Methods and compositions for treating mitochondrial diseases or disorders and heteroplasmy

Abstract

To provide: compositions of cells with mitochondrial DNA reduction and / or mitochondrial DNA replacement; methods for producing the same; and methods for treating various diseases associated with genetic or age-related mitochondrial dysfunction.SOLUTION: A method for producing mitochondria replaced cells includes: (a) contacting recipient cells with an agent that reduces 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 the step (b) in which the endogenous mtDNA has been partially reduced with (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 producing the mitochondria replaced cells.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] This application was filed on August 14, 2018, and is incorporated herein by reference in its entirety. U.S. Provisional Application No. 62 / 718,891, U.S. Provisional Application No. 62 / 731,731, filed September 14, 2018, and and claims the benefit of U.S. Provisional Application No. 62 / 817,987, filed March 13, 2019.

[0002] (Sequence Listing) This application has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. This ASCII copy, created on August 13, 2019, is available under 14595-001-228_ It is named SL.txt and is 12,905 bytes in size.

[0003] 1. FIELD OF THE INVENTION The present invention relates to cells with mitochondrial DNA reduction and / or mitochondrial DNA replacement. Compositions of the invention, methods for their production, and methods for treating genetic or age-related mitochondrial dysfunction Methods for treating various diseases are provided. [Background technology]

[0004] 2. BACKGROUND OF THE INVENTION Mitochondria play a major and important role in cellular homeostasis. It is involved in various disease processes, including intracellular signaling and apoptosis. and pyruvate oxidation, the Krebs cycle, and amino acids, fatty acids, and nucleotides. It performs numerous biochemical tasks, including metabolism of the blood, kidney, and steroids. A key function of ATP is its role in cellular energy metabolism, which includes the electron transport chain and the acid The mitochondrial respiratory chain includes the beta-oxidation of fatty acids via metabolic phosphorylation and the production of ATP. Complex I (NADH-ubiquinone oxidoreductase), Complex II (succinate-ubiquinone oxidoreductase), Oxidoreductase), Complex III (ubiquinol-ferricytochrome c oxidoreductase) Complex IV (cytochrome c oxidoreductase), and Complex V (FIFO ATPase): It consists of five multisubunit protein complexes embedded in the inner membrane.

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

[0006] Mitochondrial diseases or disorders are clinically heterogeneous disorders characterized by dysfunctional mitochondria. The onset of the disease can occur at any age and is characterized by a wide variety of clinical symptoms. Mitochondrial diseases or disorders can affect any organ or tissue. , characteristically involving multiple strains that generally affect organs highly dependent on aerobic metabolism. It can be devastating and often progresses relentlessly with high morbidity and mortality. Chondriac diseases or disorders are the most common group of inherited metabolic disorders and inherited neurological disorders. It is one of the most common forms of

[0007] Mitochondrial diseases or disorders include those affecting structural mitochondrial proteins or mitochondria. Nuclear DNA (nDNA) and / or mitochondrial DNA (mtDNA) encoding proteins involved in function Some mitochondrial disorders can occur in a single organ (e.g., Although most cases affect only the eyes (e.g., in Leber's hereditary optic neuropathy [LHON]), It affects multiple organ systems and often shows prominent neuropathic and myopathic features. Although tissues with high energy demands, such as the brain and eyes, are more frequently affected, The phenotype of an individual can be highly variable and heterogeneous. This variation is due in part to gene regulation (nDNA and mtDNA), levels of heteroplasmy (in single cells and tissues), Factors such as percentage of mutant DNA, tissue energy demand, maternal inheritance, and mitotic segregation Which depends on several factors.

[0008] Many patients with mitochondrial diseases or disorders have a mixture of mutant and wild-type mtDNA. have a mixture of mutant and wild-type mtDNA (known as heteroplasmy); It is the key factor that determines whether a cell will develop a biochemical defect. The majority of mutations are heteroplasmic, meaning that individual cells contain both mutant mtDNA and wild-type mtDNA. High levels of heteroplasmy result in high levels of mutant mtDNA and refers to cells with low levels of wild-type mtDNA, whereas low levels of heteroplasmy Refers to cells that have low levels of mutant mtDNA and high levels of wild-type mtDNA. Studies of single cells from patients with a disease or disorder have shown that mutant mtDNA and wild-type mtD It has been shown that the level of NA is crucial for determining cell phenotype. For example, cells It is thought that the high levels of mutant mtDNA and low levels of wild-type mtDNA (i.e., high levels of heterozygotes) If the blood contains thrombus, respiratory failure occurs. The threshold at which this failure occurs is determined by the exact sudden Dependent on mutation and cell type. Usually, a high percentage level of mutant mtDNA (>50%) ) is required to produce the cell defect, but certain mtDNA mutations (usually mt tRNA mutations) ) cause defects only when present at very high levels, and other mtDNA mutations (e.g., A single large mtDNA deletion) results in a defect when ~60% of the mtDNA is deleted. For example, a higher percentage of individuals carrying the m.8993T>G pathogenic variant had higher levels of Mutant mtDNA is found in individuals with neurasthenia with ataxia and retinitis pigmentosa (NARP). are also seen in individuals with Leigh syndrome. Furthermore, the clinical phenotype in MELAS and MERRF is Correlates with heteroplasmy (see, e.g., Chinnery, PF et al., Brain 120(Pt 10), 17 13-1721(1997)).

[0009] Advances in next-generation sequencing technology have led to the identification of mitochondrial diseases or disorders Furthermore, studies of other organisms, such as the nematode C. elegans, have revealed many mutations. For example, in C. elegans, some of the proteins involved in heteroplasmy have been identified. Recent studies using mitochondrial unfolded protein response (UPRmt) have shown that to maintain heteroplasmy and propagate mutant mtDNA after disruption of the original mtDNA. It has been shown that the ATPase functions in the nucleus of the nucleus (e.g., Lin, YF et al., Nature 533, 416-419, doi:10 However, heteroplasmy in mammals The mechanisms underlying maintenance and proliferation remain unclear.

[0010] The management and treatment of patients with mitochondrial diseases or disorders remains challenging. For the vast majority of patients, the condition progresses inexorably, resulting in considerable morbidity and ultimately Even in severely affected patients, this can result in death. The method involves the cytotoxicity of cells with low concentrations of ethidium bromide (EtBr), a known carcinogen and teratogen. This involves long-term treatment, limiting its therapeutic use. In addition to the difficulty, EtBr protocols can take several months, which limits their clinical use. Furthermore, mitochondrial import protocols typically require the addition of exogenous mitochondria. Before the migration of the rhesus, cells called rho(ρ)0 cells are completely depleted of endogenous mtDNA. Complete depletion of ATP severely hinders the ability of cells to ingest exogenous mitochondria.

[0011] Other mitochondrial transfer protocols have been shown to transfer mitochondria without depleting endogenous mtDNA. However, this approach is inefficient and harmful to the cells. For example, mitochondrial import using simple co-incubation was found to It has been reported that it is ineffective and not equally efficient across various cell types. Additional techniques for transfer involve invasive instruments or nanoparticles that are harmful to the recipient cells. Although these require injections using other invasive devices such as Nobraid, all involve co-incubation. This was less efficient than the conventional method (Caicedo et al., Stem Cells International, (2017), v ol. 2017, Article ID 7610414, page 23).

[0012] Therefore, current mitochondrial transfer methods are not only impractical for clinical settings, but also are inefficient, harmful to recipient cells, and / or very time-consuming. Therefore, mitochondrial diseases or disorders and malfunctioning of the mitochondria are also important. having or suffering from a disease or disorder associated with poor or dysfunctional mitochondria or modifications that can be optionally used in the treatment of subjects suspected of having a disorder. Improved mitochondrial transfer methods and methods for studying mitochondrial diseases or disorders There is a great unmet need to develop improved models. Summary of the Invention

[0013] (3. Summary of the Invention) In one aspect, provided herein are methods for producing mitochondria-replaced cells. (a) contacting recipient cells with an agent that reduces endogenous mtDNA copy number; (b) the agent partially reduces the endogenous mtDNA copy number in the recipient cells; (c) incubating the recipient cells for a period of time sufficient to induce the proliferation of the endogenous The recipient cells from step (b) in which the sexual mtDNA is partially reduced and (2) the recipient cells from a healthy donor The exogenous mitochondria are then non-invasively transferred into the recipient cells. The cells are then co-incubated for a period of time sufficient for the transfer of mitochondria. A method including: making.

[0014] In another aspect, provided herein is a method for treating a patient in need of mitochondrial replacement. A method of treating a subject, comprising: (a) (i) treating recipient cells with a reduced mtDNA copy number; (ii) contacting the recipient cells with an agent, wherein the agent increases the mtDNA copy number in the recipient cells; incubating the recipient cells for a period of time sufficient to partially reduce the and (iii) (1) the recipient cells from step (ii) in which the endogenous mtDNA is partially reduced; 2) Exogenous mitochondria from a healthy donor are introduced into the recipient. The cells are co-incubated for a period of time sufficient to non-invasively transfect the cells, thereby The method comprises the steps of producing mitochondrial-exchanged cells ex vivo or in vivo. (b) producing the mitochondrial exchange recipient cells from step (a) in vitro; and administering a therapeutically effective amount of said mitochondrial replacement to a subject in need thereof. is.

[0015] In yet another aspect, provided herein are methods for treating a patient with an age-related disease or at an age 1. A method of treating a subject suspected of having a related disease, comprising: (a) (i) recipient cells; (ii) contacting the recipient with an agent that reduces mtDNA copy number; the recipient cells for a period of time sufficient to partially reduce the mtDNA copy number in the cells. and (iii) incubating the endogenous mtDNA in step (ii)(i) (2) the recipient cells derived from the donor and (3) the exogenous mitochondria derived from the donor. Co-incubation is continued for a period of time sufficient to non-invasively transfer mitochondrial material into the recipient cells. and transferring the mitochondria to the cells, thereby producing mitochondria-exchanged cells. (b) generating replacement cells ex vivo or in vitro; and (b) generating replacement cells from step (a). and administering a therapeutically effective amount of endogenous replacement recipient cells to a subject suffering from the age-related disease or to a subject suffering from the age-related disease. administering to a subject suspected of having the disease.

