Methods and compositions for treating mitochondrial diseases or disorders and heteroplasmy

Abstract

The present invention provides methods and compositions for the production of mitochondrial replacement cells (MirCs), as well as therapeutic methods using such compositions to treat subjects with age-related diseases or syndromes, mitochondrial diseases or disorders, or otherwise in need of mitochondrial replacement. Methods and compositions for producing recipient cells with mitochondrial diseases or disorders, and methods and compositions for producing or enhancing the production of populations of induced pluripotent stem cells (iPSCs) are also provided. Additionally, methods and compositions for enhancing mitochondrial import are included. [Selected Figure] Figure 1A

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 718,891, filed Aug. 14, 2018, U.S. Provisional Application No. 62 / 731,731, filed Sep. 14, 2018, and U.S. Provisional Application No. 62 / 817,987, filed Mar. 13, 2019, which are hereby incorporated by reference in their entirety.

[0002] (Sequence Listing) This application is being filed electronically in ASCII format and includes a sequence listing that is hereby incorporated by reference in its entirety. The ASCII copy created on Aug. 13, 2019 is named 14595-001-228_SL.txt and is 12,905 bytes in size.

[0003] (1. Field of the Invention) The present invention provides compositions of cells having reduced and / or exchanged mitochondrial DNA, methods for producing the same, and methods for treating various diseases associated with hereditary or age-related mitochondrial dysfunction.

Background Art

[0004] (2. Background of the Invention) Mitochondria play a major and important role in cellular homeostasis and are involved in various disease processes. It is involved in intracellular signaling and apoptosis and performs numerous biochemical tasks such as pyruvate oxidation, the Krebs cycle, and the metabolism of amino acids, fatty acids, nucleotides, and steroids. One extremely important task is its role in cellular energy metabolism. This includes β-oxidation of fatty acids and the production of ATP by 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, including 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).

[0005] The mammalian mitochondrial genome is a small circular double-stranded molecule containing 37 genes, including 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and 2 ribosomal RNA (rRNA) genes. Of these, 24 (22 tRNAs and 2 rRNAs) are required for mitochondrial DNA translation, and 13 encode subunits of the respiratory chain complexes. Furthermore, nuclear DNA (nDNA) encodes the majority of the approximately 900 gene products within mitochondria.

[0006] Mitochondrial diseases or disorders are a clinically heterogeneous group of disorders characterized by dysfunctional mitochondria. Disease onset can occur at any age and may present with a wide variety of clinical symptoms. Mitochondrial diseases or disorders can affect any organ or tissue and characteristically can affect multiple systems, generally those organs that are extremely dependent on aerobic metabolism, and often progress relentlessly with high morbidity and mortality. Mitochondrial diseases or disorders are the most common group of inherited metabolic disorders and one of the most common forms of inherited neuropathy.

[0007] Mitochondrial diseases or disorders can be caused by mutations in genes of nuclear DNA (nDNA) and / or mitochondrial DNA (mtDNA) that encode structural mitochondrial proteins or proteins involved in mitochondrial function. Some mitochondrial disorders affect only a single organ (e.g., the eye in Leber hereditary optic neuropathy [LHON]), but many affect multiple organ systems and often exhibit prominent neurological and myopathic features. Despite the higher frequency of involvement of energy-demanding tissues such as the brain, muscle, and eye, the patient phenotype can be extremely variable and heterogeneous. This variability is due in part to several factors such as dual genetic control (nDNA and mtDNA), the level of heteroplasmy (percentage of mutant DNA in a single cell and tissue), tissue energy demand, maternal inheritance, and mitotic segregation.

[0008] Many patients with mitochondrial diseases or disorders have a mixture of mutant and wild-type mtDNA (known as heteroplasmy); the ratio of mutant to wild-type mtDNA is an important factor in determining whether a cell will express a biochemical defect. Most pathogenic mtDNA mutations are heteroplasmic, with a mixture of mutant and wild-type mtDNA within individual cells. High levels of heteroplasmy refer to cells with high levels of mutant mtDNA and low levels of wild-type mtDNA, whereas low levels of heteroplasmy refer to cells with low levels of mutant mtDNA and high levels of wild-type mtDNA. Studies of single cells from patients with mitochondrial diseases or disorders have shown that the levels of mutant and wild-type mtDNA are highly important in determining cell phenotype. For example, a cell will have a respiratory deficit if it contains high levels of mutant mtDNA and low levels of wild-type mtDNA (i.e., high levels of heteroplasmy). The threshold at which this deficit occurs is determined by the exact mutation and cell type. Usually, a high percentage level of mutant mtDNA (>50%) is required to cause a cell deficit, although some mtDNA mutations (usually mt tRNA mutations) cause a deficit only when present at very high levels, and other mtDNA mutations (e.g., a single large-scale mtDNA deletion) cause a deficit when ~60% of the mtDNA is deleted. For example, in individuals carrying the m.8993T>G pathogenic variant, a higher percentage level of mutant mtDNA is seen in individuals with Leigh syndrome than in those with neuropathy, ataxia, and retinitis pigmentosa (NARP). Furthermore, the clinical phenotypes in MELAS and MERRF correlate with heteroplasmy (see, e.g., Chinnery, P. F. et al., Brain 120(Pt 10), 1713-1721(1997)).

[0009] Advances in next-generation sequencing technologies have revealed many mutations that cause mitochondrial diseases or disorders. Further, studies of other organisms such as the nematode worm (C. elegans) have revealed some of the proteins involved in heteroplasmy. For example, recent studies using C. elegans have shown that the mitochondrial unfolded protein response (UPRmt) functions to maintain heteroplasmy and to expand mutant mtDNA after disruption of the original mtDNA (see, e.g., Lin, Y. F. et al., Nature 533, 416-419, doi:10.1038 / nature17989 (2016)). However, the mechanisms underlying the maintenance and expansion of heteroplasmy in mammals remain unknown.

[0010] The management and treatment of patients with mitochondrial diseases or disorders remains difficult. For the vast majority of patients, the condition progresses relentlessly, resulting in significant morbidity and, in the most severely affected patients, death. Classical methods for removing endogenous mtDNA involve long-term treatment of cells with low concentrations of ethidium bromide (EtBr), which are known carcinogens and teratogens, limiting their use for therapeutic purposes. In addition to the potential for unwanted side effects, EtBr protocols can take several months, further restricting their clinical use. Furthermore, mitochondrial transfer protocols typically involve complete depletion of endogenous mtDNA in cells called rho(ρ)0 cells prior to transfer of exogenous mitochondria. This complete depletion of mtDNA severely impairs the ability of cells to take up exogenous mitochondria.

[0011] Other mitochondrial transfer protocols have attempted to add mitochondria without depleting endogenous mtDNA, but this approach has been found to be inefficient and harmful to cells. For example, mitochondrial transfer using simple co-incubation has been reported to be ineffective and not equally efficient among different cell types. Additional techniques for transfer require invasive instruments that harm recipient cells, or injections using other invasive instruments such as nanoblades, but all were less efficient than co-incubation (Caicedo et al., Stem Cells International, (2017), vol. 2017, Article ID 7610414, page 23).

[0012] Therefore, recent mitochondrial transfer methods are not only not practical for the clinical setting, but are also inefficient, harmful to recipient cells, and / or very time-consuming. Accordingly, there is a great unmet need to develop improved mitochondrial transfer methods that can be optionally used in the treatment of subjects having or suspected of having a mitochondrial disease or disorder and diseases or disorders associated with non-functional or malfunctioning mitochondria, as well as improved models for studying mitochondrial diseases or disorders. SUMMARY OF THE INVENTION

[0013] (3. Summary of the Invention) In one aspect, provided herein is a method of generating mitochondrial replacement cells, comprising: (a) contacting a recipient cell with an agent that reduces the endogenous mtDNA copy number; (b) incubating the recipient cell for a period sufficient 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 is partially reduced and (2) exogenous mitochondria from a healthy donor for a period sufficient to non-invasively transfer the exogenous mitochondria into the recipient cell, thereby generating mitochondrial replacement cells.

[0014] In another aspect, provided herein is a method of treating a subject in need of mitochondrial replacement, comprising: (a) generating mitochondrial replacement cells ex vivo or in vitro, comprising: (i) contacting a recipient cell with an agent that reduces the mtDNA copy number; (ii) incubating the recipient cell for a period sufficient 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 is partially reduced and (2) exogenous mitochondria from a healthy donor for a period sufficient to non-invasively transfer the exogenous mitochondria into the recipient cell, thereby generating mitochondrial replacement cells; and (b) administering a therapeutically effective amount of the mitochondrial replacement recipient cells from step (a) to the subject in need of mitochondrial replacement.

[0015] In another aspect, provided herein is a method of treating a subject having or suspected of having an age-related disease, the method comprising: (a) (i) contacting recipient cells with an agent that reduces the mtDNA copy number; (ii) incubating the recipient cells for a period sufficient for the agent to partially reduce the mtDNA copy number in the recipient cells; and (iii) (1) co-incubating the recipient cells from step (ii) in which the endogenous mtDNA is partially reduced with (2) exogenous mitochondria from a healthy donor for a period sufficient to non-invasively transfer the exogenous mitochondria into the recipient cells, thereby producing mitochondrial exchange cells: producing the mitochondrial exchange cells ex vivo or in vitro; and (b) administering a therapeutically effective amount of the mitochondrial exchange recipient cells from step (a) to a subject having or suspected of having the age-related disease.

[0016] In a further aspect, provided herein is a method of treating a subject having or suspected of having a mitochondrial disease or disorder, the method comprising: (a) (i) contacting recipient cells with an agent that reduces the mtDNA copy number; (ii) incubating the recipient cells for a period sufficient for the agent to partially reduce the mtDNA copy number in the recipient cells; and (iii) (1) co-incubating the recipient cells from step (ii) in which the endogenous mtDNA is partially reduced with (2) exogenous mitochondria from a healthy donor for a period sufficient to non-invasively transfer the exogenous mitochondria into the recipient cells, thereby producing mitochondrial exchange cells: producing the mitochondrial exchange recipient cells ex vivo or in vitro; and (b) administering a therapeutically effective amount of the mitochondrial exchange recipient cells from step (a) to a subject having or suspected of having the mitochondrial disease or disorder.

[0017] In some embodiments of the methods provided herein, the exogenous mitochondria are functional mitochondria. In certain embodiments, the exogenous mitochondria contain wild-type mtDNA. In specific embodiments, the exogenous mitochondria are isolated mitochondria. In further embodiments, the isolated mitochondria are intact mitochondria. In some embodiments, the exogenous mitochondria are allogeneic.

[0018] Also provided herein is a method of generating a mitochondrial exchange cell, comprising: (a) contacting a recipient cell with an agent that reduces the endogenous mtDNA copy number; (b) incubating the recipient cell for a period sufficient 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 is partially reduced and (2) exogenous mtDNA from a healthy donor for a period sufficient to non-invasively transfer the exogenous mtDNA into the recipient cell, thereby generating a mitochondrial exchange cell.

[0019] The present disclosure provides a method for treating a subject in need of mitochondrial replacement, comprising: (a) (i) contacting recipient cells with an agent that reduces the mtDNA copy number; (ii) incubating the recipient cells for a period sufficient for the agent to partially reduce the mtDNA copy number in the recipient cells; and (iii) co-incubating (1) the recipient cells from step (ii) in which the endogenous mtDNA is partially reduced and (2) exogenous mtDNA from a healthy donor for a period sufficient to non-invasively transfer the exogenous mtDNA into the recipient cells, thereby generating mitochondrial replacement cells: wherein the mitochondrial replacement cells are generated ex vivo or in vitro; and (b) administering a therapeutically effective amount of the mitochondrial replacement recipient cells from step (a) to a subject in need of mitochondrial replacement.

[0020] In another aspect, provided herein is a method for treating a subject having or suspected of having an age-related disease, comprising: (a) (i) contacting recipient cells with an agent that reduces the mtDNA copy number; (ii) incubating the recipient cells for a period sufficient for the agent to partially reduce the mtDNA copy number in the recipient cells; and (iii) co-incubating (1) the recipient cells from step (ii) in which the endogenous mtDNA is partially reduced and (2) exogenous mtDNA from a healthy donor for a period sufficient to non-invasively transfer the exogenous mtDNA into the recipient cells, thereby generating mitochondrial replacement cells: wherein the mitochondrial replacement cells are generated ex vivo or in vitro; and (b) administering a therapeutically effective amount of the mitochondrial replacement recipient cells from step (a) to a subject having or suspected of having an age-related disease.

[0021] In another aspect, provided herein is a method of treating a subject having or suspected of having a mitochondrial disease or disorder, the method comprising: (a) (i) contacting recipient cells with an agent that reduces the mtDNA copy number; (ii) incubating the recipient cells for a period of time sufficient for the agent to partially reduce the mtDNA copy number in the recipient cells; and (iii) (1) co-incubating the recipient cells from step (ii) in which the endogenous mtDNA is partially reduced with (2) exogenous mtDNA from a healthy donor for a period of time sufficient to non-invasively transfer the exogenous mtDNA into the recipient cells, thereby creating mitochondrial exchange cells: producing the mitochondrial exchange recipient cells ex vivo or in vitro; and (b) administering a therapeutically effective amount of the mitochondrial exchange recipient cells from step (a) to a subject having or suspected of having the mitochondrial disease or disorder.

[0022] In certain embodiments of the methods provided herein, the agent that reduces the endogenous mtDNA copy number is selected from the group consisting of a polynucleotide encoding a fusion protein comprising a mitochondrial targeting sequence (MTS) and an endonuclease, a polynucleotide encoding an endonuclease, and a small molecule. In some embodiments, the small molecule is a nucleoside reverse transcriptase inhibitor (NRTI). In other embodiments, the polynucleotide is composed of messenger ribonucleic acid (mRNA) or deoxyribonucleic acid (DNA). In further embodiments, the recipient cell transiently expresses the fusion protein. In still further embodiments, 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). In some embodiments, the MTS targets a mitochondrial matrix protein. In a specific embodiment, 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.

[0023] In some embodiments of the methods provided herein, the agent that reduces the endogenous mtDNA copy number reduces the endogenous mtDNA copy number by about 5% to about 99%. In one embodiment, the agent that reduces the endogenous mtDNA copy number reduces the endogenous mtDNA copy number by about 30% to about 70%. In further embodiments, the agent that reduces the endogenous mtDNA copy number reduces the endogenous mtDNA copy number by about 50% to about 95%. In still further embodiments, the agent that reduces the endogenous mtDNA copy number reduces the endogenous mtDNA copy number by about 60% to about 90%. In some embodiments, the agent that reduces the endogenous mtDNA copy number reduces mitochondrial aggregates.

[0024] Also provided herein is a method of producing mitochondrial exchange cells, comprising: (a) contacting a recipient cell with an agent that reduces mitochondrial function; (b) incubating the recipient cell for a period sufficient 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) with partially reduced endogenous mitochondrial function and (2) exogenous mitochondria from a healthy donor for a period sufficient to non-invasively transfer the exogenous mitochondria into the recipient cell, thereby producing mitochondrial exchange cells.

[0025] The present disclosure also provides a method of producing mitochondrial exchange cells, comprising: (a) contacting a recipient cell with an agent that reduces mitochondrial function; (b) incubating the recipient cell for a period sufficient 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) with partially reduced endogenous mitochondrial function and (2) exogenous mtDNA from a healthy donor for a period sufficient to non-invasively transfer the exogenous mtDNA into the recipient cell, thereby producing mitochondrial exchange cells.

[0026] In some embodiments of the methods provided herein, 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.

[0027] In certain embodiments of the methods provided herein, the subject in need of mitochondrial replacement has dysfunctional mitochondria; a disease selected from the group consisting of age-related diseases, mitochondrial diseases or disorders, neurodegenerative diseases, retinal diseases, diabetes, hearing impairments, genetic diseases; or a combination thereof. In some embodiments, 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. In a specific embodiment, the retinal disease is selected from the group consisting of age-related macular degeneration, macular edema, and glaucoma.

[0028] In some embodiments of the methods provided herein, the age-related disease is selected from the group consisting of autoimmune diseases, metabolic diseases, genetic diseases, cancer, neurodegenerative diseases, and immune senescence. In certain embodiments of the methods provided herein, the metabolic disease is diabetes. In further embodiments, the neurodegenerative disease is Alzheimer's disease, or Parkinson's disease. In still further embodiments, the genetic disease is selected from the group consisting of Hutchinson-Gilford progeria syndrome, Werner syndrome, and Huntington's disease.

[0029] In certain embodiments of the methods provided herein, mitochondrial diseases or disorders are caused by mitochondrial DNA abnormalities, nuclear DNA abnormalities, or both. In specific embodiments, mitochondrial diseases or disorders caused by mitochondrial DNA abnormalities include chronic progressive external ophthalmoplegia (CPEO), Pearson syndrome, Kearns-Sayre syndrome (KSS), diabetes, and deafness-dystonia (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 / substantia nigra degeneration (FBSN), Luft disease, aminoglycoside-induced deafness (AID), and mitochondrial DNA multiple deletion syndrome, and are selected from the group consisting of.In yet other specific embodiments, mitochondrial diseases or disorders caused by nuclear DNA abnormalities 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 ataxic neuropathy dysarthria ophthalmoparesis (SANDO), disorders of the brainstem and spinal cord, and leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL), coenzyme Q10 deficiency, Leigh syndrome, mitochondrial complex abnormalities, fumarase deficiency, alpha - ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl - CoA ligase deficiency, pyruvate dehydrogenase complex deficiency (PDHC), pyruvate carboxylase deficiency (PCD), carnitine palmitoyltransferase I (CPT I) deficiency, carnitine palmitoyltransferase II (CPT II) deficiency, carnitine - acyl - carnitine (CACT) deficiency, autosomal dominant / autosomal recessive progressive external ophthalmoplegia (ad - / ar - PEO), infantile - onset spinocerebellar ataxia (IOSCA), mitochondrial myopathy (MM), spinal muscular atrophy (SMA), growth retardation, aminoaciduria, cholestasis, iron overload, early death (GRACILE), and Charcot - Marie - Tooth disease type 2A (CMT2A), and are selected from the group consisting of these.

[0030] In some embodiments of the methods provided herein, endogenous mtDNA encodes dysfunctional mitochondria. In specific embodiments, endogenous mtDNA contains mutant mtDNA. In other embodiments, endogenous mtDNA in recipient cells contains wild - type mtDNA. In yet further embodiments, endogenous mtDNA contains mtDNA associated with mitochondrial diseases or disorders. In some embodiments, endogenous mtDNA is heteroplasmic. In specific embodiments, recipient cells have endogenous mitochondria that are dysfunctional.

[0031] In certain embodiments of the methods provided herein, the mitochondrial replacement cells have a total mtDNA copy number that is no more than about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, or greater than the total mtDNA copy number of the recipient cells prior to contact with an agent that reduces the endogenous mtDNA copy number.

[0032] In some embodiments, the recipient cells are animal cells or plant cells. In certain embodiments, the animal cells are mammalian. In specific embodiments, the recipient cells are somatic cells. In other embodiments, the recipient cells are bone marrow cells. In some embodiments, the bone marrow cells are hematopoietic stem cells (HSCs) or mesenchymal stem cells (MSCs). In other embodiments, the recipient cells are cancer cells. In further embodiments, the recipient cells are primary cells. In still further embodiments, the recipient cells are immune cells. In specific embodiments, the immune cells are selected from the group consisting of T cells, phagocytes, microglial cells, and macrophages. In further embodiments, the T cells are CD4+ T cells. In other embodiments, the T cells are CD8+ T cells. In certain embodiments, the T cells are chimeric antigen receptor (CAR) T cells.

[0033] In another embodiment of the methods provided herein, the exogenous mitochondria and / or exogenous mtDNA are stable. In some embodiments, the exogenous mtDNA changes heteroplasmy in the recipient cells.

[0034] In some aspects of the methods provided herein, the method further comprises delivering a small molecule, peptide, or protein.

[0035] The present disclosure also provides a method as provided herein, further comprising contacting a recipient cell with a second active agent prior to incubating the recipient cell with exogenous mitochondria and / or exogenous mtDNA. In certain embodiments, the second active agent is selected from the group consisting of macromolecules, 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, batquinone (Epi743), thioctic acid, A0001 (α - tocopherol quinone), mitochondrial CoQ10 (MitoQ), SkQ1 (bisomithine), resveratrol, curcumin, ketogenic diet therapy, hypoxia, and activators of endocytosis. In some embodiments, the activator of endocytosis is a modulator of cell metabolism. In a specific embodiment, the modulator of cell metabolism includes nutrient starvation, chemical inhibitors, or small molecules. In a further embodiment, the chemical inhibitor or small molecule is an mTOR inhibitor. In an even further embodiment, the mTOR inhibitor includes rapamycin or a derivative thereof.

[0036] The present disclosure provides a composition comprising one or more mitochondrial exchange cells obtained by a method comprising: (a) contacting a recipient cell with an agent that reduces the endogenous mtDNA copy number; (b) incubating the recipient cell for a period sufficient 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 is partially reduced and (2) exogenous mitochondria from a healthy donor for a period sufficient to non - invasively transfer the exogenous mitochondria into the recipient cell, thereby producing a mitochondrial exchange cell, wherein the mitochondrial exchange cell comprises more than 5% exogenous mtDNA.

[0037] The present disclosure further provides a composition of one or more mitochondrial replacement cells obtained by a method of creating mitochondrial replacement cells, which method comprises: (a) contacting recipient cells with an agent that reduces the endogenous mtDNA copy number; (b) incubating the recipient cells for a period sufficient for the agent to partially reduce the endogenous mtDNA copy number in the recipient cells; and (c) co-incubating (1) the recipient cells from step (b) in which the endogenous mtDNA is partially reduced and (2) exogenous mtDNA from a healthy donor for a period sufficient to non-invasively transfer the exogenous mtDNA into the recipient cells, whereby the mitochondrial replacement cells contain more than 5% exogenous mtDNA. In some embodiments of the compositions provided herein, the one or more mitochondrial replacement cells contain a total mtDNA copy number that does not exceed about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, or more than that compared to the total mtDNA copy number of the recipient cells prior to contact with the agent that reduces the endogenous mtDNA copy number.

[0038] In another aspect, provided herein is a composition for use in a method of creating one or more mitochondrial replacement cells, the composition comprising an agent that reduces the endogenous mtDNA copy number and a second active agent. In some embodiments, the composition further comprises one or more recipient cells or combinations thereof. In one embodiment, the composition further comprises exogenous mtDNA, exogenous mitochondria, and / or exogenous mitochondria.

[0039] In certain embodiments of the compositions provided herein, the agent that reduces the endogenous mtDNA copy number is a small molecule or a fusion protein. In some embodiments, the small molecule is a nucleoside reverse transcriptase inhibitor (NRTI). In other embodiments, the fusion protein comprises an endonuclease that cleaves mtDNA and a mitochondrial targeting sequence (MTS). In some embodiments, the endonuclease cleaves wild-type mtDNA. In a specific embodiment, 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). In some embodiments, the MTS targets a mitochondrial matrix protein. In a further embodiment, the mitochondrial matrix protein is cytochrome c oxidase subunit IV, cytochrome c oxidase subunit VIII, and cytochrome c oxidase subunit X. In a specific embodiment, the fusion protein is transiently expressed.

[0040] In some embodiments of the compositions provided herein, the reduction in the endogenous mtDNA copy number is a partial reduction. In one embodiment, the partial reduction is a reduction of about 5% to about 99% of the endogenous mtDNA. In a specific embodiment, the partial reduction is a reduction of about 50% to about 95% of the endogenous mtDNA copy number. In a further embodiment, the partial reduction is a reduction of about 60% to about 90% of the endogenous mtDNA copy number.

[0041] The present disclosure provides a method of obtaining one or more mitochondrial exchange cells, the method comprising: (a) contacting recipient cells with an agent that reduces mitochondrial function; (b) incubating the recipient cells for a period sufficient for the agent to partially reduce endogenous mitochondrial function in the recipient cells; and (c) co-incubating (1) the recipient cells from step (b) in which the endogenous mitochondrial function is partially reduced and (2) exogenous mitochondria from a healthy donor for a period sufficient to non-invasively transfer the exogenous mitochondria into the recipient cells, thereby producing mitochondrial exchange cells. Also provided is a composition comprising one or more mitochondrial exchange cells obtained by the method, wherein the mitochondrial exchange cells comprise more than 5% exogenous mtDNA.

[0042] In another aspect, provided herein is a composition of one or more mitochondrial exchange cells obtained by a method comprising: (a) contacting recipient cells with an agent that reduces mitochondrial function; (b) incubating the recipient cells for a period sufficient for the agent to partially reduce endogenous mitochondrial function in the recipient cells; and (c) co-incubating (1) the recipient cells from step (b) in which the endogenous mitochondrial function is partially reduced and (2) exogenous mtDNA from a healthy donor for a period sufficient to non-invasively transfer the exogenous mtDNA into the recipient cells, thereby producing mitochondrial exchange cells, wherein the mitochondrial exchange cells comprise more than 5% exogenous mtDNA. In some embodiments, the one or more mitochondrial exchange cells comprise a total mtDNA copy number that is no more than about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, or more than the total mtDNA copy number of the recipient cells prior to contact with an agent that reduces the endogenous mtDNA copy number.

[0043] The present disclosure also provides a composition for use in a method of generating one or more mitochondrial exchange cells, the composition comprising an agent that reduces mitochondrial function and a second active agent. In some embodiments, the composition further comprises exogenous mitochondria, one or more recipient cells, or a combination thereof. In further embodiments, the composition further comprises exogenous mtDNA.

[0044] In some embodiments of the compositions provided herein, the one or more mitochondrial exchange cells comprise wild-type exogenous mtDNA.

[0045] Also provided herein is a composition further comprising a second active agent. In some embodiments, the second active agent is selected from the group consisting of macromolecules, 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, batquinone (Epi743), thioctic acid, A0001 (α-tocopherol quinone), mitochondrial CoQ10 (MitoQ), SkQ1 (bisomithine), resveratrol, curcumin, ketogenic diet therapy, hypoxia, and activators of endocytosis. In a specific embodiment, the activator of endocytosis is an activator of the clathrin-independent endocytosis pathway. In some embodiments, the activator of endocytosis is an activator of the clathrin-independent endocytosis pathway. In a further embodiment, the clathrin-independent endocytosis pathway is selected from the group consisting of the CLIC / GEEC endocytosis pathway, Arf6-dependent endocytosis, flotillin-dependent endocytosis, macropinocytosis, circular doral ruffles, phagocytosis, and trans-endocytosis. In a still further embodiment, the clathrin-independent endocytosis pathway is macropinocytosis. In a specific embodiment, the activator of endocytosis comprises nutrient stress and / or an mTOR inhibitor. In some embodiments, the mTOR inhibitor comprises rapamycin or a derivative thereof.

[0046] In certain embodiments, the present disclosure further provides a composition in which the total mtDNA copy number of one or more mitochondrial replacement cells contains more than 5% exogenous mtDNA. In some embodiments, the total mtDNA copy number of one or more mitochondrial replacement cells contains more than 30% exogenous mtDNA. In specific embodiments, the total mtDNA copy number of one or more mitochondrial replacement cells contains more than 50% exogenous mtDNA. In further embodiments, the total mtDNA copy number of one or more mitochondrial replacement cells contains more than 75% exogenous mtDNA.

[0047] In some embodiments of the compositions provided herein, the exogenous mitochondria are isolated mitochondria. In specific embodiments, the isolated mitochondria are intact. In some embodiments, the exogenous mitochondria and / or exogenous mtDNA are allogeneic. In specific embodiments, the exogenous mitochondria further contain exogenous mtDNA.

[0048] In certain embodiments of the compositions provided herein, the one or more cells are animal cells or plant cells. In some embodiments, the animal cells are mammals. In specific embodiments, the cells are somatic cells. In further embodiments, the somatic cells are epithelial cells. In still further embodiments, the epithelial cells are thymic epithelial cells (TEC). In other embodiments, the somatic cells are immune cells. In certain embodiments, the immune cells are T cells. In specific embodiments, the T cells are CD4+ T cells. In other embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are chimeric antigen receptor (CAR) T cells. In other embodiments, the immune cells are phagocytes. In certain embodiments, the one or more mitochondrial replacement cells are bone marrow cells. In specific embodiments, the bone marrow cells are hematopoietic stem cells (HSC) or mesenchymal stem cells (MSC).

[0049] In some embodiments of the compositions provided herein, one or more mitochondrial replacement cells are more viable than isogenic cells having homoplasmic endogenous mtDNA. In other embodiments, one or more mitochondrial replacement cells are effective for the killing of cancer cells, the treatment of age-related diseases, the treatment of mitochondrial diseases or disorders, the treatment of neurodegenerative diseases, the treatment of diabetes or genetic diseases.

[0050] In certain embodiments of the compositions provided herein, the composition further comprises a small molecule, a peptide, or a protein.

[0051] Also provided herein is a composition for use in delaying cellular aging and / or extending cellular lifespan, the composition comprising: (a) aged or potentially aged cells having endogenous mitochondria; (b) isolated exogenous mitochondria from non-aged cells; and (c) an agent that reduces the endogenous mtDNA copy number. In some embodiments, the agent is a fusion protein. In one embodiment, the fusion protein comprises an endonuclease that cleaves mtDNA and a mitochondrial targeting sequence (MTS). In a specific embodiment, the endonuclease cleaves wild-type mtDNA. In some embodiments, 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). In a further embodiment, the MTS targets a mitochondrial matrix protein. In yet a further embodiment, 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. In one embodiment, the fusion protein is transiently expressed in the aged or potentially aged cells.

[0052] The present disclosure further provides a composition for use in delaying senescence and / or extending the lifespan in cells, the composition comprising: (a) senescent or potentially senescent cells having endogenous mitochondria; (b) isolated exogenous mitochondria derived from non-senescent cells; and (c) an agent that reduces mitochondrial function. In some embodiments, 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. In some embodiments, the exogenous mitochondria derived from non-senescent cells have enhanced function compared to the endogenous mitochondria.

[0053] In some embodiments, a composition for use in delaying senescence and / or extending the lifespan in cells further comprises a second active agent. In a specific embodiment, the second active agent is selected from the group consisting of macromolecules, 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, batquinone (Epi743), thioctic acid, A0001 (α-tocopherol quinone), mitochondrial CoQ10 (MitoQ), SkQ1 (bisomithine), resveratrol, curcumin, ketogenic diet therapy, hypoxia, and activators of endocytosis. In some embodiments, the activator of endocytosis is an activator of the clathrin-independent endocytosis pathway. In a specific embodiment, the clathrin-independent endocytosis pathway is selected from the group consisting of the CLIC / GEEC endocytosis pathway, Arf6-dependent endocytosis, flotillin-dependent endocytosis, macropinocytosis, circular doral ruffles, phagocytosis, and trans-endocytosis. In a further embodiment, the clathrin-independent endocytosis pathway is macropinocytosis. In some embodiments, the activator of endocytosis comprises nutrient stress and / or an mTOR inhibitor. In one embodiment, the mTOR inhibitor comprises rapamycin or a derivative thereof.

