GFP1, 2, and 3, respectively) or pmax GFP as a positive control or Ctl EP as a negative control by microscopy ( FIG. 19A ) or FACS ( FIG. 19B ), and show that MTS-GFP1 was the optimal protocol for electroporating HSCs.
  • HSCs hematopoietic cells
  • FIG. 19C depicts 3-D confocal fluorescent imaging of the bone marrow-derived Sca-1 cells 48 hours after the co-incubation with DsRed-labeled mitochondria from EPC100 cells, and showed that the exogenous mitochondria were engulfed.
  • FIG. 19D depicts quantification of the mitochondrial transfer efficiency by FACS analysis of DsRed fluorescence, and revealed that a subpopulation of about 10% of the Sca-1 exhibited a right ward shift of the fluorescent.
  • FIG. 19E depicts the scheme used to generate HSC derived MirC by coincubating with exogenous mitochondria on day 4 and analyzing the MirC on day 6 by SNP assay.
  • FIG. 19F depicts the FACS sorting for the c-kit+, Sca-1+, Lineage ⁇ , CD34 ⁇ (called as KSLC) fraction of cells.
  • FIG. 19G depicts that the doubling time of the KSLC fraction was 19 hours.
  • FIG. 19I depicts the scheme used to evaluate HSC derived MirC.
  • FIG. 19I depicts quantification of the murine mtND1 heteroplasmy level percentage in murine KSLC-derived MirC or the parental recipient BL6 cells or NZB donor cells, and demonstrated that the MirC derived HSC expressed 99.9% of the polymorphism genotype of the donor cells on day 6 following the MTS-XbaI mRNA transfer with electroporation.
  • FIG. 20A depicts a 2-D plot of droplet digital PCR results in which sequences for mutated mtDNA and non-mutated mtDNA were analyzed in normal human dermal fibroblasts for tRNA Leu 3243 A>G, and show only detection of the non-mutant sequence (lower right quadrant) and no detection of the mutant sequence (upper left quadrant).
  • FIG. 20B depicts a 2-D plot of droplet digital PCR results in which sequences for mutated mtDNA and non-mutated mtDNA were analyzed in normal human dermal fibroblasts for ND3 10158 T>C, and show only detection of the non-mutant sequence (lower right quadrant) and no detection of the mutant sequence (upper left quadrant).
  • FIG. 20C depicts a 2-D plot of droplet digital PCR results in which sequences for mutated mtDNA and non-mutated mtDNA were analyzed in normal human dermal fibroblasts for ATP6 9185 T>C, and show only detection of the non-mutant sequence (lower right quadrant) and no detection of the mutant sequence (upper left quadrant).
  • FIG. 20D depicts a 2-D plot of droplet digital PCR results in which sequences for mutated mtDNA and non-mutated mtDNA were analyzed in primary skin fibroblasts from a patient with MELAS having an mtDNA A3243G mutation, and show the majority of cells had homoplasmy of mutated mtDNA (upper left quadrant).
  • FIG. 20E depicts a 2-D plot of droplet digital PCR results in which sequences for mutated mtDNA and non-mutated mtDNA were analyzed in primary skin fibroblasts from a patient with Leigh Syndrome having a mtDNA T10158C mutation of Complex I, ND3 gene, and showed a minor portion of double positive cells with heteroplasmy in a single cell level (upper right quadrant), a major population of homoplasmy of mutated mtDNA (lower right), and no population with homoplasmy of non-mutated mtDNA (upper left).
  • compositions that include one or more mitochondria replaced cells obtained by the methods provided herein.
  • the compositions can also include a second active agent that enhances the uptake of exogenous mitochondria, exogenous mtDNA, or a combination thereof, and/or an agent that reduces endogenous mtDNA copy number or reduces endogenous mitochondrial function.
  • the compositions can also include exogenous mitochondria and/or exogenous mtDNA, one or more recipient cells, or a combination thereof.
  • provided herein are methods and compositions for use in the treatment of a disease or disorder associated with dysfunctional mitochondria.
  • the methods and compositions provided herein can also be used to delay senescence, extend the lifespan, or enhance the function of a cell that has functional mitochondria, and is not limited to replacement of dysfunctional mitochondria.
  • the methods and compositions provided herein can also be used to replace functional mitochondria with exogenous mitochondria that is dysfunctional or exhausted, for example, to generate a disease model.
  • mitochondria replaced cell or MirC is intended to mean a cell having the substitution of endogenous mitochondria and/or mtDNA with exogenous mitochondria and/or mtDNA.
  • an exemplary mitochondria replaced cell involves the substitution of endogenous mtDNA that encodes dysfunctional mitochondria, such as mtDNA originating from a subject having a mitochondrial disease or disorder, with exogenous mtDNA that encodes functional mitochondria, such as mtDNA originating from a healthy subject.
  • exemplary MirC can also include a cell with endogenous mitochondria substituted with exogenous mitochondria.
  • the substitution of the endogenous mitochondria and/or mtDNA can also include, for example, functional endogenous mtDNA from one cell, such as from an old cell, that is substituted with functional exogenous mtDNA from a different cell, such as from a healthier cell that is from a young subject. It is further understood that healthy endogenous mitochondria and/or mtDNA can also be substituted with dysfunctional exogenous mitochondria and/or exogenous mtDNA such as, for example, to mimic a mitochondrial disease or disorder.
  • exemplary mitochondria and/or mtDNA replacement involves substitution of about 5% of more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 95% or more of the endogenous mitochondria and/or mtDNA.
  • the term “recipient cell,” “acceptor cell,” and “host cell” are interchangeable and refer to a cell receiving the exogenous mitochondria and/or mtDNA.
  • the exogenous mitochondria and/or mtDNA is from isolated mitochondria from a donor cell.
  • the donor cells and the recipient cells may be different or identical.
  • the donor cells and the recipient cells come from different or the same species.
  • the donor cells and the recipient cells come from different or the same tissues.
  • a healthy donor is intended to mean a donor that does not have a mitochondrial disease or disorder, age-related disease, or otherwise dysfunctional mitochondria.
  • a healthy donor has a wild-type mtDNA sequence, relative to the Cambridge Reference Sequence of the mitochondrial genome.
  • the terms “treat,” “treating,” and “treatment” refer to reduction in severity, progression, spread, and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage. “Treatment” is meant to include therapeutic treatment as well as prophylactic, or suppressive measures for the condition, disease or disorder.
  • agents when used in reference to depleting reducing mtDNA refers to an enzyme or compound that is capable of reducing mtDNA.
  • Preferred agents include restriction enzymes, such as XbaI, that cleave mtDNA at one or more sites, without producing toxicity in the recipient cell.
  • agents can also include an enzyme or compound that inhibit mtDNA synthesis or selectively promote degradation of the mitochondria.
  • the terms “reduce,” or “decrease” generally means a decrease of at least 5%, for example a decrease by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or any decrease between 5%-99% as compared to a reference level, as that term is defined herein. It is understood that a partial reduction or an agent that partially reduces endogenous mtDNA or decrease, as used herein, does not result in a complete depletion of all endogenous mtDNA (i.e., ⁇ 0 cells).
  • increase generally means an increase of at least 5%, for example an increase by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or more than 90%.
  • endogenous refers to originating or derived internally.
  • endogenous mitochondria are mitochondria that are native to a cell.
  • exogenous refers to cellular material (e.g., mitochondria or mtDNA) that is non-native to the host, such as cellular material that is derived externally. “Externally” typically means from a different source.
  • mitochondrial genomes are exogenous to host cells or host mitochondria when the mitochondrial genomes originate from different cell types or different species than the host cells or host mitochondria.
  • exogenous can also refer to mitochondrial genomes that are removed from mitochondria, manipulated, and returned to the same mitochondria.
  • the term “sufficient period of time” refers to an amount of time that produces the desired results. It is understood that the sufficient period of time will vary according to the experimental conditions, including but not limited to, the temperature, the amount of reagent used, and the cell type. Exemplary protocols are provided throughout as guidelines for the “sufficient period of time,” and a person skilled in the art would be able to identify the period of time that is sufficient without undue experimentation.
  • the term “majority” is intended to mean the greatest amount, relative to the other amounts being compared.
  • An exemplary majority when comparing two groups is an amount that is any integer greater than about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more, or about 95% or more, of the total population, including any integer in-between. It is understood that the majority will depend on the total population being compared, and can be amounts lower than 50% when there are three or more groups being compared.
  • non-invasively when used in reference to the transfer of exogenous material is intended to mean without the use of invasive instruments (e.g., nanoblade or electroporation), physical force (e.g., centrifugation), or harmful culture conditions (e.g., thermal shock).
  • invasive instruments e.g., nanoblade or electroporation
  • physical force e.g., centrifugation
  • harmful culture conditions e.g., thermal shock
  • the term “subject in need of mitochondrial replacement,” is intended to mean a subject that has or is predisposed to having a dysfunctional mitochondria.
  • the subject in need of mitochondrial replacement may be asymptomatic and in need of preventative care.
  • the subject in need of mitochondrial replacement may also be symptomatic and in need of treatment.
  • the subject in need of mitochondrial replacement has dysfunctional mitochondria that is not the result of an age-related disease or a mitochondrial disease or disorder.
  • the term “subject” is intended to mean a mammal.
  • a subject can be a human or a non-human mammal, such as a dog, cat, bovid, equine, mouse, rat, rabbit, or transgenic species thereof. It is understood that a “subject” can also refer to a “patient,” such as a human patient.
  • an effective amount refers to the amount of a composition of the invention effective to modulate, treat, or ameliorate any disease or disorder associated with heteroplasmy and/or dysfunctional mitochondria.
  • an effective amount can include, for example, a therapeutically effective amount, which refers to an effective amount in a therapy, or a biologically effective amount, which refers to an effective amount for a biological effect.
  • therapeutically effective amount and “effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent.
  • a given composition that will correspond to such an amount will vary depending upon various factors, such as the given composition, the pharmaceutical formulation, the route of administration, the type of condition, disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.
  • a therapeutically effective amount of an agent may be readily determined by one of ordinary skill by routine methods known in the art.
  • age-related disease refers to any number of conditions attributable to advancement in age. These conditions include, without limitation, osteoporosis, bone loss, arthritis, stiffening joints, cataracts, macular degeneration, metabolic diseases including diabetes mellitus, neurodegenerative diseases including Alzheimer's Disease and Parkinson's Disease, immunosenescence, and heart disease including atherosclerosis and dyslipidemia.
  • age related disease further encompasses neurodegenerative diseases, such as Alzheimer's Disease and related disorders, ALS, Huntington's disease, Parkinson's Disease, and cancer.
  • autoimmune disease is intended to mean a disease or disorder arising from immune reactions directed against an individual's own tissues, organs or a manifestation thereof or a resulting condition therefrom.
  • An autoimmune disease can refer to a condition that results from, or is aggravated by, the production of autoantibodies that are reactive with an autoimmune antigen or epitope thereof.
  • An autoimmune disease can be tissue- or organ-specific, or it can be a systemic autoimmune disease.
  • Systemic autoimmune diseases include connective tissue diseases (CTD), such as systemic lupus erythematosus (lupus; SLE), mixed connective tissue disease systemic sclerosis, polymyositis (PM), dermatomyositis (DM), and Sjögren's syndrome (SS). Additional exemplary autoimmune diseases further include rheumatoid arthritis, and anti-neutrophil cytoplasmic antibody (ANCA) polyangiitis.
  • CTD connective tissue diseases
  • SLE systemic lupus erythematosus
  • PM polymyositis
  • DM dermatomyositis
  • SS Sjögren's syndrome
  • Additional exemplary autoimmune diseases further include rheumatoid arthritis, and anti-neutrophil cytoplasmic antibody (ANCA) polyangiitis.
  • ANCA anti-neutrophil cytoplasmic antibody
  • genetic disease refers to a disease caused by an abnormality, such as a mutation, in the nuclear genome.
  • exemplary genetic diseases include, but are not limited to, Hutchinson-Gilford Progeria Syndrome, Werner Syndrome, and Huntington's disease.
  • cancer includes but is not limited to, solid cancer and blood borne cancer.
  • cancer and cancer refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • mitochondrial disease or disorder and “mitochondrial disorder” are interchangeable and refer to a group of conditions caused by inherited or acquired damage to the mitochondria causing an energy shortage within those areas of the body.
  • exemplary organs effected by mitochondrial disease or disorder include those that consume large amounts of energy such as the liver, muscles, brain, eye, ear, and the heart. The result is often liver failure, muscle weakness, fatigue, and problems with the heart, eyes, and various other systems.
  • mitochondrial DNA abnormalities refer to mutations in mitochondrial genes whose products localize to the mitochondrion, and not observed in the cells of healthy subjects.
  • exemplary diseases associated with mitochondrial DNA abnormalities include, for example, chronic progressive external ophthalmoplegia (CPEO), Pearson syndrome, Kearns-Sayre syndrome (KSS), diabetes and deafness (DAD), leber hereditary optic neuropathy (LHON), LHON-plus, neuropathy, ataxia, and retinitis pigmentosa syndrome (NARP), maternally-inherited Leigh syndrome (MILS) also known as Leigh syndrome caused by mutant mtDNA, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), myoclonic epilepsy and ragged-red fiber disease (MERRF), familial bilateral striatal necrosis/striatonigral degeneration (FBSN), Lucas disease, aminoglycoside-induced Deafness (AID), and multiple deletions
  • CPEO chronic progressive external
  • nuclear DNA abnormalities within the context of mitochondrial disease or disorder refer to mutations or changes in the coding sequence of nuclear genes whose products localize to the mitochondrion.
  • mitochondrial disease or disorders associated with nuclear mutations include Mitochondrial DNA depletion syndrome-4A, mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase gamma (POLG)-related disorders, sensory ataxia neuropathy dysarthria ophthalmoplegia (SANDO), leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL), co-enzyme Q10 deficiency, Leigh syndrome (caused by nuclear mutations), mitochondrial complex abnormalities, fumarase deficiency, ⁇ -ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl-CoA ligase de
  • disfunctional mitochondria refer to mitochondria that are in opposition to functional mitochondria.
  • exemplary dysfunctional mitochondria include mitochondria that are incapable of synthesizing or synthesize insufficient amounts of ATP by oxidative phosphorylation.
  • functional mitochondria refers to mitochondria that consume oxygen and produce ATP.
  • mutation refers to any changing of the structure of a gene, resulting in a variant (also called “mutant”) form. Mutations in a gene may be caused by the alternation of single base in DNA, or the deletion, insertion, or rearrangement of larger sections of genes or chromosomes. In some embodiments, the mutation can affect the function or the resulting protein. For example, a mutation in a single nucleotide of DNA (i.e., point mutation) in the coding region of a protein can result in a codon that encodes for a different amino acid (i.e., missense mutation). It is understood that this different amino acid can alter the structure of the protein, and that in certain circumstances, as described herein, can alter the function of the organelle, such as the mitochondrion.
  • heteroplasmy and “heteroplasmic” refer to the occurrence of more than one type of mitochondrial DNA genome in an individual or sample. Varying degrees of heteroplasmy are associated with varying degrees of the physiological conditions described herein. Heteroplasmy may be identified by means known to the art, and the severity of the physiological condition associated with specific nucleotide alleles is expected to vary with the percentage of such associated alleles within the individual.
  • wild-type when used in the context of mitochondrial DNA refers to the genotype of the typical form of a species as it occurs in nature.
  • An exemplary reference genome for the wild-type human mtDNA genome includes the Cambridge Reference Sequence (CRS).
  • the term “old” or “older” is intended to mean that the source of the mtDNA is from a subject that is greater in age than the recipient cell, or from a cell in a population of cells that have doubled their population a greater number of times since their culture in vitro (i.e., population doubling level, PDL) relative to the recipient cell.
  • the term “young” or “younger” is intended to mean that the source of the mtDNA is from a subject that is lower in age than the recipient cell, or from a cell in a population of cells that have doubled their population a fewer number of times since their culture in vitro (i.e., population doubling level, PDL) relative to the recipient cell.
  • mitochondria As used herein, the term “isolated” when used in reference to mitochondria refers to mitochondria that have been physically separated or removed from the other cellular components of its natural biological environment.
  • intact and “intact mitochondria” refers to mitochondria comprising an outer and an inner membrane, an inter-membrane space, the cristae (formed by the inner membrane) and the matrix.
  • exemplary intact mitochondria contain mtDNA.
  • intact mitochondria are functional mitochondria.
  • intact dysfunctional mitochondria can also be used in the present invention.
  • autologous is intended to mean biological compositions obtained from the same subject.
  • allogeneic is intended to mean biological compositions obtained from the same species, but a different genotype than that of the subject receiving the biological composition.
  • animal cell is intended to mean any cell from a eukaryotic organism. It is understood that an animal cell can include mammalian and non-mammalian species, such as amphibians, fish, insects (e.g., Drosophila ), and worms (e.g., Caenorhabditis elegans ).
  • fusion protein refers to a sequence of amino acids, predominantly, but not necessarily, connected to each other by peptidic bonds, wherein a part of the sequence is derived (i.e., has sequence similarity to sequences) from one origin (native or synthetic) and another part of the sequence is derived from one or more other origin.