[0016] In a further aspect, provided herein is a method for treating a mitochondrial disease or disorder. and treating a subject having or suspected of having a mitochondrial disease or disorder. (a)(i) contacting recipient cells with an agent that reduces mtDNA copy number; (ii) causing the agent to partially reduce mtDNA copy number in the recipient cells; and (iii) incubating the recipient cells for a period of time sufficient to induce the expression of the endogenous The recipient cells from step (ii) in which the virion mtDNA is partially reduced and (ii) the recipient cells from a healthy donor The exogenous mitochondria are then non-invasively transferred into the recipient cells. The cells are then co-incubated for a period of time sufficient for the transfer of mitochondria. preparing mitochondria exchange recipient cells ex vivo or in vitro. (b) producing the mitochondria-exchanged recipient cells from step (a); and (b) treating the mitochondria-exchanged recipient cells from step (a). A therapeutically effective amount is administered to a subject having the mitochondrial disease or disorder or who has no mitochondrial disease or disorder. or administering to a subject suspected of having the disorder.

[0017] In some embodiments of the methods provided herein, the exogenous mitochondria are: In some embodiments, the exogenous mitochondria are wild-type mitochondria. In a specific embodiment, the exogenous mitochondria comprise isolated mitochondria. In a further embodiment, the isolated mitochondria are intact mitochondria. In some embodiments, the exogenous mitochondria are allogeneic. It is something.

[0018] Also provided herein is a method for producing a mitochondrial replacement cell, comprising: (b) contacting the recipient cells with an agent that reduces the endogenous mtDNA copy number; The agent is sufficient to partially reduce the endogenous mtDNA copy number in the recipient cells. and (c) (1) incubating the recipient cells for a period of time sufficient to induce the expression of the endogenous mtDNA. (2) the recipient cells from step (b) that are gradually reduced in exogenous mtDNs from a healthy donor; A is co-inoculated with the exogenous mtDNA for a period of time sufficient to non-invasively transfer the exogenous mtDNA into the recipient cells. and incubating the cells with mitochondrial replacement, thereby producing mitochondria-exchanged cells.

[0019] The present disclosure provides a method of treating a subject in need of mitochondrial replacement, comprising: (a)(i) (ii) contacting the recipient cells with an agent that reduces mtDNA copy number; for a period of time sufficient for the agent to partially reduce mtDNA copy number in the recipient cells; (iii) incubating the recipient cells; and (ii) (i) the endogenous mtDNA is partially depleted. (ii) the recipient cells from the step (ii) and (ii) exogenous mtDNA from a healthy donor; Co-incubation is continued for a period of time sufficient to non-invasively transfer exogenous mtDNA into the recipient cells. and transferring the mitochondria to the cells, thereby producing mitochondria-exchanged cells. (b) generating replacement cells ex vivo or in vitro; and (b) generating replacement cells from step (a). A therapeutically effective amount of mitochondrial replacement recipient cells is administered to a subject in need of said mitochondrial replacement. Also provided are methods comprising administering to a subject.

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

[0021] In yet another aspect, provided herein are methods for treating mitochondrial diseases or disorders. and treating a subject having or suspected of having a mitochondrial disease or disorder. (a)(i) contacting recipient cells with an agent that reduces mtDNA copy number; (ii) causing the agent to partially reduce mtDNA copy number in the recipient cells; and (iii) incubating the recipient cells for a period of time sufficient to induce the expression of the endogenous The recipient cells from step (ii) in which the virion mtDNA is partially reduced and (ii) the recipient cells from a healthy donor The exogenous mtDNA is transferred to the recipient cells in a manner sufficient to non-invasively transfer the exogenous mtDNA into the recipient cells. and co-incubating the cells for a period of time, thereby producing mitochondria-exchanged cells. mitochondrial exchange recipient cells are generated ex vivo or in vitro; and (b) administering a therapeutically effective amount of the mitochondrial exchange recipient cells from step (a) to the mitochondrial exchange recipient cells. having an endocrine disease or disorder or having a mitochondrial disease or disorder administering the compound to a subject suspected of having the compound.

[0022] In certain embodiments of the methods provided herein, the endogenous mtDNA copy number is reduced. The active agent is a fusion protein containing a mitochondrial targeting sequence (MTS) and an endonuclease. Polynucleotides encoding proteins, polynucleotides encoding endonucleases In some embodiments, the small molecule is selected from the group consisting of: In other embodiments, the polynucleotide is a nucleoside reverse transcriptase inhibitor (NRTI). Otides are composed of messenger ribonucleic acid (mRNA) or deoxyribonucleic acid (DNA). In a further embodiment, the recipient cells transiently express the fusion protein. In still further embodiments, the endonuclease is XbaI, EcoRI, BamHI, HindIII , PstI, Cas9, zinc finger nucleases (ZFNs), and transcription activator-like effectors. In some embodiments, the M is selected from the group consisting of TALENs. The TS targets a mitochondrial matrix protein. , a mitochondrial matrix protein, is a cytochrome c oxidase subunit IV , cytochrome c oxidase subunit VIII, and cytochrome c oxidase subunit and X.

[0023] In some embodiments of the methods provided herein, the endogenous mtDNA copy number is reduced. The agent that reduces the endogenous mtDNA copy number reduces from about 5% to about 99%. In this study, agents that reduce endogenous mtDNA copy number reduced the endogenous mtDNA copy number by approximately 30%. In a further embodiment, the endogenous mtDNA copy number is reduced by about 70%. The agent reduces the endogenous mtDNA copy number by about 50% to about 95%. In these cases, agents that reduce endogenous mtDNA copy number reduce approximately 60% of the endogenous mtDNA copy number. In some embodiments, the endogenous mtDNA copy number is reduced by about 90%. Agents that increase mitochondrial mass reduce mitochondrial clumping.

[0024] Also provided herein is a method for producing a mitochondrial replacement cell, comprising: (b) contacting the recipient cells with an agent that reduces mitochondrial function; The agent partially reduces the endogenous mitochondrial function in the recipient cells. and (c)(1) incubating the recipient cells for a period of time sufficient to induce the expression of the endogenous miR-1 receptor. (2) the recipient cells from step (b) in which mitochondrial function is partially reduced; Donor-derived exogenous mitochondria are introduced into the recipient cells. The cells were co-incubated for a period of time sufficient for invasive transfer, thereby allowing mitochondrial exchange. producing a recombinant cell.

[0025] The present disclosure provides a method for producing mitochondria-exchanged cells, comprising: (a) transferring recipient cells to a cell culture medium; (b) contacting the recipient with an agent that reduces mitochondrial function; and administering the mitochondrial enzyme to the mouse cells for a period of time sufficient to partially reduce the endogenous mitochondrial function in the mouse cells. and (c) (1) incubating the recipient cells with the endogenous mitochondrial function. (2) the recipient cells from step (b) that are differentially reduced in number and (3) the exogenous mt cells from a healthy donor. The DNA is co-infused for a period of time sufficient to non-invasively transfer the exogenous mtDNA into the recipient cells. and incubating the cells with the mitochondrial exchanger, thereby producing the mitochondrial exchanger. Provide.

[0026] In some embodiments of the methods provided herein, the method comprises reducing mitochondrial function. The agent that reduces endogenous mitochondrial function transiently reduces endogenous mitochondrial function. Therefore, agents that reduce mitochondrial function permanently disrupt endogenous mitochondrial function. to reduce it to.

[0027] In certain embodiments of the methods provided herein, the mitochondrial replacement is Subjects with dysfunctional mitochondria; age-related diseases; mitochondrial diseases or disorders neurodegenerative diseases, retinal diseases, diabetes, hearing impairment, and genetic disorders In some embodiments, the neurodegenerative disease is a muscle Amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, Dreich's ataxia, Charcot-Marie-Tooth disease, and leukodystrophies In a specific embodiment, the retinal disease is selected from the group consisting of age-related macular degeneration, macular edema, and the like. and glaucoma.

[0028] In some embodiments of the methods provided herein, the age-related disease is an autoimmune a disease, a metabolic disease, a genetic disease, cancer, a neurodegenerative disease, and immunosenescence. In certain embodiments of the methods provided herein, the metabolic disease is diabetes. In a further embodiment, the neurodegenerative disease is Alzheimer's disease or Parkinson's disease. In still further embodiments, the genetic disease is a Hutchinson-Gilford Selected from the group consisting of Progeria syndrome, Werner syndrome, and Huntington's disease .

[0029] In certain embodiments of the methods provided herein, the mitochondrial disease or disorder is: It is caused by mitochondrial DNA abnormalities, nuclear DNA abnormalities, or both. Mitochondrial diseases or disorders caused by mitochondrial DNA abnormalities in Harm is associated with chronic progressive external ophthalmoplegia (CPEO), Pearson syndrome, and Kearns-Sayre syndrome (KSS). , diabetes, and hearing loss (DAD), mitochondrial diabetes, Leber's hereditary optic neuropathy (LHON) , LHON-plus, Neuropathy, Ataxia, and Retinitis Pigmentosa Syndrome (NARP), Maternal Hereditary Leigh syndrome (MILS), mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (M ELAS), Myoclonic Epilepsy and Ragged Red Fiber Disease (MERRF), Familial Bilateral Striatal Necrosis / Striatonigral degeneration (FBSN), Luft's disease, aminoglycoside-induced hearing loss (AID), and mitochondrial deafness In another specific embodiment, the gene is selected from the group consisting of multiple deletion syndromes of endorphin DNA. Mitochondrial diseases or disorders caused by nuclear DNA abnormalities are Mitochondrial DNA depletion syndrome-4A, Mitochondrial recessive ataxia syndrome (MIRAS), Mitochondrial neuropathies Mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase gamma (P OLG-related disorders, sensory ataxic neuropathy dysarthria ophthalmoplegia (SANDO), brainstem and spinal cord disorders Leukoencephalopathy with spinal cord damage and elevated lactate (LBSL), coenzyme Q10 deficiency, Leigh syndrome, mitochondrial dysfunction, andria complex abnormalities, fumarase deficiency, α-ketoglutarate dehydrogenase complex (KG DHC deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenase complex deficiency (PDHC), pyruvate carboxylase deficiency (PCD), carnitine palmitoyltransferase Carnitine palmitoyltransferase I (CPT I) deficiency, Carnitine palmitoyltransferase II (CPT II) deficiency , Carnitine-acyl-carnitine (CACT) deficiency, autosomal dominant / autosomal recessive progressive extraocular Muscle paralysis (ad- / ar-PEO), infantile-onset spinocerebellar atrophy (IOSCA), mitochondrial myopathy (MM) ) Spinal muscular atrophy (SMA), growth retardation, aminoaciduria, cholestasis, iron overload, early death (GRACI) LE), and Charcot-Marie-Tooth disease type 2A (CMT2A).