[0054] In another aspect, the present disclosure provides a pharmaceutical composition comprising a population of isolated mitochondrial replacement cells having exogenous mitochondria from a healthy donor, wherein the cells are obtained by any of the methods provided herein for obtaining mitochondrial replacement cells. In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a population of isolated mitochondrial replacement cells having exogenous mtDNA from a healthy donor, wherein the cells are obtained by any of the methods provided herein for obtaining mitochondrial replacement cells. In some embodiments, a pharmaceutical composition comprising a population of isolated mitochondrial replacement cells having exogenous mtDNA from a healthy donor further comprises exogenous mitochondria.

[0055] For example, in some embodiments, a pharmaceutical composition comprising exogenous mitochondria from a healthy donor is obtained by a method for generating mitochondrial replacement cells comprising: (a) contacting recipient cells with an agent that reduces the endogenous mtDNA copy number; (b) incubating the recipient cells for a period of time sufficient for the agent to partially reduce the endogenous mtDNA copy number in the recipient cells; and (c) co-incubating (1) the recipient cells from step (b) in which the endogenous mtDNA is partially reduced and (2) exogenous mitochondria from a healthy donor for a period of time sufficient to non-invasively transfer the exogenous mitochondria into the recipient cells, thereby generating mitochondrial replacement cells. In one embodiment, the cells are obtained by a method comprising: (a) contacting recipient cells with an agent that reduces mitochondrial function; (b) incubating the recipient cells for a period of time sufficient for the agent to partially reduce the endogenous mitochondrial function in the recipient cells; and (c) co-incubating (1) the recipient cells from step (b) in which the endogenous mitochondrial function is partially reduced and (2) exogenous mitochondria from a healthy donor for a period of time sufficient to non-invasively transfer the exogenous mitochondria into the recipient cells, thereby generating mitochondrial replacement cells.

[0056] In other embodiments, the cell is obtained by a method comprising: (a) contacting a recipient cell with an agent that reduces the endogenous mtDNA copy number; (b) incubating the recipient cell for a period of time sufficient 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 is partially reduced and (2) exogenous mtDNA from a healthy donor for a period of time sufficient to non-invasively transfer the exogenous mtDNA into the recipient cell, thereby producing a mitochondrial exchange cell. In other embodiments, the cell is obtained by a method comprising: (a) contacting a recipient cell with an agent that reduces mitochondrial function; (b) incubating the recipient cell for a period of time sufficient 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 is partially reduced and (2) exogenous mtDNA from a healthy donor for a period of time sufficient to non-invasively transfer the exogenous mtDNA into the recipient cell, thereby producing a mitochondrial exchange cell.

[0057] In certain embodiments of the pharmaceutical compositions provided herein, the cells are obtained by a method further comprising contacting the recipient cells with a second activating agent before incubating the recipient cells with exogenous mitochondria and / or exogenous mtDNA. In some embodiments, the second activating agent is selected from the group consisting of macromolecules, small molecules, or cell therapies, and the second activating agent is optionally selected from the group consisting of rapamycin, NR (nicotinamide riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxolone (RTA408), KH176, batquinone (Epi743), thioctic acid, A0001 (α-tocopherol quinone), mitochondrial CoQ10 (MitoQ), SkQ1 (bisomithine), resveratrol, curcumin, ketogenic diet therapy, hypoxia, and activators of endocytosis. In a specific embodiment, the activator of endocytosis is a modulator of cell metabolism. In other embodiments, the modulator of cell metabolism includes nutrient starvation, chemical inhibitors, or small molecules. In further embodiments, the chemical inhibitor or small molecule is an mTOR inhibitor. In still further embodiments, the mTOR inhibitor includes rapamycin or a derivative thereof.

[0058] In certain embodiments of the pharmaceutical compositions provided herein, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0059] In some embodiments of the pharmaceutical compositions provided herein, the cells are T cells. In other embodiments, the cells are hematopoietic stem cells.

Brief Description of the Drawings

[0060] (4. Brief Description of the Drawings)

Figure 1

[0061] Figure 1B shows the plasmid construct of the mitochondrial targeting sequence (MTS)-XbaI restriction enzyme (XbaIR) plasmid.

[0062] Figure 1C shows that isolated mitochondrial DNA was digested at multiple sites by the XbaI restriction enzyme, whereas digestion of mitochondrial DNA with NotI yielded a single fragment as predicted by the Cambridge reference sequence (CRS) of mitochondrial DNA.

[0063] Figure 1D shows five XbaIR endonuclease sites (1193, 2953, 7440, 8286, 10256) on human mitochondrial DNA predicted by the Cambridge reference sequence (CRS).

[0064] Figure 1E shows microscopy of human skin fibroblasts under phase contrast (left), immunofluorescence of green fluorescent protein (center), and merged field (right) after uptake of the fusion MTS-green fluorescent protein (GFP) plasmid using a Nucleofector. Top, low magnification. Bottom, high magnification.

[0065] Figure 1F shows the constructs of the pCAGGS-MTS-EGFP-PuroR and pCAGGS-MTS-XbaIR-PuroR plasmids.

[0066] Figure 1G shows the localization of the exogenous gene product MTS-EGFP in mitochondria by mitochondrial-specific staining using tetramethylrhodamine methyl ester (TMRM).

[0067]

Figure 2

[0068] Figure 2B shows the quantification of human β-actin (Actb) (left column) and mitochondrial DNA (mtDNA) (right column) after exposure to either the MTS-XbaI R endonuclease method or ethidium bromide treatment, compared to non-contact cells. XbaI R resulted in a greater decline in mtDNA compared to EtBr treatment. Actb was used as a housekeeping gene.

[0069] Figure 2C shows a greater decline in mitochondria after exposure to gene transfer of MTS-XbaI R, compared to EtBr treatment, based on DsRed fluorescence expressed in mitochondria.

[0070] Figure 2D shows the semi-quantification of mitochondrial membrane potential (a surrogate marker for mitochondrial content) in cells exposed to gene transfer of MTS-XbaI R or EtBr, using FACS analysis with the use of TMRM, indicating that MTS-XbaI R resulted in a greater decline in mitochondria.

[0071] Figure 2E shows the time-course quantification of transgene expression in the gene transfer system over 14 days.

[0072] Figures 2F and 2G show fluorescence images (Figure 2F) after the transfer of a plasmid carrying GFP before puromycin selection ("pre") and after puromycin selection ("post"), and quantification of the GFP / mitochondria ratio (Figure 2G) shows enrichment of the GFP plasmid after puromycin selection.

[0073]

Figure 3

[0074] Figure 3B shows the decrease in mitochondria on day 6 after gene transfer of XbaIR (top), rather than after transfer of the negative control vector expressing GFP (bottom), as measured by TMRM staining.

[0075] Figure 3C shows the quantification of mitochondrial DNA copy number estimated by qPCR of human 12S rRNA compared to nuclear β-actin levels in NHDF cells after gene transfection of XbaIR or GFP transfection. When indicated (「Mt Tx」), mitochondria were transferred into recipient cells. XbaIR resulted in a significant decrease in mitochondrial DNA. This could be rescued to a level equivalent to that of control-treated cells after transfer of exogenous mitochondria. N = 3, * p < 0.01.

[0076] Figure 3D shows photographs from a time-lapse video: upper left: co-culture with mitochondria isolated from ρ(−) cells and marked with DsRed; upper right: ρ(−) cells as a control; lower left: co-culture of NHDF with mitochondria; lower right: co-culture of mock transfectants of NHDF with mitochondria;

[0077] Figure 3E shows a series of 10 static images from the time-lapse video shown in Figure 3D arranged horizontally in chronological order;

[0078] Figure 3F shows the measurement of DsRed-labeled mitochondria by FACS analysis, which reveals that the present invention (「DsRed-Mt EPC100」) results in an increase in the uptake of exogenous mitochondria compared to previously described methods.

[0079] Figures 3G and 3H show microscopic images of DsRed-labeled mitochondria (Figure 3G) and phase contrast (Figure 3H) after mitochondrial import in ρ(0) cells treated or not treated with antimycin, indicating that the uptake of exogenous mitochondria did not occur in cells with complete destruction of mitochondria;

[0080] Figure 3I shows a series of five static images arranged horizontally in chronological order from the time-lapse video shown in Figure 3G.

[0081] Figure 3J shows the quantification of the fluorescence intensity of DsRed-labeled isolated exogenous mitochondria measured every 24 hours in ρ(-) cells or ρ(-) mock-transfected cells, or untreated cells (added Mt) incubated with Ds-Red mitochondria.

[0082]

Figure 4

[0083] Figure 4B shows a representative image from a video for observing ingested exogenous mitochondria (shown in red) in recipient cells with mitochondria marked with GFP. The video was recorded by using super-resolution microscopy, and almost no merged images were recognized. Most of the donor mitochondria exist separately from the existing mitochondria.

[0084] Figure 4C shows a three-dimensional reconstructed photograph of the fusion body.

[0085] Figure 4D shows a photograph of NHDF into which the gene encoding DsRed fused with the mitochondrial import signal was transfected.

[0086] Figure 4E shows a photograph of EPC100 into which a gene encoding EGFP fused with TFAM has been transferred.

[0087] Figure 4F shows the time course of mitochondrial transfer using cells marked with DsRed as recipients and TFAM-targeted EGFP as donor mitochondria.

[0088] Figure 4G shows that after exogenous mitochondria transiently contacted recipient cells, exogenous TFAM was stably incorporated into existing mitochondria, suggesting that mitochondrial nucleoids containing TFAM were transferred into existing mitochondria through a transient contact similar to phagocytosis.

[0089]

Figure 5

[0090] Figure 5B shows DNA sequencing data of nucleotides surrounding hmt16362 in NHDF ctrl recipient cells (SEQ ID NO: 1), EPC100 ctrl donor cells (SEQ ID NO: 2), NHDF-derived ρ(-) cells without mitochondrial exchange (SEQ ID NO: 3), and NHDF-derived ρ(-) cells with mitochondrial exchange (SEQ ID NO: 4), whereby it was shown that NHDF-derived ρ(-) cells with mitochondrial exchange (SEQ ID NO: 4) changed from A of the original recipient cells to G of the donor mtDNA at hmt16362.

[0091] Figure 5C shows the set of primers hmt16318-F (SEQ ID NO: 6) and hmt16414-R (SEQ ID NO: 9) used for amplifying the HV1 region of the human mitochondrial DNA D-loop (SEQ ID NO: 8) surrounding hmt16362, as well as the NHDF-specific probe (SEQ ID NO: 5) and EPC100-specific probe (SEQ ID NO: 7) designed for TaqMan SNP genotyping assay.

[0092] Figure 5D shows the quantification of NHDF-specific hmtDNA (left) and EPC100-specific hmtDNA (right) in parental NHDF and EPC100 cell lines, or NHDF cells with mitochondria from EPC100 cells (XbaIR Mt+) treated with XbaIR or NHDF cells without mitochondria from EPC100 cells (XbaIR Mt-) treated with XbaIR. This reveals that EPC100 mitochondria are successfully transferred into XbaIR Mt+ cells when evaluated using a single nucleotide polymorphism assay (SNP).

[0093]

Figure 6

[0094] Figure 6B shows that mitochondrial exchange in NHDF cells (ρ(-)Mt) restores mitochondrial function compared to NHDF control cells and NHDF (ρ(-)) without mtDNA exchange, as indicated by respiratory flux (routine, electron transport system (ETS), ROX), free routine activity (mitochondrial ATP production), proton leak, and coupling efficiency at each stage.

[0095] Figure 6C shows a time-lapse micrograph enabling an estimation of the continuous cell number based on the cell surface area, by which it was shown that ρ(-) cells enter a resting state for 3 to 12 days, while mitochondrial exchange cells regain their proliferative ability after 6 days.

[0096] Figure 6D shows a scheme of the protocol used to investigate the molecular mechanism of macropinocytosis. This protocol included transfecting NHDF cells with the MTS-XbaIR-P2A-PuroR plasmid, selecting with puromycin, and then either starving the cells for 60 minutes or treating the cells with palmitic acid (PA) or rapamycin for 24 hours.

[0097] Figures 6E - 6H show the quantification of WES™ analysis for the phosphorylation of S6 kinase (Figure 6E) and the phosphorylation of AMPK (Figure 6G), as well as the corresponding WES™ blots (Figure 6F) and (Figure 6H), respectively, by which it was shown that AMPK is activated and mTOR is completely suppressed in ρ(-) cells. Rapa: Rapamycin, PA: Palmitic acid, EAA-: Essential amino acid deficiency.

[0098] Figure 6I shows the protocol used to investigate the effect of mTOR-mediated macropinocytosis in the setting of MirC production protocol.

[0099] Figures 6J - 6L show the quantification (Figures 6J and 6K) and FACS analysis (Figure 6L) of DsRed-labeled mitochondrial uptake in control (top), mock-transfected cells (center), and ρ(-) cells with or without rapamycin treatment or with or without palmitic acid (PA) treatment. ρ(-) cells showed a greater uptake of mitochondria compared to control or mock TF cells, and the mitochondrial uptake increased significantly after rapamycin treatment, while palmitic acid decreased the mitochondrial uptake in ρ(-) cells.

[0100]

Figure 7

[0101] Figure 7B shows DNA sequencing data of the nucleotides surrounding hmt10158 in ND3 of EPC100 cells (top; SEQ ID NO: 10) and the nucleotides surrounding hmt10158 in ND3 of Leigh syndrome (7SP) fibroblasts (bottom; SEQ ID NO: 11), thereby revealing the mutation 10158T>C, which is a mosaic of the major wave C and the minor wave T, indicating heteroplasmy.

[0102] Figure 7C shows photographs from a time-lapse video showing similar behavior in both ρ(−)7SP fibroblasts with exogenous mitochondria and ρ(−)7SP fibroblasts without exogenous mitochondria, as seen in the NHDF experiment.

[0103] Figure 7D shows the quantification of mitochondrial DNA copy number estimated by qPCR of human 12S rRNA compared to nuclear β-actin levels in NHDF cells after gene transfection with XbaIR or mock transfection. When indicated, mitochondria were transferred into recipient cells. XbaIR resulted in a significant decrease in mitochondrial DNA, which could be rescued by the transfer of exogenous mitochondria (n = 3).

[0104] Figure 7E shows DNA sequencing data of nucleotides surrounding hmt10158 in 7SP ctrl recipient cells (SEQ ID NO: 14), EPC100 ctrl donor cells (SEQ ID NO: 12), 7SP-derived ρ(-) cells without mitochondrial exchange (SEQ ID NO: 13), and 7SP-derived ρ(-) cells with mitochondrial exchange (SEQ ID NO: 15). Thereby, while 7SP ctrl cells are heteroplasmic (majority 10158C; SEQ ID NO: 14), it was revealed that EPC100 has only T at the same position of mitochondrial DNA (SEQ ID NO: 12). The ρ(-) cell stem derived from 7SP cells represented the same wave as the original (SEQ ID NO: 13), while the mitochondrial exchange 7SP cells showed T as the major wave (SEQ ID NO: 15).

[0105] Figure 7F shows the set of primers hmt10085-F (SEQ ID NO: 17) and hmt10184-R (SEQ ID NO: 20) used for amplification of ND3 of human mitochondrial DNA (SEQ ID NO: 16) surrounding the Leigh syndrome-related SNP of hmt10158, and the EPC100-specific probe (SEQ ID NO: 18) and 7SP-specific probe (SEQ ID NO: 19) designed for TaqMan SNP genotyping assay. The ND3 peptide sequence is also shown (SEQ ID NO: 46).

[0106] Figure 7G shows the quantification of the percentage of hmt10158 heteroplasmy in each cell group evaluated by SNP assay, whereby it was revealed that the exogenous normal sequence (“healthy”) occupies up to 80% in mitochondrial exchange 7SP cells, although the original heteroplasmy of the mutant sequence is more than 90%. In the case of mock transfectants, the heteroplasmy did not change significantly and maintained approximately the same ratio.

[0107] Figures 7H and 7I show the quantification of the percentage of heteroplasmy level (Figure 7H) and the absolute mtDNA copy number (Figure 7I) in three independent experiments in 7SP cells treated with mock control and subjected to mitochondrial transfer.

[0108] Figure 7J shows a series of 10 static images from the time-lapse video shown in Figure 7C, arranged horizontally in chronological order.

[0109]

Figure 8

[0110] Figure 8B shows time-lapse estimated cell proliferation in 7SP fibroblasts with mitochondrial exchange, ρ(-) 7SP fibroblasts, and ρ(-) 7SP fibroblasts, revealing that while ρ(-) 7SP fibroblasts are in a quiescent state, mitochondrial exchange 7SP cells recover cell proliferation to a level equivalent to that of the original 7SP fibroblasts around day 12.

[0111] Figure 8C shows senescence in 7SP fibroblasts at approximately population doubling level (PDL) 25, which was extended to approximately PDL 63 in ρ(-) 7SP fibroblasts where healthy mitochondrial exchange was performed at PDL 8, indicating lifespan extension of ρ(-) 7SP fibroblasts with healthy mitochondrial exchange.

[0112] Figure 8D shows that an increase in PDL results in an increase in cell size (left), which reverses after mitochondrial exchange and is maintained even beyond PDL 50 (right).

[0113] Figure 8E shows a short tandem repeat (STR) assay that discriminates cells of different origins and identifies contamination of different types of cells. The STR pattern in mitochondrial exchange cells at various time points was identical to the STR pattern in the original 7SP fibroblasts.

[0114] Figure 8F shows the RT-PCR quantification of telomerase in 7SP fibroblasts and mitochondrial exchange cells for various PDLs compared to HeLa and EPC100, indicating that the cells did not transform into cancer cells.

[0115]

Figure 9

[0116] Figures 9B and 9C show that after approximately PDL30, almost control levels were restored in mitochondrial exchange cells (ρ(-)Mt) as indicated by respiratory fluxes (routine, electron transport system (ETS), ROX), free routine activity (mitochondrial ATP production), proton leak, and coupling efficiency (Figure 9B), as well as flux control ratios (FCR), ROX / E, L / E, R / E, and (R - L) / E (Figure 9C).

[0117]

Figure 10

[0118] Figures 10B - 10D show the FACS analysis of annexin V-positive and propidium iodide (PI)-positive cells (Figure 10B) and the quantification of annexin V-positive cells (Figure 10C) and propidium iodide-positive cells (PI; Figure 10D) after untreated or H2O2 treatment, indicating that 7SP cells are more sensitive to H2O2 compared to NHDF cells, while 7SP MirC is not.

[0119] Figure 10E shows microscopic examination images of NHDF, 7SP, and 7SP MirC cells under basic conditions or after starvation conditions (EAA-), indicating that 7SP cells are more sensitive to starvation conditions compared to NHDF cells, while 7SP MirC is not.

[0120] Figures 10F - 10H show FACS analysis of annexin V - positive cells and PI - positive cells (Figure 10F), quantification of annexin V - positive cells (Figure 10G), and quantification of PI - positive cells (Figure 10H) of untreated or starved cells, indicating that 7SP cells are more sensitive to starvation conditions compared to NHDF cells, while 7SP MirC is not.

[0121]

Figure 11

[0122]

Figure 12

[0123] Figures 12B - 12D show alkaline phosphatase (AP) staining and quantification as an indicator of iPSCs generated from any of 7SP fibroblasts, MirC derived from 7SP fibroblasts, or mock transfectants derived from 7SP fibroblasts. Microscopic examination of AP-stained cells (left panel in Figure 12B; 7SP fibroblasts, middle panel; MirC derived from 7SP fibroblasts, right panel: mock transfectant of 7SP fibroblasts) and macroscopic examination (left panel in Figure 12C; 7SP fibroblasts, middle panel; MirC derived from 7SP fibroblasts, right panel: mock transfectant of 7SP fibroblasts), as well as quantification of AP-stained cells (Figure 12D), revealed that mitochondrial exchange in either NHDF or 7SP fibroblasts after XbaI R treatment results in an increase in AP staining.

[0124] Figure 12E shows colony formation of iPSCs derived from mitochondrial exchange 7SP fibroblasts. Photographs of three representative colonies 75 days and 170 days after gene transfer of reprogramming factors.

[0125] Figure 12F shows immunohistochemical staining of OCT3 / 4, NANOG, TRA1 - 80, and TRA - 160 (which are representative markers of pluripotent stem cells) in iPSCs generated from 7SP fibroblasts after mitochondrial exchange;

[0126] Figure 12G shows mitochondrial DNA copy numbers in iPSCs derived from MirC of 7SP fibroblasts compared to the original 7SP fibroblasts as a reference and standard human iPSCs (201B7), revealing that the iPSCs have a limited number of mitochondrial DNA similar to that of standard human iPSCs (201B7).

[0127] Figures 12H and 12I show the percentage of heteroplasmy (Figure 12H) and the absolute mtDNA copy number (Figure 12I) in iPSCs derived from 7SP fibroblasts-derived MirC 170 days after reprogramming treatment. This reveals that 7SP fibroblasts-derived MirC forming iPSCs show at least three colonies with negligible levels of mutant genomic sequences, a decrease in total mtDNA, and donor mtDNA approaching 100%. It was also suggested that changes in heteroplasmy in MirC return to their original state and may differ from mitochondrial replacement therapy in IVF.

[0128]

Figure 13

[0129] Figure 13B shows DNA sequencing data of nucleotides surrounding hmt16145 in NHDF ctrl recipient cells (SEQ ID NO: 21) with genotype hmt16145 A and TIG1 ctrl donor cells (SEQ ID NO: 22) with genotype hmt16145 G.

[0130] Figure 13C shows the quantification of hmt16145 heteroplasmy level (%) by SNP assay in cells derived from mitochondrial exchange cells (MirC) (senescent NHDF recipient cells with mitochondrial transfer of mitochondria from "young" TIG1 donor cells). This shows that over 90% of the mtDNA in NHDF-derived MirC cells with mitochondrial exchange from TIG1-derived mitochondrial donor cells is hmt16145 G (i.e., from TIG1 mtDNA), whereas 100% of the mtDNA in NHDF ctrl cells is hmt16145 A.

[0131] Figure 13D shows the quantification of population doubling level (PDL) vs. time (days) (left) and doubling time (hours) vs. population doubling level (right) in recipient NHDF cells transfected with MTS-GFP (“Mock”) or MTS-XbaIR (“MirC”), incubated with exogenous mitochondria from TIG1 donor cells, or not transfected (“Ctrl”). As shown by the upward movement of PDL (left) and the rightward movement of PDL (right), MirC with “young” donor TIG1 embryonic lung cells (PDL 10) into “aged” normal human dermal fibroblast (NHDF) recipient cells (PDL 41) showed an extended lifespan.

[0132] Figure 13E shows the quantification of population doubling level (PDL) vs. time (days) (left) and doubling time (hours) vs. population doubling level (right) in normal human dermal fibroblasts transfected with MTS-GFP and mitochondrially imported (“Mock”), transfected with MTS-XbaIR and mitochondrially imported (“MirC”), or not transfected (“Ctrl”). As shown by the downward movement of PDL (left) and the leftward movement of PDL (right), mitochondrial import from “aged” donor cells (PDL 49) to “young” recipient cells (PDL <21) showed a decreased lifespan.

[0133]

Figure 14

[0134] Figure 14B shows strong expression of the transgene MTS-GFP in the mitochondria of T cells 24 hours after electroporation.

[0135] Figure 14C shows the FACS analysis of GFP expression in T cells after transfection of MTS-GFP mRNA by electroporation, revealing that GFP expression is present in almost all T cells.

[0136] Figure 14D shows the FACS analysis of DsRed-labeled mitochondria, indicating that the MTS-XbaIR construct strongly degraded endogenous mitochondria, while MTS-GFP did not.

[0137] Figure 14E shows the scheme of the protocol design for determining the optimal period of mitochondrial co-incubation.

[0138] Figure 14F shows the fluorescence images of electroporated control cells (upper panel) and electroporated MTS-GFP cells (lower panel) at 4 hours, 2 days, 4 days, 6 days, and 8 days after electroporation (EP), showing that the MTS-GFP construct showed high expression within 4 hours after electroporation and was almost absent by day 6.

[0139] Figures 14G and 14H show the electrophoresis (Figure 14H) and quantification (Figure 14G) of GFP in cells that received MTS-GFP mRNA compared to GAPDH. Peak expression occurred on day 4, and the expression disappeared by day 6.

[0140] Figure 14I shows the quantification of XbaIR transcript levels at 4 hours, 2 days (d2), 4 days (d4), 6 days (d6), and 8 days (d8), indicating that the transcript expression of the endonuclease was highest 4 hours after gene transfer.

[0141] Figure 14J shows the quantification of mitochondrial content (12S rRNA) in cells subjected to MTS-XbaI, indicating that mitochondria decreased to approximately 30% by day 2 and were maintained below 20% throughout the experimental period.

[0142]

Figure 15

[0143] Figure 15B shows DNA sequencing data of the nucleotides surrounding hmtDNA 218 and hmtDNA 224 in the HV1 region of the human mitochondrial DNA D-loop in human primary NH T cell recipient cells (top; SEQ ID NO: 23) and EPC100 donor cells (bottom; SEQ ID NO: 24). hmtDNA 218 and hmtDNA 224 are C / C (SEQ ID NO: 23) and T / T (SEQ ID NO: 24) for T cells and EPC100 cells, respectively.

[0144] Figure 15C shows the set of primers hmtHV1-F (SEQ ID NO: 26) and hmtHV1-R (SEQ ID NO: 27) for amplifying the HV1 region of the human mitochondrial DNA D-loop (SEQ ID NO: 25) surrounding the SNPs of hmtDNA 218 and hmtDNA 224, as well as SNP assay primers 1-F (SEQ ID NO: 40), SNP assay - primer 1-R (SEQ ID NO: 41), N-terminal VIC-labeled EPC100-specific probe (SEQ ID NO: 38), and N-terminal FAM-labeled T cell-specific probe (SEQ ID NO: 39) designed for TaqMan SNP genotyping assay.

[0145] Figure 15D shows the quantification of the amount of exogenous mtDNA present in recipient cells on days 7 and 12 for mock (MTS-GFP) or MTS-XbaIR (XbaIR)-treated cells after co-incubation with exogenous mitochondria from donor EPC100 cells. Quantification of recipient and donor cells was performed as a positive control.

[0146] Figure 15E shows the quantification of a respirometry experiment performed using the Oroboros O2k, which showed the restoration of ATP production and coupling efficiency in MirC derived from human T cells, whereas ρ(-) human T cells generated by electroporation-mediated transfer of XbaIR mRNA maintained the loss of ATP production throughout the experimental period.

[0147] Figures 15F and 15G show representative raw data using the coupling control protocol (CCP), indicating that MirC T cells are able to restore mitochondrial respiration.

[0148]

Figure 16

[0149] Figure 16B shows the quantitative analysis of GFP expression in T cells (middle) after electroporation (EP) of pmax GFP or T cells (right) after electroporation (EP) of MTS-GFP, or T cells without electroporation (left) 6 hours after EP (upper left panel), 2 days after EP (upper right panel), 4 days after EP (lower left panel), and 6 days after EP (lower right panel). The survival rate was not significantly affected by EP with MTS-GFP at 2 or 4 days.

[0150] Figure 16C shows the qPCR quantification of XbaIR levels in T cells electroporated with the MTS-XbaIR vector 4 hours, 2 days, 4 days, and 6 days after electroporation, indicating that XbaIR expression decreases slowly.

[0151] Figure 16D shows the quantification of 12S rRNA levels in T cells electroporated with MTS-XbaIR, indicating that mouse mtDNA decreased by approximately 60% by day 4.

[0152] Figure 16E shows the scheme of the protocol used for MirC generation in T cells using mitochondrial co-incubation on day 5.

[0153] Figure 16F shows the 48-hour FACS analysis of ingested DsRed-labeled mitochondria in recipient T cells after co-incubation with isolated DsRed-labeled mitochondria, revealing a significant positive rate (9.73%) of T cells expressing exogenous mitochondria with MTS-XbaIR compared to 0.43% of control cells without electroporation (i.e., "addition").

[0154]

Figure 17

[0155] Figure 17B shows the set of primers 2716-F (SEQ ID NO: 28) and 2883-R (SEQ ID NO: 33), the BL6-specific probe (SEQ ID NO: 29) and the NZB-specific probe (SEQ ID NO: 31), and the BamH1-mND1-F primer (SEQ ID NO: 30) used for cloning the nucleotide sequence into a plasmid for generating a standard curve enabling absolute quantification, which were used for amplification of ND1 of mouse mitochondrial DNA (SEQ ID NO: 32) surrounding polymorphic nucleotides mmt2766 and mmt2767 designed for TaqMan SNP genotyping assay. The ND1 peptide sequence is also shown (SEQ ID NO: 47).

[0156] Figure 17C shows the quantification of mouse mtND1 heteroplasmy levels in BL6 recipient cells 7 and 12 days after control electroporation (columns 1 and 2, respectively) or co-incubation with isolated mitochondria from MTS-XbaI electroporation and NZB cells (columns 3 and 4, respectively). The basal levels of BL6 (column 5) and NZB (column 6) cells were measured as controls.

[0157] Figure 17D shows the measurement of telomere length (young to old: Y to O) after treatment of aged mouse cells with MTS-XbaIR mRNA for generating MirC and co-incubation with exogenous mitochondria from young donor cells, thereby revealing an increase in telomere length of MirC compared to the "aged" parental cells.

[0158] Figure 17E shows the measurement of SASP-related cytokines CXCL1, ICAM1, IL-6, and IL-8 in aged parental T cells or T cells derived from MirC, thereby revealing that CXCL1 and IL6 are less in T cells derived from MirC.

[0159] Figure 17F shows the measurement of DNA damage response in MirC and the original T cells using histone 2A (H2A) phosphorylation antibody, indicating that the positive rate of DDR is lower in MirC (1.53%) compared to the original T cells (4.75%).

[0160]

Figure 18

[0161] Figure 18B shows representative images of tumor growth imaging performed during the experimental protocol.

[0162] Figure 18C shows the body weights of the sham group, young T cell group, or MirC group, revealing that no significant difference was observed among the three groups during the 25-day experimental period.