  • exemplary fusion proteins can be prepared by construction of an expression vector that codes for the whole of the fusion protein (coding for both sections, such as a mitochondrial-targeted sequence and an endonuclease) so that essentially all the bonds are peptidic bonds. It is also understood that the fusion may be made by chemical conjugation, such as by using any of the known methodologies used for conjugating peptides.
  • mitochondria-targeted sequence and “mitochondrial targeting sequence (MTS)” are interchangeable and refer to any amino acid sequence capable of causing the transport of an enzyme, peptide, sequence, or compound attached to it into the mitochondria.
  • the MTS is a human MTS. In another embodiment, the MTS is from another species.
  • Non-limiting examples of such sequences are the cytochrome c oxidase subunit X (COX10) MTS (MAASPHTLSSRLLTGCVGGSVWYLERRT, SEQ ID NO: 36), and the cytochrome c oxidase subunit VIII (COX8) MTS (MSVLTPLLLRSLTGSARRLMVPRA, SEQ ID NO: 37).
  • Additional non-limiting examples of MTS sequences are the natural MTS of each individual mitochondrial protein that is encoded by the nuclear DNA, translated (produced) in the cytoplasm and transported into the mitochondria, as well as citrate synthase (cs), lipoamide deydrogenase (LAD), and C6ORF66 (ORF).
  • the various MTS may be exchangeable for each mitochondrial enzyme among themselves. Each possibility represents a separate embodiment of the fusion protein for use of the present invention.
  • small molecule refers to a compound that affects a biological process and has molecular weight of about 900 Daltons or lower.
  • An exemplary small molecule had a molecular weight between about 300 and about 700 daltons.
  • the terms “about” or “approximately” when used in conjunction with a number refer to any number within 1, 5, 10, 15 or 20% of the referenced number.
  • somatic cell refers to any differentiated cell forming the body of an organism, apart from stem cells, progenitor cells, and germline cells (i.e., ovogonies and spermatogonies) and the cells derived therefrom (e.g., oocyte, spermatozoa).
  • stem cells progenitor cells
  • germline cells i.e., ovogonies and spermatogonies
  • the cells derived therefrom e.g., oocyte, spermatozoa
  • oocyte spermatozoa
  • Somatic cells are obtained from animals, preferably human subjects, and cultured according to standard cell culture protocols available to those of ordinary skill in the art.
  • endocytosis pathway refers to the cellular process in which cells take in molecules from their surroundings.
  • the endocytosis pathway can be “clathrin-dependent,” which requires the recruitment of clathrin to help curve the plasma membrane into the vesicle which absorbs the molecules, or “clathrin-independent,” which does not require the recruitment of clathrin.
  • An exemplary type of clathrin-independent endocytosis includes, for example, macropinocytosis.
  • activator of endocytosis refers to agents that, e.g., induce or activate the endocytosis pathway, or process, such that the endocytosis pathway is increased.
  • An exemplary “activator of endocytosis” increases mitochondrial uptake from the extracellular environment.
  • macropinocytosis refers to a clathrin-independent form of endocytosis that mediates the non-selective uptake of solute molecules, nutrients and antigens.
  • the term “compound” refers to a compound capable of effecting a desired biological function.
  • the term includes, but is not limited to, DNA, RNA, protein, polypeptides, and other compounds including growth factors, cytokines, hormones or small molecules.
  • peptide As used herein, the term “peptide” the terms “peptide,” “polypeptide” and “protein” are used interchangeably and in their broadest sense to refer to constrained (that is, having some element of structure as, for example, the presence of amino acids which initiate a ⁇ turn or ⁇ pleated sheet, or for example, cyclized by the presence of disulfide bonded Cys residues) or unconstrained (e.g., linear or unstructured) amino acid sequences.
  • the amino acids making up the polypeptide may be naturally derived, or may be synthetic.
  • the polypeptide can be purified from a biological sample.
  • polypeptide, protein, or peptide also encompasses modified polypeptides, proteins, and peptides, e.g., glycopolypeptides, glycoproteins, or glycopeptides; or lipopolypeptides, lipoproteins, or lipopeptides.
  • modulate As used herein, the terms “modulate,” “modulation,” “modulator,” and “modulating” are intended to mean a change in the character or composition of the basal, homeostatic state.
  • An exemplary modulation includes altering cellular metabolism by disrupting the homeostasis, such that cellular metabolism is significantly reduced.
  • modulator includes inhibitors and activators.
  • Inhibitors are agents that, e.g., inhibit expression or modification of a desired protein, pathway, or process, or bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of the described target protein, pathway, or process.
  • inhibitors are antagonists of the target protein, pathway, or process.
  • Activators are agents that, e.g., induce or activate the expression or modification of a described target protein, pathway, or process, or bind to, stimulate, increase, open, activate, facilitate, enhance activation of inhibitor activity, sensitize or up regulate the activity of described target protein (or encoding polynucleotide), pathway, or process.
  • an activator is an agonist of the target protein, pathway, or process.
  • Modulators include naturally occurring and synthetic ligands, antagonists and agonists (e.g., small chemical molecules, antibodies and the like that function as either agonists or antagonists). It is further understood that modulators can be biological (e.g., antibodies), or chemical.
  • the term “prior to” is intended to mean a period of time preceding the initiation of an event, such that it is a sufficient length of time to achieve and sustain a desired result (e.g., antibiotic selection) or effect (e.g., biological effect) without the desired result or effect completely dissipating before the intended event is initiated.
  • a desired result e.g., antibiotic selection
  • effect e.g., biological effect
  • modulating cellular metabolism prior to transfer of an exogenous mitochondria and/or exogenous mtDNA would involve a sufficient period of time to, for example, exhibit a desired biological effect (e.g., increase phosphorylation of S6 kinase), without the biological effect reverting back to the homeostatic state before the transfer of exogenous mitochondria and/or exogenous mtDNA occurs.
  • a desired biological effect e.g., increase phosphorylation of S6 kinase
  • nutrient stress refers to nutrient deficiency or nutrient starvation conditions sufficient to produce perturbations in the cellular homeostasis, such as induction of autophagy, AMPK signaling, and/or mTOR signaling pathways.
  • exemplary nutrient stress conditions include serum starvation, removal of essential amino acids, and/or disruption of metabolic pathways.
  • nucleic acid and “polynucleotide” are used interchangeably herein to describe a polymer of any length composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, or compounds produced synthetically, which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions.
  • bases are synonymous with “nucleotides” (or “nucleotide”), i.e., the monomer subunit of a polynucleotide.
  • A when used in reference to a nucleotide is intended to mean adenine (A).
  • G when used in reference to a nucleotide is intended to mean Guanine (G).
  • C when used in reference to a nucleotide is intended to mean Cytosine (C).
  • T when used in reference to a nucleotide is intended to mean Thymine (T).
  • pharmaceutically acceptable when used in reference to a carrier, is intended to mean that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • the present invention is based, in part, on the discovery that any agent that reduces the function of endogenous mitochondria, including an agent that reduces endogenous mitochondrial DNA (mtDNA), may enhance the non-invasive transfer of exogenous mitochondria.
  • mtDNA endogenous mitochondrial DNA
  • the complete depletion of the endogenous mtDNA such as with ⁇ (0) cells, prevents this enhancement. This is because the non-invasive transfer of exogenous mitochondria is energy dependent, and a complete depletion of the endogenous mtDNA greatly limits the energy available to facilitate the non-invasive transfer process.
  • the non-invasive transfer of exogenous mitochondria is also inefficient when the mitochondria function and/or mtDNA is unperturbed, for example, when mitochondria is merely co-incubated (i.e., “add-on”) or added by centrifugation.
  • a mitochondria replaced cell can include (a) contacting a recipient cell with an agent that reduces endogenous mtDNA copy number or an agent that reduces mitochondrial function; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mtDNA copy number or partially reduce the endogenous mitochondrial function in the recipient cell, respectively; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mtDNA, or the endogenous mitochondrial function, respectively, has been partially reduced, and (2) exogenous mitochondria from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell.
  • a method of generating a mitochondria replaced cell that includes performing steps (a) and (b) as described above, and then (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mtDNA or the endogenous mitochondrial function, respectively, has been partially reduced, and (2) exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mtDNA into the recipient cell, thereby generating a mitochondria replaced cell.
  • the exogenous mtDNA is transferred via exogenous mitochondria.
  • the generation of a MirC can be a useful strategy for a variety of applications.
  • the transfer of exogenous mitochondria, exogenous mtDNA, or a combination thereof into a recipient cell can be useful in, for example, replacing endogenous mitochondria that is dysfunctional and/or comprised of mutant mtDNA with functional mitochondria, such as mitochondria comprised of wild-type mtDNA.
  • the methods provided herein are performed in a recipient cell that has endogenous mtDNA that encodes for dysfunctional mitochondria.
  • the endogenous mtDNA is mutant mtDNA.
  • the endogenous mtDNA is heteroplasmic and comprised of both wild-type mtDNA and mutant mtDNA.
  • the transfer of exogenous mitochondria, exogenous mtDNA, or a combination thereof can involve the transfer of functional mitochondria or wild-type mtDNA to replace endogenous mitochondria that is, for example, dysfunctional or comprised of mutant mtDNA.
  • the exogenous mtDNA is wild-type mtDNA.
  • the endogenous mitochondria of the recipient cell has wild-type mtDNA, and dysfunctional endogenous mitochondria.
  • exemplary dysfunctional mitochondria of the recipient cell with wild-type mtDNA can include mutant nuclear DNA that encode for mitochondrial proteins, or dysfunctional mitochondria that arises due to a secondary effect, such as aging or disease.
  • Mitochondrial dysfunction can occur as a result of many factors. Non-limiting examples include mitochondrial dysfunction due to a disease (e.g., an age-related disease, a mitochondrial disease or disorder, a neurodegenerative disease, a retinal disease, a genetic disease), diabetes, a hearing disorder, or any combination thereof. Mitochondrial dysfunction can involve the function of the endogenous mitochondria being reduced by greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. Therefore, in some embodiments, the endogenous mitochondria includes mitochondria with reduced function of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.
  • the methods provided herein are applicable to both homoplasmic and heteroplasmic mtDNA.
  • the endogenous mtDNA is a single type of mtDNA (i.e., the endogenous mtDNA is homoplastic).
  • the endogenous mtDNA includes more than one type of mtDNA (i.e., the endogenous mtDNA is heteroplasmic).
  • the heteroplasmic mtDNA includes both wild-type mtDNA and mutant mtDNA. Generally, the proportion of mutant mtDNA determines the severity of the phenotype and can influence the degree to which mitochondrial function is reduced.
  • the heteroplasmic mtDNA is 5% mutant mtDNA and 95% wild-type mtDNA, and the mitochondrial function is reduced 5%.
  • the heteroplasmic mtDNA is 55% mutant mtDNA and 45% wild-type mtDNA, and the mitochondrial function is reduced 55%.
  • the percentage of mutant mtDNA need not be proportional to the mitochondrial function.
  • Dysfunctional mitochondria is generally characterized by a loss of efficiency in the electron transport chain and reductions in the synthesis of high-energy molecules, such as adenosine-5′-triphosphate (ATP), the leakage of deleterious reactive oxygen species (ROS), and/or disrupted cellular respiration.
  • ATP adenosine-5′-triphosphate
  • ROS deleterious reactive oxygen species
  • a person skilled in the art would understand how to evaluate mitochondrial function.
  • cell-based assays such as the Seahorse Bioscience XF Extracellular Flux Analyzer, can used performed for the determination of basal oxygen consumption, glycolysis rates, ATP production, and respiratory capacity in a single experiment to assess mitochondrial dysfunction.
  • the Oroboros 02K respirometer can also be used to establish quantitative functional mitochondrial diagnosis. It is understood that the assay examples described above are exemplary and are not inclusive of all methods to evaluate mitochondrial function.
  • functional mitochondria have an intact outer membrane. In some embodiments, functional mitochondria are intact mitochondria. In another embodiment, functional mitochondria consume oxygen at an increasing rate over time. In another embodiment, the functionality of mitochondria is measured by oxygen consumption. In another embodiment, oxygen consumption of mitochondria may be measured by any method known in the art such as, but not limited to, the MitoXpress fluorescence probe (Luxcel). In some embodiments, functional mitochondria are mitochondria which display an increase in the rate of oxygen consumption in the presence of ADP and a substrate such as, but not limited to, glutamate, malate or succinate. Each possibility represents a separate embodiment of the present invention. In another embodiment, functional mitochondria are mitochondria that produce ATP.
  • a MirC is generated using a recipient cell with functional endogenous mitochondria, wild-type mtDNA, or a combination thereof, and the exogenous mitochondria is also functional, contains wild-type mtDNA, or a combination thereof.
  • endogenous wild-type mtDNA can be reduced using the methods provided herein and exogenous wild-type mtDNA can be transferred into the recipient cell, such as mitochondrial replacement in an “old” recipient cell (e.g., a cell from an aged subject or a cell with relatively high population doubling level (PDL)) with exogenous mtDNA from a healthy donor cell (e.g., a young cell with relatively low PDL).
  • the exogenous mtDNA is from a donor cell that is a healthy donor cell, for example a donor cell that is younger than the recipient cell.
  • the donor and recipient cell have a difference in PDL of about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 4 fold, about 5 fold, or greater than 5 fold.
  • the donor and recipient cells are from subjects that are separated in age by about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 4 fold, about 5 fold, or greater than 5 fold.
  • a difference in age between the donor cell and the recipient cell is not a requirement.
  • the donor and recipient cell are the same age, and the donor cell is a heathy cell.
  • the generation of a MirC is performed in a recipient cell with functional endogenous mitochondria, such as wild-type endogenous mtDNA, and the exogenous mtDNA is mutant, encodes for dysfunctional mitochondria, the exogenous mitochondria is dysfunctional, or a combination thereof.
  • the exogenous mitochondria, exogenous mtDNA, or a combination thereof is from a donor cell that is older than the recipient cell.
  • a model of a mitochondrial disease or disorder can be created by replacement of functional mitochondria in a recipient cell with exogenous mtDNA from a donor cell that is mutant and/or encodes for dysfunctional mitochondria. It is understood that the examples described herein are exemplary and are not inclusive of all combinations involving mtDNA replacement.
  • the methods of generating a MirC can be practiced using either an agent that reduces endogenous mtDNA, or an agent that reduces endogenous mitochondrial function. In certain circumstances, a combination of the two agents can be used. Agents that are capable of reducing mitochondrial function are well known in the field, and are within the skillset of a person skilled in the art.
  • Exemplary agents include inhibitors of the mitochondrial respiratory chain that block respiration in the presence of either ADP or uncouplers, such as an inhibitor of complex III (e.g., myxothiazol), an inhibitor of complex IV (e.g., sodium azide, potassium cyanide (KCN)), or an inhibitor of complex V (e.g., oligomycin); inhibitors of phosphorylation that abolish the burst of oxygen consumption after adding ADP, but have no effect on uncoupler-stimulated respiration; uncoupling agents that abolish the obligatory linkage between the respiratory chain and the phosphorylation system which is observed with intact mitochondria (e.g., dinitrophenol, CCCP, FCCP); ATP/ADP transport inhibitor, such as an adenine nucleotide translocase inhibitor (e.g., atractyloside) that either prevent the export of ATP, or the import of raw materials across the mitochondrial inner membrane; ionophores (e.g.
  • an inhibitor of complex III e.g.
  • valinomycin e.g. valinomycin, nigericin
  • a Krebs cycle inhibitor e.g. arsenite, aminooxyacetate
  • the agent that reduces endogenous mitochondrial function transiently reduces the endogenous mitochondrial function.
  • the agent that reduces endogenous mitochondrial function permanently reduces the endogenous mitochondrial function.
  • the agent that reduces endogenous mitochondrial function partially reduces the endogenous mitochondrial function.
  • the agent that reduces mtDNA is selected from a nucleic acid encoding a fusion protein comprising a mitochondrial-targeted sequence (MTS) and an endonuclease, an endonuclease, or a small molecule.
  • the small molecule is a nucleoside reverse transcriptase inhibitor (NRTI).
  • the nucleic acid can be a messenger ribonucleic acid (mRNA) or a deoxyribonucleic acid (DNA).
  • the agent that reduces mtDNA is a plasmid DNA expression vector cassette encoding an endonuclease.
  • the agent is a plasmid DNA expression vector cassette encoding an endonuclease with a MTS.
  • Various expression vector cassettes can be used, and a person skilled in the art would understand the necessary considerations required to enable successful expression of the endonuclease depending on the host cell.
  • a mammalian expression vector such as a vector having a cytomegalovirus (CMV) promoter, SV40 promoter, or CAG promoter, would be suitable for expression of the endonuclease in a mammalian cell, but not a non-mammalian cell.
  • CMV cytomegalovirus
  • viral expression vectors can also be used and a person skilled in the art would understand that such viral expression vectors may require helper plasmids (i.e., envelope and packaging plasmids) to be used in tandem with the transfer plasmid.
  • the agent is an mRNA encoding an endonuclease.
  • the agent is an mRNA encoding an endonuclease with a MTS.