[0030] In some embodiments of the methods provided herein, the endogenous mtDNA is dysfunctional. In a specific embodiment, the endogenous mtDNA encodes a mutant m In other embodiments, the endogenous mtDNA in the recipient cell comprises wild-type mtDNA. In still further embodiments, the endogenous mtDNA comprises a mitochondrial disease or In some embodiments, the endogenous mtDNA comprises a heterozygous mtDNA associated with a disorder. In a specific embodiment, the recipient cells are endogenous They have mitochondrial membranes.

[0031] In certain embodiments of the methods provided herein, the mitochondrial replacement cells are endogenous The total mtDNA copy number of recipient cells before contact with an agent that reduces the mtDNA copy number of recipient cells. - Compared to the number of have a low total mtDNA copy number.

[0032] In some embodiments, the recipient cell is an animal cell or a plant cell. In certain embodiments, the animal cell is mammalian. 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 yet a further embodiment, the recipient cells are primary cells. In a specific embodiment, the immune cell is a T cell, The cells are selected from the group consisting of phagocytes, microglial cells, and macrophages. In some embodiments, the T cells are CD4+ T cells. In other embodiments, the T cells are CD8+ T cells. In one embodiment, the T cell is a chimeric antigen receptor (CAR) T cell.

[0033] In another embodiment of the methods provided herein, exogenous mitochondria and / or The exogenous mtDNA is stable. In some embodiments, the exogenous mtDNA is stable. Alters heteroplasmy in mouse cells.

[0034] In some embodiments of the methods provided herein, the methods include administering to a subject a small molecule, a peptide, or further comprising delivering a protein.

[0035] The present disclosure provides methods for co-injecting recipient cells with exogenous mitochondria and / or exogenous mtDNA. The method further comprises contacting the recipient cells with a second active agent prior to incubating the recipient cells. Also provided herein are methods, including the use of a second active agent. is selected from the group consisting of a macromolecule, a small molecule, or a cell therapy, and a second active agent These include rapamycin, NR (nicotinamide riboside), bezafibrate, idebenone, and cis- Theamine hydrogen tartrate (RP103), elamipretide (MTP131), omaveloxolone (RTA408), K H176, batchiquinone (Epi743), thioctic acid, A0001 (α-tocopherol quinone), mitochondrion Rear CoQ10 (MitoQ), SkQ1 (Bisomitin), resveratrol, curcumin, ketogenic diet therapy, Optionally, the inhibitor is selected from the group consisting of hypoxia and activators of endocytosis. In some embodiments, the activator of endocytosis is a module of cellular metabolism. In a specific embodiment, the modulator of cellular metabolism is a modulator of nutrient starvation, In yet a further embodiment, the chemical inhibitor In yet a further embodiment, the compound or small molecule is an mTOR inhibitor. The bitters include rapamycin or a derivative thereof.

[0036] The present disclosure provides a method for (a) contacting recipient cells with an agent that reduces endogenous mtDNA copy number. (b) the agent partially reduces the endogenous mtDNA copy number in the recipient cells. and (c)(1) incubating the recipient cells for a period of time sufficient to induce the expression of the endogenous mtDNA. (2) the recipient cells from step (b) that are differentially reduced in number and (3) the exogenous mitochondria from a healthy donor. Mitochondria are used to non-invasively transfer exogenous mitochondria into the recipient cells. Co-incubate for a sufficient period of time to thereby produce mitochondria-exchanged cells: A composition comprising one or more mitochondrial replacement cells obtained by the method, Also provided is a composition wherein the doria-replaced cells contain greater than 5% exogenous mtDNA.

[0037] The present disclosure provides a method for (a) contacting recipient cells with an agent that reduces endogenous mtDNA copy number. (b) the agent partially reduces the endogenous mtDNA copy number in the recipient cells. and (c)(1) incubating the recipient cells for a period of time sufficient to induce the expression of the endogenous mtDNA. (2) the recipient cells from step (b) that are differentially reduced in number and (3) the exogenous mt cells from a healthy donor. The DNA is co-infused for a period of time sufficient to non-invasively transfer the exogenous mtDNA into the recipient cells. The mitochondrial exchange cells were then incubated with 100% ATP. The above composition of mitochondria-exchanged cells, wherein the mitochondria-exchanged cells comprise more than 5% Further provided are compositions comprising exogenous mtDNA. In one embodiment, the one or more mitochondrial replacement cells have a reduced endogenous mtDNA copy number. Approximately 1.1 times the total mtDNA copy number of recipient cells before contact with the agent that induces Contains no more than about 1.2x, 1.3x, 1.4x, 1.5x, or more of the total mtDNA copy number .

[0038] In another aspect, provided herein are methods for producing one or more mitochondrial replacement cells. A composition for use in a method for producing a medicament for the treatment of ... In some embodiments, the composition comprises one or more of a hydroxybenzoate and a second active agent. In one embodiment, the composition further comprises a recipient cell of an exogenous The composition further comprising mtDNA, exogenous mtDNA, and / or exogenous mitochondria.

[0039] In certain embodiments of the compositions provided herein, the endogenous mtDNA copy number is reduced. In some embodiments, the agent is a small molecule or a fusion protein. The fusion protein is a nucleoside reverse transcriptase inhibitor (NRTI). The protein contains an endonuclease that cleaves mtDNA and a mitochondrial targeting sequence (MTS). In some embodiments, the endonuclease cleaves wild-type mtDNA. In specific embodiments, the endonuclease is XbaI, EcoRI, BamHI, HindIII, Pst I, Cas9, zinc finger nucleases (ZFNs), and transcription activator-like effectors In some embodiments, the MTS is selected from the group consisting of: In a further embodiment, the mitochondrial matrix protein is targeted. Chondriac matrix proteins include cytochrome c oxidase subunit IV, ... and cytochrome c oxidase subunit IV. Chromium 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 endogenous mtDNA copy number The reduction is a partial reduction. In some embodiments, the partial reduction is a partial reduction of endogenous mtDNA. In a specific embodiment, the partial reduction is from about 5% to about 99%. The reduction in copy number is about 50% to about 95%. In further embodiments, the partial reduction is This is a reduction of approximately 60% to 90% in endogenous mtDNA copy number.

[0041] The present disclosure provides a method for the treatment of mitochondrial dysfunction by (a) contacting recipient cells with an agent that reduces mitochondrial function. (b) the agent partially reduces endogenous mitochondrial function in the recipient cells. (c) incubating the recipient cells for a period of time sufficient to allow the endogenous mycobacterial cells to express the endogenous mycobacterial cells; and (2) the recipient cells from step (b) in which mitochondrial function is partially reduced; Donor-derived exogenous mitochondria are introduced into the recipient cells. The cells were co-incubated for a period of time sufficient for invasive transfer, thereby allowing mitochondrial exchange. Produce a mitochondria-exchanged cell: a composition comprising one or more mitochondrial-exchanged cells obtained by the method. Also provided is a composition wherein the mitochondrial replaced cells contain greater than 5% exogenous mtDNA.

[0042] In another aspect, provided herein are methods for (a) converting recipient cells into mitochondrial cells. (b) contacting the recipient cells with an agent that reduces the recipient cells' endogenous function; The recipient cells are then transfected for a period of time sufficient to partially reduce mitochondrial function. and (c) (1) incubating the cells from step (b) in which the endogenous mitochondrial function is partially reduced. (2) exogenous mtDNA from a healthy donor is transfected into the recipient cells. and co-incubating for a period of time sufficient to non-invasively transfect the recipient cells, thereby Produce mitochondrial exchange cells: One or more mitochondrial exchange cells obtained by the method A composition of cells, wherein the mitochondrial replacement cells contain more than 5% exogenous mtDNA. In some embodiments, the one or more mitochondrial replacement cells contain endogenous mtDNA. The total mtDNA copy number of recipient cells was compared to that before contact with the agent that reduces the copy number. In total, the total mtDNA does not exceed about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, or more. Includes copy number.

[0043] The present disclosure provides compositions for use in methods of producing one or more mitochondria-replaced cells. a composition comprising an agent that reduces mitochondrial function and a second active agent; In some embodiments, the composition comprises exogenous mitochondria, one or more In yet a further embodiment, the recipient cells further comprise: The composition further comprises exogenous mtDNA.

[0044] In some embodiments of the compositions provided herein, one or more mitochondria The replacement cells contain wild-type exogenous mtDNA.