[0163] Figures 18D and 18E show the quantification of individual (Figure 18D) and average (Figure 18E) cancer nodule sizes, indicating that the MirC group reduced the cancer nodule size to a level equivalent to that of the young T cell group (lower line), while the sham group increased the cancer nodules throughout the experimental period (upper line).

[0164] Figure 18F shows the scheme of the protocol used to analyze the presence of injected T cells in animals.

[0165] Figure 18G shows FACS analysis of peripheral blood (left panel) or spleen (right panel). Negative controls using C57BL / 6 mice (upper left panel) and positive controls using GFP transgenic mice (lower left panel) were created for both peripheral blood and spleen. The positive rates of T cells expressing GFP fluorescence were recognized as 0.057% and 0.9% in peripheral blood and spleen, respectively.

[0166] Figure 18H shows immunofluorescence images of infused T cells detected in mice on day 6 after transplantation.

[0167] Figure 18I shows the percentage of chimerism after injection of exogenous T cells in peripheral blood (PB) or spleen after injection of 1×10 7 cells or 2×10 7 cells.

[0168]

Figure 19

[0169] Figure 19C shows 3-D confocal fluorescence imaging of bone marrow-derived Sca-1 cells 48 hours after co-incubation with DsRed-labeled mitochondria from EPC100 cells, indicating that exogenous mitochondria were engulfed.

[0170] Figure 19D shows the quantification of mitochondrial transfer efficiency by FACS analysis of DsRed fluorescence, revealing that approximately 10% of the Sca-1 subpopulation shows a shift in fluorescence to the right.

[0171] Figure 19E shows the scheme used to generate HSC-derived MirC by incubating with exogenous mitochondria on day 4 and analyzing MirC by SNP assay on day 6.

[0172] Figure 19F shows the FACS sorting of cells in the c-kit+, Sca-1+, lineage-, CD34- (referred to as KSLC) fraction.

[0173] Figure 19G shows that the doubling time of the KSLC fraction was 19 hours.

[0174] Figure 19H shows the scheme used to evaluate HSC-derived MirC.

[0175] Figure 19I shows the quantification of the percentage of mouse mtND1 heteroplasmy levels in mouse KSLC-derived MirC or parental recipient BL6 cells or NZB donor cells, indicating that MirC-derived HSCs express 99.9% of the polymorphic genotype of donor cells 6 days after transfection of MTS-XbaI mRNA by electroporation.

[0176]

Figure 20

[0177] Figure 20B shows a 2-D plot of the results of droplet digital PCR analyzing the sequences of mutant and non-mutant mtDNA for ND3 10158 T>C in normal human skin fibroblasts, which shows only the detection of non-mutant sequences (lower right quadrant) and no detection of mutant sequences (upper left quadrant).

[0178] Figure 20C shows a 2-D plot of the results of droplet digital PCR analyzing the sequences of mutant mtDNA and non-mutant mtDNA for ATP6 9185 T>C in normal human skin fibroblasts, which shows only the detection of non-mutant sequences (lower right quadrant) and does not show the detection of mutant sequences (upper left quadrant).

[0179] Figure 20D shows a 2-D plot of the results of droplet digital PCR analyzing the sequences of mutant mtDNA and non-mutant mtDNA in primary skin fibroblasts derived from a MELAS patient with the mtDNA A3243G mutation, which shows that most cells have homoplasmy of mutant mtDNA (upper left quadrant).

[0180] Figure 20E shows a 2-D plot of the results of droplet digital PCR analyzing the sequences of mutant mtDNA and non-mutant mtDNA in primary skin fibroblasts derived from a Leigh syndrome patient with the mtDNA T10158C mutation in complex I, ND3 gene, which shows that a small population of double-positive cells have heteroplasmy at the single-cell level (lower right quadrant), a large population have homoplasmy of mutant mtDNA (lower right), and there is no population with homoplasmy of non-mutant mtDNA (lower left).

Modes for Carrying Out the Invention

[0181] (5. Detailed Description of the Invention) Provided herein is a novel and improved method for generating mitochondrial exchange cells (MirC) that does not require complete removal of endogenous mtDNA and can be optionally carried out using reagents compatible with clinical use. Further, in certain embodiments, provided herein is a therapeutic method comprising administering a therapeutically effective amount of MirC generated using the method provided herein.

[0182] Also provided is a composition comprising one or more mitochondrial exchange cells obtained by the methods provided herein. In certain embodiments, the composition 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 decreases the endogenous mtDNA copy number or decreases the endogenous mitochondrial function. In further embodiments, the composition can include exogenous mitochondria and / or exogenous mtDNA, one or more recipient cells, or a combination thereof. In one specific embodiment, provided herein are methods and compositions for use in the treatment of diseases or disorders associated with dysfunctional mitochondria. However, it is understood that the methods and compositions provided herein can also be used to delay aging, extend lifespan, or enhance the function of cells having functional mitochondria and are not limited to the replacement of dysfunctional mitochondria. Further, for example, to generate disease models, the methods and compositions provided herein can be used to replace functional mitochondria with exogenous mitochondria that are dysfunctional or exhausted.

[0183] (5.1 Definitions) Unless defined otherwise, all terms including technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and in the following experimental methods is widely known and commonly used in the relevant art.

[0184] As used herein, the term "mitochondrial replacement cell" or "MirC" is intended to mean a cell having a replacement of endogenous mitochondria and / or mtDNA with exogenous mitochondria and / or mtDNA. For example, an exemplary mitochondrial replacement cell (MirC) involves the replacement of endogenous mtDNA encoding dysfunctional mitochondria, e.g., mtDNA derived from a subject having a mitochondrial disease or disorder, with exogenous mtDNA encoding functional mitochondria, e.g., mtDNA derived from a healthy subject. An exemplary MirC can also include a cell in which the endogenous mitochondria have been replaced with exogenous mitochondria. However, it is understood that the replacement of endogenous mitochondria and / or mtDNA can also include the replacement of functional endogenous mtDNA derived from a cell, e.g., an aged cell, with functional exogenous mtDNA derived from a different cell, e.g., a healthier cell derived from a younger subject. For example, it is further understood that healthy endogenous mitochondria and / or mtDNA can be replaced with dysfunctional exogenous mitochondria and / or exogenous mtDNA in order to mimic a mitochondrial disease or disorder. The exchange need not result in a complete replacement of all of the endogenous mitochondria within the cell, and exemplary mitochondrial and / or mtDNA exchanges involve replacement of endogenous mitochondria and / or mtDNA of about 5% or 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 greater.

[0185] As used herein, the terms "recipient cell", "acceptor cell", and "host cell" are interchangeable and refer to a cell that has received exogenous mitochondria and / or mtDNA. In some embodiments, the exogenous mitochondria and / or mtDNA are derived from isolated mitochondria from donor cells. In some embodiments, the donor cell and the recipient cell may be different or the same. In some embodiments, the donor cell and the recipient cell are from different species or the same species. In some embodiments, the donor cell and the recipient cell are from different tissues or the same tissue.

[0186] As used herein, the term "healthy donor" is intended to mean a donor that does not have a mitochondrial disease or disorder, an age-related disease, or otherwise dysfunctional mitochondria. In a preferred embodiment, the healthy donor has a wild-type mtDNA sequence compared to the Cambridge reference sequence of the mitochondrial genome.

[0187] As used herein, the terms "treat", "treating", and "treatment" refer to a reduction in the severity, progression, spread, and / or frequency of symptoms, the disappearance of symptoms and / or the underlying cause, the prevention of the occurrence of symptoms and / or its underlying cause, and the improvement or repair of damage. "Treatment" is intended to include therapeutic treatment of a disease, disorder, or condition, as well as prophylactic or suppressive measures.

[0188] As used herein, the term "agent", when used with respect to depletion reduction of mtDNA, refers to an enzyme or compound that can reduce mtDNA. Preferred agents include restriction enzymes such as XbaI that cleave mtDNA at one or more sites without causing toxicity in recipient cells. However, agents can also include enzymes or compounds that inhibit mtDNA synthesis or selectively promote mitochondrial degradation.

[0189] As used herein, the terms "reduce" or "decrease", when so defined herein, generally mean a reduction of at least 5%, such as 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 reduction between 5% and 99%, compared to a reference level. It is understood that a partial reduction or an agent or reduction that partially reduces endogenous mtDNA, as used herein, does not result in complete depletion of all endogenous mtDNA (i.e., ρ0 cells). The term "increase", as used herein, generally means an increase of at least 5%, such as 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 an increase of more than 90%.

[0190] As used herein, the term "endogenous" refers to arising from within or being derived from within. For example, endogenous mitochondria are mitochondria that are native to the cell.

[0191] As used herein, the term "exogenous" refers to cell material (e.g., mitochondria or mtDNA) that is not native to the host, e.g., cell material that is derived from outside. "From outside" generally means from a different source. For example, when a mitochondrial genome is derived from a cell type or species different from the host cell or host mitochondria, the mitochondrial genome is exogenous to the host cell or host mitochondria. Further, "exogenous" can also refer to a mitochondrial genome that is removed from a mitochondrion, manipulated, and returned to the same mitochondrion.

[0192] As used herein, the term "sufficient period" refers to an amount of time that yields the desired result. It is understood that the sufficient period will vary depending on experimental conditions, including but not limited to temperature, the amount of reagent used, and cell type. Exemplary protocols are provided throughout as a guide for a "sufficient period," and one of ordinary skill in the art will be able to identify a period that is sufficient without undue experimentation.

[0193] As used herein, the term "majority" is intended to mean the greatest amount as compared to other amounts being compared. Exemplary majorities when comparing two groups are amounts that are greater than about 50%, about 60%, about 70%, about 80%, or about 90%, or any integer greater than about 95% of the total population, including any integer therebetween. The majority is determined by the total population being compared and it is understood that when more than three groups are being compared, the amount can be less than 50%.

[0194] As used herein, the term "non-invasively" when used in connection with the introduction of exogenous materials is intended to mean not using invasive instruments (e.g., nanoblades or electroporation), physical forces (e.g., centrifugation), or detrimental culture conditions (e.g., heat shock). In a preferred embodiment, the non-invasive introduction procedure involves co-incubation of recipient cells and donor mitochondria.

[0195] As used herein, the term "subject in need of mitochondrial replacement" is intended to mean a subject that has dysfunctional mitochondria or is predisposed to having dysfunctional mitochondria. A subject in need of mitochondrial replacement may be asymptomatic and in need of preventive care. A subject in need of mitochondrial replacement may also be asymptomatic and in need of treatment. In certain embodiments, a subject in need of mitochondrial replacement has dysfunctional mitochondria that are not the result of an age-related disease or a mitochondrial disease or disorder.

[0196] As used herein, the term "subject" is intended to mean a mammal. The subject can be a human or non-human mammal, such as a dog, cat, cow, horse, mouse, rat, rabbit, or transgenic species thereof. It is understood that "subject" can also refer to a "patient," e.g., a human patient.

[0197] As used herein, the term "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. Thus, an effective amount can include, for example, a therapeutically effective amount, which refers to an effective amount in treatment, or a biologically effective amount, which refers to an effective amount for a biological effect. The terms "therapeutically effective amount" and "effective amount" can encompass an amount that improves the overall treatment, reduces or avoids the symptoms or causes of a disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent. The amount of a given composition corresponding to such an amount will vary depending on various factors, such as the given composition, pharmaceutical formulation, route of administration, type of disease, disorder, or condition, the identity of the subject or host being treated, etc., but can nevertheless be routinely determined by one of ordinary skill in the art. As defined herein, the therapeutically effective amount of an agent can be readily determined by one of ordinary skill in the art by routine methods known in the art.

[0198] As used herein, the term "age-related disease" refers to any number of diseases resulting from aging. These diseases include, but are not limited to, osteoporosis, osteopenia, arthritis, joint stiffness, cataracts, macular degeneration, metabolic diseases including type 2 diabetes, neurodegenerative diseases including Alzheimer's disease and Parkinson's disease, immunosenescence, and heart diseases including atherosclerosis and dyslipidemia. The phrase "age-related disease" further encompasses neurodegenerative diseases, such as Alzheimer's disease and related disorders, ALS, Huntington's disease, Parkinson's disease, and cancer.

[0199] As used herein, the term "autoimmune disease" is intended to mean a disease or disorder, or its manifestation, or a condition resulting therefrom, that is caused by an immune reaction against an individual's own tissues or organs. An autoimmune disease can also refer to a condition that is caused by, or exacerbated by, the production of autoantibodies that react with an autoimmune antigen or its epitope. An autoimmune disease can be tissue- or organ-specific, or it can be a systemic autoimmune disease. Examples of systemic autoimmune diseases include connective tissue diseases (CTDs), such as systemic lupus erythematosus (lupus; SLE), mixed connective tissue disease, systemic sclerosis, polymyositis (PM), dermatomyositis (DM), and Sjögren's syndrome (SS). Further exemplary autoimmune diseases include rheumatoid arthritis and antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis.

[0200] As used herein, the term "genetic disease" refers to a disease caused by an abnormality, e.g., 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.

[0201] As used herein, the term "cancer" includes, but is not limited to, solid cancers and hematologic cancers. The terms "cancer" and "cancerous" refer to, or describe, a mammalian physiological state typically characterized by uncontrolled cell proliferation.

[0202] As used herein, the terms "mitochondrial disease or disorder" and "mitochondrial disorder" are interchangeable and refer to a group of diseases caused by genetic or acquired damage to mitochondria that results in an energy deficiency within a region of the body. Exemplary organs affected by mitochondrial disease or disorder include organs that consume large amounts of energy, such as the liver, muscle, brain, eyes, ears, and heart. The consequences are often liver failure, muscle weakness, fatigue, and problems related to the heart, ears, and various other systems.

[0203] As used herein, the term "mitochondrial DNA abnormality" refers to a mutation of a mitochondrial gene whose product is localized in mitochondria and is not observed in the cells of a healthy subject. 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), Luft disease, aminoglycoside-induced deafness (AID), and mitochondrial DNA multiple deletion syndrome.

[0204] As used herein, the term "nuclear DNA abnormality" in the context of a mitochondrial disease or disorder refers to a mutation or change in the coding sequence of a nuclear gene whose product is localized in the mitochondria. Exemplary mitochondrial diseases 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 ataxic neuropathy dysarthria ophthalmoparesis (SANDO), disorders of the brainstem and spinal cord and leukoencephalopathy with elevated lactate (LBSL), coenzyme Q10 deficiency, Leigh syndrome (caused by nuclear mutations), mitochondrial complex abnormalities, fumarase deficiency, alpha - ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl - CoA ligase deficiency, pyruvate dehydrogenase complex deficiency (PDHC), pyruvate carboxylase deficiency (PCD), carnitine palmitoyltransferase I (CPT I) deficiency, carnitine palmitoyltransferase II (CPT II) deficiency, carnitine - acyl - carnitine (CACT) deficiency, autosomal dominant / autosomal recessive progressive external ophthalmoplegia (ad - / ar - PEO), infantile - onset spinocerebellar ataxia (IOSCA), mitochondrial myopathy (MM) spinal muscular atrophy (SMA), growth retardation, aminoaciduria, cholestasis, iron overload, early death (GRACILE), and Charcot - Marie - Tooth disease type 2A (CMT2A).

[0205] As used herein, the term "dysfunctional mitochondria" refers to mitochondria that are the opposite of functional mitochondria. Exemplary dysfunctional mitochondria include those that cannot synthesize ATP by oxidative phosphorylation or synthesize an insufficient amount of ATP. As used herein, the term "functional mitochondria" refers to mitochondria that consume oxygen and produce ATP.

[0206] As used herein, the term "mutation" refers to any change in the genetic structure that results in a mutant (also referred to as a "mutant") form. Mutations in genes can be caused by changes in a single base of DNA, or deletions, insertions, or rearrangements of larger portions of genes or chromosomes. In some embodiments, a mutation can affect the function or resulting protein. For example, a single nucleotide mutation in DNA within the coding region of a protein (i.e., a point mutation) can result in a codon that encodes a different amino acid (i.e., a missense mutation). It is understood that this different amino acid can change the structure of the protein and, in certain circumstances as described herein, can change the function of an organelle such as a mitochondrion.

[0207] As used herein, the terms "heteroplasmy" and "heteroplasmic" refer to the occurrence of multiple types of mitochondrial DNA genomes in an individual or sample. Various degrees of heteroplasmy are associated with various degrees of physiological states described herein. Heteroplasmy can be identified by means known in the art, and the severity of a physiological state associated with a particular nucleotide allele is thought to vary depending on the percentage of such associated alleles within an individual.

[0208] As used herein, the term "wild-type" when used in connection with 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 is the Cambridge Reference Sequence (CRS).

[0209] As used herein, the terms "aged" or "older" are intended to mean that the source of the mtDNA is from a subject that is older than the recipient cells, or that the cells within the population of cells that have doubled the population have been doubled a greater number of times (i.e., population doubling level, PDL) since its in vitro culture compared to the recipient cells.

[0210] As used herein, the terms "young" or "younger" are intended to mean that the source of the mtDNA is from a subject that is younger than the recipient cells, or that the cells within the population of cells that have doubled the population have been doubled a lesser number of times (i.e., population doubling level, PDL) since its in vitro culture compared to the recipient cells.

[0211] As used herein, the term "isolated", when used in connection with mitochondria, refers to mitochondria that have been physically separated or removed from other cellular components of their natural biological environment.

[0212] As used herein, the terms "intact" and "intact mitochondria" refer to mitochondria that include an outer membrane, an inner membrane, an intermembrane space, cristae (formed by the inner membrane), and a matrix. Exemplary intact mitochondria contain mtDNA. In preferred embodiments, the intact mitochondria are functional mitochondria. However, it is understood that intact dysfunctional mitochondria can also be used in the present invention.

[0213] As used herein, the term "autologous" is intended to mean a biological composition obtained from the same subject.

[0214] As used herein, the term "allogeneic" is intended to mean a biological composition obtained from the same species but having a genotype different from that of the subject receiving the biological composition.

[0215] As used herein, the term "animal cell" is intended to mean any cell derived from a eukaryote. It is understood that animal cells can include mammalian and non-mammalian species such as amphibians, fish, insects (e.g., Drosophila), and worms (e.g., Caenorhabditis elegans).

[0216] As used herein, the term "fusion protein" refers, primarily but not necessarily, to a sequence of amino acids connected to each other by peptide bonds, where a portion of the sequence is derived from one origin (natural or synthetic) (i.e., has sequence similarity to the sequence), and another portion of the sequence is derived from one or more other origins. Exemplary fusion proteins can be prepared by constructing an expression vector that encodes the entire fusion protein such that essentially all linkages are peptide bonds (both portions, e.g., encoding a mitochondrial targeting sequence and an endonuclease). It is also understood that the fusion can be made by chemical conjugation using any of the known methods used to conjugate peptides.

[0217] As used herein, the terms "mitochondrial-targeted sequence (MTS)" and "mitochondrial targeting sequence (MTS)" are interchangeable and refer to any amino acid sequence that causes the transport of an enzyme, peptide, sequence, or compound attached thereto to the mitochondria. In certain embodiments, the MTS is a human MTS. In another embodiment, the MTS is from another species. Non-limiting examples of such sequences are cytochrome c oxidase subunit X (COX10) MTS

Chemical formula

[0218] As used herein, the term "small molecule" refers to a compound that affects a biological process and has a molecular weight of about 900 Daltons or less. Exemplary small molecules have a molecular weight of about 300 to about 700 Daltons.

[0219] As used herein, the term "about" or "approximately" when used in conjunction with a number refers to any number within 1, 5, 10, 15, or 20% of the recited number.

[0220] As used herein, the term "somatic cell" refers to any differentiated cell that forms the body of an organism, apart from stem cells, progenitor cells, and germ line cells (i.e., oogonia and spermatogonia) and cells derived therefrom (e.g., oocytes, sperm). For example, internal organs, skin, bone, blood, and connective tissue are all composed of somatic cells. Somatic cells are obtained from an animal, preferably a human subject, and cultured according to standard cell culture protocols available to those skilled in the art.

[0221] As used herein, the term "endocytosis pathway" refers to the cellular process by which a cell takes in molecules from its surroundings. The endocytosis pathway can be "clathrin-dependent", which requires the mobilization of clathrin to help bend the plasma membrane into vesicles that absorb molecules, or "clathrin-independent", which does not require the mobilization of clathrin. An exemplary type of clathrin-independent endocytosis is, for example, macropinocytosis. As used herein, the term "endocytosis activator" refers to an agent that induces or activates an endocytosis pathway or process, such that, for example, the endocytosis pathway is increased. An exemplary "endocytosis activator" increases mitochondrial uptake from the extracellular environment.

[0222] As used herein, the term "macropinocytosis" refers to a clathrin-independent form of endocytosis that mediates the non-selective uptake of solute molecules, nutrients, and antigens.

[0223] As used herein, the term "compound" refers to a compound that can effect a desired biological function. The term includes, but is not limited to, DNA, RNA, proteins, polypeptides, and other compounds including growth factors, cytokines, hormones, or small molecules.

[0224] As used herein, the terms "peptide," "peptides," "polypeptides," and "proteins" are used interchangeably and in their broadest sense to refer to amino acid sequences that are constrained (i.e., having some elements of structure, such as the presence of amino acids that cause a β-turn or β-pleated sheet, or cyclized, e.g., by the presence of disulfide-bonded Cys residues) or unconstrained (e.g., linear or unstructured). The amino acids that make up a polypeptide may be of natural origin or may be synthetic. A polypeptide can be purified from a biological sample. Polypeptides, proteins, or peptides also include modified polypeptides, proteins, and peptides, such as glycopolypeptides, glycoproteins, or glycopeptides; or lipopolypeptides, lipoproteins, or lipopeptides.

[0225] As used herein, the terms "modulate", "modulation", "modulator", and "modulating" are intended to mean a change in the nature or composition of a basal homeostatic state. Exemplary modulations include changes in cellular metabolism due to disruption of homeostasis such that cellular metabolism is significantly decreased. The term "modulator" includes inhibitors and activators. An inhibitor, for example, inhibits, binds to, partially or completely blocks stimulation, decreases activation, interferes with, delays, inactivates, desensitizes, or down-regulates the expression or modification of a desired protein, pathway, or process, or the activity of the target protein, pathway, or process as described. In certain embodiments, the inhibitor is an antagonist of the target protein, pathway, or process. An activator, for example, induces or activates the expression or modification of the target protein, pathway, or process as described, or binds to, stimulates, increases, initiates, activates, promotes, enhances, sensitizes, or up-regulates the activation of inhibitor activity, or the activity of the target protein (or the encoding polynucleotide), pathway, or process as described. In certain embodiments, the activator is an agonist of the target protein, pathway, or process. Modulators include natural and synthetic ligands, antagonists, and agonists (e.g., small chemical molecules, antibodies, etc. that function as either an agonist or an antagonist). It is further understood that the modulator can be biological (e.g., an antibody) or chemical.

[0226] As used herein, the term "prior to" is intended to mean a period preceding the onset of an event that is sufficient for achieving and sustaining a desired result (e.g., antibiotic selection) or effect (e.g., biological effect) without the complete dissipation of the desired result or effect before the intended event is initiated. For example, in an exemplary situation, modulating cellular metabolism prior to the introduction of exogenous mitochondria and / or exogenous mtDNA would, for example, result in a desired biological effect (e.g., increasing phosphorylation of S6 kinase) without reverting the biological effect to a homeostatic state prior to the introduction of exogenous mitochondria and / or exogenous mtDNA.

[0227] As used herein, the term "nutritional stress" refers to a lack of nutrients or a state of nutritional starvation sufficient to cause disruption of cellular homeostasis, such as induction of autophagy, AMPK signaling, and / or mTOR signaling pathways. Exemplary nutritional stress states include serum starvation, removal of essential amino acids, and / or disruption of metabolic pathways.

[0228] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to describe a polymer of any length composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, or a compound produced synthetically, that can hybridize with a natural nucleic acid in a sequence-specific manner similar to that of two natural nucleic acids, e.g., can participate in Watson-Crick type base pairing interactions. As used herein in the context of polynucleotide sequences, the term "bases" (or "base") is synonymous with "nucleotides" (or "nucleotide"), i.e., the monomer subunits of a polynucleotide. The abbreviation "A" when used in the context of nucleotides is intended to mean adenine (A). The abbreviation "G" when used in the context of nucleotides is intended to mean guanine (G). The abbreviation "C" when used in the context of nucleotides is intended to mean cytosine (C). The abbreviation "T" when used in the context of nucleotides is intended to mean thymine (T).

[0229] As used in the context of a carrier, the term "pharmaceutically acceptable" is intended to mean that the carrier, diluent, or excipient must be compatible with the other ingredients of the formulation and not injurious to its recipient.

[0230] The practice of the embodiments provided herein, unless otherwise indicated, utilizes conventional techniques of molecular biology, microbiology, and immunology, which are within the skill of those in the art. Such techniques are sufficiently explained in the literature.Examples of particularly suitable reference texts include the following: Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); Glover, ed., DNA Cloning, Volumes I and II (1985); Gait, ed., Oligonucleotide Synthesis (1984); Hames and Higgins, eds., Nucleic Acid Hybridization (1984); Hames and Higgins, eds., Transcription and Translation (1984); Freshney, ed., Animal Cell Culture: Immobilized Cells and Enzymes (IRL Press, 1986); Kallen et al., Plant Molecular Biology - A Laboratory Manual (edited by Melody S. Clark; Springer-Verlag, 1997); Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, Protein Purification: Principles and Practice (Springer Verlag, N.Y., 2nd Edition, 1987); and Weir and Blackwell, eds., Handbook of Experimental Immunology, Volumes I - IV (1986).

[0231] (Method for producing mitochondrial exchange cells (MirC) with 5.2) The present invention is based in part on the discovery that any agent that reduces the function of endogenous mitochondria, including agents that reduce endogenous mitochondrial DNA (mtDNA), can enhance the non-invasive transfer of exogenous mitochondria. However, complete depletion of endogenous mtDNA, as in the case of ρ(0) cells, impedes this enhancement. This is because the non-invasive transfer of exogenous mitochondria is energy-dependent, and complete depletion of endogenous mtDNA greatly limits the energy available for promoting the non-invasive transfer process. Similarly, non-invasive transfer of exogenous mitochondria is inefficient when mitochondrial function and / or mtDNA is not disrupted, for example, when mitochondria are simply co-incubated (i.e., "added") or added by centrifugation.

[0232] Accordingly, provided herein is a method of generating mitochondrial replacement cells (MirC), comprising: (a) contacting a recipient cell with an agent that reduces the endogenous mtDNA copy number or an agent that reduces mitochondrial function; (b) incubating the recipient cell for a period sufficient 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) (1) co-incubating the recipient cell derived from step (b) with a partially reduced endogenous mtDNA or endogenous mitochondrial function, respectively, and (2) exogenous mitochondria from a healthy donor for a period sufficient to non-invasively transfer the exogenous mitochondria into the recipient cell, thereby generating mitochondrial replacement cells. Also provided herein is a method of generating mitochondrial replacement cells, comprising performing steps (a) and (b) above, and then (c) (1) co-incubating the recipient cell derived from step (b) with a partially reduced endogenous mtDNA or endogenous mitochondrial function, respectively, and (2) exogenous mtDNA from a healthy donor for a period sufficient to non-invasively transfer the exogenous mtDNA into the recipient cell, thereby generating mitochondrial replacement cells. In certain embodiments, the exogenous mtDNA is transferred by exogenous mitochondria.

[0233] The generation of MirC can be a strategy useful for various applications. As an example, the transfer of exogenous mitochondria, exogenous mtDNA, or a combination thereof into recipient cells can be useful, for example, in replacing endogenous mitochondria that are dysfunctional and / or composed of mutant mtDNA with functional mitochondria, for example, mitochondria composed of wild-type mtDNA. In certain embodiments, the methods provided herein are performed in recipient cells having endogenous mtDNA encoding dysfunctional mitochondria. In a specific embodiment, the endogenous mtDNA is mutant mtDNA. In certain embodiments, the endogenous mtDNA is heteroplasmic and composed of both wild-type mtDNA and mutant mtDNA.

[0234] As described above, in certain applications, the transfer of exogenous mitochondria, exogenous mtDNA, or a combination thereof can involve, for example, the transfer of functional mitochondria or wild-type mtDNA to replace endogenous mitochondria that are dysfunctional or composed of mutant mtDNA. Thus, in certain embodiments, the exogenous mtDNA is wild-type mtDNA. In other embodiments, the recipient cell's endogenous mitochondria have wild-type mtDNA and dysfunctional endogenous mitochondria. For example, exemplary dysfunctional mitochondria in recipient cells having wild-type mtDNA can include mutant nuclear DNA encoding mitochondrial proteins, or dysfunctional mitochondria resulting from secondary effects such as aging or disease.

[0235] Therefore, endogenous mitochondria that are dysfunctional, composed of mutant mtDNA, or a combination thereof can be replaced using the methods described herein. Mitochondrial dysfunction can occur as a result of many factors. Non-limiting examples include mitochondrial dysfunction due to disease (e.g., age-related disease, mitochondrial disease or disorder, neurodegenerative disease, retinal disease, genetic disease), diabetes, hearing impairment, or any combination thereof. Mitochondrial dysfunction can include a function of endogenous mitochondria that is reduced by more than 5%, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90%. Therefore, in some embodiments, the endogenous mitochondria include mitochondria whose function is reduced by 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%.

[0236] The methods provided herein are applicable to both homoplasmic mtDNA and heteroplasmic mtDNA. In a specific embodiment, the endogenous mtDNA is a single type of mtDNA (i.e., the endogenous mtDNA is homoplasmic). In other specific embodiments, the endogenous mtDNA includes multiple types of mtDNA (i.e., the endogenous mtDNA is heteroplasmic). In some embodiments, the heteroplasmic mtDNA includes both wild-type mtDNA and mutant mtDNA. Usually, the ratio of mutant mtDNA can determine the severity of the phenotype and can affect the degree to which mitochondrial function is reduced. For example, in some embodiments, the heteroplasmic mtDNA is 5% mutant mtDNA and 95% wild-type mtDNA, and mitochondrial function is reduced by 5%. In other embodiments, the heteroplasmic mtDNA is 55% mutant mtDNA and 45% wild-type mtDNA, and mitochondrial function is reduced by 55%. However, it is understood that the percentage of mutant mtDNA need not be proportional to mitochondrial function.