  • the agent is an endonuclease that is a recombinant protein.
  • the agent is a small molecule, such as, for example, a small molecule that disrupts synthesis of mtDNA. Techniques for generating any of the expression methods are known to those skilled in the art, and can be readily performed without undue experimentation. In preferred embodiments, the agent is suitable for clinical use.
  • the endonuclease can be a restriction enzyme that cleaves DNA double helices into fragments at specific sites, such as XbaI, which cleaves the following sequence of DNA:
  • the endonuclease can also include, for example, restriction enzymes other than XbaI, such as EcoRI, BamHI, HindIII, or PstI, which all digest mtDNA at multiple sites. Endonucleases have defined recognition sites, which allows prediction of their sensitivity on mtDNA. The defined recognition sites of restriction enzymes, such as, for example, XbaI, EcoRI and SmaI, are specific to a given nucleic acid sequence. Accordingly, in some embodiments, the reduction of endogenous mtDNA can be performed using zinc fingers and transcription activator-like effectors (TALEs) that have been combined to DNA nucleases.
  • TALEs transcription activator-like effectors
  • the endonuclease can be a programmable nuclease, such as a RNA-guided DNA endonuclease (e.g., Cas9), zinc finger nuclease (ZFN), or transcription activator-like effector nuclease (TALEN).
  • a RNA-guided DNA endonuclease e.g., Cas9
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • nucleases described above are non-limiting, and that a person skilled in the art can readily identify suitable endonucleases using techniques known in the art.
  • the Cambridge Reference Sequence or similar consensus sequence can be used to identify suitable endonucleases that recognize the mtDNA sequence by, for example, an in silico analysis.
  • the endonuclease cleaves a wild-type sequence of mtDNA. In other embodiments, the endonuclease cleaves a mutant sequence of mtDNA.
  • agent that reduces endogenous mtDNA need not be an endonuclease and that any agent capable of reducing mtDNA can be employed, including an agent that inhibits the biosynthesis of mtDNA, such as ethidium bromide.
  • agents such as, for example, Urolithin A or the small molecule p62-mediated mitophagy inducer (PMI), that induce autophagy in order to promote the selective degradation of endogenous mitochondria (i.e., mitophagy agonist).
  • PMI mitophagy inducer
  • the present invention can also be practiced using a nucleoside reverse transcriptase inhibitor (NRTI) as an agent that reduces mtDNA.
  • NRTI nucleoside reverse transcriptase inhibitor
  • the expression vector cassette can include one or more antibiotic resistance genes to enable selection of a population of cells that express the expression vector cassette.
  • the expression vector can include the puromycin N-acetyl-transferase gene (pac) from Streptomyces , and cells can be selected using puromycin.
  • pac puromycin N-acetyl-transferase gene
  • the selection can be brief (e.g., 24-48 hours) to limit long term exposure to the drug.
  • the expression vector cassette can include other antibiotics resistance genes, such as, for example, the bsr, bls, or BSD gene for selection with Blasticidin, or the hph gene for selection with hydromycin B. It is generally understood that the concentration of antibiotic used for selection will depend on the type of antibiotic and the cell type, and would be readily obtainable to one skilled in the art without undue experimentation. It is further understood that selection can be produced by any means known in the art, and need not involve antibiotic resistance. For example, in some embodiments, selection of the cells can be performed by, for example, fluorescence-activated cell sorting (FACS) of a cell surface marker or expression of a fluorescent protein encoded by the expression.
  • FACS fluorescence-activated cell sorting
  • selection can be performed according to the cell's phenotype.
  • the successful deletion of mutant endogenous mtDNA in a cell with heteroplasmy can result in a phenotypic response that is selectable, such as, for example, cell survival.
  • the cells are selected after introducing an expression vector cassette that contains an endonuclease that degrades mtDNA.
  • the cells are selected to obtain a homogenous population of cells that express an endonuclease that degrades mtDNA.
  • the cells are selected after introducing an expression vector cassette that contains an endonuclease that degrades mtDNA, and a homogenous, stable cell line is generated.
  • the cells are selected to enrich for a population of cells that express an endonuclease that degrades mtDNA. As described above, this enrichment by selection can involve a brief exposure to an antibiotic.
  • the enriched cells can stably express the endonuclease or transiently express the endonuclease depending on the extent and/or manner of the selection pressure. It is understood that an enriched population need not be homogenous, and that an enriched population of cells that express an endonuclease that degrades mtDNA contains a higher percentage of cells with the endonuclease, relative to an unselected population of cells, but may also contain some cells that do not express the endonuclease.
  • the cells are not selected after introducing an expression vector that contains an endonuclease that degrades mtDNA. In specific embodiments, the cells are not selected after introducing an expression vector that contains an endonuclease that degrades mtDNA, and the endonuclease is transiently expressed.
  • the plasmid DNA expression vector cassette is introduced by electroporation.
  • the electroporation method is flow electroporation, such as MaxCyte Flow Electroporation.
  • the electroporation method includes the nucleofection technology, such as Lonza's NucleofectorTM technology.
  • the plasmid DNA expression vector cassette is introduced by cationic lipid transfection.
  • the plasmid DNA expression vector cassette is introduced by viral transduction. It is understood that the methods described above for introducing the expression vector cassette are non-limiting and merely intended to be exemplary methods, and that any method known in the art can be used for introducing the DNA expression vector cassette.
  • expression of the endonuclease can also involve introduction of mRNA encoding the endonuclease or introducing the endonuclease as a recombinant protein.
  • the MaxCyte electroporator can be used for mRNA transfection, particularly in the clinical setting, which has cleared the standards of Good Manufacturing Practice and Good Clinical Practice. The transfection can be performed using the MaxCyte electroporator according to the manufacturer's protocol. It is further understood that the methods described above are merely exemplary and that any means of introducing mRNA and/or recombinant protein can be used.
  • MTS mitochondrial targeting sequence
  • MTS to the mitochondrial matrix can be used, such as the MTS that is a targeting peptide from the cytochrome c oxidase subunit IV (COX 4), subunit VIII (COX 8), or subunit X (COX 10).
  • MTS cytochrome c oxidase subunit IV
  • COX 8 subunit VIII
  • COX 10 subunit X
  • any target sequence derived from any nuclear encoded mitochondrial matrix or inner membrane enzyme or an artificial sequence that is capable of rendering the fusion protein into a mitochondrial imported protein hydrophobic moment greater than 5.5, at least two basic residues, amphiphilic alpha-helical conformation; see, e.g., Bedwell et al., Mol Cell Biol. 9(3) (1989), 1014-1025) is useful for the purposes of the present invention.
  • the MTS is a human MTS. In another embodiment, the MTS is from another species.
  • Non-limiting examples of such sequences are the cytochrome c oxidase subunit X (COX 10) MTS (MAASPHTLSSRLLTGCVGGSVWYLERRT, SEQ ID NO: 36), and the cytochrome c oxidase subunit VIII (COX 8) MTS (MSVLTPLLLRSLTGSARRLMVPRA, SEQ ID NO: 37).
  • MTS sequences are the natural MTS of each individual mitochondrial protein that is encoded by the nuclear DNA, translated (produced) in the cytoplasm and transported into the mitochondria, as well as citrate synthase (cs), lipoamide deydrogenase (LAD), and C6ORF66 (ORF).
  • the various MTS may be exchangeable for each mitochondrial enzyme among themselves.
  • the MTS targets a mitochondrial matrix protein.
  • the mitochondrial matrix protein is subunit VIII of human cytochrome C oxidase. Each possibility represents a separate embodiment of the fusion protein for use of the present invention.
  • the recipient cell Upon contacting a recipient cell with an agent that reduces endogenous mtDNA copy number or an agent that reduces endogenous mitochondrial function, the recipient cell is incubated for a sufficient period of time for the agent to partially reduce the endogenous mtDNA copy number in the recipient cell or partially reduce the endogenous mitochondrial function in the recipient cell, respectively. Identifying the “sufficient period of time” to allow the agent to reduce partially reduce the endogenous mtDNA copy number or partially reduce the endogenous mitochondrial function is within the skill of those in the art.
  • the sufficient or proper time period will vary according to various factors, including but not limited to, the particular type of cells, the amount of starting material (e.g., the number of recipient cells and/or amount of mtDNA to be reduced), the amount and type of agent(s), the plasmid promoter regulator(s), and/or the culture conditions.
  • the sufficient period of time to allow a partial reduction of the endogenous mtDNA copy number in a recipient cell is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 1-2 weeks, about 2-3 weeks or about 3-4 weeks.
  • the sufficient period of time will be long enough that the resulting recipient cell has a reduction in a majority of the endogenous mtDNA copy number or a reduction in the function of a majority of the endogenous mitochondria and is also substantially free of the agent that reduces endogenous mtDNA or the agent that reduces endogenous mitochondrial function before incubating the recipient cell with an exogenous mtDNA and/or exogenous mitochondria.
  • An important and novel aspect of the present invention is the finding that mitochondrial transfer efficiency is severely reduced in cells with a complete depletion of endogenous mitochondria (i.e., ( ⁇ ) 0 cells), but can be greatly improved when the endogenous mtDNA copy number is reduced but not completely depleted (i.e., ( ⁇ ) ⁇ cells). Furthermore, the present invention also demonstrates that simple add-on or centrifugation protocols are inefficient without partial reduction in the endogenous mtDNA copy number. Accordingly, in preferred embodiments, the reduction of the endogenous mtDNA copy number in the recipient cell is less than a 100% depletion of the endogenous mtDNA.
  • the endogenous mtDNA copy number in the recipient cell is reduced by about 5% to about 99%.
  • the agent that reduces endogenous mtDNA copy number reduces about 30% to about 70% of the endogenous mtDNA copy number.
  • the agent that reduces endogenous mtDNA copy number reduces about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more, or about 95% or more of the endogenous mtDNA copy number.
  • the agent that reduces endogenous mtDNA copy number reduces about 60% to about 90% of the endogenous mtDNA copy number. It is also understood that in some embodiments, the agent that reduces endogenous mtDNA copy number reduces mitochondrial mass.
  • the exogenous mtDNA is contained in isolated exogenous mitochondria from a donor cell. Mitochondrial isolation may be accomplished by any of a number of well-known techniques including but not limited to those described herein, and in the cited references.
  • the exogenous mitochondria for use in mitochondrial transfer is isolated using a commercial kit, such as, for example, the Qproteum mitochondria isolation kit (Qiagen, USA), the MITOISO2 mitochondria isolation kit (Sigma, USA), or Mitochondria Isolation Kit for Cultured Cells (Thermo Scientific). In other embodiments, the exogenous mitochondria for use in mitochondrial transfer is isolated manually.
  • an exemplary manual isolation of mitochondria includes isolating the mitochondria from donor cells by pelleting the donor cells, washing the cell pellet of 1-2 mL derived from approximately 10 9 cells grown in culture, swelling the cells in a hypotonic buffer, rupturing the cells with a Dounce or Potter-Elvehjem homogenizer using a tight-fitting pestle, and isolating the mitochondria by differential centrifugation.
  • Manual isolation can also include, for example, sucrose density gradient ultracentrifugation, or free-flow electrophoresis.
  • the isolated donor mitochondria is substantially pure of other organelles.
  • the isolated mitochondria can contain impurities and is enriched for mitochondria.
  • the isolated mitochondria are about 90% pure, about 80% pure, about 70% pure, about 60%, pure, about 50%, pure, or any integer in-between.
  • any impurities contained with the isolated donor mitochondria will not affect the viability or function of the recipient cell upon mitochondrial transfer.
  • the transfer of the exogenous mitochondria, exogenous mtDNA, or a combination thereof does not involve transfer of non-mitochondrial organelles.
  • the quantity and quality of isolated mitochondria can easily be determined by a number of well-known techniques including but not limited to those described herein, and in the cited references.
  • the quantity of isolated mitochondria is determined by assessment of total protein content.
  • Various methods are available for measurement of total protein content, such as the Biuret and Lowry procedures (see, e.g., Hartwig et al., Proteomics, 2009 June; 9(11):3209-14).
  • the quantity of isolated mitochondria is determined by mtDNA copy number.
  • the isolated mitochondria are functional mitochondria. In further embodiments, the isolated mitochondria are dysfunctional mitochondria. In some embodiments, the mitochondrial function can be assessed in the donor cell prior to isolation. In other embodiments, the mitochondrial function can be assayed from the isolated mitochondria.
  • the preservation of mitochondrial membrane integrity is another important factor during mitochondria isolation.
  • the mtDNA used in the methods provided herein is from intact mitochondria. In specific embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or greater than 90% of the isolated mitochondria are intact.
  • Mitochondrial membrane integrity can be accomplished by any of a number of well-known techniques including but not limited to those described herein, and in the cited references. For example, TMRM, Rhod123, JC-1 and DiOC6 are typical probes for measurement of mitochondrial membrane potential (see, e.g., Perry et al., Biotechniques, 2011 February; 50(2):98-115).
  • JC-1 is a widely used dye for measurement of inner-membrane potential of isolated mitochondria, and is based on electrochemical proton gradient of mitochondrial inner membrane.
  • the recipient having a partial reduction of endogenous mtDNA in co-incubated with exogenous mitochondria from a healthy donor for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell.
  • the recipient having a partial reduction of endogenous mtDNA in co-incubated with exogenous mtDNA from a healthy donor for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell. Identifying the “sufficient period of time” to non-invasively transfer exogenous mitochondria and/or exogenous mtDNA into the recipient cell is within the skill of those in the art.
  • the sufficient or proper time period will vary according to various factors, including but not limited to, the particular type of cells, the amount of starting material (e.g., the number of recipient cells and/or amount of endogenous mtDNA to be replaced), the amount of donor material (e.g., the quantity, quality, and/or purity of exogenous mtDNA) and/or the culture conditions.
  • the sufficient period of time to non-invasively transfer exogenous mitochondria and/or exogenous mtDNA into a recipient cell is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 1-2 weeks, about 2-3 weeks or about 3-4 weeks.
  • the recipient cells will have a majority of the exogenous mtDNA and be substantially free of any exogenous mitochondria organelles.
  • Another feature of the current invention is the finding that the total mtDNA copy number in the MirC does not substantially increase, relative to the original recipient cell.
  • other less efficient methods have attempted to add on mitochondria without modulating the recipient cell before the co-incubation step, or transfer exogenous mitochondria using centrifugation without modulating the recipient cell prior to the centrifugation. Consequently, the resultant cell populations using the inefficient methods tend to have large increase in the total mtDNA copy number.
  • the mitochondria replaced cell has a total mtDNA copy number no greater than about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, or more, relative to the total mtDNA copy number of the recipient cell prior to contacting with the agent that reduces endogenous mtDNA copy number.
  • non-invasive transfer is another unique aspect of the present invention.
  • Previous methods have employed invasive instruments to inject exogenous mitochondria, physically force the mitochondria into the cells by centrifugation, or similar harsh conditions that are harmful to the recipient cells.
  • the use of the non-invasive transfer is a beneficial feature of this invention, which lends itself to use in the clinical setting.
  • the exogenous mitochondria, exogenous mtDNA, or a combination thereof can be autologous or allogeneic to the recipient cell.
  • the exogenous mtDNA is allogeneic, relative to the recipient cell.
  • the exogenous mtDNA can be obtained from the same species as the recipient cell, and have a different genotype than that of the recipient cell.
  • the exogenous mitochondria, exogenous mtDNA, or a combination thereof is autologous.
  • an exemplary autologous exogenous mtDNA can include mtDNA from a healthy donor cell, for example a “young” donor cell such as from umbilical cord blood, and the recipient cell can be from the same subject, and be an “old” recipient cell, where the terms “young” and “old” refer to the total number of times the cells in the population have doubled or the age of the subject from which the cells are taken.
  • Another exemplary autologous exogenous mtDNA can include, for example, donor mtDNA that has been isolated from the same subject as the recipient cell and modified prior to replacing it with the recipient cell.
  • only the mtDNA and/or mitochondria are allogenic and the recipient cell is autogenic to the subject in need of an exogenous mtDNA and/or exogenous mitochondria.
  • the replacement of mtDNA in the recipient cell can be evaluated by sequencing the DNA sequences of hyper variable region (HVR) of mtDNA, for example, the HV1 and/or HV2 of the D-loop, and comparing it to the sequence of both the donor mitochondria and the recipient cells.
  • HVR hyper variable region
  • the differences in sequences between the recipient cell and the donor mitochondria can be identified by a Single Nucleotide Polymorphism assay.
  • the amplified sequences of the mtDNA from the recipient cell and the donor mitochondria can be cloned into a plasmid for use as a standard for quantification.
  • the cells are animal cells or plant cells.
  • the cells are mammalian cells.
  • the cells are isolated from a mammalian subject who is selected from a group consisting of: a human, a horse, a dog, a cat, a mouse, a rat, a cow and a sheep.
  • the cells are human cells.
  • the cells are cells in culture. The cells may be obtained directly from a mammal (preferably human), or from a commercial source, or from tissue, or in the form for instance of cultured cells, prepared on site or purchased from a commercial cell source and the like.
  • the cells are primary cells (i.e., cells obtained directly from living tissue, for example, biopsy material).