[0045] Also provided herein are compositions further comprising a second active agent. In some embodiments, the second active agent is selected from the group consisting of a large molecule, a small molecule, or a cell therapy. and the second active agent is selected from the group consisting of rapamycin, NR (nicotinamide adenine dinucleotide analogue), Boside), bezafibrate, idebenone, cysteamine hydrogen tartrate (RP103), elamipril Retide (MTP131), omaveloxolone (RTA408), KH176, vaquiquinone (Epi743), thioctic acid , A0001 (α-tocopherol quinone), mitochondrial CoQ10 (MitoQ), SkQ1 (bisomitin), Resveratrol, curcumin, ketogenic diet treatment, hypoxia, and endocytic activity In a specific embodiment, the endothelial cell is optionally selected from the group consisting of: Activators of clathrin-independent endocytosis pathways In some embodiments, the activator of endocytosis is a cla. In a further embodiment, the ATP-dependent endocytic pathway is an activator of the thrombin-independent endocytic pathway. The clathrin-independent endocytic pathway is similar to the CLIC / GEEC endocytic pathway. tract, Arf6-dependent endocytosis, flotillin-dependent endocytosis, macropinocytosis Cytosis, circular doral ruffles, phagocytosis, and trans-endocytosis. In this study, the clathrin-independent endocytic pathway is macropinocytosis. In a specific embodiment, the activator of endocytosis is a nutrient stress and and / or an mTOR inhibitor. In some embodiments, the mTOR inhibitor is Includes rapamycin or its derivatives.

[0046] In certain embodiments, the present disclosure provides a method for determining the total mtDNA copy number of one or more mitochondrial replacement cells. Further provided are compositions comprising more than 5% exogenous mtDNA. The total mtDNA copy number of these mitochondrial replacement cells contains more than 30% exogenous mtDNA. In one embodiment, the total mtDNA copy number of the one or more mitochondrial replacement cells is greater than 50%. In a further embodiment, the total mtDNA of one or more mitochondrial replacement cells is The DNA copy number includes more than 75% exogenous mtDNA.

[0047] In some embodiments of the compositions provided herein, the exogenous mitochondria In a specific embodiment, the isolated mitochondrion In some embodiments, the exogenous mitochondria and / or exogenous mitochondria are intact. In a specific embodiment, the exogenous mtDNA is allogeneic. A further contains exogenous mtDNA.

[0048] In certain embodiments of the compositions provided herein, the one or more cells are animal cells or In some embodiments, the animal cell is a mammalian cell. In an embodiment, the cell is a somatic cell. In a further embodiment, the somatic cell is an epithelial cell. In still further embodiments, the epithelial cells are thymic epithelial cells (TECs). In other embodiments, the somatic cells are immune cells. In certain embodiments, the immune cells are In a specific embodiment, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are chimeric In another embodiment, the immune cell is a phagocyte. In an embodiment, the one or more mitochondria-replaced cells are bone marrow cells. In embodiments, the bone marrow cells are hematopoietic stem cells (HSCs) or mesenchymal stem cells (MSCs).

[0049] In some embodiments of the compositions provided herein, one or more mitochondria The replacement cells are more viable than syngeneic cells with homoplasmic endogenous mtDNA. In other embodiments, the one or more mitochondrial replacement cells are used to treat cancer cell death, age-related Treatment of diseases, treatment of mitochondrial diseases or disorders, treatment of neurodegenerative diseases, diabetes or genetic disorders It is effective in treating sexual disorders.

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

[0051] Also provided herein are methods for delaying aging and / or extending lifespan in cells. (a) a composition for use in treating senescent or senescent mitochondrial disease having endogenous mitochondria; (b) isolated exogenous mitochondria from non-senescent cells; and (c) endogenous mitochondria. In some embodiments, the composition comprises: an agent that reduces the mtDNA copy number of a target gene. In some embodiments, the agent is a fusion protein. It contains an endonuclease that cleaves mtDNA and a mitochondrial targeting sequence (MTS). In some embodiments, the endonuclease cleaves wild-type mtDNA. In this study, endonucleases such as XbaI, EcoRI, BamHI, HindIII, PstI, Cas9, and GIN Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases ( In a further embodiment, the MTS is selected from the group consisting of mitochondrial In yet a further embodiment, the target is a mitochondrial matrix protein. Matrix proteins include cytochrome c oxidase subunit IV, cytochrome c oxidase oxidase subunit VIII, and cytochrome c oxidase subunit X In some embodiments, the fusion protein is selected from the senescent or senescent-prone cells. It is transiently expressed in

[0052] The present disclosure provides compositions for use in delaying aging and / or extending lifespan in cells. (a) senescent or senescent cells having endogenous mitochondria; (b) senescent cells (c) isolated exogenous mitochondria from cells that have not undergone mitochondrial function reduction; and In some embodiments, the composition further comprises: Factors that reduce chondrial function transiently reduce endogenous mitochondrial function. In other embodiments, the agent that reduces mitochondrial function is an agent that reduces endogenous mitochondrial function. In some embodiments, non-senescent cells permanently reduce endogenous function. Exogenous mitochondria derived from cerebrospinal fluid have enhanced function compared to endogenous mitochondria. do.

[0053] In some embodiments, in delaying senescence and / or extending lifespan in cells The composition for use further comprises a second active agent. the second active agent is selected from the group consisting of a macromolecule, a small molecule, or a cell therapy; The second active agent is rapamycin, NR (nicotinamide riboside), bezafibril, Idebenone, cysteamine hydrogen tartrate (RP103), elamipretide (MTP131), omabe Loxolone (RTA408), KH176, vaquiquinone (Epi743), thioctic acid, A0001 (α-tocopherol CoQ10 (MitoQ), SkQ1 (Bisomitin), resveratrol, From a group consisting of cumin, ketogenic diet, hypoxia, and endocytosis activators In some embodiments, the activator of endocytosis is It is an activator of the clathrin-independent endocytic pathway. In embodiments, the clathrin-independent endocytosis pathway is a CLIC / GEEC endocytosis pathway. ectosis pathway, Arf6-dependent endocytosis, flotillin-dependent endocytosis, Macropinocytosis, circular doral ruffles, phagocytosis endocytosis, and trans-endocytosis. In embodiments, the clathrin-independent endocytosis pathway is macropinocytosis In some embodiments, the activator of endocytosis is a trophic factor. stress and / or mTOR inhibitors. In certain embodiments, the mTOR inhibitor The compounds include rapamycin or its derivatives.

[0054] In another aspect, the present disclosure provides an isolated mitochondrial cell line comprising exogenous mitochondria derived from a healthy donor. a pharmaceutical composition comprising a population of mitochondrial exchanged cells, the cells comprising mitochondria exchanged A composition obtained by any of the methods provided herein for obtaining apoptotic cells. In another aspect, the present disclosure provides a method for producing a human hamster ovarian tumor cell line comprising: 1. A pharmaceutical composition comprising a population of isolated mitochondrial exchange cells, the cells being mitochondrial exchange cells. obtained by any of the methods provided herein for obtaining endonuclearis-exchanged cells, In some embodiments, pharmaceutical compositions are provided, comprising exogenous mtDNA derived from a healthy donor. A pharmaceutical composition comprising a population of isolated mitochondrial replacement cells having exogenous mitochondria. Also includes Doria.

[0055] For example, in some embodiments, exogenous mitochondria from healthy donors are included. The pharmaceutical composition comprises: (a) a compound that induces a reaction in a recipient cell with an agent that reduces endogenous mtDNA copy number; (b) contacting the recipient cells with the agent to partially increase the endogenous mtDNA copy number in the recipient cells; and (c)(1) incubating the recipient cells for a period of time sufficient to reduce the (2) the recipient cells from step (b) in which the endogenous mtDNA has been partially reduced and (3) healthy recipient cells; The exogenous mitochondria derived from the donor are introduced into the recipient cells in a manner that is non-invasive. The cells were co-incubated for a period of time sufficient for rapid transfer of the mitochondrial The mitochondria are obtained by a method for producing mitochondria-exchanged cells, the method comprising producing cells. In one embodiment, the cells comprise: (a) a recipient cell that has reduced mitochondrial function; (b) contacting the recipient cell with an agent that activates the endogenous mitochondria in the recipient cell; The recipient cells are incubated for a period of time sufficient to partially reduce chondrial function. and (c) (1) detecting a cell-derived cell line derived from step (b) in which endogenous mitochondrial function is partially reduced. (1) The recipient cells and (2) exogenous mitochondria from a healthy donor were transfected into each other. Co-incubation is continued for a period of time sufficient to non-invasively transfer chondria into the recipient cells. and thereby producing mitochondrial-exchanged cells. .

[0056] In other embodiments, the cells are (a) recipient cells that have a low endogenous mtDNA copy number; (b) contacting the recipient cell with an agent that disrupts the endogenous mtDNA in the recipient cell; Incubating the recipient cells for a period of time sufficient to partially reduce the copy number. and (c)(1) the recipient from step (b) in which the endogenous mtDNA is partially reduced. (2) exogenous mtDNA from a healthy donor is introduced into the recipient cells without introducing the exogenous mtDNA into the recipient cells. The cells were co-incubated for a period of time sufficient for rapid transfer of the mitochondrial In another embodiment, the cells are obtained by a method comprising producing the cells. (b) contacting the recipient cells with an agent that reduces mitochondrial function; The agent partially reduces the endogenous mitochondrial function in the recipient cells. and (c)(1) incubating the recipient cells for a period of time sufficient to induce the expression of the endogenous miR-1 receptor. (2) the recipient cells from step (b) in which mitochondrial function is partially reduced; The exogenous mtDNA from the donor is transferred non-invasively into the recipient cells. The cells are then co-incubated for a period of time sufficient to produce mitochondria-exchanged cells. and:

[0057] In certain embodiments of the pharmaceutical compositions provided herein, the cells are recipient cells. Before co-incubating the cells with exogenous mitochondria and / or exogenous mtDNA, the cells were incubated with the recipe. and a method further comprising contacting the patient cells with a second active agent. In some embodiments, the second active agent is a macromolecule, a small molecule, or a or cell therapy, and the second active agent is selected from the group consisting of rapamycin, NR (nicotinamide riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103), elamipretide (MTP131), omaveloxolone (RTA408), KH176, vatiquinone (Epi 743), thioctic acid, A0001 (α-tocopherol quinone), mitochondrial CoQ10 (MitoQ), Sk Q1 (bisomitin), resveratrol, curcumin, ketogenic diet, hypoxia, and endoscopy In a specific embodiment, the activator of mitosis is optionally selected from the group consisting of: Therefore, activators of endocytosis are modulators of cellular metabolism. In embodiments, the modulator of cellular metabolism is nutrient starvation, a chemical inhibitor, or a small molecule. In further embodiments, the chemical inhibitor or small molecule is an mTOR inhibitor. In still further embodiments, the mTOR inhibitor is rapamycin or its derivatives. Includes derivatives of.