[0237] Dysfunctional mitochondria are characterized by routine, loss of efficiency in the electron transport chain and decreased synthesis of high-energy molecules such as adenosine-5'-triphosphate (ATP), leakage of harmful reactive oxygen species (ROS), and / or disruption of cellular respiration. Those skilled in the art will understand how to evaluate mitochondrial function. For example, mitochondrial dysfunction can be evaluated using cell-based assays such as the Seahorse Bioscience XF Extracellular Flux Analyzer, which is performed to determine basal oxygen consumption, glycolysis rate, ATP production, and respiratory capacity in a single experiment. Similarly, a quantitative functional mitochondrial diagnosis can also be established using an Oroboros 02K respirometer. It is understood that the above assay examples are illustrative and do not include all methods for evaluating mitochondrial function.

[0238] In some embodiments, functional mitochondria have an intact outer membrane. In some embodiments, functional mitochondria are intact mitochondria. In another embodiment, functional mitochondria consume oxygen at a rate that increases over time. In another embodiment, mitochondrial functionality is measured by oxygen consumption. In another embodiment, mitochondrial oxygen consumption can be measured by any method known in the art, including but not limited to the MitoXpress fluorescent probe (Luxcel). In some embodiments, functional mitochondria are mitochondria that show an increase in the rate of oxygen consumption in the presence of substrates such as ADP and, without limitation, glutamate, malate, or succinate. Each possibility represents a separate embodiment of the invention. In another embodiment, functional mitochondria are mitochondria that produce ATP.

[0239] The methods provided herein may be useful in generating MirC from recipient cells having dysfunctional mitochondria, mutant mtDNA, or a combination thereof, although it is understood that the generation of MirC need not be performed in recipient cells having dysfunctional mitochondria. In some embodiments, MirC is generated using recipient cells having functional endogenous mitochondria, wild-type mtDNA, or a combination thereof, and the exogenous mitochondria are also functional, contain wild-type mtDNA, or a combination thereof. For example, endogenous wild-type mtDNA can be reduced using the methods provided herein, and exogenous wild-type mtDNA can be transferred into recipient cells, such as mitochondrial exchange in "aged" recipient cells (e.g., cells from an aged subject or cells having a relatively high population doubling level (PDL)) using exogenous mtDNA from a healthy donor cell (e.g., a young cell having a relatively low PDL). Thus, in one embodiment, the exogenous mtDNA is from a donor cell that is a healthy donor cell, e.g., a donor cell that is younger than the recipient cell. In one embodiment, the donor and recipient cells 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. In other embodiments, the donor and recipient cells are from subjects that are 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 apart in age. However, it is understood that a difference in age between the donor and recipient cells is not a requirement. In some embodiments, the donor and recipient cells are of the same age and the donor cells are healthy cells.

[0240] In other embodiments, the generation of MirC is performed in recipient cells with functional endogenous mitochondria, such as wild-type endogenous mtDNA, where the exogenous mtDNA is a mutant, encodes dysfunctional mitochondria, the exogenous mitochondria are dysfunctional, or a combination thereof. In other embodiments, the exogenous mitochondria, exogenous mtDNA, or a combination thereof are from donor cells that are older than the recipient cells. For example, in some embodiments, a model of a mitochondrial disease or disorder can be created by the exchange of functional mitochondria of the recipient cells with exogenous mtDNA from donor cells that are mutant and / or encode dysfunctional mitochondria. It is understood that the examples described herein are illustrative and do not include all combinations involving mtDNA exchange.

[0241] As provided herein, the method of generating MirC can be carried out using either an agent that reduces endogenous mtDNA or an agent that reduces endogenous mitochondrial function. In certain situations, a combination of the two agents can be used. Agents that can reduce mitochondrial function are well known in the art and are within the skill of those in the art. Exemplary agents include any of ADP or uncoupling agents, such as inhibitors of Complex III (e.g., myxothiazol), inhibitors of Complex IV (e.g., sodium azide, potassium cyanide (KCN)), or inhibitors of Complex V (e.g., oligomycin); inhibitors of phosphorylation that abrogate the burst of oxygen consumption after addition of ADP but have no effect on respiration stimulated by uncoupling agents; uncoupling agents (e.g., dinitrophenol, CCCP, FCCP) that abrogate the forced coupling between the respiratory chain and the phosphorylation system observed in intact mitochondria; ATP / ADP transporter inhibitors (e.g., adenine nucleotide translocase inhibitors such as atractyloside) that interfere with either the efflux of ATP or the influx of substrates across the inner mitochondrial membrane; ionophores (e.g., valinomycin, nigericin) that allow compounds that normally cannot cross the inner membrane to permeate; or inhibitors of the mitochondrial respiratory chain that block respiration in the presence of one or more TCA cycle enzymes or Krebs cycle inhibitors (e.g., arsenite, aminooxyacetate) that block associated reactions. It is understood that the above agents that can reduce mitochondrial function are non-limiting and that those skilled in the art can readily identify suitable agents that can reduce mitochondrial function using techniques known in the art.

[0242] In a specific embodiment, the agent that reduces the intrinsic mitochondrial function transiently reduces the intrinsic mitochondrial function. In other embodiments, the agent that reduces the intrinsic mitochondrial function permanently reduces the intrinsic mitochondrial function. In a preferred embodiment, the agent that reduces the intrinsic mitochondrial function partially reduces the intrinsic mitochondrial function.

[0243] Using various agents, mtDNA can be reduced. In certain embodiments, the agent for reducing mtDNA is selected from a fusion protein comprising a mitochondrial targeting sequence (MTS) and an endonuclease, an endonuclease, or a nucleic acid encoding a small molecule. In certain embodiments, the small molecule is a nucleoside reverse transcriptase inhibitor (NRTI). The nucleic acid can be messenger ribonucleic acid (mRNA) or deoxyribonucleic acid (DNA). In certain embodiments, the agent for reducing mtDNA is a plasmid DNA expression vector cassette encoding an endonuclease. In a preferred embodiment, the agent is a plasmid DNA expression vector cassette encoding an endonuclease together with an MTS. Various expression vector cassettes can be used, and one skilled in the art will understand the necessary considerations required to enable successful expression of the endonuclease depending on the host cell. For example, mammalian expression vectors such as vectors having a cytomegalovirus (CMV) promoter, an SV40 promoter, or a CAG promoter would be suitable for expression of the endonuclease in mammals rather than non-mammalian cells. Similarly, it is understood that viral expression vectors can also be used, and one skilled in the art will understand that such viral expression vectors may require helper plasmids (i.e., envelope and packaging plasmids) used in tandem with the transfer plasmid. In other embodiments, the agent is an mRNA encoding an endonuclease. In other preferred embodiments, the agent is an mRNA encoding an endonuclease together with an MTS. In yet further embodiments, the agent is an endonuclease that is a recombinant protein. In other embodiments, the agent is a small molecule such as, for example, a small molecule that disrupts mtDNA synthesis. Techniques for creating any of the expression methods are known to those skilled in the art and can be readily implemented without undue experimentation. In a preferred embodiment, the agent is suitable for clinical use.

[0244] In a specific embodiment, the endonuclease is, for example, the following DNA sequences: [Chemical formula] It can be a restriction enzyme that cleaves DNA double helix at specific sites, such as XbaI, to produce fragments. Examples of endonucleases include restriction enzymes other than XbaI, such as EcoRI, BamHI, HindIII, or PstI, all of which digest mtDNA at multiple sites. Endonucleases have defined recognition sites, which enables prediction of their sensitivity to mtDNA. For example, the defined recognition sites of restriction enzymes such as XbaI, EcoRI, and SmaI are specific to a given nucleic acid sequence. Thus, in some embodiments, reduction of endogenous mtDNA can be achieved using zinc fingers and transcription activator-like effectors (TALEs) that are combined with DNA nucleases. These two types of DNA-binding proteins can be modified to have specificity for a new target DNA sequence. Similarly, clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 protein can be introduced into cells by addition of the corresponding coding gene. Therefore, in some embodiments, the endonuclease can be a programmable nuclease, such as an RNA-guided DNA endonuclease (e.g., Cas9), zinc finger nuclease (ZFN), or transcription activator-like effector nuclease (TALEN). It is understood that the above nucleases are non-limiting and that those skilled in the art can readily identify suitable endonucleases using techniques known in the art. For example, a suitable endonuclease that recognizes the mtDNA sequence can be identified by, for example, in silico analysis using a Cambridge reference sequence or a similar consensus sequence. In a specific embodiment, the endonuclease cleaves the wild-type sequence of mtDNA. In other embodiments, the endonuclease cleaves the mutant sequence of mtDNA. It is also understood that the agent for reducing endogenous mtDNA does not have to be an endonuclease and can utilize any mtDNA that can be reduced, including agents that inhibit mtDNA biosynthesis, such as ethidium bromide.Also contemplated herein are agents (i.e., mitophagy agonists) such as urolithin A or small molecule p62-mediated mitophagy inducer (PMI) that induce autophagy to promote, for example, the selective degradation of endogenous mitochondria. The present invention can also be practiced using a nucleoside reverse transcriptase inhibitor (NRTI) as an agent that reduces mtDNA.

[0245] Furthermore, in some embodiments, 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. For example, in some embodiments, the expression vector can include the puromycin N-acetyl-transferase gene (pac) from Streptomyces, and cells can be selected using puromycin. In situations where selection is performed using an antibiotic, such as puromycin, the selection can be for a short time (e.g., 24 - 48 hours) to limit long-term exposure to the drug. However, it is understood that the examples provided above are merely illustrative, and the expression vector cassette can include other antibiotic resistance genes, such as the BSD gene for selection by bsr, bls, or blasticidin, or the hph gene for selection by hygromycin B. The concentration of the antibiotic used for selection is determined by the type of antibiotic and the type of cell, and it is generally understood that it is readily available to those skilled in the art without undue experimentation. It is further understood that the selection can be brought about by any means known in the art and does not necessarily involve antibiotic resistance. For example, in some embodiments, cell selection can be performed by fluorescence-activated cell sorting (FACS) of cell surface markers or by expression of a fluorescent protein encoded by the expression. In yet further embodiments, the selection can be performed according to the phenotype of the cells. For example, in some embodiments, mutant endogenous mtDNA in cells with heteroplasmy can result in a selectable phenotypic response, such as cell survival.

[0246] Thus, in some embodiments, cells are selected after introduction of an expression vector cassette containing an endonuclease that degrades mtDNA. In some embodiments, cells are selected to obtain a homogeneous cell population that expresses an endonuclease that degrades mtDNA. In a specific embodiment, cells are selected after introduction of an expression vector cassette containing an endonuclease that degrades mtDNA, and a homogeneous stable cell line is produced. In other embodiments, cells are selected to enrich a population of cells that express an endonuclease that degrades mtDNA. As noted above, this selection-based enrichment can involve a short exposure to an antibiotic. The cells to be enriched can stably express the endonuclease or transiently express the endonuclease, depending on the degree and / or pattern of the selection pressure. It is understood that the enriched population need not be homogeneous and that the enriched cell population that expresses an endonuclease that degrades mtDNA contains a higher percentage of cells with the endonuclease than the unselected cell population, but may also contain some cells that do not express the endonuclease.

[0247] In other embodiments, cells are not selected after introduction of an expression vector containing an endonuclease that degrades mtDNA. In a specific embodiment, cells are not selected after introduction of an expression vector containing an endonuclease that degrades mtDNA, and the endonuclease is transiently expressed.

[0248] Various methods for introducing plasmid DNA expression vector cassettes, mRNA, and / or recombinant proteins are known in the art. In some embodiments, the plasmid DNA expression vector cassette is introduced by electroporation. In a specific embodiment, the electroporation method is flow electroporation, for example, MaxCyte Flow Electroporation. In other specific embodiments, the electroporation method includes nucleofection technology, for example, Lonza's Nucleofector™ technology. In other embodiments, the plasmid DNA expression vector cassette is introduced by cationic lipid transfection. In yet further embodiments, the plasmid DNA expression vector cassette is introduced by viral transduction. The above methods for introducing the expression vector cassette are non-limiting and are merely intended to be exemplary methods, and it is understood that any method known in the art can be used to introduce the DNA expression vector cassette.

[0249] When the agent for reducing endogenous mitochondria contains an endonuclease, the expression of the endonuclease can also involve the introduction of the mRNA encoding the endonuclease or the introduction of the endonuclease as a recombinant protein. In one embodiment, the MaxCyte electroporator can be used for mRNA transfection at the clinical site, particularly having cleared the standards of good manufacturing practice and good clinical practice. The transfection can be carried out using the MaxCyte electroporator according to the manufacturer's protocol. It is further understood that the above methods are merely exemplary and that any means for introducing mRNA and / or recombinant protein can be used.

[0250] The specific targeting of endonucleases to mitochondria can be achieved by incorporating a mitochondrial targeting sequence (MTS) adjacent to the endonuclease coding sequence, which results in a fusion protein that targets mitochondria. Potent MTSs have been identified and shown to be able to target proteins to specific compartments when fused to their N-terminus, and are referred to as mitochondrial targeting sequences. MTSs suitable for the methods of the present invention are well known to those skilled in the art (see, for example, U.S. Patent No. 8,039,587B2, which is hereby incorporated by reference in its entirety). For example, MTSs that are targeting peptides derived from cytochrome c oxidase subunit IV (COX 4), subunit VIII (COX 8), or subunit X (COX 10) can be used, such as MTSs for the mitochondrial matrix. In principle, any targeting sequence or fusion protein derived from any nuclear-encoded mitochondrial matrix or inner membrane enzyme can be made into a mitochondrial import protein (hydrophobic moment greater than 5.5, at least two basic residues, amphipathic α-helix conformation; see, for example, Bedwell et al., Mol Cell Biol. 9(3)(1989), 1014-1025), and artificial sequences can be useful for the purposes of the present invention.

[0251] In certain embodiments, the MTS is a human MTS. In another embodiment, the MTS is from another species. Non-limiting examples of such sequences are cytochrome c oxidase subunit X (COX 10) MTS

Chemical formula

Chemical formula

[0252] When a recipient cell is contacted with an agent that reduces the endogenous mtDNA copy number or an agent that reduces the endogenous mitochondrial function, the recipient cell is incubated for a period sufficient for the agent to partially reduce the endogenous mtDNA copy number or partially reduce the endogenous mitochondrial function in the recipient cell, respectively. Identifying a "sufficient period" that enables the agent to 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 appropriate period varies depending on various factors including, but not limited to, the specific type of cell, the amount of starting material (e.g., the number of recipient cells and / or the amount of mtDNA to be reduced), the amount and type of agent, plasmid promoter regulators, and / or culture conditions. In various embodiments, a sufficient period that enables a partial reduction of the endogenous mtDNA copy number in the 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. In a preferred embodiment, the sufficient period is long enough such that the resulting recipient cells have a substantial reduction in most of the endogenous mtDNA copy number or most of the endogenous mitochondrial function and are substantially free of the agent that reduces the endogenous mtDNA or the agent that reduces the endogenous mitochondrial function before incubating the recipient cells with exogenous mtDNA and / or exogenous mitochondria.

[0253] An important and novel aspect of the present invention is that the mitochondrial import efficiency is severely reduced in cells with complete depletion of endogenous mitochondria (i.e., (ρ)0 cells), but when the endogenous mtDNA copy number is reduced but not completely depleted (i.e., (ρ) -It has been discovered that it can be greatly improved. Furthermore, the present invention also shows that a mere addition or centrifugation protocol is inefficient without a partial reduction in the endogenous mtDNA copy number. Thus, in a preferred embodiment, the reduction in the endogenous mtDNA copy number in the recipient cells is less than 100% depletion of the endogenous mtDNA. In some embodiments, the endogenous mtDNA copy number in the recipient cells has decreased by about 5% to about 99%. In a specific embodiment, the agent that reduces the endogenous mtDNA copy number reduces about 30% to about 70% of the endogenous mtDNA copy number. In other embodiments, the agent that reduces the endogenous mtDNA copy number reduces by 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. In yet further embodiments, the agent that reduces the endogenous mtDNA copy number reduces about 60% to about 90% of the endogenous mtDNA copy number. In some embodiments, it is also understood that the agent that reduces the endogenous mtDNA copy number reduces mitochondrial aggregates.

[0254] In one embodiment, the exogenous mtDNA is contained in isolated exogenous mitochondria from donor cells. Mitochondrial isolation can be achieved by any of several well-known techniques including, but not limited to, the techniques described herein and in the cited references. In one embodiment, the exogenous mitochondria for use in mitochondrial transfer are isolated using a commercially available kit such as, for example, the Qproteum Mitochondria Isolation Kit (Qiagen, USA), the MITOISO2 Mitochondria Isolation Kit (Sigma, USA), or the Mitochondria Isolation Kit for Cultured Cells (Thermo Scientific). In other embodiments, the exogenous mitochondria for use in mitochondrial transfer are isolated manually. For example, an exemplary manual isolation of mitochondria pelleted the donor cells and grew them under culture for about 10 9Isolating mitochondria from donor cells involves washing 1-2 mL of cell pellets obtained from individual cells, swelling the cells in a hypotonic buffer, rupturing the cells with a Dounce or Potter-Elvehjem homogenizer using a pestle that fits snugly, and isolating the mitochondria by differential centrifugation. Manual isolation can also include, for example, sucrose density gradient ultracentrifugation or free flow electrophoresis. Without being bound by any particular theory, it is understood that the kits and manual methods described herein are exemplary, and any method of mitochondrial isolation can be used and is within the skill of the art.

[0255] In some embodiments, the isolated donor mitochondria are substantially pure from other organelles. In other embodiments, the isolated mitochondria can contain impurities and are enriched for mitochondria. For example, in some embodiments, the isolated mitochondria are about 90% pure, about 80% pure, about 70% pure, about 60% pure, about 50% pure, or any integer in between. Generally, it is understood that any impurities contained in the isolated donor mitochondria do not affect the survival or function of recipient cells upon mitochondrial transfer. In specific embodiments, the transfer of exogenous mitochondria, exogenous mtDNA, or combinations thereof is not accompanied by the transfer of non-mitochondrial organelles.

[0256] The amount and quality of isolated mitochondria can be readily determined by several well-known techniques including, but not limited to, the techniques described herein and in the cited references. For example, in some embodiments, the amount of isolated mitochondria is determined by assessment of the total protein content. A variety of methods are available for measuring the total protein content, such as the Biuret and Lowry methods (see, e.g., Hartwig et al., Proteomics, 2009 Jun; 9(11):3209-14). In other embodiments, the amount of isolated mitochondria is determined by the mtDNA copy number.

[0257] In some embodiments, the isolated mitochondria are functional mitochondria. In further embodiments, the isolated mitochondria are dysfunctional mitochondria. In some embodiments, mitochondrial function can be evaluated in the donor cells prior to isolation. In other embodiments, mitochondrial function can be assayed from the isolated mitochondria.

[0258] Maintenance of mitochondrial membrane integrity is another important factor during mitochondrial isolation. In some embodiments, 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 more than 90% of the isolated mitochondria are intact. Mitochondrial membrane integrity can be achieved by any of several well-known techniques including, but not limited to, the techniques described herein and in the cited references. For example, TMRM, Rhod123, JC-1, and DiOC6 are typical probes for measuring mitochondrial membrane potential (see, e.g., Perry et al., Biotechniques, 2011 Feb;50(2):98-115). JC-1 is a widely used dye for measuring the inner membrane potential of isolated mitochondria and is based on the electrochemical proton gradient across the inner mitochondrial membrane.

[0259] In certain embodiments of the methods provided herein, a recipient having a partial reduction of endogenous mtDNA is incubated with exogenous mitochondria from a healthy donor for a period of time sufficient to non-invasively transfer the exogenous mitochondria into the recipient cells, thereby creating mitochondrial exchange cells. In other embodiments, a recipient having a partial reduction of endogenous mtDNA is incubated with exogenous mtDNA from a healthy donor for a period of time sufficient to non-invasively transfer the exogenous mtDNA into the recipient cells, thereby creating mitochondrial exchange cells. Identifying a "sufficient period" for non-invasively transferring exogenous mitochondria and / or exogenous mtDNA into recipient cells is within the skill of the art. The sufficient or appropriate period can vary depending on various factors including, but not limited to, the specific type of cell, the amount of starting material (e.g., the number of recipient cells and / or the amount of endogenous mtDNA being exchanged), the amount of donor material (e.g., the amount, quality, and / or purity of the exogenous mtDNA), and / or the culture conditions. In various embodiments, a sufficient period for non-invasively transferring exogenous mitochondria and / or exogenous mtDNA into recipient cells 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. In certain embodiments, at the end of the co-incubation, the recipient cells have most of the exogenous mtDNA and substantially no exogenous mitochondrial organelles.

[0260] Another feature of the present invention is the discovery that the total mtDNA copy number in MirC does not substantially increase compared to the original recipient cells. In contrast, other less efficient methods have attempted to transfer exogenous mitochondria using centrifugation without conditioning the recipient cells prior to the co-incubation step or without conditioning the recipient cells prior to centrifugation. As a result, cell populations obtained using inefficient methods tend to have a large increase in the total mtDNA copy number. Thus, in certain embodiments, the mitochondrial exchange cells have a total mtDNA copy number that does not exceed about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, or more compared to the total mtDNA copy number of the recipient cells prior to contact with an agent that reduces the endogenous mtDNA copy number.

[0261] The use of non-invasive transfer is another unique aspect of the present invention. Previous methods have used invasive instruments to inject exogenous mitochondria and physically force the mitochondria into the cells by centrifugation or similar harsh conditions that are harmful to the recipient cells. In a clinical setting, especially when the number of recipient cells is limited, such as in the case of hematopoietic stem cells or T cells, harsh cell manipulation is undesirable. Therefore, the use of non-invasive transfer is a beneficial feature of the present invention that is suitable for use in a clinical setting.

[0262] As provided herein, the exogenous mitochondria, exogenous mtDNA, or a combination thereof can be autologous or allogeneic to the recipient cell. In some embodiments, the exogenous mtDNA is allogeneic to the recipient cell. For example, the exogenous mtDNA can be obtained from the same species as the recipient cell and have a genotype different from that of the recipient cell. In other embodiments, the exogenous mitochondria, exogenous mtDNA, or a combination thereof is autologous. As an example, exemplary autologous exogenous mtDNA can include mtDNA derived from healthy donor cells, such as "young" donor cells, such as those derived from umbilical cord blood, where the recipient cells can be from the same subject and be "aged" recipient cells, where the terms "young" and "aged" refer to the total number of times cells in the population double or the age of the subject from which the cells are harvested. Another exemplary autologous exogenous mtDNA can include, for example, donor mtDNA isolated from the same subject as the recipient cell and modified prior to exchanging it with the recipient cell. In certain embodiments, only the mtDNA and / or mitochondria are allogeneic, and the recipient cells are autologous to the subject in need of the exogenous mtDNA and / or exogenous mitochondria.

[0263] In certain embodiments, the exchange of mtDNA in recipient cells can be evaluated by sequencing the hypervariable regions (HVRs) of mtDNA, such as the DNA sequences of HV1 and / or HV2 of the D-loop, and comparing it to the sequences of both donor mitochondria and recipient cells. In a specific embodiment, the differences between the recipient cell and donor mitochondrial sequences can be identified by a single nucleotide polymorphism assay. For example, the amplified sequences of mtDNA from recipient cells and donor mitochondria can be cloned into a plasmid for use as a standard for quantification.

[0264] In some embodiments, the cells (i.e., donor cells and recipient cells) are animal cells or plant cells. In a specific embodiment, the cells are mammalian. In some embodiments, the cells are isolated from a mammalian subject selected from the group consisting of human, horse, dog, cat, mouse, rat, cow, and sheep. In some embodiments, the cells are human cells. In some embodiments, the cells are cells under culture. The cells can be obtained directly from a mammalian (preferably human) subject, or from a commercial source, or from a tissue, or in the form of, for example, cultured cells, and can also be prepared in situ or purchased from a commercial cell source. In one embodiment, the cells are primary cells (i.e., cells obtained directly from living tissue, e.g., biopsy material). The cells may be derived from any organ, including but not limited to blood or the lymphatic system, muscle, any organ, gland, skin, or brain. In one embodiment, the cells are somatic cells. In some embodiments, the cells are selected from the group consisting of epithelial cells, nerve cells, epithelial 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, spleen cells, and pancreatic beta cells.

[0265] As provided herein, in certain embodiments, the donor cells are commercially available cells cultured under current Good Manufacturing Practice (cGMP) standards. For example, the donor cells can be obtained from a cell repository such as Waisman Biomanufacturing, or similar commercial resources, such as a commercial source that produces cGMP-compliant cells. In some embodiments, the donor cells are bone marrow-derived mesenchymal stromal cells (BM-MSCs) manufactured under cGMP. In other embodiments, the cells are cGMP-grade human hepatocytes. Thus, it is understood that the donor cells can be frozen cells that are thawed prior to mitochondrial isolation. However, the mitochondria need not be isolated after the cells are frozen and can be isolated from fresh cells and used immediately, or in some embodiments, the mitochondria can be isolated and then frozen prior to transfer into recipient cells.

[0266] In some embodiments, the cells are cancer cells. Typically, 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 tumor, primary brain tumor, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head and neck cancer, breast cancer, ovarian cancer, lung cancer, small cell lung cancer, Wilms tumor, cervical cancer, testicular cancer, bladder cancer, pancreatic cancer, stomach cancer, colon cancer, prostate cancer, genitourinary cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, adrenocortical carcinoma, malignant pancreatic insulinoma, malignant carcinoid cancer, choriocarcinoma, fungating polypoid tumor, malignant hypercalcemia, cervical dysplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi sarcoma, polycythemia vera, essential thrombocythemia, Hodgkin's disease, non-Hodgkin lymphoma, soft tissue sarcoma, osteosarcoma, primary macroglobulinemia, and retinoblastoma.

[0267] In some embodiments, the cells are stem cells. As used herein, the term "stem cell" refers to undifferentiated cells that can be induced to proliferate. Stem cells can self-maintain or self-renew, which means that with each cell division, one daughter cell also becomes a stem cell. Stem cells can be obtained from embryonic, postnatal, juvenile, or adult tissues. Stem cells can be pluripotent or multipotent. As used herein, the term "progenitor cell" refers to undifferentiated cells that are derived from stem cells and are not themselves stem cells. Some progenitor cells can give rise to progeny that can differentiate into multiple cell types. Stem cells include pluripotent stem cells that can form cells of any of the body's tissue lineages: mesoderm, endoderm, and ectoderm. Thus, for example, stem cells can be selected from human embryonic stem (ES) cells; human inner cell mass (ICM) / epiblast cells; human primitive ectoderm cells, human primitive endoderm cells; human primitive mesoderm cells; and human primordial germ (EG) cells. Stem cells also include multipotent stem cells that can form a number of cell lineages that make up an entire tissue or multiple tissues, such as, but not limited to, hematopoietic stem cells or neural progenitor cells. Stem cells also include totipotent stem cells that can form an entire organism. In some embodiments, the stem cells are mesenchymal stem cells. The term "mesenchymal stem cell" or "MSC" is used interchangeably for cells that are not terminally differentiated or that can divide to give rise to cells that are either stem cells or that irreversibly differentiate in response to various influences from bioactive factors such as cytokines, into cells of the mesenchymal cell lineage, such as adipogenic, osteogenic, chondrogenic, elastic, and fibronectin tissues, myoblasts), and adult cells that give rise to tissues other than those derived from embryonic mesoderm (e.g., nerve cells). In some embodiments, the stem cells are partially differentiated or differentiated cells. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs) that have been reprogrammed or de-differentiated. In a specific embodiment, the recipient cells are iPSCs.In other embodiments, the recipient cells are hematopoietic stem cells (HSCs) or MSCs. The stem cells can be obtained from embryonic, fetal, or adult tissues.

[0268] In other embodiments, the cells are immune cells. In specific embodiments, the recipient cells are immune cells. In some embodiments, the immune cells are selected from the group consisting of T cells, phagocytes, microglial cells, and macrophages. In a specific embodiment, the T cells are CD4+ T cells. In other embodiments, the T cells are CD8+ T cells. In yet further embodiments, the T cells are chimeric antigen receptor (CAR) T cells. In a specific embodiment, the recipient cells are exhausted or nearly exhausted T cells in a state of T cell dysfunction or a state close thereto.

[0269] (5.3 Method for enhancing mitochondrial transfer) Also provided herein is a method for the transfer of mtDNA and / or mitochondria, involving the use of a second active agent in combination with any of the methods described in Section 5.2. It has been reported that mitochondrial transfer is related to the endocytosis pathway, which is an ATP-dependent process. For example, under certain cell culture conditions, mitochondria have been observed to be engulfed by macropinocytosis (e.g., Kitani et al., J Cell Mol Med., 2014, 18(8):1694-1703). Therefore, the present invention also relates to the novel discovery that the use of a second active agent prior to incubating recipient cells with exogenous mitochondria and / or exogenous mtDNA can promote the uptake of exogenous mitochondria and / or exogenous mtDNA.

[0270] Various types of agents can be used to promote the uptake of exogenous mitochondria and / or exogenous mtDNA. In some embodiments, the second active agent is selected from the group consisting of macromolecules, small molecules, or cell therapies, and the second active agent is rapamycin, NR (nicotinamide riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxolone (RTA408), KH176, batquinone (Epi743), thioctic acid, A0001 (α-tocopherol quinone), mitochondrial CoQ10 (MitoQ), SkQ1 (bisomithine), resveratrol, curcumin, ketogenic diet therapy, hypoxia, and activators of endocytosis, optionally selected from the group consisting of. In a specific embodiment, the activator of endocytosis is a modulator of cell metabolism. Cell metabolism can be prepared using various methods known to those skilled in the art. In one embodiment, the regulation of cell metabolism includes nutrient starvation, chemical inhibitors, or small molecules.

[0271] As described above, it has been reported that the transfer of intact mitochondria occurs via the endocytosis pathway. For example, exogenous mitochondria and / or exogenous mtDNA are transferred by the uptake of intact mitochondria via the endocytosis pathway. The endocytosis pathway can be subdivided into four categories: 1) clathrin-mediated endocytosis, 2) caveolae, 3) macropinocytosis, and 4) phagocytosis. Clathrin-mediated endocytosis is mediated by small vesicles (about 100 nm in diameter) with a morphologically distinct coat composed of a protein complex mainly associated with the cytoplasmic protein clathrin. Thus, in one embodiment, the endocytosis pathway for mitochondrial transfer is the clathrin-dependent endocytosis pathway. In other embodiments, the endocytosis pathway for mitochondrial transfer is a clathrin-independent pathway. In a specific embodiment, the endocytosis pathway is macropinocytosis.