  • the cells may come from any organ including, but not limited to, the blood or lymph system, from muscles, any organ, gland, the skin, or the brain.
  • the cells are somatic cells.
  • the cells are selected from the group consisting of epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells (e.g., bone marrow cells), melanocytes, chondrocytes, hepatocytes, B-cells, T cells, erythrocytes, macrophages, monocytes, fibroblasts, muscle cells, vascular smooth muscle cells, hepatocytes, splenocytes, and pancreatic R cells.
  • epithelial cells e.g., neural cells, epidermal cells, keratinocytes, hematopoietic cells (e.g., bone marrow cells), melanocytes, chondrocytes, hepatocytes, B-cells, T cells, erythrocytes, macrophages, monocytes, fibroblasts, muscle cells, vascular smooth muscle cells, hepatocytes, splenocytes, and pancreatic R cells.
  • the donor cells are commercially available cells cultured under Current Good Manufacturing Practices (cGMP).
  • the donor cells can be obtained from a cell repository, such as Waisman Biomanufacturing, or similar commercial resource, such as a commercial source that generates cGMP compliant cells.
  • the donor cells are cGMP manufactured bone-marrow derived Mesenchymal Stromal Cells (BM-MSCs).
  • BM-MSCs bone-marrow derived Mesenchymal Stromal Cells
  • the cells are cGMP grade human hepatocytes.
  • the donor cells can be frozen cells that are thawed prior to isolating the mitochondria.
  • the mitochondria need not be isolated after freezing the cells, and can be isolated from fresh cells and used immediately, or, in certain embodiments, the mitochondria can be isolated and then frozen before transferring into the recipient cell.
  • the cells are cancer cells.
  • the cancer cells are isolated from a cancer selected from the group consisting of breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia
  • the cells are stem cells.
  • stem cell refers to an undifferentiated cell that can be induced to proliferate.
  • the stem cell is capable of self-maintenance or self-renewal, meaning that with each cell division, one daughter cell will also be a stem cell.
  • Stem cells can be obtained from embryonic, post-natal, juvenile, or adult tissue.
  • Stem cells can be pluripotent or multipotent.
  • progenitor cell refers to an undifferentiated cell derived from a stem cell, and is not itself a stem cell. Some progenitor cells can produce progeny that are capable of differentiating into more than one cell type.
  • Stem cells include pluripotent stem cells, which can form cells of any of the body's tissue lineages: mesoderm, endoderm and ectoderm. Therefore, for example, stem cells can be selected from a human embryonic stem (ES) cell; a human inner cell mass (ICM)/epiblast cell; a human primitive ectoderm cell, a human primitive endoderm cell; a human primitive mesoderm cell; and a human primordial germ (EG) cell.
  • ES human embryonic stem
  • ICM inner cell mass
  • EG human primordial germ
  • Stem cells also include multipotent stem cells, which can form multiple cell lineages that constitute an entire tissue or tissues, such as but not limited to hematopoietic stem cells or neural precursor cells.
  • Stem cells also include totipotent stem cells, which can form an entire organism.
  • the stem cell is a mesenchymal stem cell.
  • mesenchymal stem cell or “MSC” is used interchangeably for adult cells which are not terminally differentiated, which can divide to yield cells that are either stem cells, or which, irreversibly differentiate to give rise to cells of a mesenchymal cell lineage, e.g., adipose, osseous, cartilaginous, elastic and fibrous connective tissues, myoblasts) as well as to tissues other than those originating in the embryonic mesoderm (e.g., neural cells) depending upon various influences from bioactive factors such as cytokines.
  • the stem cell is a partially differentiated or differentiating cell.
  • the stem cell is an induced pluripotent stem cell (iPSC), which has been reprogrammed or de-differentiated.
  • the recipient cell is an iPSC.
  • the recipient cell is a hematopoietic stem cell (HSC) or a MSC.
  • HSC hematopoietic stem cell
  • Stem cells can be obtained from embryonic, fetal or adult tissues.
  • the cells are immune cells.
  • the recipient cell is an immune cell.
  • the immune cell is selected from the group consisting of a T cell, a phagocyte, a microglial cell, and a macrophage.
  • the T cell is a CD4+ T cell.
  • the T cell is a CD8+ T cell.
  • the T cell is a chimeric antigen receptor (CAR) T cell.
  • the recipient cell is an exhausted or near exhausted T cell in a state or near a state of T cell dysfunction.
  • the transfer of mitochondria has been reported to involve the endocytosis pathway, which is an ATP-dependent process.
  • mitochondria have been observed to be engulfed via macropinocytosis (see, e.g., Kitani et al., J Cell Mol Med., 2014, 18(8):1694-1703).
  • the present invention also relates to the novel findings that the use of a second active agent prior to co-incubating the recipient cell with exogenous mitochondria and/or exogenous mtDNA can promote the uptake of the exogenous mitochondria and/or exogenous mtDNA.
  • the second active agent is selected from the group consisting of large molecules, small molecules, or cell therapies, and the second active agent is optionally selected from the group consisting of rapamycin, NR (Nicotinamide Riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxolone (RTA408), KH176, Vatiquinone (Epi743), thioctic acid, A0001 (alpha-tocopherolquinone), mitochondrial CoQ10 (MitoQ), SkQ1 (Visomitin), resveratrol, curcumin, ketogenic treatment, hypoxia, and an activator of endocytosis.
  • rapamycin NR (Nicotinamide Riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxo
  • the activator of endocytosis is a modulator of cellular metabolism.
  • Cellular metabolism can be modulated using various methods known to one skilled in the art.
  • modulation of cellular metabolism comprises nutrient starvation, a chemical inhibitor, or a small molecule.
  • endocytosis pathway As described above, transfer of intact mitochondria has been reported to occur by an endocytosis pathway.
  • the exogenous mitochondria and/or exogenous mtDNA can be transferred by uptake of intact mitochondria via the endocytosis pathway.
  • the endocytosis pathways can be subdivided into four categories: 1) clathrin-mediated endocytosis, 2) caveolae, 3) macropinocytosis, and 4) phagocytosis.
  • Clathrin-mediated endocytosis is mediated by small (approx. 100 nm in diameter) vesicles that have a morphologically characteristic coat made up of a complex of proteins that are mainly associated with the cytosolic protein clathrin.
  • the endocytosis pathway for mitochondrial transfer is a clathrin-dependent endocytosis pathway.
  • the endocytosis pathway for mitochondrial transfer is a clathrin-independent pathway.
  • the endocytosis pathway is macropinocytosis.
  • Macropinocytosis has been suggested to be an important process in nutrient-deprived environments. As a result, it was hypothesized that a shortage of cellular nutrients or an inhibition of the pathways or target molecules that are activated with sufficient nutrition such as mTOR could be a strategy to augment the cellular engulfment of intact mitochondria into the cytosol. Specifically, as provided herein, it was discovered that a suppression of mTOR can enhance the uptake of exogenous mitochondria. mTOR is an essential sensor of amino acids, energy, oxygen, and growth factors, and a key regulator of protein, lipid, and nucleotide synthesis that is involved in uptake of extracellular nutrients.
  • the methods provided herein further comprise contacting a recipient cell with a small compound, a peptide, or a protein that can increase macropinocytosis.
  • the methods provided herein further comprise modulating cellular metabolism of a recipient cell prior to transfer of the exogenous mitochondria and/or exogenous mtDNA.
  • the modulating cellular metabolism is performed using the same small compound, a peptide, or a protein that can increase macropinocytosis.
  • Modulating cellular metabolism may be accomplished by any of a number of well-known techniques including but not limited to those described herein, and in the cited references. For example, in some embodiments, modulating cellular metabolism is performed by nutrient starvation or nutrient deprivation. In other embodiments, modulating cellular metabolism is performed by a chemical inhibitor or small molecule. In specific embodiments, the chemical inhibitor or small molecule is an mTOR inhibitor.
  • Rapamycin also known as sirolimus (CAS Number 53123-88-9; C 51 H 79 NO 13 ), and rapamycin derivatives (e.g., rapamycin analogs, also known as “rapalogs”).
  • Rapamycin derivatives include, for example, temsirolimus (CAS Number 162635-04-3; C 56 H 87 NO 16 ), everolimus (CAS Number 159351-69-6; C 53 H 83 NO 14 ), and ridaforolimus (CAS Number 572924-54-0; C 53 H 84 NO 14 P).
  • the methods provided herein for mitochondrial transfer further comprise modulating cellular metabolism of a recipient cell prior to transfer of the exogenous mitochondria and/or exogenous mtDNA using rapamycin or a derivative thereof. It is understood that the embodiments described above for modulating cellular metabolism are non-limiting, and modulating cellular metabolism need not involve a chemical compound or small molecule.
  • rapamycin or a derivative thereof which include clinically approved drugs, can be utilized to increase the transfer efficiency of exogenous mitochondria, either as a stand-alone method or in combination with any of the methods provided herein, such as methods involving the partial reduction in the endogenous mitochondria of the recipient cells.
  • the mtDNA can be delivered by clathrin-dependent endocytosis, or clathrin-independent endocytosis.
  • the clathrin-independent pathway can be, for example, CLIC/GEEC endocytic pathway, Arf6-dependent endocytosis, flotillin-dependent endocytosis, macropinocytosis, circular doral ruffles, phagocytosis, or trans-endocytosis.
  • exogenous mitochondria and/or exogenous mtDNA can be enhanced by the use of any compound that stimulates mitochondrial delivery, such as an activator of endocytosis.
  • Non-limiting exemplary compounds suitable for activating endocytosis include, for example, phorbol-12-myristate-13-acetate (PMA) (C 36 H 56 O 8 ), 12-O-tetradecanoylphorbol 13-acetate (TPA) (C 36 H 56 O 8 ), tanshinone IIA sodium sulfonate (TSN-SS) (C 19 H 17 O 6 S.Na), and phorbol-12,13-dibutyrate, or derivatives thereof.
  • non-endocytosis mediated transfer of mtDNA and/or mitochondria can be used, including methods that bypass endocytosis and/or cell fusion.
  • compositions for the treatment of conditions associated with mutant mtDNA and/or dysfunctional mitochondria uses of compositions for the treatment of conditions associated with mutant mtDNA and/or dysfunctional mitochondria, and uses of compositions in the manufacture of medicaments for the treatment of conditions associated with mutant mtDNA and/or dysfunctional mitochondria.
  • methods of treatment involving the use of exogenous mitochondria and/or exogenous mtDNA to restore or enhance the function of endogenous mitochondria, uses of compositions to restore or enhance the function of endogenous mitochondria, and uses of compositions in the manufacture of medicaments for the treatment of a subject in need of mitochondrial replacement.
  • the treatment involves prevention of mitochondrial dysfunction.
  • provided herein are methods of treating a subject having or suspected of having an age-related disease involving any of methods described in Section 5.2 and/or Section 5.3.
  • methods of treating a subject having or suspected of having an age-related disease involving generating a mitochondria replaced cell ex vivo or in vitro by contacting a recipient cell with an agent that reduces endogenous mtDNA or reduces endogenous mitochondrial function, incubating the recipient cell for a sufficient period of time for the agent to partially reduce the mtDNA copy number in the recipient cell or partially reduce the endogenous mitochondrial function, co-incubating (1) the recipient cell in which the endogenous mtDNA or endogenous mitochondrial function has been partially reduced, and (2) exogenous mitochondria and/or exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell, and then administering a therapeutically effective amount of the mitochondria replaced recipient
  • the age-related disease includes an autoimmune disease, a metabolic disease, a genetic disease, cancer, a neurodegenerative disease, and immunosenescence.
  • the metabolic disease can include diabetes.
  • Non-limiting examples of neurodegenerative diseases that can be treated by the methods provided herein include Alzheimer's disease, or Parkinson's disease.
  • the genetic disease capable of being treated include Hutchinson-Gilford Progeria Syndrome, Werner Syndrome, and Huntington's disease. Additional age-related diseases that involve dysfunctional mitochondria are also contemplated.
  • the methods of treating a subject having or suspected of having an age-related disease involves generating a MirC, where the recipient cell used to generate the MirC is a T cell or a hematopoietic stem cell (HSC).
  • the recipient cell used to generate the MirC is a T cell or a hematopoietic stem cell (HSC).
  • HSC hematopoietic stem cell
  • endogenous mtDNA, endogenous mitochondria, or a combination thereof in a senescent T cell or hematopoietic stem cell (HSC) can be replaced for rejuvenation.
  • the in vitro or ex vivo mitochondrial replacement can be a feasible option for the treatment using human T cells and/or hematopoietic stem cells with diseased patients.
  • the methods provided herein can be used to delay senescence and/or extending lifespan in a cell by non-invasively transferring isolated exogenous mitochondria from a healthy, non-senescent cell into a senescent or near senescent cell to rejuvenate the recipient cell, and the resulting rejuvenated MirC can then be administered to a patient having or suspected of having an age-related disease.
  • the rejuvenation of senescent T cells is one possible embodiment by which the present invention can be used to treat a subject having an age-related disease, such as cancer.
  • an old T cell exhibiting a Senescence Associated Secretory Phenotype (SASP) consisting of inflammatory cytokines, growth factors, and proteases, reduced and/or slower rates of cell population doublings, shortened telomeres, increased DNA damage response (DDR), or a combination thereof can be rejuvenated by using the methods provided herein to non-invasively transfer, for example, isolated mitochondria from a young, healthy T cell that is autologous to a subject having an age-related disease, such as cancer.
  • the T cell-derived MirC with characteristics of a young, non-senescent cell can then be administered to the subject for treatment of the age-related disease.
  • the methods of treating a subject having or suspected of having an age-related disease involves generation of a MirC where the recipient cell is a T cell.
  • T cell fate is regulated by the metabolic pathway, with either glycolysis or oxidative phosphorylation (OXPHOS) being responsible for providing a majority of the energy to T cells.
  • OXPHOS oxidative phosphorylation
  • Glycolysis dominant T cells select to differentiate into effector T cells, whereas OXPHOS dominant T cells for memory T cells.
  • exogenous mitochondria and/or mtDNA can be used to modulate T cell fate. For example, in the case of allergy, exogenous mitochondria and/or mtDNA could be used to calm hyper-activated T cells.
  • exogenous mitochondria and/or mtDNA could empower anti-tumor T cells to allow the T cells to persist for a longer time, or facilitate T cell lytic capacity and/or reduce tumor burden.
  • emerging treatments using chimeric antigen receptor T cells utilize autologous T cells. Those CAR T cells might be in fatigue due to aging or malnutrition such as cachexia which is frequently seen in a severe pathologic stage of cancer.
  • the mitochondrial replacement technology may energize and rejuvenate CAR T to provide more ATP leading to better outcomes.
  • the methods of treating a subject involve a recipient cell that is a T cell.
  • the T cell can be a CD4+ T cell, a CD8+ T cell, or a CAR T cell.
  • the mitochondrial replacement in the recipient T cell results in a T cell with a prolonged lifespan.
  • the lifespan can be increased about 1.5 fold, about 2 fold, about 3 fold, about 4 fold, about 5 fold, or greater than 5 fold.
  • the mitochondrial replacement in the recipient T cell inhibits or delays senescence of the recipient T cells, as compared to a T cell without mitochondrial replacement.
  • lifespan can be prolonged by performing mtDNA replacement using exogenous mitochondria and/or exogenous mtDNA from a donor cell that is younger than the recipient cell.
  • the donor and recipient cell have a difference in PDL of about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 4 fold, about 5 fold, or greater than 5 fold.
  • the donor and recipient cells are from subjects that are separated in age by about 5 years, about 10 years, about 15 years, about 20 years, or greater than 20 years.
  • the mitochondrial replacement in the recipient T cell results in T cells having increased lytic capacity, relative to T cells not having mitochondrial replacement.
  • the mitochondrial replacement in the T cells results in reduced tumor burden.
  • plasmid-based gene transfection can be used to generate a T cell with exogenous mitochondria and/or exogenous mtDNA
  • mRNA transfection can be used.
  • the use of mRNA transfection can decrease the chance of the RNA sequence being integrated into the host genome, and can also have minimal long-term gene expression that would cause the endogenous mtDNA reduction.
  • the MaxCyte electroporator can be used for mRNA transfection, particularly in the clinical setting, which has cleared the standards of Good Manufacturing Practice and Good Clinical Practice.
  • the transfection can be performed using the MaxCyte electroporator according to the manufacturer's protocol.
  • the methods of treating a subject having or suspected of having an age-related disease can also involve the generation of a MirC using the methods provided herein, where the recipient cell is a hematopoietic stem cell (HSC).
  • HSC hematopoietic stem cells
  • HSC hematopoietic stem cells
  • malfunctions of HSCs have been reported to be involved in senescence throughout the whole body. Therefore, it is contemplated that HSC-derived MirC can be used as a method of treatment in any age-related disease.
  • allogenic HSC transplantation can result in rejection of the transplant or even graft-versus-host disease.
  • Autologous HSC transplantation is often a safer and more practical measure for disease intervention.