[0058] In certain embodiments of the pharmaceutical compositions provided herein, the pharmaceutical composition is It further comprises an acceptable carrier.

[0059] In some embodiments of the pharmaceutical compositions provided herein, the cell is a T cell. In other embodiments, the cells are hematopoietic stem cells. [Brief explanation of the drawings]

[0060] (4. Brief description of the drawings) [Figure 1] FIG. 1A shows a scheme for the generation of mitochondrial exchange cells (MirCs).

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

[0062] Figure 1C shows isolated mitochondrial DNA digested at multiple sites with XbaI restriction enzyme. whereas NotI digestion of mitochondrial DNA is performed using the Cambridge Reference Sequence for Mitochondrial DNA. The results show that the sequence (CRS) yielded a single fragment as predicted.

[0063] Figure 1D shows the 5′ sequence of human mitochondrial DNA predicted by the Cambridge Reference Sequence (CRS). The four XbaIR endonuclease sites (1193, 2953, 7440, 8286, 10256) are shown.

[0064] FIG. 1E shows the fusion of MTS-green fluorescent protein using an electroporator (Nucleofector). Human dermal fibroblasts under phase contrast after uptake of (GFP) plasmid (left), green fluorescent protein Immunofluorescence (center) and composite field microscopy (right) are shown. Top, low magnification. Bottom, high magnification. rate.

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

[0066] Figure 1G shows mitochondrial-specific immunoprecipitation using tetramethylrhodamine methyl ester (TMRM). Staining shows localization of the exogenous transgene product MTS-EGFP within mitochondria.

[0067] [Figure 2] Figure 2A shows a scheduling scheme for comparing the MTS-XbaIR endonuclease method (top) with the conventional method using ethidium bromide (EtBr) (middle) compared to non-contacted cells.

[0068] Figure 2B shows the difference between the MTS-XbaIR endonuclease method and ethidium bromide treatment compared to non-contacted cells. Human β-actin (Actb) (left column) and mitochondrial DNA (mtDNA) (left column) after exposure to either of the following conditions: The right column shows the quantification of mtDNA. XbaIR also caused a greater reduction in mtDNA compared to EtBr treatment. Actb was used as a housekeeping gene.

[0069] Figure 2C shows the effect of EtBr treatment on the expression of DsRed in mitochondria compared to M. 10 shows a greater reduction in mitochondria after exposure to TS-XbaIR gene transfer.

[0070] Figure 2D shows the results of gene transfer of MTS-XbaIR or EtBr using FACS analysis with TMRM. We demonstrated semiquantitation of mitochondrial membrane potential (a surrogate marker of mitochondrial content) in cells. This indicates that MTS-XbaIR resulted in a greater reduction in mitochondria.

[0071] Figure 2E shows the time course quantification of transgene expression in the gene transfer system over a 14-day period. There are.

[0072] Figures 2F and 2G show the results before ("pre") and after ("po") puromycin selection. Fluorescence images (Figure 2F) are shown after transfection of a plasmid carrying GFP (st) and show the GFP / mitochondrial expression. Quantification of the Doria ratio (Figure 2G) demonstrated enrichment of the GFP plasmid after puromycin selection. are.

[0073] [Figure 3]Figure 3A shows a scheme of the mitochondrial exchange schedule. TF: XbaIR or mock gene transfection; Puro: puromycin for enrichment of transfected cells; U+: addition of uridine to rescue ρ(-) cells lacking mitochondrial ATP production; Mt Tx: mitochondrial transfer; NHDF: normal human dermal fibroblasts, EPC100: placental vein endothelial-derived cell line.

[0074] Figure 3B shows the difference in the number of cells expressing GFP after transfection with a negative control vector (bottom) as measured by TMRM staining. (above) shows a decline in mitochondria at 6 days after XbaIR gene transfer.

[0075] Figure 3C shows NHDF cells after gene transfection of XbaIR or GFP transfection. Mitochondrial levels estimated by qPCR of human 12S rRNA compared with nuclear β-actin levels in vacuoles Quantification of mitochondrial DNA copy number is shown. Where indicated ("Mt Tx"), mitochondrial DNA copy number is also shown. XbaIR was transferred into recipient cells. XbaIR caused a significant reduction in mitochondrial DNA. This resulted in a similar level of respirability to control-treated cells after the transfer of exogenous mitochondria. N=3, * p<0.01.

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

[0077] Figure 3E shows a series of images from the time-lapse movie shown in Figure 3D, arranged horizontally in chronological order. Showing 10 still images of;

[0078] Figure 3F shows the measurement of DsRed-labeled mitochondria by FACS analysis, which revealed The present invention ("DsRed-Mt EPC100") provides a novel method for the detection of exogenous mitochondrial DNA compared to previously described methods. It has been shown that this results in increased uptake of endorphins.

[0079] Figures 3G and 3H show the effect of antimycin on the proliferation of ρ(0) cells treated with or without antimycin. Microscopic images of DsRed-labeled mitochondria (Figure 3G) and phase contrast (Figure 3H) after mitochondrial transfer. This indicates that engulfment of exogenous mitochondria does not result in complete destruction of mitochondria. It has been shown that this did not occur in cells with

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

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

[0082] [Figure 4] FIG. 4A shows a scheme for measuring the fate of donor mitochondria after engulfment by recipient cells, using DsRed-marked mitochondria as donors and EGFP-marked cells as recipients.

[0083] Figure 4B shows the endothelial cell division in recipient cells with GFP-marked mitochondria. A sample of the mitochondria from the video was taken to observe the embedded exogenous mitochondria (shown in red). Representative images are shown. The video was recorded using ultra-precise microscopy and fused. The merged image is barely recognizable, and most of the donor mitochondria are located in the existing mitochondria. and exist separately.

[0084] FIG. 4C shows a three-dimensional reconstruction of the fusion.

[0085] Figure 4D shows the transfected cells carrying the gene encoding DsRed fused to the mitochondrial import signal. Figure 1 shows a photograph of the NHDF.

[0086] Figure 4E shows a photograph of EPC100 cells transfected with a gene encoding EGFP fused to TFAM. There are.

[0087] Figure 4F shows the results of DsRed-marked cells as recipients and TFAM-targeted EGFP recipients. Figure 1 shows the time course of mitochondrial import using mitochondrial as the host mitochondria.

[0088] Figure 4G shows exogenous TFAM transfection after exogenous mitochondria transiently contacted recipient cells. The TFAM-containing mitochondrial nuclei were stably engulfed by existing mitochondria. The mitochondria were transferred to existing mitochondria via transient contact similar to oral ingestion. This suggests that...

[0089] [Figure 5] Figure 5A shows the entire circular mitochondrial DNA with the Cambridge Reference Sequence (CRS) of human mitochondrial DNA showing hypervariable ("HV") regions 1 / 2 and five primers to identify differences between NHDF and EPC100;

[0090] Figure 5B shows the results of NHDF ctrl recipient cells (SEQ ID NO: 1), EPC100 ctrl donor cells (SEQ ID NO: 2). NHDF-derived ρ(-) cells without mitochondrial replacement (SEQ ID NO: 3), and NHDF-derived ρ(-) cells with mitochondrial replacement The DNA sequence of nucleotides surrounding hmt16362 in NHDF-derived ρ(-) cells (SEQ ID NO: 4) The sequencing data show that NHDF-derived ρ(- ) cells (SEQ ID NO: 4) in hmt16362, A of the original recipient cells was converted to G of the donor mtDNA. has been shown to change.

[0091] FIG. 5C shows the HV1 region of the human mitochondrial DNA D-loop (SEQ ID NO: 8) surrounding hmt16362. The primers used for amplification, hmt16318-F (SEQ ID NO: 6) and hmt16414-R (SEQ ID NO: 9), were NHDF-specific probes ( 5) and EPC100-specific probe (SEQ ID NO: 7).

[0092] Figure 5D shows mitochondria derived from parental NHDF and EPC100 cell lines, or EPC100 cells treated with XbaIR. Mitochondria-bearing NHDF cells (XbaIR Mt+) or XbaIR-treated EPC100 cells NHDF-specific hmtDNA (left) and EPC100-specific hmtDNA (right) in NHDF cells (XbaIR Mt-) lacking endonucleases. The figure shows the quantification of target hmtDNA (right), which allows for the use of single nucleotide polymorphism assays (SNPs). EPC100 mitochondria were successfully transferred into XbaIR Mt+ cells when assessed using It became clear.

[0093] [Figure 6] Figure 6A shows representative oxigraphies from mitochondrial function assays performed using an Oroboros Oxygraph-2k, demonstrating that NHDF cells with replaced mitochondria (ρ(-)Mt) (bottom) regain mitochondrial function compared with control NHDF cells (top) and ρ(-) NHDF cells without mitochondrial replacement (center). The instrument displays respiratory flow (pmol / sec / 1 x 106 cells, right axis) and oxygen concentration (μM, left axis) in red and blue, respectively.

[0094] Figure 6B shows the respiratory flow (routine, electron transport system (ETS), ROX) and free routine activity at each stage. Mitochondrial ATP production, proton leakage, and coupling efficiency were measured in NHDF cells (ρ(-)Mt). Mitochondrial exchange in NHDF cells was significantly higher than in control NHDF cells and NHDF cells without mtDNA exchange (ρ(-)). , which has been shown to restore mitochondrial function.

[0095] Figure 6C shows time-lapse microscopy allowing for continuous cell number estimation based on cell surface area. The photograph shows that ρ(-) cells remain quiescent for 3 to 12 days, whereas Mitochondria-exchanged cells were shown to regain their proliferative capacity after 6 days.