[0272] Macropinocytosis has been suggested to be an important process in nutrient-deprived environments. As a result, a hypothesis has been put forward that inhibition of pathways or target molecules activated by nutrient starvation or sufficient nutrient uptake, such as inhibition of mTOR, is a strategy to enhance the macropinocytic uptake of intact mitochondria into the cytosol. Specifically, as provided herein, it has been discovered that inhibition of mTOR can enhance the uptake of exogenous mitochondria. mTOR is an essential sensor of amino acids, energy, oxygen, and growth factors, and an important regulator of protein, lipid, and nucleotide synthesis involved in the uptake of extracellular nutrients. Thus, in some embodiments, the methods provided herein further comprise contacting the recipient cells with small compounds, peptides, or proteins that can increase macropinocytosis. In specific embodiments, the methods provided herein further comprise modulating the cellular metabolism of the recipient cells prior to the transfer of exogenous mitochondria and / or exogenous mtDNA. In one embodiment, modulating the cellular metabolism is performed using the same small compounds, peptides, or proteins that can increase macropinocytosis.

[0273] Modulating cellular metabolism can be achieved by any of several 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 chemical inhibitors or small molecules. In a specific embodiment, the chemical inhibitor or small molecule is an mTOR inhibitor.

[0274] Rapamycin, also known as sirolimus (CAS number 53123-88-9; C 51 H 79 NO 13And various compounds, including rapamycin and its derivatives (e.g., rapamycin analogs, also known as "rapalogs"), are known to inhibit mTOR. Examples of 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). Thus, in some embodiments, the method of mitochondrial import provided herein further comprises modulating the cellular metabolism of the recipient cells prior to the import of exogenous mitochondria and / or exogenous mtDNA using rapamycin or its derivatives. It is understood that the above embodiments for modulating cellular metabolism are non-limiting and that modulating cellular metabolism need not involve chemical compounds or small molecules.

[0275] Thus, in some embodiments, rapamycin or its derivatives, including clinically approved drugs, can be utilized to increase the efficiency of exogenous mitochondrial import either as an independent method or in combination with any of the methods provided herein, e.g., methods involving partial reduction of the recipient cells' endogenous mitochondria.

[0276] One of ordinary skill in the art would understand that additional delivery methods can be used to introduce exogenous mitochondria and / or exogenous mtDNA, and that macropinocytosis is an exemplary pathway. In some embodiments, mtDNA can be delivered by clathrin-dependent endocytosis or clathrin-independent endocytosis. In a specific embodiment, the clathrin-independent pathway can be, for example, the CLIC / GEEC endocytosis pathway, Arf6-dependent endocytosis, flotillin-dependent endocytosis, macropinocytosis, circular doral ruffles, phagocytosis, or trans-endocytosis. Delivery of 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 and exemplary compounds suitable for activating endocytosis include, for example, phorbol-12-myristate-13-acetate (PMA) (C 36 H 56 O8), 12-O-tetradecanoylphorbol 13-acetate (TPA) (C 36 H 56 O8), sodium tanshinone IIA sulfonate (TSN-SS) (C 19 H 17 O6S.Na), and phorbol-12,13-dibutyrate, or derivatives thereof. It is further understood that non-endocytosis-mediated transfer of mtDNA and / or mitochondria can be used, including methods that bypass endocytosis and / or cell fusion.

[0277] (5.4 Treatment Methods) Provided herein are various methods for the treatment of diseases associated with mutant mtDNA and / or dysfunctional mitochondria, the use of compositions for the treatment of diseases associated with mutant mtDNA and / or dysfunctional mitochondria, and the use of compositions in the manufacture of medicaments for the treatment of diseases associated with mutant mtDNA and / or dysfunctional mitochondria. Also provided are the use of exogenous mitochondria and / or exogenous mtDNA for restoring or enhancing the function of endogenous mitochondria, the use of compositions for restoring or enhancing the function of endogenous mitochondria, and a treatment method involving the use of compositions in the manufacture of medicaments for the treatment of subjects in need of mitochondrial replacement. In certain embodiments, the treatment involves preventing mitochondrial dysfunction.

[0278] (5.4.1 Method for treating age-related diseases) In certain embodiments, provided herein is a method of treating a subject having or suspected of having an age-related disease, the method comprising any of the methods described in Section 5.2 and / or Section 5.3. In some embodiments, provided herein is a method of treating a subject having or suspected of having an age-related disease, the method comprising contacting recipient cells with an agent that reduces endogenous mtDNA or reduces endogenous mitochondrial function, incubating the recipient cells for a period of time sufficient for the agent to partially reduce the mtDNA copy number or partially reduce the endogenous mitochondrial function in the recipient cells, co-incubating (1) recipient cells with a partially reduced endogenous mtDNA or endogenous mitochondrial function and (2) exogenous mitochondria and / or exogenous mtDNA from a healthy donor for a period of time sufficient to non-invasively transfer the exogenous mitochondria into the recipient cells, thereby generating mitochondrial exchange cells ex vivo or in vitro, and then administering a therapeutically effective amount of the mitochondrial exchange recipient cells to a subject having or suspected of having an age-related disease.

[0279] In certain embodiments, age-related diseases include autoimmune diseases, metabolic diseases, genetic diseases, cancer, neurodegenerative diseases, and immunosenescence. Metabolic diseases 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. Further, genetic diseases that can be treated include Hutchinson-Gilford progeria syndrome, Werner syndrome, and Huntington's disease. Additional age-related diseases involving dysfunctional mitochondria are also contemplated.

[0280] In one embodiment, a method of treating a subject having or suspected of having an age-related disease includes generating a MirC, wherein the recipient cells used to generate the MirC are T cells or hematopoietic stem cells (HSCs). For example, endogenous mtDNA, endogenous mitochondria, or a combination thereof in aged T cells or hematopoietic stem cells (HSCs) can be replaced for rejuvenation. Mitochondrial replacement in vitro or ex vivo can be a viable option for treatment of affected patients using human T cells and / or hematopoietic stem cells. Thus, in some embodiments, using the methods provided herein, isolated exogenous mitochondria from healthy, non-aged cells are non-invasively transferred into aged or aging cells to delay aging and / or extend the lifespan of the cells, rejuvenating the recipient cells, and then the resulting rejuvenated MirC can be administered to a patient having or suspected of having an age-related disease.

[0281] As shown herein, rejuvenation of aged T cells is one possible embodiment that can be used to treat a subject having an age-related disease, such as cancer, using the present invention. By way of example, aged T cells exhibiting an aging-related secretory phenotype (SASP) consisting of inflammatory cytokines, growth factors, and proteases, a decreased and / or delayed rate of cell population doubling, telomere shortening, an increased DNA damage response (DDR), or a combination thereof can be rejuvenated using the methods provided herein by non-invasively transferring isolated mitochondria from young, healthy T cells that are autologous to the subject having an age-related disease, such as cancer. Subsequently, T cell-derived MirC having the characteristics of young, non-aged cells can be administered to a subject for the treatment of an age-related disease.

[0282] Thus, in a specific embodiment, a method of treating a subject having or suspected of having an age-related disease involves the generation of MirC, where the recipient cells are T cells. T cell fate is regulated by metabolic pathways, and either glycolysis or oxidative phosphorylation (OXPHOS) is involved in providing most of the energy to T cells. Glycolysis-dominant T cells are selected to differentiate into effector T cells, whereas OXPHOS-dominant T cells are for memory T cells. Thus, exogenous mitochondria and / or mtDNA can be used to regulate T cell fate. For example, in the case of allergy, exogenous mitochondria and / or mtDNA can be used to quiet over-activated T cells. In other situations such as cancer immunotherapy, exogenous mitochondria and / or mtDNA can empower anti-tumor T cells to enable the T cells to persist for a long time, or promote T cell lytic ability and / or reduce tumor burden. Further, in a new treatment using chimeric antigen receptor T cells (CAR T), autologous T cells are used. These CAR T cells may be in a fatigued state due to malnutrition such as aging or cachexia frequently seen in severe pathological stages of cancer. Mitochondrial exchange technology can activate CAR T, rejuvenate it, and result in better outcomes by providing more ATP.

[0283] Thus, in certain embodiments, a method of treating a subject includes recipient cells that are T cells. The T cells can be CD4+ T cells, CD8+ T cells, or CAR T cells. In specific embodiments, mitochondrial exchange in recipient T cells results in T cells with extended lifespan. For example, the lifespan can be increased by about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, or more than 5-fold. In specific embodiments, mitochondrial exchange in recipient T cells inhibits or delays the aging of recipient T cells compared to T cells without mitochondrial exchange. As described in Section 5.2, lifespan can be extended by performing mtDNA exchange using exogenous mitochondria and / or exogenous mtDNA from donor cells that are younger than the recipient cells. In certain embodiments, the donor and recipient cells 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 more than 5-fold. In other embodiments, the donor and recipient cells are from subjects whose ages are separated by about 5 years, about 10 years, about 15 years, about 20 years, or more than 20 years. In other specific embodiments, mitochondrial exchange in recipient T cells results in T cells with increased lytic ability compared to T cells without mitochondrial exchange. In yet further embodiments, mitochondrial exchange in T cells results in a decrease in tumor burden.

[0284] In certain embodiments, plasmid-based gene transfection can be used to generate T cells having exogenous mitochondria and / or exogenous mtDNA, while in other embodiments, mRNA transfection can be used. The use of mRNA transfection can reduce the chance that the RNA sequence is integrated into the host genome and can also have minimal long-term gene expression that causes a decrease in endogenous mtDNA.

[0285] In certain embodiments, the MaxCyte electroporator can be used for mRNA transfection in a clinical setting, particularly one that meets the standards of Good Manufacturing Practice and Good Clinical Practice. Transfection can be performed using the MaxCyte electroporator according to the manufacturer's protocol.

[0286] Methods for treating a subject having or suspected of having an age-related disease can also include the generation of MirC using the methods provided herein, where the recipient cells are hematopoietic stem cells (HSCs). Hematopoietic stem cells (HSCs) supply not only blood cells, but also, for example, endothelium that can replenish resident cells damaged in remote organs through transdifferentiation. Furthermore, HSC dysfunction has been reported to be involved in systemic aging. Therefore, it is contemplated that HSC-derived MirC can be used as a method for treating any age-related disease.

[0287] Furthermore, allogeneic HSC transplantation can sometimes cause graft rejection or even graft-versus-host disease. Autologous HSC transplantation is often a safer and more practical means for disease intervention. For example, autologous HSC transplantation usually does not require pretreatment with immunosuppressive agents such as radiation and chemicals. Therefore, in vitro or ex vivo generation of MirC using healthy and young exogenous mtDNA derived from mitochondria in autologous HSCs that are later returned to the patient's body is contemplated using the methods provided herein.

[0288] In certain embodiments, the HSCs are autologous to a subject in need of mitochondrial and / or mtDNA exchange, and the exogenous mtDNA is allogeneic. As provided herein, mtDNA exchange in HSCs can result in differentiated cells having functional mitochondria and / or differentiated cells having improved function. Therefore, the methods provided herein can be used in the context of HSC transplantation.

[0289] Aging alters biological processes, leading to the development of degenerative disorders such as Alzheimer's disease, atherosclerosis, osteoporosis, type 2 diabetes mellitus, and tissue fibrosis that contributes to chronic kidney disease and chronic obstructive pulmonary disease. Mitochondria can play a role in aging through reactive oxygen species generated by mitochondria, which may affect the aging process. Mitochondrial dysfunction in aging is thought to be due to the over-regulation of nicotinamide adenine dinucleotide (NAD) caused by the downregulation of nicotinamide phosphoribosyltransferase (NAMPT) and the overactivation of poly(ADP-ribose) polymerase 1 (PARP1). + ) deficiency of NAD + This leads to a vicious cycle of deregulation of nutrient sensing, resulting in the inhibition of the NAD-dependent deacetylase sirtuin 1 (SIRT1). This is then followed by the acetylation-dependent inactivation of PGC1α, resulting in the deacetylation of NAD + Low PGC1α activity leads to a downregulation of the expression of not only nuclear-encoded mitochondrial proteins but also the mitochondrial transcription factor TFAM, which is located on the periphery of mitochondrial DNA.

[0290] In addition to two core aging regulatory pathways involving p53 and p16 / Rb, the senescence-associated secretory phenotype (SASP), in which many inflammatory cytokines, chemokines, and proteases such as IL-1, IL-6 / VEGF, IL-8, and CXCL9 / MMP are released, is one of the most characterized phenomena in aging. The transcription factor GATA4 is degraded along with the association of the autophagy adapter p62 by selective autophagy under normal conditions, whereas DNA damage response (DDR) kinases ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3 related) receptor-type aging signals promote the dissociation of GATA4 and p62, stabilize GATA4, and then activate NF-kB through TRAF3IP2 (tumor necrosis factor receptor-associated factor interacting protein 2) and IL1A to support SASP. SASP is completely blocked in rho0 cells (cells without mtDNA established by forced mitophagy). Mitochondrial exchange of oocytes derived from the elderly in experimental IVF reliably enhanced the success rates of fertilization, embryo development and implantation, and offspring production.

[0291] Impairment of protein homeostasis (protein homeostasis) is another feature of aging. The integrity of protein homeostasis is strictly maintained by translational regulation, protein folding chaperones, the ubiquitin-proteasome system (UPS), and the autophagy-lysosome system. Since chaperones are ATP-dependent, the age-associated decrease in bioenergy exposes the function for accurate protein folding to risk. Both the UPS and the autophagy-lysosome system, including mitophagy, decrease over time. Changes in these three systems generate aggregates that are not recycled in the cytosol, resulting in degenerative disorders. In the mitochondrial matrix, the accumulation of abnormal proteins not only activates this system to degrade it, but also the mitochondrial unfolded protein response (UPR mtIt provides an opportunity to restore mitochondrial functions that communicate with the nucleus, which is called []. All of the above-mentioned pathways involve mitochondria. In the case of mitochondrial exchange in somatic cells, it can disrupt the deteriorating harmful aging cycle, slow down the aging process, and even rejuvenate cells.

[0292] Therefore, the method provided herein provides a clinically feasible method for treating heteroplasmy and / or treating various diseases such as diseases associated with aging by replacing endogenous dysfunctional mitochondria, for example, endogenous mitochondria having mutant mtDNA, with young and / or healthy mitochondria that can have either autologous or allogeneic origin.

[0293] In some embodiments, the method of mitochondrial exchange provided herein can be used for the treatment of mitochondrial diseases or disorders, as well as aging, cancer, and immune system deficiencies.

[0294] (5.4.2 Method for Treating Mitochondrial Diseases or Disorders) Also provided herein is a method for treating a subject having or suspected of having a mitochondrial disease or disorder according to any of the methods described in Section 5.2 and / or Section 5.3. In some embodiments, the method for treating a subject having or suspected of having a mitochondrial disease or disorder comprises preparing 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 mitochondrial exchange recipient cells to a subject having or suspected of having a mitochondrial disease or disorder.

[0295] A variety of mitochondrial diseases or disorders are known and can all be treated using the methods provided herein. For example, a mitochondrial disease or disorder treatable using the methods provided herein can be complex I deficiency (OMIM: 252010). Complex I deficiency can be caused by a mutation in any of its subunits. In another embodiment, 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).

[0296] Furthermore, a mitochondrial disease or disorder treatable using the methods provided herein can be complex IV deficiency (cytochrome c oxidase; OMIM: 220110). Complex IV deficiency can be caused by a mutation in any of its subunits. In certain circumstances, 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).

[0297] Mitochondrial diseases or disorders can be caused by or associated with mutations. The mutations can be point mutations, missense mutations, deletions, and insertions. Identification of mutations in mtDNA or nDNA is understood to be within the skill of those of ordinary skill in the art, and exemplary methods such as single nucleotide polymorphism (SNP) assays or droplet digital PCR are provided herein.

[0298] Non-limiting examples of specific types of mitochondrial diseases or disorders that can be treated using the methods provided herein include ornithine transcarbamylase deficiency (hyperammonemia) (OTCD), carnitine O-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), very long-chain acyl-CoA dehydrogenase deficiency (LCAD), trifunctional protein deficiency, progressive external ophthalmoplegia with mitochondrial DNA deletions (POLG), DGUOK, TK2, pyruvate decarboxylase deficiency, and Leigh syndrome (LS).In another embodiment, the mitochondrial disease or disorder is selected from the group consisting of: Alpers disease; Barth syndrome; β-oxidation defect; carnitine-acyl-carnitine deficiency; carnitine deficiency; coenzyme Q10 deficiency; complex II deficiency (OMIM: 252011), complex III deficiency (OMIM: 124000), complex V deficiency (OMIM: 604273), LHON-Leber hereditary optic atrophy; MM-mitochondrial myopathy; LIMM-lethal infantile mitochondrial myopathy; MMC-maternally inherited myopathy and cardiomyopathy; NARP-neurogenic muscle weakness, ataxia, and retinitis pigmentosa; Leigh disease; FICP-lethal infantile cardiomyopathy plus, MELAS-related cardiomyopathy; MELAS-mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes; LDYT-Leber hereditary optic neuropathy and dystonia; MERRF-myoclonus epilepsy and ragged-red muscle fibers; MHCM-maternally inherited hypertrophic cardiomyopathy; CPEO-chronic progressive external ophthalmoplegia; KSS-Kearns-Sayre syndrome; DM-maturity-onset diabetes of the young; DMDF-maturity-onset diabetes of the young + deafness; CIPO-chronic pseudo-obstruction with myopathy and ophthalmoplegia; DEAF-maternally inherited deafness; PEM-progressive encephalopathy; SNHL-sensorineural hearing loss; encephalomyopathy; mitochondrial cytopathy; DEMCHO-dementia and chorea; AMDF-ataxia, myoclonus; ESOC-epilepsy; optic atrophy; FBSN-familial bilateral striatal necrosis; FSGS-focal segmental glomerulosclerosis; LIMM-lethal infantile mitochondrial myopathy; MDM-myopathy and maturity-onset diabetes of the young; MEPR-myoclonus epilepsy and psychomotor regression; MERME-MERRF / MELAS overlap disorder; MHCM-maternally inherited hypertrophic cardiomyopathy; MICM-maternally inherited cardiomyopathy; MILS-maternally inherited Leigh syndrome; mitochondrial encephalomyopathy; multi-system mitochondrial disorder (myopathy, encephalopathy, blindness, hearing loss, peripheral neuropathy); NAION-non-arteritic anterior ischemic optic neuropathy; PEM-progressive encephalopathy; PME-progressive myoclonus epilepsy; RTT-Rett syndrome; SIDS-sudden infant death syndrome; and MIDD-maternally inherited diabetes and deafness.

[0299] The methods provided herein for treating mitochondrial diseases or disorders can, in certain embodiments, also include mitochondrial diseases or disorders 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-dystonia (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), Luft disease, aminoglycoside-induced deafness (AID), and mitochondrial DNA multiple deletion syndrome.

[0300] mtDNA mutations are thought to be associated with numerous clinical disorders. In adults, these include neurological disorders (e.g., migraine, stroke, epilepsy, dementia, myopathy, peripheral neuropathy, diplopia, ataxia, language disorders, and sensorineural hearing loss), gastrointestinal disorders (e.g., constipation, irritable bowel, and dysphagia), heart diseases (e.g., heart failure, heart block, and cardiomyopathy), respiratory diseases (e.g., respiratory failure, nocturnal hypoventilation, recurrent aspiration, and pneumonia), endocrine disorders (e.g., diabetes, thyroid disease, parathyroid disease, and ovarian insufficiency), ophthalmic diseases (e.g., optic atrophy, cataract, ophthalmoplegia, and ptosis). In children, disorders thought to be associated with mtDNA mutations include neurological disorders (e.g., epilepsy, myopathy, psychomotor retardation, ataxia, spasticity, dystonia, and sensorineural hearing loss), gastrointestinal disorders (e.g., vomiting, failure to thrive, and dysphagia), heart diseases (e.g., biventricular hypertrophic cardiomyopathy and arrhythmia), respiratory diseases (e.g., central hypoventilation and apnea), hematological diseases (e.g., anemia and pancytopenia), kidney diseases (e.g., tubular abnormalities), liver diseases (e.g., liver failure), endocrine disorders (e.g., diabetes and adrenal insufficiency), and ophthalmic diseases (e.g., optic atrophy). Accordingly, the methods and compositions provided herein are contemplated for use in the treatment or prevention of diseases and disorders associated with mtDNA mutations.

[0301] In other specific embodiments, the methods provided herein enable the treatment of mitochondrial diseases or disorders, where the mitochondrial disease or disorder is caused by a nuclear DNA abnormality, and the nuclear DNA abnormality is 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 ataxic neuropathy dysarthria ophthalmoparesis (SANDO), disorders of the brainstem and spinal cord, and leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL), coenzyme Q10 deficiency, Leigh syndrome, mitochondrial complex abnormalities, fumarase deficiency, alpha-ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenase complex deficiency (PDHC), pyruvate carboxylase deficiency (PCD), carnitine palmitoyltransferase I (CPT I) deficiency, carnitine palmitoyltransferase II (CPT II) deficiency, carnitine-acylcarnitine (CACT) deficiency, autosomal dominant / autosomal recessive progressive external ophthalmoplegia (ad- / ar-PEO), infantile-onset spinocerebellar ataxia (IOSCA), mitochondrial myopathy (MM), spinal muscular atrophy (SMA), growth retardation, aminoaciduria, cholestasis, iron overload, early death (GRACILE), and Charcot-Marie-Tooth disease type 2A (CMT2A).

[0302] Many individuals with mtDNA mutations exhibit clinical feature groups that are classified into distinct clinical syndromes, such as Kearns-Sayre syndrome (KSS), chronic progressive external ophthalmoplegia (CPEO), mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), myoclonic epilepsy with ragged red fibers (MERRF), neuropathy, ataxia, and retinitis pigmentosa (NARP), or Leigh syndrome (LS). However, there is significant clinical variability, and many individuals do not neatly fit into one specific category that can be well explained by the various overlapping disease phenotypes (including mitochondrial recessive ataxia syndrome (MIRAS) due to mutations in the nuclear gene POLG that emerged as a major cause of mitochondrial diseases or disorders).

[0303] Exemplary diseases in which mitochondrial disorders are known to play important roles include, but are not limited to, the onset of many neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Furthermore, mitochondrial diseases or disorders are subclassified into several syndromes according to symptoms rather than the type of mutation. For example, mitochondrial syndromes include mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), myoclonic epilepsy with ragged red fibers (MERRF), and Leigh syndrome.

[0304] (5.4.3 Method of Treating a Subject in Need of Mitochondrial Replacement) Also provided herein is a method of treating a subject in need of mitochondrial replacement according to any of the methods described in Section 5.2 and / or Section 5.3. In some embodiments, the method of treating a subject in need of mitochondrial replacement comprises generating 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 mitochondrial replacement recipient cells to the subject in need of mitochondrial replacement.

[0305] Subjects in need of mitochondrial replacement include any subject having dysfunctional mitochondria. In certain embodiments, 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 impairment, a genetic disease, or a combination thereof. Neurodegenerative diseases that may 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. Retinal diseases can be wet or dry age-related macular degeneration, macular edema, or glaucoma. Other exemplary diseases, such as age-related diseases and / or mitochondrial diseases or disorders, are described in more detail in Sections 5.4.1 and 5.4.2.

[0306] Subjects in need of mitochondrial replacement may include subjects who have a predisposition to mitochondrial dysfunction and are asymptomatic. For example, the subject may have mutant mtDNA, but for example, show no signs of a mitochondrial disease. This is because the disease is an adult-onset disease. Therefore, by treating a subject in need of mitochondrial replacement using the methods provided herein, it is also possible to prevent any of the diseases described herein.

[0307] (5.5 Methods for generating iPSCs) The present invention also provides the methods described in Sections 5.2 and 5.3 for producing or enhancing the production of induced pluripotent stem cells (iPSCs) from non-pluripotent cells. iPSCs have been shown to be produced from non-pluripotent cells using the exogenous expression of stem cell factors such as Oct3 / 4, Klf4, Sox2, and c-Myc. Furthermore, although a small number of mitochondrial DNA (mtDNA) copies have been detected in undifferentiated ESCs, this number increases along with the level of mitochondrial maturation upon differentiation (Facucho-Oliveira JM et al., J Cell Sci 2007;120(Pt 22):4025-4034). Thus, the present invention uses the methods provided herein to contact non-pluripotent recipient cells with an agent that reduces endogenous mtDNA and incubate the non-pluripotent recipient cells for a period sufficient for the agent to partially reduce the endogenous mtDNA of the non-pluripotent cells, thereby reducing the endogenous mtDNA in non-pluripotent ones, and then introducing one or more expression cassettes for the expression of Oct3 / 4, Klf4, Sox2, and c-Myc, thereby confirming that the production of iPSCs can be enhanced. In certain embodiments, exogenous mtDNA and / or exogenous mitochondria are non-invasively transferred into recipient cells.

[0308] It is understood that the introduction of one or more expression cassettes for the expression of Oct3 / 4, Klf4, Sox2, and c-Myc can occur before, during, or after the introduction of an agent that reduces endogenous mtDNA. Thus, in some embodiments, a method of producing iPSCs comprises introducing one or more expression cassettes for the expression of Oct3 / 4, Klf4, Sox2, and c-Myc, contacting non-pluripotent recipient cells with an agent that reduces endogenous mtDNA, and incubating the non-pluripotent recipient for a period sufficient for the agent to partially reduce the endogenous mtDNA of the recipient cells.

[0309] In certain embodiments, the method further includes incubating the recipient cells with exogenous mitochondria and / or exogenous mtDNA for a period sufficient to non-invasively transfer the exogenous mitochondria and / or exogenous mtDNA into the recipient cells. In specific embodiments, the method further includes incubating the recipient cells with exogenous mitochondria and / or exogenous mtDNA for a period sufficient to replace a majority of the endogenous mtDNA. Methods of producing iPSCs from non-pluripotent cells 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.

[0310] Since a small number of mitochondrial DNA (mtDNA) copies have been detected in undifferentiated embryonic stem cells (ESCs), the methods provided herein can also be used to promote pluripotency of non-pluripotent stem cells and reduce the number of exogenous genes required to generate iPSCs. For example, in some embodiments, using the methods provided herein, a non-pluripotent recipient cell is contacted with an agent that reduces endogenous mtDNA, and the non-pluripotent recipient cell is incubated for a period sufficient for the agent to partially reduce the endogenous mtDNA of the non-pluripotent cell, thereby reducing the endogenous mtDNA in the non-pluripotent one, and then one or more of Oct3 / 4, Klf4, Sox2, and c-Myc are introduced into the non-pluripotent cell, thereby generating a pluripotent stem cell, thereby generating iPSCs. In some embodiments, iPSCs can be generated using only small molecule agents and without exogenous factors.

[0311] In certain embodiments, the iPSCs can contain mutant mtDNA. For example, the mutant mtDNA can contain point mutations such as point mutations in tRNA (e.g., MELAS). The mutant mtDNA can also include mtDNA having a long deletion of mtDNA. In other embodiments, the non-pluripotent cells used to produce the iPSCs are heteroplasmic. The incorporation of mutant mtDNA can, for example, facilitate the generation of disease models.

[0312] In some embodiments, the non-pluripotent recipient cells are somatic cells. In a specific embodiment, the non-pluripotent cells are fibroblasts.

[0313] The culturing conditions, identification, and establishment of iPSCs are within the skill of those in the art. For example, the methods include those provided in U.S. Pat. Nos. 8,058,065 and 8,278,104, which are hereby incorporated by reference in their entirety.

[0314] (5.6 Assays for Measuring Heteroplasmy) As previously disclosed, mutant mtDNA and / or heteroplasmy can give rise to dysfunctional mitochondria. Therefore, assays useful for assessing mitochondrial function and / or mtDNA mutations in the context of the methods provided herein for mtDNA exchange include any assay known to those of skill in the art that can be used to determine or predict the functionality of mitochondria and / or mtDNA mutations.

[0315] As an example, assays for determining mitochondrial function include, for example, the following: secreted factors associated with aging (e.g., pro-inflammatory cytokines, proteases, and growth and angiogenesis factors, e.g., IL-1, IL-6 / VEGF, IL-8, and CXCL9 / MMP); mitochondrial function by using Oroboros; mitophagy by using Keima-Red; mitochondrial permeability; mitochondrial membrane potential; cytochrome c level; reactive oxygen species; cellular respiration; transcriptomics and proteomics for measuring activated innate immunity, inactivation of over-activated glycolysis, alleviation of ER stress, suppression of the mTOR-S6 pathway, and cell cycle; mitochondrial dynamics observed by super-resolution microscopy and quantified by specialized software, e.g., measurement of either one of fission and fusion; or any assay known in the art for measuring mitochondrial function.

[0316] Various sequencing methods can be used in combination with any of the methods provided herein to (1) detect mutant mtDNA, (2) quantify heteroplasmy, and / or (3) evaluate or confirm the transfer of exogenous mitochondria and / or exogenous mtDNA. A stretch of approximately 1,100 nucleotides contains no genes and is referred to as the D-loop, displacement loop, and control region. The D-loop contains two regions where mutations accumulate at a higher frequency than at other locations in the mitochondrial genome. These regions are referred to as hypervariable regions HV1 and HV2, respectively. Thus, in some embodiments, mtDNA mutations can be identified in the context of the methods provided herein by sequencing the hypervariable regions (HV) of the D-loop of mtDNA (i.e., HV1 and / or HV2). mtDNA sequencing can be performed using any sequencing method known in the art. In a specific embodiment, the sequencing method includes a single nucleotide polymorphism (SNP) assay. In other embodiments, the sequencing method includes digital PCR. In a specific embodiment, the digital PCR is droplet digital PCR.

[0317] (5.7 Composition) Also provided herein is a composition of cells obtained by any of the methods described in Sections 5.2 - 5.5. In certain embodiments, provided herein is a composition comprising one or more mitochondria - exchanged cells obtained by a method comprising: (a) contacting a recipient cell with an agent that reduces the endogenous mtDNA copy number; (b) incubating the recipient cell for a period of time sufficient 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 is partially reduced and (2) exogenous mitochondria for a period of time sufficient to non - invasively transfer the exogenous mitochondria into the recipient cell, thereby producing a mitochondria - exchanged cell, wherein the mitochondria - exchanged cell comprises more than 5% exogenous mtDNA. In other embodiments, provided herein is a composition comprising one or more mitochondria - exchanged cells obtained by a method comprising: (a) contacting a recipient cell with an agent that reduces the endogenous mtDNA copy number; (b) incubating the recipient cell for a period of time sufficient 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 is partially reduced and (2) exogenous mtDNA from a healthy donor for a period of time sufficient to non - invasively transfer the exogenous mtDNA into the recipient cell, thereby producing a mitochondria - exchanged cell, wherein the mitochondria - exchanged cell comprises more than 5% exogenous mtDNA.