  • autologous HSC transplantation typically does not require the preconditioning with immunosuppressive agents, such as radiation and chemicals. Accordingly, in vitro or ex vivo generation of a MirC using exogenous mtDNA from a healthy young mitochondria in an autologous HSC that is then returned back to the patients' bodies is envisioned using the methods provided herein.
  • the HSC is autologous to the subject in need of mitochondrial and/or mtDNA replacement, and the exogenous mtDNA is allogenic.
  • the mtDNA replacement in an HSC can result in a differentiated cell with functional mitochondria and/or a differentiated cell with improved function. Accordingly, the methods provided herein can be used in the setting of HSC transplantation.
  • Aging alters the biological processes and leads to development of degenerative disorders, such as Alzheimer's disease, atherosclerosis, osteoporosis, type 2 diabetes mellitus, and tissue fibrosis which is causative for chronic kidney disease and chronic obstructive pulmonary disease.
  • Mitochondria can play a role in senescence, via reactive oxygen species generated by mitochondria, which can impact the ageing process.
  • Mitochondrial dysfunction in aging is in a vicious cycle related to a deregulated nutrient sensing where a shortage of nicotinamide adenine dinucleotide (NAD + ), caused by downregulation of nicotinamide phosphoribosyltransferase (NAMPT) and hyperactivation of poly (ADP-ribose) polymerase 1 (PARP1), leads to an inhibition of NADtdependent deacetylase sirtuin 1 (SIRT1). It then relays to the acetylation-dependent inactivation of PGC1 ⁇ consequently resulting in a depressed mitochondrial biogenesis that exaggerates the NAD + availability.
  • the low activity of PGC1 ⁇ yields downregulated the expressions not only of mitochondrial proteins encoded in nucleus but also of the mitochondrial transcription factor TFAM neighboring the mitochondrial DNA.
  • SASP senescence-associated secretory phenotype
  • the transcription factor GATA4 is degraded with the association of the autophagic adaptor p62 by selective autophagy under normal condition, whereas DNA damage response (DDR) kinases ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related) received senescence signals facilitate the dissociation between GATA4 and p62 and stabilize GATA4, in turn activate NF-kB through TRAF3IP2 (tumor necrosis factor receptor-associated factor interacting protein 2) and ILIA and support SASP.
  • SASP is completely hampered in rho0 cells (mtDNA free cells established by a forced mitophagy). Mitochondrial replacement of oocytes derived from the old in an experimental IVF surely promoted the success rate for zygote formation, development and embedding of embryo, and bearing offspring.
  • Impaired proteostasis is another characteristics in aging.
  • the integrity of proteostasis is strictly maintained by translational regulation, protein folding chaperon, ubiquitin-proteasome system (UPS), and the autophagy-lysosome system.
  • chaperones depend upon ATP, decrease of bioenergy with aging jeopardize the function to correct protein folding.
  • UPS and the autophagy-lysosome system, including mitophagy decline with time. The alternations of these three systems generate aggregates which are not recycled in cytosol leading to degenerative disorders.
  • mitochondrial unfolded protein response URR′
  • All the above mentioned pathways involve mitochondria.
  • the mitochondrial replacement in somatic cells could break the deleterious worsening cycle of aging, slow the senescent process, and even rejuvenate cells.
  • the methods provided herein offer clinically viable methods to treat heteroplasmy, and/or treat various diseases, such as diseases associated with senescence, by replacing endogenous dysfunctional mitochondria, such as endogenous mitochondria with mutant mtDNA, with young and/or healthy mitochondria that can have either an autologous or allogeneic origin.
  • the methods provided herein for mitochondria replacement can be used for the treatment of mitochondrial disease or disorder, as well as senescence, cancer, and immune system deficiencies.
  • the methods of treating a subject having or suspected of having mitochondrial disease or disorder include generating a MirC according to any of the methods described in Section 5.2 and/or Section 5.3, and then administering a therapeutically effective amount of the mitochondria replaced recipient cell to the subject having or suspected of having a mitochondrial disease or disorder.
  • mitochondrial diseases or disorders are known, and all are capable of being treated using the methods provided herein.
  • the mitochondrial disease or disorder capable of being treated using the methods provided herein can be a Complex I deficiency (OMIM:252010).
  • Complex I deficiency can be caused by a mutation in any of the subunits thereof.
  • the Complex I deficiency is caused by a mutation in a gene selected from the group consisting of NDUFV1 (OMIM:161015), NDUFV2 (OMIM:600532), NDUFS1 (OMIM:157655), NDUFS2 (OMIM:602985), NDUFS3 (OMIM:603846), NDUFS4 (OMIM:602694), NDUFS6 (OMIM:603848), NDUFS7 (OMIM:601825), NDUFS8 (OMIM:602141), and NDUFA2 (OMIM:602137).
  • NDUFV1 NDUFV1
  • NDUFV2 NDUFV2
  • NDUFS1 OMIM:157655
  • NDUFS2 OMIM:602985
  • NDUFS3 OMIM:603846
  • NDUFS4 OMIM:602694
  • NDUFS6 OMIM:603848
  • NDUFS7 OMIM:601825
  • the mitochondrial disease or disorder capable of being treated using the methods provided herein can be a Complex IV deficiency (cytochrome c oxidase; OMIM:220110).
  • Complex IV deficiency can be caused by a mutation in any of the subunits thereof.
  • the Complex IV deficiency is caused by a mutation in a gene selected from the group consisting of MTCO1 (OMIM:516030), MTCO2 (OMIM:516040), MTCO3 (OMIM:516050), COX10 (OMIM:602125), COX6B1 (OMIM:124089), SCO1 (OMIM:603644), FASTKD2 (OMIM:612322), and SCO2 (OMIM:604272).
  • MTCO1 OMIM:516030
  • MTCO2 OMIM:516040
  • MTCO3 OMIM:516050
  • COX10 OMIM:602125
  • COX6B1 OMIM:124089
  • SCO1 OMIM:603644
  • FASTKD2 OMIM:612322
  • SCO2 SCO2
  • Mitochondrial diseases or disorders can be caused by or associated with a mutation.
  • the mutation can be a point mutation, a missense mutation, a deletion, and an insertion. It is understood that the identification of mutations in mtDNA or nDNA is within the skill of those in the art, and exemplary methods are provided herein, such as, for example, a single nucleotide polymorphism (SNP) assay or a droplet digital PCR.
  • SNP single nucleotide polymorphism
  • Non-limiting examples of specific types of mitochondrial diseases or disorders capable of being treated using the methods provided herein include Ornithine Transcarbamylase deficiency (hyperammonemia) (OTCD), Carnitine 0-palmitoyltransferase II deficiency (CPT2), Fumarase deficiency, Cytochrome c oxidase deficiency associated with Leigh syndrome, Maple Syrup Urine Disease (MSUD), Medium-Chain Acyl-CoA Dehydrogenase deficiency (MCAD), Acyl-CoA Dehydrogenase Very Long-Chain deficiency (LCAD), Trifunctional Protein deficiency, Progressive External Ophthalmoplegia with Mitochondrial DNA Deletions (POLG), DGUOK, TK2, Pyruvate Decarboxylase deficiency, and Leigh Syndrome (LS).
  • OTD Ornithine Transcarbamylase deficiency
  • CPT2 Carnitine 0-
  • the mitochondrial disease or disorder is selected from the group consisting of Alpers Disease; Barth syndrome; (3-oxidation defects; carnitine-acyl-camitine deficiency; carnitine deficiency; co-enzyme Q10 deficiency; Complex II deficiency (OMIM:252011), Complex III deficiency (OMIM:124000), Complex V deficiency (OMIM:604273), LHON-Leber Hereditary Optic Neuropathy; MM-Mitochondrial Myopathy; LIMM-Lethal Infantile Mitochondrial Myopathy; MMC-Maternal Myopathy and Cardiomyopathy; NARP-Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa; Leigh Disease; FICP-Fatal Infantile Cardiomyopathy Plus, a MELAS-associated cardiomyopathy; MELAS-Mitochondrial Encephalomyopathy with Lactic Acidosis and Strokelike episodes; LDYT-Leber's hereditary
  • the methods provided herein for treating a mitochondrial disease or disorder can also include, in specific embodiments, a mitochondrial disease or disorder caused by mitochondrial DNA abnormalities, where the mitochondrial DNA abnormalities are selected from the group consisting of chronic progressive external ophthalmoplegia (CPEO), Pearson syndrome, Kearns-Sayre syndrome (KSS), diabetes and deafness (DAD), leber hereditary optic neuropathy (LHON), LHON-plus, neuropathy, ataxia, and retinitis pigmentosa syndrome (NARP), maternally-inherited Leigh syndrome (MILS), mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), myoclonic epilepsy and ragged-red fiber disease (MERRF), familial bilateral striatal necrosis/striatonigral degeneration (FBSN), ships disease, aminoglycoside-induced Deafness (AID), and multiple deletions of mitochondrial DNA syndrome.
  • CPEO chronic progressive external ophthalmoplegia
  • Mutations in mtDNA are thought to be associated with numerous clinical disorders. In adults, these include neurological diseases (e.g., migraine, strokes, epilepsy, dementia, myopathy, peripheral neuropathy, diplopia, ataxia, speech disturbances, and sensorineural deafness), gastrointestinal diseases (e.g., constipation, irritable bowel, and dysphagia), cardiac diseases (e.g., heart failure, heart block, and cardiomyopathy), respiratory diseases (e.g., respiratory failure, nocturnal hypoventilation, recurrent aspiration, and pneumonia), endocrine diseases (e.g., diabetes, thyroid disease, parathyroid disease, and ovarian failure), ophthalmological diseases (e.g., optic atrophy, cataract, ophthalmoplegia, and ptosis).
  • neurological diseases e.g., migraine, strokes, epilepsy, dementia, myopathy, peripheral neuropathy, diplopia, ataxia, speech disturbances, and sensorineural deafness
  • disorders thought to be associated with mtDNA mutations include neurological diseases (e.g., epilepsy, myopathy, psychomotor retardation, ataxia, spasticity, dystonia, and sensorineural deafness), gastrointestinal diseases (e.g., vomiting, failure to thrive, and dysphagia), cardiac diseases (e.g., biventricular hypertrophic cardiomyopathy and rhythm abnormalities), respiratory diseases (e.g., central hypoventilation and apnea), hematological diseases (e.g., anemia and pancytopenia), renal diseases (e.g., renal tubular defects), liver diseases (e.g., hepatic failure), endocrine diseases (e.g., diabetes and adrenal failure), and ophthalmological diseases (e.g., optic atrophy).
  • neurological diseases e.g., epilepsy, myopathy, psychomotor retardation, ataxia, spasticity, dystonia, and sensorineural deafness
  • gastrointestinal diseases e.g., vomiting, failure to thrive
  • the methods provided herein allow for treating a mitochondrial disease or disorder where the mitochondrial disease or disorder is caused by nuclear DNA abnormalities, and the nuclear DNA abnormalities are selected from the group consisting of Mitochondrial DNA depletion syndrome-4A, mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase gamma (POLG)-related disorders, sensory ataxia neuropathy dysarthria ophthalmoplegia (SANDO), leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL), co-enzyme Q10 deficiency, Leigh syndrome, mitochondrial complex abnormalities, fumarase deficiency, ⁇ -ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenase complex deficiency (PD
  • KSS Kearns-Sayre syndrome
  • CPEO chronic progressive external ophthalmoplegia
  • MELAS mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes
  • MERRF myoclonic epilepsy with ragged-red fibers
  • NARP neurogenic weakness with ataxia and retinitis pigmentosa
  • LS Leigh syndrome
  • Exemplary diseases where mitochondrial impairment is known to play an important role include, but are not limited to, the pathogenesis of many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis.
  • mitochondrial disease or disorders are subtyped into a number of syndromes according to the symptoms rather than the types of mutations.
  • mitochondrial syndromes include Mitochondrial myopathy, Encephalomyopathy, Lactic acidosis, Stroke-like symptoms (MELAS), Myoclonic Epilepsy with Ragged Red Fibers (MERRF), and Leigh syndrome.
  • the methods of treating a subject in need of mitochondrial replacement include generating a MirC according to any of the methods described in Section 5.2 and/or Section 5.3, and then administering a therapeutically effective amount of the mitochondria replaced recipient cell to the subject in need of mitochondrial replacement.
  • a subject in need of mitochondrial replacement includes any subject that has a dysfunctional mitochondria.
  • the subject in need of mitochondrial replacement has an age-related disease, a mitochondrial disease or disorder, a neurodegenerative disease, a retinal disease, diabetes, a hearing disorder, a genetic disease, or a combination thereof.
  • Neurodegenerative diseases that can benefit from mitochondrial replacement include, but are not limited to, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, Friedreich's ataxia, Charcot Marie Tooth disease and leukodystrophy.
  • the retinal disease can be wet or dry age-related macular degeneration, macular edema, or glaucoma.
  • Other exemplary diseases, such as age-related diseases, and/or mitochondrial disease or disorder are described in more detail in Section 5.4.1 and 5.4.2.
  • a subject in need of mitochondrial replacement can also include a subject that is predisposed to mitochondrial dysfunction, and is asymptomatic.
  • the subject may have mutant mtDNA, but be without manifestations of, for example, a mitochondrial disease because the disease is an adult-onset disease. Therefore, the methods provided herein can be used to also prevent any of the diseases described herein by treating a subject in need of mitochondrial replacement.
  • the current invention also provides methods, as described in Section 5.2 and Section 5.3, for producing or enhancing the production of an induced pluripotent stem cell (iPSC) from a non-pluripotent cell.
  • iPSCs have been demonstrated to be produced from non-pluripotent cells using exogenous expression of stemness factors, such as Oct3/4, Klf4, Sox2, and c-Myc.
  • stemness factors such as Oct3/4, Klf4, Sox2, and c-Myc.
  • mtDNA mitochondrial DNA
  • the present invention has also identified that the methods provided herein can be used to enhance the generation of iPSC by reducing endogenous mtDNA in a non-pluripotent by contacting a recipient non-pluripotent cell with an agent that reduces endogenous mtDNA, incubating the recipient non-pluripotent cell for a sufficient period of time for the agent to partially reduce the endogenous mtDNA in the non-pluripotent cell, and then introducing one or more expression cassettes for expression of Oct3/4, Klf4, Sox2, and c-Myc.
  • exogenous mtDNA and/or exogenous mitochondria is non-invasively transferred into the recipient cells.
  • the methods for producing an iPSC includes introducing one or more expression cassettes for expression of Oct3/4, Klf4, Sox2, and c-Myc, contacting a recipient non-pluripotent cell with an agent that reduces endogenous mtDNA, and incubating the recipient non-pluripotent cell for a sufficient period of time for the agent to partially reduce the endogenous mtDNA in the recipient cell.
  • the method further comprises incubating the recipient cell with an exogenous mitochondria and/or exogenous mtDNA for a sufficient period of time to non-invasively transfer the exogenous mitochondria and/or exogenous mtDNA into a recipient cell.
  • the method further comprises incubating the recipient cell with an exogenous mitochondria and/or exogenous mtDNA for a sufficient period of time to replace a majority of the endogenous mtDNA.
  • the methods of producing an iPSC from a non-pluripotent cell that include transferring exogenous mitochondria and/or exogenous mtDNA and/or exogenous mitochondria can also include any of the embodiments described in Section 5.3.
  • mtDNA mitochondrial DNA
  • the methods provided herein can be used to generate an iPSC by reducing endogenous mtDNA in a non-pluripotent by contacting a recipient non-pluripotent cell with an agent that reduces endogenous mtDNA, incubating the recipient non-pluripotent cell for a sufficient period of time for the agent to partially reduce the endogenous mtDNA in the non-pluripotent cell, and then introducing one or more of Oct3/4, Klf4, Sox2, and c-Myc into the non-pluripotent cell, thereby generating a pluripotent stem cell.
  • the iPSC can even be generated using only small molecule agents and no exogenous factors.
  • the iPSC contains mutant mtDNA.
  • the mutant mtDNA can contain a point mutation, such as, for example, a point mutation in tRNA (e.g., MELAS).
  • the mutant mtDNA can also include mtDNA with a long deletion of mtDNA.
  • the non-pluripotent cell for use in producing iPSC is heteroplasmic. The incorporation of mutant mtDNA can facilitate, for example, generation of disease models.
  • the non-pluripotent recipient cells are somatic cells. In specific embodiments, the non-pluripotent cells are fibroblasts.
  • iPSCs Culture conditions, identification, and establishment of iPSCs is within the skill of those in the art.
  • methods include those provided in U.S. Pat. Nos. 8,058,065, and 8,278,104, which are hereby incorporated by reference in their entireties.
  • assays useful for assessing mitochondrial function and/or mtDNA mutations in connection with the methods provided herein for mtDNA replacement include any assays known to a person skilled in the art that can be used to determine or predict the functionality of mitochondria and/or mtDNA mutations.