[0096] Figure 6D shows the protocol used to investigate the molecular mechanism of macropinocytosis. This protocol involves transfecting NHDF cells with the MTS-XbaIR-P2A-PuroR promoter. Transfected with smid, selected with puromycin, and then serum-starved the cells for 60 min. By starving or treating cells with palmitic acid (PA) or rapamycin for 24 hours, This included:

[0097] Figures 6E to 6H show the WES ( Quantification of the WES™ analysis and the corresponding WES™ blots (Figure 6F) and (Figure 6H), respectively, are shown. This results in AMPK activation and complete suppression of mTOR in ρ(-) cells. Rapa: rapamycin, PA: palmitic acid, EAA-: essential amino acid deficiency.

[0098] Figure 6I examines the effect of mTOR-mediated macropinocytosis in the setting of the mirC preparation protocol. The figure shows the protocol used to

[0099] 6J to 6L show the results with or without rapamycin treatment, or with or without palmitic acid (PA) treatment. DsR in control (top), mock-transfected (middle), and ρ(-) cells without or with Quantification of ed-labeled mitochondrial uptake (FIG. 6J and FIG. 6K) and FACS analysis (FIG. 6L) are shown. ρ(-) cells showed greater uptake of mitochondria compared to control or mock TF cells. Mitochondrial uptake was significantly increased after rapamycin treatment, whereas palmitate Phosphate reduced mitochondrial uptake in ρ(-) cells.

[0100] [Figure 7] Figure 7A shows the complete mtDNA sequence showing the Leigh syndrome-associated mutation 10158T>C in the respiratory chain complex I (CI) subunit of the ND3 gene of mitochondrial DNA.

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

[0102] Figure 7C shows the ρ(-)7SP fibroblasts with exogenous mitochondria as seen in the NHDF experiment. The results showed similar behavior in both ρ(-)7SP fibroblasts and ρ(-)7SP fibroblasts lacking exogenous mitochondria. The images shown are from a time-lapse video.

[0103] Figure 7D shows NHDF after gene transfection of XbaIR or mock transfection. Mitochondrial levels estimated by qPCR of human 12S rRNA relative to nuclear β-actin levels in cells Quantification of chondrial DNA copy number is shown. Mitochondria, where indicated, XbaIR was introduced into recipient cells, resulting in a significant reduction in mitochondrial DNA. This could be rescued by the transfer of exogenous mitochondria (n=3).

[0104] Figure 7E shows the results of the 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 mitochondrial exchange The DNA sequence of the nucleotides surrounding hmt10158 in 7SP-derived ρ(-) cells (SEQ ID NO: 15) of the ant We present sequencing data that demonstrate that 7SP ctrl cells are heteroplasmic. (majority 10158C; SEQ ID NO: 14), whereas EPC100 has a T at the same position in mitochondrial DNA. It was revealed that the ρ(-) cell stem derived from 7SP cells only had the nucleotide sequence (SEQ ID NO: 12). (SEQ ID NO: 13), whereas mitochondrial-exchanged 7SP cells showed a major wave T was shown as (SEQ ID NO: 15).

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

[0106] Figure 7G shows the hmt10158 heterozygotes in each cell group assessed by SNP assay. The figure shows the quantification of the percentage of mutant sequences that are heterozygous for the original heterozygous sequence. Despite >90% plasminogen activator, exogenous normal sequences ("healthy") are present in mitochondria. The exchange rate was found to be up to 80% in 7SP cells. In the case of the control, heteroplasmy did not change significantly and remained at approximately the same ratio.

[0107] Figures 7H and 7I show three mitochondrial transfer events in 7SP cells treated with mock controls and subjected to mitochondrial transfer. Heteroplasmy level percentage (Figure 7H) and absolute mtDNA heteroplasmy in independent experiments Quantitation of copy number (Figure 7I) is shown.

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

[0109] [Figure 8] Figure 8A shows photomicrographs of ρ(-) mitochondrial-exchanged 7SP fibroblasts over time compared with original 7SP fibroblasts and ρ(-) 7SP fibroblasts, revealing that proliferation of mitochondrial-exchanged cells was restored to near control levels.

[0110] Figure 8B shows 7SP fibroblasts with mitochondrial exchange, ρ(-) 7SP fibroblasts, and ρ(-) Time-lapse estimated cell proliferation in 7SP fibroblasts, which allows for the identification of ρ(-)7SP While the fibroblasts were in a quiescent state, the mitochondria-exchanged 7SP cells were in a quiescent state around day 12. It was found that the cell proliferation was restored to a level equivalent to that of 7SP fibroblasts.

[0111] Figure 8C shows senescence in 7SP fibroblasts at approximately population doubling level (PDL) 25, which In ρ(-)7SP fibroblasts, the replacement of healthy mitochondria was performed at PDL 8 and extended to approximately PDL 63. These results demonstrate that ρ(-)7SP fibroblasts with healthy mitochondrial exchange exhibited increased lifespan.

[0112] Figure 8D shows that increased PDL leads to increased cell size (left), which in turn leads to increased mitochondrial exchange. This shows that the effect later reverses and is maintained even after PDL 50 (right).

[0113] Figure 8E shows a short timeline that distinguishes cells of different origins and identifies contamination with different cell types. Dem repeat (STR) assays are shown. The STR pattern in the 7SP fibroblasts was completely identical to that in the original 7SP fibroblasts. Ta.

[0114] Figure 8F shows the results of 7SP fibroblasts and mitochondria for various PDLs compared to HeLa and EPC100. RT-PCR quantification of telomerase in rearranged cells was shown, which indicates that the cells are cancer cells. It was shown that the cells were not transformed.

[0115] [Figure 9] Figure 9A shows oximetry in 7SP fibroblasts at various PDLs after mitochondrial replacement with Oroboros O2k according to the controlled coupling protocol (CCP). The kinetics showed that mitochondrial function declined early in the PDL, then gradually recovered, eventually surpassing that of intact 7SP fibroblasts as a control.

[0116] 9B and 9C show respiratory flow (routine, electron transport system (ETS), ROX), free routine activity (min, ATP production), proton leakage and coupling efficiency (Figure 9B), and flux control ratio (F After approximately PDL30, mitochondrial exchange was observed by CR), ROX / E, L / E, R / E, and (RL) / E (Figure 9C). This shows that the control level was almost restored in the cells (ρ(-)Mt).

[0117] [Figure 10] Figure 10A shows microscopy images of NHDF, 7SP, and 7SP MirC cells under basal conditions or after reperfusion with H2O2, demonstrating that 7SP cells are more sensitive to H2O2 than NHDF cells, whereas 7SP MirC cells are not.

[0118] Figures 10B-10D show Annexin V-positive cells and proiodide after untreated or treatment with H2O2. FACS analysis of pydium iodide (PI)-positive cells (Fig. 10B) and quantification of annexin V-positive cells (Fig. 10C) Quantification of propidium iodide-positive cells (PI; Fig. 10D) shows that 7SP cells were NHDF cells. This indicates that the 7SP MirC is more sensitive to H2O2 than the 7SP MirC. do.

[0119] FIG. 10E shows microscopic images of NHDF, 7SP, and 7SP MirC cells under basal or starvation conditions (EAA-). This indicates that 7SP cells are more sensitive to starvation conditions than NHDF cells. However, 7SP MirC shows that this is not the case.

[0120] Figures 10F-10H show FACS analysis of Annexin V-positive and PI-positive cells untreated or after starvation ( Figure 10F) and quantification of Annexin V-positive cells (Figure 10G) and PI-positive cells (Figure 10H). This is because 7SP cells are more sensitive to starvation conditions than NHDF cells, whereas 7SP cells are more sensitive to starvation conditions than NHDF cells. MirC shows that this is not the case.

[0121] [Figure 11] Figure 11 shows quantification of the expression levels of representative SASP cytokines IL-6 and IL-8, the chemokine CXCL-1, and the growth factor ICAM1 in NHDFs, 7SP fibroblasts, and 7SP fibroblast-derived MirC cells, whose PDLs are similar (approximately 15–20). This shows a significant decrease in IL-6, indicating restoration of SASP in MirCs. GAPDH was used for normalization.

[0122] [Figure 12] FIG. 12A shows a scheme for the generation of induced pluripotent stem cells (iPSCs) from mitochondrial-exchanged 7SP fibroblasts.

[0123] 12B to 12D show the results of 7SP fibroblasts, 7SP fibroblast-derived MirC, or 7SP fibroblast-derived MirC. Alkaline phosphatase activity as an indicator of iPSCs generated from either the mock transfectants or the Microscopic examination of AP-stained cells (Fig. 12B, left) shows the ATP staining and quantification. Panel: 7SP fibroblasts, middle panel: 7SP fibroblast-derived MirC, right panel: 7SP line fibroblast mock transfectants) and macroscopic microscopy (Figure 12C, left panel; 7SP line Fibroblasts, middle panel; 7SP fibroblast-derived MirC, right panel: mock 7SP fibroblasts Transfectants) and quantification of AP-stained cells (Fig. 12D) showed that NHDFs or We found that mitochondrial exchange in either 7SP fibroblasts resulted in an increase in AP staining. It was revealed that:

[0124] Figure 12E shows colony formation of iPSCs derived from mitochondrial-exchanged 7SP fibroblasts. Three representative colonies 75 and 170 days after transfection of reprogramming factors. photograph.

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

[0126] Figure 12G shows the effect of 7SP fibroblasts on the cellular fate of iPSCs in comparison with the original 7SP fibroblasts and standard human iPSCs (201B7) as a reference. Mitochondrial DNA copy number in iPSCs derived from SP fibroblast-derived MirCs is shown. This allows iPSCs to retain a limited number of mitochondrial DNA fragments similar to those in standard human iPSCs (201B7). It was revealed that they have mitochondrial DNA of

[0127] Figures 12H and 12I show the results of 7SP fibroblast-derived MirC after 170 days of reprogramming treatment. The percentage of heteroplasmy (Figure 12H) and absolute mtDNA copy number ( Figure 12I) which shows that iPSC-forming 7SP fibroblast-derived MirCs are at least Three colonies also showed negligible levels of mutant genome sequences, total mtDNA degradation, and Nearly 100% donor mtDNA was found, and heteroplasmy in MirC was observed. It has been suggested that this may be different from mitochondrial replacement therapy in IVF, as the mitochondrial conversion rate returns to the original state. It was.