[0318] The composition can also be obtained by a method that includes contacting cells with an agent that reduces mitochondrial function, and then incubating the recipient cells for a period sufficient for the agent to partially reduce the endogenous mitochondrial function in the recipient cells. In some embodiments, the recipient cells with partially reduced endogenous mitochondrial function are then co-incubated with any exogenous mitochondria from a healthy donor for a period sufficient to non-invasively transfer the exogenous mitochondria into the recipient cells, thereby creating mitochondrial exchange cells. In other embodiments, the recipient cells with partially reduced endogenous mitochondrial function are then co-incubated with any exogenous mtDNA from a healthy donor for a period sufficient to non-invasively transfer the exogenous mitochondria into the recipient cells, thereby creating mitochondrial exchange cells. In some embodiments, the mitochondrial exchange cells produced by the above method contain more than 5% exogenous mtDNA.

[0319] As described above, exogenous mitochondria can be composed of exogenous mtDNA. Therefore, in some embodiments, both exogenous mitochondria and exogenous mtDNA are transferred into the recipient cells, and MirC has both exogenous mitochondria and exogenous mtDNA. In other embodiments, the exogenous mtDNA is transferred into the recipient cells via exogenous mitochondria, and then the exogenous mtDNA is delivered to the endogenous mitochondria. In certain situations, the exogenous mitochondria are removed from the cells after the exogenous mtDNA has been delivered to the endogenous mitochondria. Thus, in some embodiments, MirC has exogenous mtDNA and no exogenous mitochondria.

[0320] Since the endogenous mtDNA of the recipient cells is partially degraded, MirC, which contains exogenous mitochondria, exogenous mtDNA, or a combination thereof, can contain both exogenous mtDNA and endogenous mtDNA. Similarly, in a scenario where exogenous mitochondria are transferred into recipient cells, MirC can contain both exogenous and endogenous mitochondria. Thus, in a specific embodiment, the composition of one or more mitochondrial exchange cells obtained by the methods provided herein has a combination of endogenous and exogenous mitochondria. In other embodiments, the composition of one or more mitochondrial exchange cells obtained by the methods provided herein has a combination of endogenous mtDNA and exogenous mtDNA (i.e., heteroplasmic mtDNA). In yet further embodiments, the one or more mitochondrial exchange cells have a total mtDNA copy number that is no more than about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, or more than that compared to the total mtDNA copy number of the recipient cells before contact with an agent that reduces the endogenous mtDNA copy number.

[0321] The present invention also includes compositions for use in a method of creating mitochondrial exchange that includes an agent that reduces endogenous mtDNA or an agent that reduces mitochondrial function and a second active agent. In certain embodiments, the composition can further include exogenous mitochondria, one or more recipient cells, or a combination thereof. In yet further embodiments, the composition can further include exogenous mtDNA.

[0322] As described in Section 5.3, various second active agents can be used in a method for generating one or more mitochondrial exchange cells. For example, in some embodiments, the second active agent includes macromolecules, small molecules, or cell therapies, and the second active agent is arbitrarily selected from rapamycin, NR (nicotinamide riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxolone (RTA408), KH176, batiquinone (Epi743), thioctic acid, A0001 (α-tocopherol quinone), mitochondrial CoQ10 (MitoQ), SkQ1 (bisomithine), resveratrol, curcumin, ketogenic diet therapy, hypoxia, and activators of endocytosis.

[0323] The use of an endocytosis activator was found to enhance the uptake of exogenous mitochondria in cells treated with the MTS-XbaI R plasmid, but had no effect on promoting uptake in "plus" or mock-transfected cells, indicating that this mechanism of importing exogenous mitochondria is specific to the methods provided herein. Non-limiting and exemplary compounds suitable for activating endocytosis include, for example, phorbol-12-myristate-13-acetate (PMA) (C 36 H 56 O8), 12-O-tetradecanoylphorbol 13-acetate (TPA) (C 36 H 56 O8), sodium tanshinone IIA sulfonate (TSN-SS) (C 19 H 17 O6S.Na), and phorbol-12,13-dibutyrate, or derivatives thereof. In some embodiments, the endocytosis activator includes a modulator of cell metabolism.

[0324] Regulating cell metabolism can be achieved by any of several well-known techniques including, but not limited to, the techniques described herein and in the cited references. For example, in some embodiments, regulating cell metabolism is effected by nutrient starvation or deprivation. In other embodiments, regulating cell metabolism is effected by chemical inhibitors or small molecules. In a specific embodiment, the chemical inhibitor or small molecule is an mTOR inhibitor.

[0325] Rapamycin, also known as sirolimus (CAS number 53123-88-9; C 51 H 79 NO 13 ) and various compounds including rapamycin derivatives (e.g., rapamycin analogs, also known as "rapalogs") are known to inhibit mTOR. Examples of 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). Thus, in some embodiments, the compositions provided herein include rapamycin or a derivative thereof. It is understood that the above embodiments for regulating cell metabolism are non-limiting and that regulating cell metabolism can be effected without the need for chemical compounds or small molecules and can include the regulation of other pathways beyond mTOR. It is also understood that the compositions can optionally include activators of endocytosis and that it is not a required component. Further, in some embodiments, the invention provided herein can include, for example, non-endocytosis-mediated transfer of mtDNA and / or mitochondria in a non-clinical setting.

[0326] As described in Section 5.5, the present invention provides, in certain embodiments, a composition for use in a method of producing induced pluripotent stem cells (iPSCs) from non-pluripotent cells, comprising an agent that reduces endogenous mtDNA, one or more expression cassettes for the expression of Oct3 / 4, Klf4, Sox2, and c-Myc, and recipient cells, wherein the recipient cells are non-pluripotent cells, and wherein the agent that reduces endogenous mtDNA is present in an amount effective to increase the efficiency of producing induced pluripotent stem cells (iPSCs) from non-pluripotent cells as compared to non-pluripotent cells not treated with an agent that reduces endogenous mtDNA. In some embodiments, the agent that reduces endogenous mtDNA is present in an amount effective to increase the efficiency of producing induced pluripotent stem cells (iPSCs) from non-pluripotent cells as compared to non-pluripotent cells not treated with an agent that reduces endogenous mtDNA. This is based, in part, on the observation that pluripotent cells have a reduced mtDNA copy number. In a specific embodiment, the composition for use in a method of producing iPSCs further comprises exogenous mitochondria and / or exogenous mtDNA.

[0327] The present invention also includes pharmaceutical compositions for use in the treatment of any subject in need of mitochondrial replacement, age-related diseases, mitochondrial diseases or disorders, neurodegenerative diseases, diabetes, genetic diseases, or mitochondrial replacement as described in Section 5.4. In certain embodiments, provided herein is a pharmaceutical composition comprising a population of isolated mitochondrial replacement cells having exogenous mitochondria from a healthy donor, wherein the cells are obtained by the methods described herein, for example, in Sections 5.2-5.3. In other embodiments, the pharmaceutical composition comprises a population of isolated mitochondrial replacement cells having exogenous mitochondria and / or exogenous mtDNA from a healthy donor, and the cells are obtained by the methods described herein, for example, in Sections 5.2-5.3. For example, in some embodiments, mitochondrial replacement cells having exogenous mtDNA can optionally further comprise exogenous mitochondria. In other embodiments, the exogenous mtDNA is transferred into the cell via exogenous mitochondria that are delivered to the endogenous mitochondria, and then the exogenous mitochondria are removed from the recipient cell.

[0328] The present disclosure also provides a pharmaceutical composition comprising a population of isolated mitochondrial replacement cells having exogenous mitochondria from a healthy donor, wherein the cells are obtained by any of the methods provided herein for obtaining mitochondrial replacement cells. In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a population of isolated mitochondrial replacement cells having exogenous mtDNA from a healthy donor, wherein the cells are obtained by any of the methods provided herein for obtaining mitochondrial replacement cells. In some embodiments, a pharmaceutical composition comprising a population of isolated mitochondrial replacement cells having exogenous mtDNA from a healthy donor further comprises exogenous mitochondria.

[0329] For example, in some embodiments, a pharmaceutical composition comprising exogenous mitochondria from a healthy donor is obtained by a method that includes contacting cells with an agent that reduces the mtDNA copy number and then incubating the recipient cells for a period sufficient for the agent to partially reduce the endogenous mtDNA copy number of the recipient cells. In some embodiments, the recipient cells having a partially reduced endogenous mtDNA copy number are then co-incubated with any exogenous mitochondria from a healthy donor for a period sufficient to non-invasively transfer the exogenous mitochondria into the recipient cells, thereby producing mitochondrial exchange cells. In other embodiments, the recipient cells having a partially reduced endogenous mtDNA copy number are then co-incubated with any exogenous mtDNA from a healthy donor for a period sufficient to non-invasively transfer the exogenous mitochondria into the recipient cells, thereby producing mitochondrial exchange cells. In some embodiments, the mitochondrial exchange cells produced by the above method contain more than 5% exogenous mtDNA.

[0330] In other embodiments, the cells are obtained by a method comprising contacting the cells with an agent that reduces mitochondrial function, and then incubating the recipient cells for a period of time sufficient for the agent to partially reduce the endogenous mitochondrial function in the recipient cells. In some embodiments, the recipient cells having partially reduced endogenous mitochondrial function are then co-incubated with any exogenous mitochondria from a healthy donor for a period of time sufficient to non-invasively transfer the exogenous mitochondria into the recipient cells, thereby producing mitochondrial exchange cells. In other embodiments, the recipient cells having partially reduced endogenous mitochondrial function are then co-incubated with any exogenous mtDNA from a healthy donor for a period of time sufficient to non-invasively transfer the exogenous mtDNA into the recipient cells, thereby producing mitochondrial exchange cells. In some embodiments, the mitochondrial exchange cells produced by the above method contain more than 5% exogenous mtDNA. The agent that reduces mitochondrial function can transiently or permanently reduce mitochondrial function. It is within the skill of the art to determine whether the agent transiently (e.g., reversible inhibitor) or permanently (e.g., irreversible inhibitor) reduces mitochondrial function.

[0331] In certain embodiments of the pharmaceutical compositions provided herein, the cells are obtained by a method that further comprises contacting the recipient cells with a second active agent before incubating the recipient cells with exogenous mitochondria and / or exogenous mtDNA. In some embodiments, the second active agent is selected from the group consisting of macromolecules, 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, batquinone (Epi743), thioctic acid, A0001 (α-tocopherol quinone), mitochondrial CoQ10 (MitoQ), SkQ1 (bisomithine), resveratrol, curcumin, ketogenic diet therapy, hypoxia, and activators of endocytosis. In a specific embodiment, the activator of endocytosis is a modulator of cell metabolism. In other embodiments, the modulator of cell metabolism comprises nutrient starvation, chemical inhibitors, or small molecules. In a further embodiment, the chemical inhibitor or small molecule is an mTOR inhibitor. In yet a further embodiment, the mTOR inhibitor comprises rapamycin or a derivative thereof.

[0332] As described in Section 5.2 above, various types of cells can be used as recipient cells and donor cells. For example, the present disclosure describes numerous examples where the recipient cells are mammals. However, it is also understood that any cell having mitochondria can be a recipient cell. Therefore, the recipient cells can also be plant cells.

[0333] In some embodiments, the animal cells are mammals. In a specific embodiment, the cells are somatic cells. In a further embodiment, the somatic cells are epithelial cells. In yet a further embodiment, the epithelial cells are thymic epithelial cells (TEC).

[0334] The present disclosure also provides a composition in which somatic cells are immune cells. For example, the composition can include immune cells, where the immune cells are T cells, for example, exhausted T cells. In some embodiments, the composition includes rejuvenated T cells containing exogenous mitochondria and / or exogenous mtDNA. For example, aged T cells or T cells likely to be aged (e.g., immunosenescent ones) can serve as recipient cells, and T cell-derived MirC can be produced using the methods provided herein for producing T cells having healthy exogenous mitochondria and / or exogenous mtDNA. In a specific embodiment, the T cell is a CD4+ T cell. In other embodiments, the T cell is a CD8+ T cell. In some embodiments, the T cell is a chimeric antigen receptor (CAR) T cell. For example, in some embodiments, the present disclosure provides a MirC that is a CAR-T cell effective in killing cancer cells. MirC-derived CART can have a long-term survival that enables increased immune surveillance and enhanced cancer cell killing. In other embodiments, the immune cell is a phagocyte.

[0335] As described above, the compositions provided herein can also include compositions for use in delaying aging and / or extending the lifespan in cells. The composition can include aged or aging cells having endogenous mitochondria, isolated exogenous mitochondria from non-aged cells, and an agent that reduces the endogenous mtDNA copy number. The composition can also include aged or aging cells having endogenous mitochondria, isolated exogenous mitochondria from non-aged cells, and an agent that reduces mitochondrial function.

[0336] Also provided herein is a composition comprising one or more mitochondrial exchange cells derived from recipient cells that are bone marrow cells. In a specific embodiment, the bone marrow cells are hematopoietic stem cells (HSCs) or mesenchymal stem cells (MSCs). For example, HSCs or MSCs can be isolated from a subject having a mitochondrial disease, an age-related disease, or suspected of having a mitochondrial disease or an age-related disease, or otherwise in need of mitochondrial exchange and having endogenous mitochondria that have been exchanged with exogenous mitochondria. Thereafter, the HSC- or MSC-derived MirC can be transplanted back into the subject in need of such mitochondrial exchange. In a further embodiment, the recipient cells are iPS cells. The composition can be used at the point of care and can be effective in the treatment of age-related diseases, mitochondrial diseases or disorders, neurodegenerative diseases, diabetes, or genetic diseases. For example, in some embodiments, the iPSCs can be differentiated into specific cell types using methods known in the art prior to administration back to the subject.

[0337] In other embodiments, provided herein is a pharmaceutical composition comprising a population of isolated pluripotent cells having a reduced amount of endogenous mtDNA, wherein the cells are obtained by any of the embodiments described in Section 5.5. In a specific embodiment, the population of isolated pluripotent cells is iPS cells.

[0338] The administration of the cells or compounds described herein is by any of the routes commonly used to introduce agents. The pharmaceutical compositions of the invention can include a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized foreign pharmacopeias for use in animals, more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. A pharmaceutically acceptable carrier is somewhat determined by the particular composition being administered and by the method used to administer the composition. Thus, there are a variety of suitable formulations of the pharmaceutical compositions of the invention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1985).

[0339] Formulations suitable for administration can include aqueous and non-aqueous solutions, isotonic sterile solutions, and aqueous and non-aqueous sterile suspensions that can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and can include suspending, solubilizing, thickening, stabilizing, and preserving agents. In the practice of the invention, the compositions can be administered, for example, orally, intranasally, topically, intravenously, intraperitoneally, intrathecally, or intravitreally (e.g., by instillation or injection). The formulations of the compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0340] In the context of the present invention, the dosage administered to a patient should be sufficient to induce a beneficial response over time in the subject, i.e., sufficient to prevent, ameliorate, or reverse the subject's disease. The optimal dosage level for any patient will be determined by a variety of factors including the potency of the particular modulator being utilized, the patient's age, weight, physical activity, and diet, as well as possible combinations with other drugs. The size of the dosage will also be determined by the presence, nature, and extent of any adverse side effects associated with the administration of the particular compound or vector in a particular subject. Administration can be accomplished by single or divided doses.

[0341] The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0342] All patents, applications, published applications, and other publications cited herein are hereby incorporated by reference in their entirety into this specification. In case of any conflict between any description of the terms herein and any document incorporated by reference herein, any description of the terms herein shall prevail.

[0343] Throughout this application, various publications are referenced. The complete disclosure of these publications is hereby incorporated by reference in this application to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples provided above, it should be understood that various changes can be made without departing from the spirit of the invention.

Examples

[0344] (6. Examples) The examples in this section are provided for illustrative purposes rather than for limiting purposes. The following examples are presented as exemplary embodiments of the present invention. They should not be regarded as limiting the broad scope of the present invention.

[0345] (Example I: Optimization of the MirC protocol revealed that XbaI degrades mtDNA in vitro and the MTS expression vector targets mitochondria) The scheme of the method used to generate mitochondrial exchange cells (MirC) is provided in Figure 1A. First, a mammalian expression vector used to express an XbaI restriction enzyme fused to a mitochondrial targeting sequence (MTS) was artificially created by cloning the MTS-XbaI sequence into the pCAGGS vector using standard techniques known in the art (Figure 1B). Among the reported mitochondrial import signals (MTS), the inventors utilized the ND4 signal sequence in this study. The resulting expression vector also contained a puromycin resistance gene to enable selection (Figure 1B).

[0346] XbaIR is one of the most potent endonucleases, and the standard sequence of mtDNA named according to the Cambridge reference sequence (CRS) of the human mitochondrial genome has five recognition sites targeted by specific endonucleases (Figure 1D). This was confirmed by co-incubation of isolated mtDNA with an in vitro endonuclease that was digested by XbaIR at multiple sites (Figure 1C). In contrast, NotI digestion of mtDNA showed a single fragment as predicted by the Cambridge reference sequence (CRS) of mitochondrial DNA (Figure 1C).

[0347] The gene transfer protocol of plasmid DNA into cells was optimized using normal human dermal fibroblast (NHDF) cells expressing enhanced green fluorescent protein (EGFP) by using a Nucleofector electroporation-based transfection method. One day after puromycin exposure at 2 μg / ml, an efficacy of over 90% and a survival of over 90% were achieved (Figure 1E).

[0348] To specifically evaluate the effectiveness of the MTS targeting sequence, a plasmid carrying MTS fused with EGFP was prepared by subcloning the EGFP gene instead of the XbaI R gene, and the pCAGGS-MTS-EGFP-PuroR plasmid was prepared (Figure 1F). Normal human dermal fibroblast cells (NHDF) were transfected with the MTS-EGFP expression vector, and the cells were counterstained with TMRM (tetramethylrhodamine, methyl ester), a cell-permeable dye that accumulates in active mitochondria with intact membrane potential (Figure 1G).

[0349] Collectively, these results showed that XbaI can be used to digest mitochondrial DNA, can efficiently transfect cells without affecting cell survival, and that an expression vector containing MTS can effectively target mitochondria.

[0350] (Example II: Endonuclease MTS-XbaI R treatment shows improved degradation of mtDNA compared to the conventional EtBr method) The efficiency and effectiveness of the MTS-XbaIR expression vector compared to the conventional method using ethidium bromide (EtBr) were evaluated according to the scheme shown in Figure 2A. The placental vein endothelial cell line EPC100 with DsRed-labeled mitochondria was cultured in pyruvate-free DMEM (Wako cat# 044-29765) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S). On day 0, the cells were either left untreated ("normal"), transfected with the MTS-XbaIR expression vector ("MTS-XbaIR"), or treated with 50 ng / mL of EtBr. On day 1, the cells were cultured in DMEM containing 10% FBS and 1% P / S supplemented with 100 μg / mL of pyruvate and 50 μg / mL of uridine. Quantitative polymerase chain reaction (qPCR) was performed on days 3 and 5 according to methods known in the art to measure mtDNA compared to the housekeeping gene β-actin (Actb). These results showed that XbaIR decreased the mtDNA copy number to 2715.8141, while EtBr treatment only decreased the mtDNA copy number to 5169.1258, similar to the DNA copy number of 6189.6867 in untreated cells (Figure 2B). The decrease in mtDNA in the endonuclease-treated group was superior to that in the group treated by the conventional method, but this was not a complete deletion, and approximately 30% of the endogenous mtDNA remained (Figure 2B). Cells with this partial decrease in mtDNA were designated as ρ(-) cells.

[0351] Enhanced degradation of endogenous mtDNA in the MTS-XbaIR-treated group compared to the EtBr group was further confirmed by microscopy of DsRed-labeled mitochondria (Figure 2C). The level of decrease was reflected in the remaining healthy mitochondrial capacity estimated by TMRM staining, which was lower in the XbaIR-treated group than in the group treated by the conventional method (Figure 2C). Furthermore, FACS analysis of NHDF cells showed a decrease in TMRM after treatment with XbaIR (Figure 2D).

[0352] The dynamics of XbaIR expression after plasmid gene transfer were examined by qPCR. On day 3, the expression reached a peak and then decreased to 0 by day 7 (Figure 2E). The same dynamics as XbaIR were confirmed for other genes of interest (e.g., GFP) (Figure 2E). Fluorescence images confirmed the enrichment of GFP that occurred in cells transfected with the MTS-EGFP-PuroR plasmid after puromycin selection compared to before transfection (Figures 2F and 2G). The fraction of GFP-positive cells increased significantly to almost 100% by exposing cells to puromycin for 1 day (Figure 2F).

[0353] These results showed that the decrease in mtDNA copy number in the XbaI endonuclease-treated group was superior to that in the group treated with the conventional EtBr method and did not completely delete all of the endogenous mtDNA. Furthermore, short-term selection with puromycin enabled significant enrichment of cells expressing the MTS construct.

[0354] (Example III: Partial degradation of endogenous mitochondria using the MTS-XbaIR construct in recipient cells enabled mitochondrial exchange from exogenous donor cells) To evaluate whether exogenous mitochondria from healthy donor cells can be transferred into recipient cells by XbaI-mediated deletion of mtDNA, NHDF cells were transfected with the MTS-GFP or MTS-XbaIR plasmid and selected with puromycin 48 hours later. Six days after transfection, isolated mitochondria from a human cell line derived from uterine endothelium labeled with DsRed (designated EPC100) were transferred into the donor cells. The protocol scheme is shown in Figure 3A.

[0355] After transfection with MTS-GFP or MTS-XbaI and selection with puromycin, mitochondrial content was evaluated by TMRM staining. As shown in Figure 3B, MTS-GFP transfected cells showed strong staining of TMRM, indicating high levels of mitochondria in NHDF cells. In contrast, MTS-XbaI transfected cells (ρ-) showed a decrease in mitochondrial volume when visualized by TMRM staining (Figure 3B).

[0356] The decrease in mitochondrial DNA was further confirmed by quantifying the number of mitochondrial DNA copies by qPCR of 12S-rRNA normalized to nuclear β-actin (Actb) (Figure 3C). On day 6, a significant decrease in mitochondrial DNA from MTS-XbaI transfected cells (ρ-) was seen compared to NHDF control cells transfected with MTS-GFP (Figure 3C). The significant decrease in mitochondrial DNA in ρ- cells persisted throughout the assay period and stopped on day 12. Specifically, the copy number dropped to about 1 / 3 of the original copy number on day 6 and further decreased to about 1 / 4 in ρ- cells on day 12 (Figure 3C).

[0357] Mitochondria were isolated from DsRed-Mt EMC by differential centrifugation. Briefly, cells were harvested from culture dishes containing homogenization buffer [HB; 20 mM HEPES-KOH (pH 7.4), 220 mM mannitol, and 70 mM sucrose] containing protease inhibitor mixture (Sigma-Aldrich, St. Louis, Missouri, USA). The cell pellet was resuspended in HB and incubated on ice for 5 minutes. The cells were disrupted by 10 strokes of a 27-gauge needle on ice. The homogenate was centrifuged twice (400 g, 4 °C, 5 minutes) to remove unbroken cells. Mitochondria were recovered by centrifugation (6000 g, 4 °C, 5 minutes) and resuspended in HB. The amount of isolated mitochondria was expressed as protein concentration using a Bio-Rad protein assay kit (Bio-Rad, Richmond, CA, USA). Mitochondrial transfer was performed by co-incubating the isolated mitochondria with cells in 2 ml of standard medium at 37 °C under 5% CO2 for 24 hours. Importantly, co-incubation of isolated mitochondria on day 12 with ρ(-) cells resulted in a significant increase in mtDNA copy number similar to that of control NHDF cells (Figure 3C).

[0358] Consistent with the results shown in FIGS. 2C and 2D, ρ(−) cells showed a decrease in mitochondrial content after MTS-XbaI transfection, as measured by visualization of TMRM. Importantly, the mitochondrial decrease could be rescued by contacting ρ(−) cells with isolated exogenous mitochondria, as shown by uptake of DsRed-labeled isolated mitochondria (FIGS. 3D and 3E). In contrast, co-culture 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 exogenous mitochondria aggregated around the cells and formed aggregates but were not internalized (FIG. 3D, lower panel). Although a small part of the mitochondria was engulfed, most of it was outside the cells with intact endogenous mitochondria, and the intensity of DsRed was maintained during this period. The DsRed aggregates became smaller and fewer, and the intensity of DsRed decreased, suggesting that after exogenous mitochondria were collected on the cell membrane, they were engulfed and the membrane portion of the mitochondria was rapidly digested.

[0359] Comparison with existing methods showed that the endonuclease method of the present invention is more effective in generating mitochondrial exchange cells with exogenous mitochondria (Figure 3F). For example, the endonuclease method of the present invention was compared with (1) the additional mitochondrial transfer method described in the previous studies of the present inventors (see, for example, the literature of Kitani, T. et al., J Cell Mol Med (2014) 18, 1694) or (2) a recently reported method using spinoculation of isolated mitochondria and metabolically healthy cells (see, for example, the literature of Kim, M. J. et al., Sci Rep 8, 3330, (2018)) (Figure 3F). None of the previously reported methods (i.e., mitochondrial addition; "Mt addition" or spinoculation at 800×g or 1500×g) showed significant transfer of exogenous mitochondria as measured by FACS analysis of DsRed-labeled exogenous mitochondria (Figure 3F). In contrast, the novel method provided herein that utilizes partial degradation of endogenous mitochondria mediated by MTS-XbaI followed by non-invasive transfer or exogenous mitochondria (Mt EPC100) showed a significant DsRed positive rate and increased mean fluorescence intensity after transfer of exogenous mitochondria (Figure 3F, upper right graph, rightmost line).

[0360] In previously established methods used in mitochondrial biology, ρ(0) cells, which are cells having a complete deletion of mitochondria, were utilized (see, for example, U.S. Patent Application No. 12 / 747,771, filed September 23, 2010, and published as US 2011-0008778 A1, which is hereby incorporated by reference in its entirety). However, ρ(0) cells were unable to engulf exogenous mitochondria (FIGS. 3G - 3I). Based on these results presented herein, a hypothesis was proposed that ρ(0) cells are unable to engulf exogenous mitochondria because they lack the energy required to undergo macropinocytosis. To test this hypothesis, the inventors designed genetically modified cells that generate ρ(0) cells by exposure to antimycin, which induces mitophagy, and examined the level of mitochondrial import. The results showed that the uptake of exogenous mitochondria does not occur in cells having a complete deletion of mitochondria (FIGS. 3G - 3I). Therefore, these results suggested that a partial deletion of existing mtDNA, rather than a complete deletion, is an important factor in the macropinocytosis of exogenous and extracellular mitochondria.

[0361] Furthermore, the uptake of DsRed-labeled exogenous mitochondria was monitored in ρ(−) cells treated or untreated with exogenous mitochondria, untransfected cells (Additional Mt), or cells treated with a mock MTS-GFP plasmid. The fluorescence intensity of DsRed was quantified every 24 hours using NIH Image software. The relative values to the initial intensity were shown as a bar graph (FIG. 3J). This quantification showed that simple additional mitochondrial co-incubation and mitochondrial co-incubation with mock transfectants increased the intensity at the same rate due to the aggregation of isolated mitochondria, indicating accumulation rather than uptake of Ds-red-labeled mitochondria. In contrast, the intensity of ρ(−) cells co-incubated with isolated exogenous mitochondria gradually decreased over time, suggesting that the engulfed mitochondria were degraded.

[0362] These results show that the MTS-XbaI expression vector can generate ρ(-) cells with partial deletions of endogenous mitochondria, and that mitochondrial content can be rescued by transferring isolated exogenous mitochondria from donor cells. As described herein, the methods of the present invention result in an improvement in the efficiency of mitochondrial transfer as compared to previously described methods, for example, methods implemented in combination with centrifugation, or simple "addition" of mitochondria without concomitant partial reduction of endogenous mtDNA. However, mitochondrial transfer cannot be performed in cells with complete degradation of endogenous mitochondria (ρ(0) cells), indicating that uptake of exogenous mitochondria is likely to require energy.

[0363] (Example IV: Isolated exogenous mitochondria fuse with endogenous mitochondria to transfer donor mtDNA) To further clarify how mitochondrial transfer of intact mitochondria occurs, the fate of the mitochondria transferred into cells was examined separately in the outer membrane, inner membrane, and nucleoid. In certain situations, transient inter-mitochondrial fusion events have been observed, where two mitochondria come into proximity and exchange soluble intermembrane space proteins and matrix proteins, and then separate again, maintaining their original form (see, for example, Liu X et al., EMBO J. 2009;28(20):3074-3089; Huang X et al., Proc Natl Acad Sci U S A. 2013;110(8):2846-2851). Therefore, transient inter-mitochondrial fusion events were analyzed under the conditions described herein.

[0364] Isolated mitochondria derived from EPC100 donor cells were labeled with DsRed, and recipient cells with mitochondria marked with EGFP were used. The scheme of the protocol utilized is shown in Figure 4A. From microscopic images of the transient contact between the donor and endogenous mitochondria, it became clear that extensive mitochondrial fusion was not observed (Figures 4B and 4C). Most of the donor mitochondria existed separately from the endogenous mitochondria. Furthermore, a few transient fusion images were observed, and afterwards, the donor mitochondria appeared to escape before it finally disappeared (Figure 4C).

[0365] Mitochondrial transfer was carried out according to the protocol shown in Figure 4F. Briefly, the mitochondria of recipient NHDF cells were marked with DsRed (Figure 4D), and the mitochondria derived from donor EPC100 cells were marked with TFAM, which binds to mtDNA and enables the tracking of mitochondria (Figure 4E). The recipient NHDF cells were transfected with the pCAGGS-MTS-XbaIR-P2A-PuroR expression vector and, on the second day, selected with puromycin for 24 hours. On the sixth day, mitochondrial transfer from TFAM-GFP-labeled mitochondria derived from EPC100 donor cells was carried out. Then, on the eighth day, the cells were imaged. Microscopic examination of the mitochondrial transfer revealed that the donor nucleoids were deposited in the existing mitochondrial matrix (Figure 4G). The exogenous mitochondria transiently contacted the recipient mitochondria, suggesting that the mitochondrial nucleoids containing TFAM were transferred into the existing mitochondria by the transient contact.

[0366] These results indicate that donor mitochondria are transferred into the mitochondrial matrix of recipient cells and become dominant upon the decline of existing mitochondria. Furthermore, according to these experiments, almost all isolated mitochondria were engulfed. On the other hand, the addition-type mitochondrial transfer transfectants and mock transfectants did not show strict engulfment but aggregated most of these exogenous mitochondria on the cell surface.