  • assays to determine mitochondrial function include, for example, measurement of any one of the following: secretory factors associated with senescence (e.g., pro-inflammatory cytokines, proteases, and growth and angiogenesis factors, such as IL-1, IL-6/VEGF, IL-8, and CXCL9/MMP); mitochondria function by using Oroboros; Mitophagy by using Keima-Red; mitochondrial permeability; mitochondrial membrane potential; cytochrome c levels; reactive oxygen species; cell respiration; transcriptomics and proteomics for measurement of activated innate immunity, rescission of hyperactivated glycolysis, mitigation of ER stress, repression of mTOR-S6 pathway, and activation of cell cycle; mitochondria dynamics, such as fission and fusion, observed by superfine microscopy, and quantified by a specialized software; or any assay known in the art that measures mitochondrial function
  • secretory factors associated with senescence e.g., pro-inflammatory cytokines, protea
  • Various sequencing methods can be used in combination of any of the methods provided herein to (1) detect mutant mtDNA, (2) quantify heteroplasmy, and/or (3) evaluate or confirm transfer of exogenous mitochondria and/or exogenous mtDNA.
  • a stretch of roughly 1,100 nucleotides is gene-free that been called D-Loop, Displacement Loop, and Control Region.
  • the D-Loop contains two regions within which mutations accumulate more frequently than anywhere else in the mitochondrial genome. The regions are called hypervariable regions HV1 and HV2, respectively.
  • mtDNA mutations can be identified in connection with the methods provided herein, by sequencing the hypervariable regions (HV) (i.e., HV1 and/or HV2) of the D-loop of mtDNA.
  • HV hypervariable regions
  • mtDNA sequencing can be performed using any sequencing method known in the art.
  • the sequencing method comprises a single nucleotide polymorphism (SNP) assay.
  • the sequencing method comprises digital PCR.
  • the digital PCR is droplet digital PCR.
  • compositions of cells obtained by any of the methods described in Sections 5.2-5.5 are also provided herein.
  • a composition comprising one or more mitochondria replaced cells obtained by the method of (a) contacting a recipient cell with an agent that reduces endogenous mtDNA copy number; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mtDNA copy number in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mtDNA has been partially reduced, and (2) exogenous mitochondria for a sufficient period of time to non-invasively transfer the exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell, wherein said mitochondria replaced cell comprises greater than 5% of exogenous mtDNA.
  • composition comprising one or more mitochondria replaced cells obtained by the method of (a) contacting a recipient cell with an agent that reduces endogenous mtDNA copy number; (b) incubating the recipient cell for a sufficient period of time for the agent to partially reduce the endogenous mtDNA copy number in the recipient cell; and (c) co-incubating (1) the recipient cell from step (b) in which the endogenous mtDNA has been partially reduced, and (2) exogenous mtDNA from healthy donor, for a sufficient period of time to non-invasively transfer exogenous mtDNA into the recipient cell, thereby generating a mitochondria replaced cell, thereby generating a mitochondria replaced cell, wherein said mitochondria replaced cell comprises greater than 5% of exogenous mtDNA.
  • compositions can also be obtained by a method that involves contacting a cell with an agent that reduces mitochondrial function, and then incubating the recipient cell for a sufficient period of time for the agent to partially reduce endogenous mitochondrial function in the recipient cell.
  • the recipient cell having partially reduced endogenous mitochondrial function can then be co-incubated with either exogenous mitochondria from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell.
  • the recipient cell having partially reduced endogenous mitochondrial function can then be co-incubated with either exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell.
  • the mitochondria replaced cell generated by the methods described above comprises greater than 5% of exogenous mtDNA.
  • the exogenous mitochondria can be comprised of exogenous mtDNA. Therefore, in some embodiments both exogenous mitochondria and exogenous mtDNA are transferred to the recipient cell and the MirC has both exogenous mitochondria and exogenous mtDNA. In other embodiments, the exogenous mtDNA is transferred to the recipient cell via exogenous mitochondria, and then the exogenous mtDNA is delivered to the endogenous mitochondria. Under certain circumstances the exogenous mitochondria is removed from the cell after the exogenous mtDNA is delivered to the endogenous mitochondria. Accordingly, in some embodiments, the MirC have exogenous mtDNA and does not have exogenous mitochondria.
  • the MirC that includes exogenous mitochondria, exogenous mtDNA, or a combination thereof can contain both exogenous mtDNA and endogenous mtDNA.
  • the MirC can contain both exogenous mitochondria and endogenous mitochondria.
  • the compositions of one or more mitochondria replaced cells obtained by the methods provided herein have a mixture of endogenous and exogenous mitochondria.
  • the compositions of one or more mitochondria replaced cells obtained by the methods provided herein have a mixture of endogenous mtDNA and exogenous mtDNA (i.e., heteroplasmic mtDNA).
  • the one or more mitochondria replaced cells have a total mtDNA copy number that is no greater than about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, or more, relative to the total mtDNA copy number of the recipient cell prior to contacting with the agent that reduces endogenous mtDNA copy number.
  • the present invention also includes compositions for use in a method of generating mitochondria replaced that includes an agent that reduces endogenous mtDNA or an agent that reduces mitochondrial function, and a second active agent.
  • the composition can further include an exogenous mitochondria, one or more recipient cells, or a combination thereof.
  • the composition can further include exogenous mtDNA.
  • the second active agent includes large molecules, small molecules, or cell therapies, and the second active agent is optionally selected from rapamycin, NR (Nicotinamide Riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxolone (RTA408), KH176, Vatiquinone (Epi743), thioctic acid, A0001 (alpha-tocopherolquinone), mitochondrial CoQ10 (MitoQ), SkQ1 (Visomitin), resveratrol, curcumin, ketogenic treatment, hypoxia, and an activator of endocytosis.
  • rapamycin NR (Nicotinamide Riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxolone (RTA408), KH176,
  • Non-limiting exemplary compounds suitable for activating endocytosis include, for example, phorbol-12-myristate-13-acetate (PMA) (C 36 H 56 O 8 ), 12-O-tetradecanoylphorbol 13-acetate (TPA) (C 36 H 56 O 8 ), tanshinone IIA sodium sulfonate (TSN-SS) (C 19 H 17 O 6 S.Na), and phorbol-12,13-dibutyrate, or derivatives thereof.
  • the activator of endocytosis comprises a modulator of cellular metabolism.
  • Modulating cellular metabolism may be accomplished by any of a number of well-known techniques including but not limited to those described herein, and in the cited references. For example, in some embodiments, modulating cellular metabolism is performed by nutrient starvation or nutrient deprivation. In other embodiments, modulating cellular metabolism is performed by a chemical inhibitor or small molecule. In specific embodiments, the chemical inhibitor or small molecule is an mTOR inhibitor.
  • rapamycin also known as sirolimus (CAS Number 53123-88-9; C 51 H 79 NO 13 ), and rapamycin derivatives (e.g., rapamycin analogs, also known as “rapalogs”).
  • Rapamycin derivatives include, for example, temsirolimus (CAS Number 162635-04-3; C 56 H 87 NO 16 ), everolimus (CAS Number 159351-69-6; C 53 H 83 NO 14 ), and ridaforolimus (CAS Number 572924-54-0; C 53 H 84 NO 14 P).
  • the compositions provided herein comprise rapamycin or a derivative thereof.
  • compositions described above for modulating cellular metabolism need not involve a chemical compound or small molecule, and can include modulation of other pathways beyond mTOR. It is also understood that the compositions can optionally comprise activators of endocytosis, and that it is not a required component. In addition, in some embodiments the invention provided herein can involve non-endocytosis mediated transfer of mtDNA and/or mitochondria, such as in non-clinical settings.
  • the present invention also provides, in certain embodiments, a composition for use in a method of producing an induced pluripotent stem cells (iPSC) from a non-pluripotent cell that includes an agent that reduces endogenous mtDNA, one or more expression cassettes for expression of Oct3/4, Klf4, Sox2, and c-Myc, and a recipient cell, wherein the recipient cell is a non-pluripotent cell, wherein the agent that reduces endogenous mtDNA is present in an amount effective to increase efficiency of producing an induced pluripotent stem cells (iPSC) from a non-pluripotent cell, as compared to a non-pluripotent cell not treated with an agent that reduces endogenous mtDNA.
  • iPSC induced pluripotent stem cells
  • the agent that reduces endogenous mtDNA is present in an amount effective to increase efficiency of producing an induced pluripotent stem cells (iPSC) from a non-pluripotent cell, as compared to a non-pluripotent cell not treated with an agent that reduces endogenous mtDNA. This is based in part on the observation that pluripotent cells have a reduction in mtDNA copy number.
  • the composition for use in a method of producing an iPSC further comprises exogenous mitochondria and/or exogenous mtDNA.
  • the present invention also includes pharmaceutical compositions for use in the treatment of an age-related disease, a mitochondrial disease or disorder, a neurodegenerative disease, diabetes, a genetic disease, or any subject in need of mitochondrial replacement, as described in Section 5.4.
  • pharmaceutical compositions that include an isolated population of mitochondria replaced cells that have exogenous mitochondria from a healthy donor, and the cells are obtained by the methods described herein, such as in Sections 5.2-5.3.
  • the pharmaceutical composition includes an isolated population of mitochondria replaced cells with exogenous mitochondria and/or exogenous mtDNA from a healthy donor, and the cells are obtained by the methods described herein, such as in Sections 5.2-5.3.
  • the mitochondria replaced cells that have exogenous mtDNA can optionally further include exogenous mitochondria.
  • the exogenous mtDNA is transferred into the cell via exogenous mitochondria, delivered to the endogenous mitochondria, and then the exogenous mitochondria is removed from the recipient cell.
  • the disclosure also provides a pharmaceutical composition comprising an isolated population of mitochondria replaced cells having an exogenous mitochondria from a healthy donor, wherein the cells are obtained by any of the methods provided herein for obtaining a mitochondrial replaced cell.
  • the disclosure provides a pharmaceutical composition comprising an isolated population of mitochondria replaced cells having an exogenous mtDNA from a healthy donor, wherein the cells are obtained by any of the methods provided herein for obtaining a mitochondrial replaced cell.
  • the pharmaceutical composition comprising an isolated population of mitochondria replaced cells having an exogenous mtDNA from a healthy donor further comprises exogenous mitochondria.
  • a pharmaceutical composition comprising an exogenous mitochondria from a healthy donor are obtained by a method that involves contacting a cell with an agent that reduces mtDNA copy number, and then incubating the recipient cell for a sufficient period of time for the agent to partially reduce endogenous mtDNA copy number in the recipient cell.
  • the recipient cell having partially reduced endogenous mtDNA copy number can then be co-incubated with either exogenous mitochondria from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell.
  • the recipient cell having partially reduced endogenous mtDNA copy number can then be co-incubated with either exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell.
  • the mitochondria replaced cell generated by the methods described above comprises greater than 5% of exogenous mtDNA.
  • the cells are obtained by a method that involves contacting a cell with an agent that reduces mitochondrial function, and then incubating the recipient cell for a sufficient period of time for the agent to partially reduce endogenous mitochondrial function in the recipient cell.
  • the recipient cell having partially reduced endogenous mitochondrial function can then be co-incubated with either exogenous mitochondria from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell.
  • the recipient cell having partially reduced endogenous mitochondrial function can then be co-incubated with either exogenous mtDNA from a healthy donor, for a sufficient period of time to non-invasively transfer exogenous mitochondria into the recipient cell, thereby generating a mitochondria replaced cell.
  • the mitochondria replaced cell generated by the methods described above comprises greater than 5% of exogenous mtDNA.
  • the agent that reduces mitochondrial function can either transiently or permanently reduce mitochondrial function. It is within the skillset of a person skilled in the art to be able to determine whether the agent would transiently (e.g., reversible inhibitor) or permanently (e.g., irreversible inhibitor) reduces mitochondrial function.
  • the cells are obtained by a method further comprising further comprising contacting the recipient cell with a second active agent prior to co-incubating the recipient cell with exogenous mitochondria and/or exogenous mtDNA.
  • the second active agent is selected from the group consisting of large molecules, small molecules, or cell therapies, and the second active agent is optionally selected from the group consisting of rapamycin, NR (Nicotinamide Riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxolone (RTA408), KH176, Vatiquinone (Epi743), thioctic acid, A0001 (alpha-tocopherolquinone), mitochondrial CoQ10 (MitoQ), SkQ1 (Visomitin), resveratrol, curcumin, ketogenic treatment, hypoxia, and an activator of endocytosis.
  • rapamycin NR (Nicotinamide Riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxo
  • the activator of endocytosis is a modulator of cellular metabolism.
  • the modulator of cellular metabolism comprises nutrient starvation, a chemical inhibitor, or a small molecule.
  • the chemical inhibitor or the small molecule is an mTOR inhibitor.
  • said mTOR inhibitor comprises rapamycin or a derivative thereof.
  • the present disclosure describes numerous examples where the recipient cells are mammalian cells. However, it is also understood that any cell with a mitochondria can be a recipient cell. Therefore, the recipient cell can also be a plant cell.
  • the animal cells are mammalian cells.
  • the cells are somatic cells.
  • the somatic cells are epithelial cells.
  • the epithelial cells are thymic epithelial cells (TECs).
  • compositions where the somatic cells are immune cells can comprise immune cells where the immune cells are T cells, such as exhausted T cells.
  • the composition includes rejuvenated T cells that contain exogenous mitochondria and/or exogenous mtDNA.
  • senescent T cells or near senescent T cells e.g., immunosenescent
  • a T cell-derived MirC can be generated using the methods provided herein to produce a T cell with healthy exogenous mitochondria and/or exogenous mtDNA.
  • the T cells are CD4+ T cells.
  • the T cells are CD8+ T cells.
  • the T cells are chimeric antigen receptor (CAR) T cells.
  • CAR chimeric antigen receptor
  • the disclosure provides a MirC that is CAR-T cell, which is efficacious in killing a cancer cell.
  • the MirC derived CART can have prolonged survival to enable increased immunosurveillance, and enhanced cancer cell killing.
  • the immune cells are phagocytic cells.
  • compositions provided herein can also include a composition for use in delaying senescence and/or extending lifespan in a cell.
  • the composition can include a senescent or near senescent cell having endogenous mitochondria, isolated exogenous mitochondria from a non-senescent cell, and an agent that reduces endogenous mtDNA copy number.
  • the composition can also include a senescent or near senescent cell having endogenous mitochondria, isolated exogenous mitochondria from a non-senescent cell, and an agent that reduces mitochondrial function.
  • compositions that include one or more mitochondria replaced cells that are derived from recipient cells that are bone marrow cells.
  • the bone marrow cells are a hematopoietic stem cell (HSC), or a mesenchymal stem cell (MSC).
  • HSC hematopoietic stem cell
  • MSC mesenchymal stem cell
  • an HSC or MSC can be isolated from a subject having or suspected of having a mitochondrial disease, an age-related disease, or otherwise be in need of mitochondrial replacement, and have the endogenous mitochondria replaced with exogenous mitochondria. Subsequently, the HSC or MSC derived MirC can then be transplanted back into the subject in need of mitochondrial replacement.
  • the recipient cells are iPS cells.
  • compositions can be used in the clinical setting and can be efficacious in treating an age-related disease, treating a mitochondrial disease or disorder, treating a neurodegenerative disease, treating diabetes, or a genetic disease.
  • the iPSC can be differentiated into a particular cell type prior to administering back into the subject, using methods known in the art.
  • compositions that include an isolated population of pluripotent cells having a reduced amount of endogenous mtDNA, wherein the cells are obtained by any of the embodiments described in Section 5.5.
  • the isolated population of pluripotent cells are iPS cells.
  • the pharmaceutical compositions of the invention may comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized foreign pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17 th ed. 1985).
  • Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • compositions can be administered, for example, orally, nasally, topically, intravenously, intraperitoneally, intrathecally or into the eye (e.g., by eye drop or injection).
  • the formulations of compounds can be presented in unit dose or multi-dose sealed containers, such as ampoules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the dose administered to a patient should be sufficient to induce a beneficial response in the subject over time, i.e., to prevent, ameliorate, or improve a condition of the subject.
  • the optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific modulator employed, the age, body weight, physical activity, and diet of the patient, and on a possible combination with other drug.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound or vector in a particular subject. Administration can be accomplished via single or divided doses.
  • Example I Optimization of the MirC Protocol Revealed that XbaI Degraded mtDNA In Vitro and the MTS Expression Vector Targeted Mitochondria
  • FIG. 1A A scheme of the method used to generate a mitochondria replaced cell (MirC) is provided in FIG. 1A .
  • the mammalian expression vector used to express the XbaI restriction enzyme fused to a mitochondrial-targeted sequence (MTS) was engineered by cloning the MTS-XbaI sequence into the pCAGGS vector using standard techniques known in the art ( FIG. 1B ).
  • mitochondrial transfer signals (MTS) being reported we utilized the ND4 signal sequence in this study.
  • the resultant expression vector also contained the puromycin resistance gene to allow for selection ( FIG. 1B ).
  • XbaIR is one of the most powerful endonucleases and a standard sequence of mtDNA named under Cambridge reference sequence (CRS) in human mitochondria genome has as many as five recognition sites targeted by the particular endonuclease ( FIG. 1D ). It was verified by an in vitro endonuclease co-incubation that isolated mtDNA was digested at multiple sites by XbaIR ( FIG. 1C ). In contrast, NotI digestion of mtDNA showed a single fragment, as predicted by Cambridge Reference Sequence (CRS) of mitochondrial DNA ( FIG. 1C ).