[0128] [Figure 13] FIG. 13A shows a scheme of the protocol for mitochondrial transfer from donor cells to recipient cells, where the donor and recipient cells are from different life stages.

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

[0130] Figure 13C shows the mitochondrial exchange cells (MirC) (mitochondrial exchange cells derived from "young" TIG1 donor cells). SNP uptake in cells derived from "old" NHDF recipient cells with mitochondrial import of ribosomal RNA Quantification of hmt16145 heteroplasmy levels (%) by SEI, which indicates that TIG1 In NHDF-derived MirC cells with mitochondrial exchange from donor cells More than 90% of the mtDNA in NHD is hmt16145 G (i.e., derived from TIG1 mtDNA), whereas F It was shown that 100% of the mtDNA in ctrl cells was hmt16145 A.

[0131] Figure 13D shows the T cells transfected with MTS-GFP ("mock") or MTS-XbaIR ("MirC") Co-incubated with exogenous mitochondria from IG1 donor cells or transfected with Population doubling level (PDL) in untransfected ("Ctrl") and recipient NHDF cells Quantification of doubling time (hours) versus time (days) (left) and population doubling level (right) is shown. The "old" normal PDL is shown by the PDL shift toward the left (left) and the PDL shift toward the right (right). "Young" donor TIG1 embryonic lung cells into human dermal fibroblast (NHDF) recipient cells (PDL 41) MirC with (PDL 10) showed an extension of lifespan.

[0132] Figure 13E shows the results of transfecting cells with MTS-GFP and either importing them into mitochondria ("mock"). " ), transfected with MTS-XbaIR and imported into mitochondria (" MirC "), or untransfected ("Ctrl"), normal human dermal fibroblasts. Quantification of population doubling level (PDL) versus time (days) (left) and doubling time (hours) versus population doubling level (right) is shown. As shown by the downward PDL movement (left) and the leftward PDL movement (right), Mitochondria from "old" donor cells (PDL 49) to "young" recipient cells (PDL < 21) Rear translocation showed a decrease in lifespan.

[0133] [Figure 14] FIG. 14A shows the quality assessment of mRNA produced by in vitro transcription as determined by electrophoresis of MTS-EGFP and MTS-XbaIR mRNA.

[0134] Figure 14B shows the MTS- This shows strong expression of the GFP transgene.

[0135] FIG. 14C shows T cells after transfection with MTS-GFP mRNA by electroporation. FACS analysis of GFP expression in T cells was performed, revealing that GFP expression was present in almost all T cells. It was revealed that it exists.

[0136] Figure 14D shows FACS analysis of DsRed-labeled mitochondria, which was obtained using the MTS-XbaIR conjugate. The construct robustly degraded endogenous mitochondria, whereas MTS-GFP did not. This shows that

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

[0138] FIG. 14F shows the results of electroporation (EP) at 4 hours, 2 days, 4 days, 6 days, and 8 days. Evaporated control cells (upper panel) and electroporated MTS-GFP cells (lower panel) The fluorescent images of the MTS-GFP constructs (top panel) are shown, demonstrating that the MTS-GFP constructs were electroporated. High expression was observed within 4 hours of transfection and was nearly absent by day 6.

[0139] Figures 14G and 14H show electrophoretic analysis of GFP relative to GAPDH in cells receiving MTS-GFP mRNA. The dynamics (Fig. 14H) and quantification (Fig. 14G) of the expression of α-glucan-1-phosphate dehydrogenase (α-glucan-1-phosphate dehydrogenase) are shown. Peak expression occurred on day 4, and expression continued until day 6. It has already disappeared.

[0140] Figure 14I shows the XbaIR transcript levels at 4 hours, 2 days (d2), 4 days (d4), 6 days (d6), and 8 days (d8). Quantitation of the endonuclease transcripts was shown, with the highest expression occurring 4 hours after transfection. This shows that

[0141] FIG. 14J shows quantification of mitochondrial content (12S rRNA) in cells subjected to MTS-XbaI. This resulted in a reduction of mitochondria to approximately 30% by day 2 and a decrease in mitochondria throughout the experimental period. It has been shown to be maintained at less than 20% throughout the body.

[0142] [Figure 15] Figure 15A shows a scheme of the MirC protocol for human primary T cells, including electroporation on day 0, analysis on day 2, mitochondrial (mt) transfer on day 7, SNP assays on days 9 and 14, and ddPCR heteroplasmy assay on day 14.

[0143] Figure 15B shows human primary NH T cell control recipient cells (top; SEQ ID NO: 23) and EPC100 control donor cells. hmtDNA 218 in the HV1 region of the human mitochondrial DNA D-loop in 16-cells (bottom; SEQ ID NO: 24) and DNA sequencing data for nucleotides surrounding hmtDNA 224. DNA 218 and hmtDNA 224 were C / C (SEQ ID NO: 23) for T cells and EPC100 cells, respectively. and T / T (SEQ ID NO: 24).

[0144] Figure 15C shows the human mitochondrial DNA D-loop surrounding the SNPs of hmtDNA 218 and hmtDNA 224. The primers hmtHV1-F (SEQ ID NO: 26) and hmtHV1-F (SEQ ID NO: 25) were used to amplify the HV1 region of the ribosomal RNA. HV1-R (SEQ ID NO: 27) set, as well as a set of SNP assay primer 1-F (SEQ ID NO: 40), SNP assay primer 1-R (SEQ ID NO: 41), -terminal VIC-labeled EPC100-specific probe (SEQ ID NO: 38), and N-terminal FAM-labeled T cell-specific probe The fragment (SEQ ID NO: 39) is shown.

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

[0146] Figure 15E shows the quantification of respirometry experiments performed with Oroboros O2k. The results showed that ATP production and coupling efficiency were restored in human T cell-derived MirC. ρ(-) human T cells generated by XbaIR mRNA transfection via microporation were The loss of ATP production was maintained throughout the entire period.

[0147] Figures 15F and 15G show representative raw data using the Coupling Control Protocol (CCP). This indicates that MirC T cells are able to restore mitochondrial respiration. are.

[0148] [Figure 16] Figure 16A shows the comparative viability (left panel) or CD3 expression (right panel) of mouse primary T cells cultured in RPMI1640 (top) or TexMACS (bottom) on days 2 (left side of the left panel), 4 (middle of the left panel), and 6 (right side of the left panel), demonstrating that RPMI1640 resulted in greater viability and cell numbers, as well as a slight increase in CD3 expression, compared to TexMACS culture medium.

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

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

[0151] FIG. 16D shows quantification of 12S rRNA levels in MTS-XbaIR electroporated T cells. , which showed that mouse mtDNA was reduced by approximately 60% by day 4.

[0152] Figure 16E shows MirC expression in T cells with mitochondrial co-incubation on day 5. 1 shows a scheme of the protocol used for the production of

[0153] Figure 16F shows the reaction after co-incubation with isolated DsRed-labeled mitochondria. Figure 1 shows a 48-hour FACS analysis of engulfed DsRed-labeled mitochondria in human T cells. This resulted in a 0.43% increase compared to control cells without electroporation (i.e., "addition"). Compared with the T cells expressing exogenous mitochondria in MTS-XbaIR (right), the positive rate was significantly higher (9.7 3%) was revealed.

[0154] [Figure 17] FIG. 17A shows DNA sequencing data of the nucleotides surrounding ND1 in mouse mtDNA C57BL6 recipient cells with genotypes mmt2766-A and mmt2767-T ("BL6"; top; SEQ ID NO: 34) and the nucleotides surrounding ND1 in NZB donor cells with genotypes mmt2766-G and mmt2767-C (bottom; SEQ ID NO: 35).

[0155] FIG. 17B shows the polymorphic nucleotide mmt Used for amplification of ND1 of mouse mitochondrial DNA (SEQ ID NO: 32) encompassing 2766 and mmt2767 The primer set 2716-F (SEQ ID NO: 28) and 2883-R (SEQ ID NO: 33) and the BL6 specific A differential probe (SEQ ID NO: 29) and a NZB-specific probe (SEQ ID NO: 31) were used, allowing absolute quantification. To clone the nucleotide sequence into a plasmid for generating a standard curve that enables The BamH1-mND1-F primer (SEQ ID NO: 30) used is shown. The ND1 peptide sequence is also shown. (SEQ ID NO: 47).

[0156] Figure 17C shows control electroporation (columns 1 and 2, respectively) or MTS-XbaI electroporation. Poration and co-incubation with isolated mitochondria from NZB cells (and Mouse mtND1 heterozygotes in BL6 recipient cells after 7 and 12 days (rows 3 and 4, respectively). Quantification of plasminogen activator levels is shown. Basal levels in BL6 (column 5) and NZB (column 6) cells were used as controls. and measured.

[0157] FIG. 17D shows the treatment of old mouse cells with MTS-XbaIR mRNA to generate MirC and young mouse cells. Measurement of telomere length after co-incubation with exogenous mitochondria from healthy donor cells The results show a linear correlation between the number of MirCs in the young and old cells (Y to O), which indicates the relative abundance of MirCs in the old parent cells. Increased telomere length was observed.

[0158] Figure 17E shows the SASP-related cytokines CXCL1, ICAM, and CXCL1 in aged parental T cells or MirC-derived T cells. The measurement of CXCL1, IL-6, and IL-8 was shown, which demonstrated that CXCL1 and IL-6 were more abundant in MirC-derived T cells. It became clear that there was little

[0159] Figure 17F shows the DNA damage in MirC and parental T cells using histone 2A (H2A) phosphorylation antibody. This shows that the DDR positivity rate was significantly higher than that of the original T cells (4.75%). The MirC concentration was shown to be low (1.53%).