[0367] In summary, the results of Examples III and IV show that ρ(-) cells degrade the engulfed mitochondria (Figure 3J), and exogenous mitochondria temporarily contact the existing mitochondria (Figures 4B - 4C), while exogenous mtDNA with TFAM exists in the existing mitochondria (Figure 4G).

[0368] Therefore, the hypothesis is put forward that exogenous mitochondria interact with endogenous mitochondria for a short time and can transport mtDNA during this short contact. In that case, the exogenous mitochondrial membrane complex can be decomposed in the cytosol to provide components for mitochondrial reconstruction. The mitochondria of recipient cells that receive exogenous mitochondria can gradually reconstruct the mitochondrial membrane complex and show functional recovery.

[0369] (Example V: Increase in exogenous mitochondria was detected after transfer of isolated exogenous mitochondria by SNP assay) To evaluate the origin of mtDNA after mtDNA exchange, the different nucleotides identified between NHDF and EPC100 were used by sequencing hypervariable regions 1 and 2 (Figures 5A and 5B). While NHDF retains an A at position 16362 of the CRS, EPC100 harbors a mutation at the same position that results in a change from A to G (Figure 5B). Importantly, evaluation of the mitochondrial exchange ρ(−) cells (NHDFρ(−)Mt) showed the presence of both the original nucleotide in the minor wave and the exogenous nucleotide G in the major wave, indicating that the cells are heteroplasmic (Figure 5B, lower panel).

[0370] Heteroplasmy in mitochondrial exchange NHDF was further evaluated by a single nucleotide polymorphism assay to detect differences between recipient NHDF and donor EPC100 (Figure 5C). The hmt16318-F primer

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[0371] These results indicate that the methods provided herein for the exchange of mtDNA with exogenous mitochondria and / or exogenous mtDNA are completely novel and represent an improvement over existing techniques. As described above, the provided method shows that exogenous mtDNA can be made the dominant mtDNA as a result of mitochondrial import after degradation of endogenous mitochondria mediated by MTS-XbaI.

[0372] (Example VI: The exchanged mitochondria generate energy and MirC shows restoration of a phenotype similar to that of normal target cells) Whether the exchanged mitochondria function to generate energy was examined by using an Oroboros O2k according to the manufacturer's instructions. Representative oxygen consumption rate curves for negative control cells, ρ(−) cells, and mitochondria-exchanged cells were generated, and then respiratory fluxes and control ratios were calculated (Figures 6A and 6B). Basal respiration, the maximal capacity of the electron transport system, and ATP production (free routine activity) all showed similar kinetics, indicating that these parameters were significantly decreased in ρ(−) cells (Figure 6B, upper row). Importantly, these parameters recovered to their original values in mitochondria-exchanged cells (Figures 6A and 6B). Non-mitochondrial ATP production (ROX) was upregulated, and the coupling ratio was downregulated in ρ(−) cells (Figure 6B, lower row). The energy supply mechanism in ρ(−) cells shifted from mitochondrial ATP generation to glycolysis, and these changes reverted after mtDNA exchange (Figure 6B, upper right).

[0373] Furthermore, the phenotypic recovery of mitochondria-exchanged cells (MirC) was demonstrated by their proliferative capacity (Figure 6C). Specifically, while ρ(−) cells showed low proliferative capacity, MirC recovered to a level close to that of control cells by 6 to 12 days (Figure 6C, right).

[0374] These results indicate that this method provides mtDNA exchange with clinically applicable materials and generates cells with functional mitochondria that enable phenotypic recovery of mitochondria-exchanged cells (MirC).

[0375] (Example VII: Inhibition of mTOR by Rapamycin Enhances Macropinocytosis of Exogenous Mitochondria in ρ(−) Cells) To determine a method for increasing the ability of cells to receive MirC, we investigated the mechanism that regulates the macropinocytosis of exogenous mitochondria. Since ρ(−) cells consume ATP as a result of mitochondrial decline, a hypothesis was proposed that the intracellular energy state of ρ(−) cells is similar to a starved state. In this regard, two molecular pathways: the mammalian targets of rapamycin complex 1 (mTORC1) and AMP-activated protein kinase (AMPK) were investigated. mTORC1 is an essential sensor of amino acids, energy, oxygen, and growth factors, and is an important regulator of protein, lipid, and nucleotide synthesis. AMPK is a sensor of AMP levels, and its activation leads to autophagy, mitochondrial biogenesis, glycolysis, and lipolysis. Both pathways are involved in the uptake of extracellular nutrients.

[0376] As shown in Fig. 6D, to investigate the mechanism of macropinocytosis in ρ(−) cells, starvation was used to stimulate AMPK / mTORC1, while the "drugs" palmitic acid and rapamycin were used to specifically stimulate mTORC1 activation and inhibit mTORC1, respectively. Rapamycin was added to the culture medium at a concentration of 50 ng / ml for 24 hours, and the cells were exposed to a serum-free glucose and essential amino acid-free medium for 1 hour to stimulate starvation. Palmitic acid (PA) has been reported to activate mTORC1 at a concentration of 200 μM in vivo, but titration of PA for cultured fibroblasts showed that a concentration of 50 μM and a duration of 24 hours were optimal based on cell viability. The ratio of phosphorylated AMPK to AMPK and the ratio of phosphorylated p70 S6 kinase to p70 S6 kinase (which is a downstream target of mTORC1) were examined by using capillary electrophoresis Wes™ (Protein Simple).

[0377] Treatment with PA or rapamycin did not significantly activate the AMPK pathway in ρ(−) cells (Figs. 6G and 6H), but the mTORC1 pathway was dramatically suppressed in ρ(−) cells at levels similar to those of starvation and rapamycin, as measured by pS6 / S6 (Figs. 6E–6F). These results indicated that mTORC1 corresponds to an important target of mitochondrial macropinocytosis in ρ(−) cells.

[0378] Next, the inventors examined the effects of rapamycin and palmitic acid during mitochondrial engulfment by treating cells with rapamycin or palmitic acid simultaneously during mitochondrial co-culture. The protocol scheme is shown in Fig. 6I. Briefly, 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 of ρ(−) cells expressing MTS-XbaI was performed 48 hours later. On day 6, the transfer of isolated mitochondria marked with DsRed from EPC100 cells was performed. On day 8, FACS analysis was performed to detect donor mitochondria by measuring DsRed expression in NHDF recipient cells.

[0379] As shown in Figs. 6I–6L, rapamycin treatment significantly enhanced the engulfment of isolated exogenous mitochondria labeled with DsRed, whereas palmitic acid clearly suppressed it. These experiments were repeated four times, and the positive rates were summarized, showing a statistically significant difference between rapamycin and palmitic acid for ρ(−) cells (Figs. 6I and 6K). In particular, there was no significant difference between mock transfection and additive mitochondrial transfer. Furthermore, these results indicated that the effect of regulating mTORC1 activity only affects mitochondrial transfer in ρ(−) cells and has no effect on “additive” or mock-transfected cells, indicating that this mechanism of transferring exogenous mitochondria is specific to the invention provided herein.

[0380] These results indicate that the activation of mTORC1 by rapamycin upon mitochondrial translocation can enhance mitochondrial macropinocytosis. Furthermore, these methods indicate that the efficiency of macropinocytosis for MirC production can be increased using rapamycin, a clinically available drug.

[0381] (Example VIII: mtDNA exchange with heteroplasmy reversal in fibroblasts derived from patients with Leigh syndrome) To investigate whether mitochondrial disease cells can be corrected by using in vitro mtDNA exchange techniques, primary fibroblasts (7SP) derived from a patient diagnosed with Leigh syndrome having the mtDNA T10158C mutation were used as recipient cells (Figure 7A). The same protocol described previously for NHDF cells was applied to 7SP fibroblasts. DNA sequencing of the mtDNA of the EPC100 donor mitochondria at nucleotide 10158 was confirmed to be T (Figure 7B, top), whereas 7SP fibroblasts had a mosaic of a major wave of T and a minor wave of C, indicating heteroplasmy (Figure 7B, bottom).

[0382] The dynamics of mtDNA content in 7S fibroblasts were almost the same as those of NHDF after mitochondrial exchange (Figures 7C and 7J). Time-lapse imaging revealed that ρ(−) 7SP fibroblasts exhibited the same behavior as ρ(−) NHDF cells. In particular, the aggregates of exogenous mitochondria accumulated on the surface of ρ(−) cells became smaller and fewer over time, and the intensity of DsRed in the cytosol rapidly decreased, suggesting efficient uptake into the cytosol and digestion in the cytosol, which was consistent with the results obtained using ρ(−) NHDF cells.

[0383] Importantly, the number of mtDNA copies after mitochondrial exchange recovered to the same value as that of the original 7S fibroblasts on day 12 (Figure 7D). On the other hand, the mock transfectants (additive mitochondrial transfer) of 7SP fibroblasts were unable to even increase the number of mtDNA copies, despite being in the same co-culture as the isolated mitochondria under the same conditions, indicating low transfer of exogenous mitochondria (Figure 7D, light gray bars).

[0384] By sequencing a mitochondrial genome fragment containing 10,158 nucleotides, we examined whether the mitochondria of 7S fibroblasts contained exogenous and healthy mtDNA. As shown in Figure 7E, the mtDNA sequence of 7SP cells changed from having mostly mutant heteroplasmy at the 10,158 nucleotide position (large wave of C and small wave of T) to having mostly wild-type mtDNA (large wave of T and small wave of C) in the recipient 7SPρ(−) cells after mitochondrial exchange (Figure 7E, bottom).

[0385] To provide quantitative information, a single nucleotide polymorphism (SNP) assay was performed to estimate the heteroplasmy brought about using this technique. The hmt10085-F primer

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[0386] In summary, these results show that the method for producing mitochondrial exchange cells described herein that partially reduces endogenous mitochondria using MTS-XbaI can be effectively used in cells from a subject having a mitochondrial disease or disorder to improve the heteroplasmy level and reduce the amount of mutant mtDNA.

[0387] (Example IX: mtDNA exchange in fibroblasts derived from a patient with Leigh syndrome results in improvement of cell lifespan and cell metabolism) The functional activity of the mitochondrial exchange 7SP fibroblasts was evaluated. As shown in Figures 8A and 8B, the proliferation of the mitochondrial exchange 7SP fibroblast (ρ(-)Mt) cells was able to recover to a level equivalent to that of the original 7SP fibroblast cells around the 12th day.

[0388] Furthermore, while mitochondrial replacement 7SP fibroblast (ρ(-)Mt) cells showed a dramatic extension of lifespan up to approximately 63 population doubling levels (PDL), the doubling time exceeded 120 hours, which is the threshold for growth arrest (Figure 8C). Cells that received mtDNA replacement at approximately 8 PDL and reconstructed cells with healthy mtDNA were able to continue dividing beyond 55 PDL, which is considered to be the number of times a normal human cell population divides before cell division stops (i.e., the Hayflick limit). In contrast, untreated 7S fibroblasts became senescent at 25 PDL (Figure 8C). Therefore, this experiment demonstrated that mtDNA replacement has a significant impact on the proliferation and lifespan of mitochondrial disease cells. Considering that aging increases with age and cancer cells often accompany mitochondrial dysfunction, this method may provide a decisive clue to rejuvenation and may provide a basis for new strategies for cancer therapy and the treatment of other age-related diseases.

[0389] The functional effect of mitochondrial import in 7S fibroblasts was further evaluated by measuring cell size (Figure 8D). A mutation in the 7S fibroblasts in the coding sequence of the ND4 gene of complex I in the respiratory chain resulted in a disorder of complex I that transfers electrons in combination with its function of pumping protons from the matrix to the intermembrane space. As a result, glycolysis becomes dominant over mitochondrial ATP production in 7S fibroblasts and, despite its damage and low function, results in a compensatory adaptation in which the cell size becomes larger to contain more mitochondria (Figure 8D). Compared to PDL 15 (black solid line), at PDL 25, the diameter of 7S fibroblasts was approximately 1.5 times larger than that of NHDF, and the increase in cell size doubled at PDL 35 and ultimately increased until its size was approximately 3 - 8 times larger (Figure 8D, left).

[0390] Consistent with the functional recovery of 7SP cells after mtDNA exchange, the significant increase in cell size observed in 7SP fibroblasts at early PDL was inhibited after mitochondrial exchange (Figure 8D, right). Furthermore, the size of mitochondrial exchange 7SP cells that received exogenous mitochondria at PDL 8 was maintained until the 50th PDL (Figure 8D, right). Additionally, the concentration of citrate synthase (CS) was two-fold higher in 7SP fibroblasts than in NHDF cells by the 10th PDL, which is consistent with the increased increase in size of 7SP fibroblasts (data not shown).

[0391] To confirm that the observed improvement in cell function after mitochondrial exchange was not due to contamination by other cell types, a short tandem repeat (STR) assay that can clearly identify cells with various origins was performed (Figure 8E). Importantly, the STR pattern in mitochondrial exchange cells at different time points was completely identical to the STR pattern of the original 7SP fibroblasts (Figure 8E), indicating no contamination. Furthermore, RT-PCR revealed that the transfer of exogenous mitochondria from cells expressing telomerase and E6 did not transform primary fibroblasts into cancer cells (Figure 8F).

[0392] Collectively, these results demonstrated that mitochondrial transfer of exogenous isolated mitochondria with wild-type mtDNA into 7SP cells derived from patients with Leigh syndrome increased the lifespan of 7SP cells and improved cell function. Importantly, this transfer did not transform mitochondrial exchange 7SP cells into cancer cells.

[0393] (Example X: Transfer of exogenous mitochondria into fibroblasts derived from patients with Leigh syndrome generates functional mitochondria) By using the Oroboros O2k and analyzing the respiratory function of cells, the functional effect of mitochondrial exchange in 7SP fibroblasts was further evaluated (Figure 9A). By quantifying the results, it was shown that basal respiration and ATP production (free routine activity) continued to decrease from the 10th passage to the 20th passage after the generation of mitochondrial exchange cells, and the maximum capacity of the electron transport system maintained the original level of 7SP fibroblasts (Figure 9B). After the transfer of exogenous mitochondria, by the 30th passage, all three indicators of respiratory function (normal, ETS, and free routine activity) increased and even exceeded the level of the original cells (Figure 9B). These results indicated that there was a slight delay in reconstructing the electron transport system with healthy and non-mutated complex I after mtDNA exchange. Proton leakage showed the same dynamics as the dynamics of non-mitochondrial ATP production, which was steadily improved from the initial stage (Figure 9B).

[0394] These results indicated that the transfer of exogenous mitochondria into fibroblasts derived from patients with mitochondrial diseases or disorders could generate functional mitochondria.

[0395] (Example XI: Transfer of exogenous mitochondria can eliminate chronic and persistent reactive oxygen species (ROS) generation) To characterize the properties of MirC derived from 7SP fibroblasts, both reperfusion and starvation models under culture conditions were used for MirC derived from 7SP fibroblasts, the original 7SP fibroblasts, and NHDF as a control. These stress conditions induced apoptosis in cultured cells, and the degree could be quantified by annexin V as an early marker and propidium iodide (PI) as a late marker. Among environmental disorders, reperfusion injury mainly contributes to mitochondrial dysfunction. Cells with a predisposition to mitochondrial dysfunction due to mtDNA mutations are more vulnerable to reperfusion injury than healthy cells.

[0396] Cells at 1×10 per well 5Cells were seeded in 6-well plates. The next day, 600 μM H2O2 (FUJIFILM Wako Pure Chemical) was added to the cells for the reperfusion model, or serum-free and essential amino acid-free ("-EAA") DMEM (FUJIFILM Wako Pure Chemical) was used as the culture medium for the starvation model. After 3 hours of H2O2 or 48 hours of starvation treatment, the cells were washed with PBS and collected in centrifuge tubes. Annexin V-FITC and PI solutions were added to the cells, protected from light, and allowed to react at room temperature for 30 minutes. Then, the cells were immediately subjected to FCM analysis using laser lines at 488 and 561 nm. Fluorescence data were collected using SH800 (Sony). Flow cytometry files were analyzed by using FlowJo software (TreeStar).

[0397] These results showed that 7SP cells derived from subjects with Leigh syndrome were extremely susceptible to both forms of stress (i.e., H2O2 and starvation). As shown in FIGS. 10A to 10D, 7SP cells treated with H2O2 showed significant increases in both early apoptosis and late apoptosis. In the reperfusion model (H2O2), NHDF did not show significant damage in the process of apoptosis based on Annexin V and PI staining (FIGS. 10B to 10D). However, this mild reperfusion stress induced apoptosis in 7SP fibroblasts. In contrast, the positive rates of both Annexin V and PI in MirC derived from 7SP fibroblasts were significantly lower than those in parental 7SP fibroblasts and were close to the level of NHDF cells (FIGS. 10B to 10D). Importantly, there was no significant difference between MirC derived from 7SP fibroblasts and NHDF, suggesting that MirC regained the ability to withstand this mild reperfusion injury.

[0398] A similar trend was observed using a starvation model (Figs. 10E - 10H). More apoptosis was shown in 7SP fibroblasts in both the early and late stages, while MirC derived from 7SP fibroblasts showed basal apoptosis levels similar to those of NHDF, which were significantly lower than those of the original 7SP fibroblasts (Figs. 10F - 10H). These results further support that the mitochondrial exchange method of the present invention improves the functional recovery of recipient cells.

[0399] These results showed that the transfer of exogenous mitochondria from healthy cells to cells with mutant mtDNA can improve the function of recipient cells.

[0400] (Example XII: Transfer of exogenous mitochondria to recipient cells reversed the early - stage senescence - associated secretory phenotype (SASP)) This example shows that the transfer of exogenous mitochondria to recipient cells reversed the early - stage senescence - associated secretory phenotype (SASP). The SASP, consisting of inflammatory cytokines, growth factors, and proteases, is a characteristic feature of senescent cells.

[0401] To determine whether the transfer of exogenous mitochondria to senescent cells can reverse the SASP, the expression levels of representative SASP cytokines IL - 6 and IL - 8, chemokine CXCL - 1, and growth factor ICAM1 were quantitatively measured at the transcriptional level in NHDF, 7SP fibroblasts, and MirC cells derived from 7SP fibroblasts, which had a similar PDL of approximately 15 - 20 (Fig. 11). IL - 6 was significantly higher in 7SP fibroblasts than in NHDF and MirC cells derived from 7SP fibroblasts, while the other three factors showed no significant difference among these cells. At this PDL, 7SP fibroblasts did not show a typical SASP, but only showed higher IL - 6 expression, suggesting the early stage of senescence. Importantly, the process of early - stage senescence at this PDL in MirC cells derived from 7SP fibroblasts could be reversed.

[0402] In summary, these data indicate that mitochondrial exchange can not only treat mitochondrial diseases associated with mtDNA mutations, but also rejuvenate aged cells such as those involved in various diseases including neurodegenerative, cardiovascular, metabolic, and autoimmune diseases, and even cancer.

[0403] (Example XIII: iPS cells generated from mtDNA-exchanged fibroblasts) To determine whether induced pluripotent stem cells (iPSCs) can be generated using cells derived from patients with long deletions in mtDNA, the inventors attempted to construct iPSCs from 7SP fibroblasts using a standard method using Sendai virus carrying Oct3 / 4, Klf4, Sox2, and c-Myc (OKSM), which are well-functioning in NHDF. The schematic of the protocol design is shown in Figure 12A.

[0404] Alkaline phosphatase staining (AP staining) for detecting early-stage iPSC colonies showed that colonies derived from the original 7SP fibroblasts appeared to have a fragile appearance, while colonies derived from mitochondrial exchange 7SP fibroblasts were robust on day 21 (Figure 12B). In contrast, ρ(-) 7SP fibroblasts that did not receive mitochondrial exchange did not generate colonies (Figure 12B). When measured by AP staining, several lines of iPS cells could be generated from mitochondrial exchange 7S fibroblasts (Figures 12C and 12D). The iPSC clones were stable in culture and showed similar morphology among independent colonies (Figure 12E). Immunohistochemical staining confirmed the expression of human pluripotent stem cell markers SOX2, OCT3 / 4, NANOG, SSEA4, TRA1-81, and TRA1-60 on colonies derived from mitochondrial exchange 7SP fibroblasts overexpressing OKSM (Figure 12F).

[0405] The iPSCs produced by the method described in this specification were further compared with the commercially available KYOU-DXR0109B human induced pluripotent stem (IPS) cells [201B7]. Importantly, the mitochondrial exchange 7SP fibroblasts showed the same level of efficiency as healthy fibroblasts with respect to iPS production. Furthermore, in agreement with previous studies, qPCR of 12S-rRNA normalized to nuclear β-actin showed that the iPS cells produced by mitochondrial exchange 7SP fibroblasts had half the mtDNA content compared to the control, and the mtDNA levels were shown to be similar to those of the 201B7 iPSC standard (Figure 12G).

[0406] Furthermore, the hmt10158 heteroplasmy level was less than 10% in the iPSCs produced (Figure 12H). Quantification of the absolute mtDNA copy number confirmed a decrease in the level of mtDNA and a decrease in mutant mtDNA (Figure 12I).

[0407] These results indicate that iPSCs can be produced using the mitochondrial exchange technology provided in this specification, and that throughout this procedure, only materials applicable to medical treatment are used, so it may be applicable in the clinical field.

[0408] (Example XIV: Mitochondrial exchange of donor cell-derived mitochondria changes the lifespan cells of recipient cells) This example shows that mtDNA exchange can change the lifespan of recipient cells. To test the hypothesis that mitochondrial exchange can rejuvenate aged cells, two models were estimated with respect to cell cycle capabilities, such as doubling time and PDL at growth arrest.

[0409] Models were designed using NHDF and TIG1 embryonic lung cells with early PDL (approx. 5 - 10, called "young") and late PDL (approx. 40 - 45, called "old"). One model involved young cells exchanged with mitochondria from old cells, named "O2Y", and another model involved old cells exchanged with mitochondria from young cells, named "Y2O" (Figure 13A).

[0410] The degree of mtDNA exchange was evaluated by TaqMan SNP genotyping assay based on the single nucleotide difference in mtDNA at position 16145 between NHDF and TIG1, which are A and G respectively (Figure 13B). MirC derived from NHDF clearly showed that more than 90% of the endogenous mtDNA (hmt16145 - A) was exchanged with TIG1 - derived mtDNA (hmt16145 - G) (Figure 13C). The small percentage of hmt16145 - A detected in parental TIG1 cells was considered background error (Figure 13C).

[0411] Furthermore, the Y2O model clearly showed a recovery of the lifespan of old cells to about 65 PDL (Figure 13D). Old control cells and mock transfectants showed growth arrest at 55 PDL, which is consistent with the Hayflick limit. On the other hand, O2Y showed a decrease in the lifespan of young cells at about 45 PDL (Figure 13E). The approximately 10 PDL difference in both models may be due to exogenous mtDNA. These results indicate that the transfer of exogenous mitochondria from young cells to old cells can rejuvenate the cells.

[0412] (Example XV: Optimization of Mitochondria - exchanged Cells (MirC) Derived from Human Primary T Cells Using mRNA Transfection) This example describes the generation of mitochondria - exchanged cells (MirC) derived from human primary T cells by using mRNA transfection.

[0413] Prior to the experiment, the use of human primary T cells was approved by the institutional ethics committee in the inventors' facility. Peripheral blood was collected from healthy volunteers and centrifuged at 400 g for 35 minutes at 20 °C using Percoll with a specific gravity of 1.077 to separate lymphocytes. The isolated lymphocytes at 1×10 cells per milliliter were seeded into 96-well flat-bottom plates coated with anti-CD3 and anti-CD28 antibodies. The plates were prepared by incubating overnight with 5 μg / ml anti-CD3 and 1 μg / ml anti-CD28 and pre-warmed at 37 °C for 2 hours before seeding. The day after seeding, IL-7 and IL-15 were added to the medium at concentrations of 20 μg / ml and 10 μg / ml, respectively. The medium was changed every 3 days using IL-7 and IL-15 at the same concentrations as the initial addition. 6 Transfection was performed using a MaxCyte electroporator meeting GMP / GCP standards according to the manufacturer's protocol. mRNA was prepared according to the manufacturer's protocol of the mMESSAGE mMACHINE T7 Ultra kit (Thermo Fisher) with slight modifications. Briefly, to minimize the possibility of RNase contamination as much as possible, the DNA template for mRNA was prepared from a plasmid carrying the DNA sequence without purifying the fragments after endonuclease digestion (Figure 14A).

[0414]

[0415] The results showed that the unpurified DNA template for EGFP mRNA production resulted in nearly 100% gene transfer efficiency with high expression and high viability 24 hours after gene transfection (Figures 14B and 14C). Due to the high transfection efficiency, antibiotic selection was not required using this method. Furthermore, transfection of MTS-XbaIR resulted in a decrease in mitochondrial membrane potential, which may have been due to a decrease in endogenous mtDNA (Figure 14D).

[0416] ​To determine the optimal protocol regarding the timing of co-incubation of isolated mitochondria, fluorescence images of human primary T cells that received GFP mRNA by electroporation using MaxCyte ATX were taken over 8 days as shown (Figure 14E). Fluorescence images of electroporated control cells in which the cells were transfected with the GFP plasmid (Figure 14F, upper panel) showed dynamics similar to those seen in the fluorescence images of fibroblasts. The expression reached a peak on day 2 and disappeared by day 8. In contrast, cells that received MTS-GFP mRNA (Figure 14F, lower panel) showed higher expression within 4 hours after electroporation and earlier disappearance on day 6 compared to those seen in cells into which the plasmid was introduced.

[0417] The protein expression of GFP in cells that received MTS-GFP mRNA was evaluated by Western blot analysis using capillary electrophoresis. As shown by Western blot (Figure 14H) and quantified in Figure 14G, peak expression occurred on day 4 and the expression was lost by day 6. The dynamics of the XbaI R transcript level were quantified by qPCR, which revealed that the transcript expression of the endonuclease reached a complete maximum 4 hours after gene transfer (Figure 14I). The XbaI R transcript level decreased rapidly by day 2 and was negligible by day 6 (Figure 14I). By quantifying 12S rRNA, the mitochondrial content was estimated (Figure 14J), and it was shown that the mitochondria decreased to about 30% by day 2 and were maintained at less than 20% throughout the experimental period.

[0418] In summary, these results indicate that mRNA transfection of endonucleases such as XbaI fused with MTS can efficiently degrade the host mtDNA and can be used to generate mitochondrial exchange cells (MirC) derived from human primary T cells.

[0419] (Example XVI: Generation of Mitochondria-Exchange Cells (MirC) Derived from Human Primary T Cells Using mRNA Transfection) After determining the optimal time point for mitochondrial transfer in the human primary T cells of Example XV, mitochondrial co-incubation was performed on day 7 to prevent digestion of exogenous mtDNA by the remaining endonucleases. The scheme of the MirC protocol for human primary T cells is shown in Figure 15A.

[0420] To determine the heteroplasmy of mtDNA in recipient human primary T cells after mitochondrial exchange, the differences in mtDNA between donor mitochondria and recipient cells were determined by TaqMan SNP genotyping assay. Sequencing of the D-loop of mtDNA in normal human primary T cells and EPC100 (mitochondrial donor cells) showed differences at two nucleotide positions (mtDNA at nucleotides 218 and 224), which were C / C and T / T for T cells and EPC100 cells, respectively (Figure 15B). To generate a standard curve for the TaqMan SNP genotyping assay, a fragment of the variable region containing nucleotides 218 and 224 of mtDNA was subcloned into pBluescript SK(-). Primers and probes were designed to map the polymorphic nucleotides onto the human reference sequence and amplify and target the desired region of the D-loop (in this case, the probe had FAM and VIC fluorophores) (Figure 15C). qPCR was performed using TaqMan polymerase with 5'-exonuclease activity to determine the threshold cycle (Ct value), which was fitted to a standard curve generated using several different copy numbers of the above plasmid for each sequence. After somatic mitochondrial exchange, the origin of EPC100 mtDNA predominated in human T cells on both day 7 and day 12, whereas mock transfectants electroporated without genetic material and incubated with mitochondria isolated using the same protocol as MirC showed exogenous mtDNA origin at less than 10% on day 7 and at background levels on day 12 (Figure 15D). This indicated that MTS-XbaIR mRNA promoted efficient mitochondrial import in human primary T cells.

[0421] Next, to evaluate the effect of mitochondrial transfer on the function of MirC human T cells, a respirometry experiment was performed using an Oroboros O2k. The results showed a recovery of ATP production and coupling efficiency in human T cell-derived MirC on day 7, while ρ(−) human T cells generated by electroporation-mediated XbaI R mRNA transfer maintained the loss of ATP production throughout the experiment (Figure 15E). Representative raw data using the coupling control protocol (CCP) are shown in Figures 15F and 15G, which indicate that MirC T cells are capable of restoring mitochondrial respiration.

[0422] These results demonstrated that human primary T cells can be mitochondrially exchanged to produce MirC using a GMP-grade electroporator such as the electroporator produced by MaxCyte.

[0423] (Example XVII: Generation of Mitochondrial Exchange Cells (MirC) Derived from Mouse Primary T Cells Using mRNA Transfection) Further characterization of T cell-derived MirC was performed on mouse T cells. Mouse T cells were isolated from a suspension solution obtained from the spleen using an EasySep Mouse Isolation Kit (STEM CELL Technologies), which provides a highly purified T cell population by negative selection using a magnet. The isolated mouse T cells (1×10 6 cells / ml) were seeded in a 96-well plate together with Dynabeads Mouse T-Activator CD3 / CD28 (Invitrogen) at a bead-to-cell ratio of 1:1 and 30 U / ml of recombinant IL-2. The medium for culturing mouse T cells was determined with respect to cell proliferation and CD3 expression, and it was found that RPMI1640 was superior to TexMACS (Figure 16A). For example, the survival rate and total cell number were greater in cells cultured in RPMI1640 compared to TexMACS. The medium was changed every 3 or 4 days.

[0424] Next, electroporation of mouse T cells was performed using the Nucleofector device and mRNA. The kinetics of GFP expression after mRNA transfer was the same as that in human T cells (Figure 16B). For example, it was found that almost all cells strongly expressed GFP 6 hours after electroporation with MTS-GFP mRNA (Figure 16B). This indicated that MTS-GFP was transfected with high efficiency. The intensity of GFP rapidly decreased over time and finally disappeared on day 6 after electroporation (Figure 16B).