  • CRS Cambridge Reference Sequence
  • NHDF Normal Human Dermal Fibroblast
  • EGFP enhanced green fluorescent protein
  • a plasmid carrying MTS fused with EGFP was generated by subcloning the EGFP gene in place of the XbaIR gene to generate the pCAGGS-MTS-EGFP-PuroR plasmid ( FIG. 1F ). Then normal human dermal fibroblasts (NHDF) were transfected with the MTS-EGFP expression vector and the cells were counter stained with TMRM (tetramethylrhodamine, methyl ester), which is a cell-permeant dye that accumulates in active mitochondria with intact membrane potential ( FIG. 1G ).
  • TMRM tetramethylrhodamine, methyl ester
  • the efficiency and efficacy of the MTS-XbaIR expression vector relative to the conventional method that employs ethidium bromide (EtBr) was evaluated according to the scheme illustrated in FIG. 2A .
  • the placental venous endothelium-derived cell line EPC100 with DsRed labeled mitochondria were cultured in pyruvate-free DMEM (Wako cat #044-29765) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S), and at day 0 the cells were either untreated (“normal”), transfected with the MTS-XbaIR expression vector (“MTS-XbaIR”), or treated with 50 ng/mL of EtBr.
  • DMEM pyruvate-free DMEM
  • FBS fetal bovine serum
  • P/S penicillin/streptomycin
  • qPCR Quantitative polymerase chain reaction
  • Example III Partial Degradation of Endogenous Mitochondria Using MTS-XbaIR Construct in Recipient Cell Enabled Mitochondria Replacement from Exogenous Donor Cell
  • NHDF cells were transfected with the MTS-GFP or MTS-XbaIR plasmids and selected using puromycin after 48 hours. After 6 days post-transfection, isolated mitochondria from human cell lines originated from the endothelium of the uterus (named as EPC100) that were labeled with DsRed were transferred to the donor cells. A scheme of the protocol is shown in FIG. 3A .
  • the mitochondria content was evaluated by TMRM staining.
  • the MTS-GFP transfected cells exhibited a strong staining for TMRM, indicating high levels of mitochondria in the NHDF cells.
  • the MTS-XbaI transfected cells ⁇
  • TMRM staining FIG. 3B
  • the reduction of mitochondrial DNA was further confirmed by quantifying the number of mitochondrial DNA copies by qPCR of 12S-rRNA after adjusting with ⁇ -actin (Actb) in the nucleus ( FIG. 3C ).
  • Actb ⁇ -actin
  • the significant reduction in mitochondrial DNA in the ⁇ -cells continued for the length of the assay, which was stopped on day 12. Specifically, the copy numbers dropped to about 1 ⁇ 3 of the original copy numbers on day 6 and further declined to about 1 ⁇ 4 on day 12 in the ⁇ -cells ( FIG. 3C ).
  • Mitochondria were isolated from DsRed-Mt EMCs by differential centrifugation.
  • the cells were harvested from culture dishes with homogenization buffer [HB; 20 mM HEPES-KOH (pH 7.4), 220 mM mannitol and 70 mM sucrose] containing a protease inhibitor mixture (Sigma-Aldrich, St. Louis, Mo., USA).
  • the cell pellet was resuspended in HB and incubated on ice for 5 min.
  • the cells were ruptured by 10 strokes of a 27-gauge needle on ice.
  • the homogenate was centrifuged (400 g, 4° C., 5 min.) two times to remove unbroken cells.
  • the mitochondria were harvested by centrifugation (6000 g, 4° C., 5 min.) and resuspended in HB.
  • the amounts of isolated mitochondria were expressed as protein concentration using a Bio-Rad protein assay kit (Bio-Rad, Richmond, Calif., USA).
  • Mitochondrial transfer was conducted by co-incubating isolated mitochondria with cells in 2 ml of standard medium at 37° C. under 5% CO2 for 24 h.
  • the co-incubation of isolated mitochondria with ⁇ ( ⁇ ) cells on day 12 resulted in a significant increase in mtDNA copy number, similar to the levels of control NHDF cells ( FIG. 3C ).
  • the ⁇ ( ⁇ ) cells exhibited reduced mitochondria content after MTS-XbaI transfection, as measured by visualization of TMRM.
  • the decrease in mitochondria could be rescued by contacting the ⁇ ( ⁇ ) cells with the isolated exogenous mitochondria, as indicated by the uptake of the DsRed labeled isolated mitochondria ( FIG. 3D and FIG. 3E ).
  • the co-cultivation of DsRed-marked and isolated mitochondria with either NHDF control cells or NHDF cells transfected with the mock transfectant MTS-EGFP expression vector revealed that the exogenous mitochondria gathered around the cells and formed aggregates, but failed to be internalized ( FIG. 3D , lower panels).
  • the endonuclease method of the present invention was more efficacious in generating a mitochondria replaced cell having exogenous mitochondria ( FIG. 3F ).
  • the endonuclease method of the present invention was compared with (1) the add-on mitochondria transfer method, described in our previous works (see, e.g., Kitani, T., et al, J Cell Mol Med (2014) 18, 1694) or (2) a recently reported method (see, e.g., Kim, M. J., et al., Sci Rep 8, 3330, (2016)) that employed spinoculation of isolated mitochondria with metabolically healthy cells ( FIG. 3F ).
  • the MTS-XbaI expression vector can generate ⁇ ( ⁇ ) cells that have a partial deletion of endogenous mitochondria, and the mitochondrial content can be rescued by transferring isolated exogenous mitochondria from donor cells.
  • the methods of the current invention provide improved efficiency of mitochondrial transfer, relative to previously described methods, such as those performed in combination with centrifugation, or simple “adding on” the mitochondria without partially reducing the endogenous mtDNA.
  • mitochondrial transfer was unable to be performed in cells with a complete degradation of endogenous mitochondria ( ⁇ (0) cells), which indicated that the uptake of exogenous mitochondria likely requires energy.
  • Example IV Isolated Exogenous Mitochondria Fuse with Endogenous Mitochondria to Transfer Donor mtDNA
  • transient intermitochondrial fusion events have been observed, where two mitochondria came into close apposition, exchanged soluble inter-membrane space and matrix proteins and re-separated, preserving the original morphology (see, e.g., Liu X et al., EMBO J. 2009; 28(20):3074-3089; Huang X et al. Proc Natl Acad Sci US A. 2013; 110(8):2846-2851). Therefore, transient intermitochondrial fusion events were analyzed under the conditions described herein.
  • Isolated mitochondria from EPC100 donor cells was labeled with DsRed, and recipient cells with EGFP-marked mitochondria were used.
  • a diagram of the protocol employed is illustrated in FIG. 4A .
  • Microscopy images of the temporal contact of the donor and resident mitochondria revealed that no broad mitochondrial fusion was observed ( FIG. 4B and FIG. 4C ).
  • the majority of the donor mitochondria existed separately from the endogenous mitochondria.
  • a few transient fusion images were observed, and then the donor mitochondria appeared to run away before it finally disappeared ( FIG. 4C ).
  • Mitochondrial transfer was performed according to the protocol illustrated in FIG. 4F . Briefly, the mitochondria of the recipient NHDF cells was marked with DsRed-marked ( FIG. 4D ), and mitochondria from the donor EPC100 cells was marked with TFAM, which binds to mtDNA and allows tracing of mitochondria ( FIG. 4E ). The recipient NHDF cells were transfected with the pCAGGS-MTS-XbaIR-P2A-PuroR expression vector, and selected with puromycin on day 2 for 24 hours. On day 6, mitochondrial transfer from TFAM-GFP labeled mitochondria from EPC100 donor cells was performed. Then on day 8, the cells were imaged.
  • the exogenous mitochondria are able to briefly interact with the endogenous mitochondria, and transport mtDNA during the brief contacts. Then, the exogenous mitochondrial membrane complexes can be degraded in the cytosol to provide building blocks for the reconstituted mitochondria.
  • the mitochondria of the recipient cell that receive the exogenous mitochondria are able to gradually reconstitute the mitochondrial membrane complex, and demonstrate the functional recovery.
  • Example V SNP Assay Detected Increase in Exogenous Mitochondria after Transfer of Isolated Exogenous Mitochondria
  • FIG. 5A and FIG. 5B To assess the origin of mtDNA following the mtDNA replacement, the different nucleotides identified between NHDF and EPC100 by sequencing the hypervariable region 1 and 2 were used ( FIG. 5A and FIG. 5B ). While NHDF preserves A at the position of 16362 in CRS, EPC100 harbors a mutation at the same position that resulted in a change from A to G ( FIG. 5B ). Importantly, evaluation of the mitochondria replaced ⁇ ( ⁇ ) cells (NHDF ⁇ ( ⁇ ) Mt) demonstrated the presence of both the original nucleotide in a minor wave and the exogenous nucleotide G in a major wave, which indicated that the cells were heteroplasmic ( FIG. 5B , bottom panel).
  • the heteroplasmy in the mitochondria replaced NHDF was further evaluated by the single nucleotide polymorphism assay to detect the difference between the recipient NHDF and the donor EPC100 ( FIG. 5C ).
  • the HV1 region was amplified using the hmt16318-F primer (5′-agccatttaccgtacatagcacatt-3′ (SEQ ID NO: 6)) and the hmt16414-R primer (5′-cacggaggatggtggtcaag-3′ (SEQ ID NO: 9)), and the SNP was detected using the NHDF specific probe (5′-CTTCTCGTCCCCATG-3′ (SEQ ID NO: 5)) and the EPC100 specific probe (5′-CCCTTCTCGCCCCCAT-3′ (SEQ ID NO: 7)) ( FIG.
  • Non-mitochondrial ATP production was upregulated, and the coupling ratio was downregulated in the ⁇ ( ⁇ ) cells ( FIG. 6B , lower row).
  • the phenotypic recovery of the mitochondria replaced cells was demonstrated by their proliferative capability ( FIG. 6C ). Specifically, the ⁇ ( ⁇ ) cells showed a poor proliferative capability, whereas the MirC recovered to levels near that of the control cells by days 6-12 ( FIG. 6C , right).
  • Example VII Inhibition of mTOR by Rapamycin Enhances Macropinocytosis of Exogenous Mitochondria in ⁇ ( ⁇ ) Cells
  • mTORC1 mammalian target of rapamycin complex 1
  • AMPK AMP-activated protein kinase
  • Palmitic acid (PA) was reported to activate mTORC1 at a concentration of 200 ⁇ M in vivo
  • the titration of PA for cultured fibroblasts showed the concentration of 50 ⁇ M and the duration of 24 hours was optimal based on the cellular viability.
  • the ratio of phosphorylated AMPK to AMPK and phosphorylated p70 S6 kinase to p70 S6 kinase, which is a downstream target of mTORC1 were examined by using capillary electrophoresis, WesTM (Protein Simple).
  • FIG. 6I A scheme of the protocol is illustrated in FIG. 6I .
  • the NHDF recipient cells were transfected with the MTS-XbaI expression vector and cultured with or without rapamycin, or with or without palmitic acid (PA).
  • Puromycin selection for ⁇ ( ⁇ ) cells expressing the MTS-XbaI was performed after 48 hours.
  • transfer of isolated mitochondria marked with DsRed from EPC100 cells was performed.
  • FACS analyses were performed to detect the donor mitochondria by measuring DsRed expression in the NHDF recipient cells.
  • rapamycin treatment significantly enhanced the engulfment of the DsRed-labeled isolated, exogenous mitochondria, whereas palmitic acid clearly suppressed it.
  • Example VIII mtDNA Replacement with Heteroplasmy Reversal in Fibroblasts Derived from Patient with Leigh Syndrome
  • the mitochondria in 7S fibroblasts contained exogenous and healthy mtDNA was examined by sequencing the mitochondrial genome fragment that included the 10158 nucleotide.
  • the mtDNA sequence of the 7SP cells changed from having a majority of mutant heteroplasmy at the 10158 nucleotide position (large wave of C and a small wave of T) to a majority of wild-type mtDNA (large wave of T and a small wave of C) in the recipient 7SP ⁇ ( ⁇ ) cells following mitochondria replacement ( FIG. 7E , bottom).
  • SNP single nucleotide polymorphism
  • Example IX mtDNA Replacement in Fibroblasts Derived from Patient with Leigh Syndrome Yields Improved Cell Lifespan and Cell Metabolism
  • the functional activity of mitochondrial replaced 7SP fibroblasts was evaluated. As shown in FIG. 8A and FIG. 8B , the proliferation of mitochondrial replaced 7SP fibroblasts ( ⁇ ( ⁇ ) Mt) cells was able to recover to levels equivalent to that of the original 7SP fibroblasts around day 12.
  • the mitochondrial replaced 7SP fibroblasts ( ⁇ ( ⁇ ) Mt) cells demonstrated a dramatic extension of lifespan, up to about the 63th population doubling level (PDL) while the doubling time was over 120 hours, which is the threshold of growth arrest ( FIG. 8C ).
  • the cells received the mtDNA replacement at about the 8th PDL and the reconstituted cells with the healthy mtDNA were able to continue dividing beyond the 55th PDL, which is thought to be the number of times a normal human cell population will divide before cell division stops (i.e., the Hayflick limit).
  • the na ⁇ ve 7S fibroblasts fell into senescence at the 25th PDL ( FIG. 8C ).
  • this methodology might provide a crucial clue in rejuvenation and this might provide a basis for a novel strategy for cancer therapy as well as therapies for other age-related diseases.
  • the functional effect of mitochondrial transfer in 7S fibroblasts was further evaluated by measuring the cell size ( FIG. 8D ).
  • the mutation in 7S fibroblasts in the coding sequence of the ND4 gene of Complex I in the respiratory chain resulted in a disturbance of Complex I to transfer electrons coupled with its function to pump protons up from the matrices to intermembrane space.
  • glycolysis was dominant to the mitochondrial ATP production in 7S fibroblasts and resulted in the compensatory adaptation to be bigger in cellular size to contain more mitochondria despite the damages and poor function ( FIG. 8D ).
  • STR short tandem repeat
  • Example X Transfer of Exogenous Mitochondria to Fibroblasts Derived from a Patient with Leigh Syndrome Yielded Functional Mitochondria
  • the functional effect of mitochondrial replacement in the 7SP fibroblasts was further evaluated by analyzing the cells' respiratory function by using Oroboros 02k ( FIG. 9A ). Quantification of the results indicated that the basal respiration and ATP production (Free Routine Activity) continued to decrease from the 10 th PDL to the 20th PDL after the generation of a mitochondrial replaced cell and the maximum capacity of electron transfer system kept the original levels of 7SP fibroblasts ( FIG. 9B ). By the 30th PDL after transfer of exogenous mitochondria, all three indices of the respiratory function (Routine, ETS, and Free routine activity) were elevated, and even surpassed the levels of the original cells ( FIG. 9B ).
  • Example XI Transfer of Exogenous Mitochondria can Dissipate Chronic and Sustained Reactive Oxygen Species (ROS) Generation
  • both a reperfusion and a starvation model under a culture condition were used for 7SP fibroblast-derived MirC, the original 7SP fibroblast, and NHDF as a control.
  • These stress conditions induce apoptosis in cultured cells, the extent of which can be quantified by AnnexinV as an early marker and propidium iodide (PI) as a late marker.
  • AnnexinV an early marker
  • PI propidium iodide
  • the reperfusion injuries are mainly attributed to mitochondrial dysfunction.
  • Cells predisposed to mitochondrial dysfunction due to mtDNA mutation are more fragile to the reperfusion injuries than healthy cells.
  • SASP early stage senescence-associated secretory phenotype
  • the expression levels of the representative SASP cytokines, IL-6 and IL-8, chemokine, CXCL-1, and growth factor, ICAM1 were quantitatively measured at the transcript levels for NHDF, 7SP fibroblast, and 7SP fibroblast-derived MirC cells, whose PDLs were almost the same, about 15 to 20 ( FIG. 11 ).
  • IL-6 was significantly higher in 7SP fibroblasts than those in NHDF and 7SP fibroblast-derived MirC, whereas the other three factors did not show any significant difference among these cells.
  • mitochondria replacement is able to not only treat mitochondrial diseases with mutations of mtDNA, but also rejuvenate senescent cells, such as cells involved in an array of diseases, including neurodegenerative, cardiovascular, metabolic, and autoimmune diseases, and even cancers.
  • Example XIII iPS Cells Generated from mtDNA Replaced Fibroblasts
  • iPSCs inducible pluripotent stem cells
  • the iPSC generated by the methods described herein were further compared to the commercially available KYOU-DXR0109B Human Induced Pluripotent Stem (IPS) Cells [201B7].
  • the mitochondria replaced 7SP fibroblasts showed the same level of efficiency with the iPS generation with that of healthy fibroblasts.
  • qPCR of 12S-rRNA, normalized to nuclear (3-actin, demonstrated that the iPS cells generated by mitochondrial replaced 7SP fibroblasts exhibited half of the mtDNA contents relative to control, and the mtDNA levels were similar to that of the 201B7 iPSC standard ( FIG. 12G ).
  • the hmt10158 heteroplasmy level was less than 10% in the generated iPSCs ( FIG. 12H ). Quantification of the absolute mtDNA copy number confirmed the reduced level of mtDNA and reduction in mutant mtDNA ( FIG. 12I ).
  • iPSCs can be generated using the mitochondrial replacement technology provided herein, and could be applicable in the clinical field because this whole procedure used only materials adaptable to clinics.
  • NHDFs and TIG1 embryonic lung cells with early PDL (around 5 to 10, called “young”) and late PDL (around 40 to 45, called “old”) were utilized to design the models.
  • One model involved young cells replaced with mitochondria derived from old cells, designated as “02Y,” and another model involved old cells replaced with mitochondria derived from young cells, designated as “Y20” ( FIG. 13A ).
  • Example XV Optimization of Mitochondria Replaced Cell (MirC) from Human Primary T Cells Using mRNA Transfection
  • This example describes the generation of mitochondria replaced Cell (MirC) from human primary T cells by using mRNA transfection.
  • lymphocytes Prior to the experiments, use of human primary T cells were approved by our institutional ethical committee. Peripheral blood was drawn from a healthy volunteer and centrifuged using percoll with a specific gravity of 1.077 at 400 g for 35 minutes at 20 degree to separate lymphocytes. Isolated lymphocytes of 1 ⁇ 10 6 cells per ml were seeded onto a 96-well flat plate coated with anti-CD3 and anti-CD28 antibodies. The plate was prepared by incubating with 5 ⁇ g/ml of anti-CD3 and 1 ⁇ g/ml of anti-CD28 of overnight and pre-warmed at 37° C. 2 hours prior to the seeding.
  • IL-7 and IL-15 were added to the medium at a concentration of 20 ⁇ g/ml and 10 ⁇ g/ml, respectively.
  • Medium was changed every third day with IL-7 and IL-15 at the same concentration as the initial addition.
  • mRNA was created according to the manufacturer's protocol in mMESSAGE mMACHINE T7 Ultra Kit (Thermo Fisher), with slight modifications. Briefly, a DNA template for mRNA was prepared from the plasmid carrying the DNA sequence without refining the fragment following endonuclease digestion, in order to reduce the possibility of mixing RNase up as lower as possible ( FIG. 14A ).
  • the protein expression of GFP in cells receiving the MTS-GFP mRNA was evaluated by western blotting analysis using a capillary electrophoresis. The peak expression occurred at day 4, and expression was lost by day 6, as illustrated in the western blot ( FIG. 14H ) and quantified in FIG. 14G . Quantification of kinetics of XbaIR transcript levels were performed by qPCR and revealed that the transcript expressions of the endonuclease were quite highest at 4 hours post-gene transfer ( FIG. 14I ). The XbaIR transcript levels rapidly decreased by day 2, and were negligible by day 6 ( FIG. 14I ). The mitochondrial contents were estimated by quantifying 12S rRNA ( FIG. 14J ), and demonstrated that mitochondria decreased to about 30% by day 2, and was maintained at less than 20% throughout the length of the experiment.
  • Example XVI Generation of Mitochondria Replaced Cell (MirC) from Human Primary T Cells Using mRNA Transfection
  • FIG. 15A A scheme of the MirC protocol for human primary T cells is shown in FIG. 15A .
  • the difference of mtDNAs between the donor mitochondria and the recipient cells was determined by TaqMan SNP genotyping assay. Sequencing of the D-loop of mtDNA in normal human primary T cells and EPC100 (mitochondria donor cells) showed a difference in 2 nucleotide positions (nucleotides 218 and 224 mtDNA), which were C/C and T/T for T cells and EPC100 cells, respectively ( FIG. 15B ).
  • the fragment of variable region encompassing the 218 and 224 nucleotides of mtDNA was subcloned into pBluescript SK( ⁇ ).
  • the polymorphic nucleotides were mapped on the human Cambridge Reference Sequence, and primers and probes were designed to amplify and target the desired region of the D-loop, where the probes had FAM and VIC fluorophore ( FIG. 15C ).
  • qPCR was carried out and threshold cycle (Ct value) was determined, which was fit to a standard curve created using several different copy numbers of the above mentioned plasmids for each sequence.
  • FIG. 15E Representative raw data using coupling-control protocol (CCP) are depicted in FIG. 15F and FIG. 15G , and show that MirC T cells are able to restore mitochondrial respiration.
  • CCP coupling-control protocol
  • human primary T cells are capable of mitochondria replacement to generate MirC using GMP graded electroporator, such as the electroporator produced by MaxCyte Inc.
  • T cell-derived MirC Further characterizations of T cell-derived MirC were executed for murine T cells.
  • the isolation of murine T cells from suspension solutions obtained from the spleen was performed using the EasySep Mouse Isolation Kit (STEM CELL Technologies, Inc.), which provides highly purified T cell population by negative selection using magnets.
  • Isolated murine T cells (1 ⁇ 10 6 cells per ml) were seeded onto 96-wells plate with Dynabeads mouse T-Activator CD3/CD28 (Invitrogen, Inc.) at a bead-to-cell ratio of 1:1 and recombinant IL-2 at 30 U/ml.
  • the medium to cultivate murine T cells was determined with respect to cell growth and CD3 expression, and RPMI1640 was found to be superior to TexMACS ( FIG. 16A ). For example, viability and total cell number were higher in cells cultured in RPMI1640, as compared to TexMACS. The medium was changed every third or fourth day.
  • This example demonstrates that mitochondria replacement was successful in murine T cells, and rejuvenated senescent T cells.
  • mtDNA heteroplasmy levels were of BL6 (recipient) cells and NZB (donor) cells.
  • Two-consecutive polymorphisms at 2766 and 2767 mtDNA for ND1 was verified ( FIG. 17A ).
  • the BL6 mitochondria contained AT at positions 2766 and 2767 of mtDNA
  • the NZB mitochondria contained GC at the same positions.
  • a primer set and two probes were designed to discriminate the polymorphism using a different fluorophore for each of the GC and AT polymorphisms ( FIG. 17B ).
  • two separate plasmids were generated to express the GC and AT polymorphisms, respectively, and a standard curve was generated to facilitate the quantitative estimation of heteroplasmy in MirC.
  • T cell-derived MirC was also examined for rejuvenation potential.
  • Recipient cells from old murine T cells were prepared from the spleen of mice (C57BL/6) that were more than 80 weeks old, and donor murine mitochondria were isolated from the liver of mice (C57BL/6) around 10 weeks old.
  • Telomere length has been reported to shorten with age. Therefore, telomere length was measured by using Absolute mouse Telomere Length Quantification qPCR Assay Kit (ScienCell, Inc.).
  • telomere length was observed to have a 1.7-fold increase in length, relative to the original old T cells ( FIG. 17D ). This demonstrated that the mitochondrial replaced cells exhibit characteristics of rejuvenation.
  • senescent T cells have been found to exhibit higher DNA damage response (DDR), compared with young T cells. Therefore, DDR was measured using the histone 2 A (H2A) phosphorylation antibody, for the MirC and the original T cells. The results indicated that the positive fraction for DDR was lower in the MirC (1.53%), compared with the original T cells (4.75%) ( FIG. 17F ). Thus, the MirC T cells had lower levels of DDR, indicating a reversal of senescent behavior.
  • H2A histone 2 A
  • Example XIX Tumor Growth is Mitigated by Adoptive Cell Transplantation (ACT) Using MirC Derived from Old T Cells Containing Exogenous Mitochondria from Young Mice
  • ACT adoptive cell transplantation
  • AE17 cells were subcutaneous injected into three groups of old mice (Group 1: old mice with ACT of T cells from young mouse; Group 2: old mice; or Group 3: old mice with ACT of MirC derived from a T cell of an old mouse transferred with exogenous mitochondria from a young mouse ( FIG. 18A ).
  • the old T cell-derived MirC were evaluated for their capability to suppress the tumor growth.
  • C57BL/6 mice aged 22 to 24 months were utilized in the ACT experiment.
  • the young mice used in the experiment were 2 to 3 months old mice.
  • tumor growth was measured using NIH image of photographs taken every 3 days ( FIG. 18B ).
  • AE17 inoculation was executed on day ⁇ 14 with 2 ⁇ 10 6 cells suspended in 100 ⁇ L Matrigel, and the day of T cell transfer was considered to be day 0.
  • 2 ⁇ 10 6 cells of either young T cells or old T cell-derived MirC were intravenously injected into tumor-bearing mice.
  • recombinant IL-2 (2 ⁇ g) was intraperitoneally injected once, followed by two more injections on day 2 and day 3.
  • T cells derived from GFP transgenic mice were transplanted into the syngeneic C57BL/6 mice, the peripheral blood and the spleen were examined to track the donor cells ( FIG. 18F ).
  • a two-dimensional plot with FSC versus FL-1 to detect GFP fluorescence was generated to clarify the rare population. Negative controls using C57BL/6 mice (left upper panel), and positive controls using GFP transgenic mice (left lower panel) were generated for both the peripheral blood and the spleen ( FIG. 18G ).
  • the definitive population of T cells expressing GFP fluorescence were recognized in both samples, although the fractions were 0.057% and 0.9% in the peripheral blood and the spleen, respectively ( FIG. 18G ).
  • the transferred T cells in this protocol were detected on day 6 following the transplantation ( FIG. 18H ), which validated that this protocol could be used to evaluate the capability of the transferred cell.
  • the percentage of chimerism following infusion of the exogenous T cells was found to increase when a greater amount of cells were infused ( FIG. 18I ).
  • FIG. 19A Based on the experiments for fibroblasts and T cells, the condition of Nucleofector/electroporation with mRNA was adjusted, and several conditions were examined for murine fetal liver-derived Sca-1 positive cells ( FIG. 19A ), which are considered to be an enriched population for hematopoietic stem cells (HSCs). Among several conditions, three conditions (program X-001, Y-001, and T-030 that are code number in the machine provider) were evaluated by immunofluorescence and cell viability ( FIG. 19A ). The experimental conditions were termed MTS-GFP1, 2, and 3 according to the program that was used (program X-001, Y-001, and T-030, respectively).
  • FIG. 19B Further examinations were performed by FACS analysis for the mean fluorescent intensities (MFI) on dayl following the electroporation with mRNA of GFP ( FIG. 19B ). The results indicated that the optimal condition was the X-001 program (MTS-GFP1) because although the right shift of MFI in the condition was little, it was significant compared with the others ( FIG. 19B ).
  • Murine bone marrow-derived Sca-1 cells were coincubated with mitochondria isolated from the syngeneic murine cells that are a stable gene-modified cell line expressing DsRed fluorescence. 3-D fluorescent imaging of the bone marrow-derived Sca-1 cells 48 hours after the co-incubation showed that the exogenous mitochondria were engulfed ( FIG. 19C ).
  • the mitochondrial transfer efficiency was estimated by FACS analysis for DsRed fluorescent axis, and revealed that a subpopulation of about 10% of the Sca-1 exhibited a right ward shift of the fluorescent, suggesting that BM-derived Sca-1 positive cells could undergo somatic mitochondria replacement ( FIG. 19D ).
  • the transfer of exogenous mitochondria in the MTS-GFP expression cells without depletion of endogenous mitochondria was too low for clinical application.
  • FIG. 19E The Real hematopoietic stem cell population is considered as c-kit + , Sca-1 + , Lineage ⁇ , CD34 ⁇ (called as KSLC) that is around 0.005% in the whole bone marrow cells (Wilkinson, A. C. et al. Nature, 571(7763):117-121 (2019)).
  • KSLC Lineage ⁇ , CD34 ⁇
  • the KSL cells were cultivated for 5 days in the presence of stem cell factors and TPO with polyvinyl alcohol (PVA). Macroscopically, the KSL cells maintained the morphology and exhibited a short doubling time of 19 hours ( FIG. 19G ).
  • PVA polyvinyl alcohol
  • FIG. 19H A scheme of the assay is shown in FIG. 19H .
  • Murine KSLC-derived MirC demonstrated that the exogenous mtDNA with polymorphism in NZB was 99.9% on day 6 following the endonuclease mRNA transfer with electroporation ( FIG. 19I ), which indicated that the exogenous mtDNA almost completely replaced the endogenous mtDNA of CL57BL/6. These results demonstrated that hematopoietic stem cells are permissive to this technology to generate MirC.
  • Example XXI Droplet Digital PCR (ddPCR) for Measurement of mtDNA and Heteroplasmy
  • mtDNA mitochondrial DNA
  • dPCR digital PCR
  • ddPCR Droplet Digital PCR
  • Cells from the target population were encapsulated into droplets at a concentration of one cell per droplet with the PCR mixture including primers and probes. Cell density was optimized to generate a single cell in a single droplet, and the fibroblasts were finally diluted in 1 ⁇ 10 6 cell/mL for ddPCR. After single-cell encapsulation, cell lysis and amplification of the target sequence were performed within the droplets. The number of droplets with a fluorescent signal indicated the number of cells carrying the target or reference gene.
  • the standard ddPCR master mix was a 25 ⁇ L mix that includes the aforementioned primer/probe mix, template DNA and 2 ⁇ ddPCR super mix.
  • Samples were loaded into an 8-chamber cartridge using 20 ⁇ L of the prepared qPCR sample followed by 70 ⁇ L of droplet generation oil in the adjacent wells. A rubber gasket was stretched across the top of the chambers to ensure a vacuum seal.
  • Each 8-chamber cartridge was loaded onto the QX100 droplet generator producing 20,000 droplets per sample.
  • 40 ⁇ L of the generated droplets were transferred to a 96-well plate and heat sealed with pierceable foil.
  • the plate was placed in a thermal cycler using standard 2-step qPCR thermal cycling conditions with a 50% (3° C./sec) ramp rate.
  • primer/probe sets Prior to running thermal cycling conditions, primer/probe sets were optimized using a temperature gradient to optimize the anneal/extend temperature.
  • the specificity for the probes to be designed for a mutated sequence and the sensitivity for the probes to be designed for a non-mutated sequence were evaluated by using normal human dermal fibroblasts (NHDF cells) that have a non-mutated sequence (the same as Cambridge Reference Sequence) ( FIG. 20A - FIG. 20C ).
  • Evaluation of the three different probe sets clearly detected the non-mutated sequence (lower right in BK01 ( FIG. 20A ), upper left in BK02 ( FIG. 20B ), and upper left in BK04 ( FIG. 20C )), and did not detect the mutant sequence (upper left in BK01 ( FIG. 20A ), lower right in BK02 ( FIG. 20B ) and BK04 ( FIG. 20C )).
  • ddPCR of fibroblasts obtained from BK01 indicated a few percentage of double positive population, and the majority was cells with homoplasmy of mutated mtDNA ( FIG. 20D ). There was no significant population with homoplasmy of non-mutated mtDNA in a single cell.
  • BK02 showed a minor portion of double positive cells, which indicated a heteroplasmy in a single cell level, defined as microheteroplasmy ( FIG. 20E ). The results from BK02 revealed a major population of homoplasmy of mutant mtDNA, and no population with homoplasmy of non-mutated mtDNA was not recognized.
  • results demonstrated that homoplasmy and heteroplasmy can be accurately, and quantitatively evaluated at a single cell level.
  • results demonstrate that the mtDNA of a subject with mitochondrial disease can be accurately measured, which could be useful for evaluating therapeutic compositions prior to transplantation in a subject or monitoring the mtDNA content prior to and/or after therapy.
  • Example XXII MtDNA Replacement in Recipient Hematopoietic Stem or Progenitor Cells (HSPCs) from Donor cGMP Manufactured Bone-Marrow Derived Mesenchymal Stromal Cells (BM-MSCs)
  • hematopoietic stem or progenitor cells can be ex vivo modulated using the mtDNA replacement methods provided herein for therapy.
  • HSPCs hematopoietic stem or progenitor cells
  • CD34 + cells are isolated and sent to a manufacturing facility.
  • the mitochondria is partially depleted according to the methods provided herein.
  • Donor mitochondria are isolated using current Good Manufacturing Practice (cGMP) manufactured bone-marrow derived Mesenchymal Stromal Cells (BM-MSCs) obtained from a cell repository (e.g., Waisman Biomanufacturing).
  • cGMP current Good Manufacturing Practice
  • BM-MSCs bone-marrow derived Mesenchymal Stromal Cells
  • the initial bone marrow aspirates are collected with full informed consent and in compliance with federal regulations (e.g., 21 CFR 1271).
  • the aspirates are processed under cGMPs and banked at an early passage for subsequent expansion.
  • the donor mitochondria from the BM-MSCs are transferred to cultured HSPCs, changing the heteroplasmy.
  • the modified HSPCs are sent back to the medical center for autologous transplantation (i.e., into the same subject that the HSPCs were isolated).
  • the patient Prior to transplantation the patient receives minimal treatment that can include a non-myeloablative regimen, such as partial irradiation or sublethal dose of anti-cancer drugs, such as busulfan.
  • the modified HSPCs only containing the allogenic donor mitochondria, are transfused back into the patient.
  • HSPCs can be ex vivo modulated using the mtDNA replacement methods provided herein for therapy that does not involve transplantation of allogenic HSPCs.

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