[0425] Transfection with MTS-XbaI mRNA showed that mouse T cells exhibited a more moderate decrease in XbaI R transcript expression compared to human T cells and persisted at low levels on day 6 (Figure 16C). Quantification of the 12S rRNA level as a surrogate marker for mtDNA showed that mouse mtDNA persisted at approximately 40% of the control even on day 6 (Figure 16D). Next, co-incubation of Ds-Red-labeled exogenous mitochondria and ρ(-) mouse T cells on day 5 was performed (Figure 16E). Despite the longer persistence of XbaI R and lower levels of endogenous mtDNA decline compared to human T cells, FACS analysis of ingested fluorescently labeled mitochondria 48 hours after co-incubation with isolated mitochondria revealed a significant positive rate (9.73%) of T cells expressing exogenous mitochondria (Figure 16F). The percentage of positive cells expressing exogenous mitochondria was even higher than the percentage in the fibroblast experiment, indicating that this protocol for mouse T cells may be optimal for generating T cell-derived MirC.

[0426] (Example XVIII: Transfer of exogenous mitochondria into T cells reversed aging) This example shows that mitochondrial exchange was successful in mouse T cells and rejuvenated aged T cells.

[0427] To evaluate whether exogenous mitochondria could be successfully transferred to generate mouse T cell-derived MirC, mtDNA heteroplasmy levels were determined for BL6 (recipient) and NZB (donor) cells. Two consecutive polymorphisms at 2766 and 2767 mtDNA of ND1 were identified (Figure 17A). Specifically, BL6 mitochondria contained AT at mtDNA positions 2766 and 2767, whereas NZB mitochondria contained GC at the same positions. A primer set and two probes were designed to distinguish the polymorphisms using different fluorophores for each of the GC and AT polymorphisms (Figure 17B). Furthermore, two separate plasmids were generated to express the GC and AT polymorphisms, respectively, and standard curves were generated to facilitate quantitative estimation of heteroplasmy in MirC.

[0428] Quantification of mitochondrial exchange (XbaIR Mt) in BL6 cells transfected with MTS-XbaI and co-incubated with mitochondria isolated from NZB mice showed an overwhelming predominance of exogenous mtDNA, whereas mock transfections co-incubated with mitochondria isolated after electroporation in the absence of endonuclease mRNA did not engulf exogenous mtDNA (Figure 17C), indicating that T cells tolerate mitochondrial exchange.

[0429] Based on the results described in this specification, since it was shown that fibroblast-derived MirC can undergo rejuvenation in vitro (Figure 13), the possibility of rejuvenation of T cell-derived MirC was also investigated. Recipient cells derived from T cells of aged mice were prepared from the spleens of mice (C57BL / 6) over 80 weeks old, and mitochondria of donor mice were isolated from the livers of mice (C57BL / 6) at about 10 weeks old. Telomere length has been reported to shorten with age. Therefore, telomere length was measured by using an absolute mouse telomere length quantification qPCR assay kit (ScienCell). Aged mouse cells were treated with MTS-XbaIR mRNA and co-incubated with exogenous mitochondria from young donor cells to generate MirC (young to old: Y to O). After that, it was observed that the telomere length increased 1.7-fold compared to the original aged T cells (Figure 17D). This indicates that mitochondrial exchange cells exhibit characteristics of rejuvenation.

[0430] Furthermore, SASP was evaluated using the same representative marker set described above (Figure 11). By measuring CXCL1, ICAM1, IL-6, and IL-8, it was revealed that MirC derived from mouse T cells reduces IL-6 and CXCL-1 and shows no change in ICAM-1 and IL-8 (Figure 17E). These results indicate a decrease in SASP for MirC T cells.

[0431] Furthermore, it is known that aged T cells show a greater DNA damage response (DDR) compared to young T cells. Therefore, using a histone 2 A (H2A) phosphorylation antibody, DDR was measured for MirC and the original T cells. The results showed that the positive rate of DDR was lower in MirC (1.53%) compared to the original T cells (4.75%) (Figure 17F). Therefore, MirC T cells have a lower DDR level and show a reversal of aged properties.

[0432] These in vitro results confirm somatic mitochondrial exchange with MirC mtDNA and support that this exchange has brought about many changes indicating reversal of aging in MirC T cells.

[0433] (Example XIX: Tumor growth is reduced by adoptive cell transfer (ACT) using MirC derived from aged T cells containing exogenous mitochondria from young mice) To examine the functional ability of mitochondrial exchange to rejuvenate cells, adoptive cell transfer (ACT) experiments were conducted. Tumor formation was induced in mice using the AE17 mesothelioma cell line derived from the peritoneal cavity of C57BL / 6J mice injected with asbestos fibers. Previous experiments using this model have shown that tumor growth is reduced by ACT of young syngeneic T cells but not by ACT of aged syngeneic T cells (Jackaman et al., OncoImmunology 2019; 8(4): 1-16).

[0434] To determine whether rejuvenated old T cells created by transferring isolated exogenous mitochondria from young mice into T cells from old mice exhibit functional activity, AE17 cells were subcutaneously injected into three groups of old mice: group 1: old mice with ACT of T cells from young mice; group 2: old mice; or group 3: old mice with ACT of MirC derived from T cells of old mice into which exogenous mitochondria from young mice had been transferred (Figure 18A). The ability of MirC derived from aged T cells to suppress tumor growth was evaluated. C57BL / 6 mice aged 22 - 24 months were used in the ACT experiment. The young mice used in the experiment were 2 - 3 months old. In addition to body weight measurements, tumor growth was measured using NIH images of photographs taken every three days (Figure 18B). 2 × 10 6 cells suspended in 100 μL of Matrigel were used to perform AE17 inoculation on day - 14, and the day of T cell transfer was considered day 0. On day 0, 2 × 10 6Individual cells were intravenously injected into tumor-bearing mice. On the same day, recombinant IL-2 (2 μg) was injected intraperitoneally once, and then twice more on days 2 and 3.

[0435] Body weights in each group showed no significant differences (Figure 18C). However, tumors regressed in both the first group of mice (aged mice with young T cells) and the third group of mice (aged mice with MirC derived from aged T cells), while tumors grew steadily in the second group (mock) of mice (Figure 18D). Relative mean tumor masses showed a similar trend to individual mice, indicating that MirC behaves like young T cells (Figure 18E).

[0436] To confirm the presence of injected T cells in animals, T cells derived from GFP transgenic mice were transplanted into syngeneic C57BL / 6 mice, and peripheral blood and spleens were examined to track donor cells (Figure 18F). A two-dimensional plot using FSC vs FL-1 to detect GFP fluorescence was created to identify rare populations. Negative controls using C57BL / 6 mice (upper left panel) and positive controls using GFP transgenic mice (lower left panel) were made for both peripheral blood and spleen (Figure 18G). Defined populations of T cells expressing GFP fluorescence were recognized in both samples, with fractions of 0.057% and 0.9% in peripheral blood and spleen, respectively (Figure 18G). Transferred T cells were detected on day 6 after transplantation by this protocol (Figure 18H), confirming that this protocol can be used to evaluate the ability of transferred cells. Furthermore, it was found that the percentage of chimerism after injection of exogenous T cells increased when larger amounts of cells were injected (Figure 18I).

[0437] These results clearly show that ex vivo generation of MirC using mitochondria from young mice in T cells from aged mice functions effectively in vivo and can reduce tumor burden at a level similar to that of T cells from young mice.

[0438] (Example XX: Hematopoietic stem cells can generate MirC) To date, gene transfer methods for hematopoietic stem cells have mainly involved the use of viral vectors because their targets have mainly been genetic disorders that require sustainable gene expression of the defective gene. As a result, electroporation is not used in the latest protocols for gene transfer of hematopoietic stem cells because it is necessary to produce permanent gene expression. In contrast, the purpose of the mitochondrial exchange technology provided herein is to achieve transient high expression of exonuclease.

[0439] Based on experiments on fibroblasts and T cells, the conditions for electroporation of Nucleofector / mRNA were adjusted and some conditions were examined for mouse fetal liver-derived Sca-1 positive cells, which are considered to be a population of enriched hematopoietic stem cells (HSC) (Figure 19A). Among several conditions, three conditions (programs X-001, Y-001, and T-030, which are the code numbers provided by the device provider) were evaluated by immunofluorescence and cell viability (Figure 19A). The experimental conditions were designated as MTS-GFP1, 2, and 3 according to the programs used (programs X-001, Y-001, and T-030, respectively).

[0440] Further investigation was carried out by FACS analysis of the mean fluorescence intensity (MFI) on the first day after electroporation of GFP mRNA (Figure 19B). The results showed that the optimal condition was the X-001 program (MTS-GFP1). This is because although there was little rightward movement of MFI among the conditions, it was prominent compared to others (Figure 19B). Mouse bone marrow-derived Sca-1 cells were incubated with mitochondria isolated from syngeneic mouse cells, a stable genetically modified cell line expressing DsRed fluorescence. 3-D fluorescence imaging of bone marrow-derived Sca-1 cells 48 hours after co-incubation showed that exogenous mitochondria were engulfed (Figure 19C). The mitochondrial transfer efficiency was estimated by FACS analysis along the DsRed fluorescence axis, which revealed that a subpopulation of approximately 10% of Sca-1 showed rightward movement of fluorescence, suggesting that BM-derived Sca-1 positive cells can undergo somatic mitochondrial exchange (Figure 19D). However, the transfer of exogenous mitochondria in MTS-GFP-expressing cells without deletion of endogenous mitochondria is very low for clinical applications.

[0441] Next, the inventors examined whether this mitochondrial exchange procedure via the generation of ρ(-) cells using MTS-XbaIR mRNA transfer could be applicable to hematopoietic stem cells (Figure 19E). The actual hematopoietic stem cell population is c-kit, which is about 0.005% of the total bone marrow cells + , Sca-1 + , lineage - , CD34 - (referred to as KSLC) (Wilkinson, A. C. et al., Nature, 571(7763):117-121(2019)). After FACS sorting of KSL cells derived from mouse bone marrow-derived cells (Figure 19F), the KSL cells were cultured for 5 days with polyvinyl alcohol (PVA) in the presence of stem cell factor and TPO. Macroscopically, the KSL cells maintained their morphology and showed a short doubling time of 19 hours (Figure 19G).

[0442] As described above, TaqMan SNP genotyping assay was used to evaluate heteroplasmy changes. The assay scheme is shown in Fig. 19H. Mouse KSLC-derived MirC showed that 99.9% of the exogenous mtDNA having the polymorphism in NZB on the 6th day after the introduction of endonuclease mRNA by electroporation (Fig. 19I), indicating that the exogenous mtDNA was almost completely replaced by the endogenous mtDNA of CL57BL / 6. These results showed that hematopoietic stem cells allow this technique to generate MirC.

[0443] (Example XXII: Droplet Digital PCR (ddPCR) for the Measurement of mtDNA and Heteroplasmy) This example shows that mitochondrial DNA (mtDNA) can be assayed for the presence of specific mtDNA sequences, such as mutations in mtDNA, using digital PCR (dPCR). Droplet digital PCR (ddPCR) is a method for performing digital PCR based on water-in-oil emulsion droplet technology.

[0444] Primary skin fibroblasts derived from patients with mitochondrial diseases were analyzed. Patient information is provided in Table 1 below. Table 1: Patient Information

Table 1

[0445] Cells from the target population were encapsulated in droplets at a concentration of 1 cell per droplet containing a PCR mixture containing primers and probes. The cell density was optimized so that a single cell would occur in a single droplet, and fibroblasts were finally diluted to 1×10 6 cells / mL for ddPCR. After encapsulating single cells, cell lysis and amplification of the target sequence were performed in the droplets. The number of droplets with a fluorescent signal indicated the number of cells carrying the target or reference gene.

[0446] Briefly stated, the 20× primer / probe mix was prepared as described in Table 2 below. The standard ddPCR master mix was a 25 μL mix containing the primer / probe mix, template DNA, and 2× ddPCR supermix. Table 2: dPCR Primer and Probe Mix [Table 2] Table 3: dPCR Reaction Master Mix [Table 3]

[0447] Samples were loaded into adjacent wells of an 8-chamber cartridge using 20 μL of prepared qPCR sample, followed by 70 μL of droplet generation oil. A rubber gasket was stretched across the top of the chamber to ensure vacuum sealing. Each 8-chamber cartridge was loaded into a QX100 droplet generator to generate 20,000 droplets per sample. Using a 50 μL multi-channel pipette, 40 μL 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 two-step qPCR temperature cycling conditions with a ramp rate of 50% (3 °C / second). The primer / probe set was optimized using a temperature gradient to optimize the annealing / extension temperature prior to running the temperature cycling conditions. Table 4: dPCR Cycling Conditions [Table 4]

[0448] After temperature cycling, the plate was loaded into a QX100 droplet reader and the endpoint reaction was analyzed. Absolute quantification of the target sequence was obtained by Poisson statistical analysis of the number of positive and negative droplets.

[0449] Prior to examining diseased cells, the specificity for probes designed for mutant sequences and the sensitivity for probes designed for non-mutant sequences were evaluated by using normal human dermal fibroblasts (NHDF cells) having non-mutant sequences (the same as the Cambridge reference sequence) (Figs. 20A - 20C). Dots in the lower left region indicated that there were no cells in the droplets. From the evaluation of three different probe sets, non-mutant sequences were clearly detected (lower right of BK01 (Fig. 20A), upper left of BK02 (Fig. 20B), and upper left of BK04 (Fig. 20C)), and mutant sequences were not detected (upper left of BK01 (Fig. 20A), lower right of BK02 (Fig. 20B), and lower right of BK04 (Fig. 20C)).

[0450] ddPCR of fibroblasts obtained from BK01 showed a few percent of double-positive population, and most were cells having homoplasmy of mutant mtDNA (Fig. 20D). There was no significant population of single cells having homoplasmy of non-mutant mtDNA. Furthermore, BK02 showed a small part of double-positive cells, indicating heteroplasmy at the single-cell level, defined as microheteroplasmy (Fig. 20E). From the results of BK02, the major homoplasmic population of mutant mtDNA was revealed, and no population having homoplasmy of non-mutant mtDNA was recognized.

[0451] In summary, these results showed that homoplasmy and heteroplasmy can be accurately and quantitatively evaluated at the single-cell level. Furthermore, these results showed that mtDNA of subjects with mitochondrial diseases can be accurately measured, which may be useful for evaluating therapeutic compositions prior to transplantation into subjects or for monitoring mtDNA content before and / or after treatment.

[0452] (Example XVII: mtDNA Exchange in Recipient Hematopoietic Stem or Progenitor Cells (HSPC) Derived from Donor cGMP-Manufactured Bone Marrow-Derived Mesenchymal Stromal Cells (BM-MSC)) This example shows that the mtDNA exchange method provided herein can be used therapeutically to ex vivo condition hematopoietic stem or progenitor cells (HSPCs).

[0453] Conditioning of HSPCs can be performed ex vivo in the context of stem cell transplantation. Briefly, peripheral blood stem cells are mobilized and a blood sample is obtained from the patient. Peripheral hematopoietic stem or progenitor cells (HSPCs), e.g., CD34 + cells are isolated and sent to a manufacturing facility. At the manufacturing facility, mitochondria are partially depleted according to the method provided herein.

[0454] Donor mitochondria are isolated using freshly cGMP-manufactured bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a cell repository (e.g., Waisman Biomanufacturing). The initial bone marrow aspirate was collected with adequate informed consent and in compliance with federal regulations (e.g., 21 CFR 1271). This aspirate was processed under cGMP and banked at early passage for later expansion.

[0455] Donor mitochondria from BM-MSCs are transferred into cultured HSPCs to alter heteroplasmy. The modified HSPCs are sent back to the medical center for autologous transplantation (i.e., to the same subject from whom the HSPCs were isolated). Prior to transplantation, the patient receives a non-myeloablative regimen, e.g., partial radiation or a minimal treatment containing less than a lethal dose of an anti-cancer drug, e.g., busulfan. The patient is transfused back with modified HSPCs containing only allogeneic donor mitochondria.

[0456] This example shows that the mtDNA exchange method provided herein can be used therapeutically without allogeneic HSPC transplantation to ex vivo condition HSPCs.

[0457] The above embodiments are merely intended to be illustrative, and those skilled in the art will recognize or be able to confirm many equivalents of specific compounds, materials, and procedures using only routine experimentation. All such equivalents are considered to be within the scope of the present invention and are encompassed by the accompanying claims. This application provides an invention in the following aspects. (Aspect 1) A method for producing mitochondria-exchanged cells, comprising: (a) contacting recipient cells with an agent that reduces the endogenous mtDNA copy number; (b) incubating the recipient cells for a period sufficient for the agent to partially reduce the endogenous mtDNA copy number in the recipient cells; and (c) co-incubating (1) the recipient cells from step (b) with a partially reduced endogenous mtDNA and (2) exogenous mitochondria from a healthy donor for a period sufficient to non-invasively transfer the exogenous mitochondria into the recipient cells, thereby producing mitochondria-exchanged cells : The above method. (Aspect 2) A method for treating a subject in need of mitochondria exchange, comprising: (a) (i) contacting recipient cells with an agent that reduces the mtDNA copy number; (ii) incubating the recipient cells for a period sufficient for the agent to partially reduce the mtDNA copy number in the recipient cells; and (iii) co-incubating (1) the recipient cells from step (ii) with a partially reduced endogenous mtDNA and (2) exogenous mitochondria from a healthy donor for a period sufficient to non-invasively transfer the exogenous mitochondria into the recipient cells, thereby producing mitochondria-exchanged cells : producing mitochondria-exchanged cells ex vivo or in vitro; and (b) administering a therapeutically effective amount of the mitochondria-exchanged recipient cells from step (a) to the subject in need of mitochondria exchange : The above method. (Aspect 3) A method for treating a subject having or suspected of having an age-related disease, comprising: (a) (i) contacting recipient cells with an agent that reduces the mtDNA copy number; (ii) incubating the recipient cells for a period sufficient for the agent to partially reduce the mtDNA copy number in the recipient cells; and (iii)(1) The recipient cells from step (ii) in which the endogenous mtDNA is partially reduced and (2) exogenous mitochondria from a healthy donor are co-incubated for a period sufficient to non-invasively transfer the exogenous mitochondria into the recipient cells, thereby producing mitochondrial exchange cells : producing mitochondrial exchange cells ex vivo or in vitro, comprising; and (b) administering a therapeutically effective amount of the mitochondrial exchange recipient cells from step (a) to a subject having or suspected of having an age-related disease : The method comprising the above. (Aspect 4) A method for treating a subject having or suspected of having a mitochondrial disease or disorder, comprising: (a) (i) contacting recipient cells with an agent that reduces the mtDNA copy number; (ii) incubating the recipient cells for a period sufficient for the agent to partially reduce the mtDNA copy number in the recipient cells; and (iii)(1) The recipient cells from step (ii) in which the endogenous mtDNA is partially reduced and (2) exogenous mitochondria from a healthy donor are co-incubated for a period sufficient to non-invasively transfer the exogenous mitochondria into the recipient cells, thereby producing mitochondrial exchange cells : producing mitochondrial exchange recipient cells ex vivo or in vitro, comprising; and (b) administering a therapeutically effective amount of the mitochondrial exchange recipient cells from step (a) to a subject having or suspected of having a mitochondrial disease or disorder : The method comprising the above. (Aspect 5) The method according to any one of aspects 1 to 4, wherein the exogenous mitochondria are functional mitochondria. (Aspect 6) The method according to any one of aspects 1 to 5, wherein the exogenous mitochondria contain wild-type mtDNA. (Aspect 7) The method according to any one of aspects 1 to 6, wherein the exogenous mitochondria are isolated mitochondria. (Aspect 8) The method according to aspect 7, wherein the isolated mitochondria are intact mitochondria. (Aspect 9) The method according to any one of aspects 1 to 8, wherein the exogenous mitochondria are allogeneic. (Aspect 10) A method for producing mitochondrial exchange cells, comprising: (a) contacting recipient cells with an agent that reduces the endogenous mtDNA copy number; (b) incubating the recipient cells for a period sufficient for the agent to partially reduce the endogenous mtDNA copy number in the recipient cells; and (c) co-incubating (1) the recipient cells from step (b) in which the endogenous mtDNA is partially reduced and (2) exogenous mtDNA from a healthy donor for a period sufficient to non-invasively transfer the exogenous mtDNA into the recipient cells, thereby producing mitochondrial exchange cells : The method as described above. (Aspect 11) A method for treating a subject in need of mitochondrial exchange, comprising: (a) (i) contacting recipient cells with an agent that reduces the mtDNA copy number; (ii) incubating the recipient cells for a period sufficient for the agent to partially reduce the mtDNA copy number in the recipient cells; and (iii) co-incubating (1) the recipient cells from step (ii) in which the endogenous mtDNA is partially reduced and (2) exogenous mtDNA from a healthy donor for a period sufficient to non-invasively transfer the exogenous mtDNA into the recipient cells, thereby producing mitochondrial exchange cells : producing mitochondrial exchange cells ex vivo or in vitro; and (b) administering a therapeutically effective amount of the mitochondrial exchange recipient cells from step (a) to the subject in need of mitochondrial exchange : The method as described above. (Aspect 12) A method for treating a subject having or suspected of having an age-related disease, comprising: (a) (i) contacting recipient cells with an agent that reduces the mtDNA copy number; (ii) incubating the recipient cells for a period sufficient for the agent to partially reduce the mtDNA copy number in the recipient cells; and (iii) co-incubating (1) the recipient cells from step (ii) in which the endogenous mtDNA is partially reduced and (2) exogenous mtDNA from a healthy donor for a period sufficient to non-invasively transfer the exogenous mtDNA into the recipient cells, thereby producing mitochondrial exchange cells Producing mitochondrial exchange cells ex vivo or in vitro, which cells contain [[:]]; and (b) Administering a therapeutically effective amount of the mitochondrial exchange recipient cells derived from step (a) to a subject having or suspected of having the age-related disease The method as described above, which method comprises [[:]]. (Aspect 13) A method for treating a subject having or suspected of having a mitochondrial disease or disorder, the method comprising: (a) (i) contacting recipient cells with an agent that reduces the mtDNA copy number; (ii) incubating the recipient cells for a period sufficient for the agent to partially reduce the mtDNA copy number in the recipient cells; and (iii) co-incubating (1) the recipient cells derived from step (ii) in which the endogenous mtDNA is partially reduced and (2) exogenous mtDNA from a healthy donor for a period sufficient to non-invasively transfer the exogenous mtDNA into the recipient cells, thereby producing mitochondrial exchange cells Producing mitochondrial exchange recipient cells ex vivo or in vitro, which cells contain [[:]]; and (b) Administering a therapeutically effective amount of the mitochondrial exchange recipient cells derived from step (a) to a subject having or suspected of having the mitochondrial disease or disorder The method as described above, which method comprises [[:]]. (Aspect 14) The method according to any one of Aspects 1 to 13, wherein the agent for reducing the endogenous mtDNA copy number is selected from the group consisting of a polynucleotide encoding a fusion protein comprising a mitochondrial targeting sequence (MTS) and an endonuclease, a polynucleotide encoding an endonuclease, and a small molecule. (Aspect 15) The method according to Aspect 14, wherein the small molecule is a nucleoside reverse transcriptase inhibitor (NRTI). (Aspect 16) The method according to Aspect 14, wherein the polynucleotide is composed of messenger ribonucleic acid (mRNA) or deoxyribonucleic acid (DNA). (Aspect 17) The method according to Aspect 14, wherein the recipient cells transiently express the fusion protein. (Aspect 18) The method according to aspect 14, wherein 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). (Aspect 19) The method according to any one of aspects 14, 17, or 18, wherein the MTS targets a mitochondrial matrix protein. (Aspect 20) The method according to aspect 19, wherein 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. (Aspect 21) The method according to any one of aspects 1 to 20, wherein the agent that reduces the endogenous mtDNA copy number reduces the endogenous mtDNA copy number by about 5% to about 99%. (Aspect 22) The method according to aspect 21, wherein the agent that reduces the endogenous mtDNA copy number reduces the endogenous mtDNA copy number by about 30% to about 70%. (Aspect 23) The method according to aspect 21, wherein the agent that reduces the endogenous mtDNA copy number reduces the endogenous mtDNA copy number by about 50% to about 95%. (Aspect 24) The method according to aspect 21, wherein the agent that reduces the endogenous mtDNA copy number reduces the endogenous mtDNA copy number by about 60% to about 90%. (Aspect 25) The method according to any o...

Claims

1. An ex vivo or in vitro method for producing mitochondrial replacement primary cells that exhibit restoration of a phenotype of mitochondrial function equivalent to that of normal cells, comprising: (a) contacting primary cells ex vivo or in vitro with a polynucleotide encoding a fusion protein comprising an endonuclease having a mitochondrial targeting sequence (MTS) to achieve depletion of a portion of endogenous mitochondrial DNA (mtDNA) of 99% or less; and (b) when the primary cells substantially do not contain the polynucleotide encoding the fusion protein, contacting the primary cells with exogenous mitochondria or exogenous mtDNA from a healthy donor to non-invasively transfer the exogenous mitochondria or exogenous mtDNA into the primary cells, thereby producing the mitochondrial replacement primary cells that exhibit restoration of a phenotype of mitochondrial function equivalent to that of normal cells The method as described above.

2. The exogenous mitochondria are: (i) functional mitochondria that consume oxygen and produce ATP; (ii) contain wild-type mtDNA; (iii) are isolated mitochondria; and / or (iv) are allogeneic, the method according to claim 1.

3. The method according to claim 2, wherein the isolated mitochondria are intact mitochondria.

4. (i) The MTS targets mitochondrial matrix proteins; (ii) 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); or (iii) The polynucleotide is messenger ribonucleic acid (mRNA) or deoxyribonucleic acid (DNA), the method according to any one of claims 1 to 3.

5. The method according to claim 4, wherein the endonuclease is XbaI.

6. The method according to claim 4, wherein 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.

7. The method according to any one of claims 1 to 6, wherein the primary cells transiently express the fusion protein.

8. The method according to any one of claims 1 to 7, wherein about 5% to about 99%, about 30% to about 70%, about 50% to about 95%, or about 60% to about 90% of the endogenous mtDNA copy number of the primary cells is reduced.

9. The endogenous mtDNA is: (i) encoding dysfunctional mitochondria; (ii) containing mutant mtDNA; (iii) containing mtDNA associated with mitochondrial diseases or disorders; (iv) being heteroplasmic; and / or (v) containing wild-type mtDNA, the method according to any one of claims 1 to 8.

10. The mitochondrial exchange primary cells have a total mtDNA copy number not exceeding about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, or about 1.5-fold compared to the total mtDNA copy number of the primary cells before contacting with the polynucleotide encoding the fusion protein, the method according to any one of claims 1 to 9.

11. The primary cells are: (i) senescent cells; (ii) bone marrow cells; (iii) immune cells; or (iv) somatic cells, the method according to any one of claims 1 to 10.

12. (i) the bone marrow cells are hematopoietic stem cells (HSCs) or mesenchymal stem cells (MSCs); or (ii) the immune cells are selected from the group consisting of T cells, phagocytes, microglial cells, and macrophages, the method according to claim 11.

13. The T cells are CD4+ T cells, CD8+ T cells, or chimeric antigen receptor (CAR) T cells, the method according to claim 12.

14. The transfer of the exogenous mitochondria and / or the exogenous mtDNA is stable, the method according to any one of claims 1 to 13.

15. The exogenous mtDNA changes the heteroplasmy in the primary cells, the method according to claim 14.

16. The method according to any one of claims 1 to 15, further comprising contacting the primary cells with an mTOR inhibitor or treating the primary cells by nutrient starvation before contacting the primary cells with the exogenous mitochondria and / or the exogenous mtDNA.

17. The mTOR inhibitor contains rapamycin or a derivative thereof, the method according to claim 16.

18. The method according to any one of claims 1 to 17, wherein the restoration of the phenotype is measured by proliferative ability, ATP production, cell survival, oxygen consumption, glycolysis, or respiratory capacity.

19. A composition comprising one or more mitochondrial replacement primary cells obtained by the method according to any one of claims 1 to 18, wherein the mitochondrial replacement primary cells contain more than 50% exogenous mtDNA.

20. (i) A subject in need of mitochondrial replacement; (ii) A subject having or suspected of having an age-related disease; or (iii) A subject having or suspected of having a mitochondrial disease or disorder, neurodegenerative disease, retinal disease, diabetes, hearing impairment, genetic disease, or a combination thereof A mitochondrial replacement primary cell for use in a method of treating The method comprising administering a therapeutically effective amount of the mitochondrial replacement primary cells to the subject, wherein the mitochondrial replacement primary cells are generated by the method according to any one of claims 1 to 18.

21. (i) 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; (ii) The retinal disease is selected from the group consisting of age-related macular degeneration, macular edema, and glaucoma; (iii) The age-related disease is selected from the group consisting of autoimmune diseases, metabolic diseases, genetic diseases, cancer, neurodegenerative diseases, and immunosenescence; (iv) The genetic disease is selected from the group consisting of Hutchinson-Gilford progeria syndrome, Werner syndrome, and Huntington's disease; (v) The mitochondrial disease or disorder is caused by mitochondrial DNA abnormality, nuclear DNA abnormality, or both; (vi) the mitochondrial disease or disorder is caused by mitochondrial DNA abnormality and 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), Luft disease, aminoglycoside-induced deafness (AID), and mitochondrial DNA multiple deletion syndrome; or (vii) the mitochondrial disease or disorder is caused by nuclear DNA abnormality and 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 γ (POLG) -related disorder, sensory ataxic neuropathy dysarthria ophthalmoplegia (SANDO), disorders of the brainstem and spinal cord and leukodystrophy with elevated lactate (LBSL), coenzyme Q10 deficiency, Leigh syndrome, mitochondrial complex abnormality, fumarase deficiency, α-ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenase complex deficiency (PDHC), pyruvate carboxylase deficiency (PCD), carnitine palmitoyltransferase I (CPT I) deficiency, carnitine palmitoyltransferase II (CPT II) deficiency, carnitine-acyl-carnitine (CACT) deficiency, autosomal dominant / autosomal recessive progressive external ophthalmoplegia (ad- / ar-PEO), infantile-onset spinocerebellar ataxia (IOSCA), mitochondrial myopathy (MM), spinal muscular atrophy (SMA), growth retardation, aminoaciduria, cholestasis, iron overload, early death (GRACILE), and Charcot-Marie-Tooth disease type 2A (CMT2A), the mitochondrial replacement primary cell for use according to claim 20.

22. The mitochondrial replacement primary cells for use according to claim 21, wherein the metabolic disease is diabetes and the neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

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