[0160] [Figure 18] Figure 18A shows a scheme of in vivo ACT experiments using old mice with ACT of T cells derived from young mice (group 1), old mice with ACT (group 2), or old mice with ACT of MirC derived from T cells of old mice transferred with exogenous mitochondria from young mice (group 3).

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

[0162] Figure 18C shows the body weight of the mock, young T cell, or MirC groups, which indicates the body weight at 25 days. It was revealed that no significant differences were observed among the three groups during the experimental period.

[0163] Figures 18D and 18E show quantification of individual (Figure 18D) and average (Figure 18E) cancer mass size. This suggests that the MirC group reduced the size of tumor clusters to a level comparable to that of young T cells. In contrast (lower line), the sham group increased tumor mass (upper line) throughout the experimental period. It became clear.

[0164] FIG. 18F shows the protocol used to analyze the presence of infused T cells in animals. The scheme is shown.

[0165] Figure 18G shows FACS analysis of peripheral blood (left panel) or spleen (right panel). A negative control using GFP transgenic mice (upper left panel) and a positive control using GFP transgenic mice (upper left panel) Controls (lower left panel) were made for both peripheral blood and spleen. Positive T cells expressing GFP fluorescence were identified. The incidence rates were found to be 0.057% and 0.9% in both peripheral blood and spleen, respectively.

[0166] FIG. 18H shows immunofluorescence images of transferred T cells detected in mice 6 days after transplantation.

[0167] Figure 18I shows the 1 x 10 7 or 2 x 10 7 The exogenous levels in peripheral blood (PB) or spleen after injection of 100 cells The percentage of chimerism after infusion of competent T cells is shown.

[0168] [Figure 19] Figures 19A and 19B show evaluation of MTS-GFP transfection into hematopoietic cells (HSCs) by microscopy (Figure 19A) or FACS (Figure 19B) using the X-001, Y-001, and T-030 programs (MTS-GFP1, 2, and 3, respectively) or pmax GFP as a positive control or Ctl EP as a negative control, indicating that MTS-GFP1 was the optimal protocol for electroporating HSCs.

[0169] Figure 19C shows the results of co-incubation with DsRed-labeled mitochondria from EPC100 cells at 48 hours. 3-D confocal fluorescence imaging of bone marrow-derived Sca-1 cells after incubation, which allows for the identification of exogenous It has been shown that mitochondria are engulfed.

[0170] Figure 19D shows quantification of mitochondrial import efficiency by FACS analysis of DsRed fluorescence. This revealed that approximately 10% of the Sca-1 subpopulation exhibited a rightward shift in fluorescence. .

[0171] Figure 19E shows the results of co-incubation with exogenous mitochondria on day 4 and SNP assay on day 6. Therefore, by analyzing MirC, we were able to clarify the scheme used to generate HSC-derived MirC. It shows.

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

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

[0174] FIG. 19H shows the scheme used to evaluate HSC-derived MirCs.

[0175] Figure 19I shows the expression of MirC from mouse KSLC or parental recipient BL6 cells or NZB donor cells. Quantification of the percentage of mouse mtND1 heteroplasmy levels is shown, which MirC-derived HSCs were transfected with MTS-XbaI mRNA by electroporation 6 days after donor - It has been shown to express 99.9% of the polymorphic genotypes of cells.

[0176] [Figure 20] Figure 20A shows a 2-D plot of droplet digital PCR results from analyzing mutant and non-mutated mtDNA sequences in normal human skin fibroblasts for tRNA Leu 3243 A>G, showing detection of only the non-mutant sequence (lower right quadrant) but not the mutant sequence (upper left quadrant).

[0177] Figure 20B shows the sequence of the mutated and non-mutated mtDNA compared with that of a normal human mtDNA for ND3 10158 T>C. Shown is a 2-D plot of droplet digital PCR results analyzed on skin fibroblasts. , which indicates only the detection of non-mutant sequences (lower right quadrant) and the detection of mutant sequences (lower right quadrant). Not shown (top left quadrant).

[0178] FIG. 20C shows the sequence of mutated and non-mutated mtDNA compared with normal human mtDNA for ATP6 9185 T>C. Shown is a 2-D plot of droplet digital PCR results analyzed on skin fibroblasts. , which indicates only the detection of non-mutant sequences (lower right quadrant) and the detection of mutant sequences (lower right quadrant). Not shown (top left quadrant).

[0179] Figure 20D shows the sequences of mutated and non-mutated mtDNA in MtDNA with the mtDNA A3243G mutation. 2-D projection of droplet digital PCR results analyzed on primary skin fibroblasts from an ELAS patient. This indicates that most cells have homoplasmy of the mutant mtDNA. This indicates that (top left quadrant).

[0180] Figure 20E shows the sequences of mutated and non-mutated mtDNA in complex I, mtDNA T10 of the ND3 gene. Droplets analyzed in primary skin fibroblasts from a patient with Leigh syndrome carrying the 158C mutation Figure 1 shows a 2-D plot of the results of the digital PCR, which identifies a small population of double-positive cells. The population has heteroplasmy at the single cell level (lower right quadrant), and the majority of the population has mutant mt There are populations with homoplasmy of non-mutated mtDNA (bottom right) and no populations with homoplasmy of non-mutated mtDNA. It was shown that (bottom left) DETAILED DESCRIPTION OF THE INVENTION

[0181] (5. Detailed Description of the Invention) Provided herein are methods suitable for clinical use that do not require complete removal of endogenous mtDNA. The mitochondrial exchange cells (MirCs) can be generated using a reagent that can be optionally used to Furthermore, in certain embodiments, the methods provided herein are novel and improved methods for Provided are methods for administering a therapeutically effective amount of MirC produced using the methods provided herein. This is a treatment method that includes:

[0182] Also provided are one or more mitochondria obtained by the methods provided herein. In one embodiment, the composition comprises exogenous mitochondria. a second active agent that enhances the uptake of exogenous mtDNA, exogenous mtDNA, or a combination thereof; and / or reduce endogenous mtDNA copy number or reduce endogenous mitochondrial function In a further embodiment, the composition may comprise an agent that induces exogenous mycobacterial activity. mitochondrial and / or exogenous mtDNA, one or more recipient cells, or a combination thereof. In one specific embodiment, provided herein is a method for producing a medicament comprising the steps of: Methods and compositions for use in treating diseases or disorders associated with dysfunctional mitochondria However, the methods and compositions provided herein may slow aging or , to extend the lifespan or enhance the function of cells with functional mitochondria. It is understood that the present invention can be applied to the replacement of dysfunctional mitochondria and is not limited to the replacement of dysfunctional mitochondria. Additionally, the methods and compositions provided herein can be used, for example, to generate disease models. In this study, functional mitochondria were compared with dysfunctional or exhausted exogenous mitochondria. It can also be exchanged for.

[0183] (5.1 Definition) Unless otherwise defined, this includes technical and scientific terms used in this application. All terms 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 in this specification and the experimental methods described below is widely known and and is commonly used in related fields.

[0184] As used herein, the term "mitochondrial exchange cell" or MirC refers to an endocrine replacement of endogenous mitochondria and / or mtDNA with exogenous mitochondria and / or mtDNA For example, the exemplary Mitochondrial Exchange Cell (MirC) endogenous mtDNA encoding dysfunctional mitochondria, e.g., mitochondrial disease or is composed of mtDNA derived from a subject with a disorder and exogenous mtDNA encoding functional mitochondria. , e.g., involving replacement with mtDNA from a healthy subject. An exemplary MirC is a gene encoding an endogenous mitochondrial It may also include cells in which the mitochondria have been replaced with exogenous mitochondria. However, replacement of endogenous mitochondria and / or mtDNA can be achieved, for example, by using a different cell-derived, e.g. , functional exogenous mtDNA from healthier cells from younger subjects, For example, functional endogenous mtDNA from aged cells may be replaced. For example, to mimic a mitochondrial disease or disorder, it is understood that healthy endogenous Dysfunctional exogenous mitochondria and / or mtDNA It is further understood that the replacement may also be with a replacement of all endogenous mitochondria in the cell. It is not necessary to completely replace the mitochondria, and exemplary mitochondria and / or mtD NA exchange: approx. 5% or more, approx. 10% or more, approx. 20% or more, approx. 30% or more, approx. 40% or more, approx. 50% or more , about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 95% or more It involves replacement of endogenous mitochondria and / or mtDNA.

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

[0186] As used herein, the term "healthy donor" refers to a donor who is also a patient with mitochondrial disease. or donors without disabilities, age-related diseases, or otherwise dysfunctional mitochondria. In a preferred embodiment, healthy donors are It has wild-type mtDNA sequences compared to the Cambridge reference sequence of the genome.

[0187] As used herein, the terms "treat," "treating," and "treatment" The term refers to a decrease in the severity, progression, spread, and / or frequency of symptoms, elimination of symptoms and / or underlying causes. "Cure" refers to the prevention of the occurrence of damage, symptoms and / or their underlying causes, and the amelioration or repair of damage. "Treatment" is intended to include therapeutic treatment and prophylactic or suppressive measures for a disease, disorder, or disorder. It is illustrated.

[0188] As used herein, the term "agent" refers to a compound that inhibits mtDNA depletion. As used, refers to an enzyme or compound that can degrade mtDNA. The agent may cleave mtDNA at one or more sites without causing toxicity in the recipient cell. However, the agent may also inhibit mtDNA synthesis or Alternatively, enzymes or compounds that selectively promote mitochondrial degradation may also be included.

[0189] As used herein, the terms "reduce" or "decrease" mean that As the term is defined herein, typically, a decrease of at least 5% compared to a reference level a decrease, e.g., by at least about 10%, or at least about 20%, or at least about 30%, or At least about 40%, or at least about 50%, or at least about 60%, or at least about 70% %, or at least about 80%, or at least about 90% reduction, or any value between 5% and 99% As used herein, partial reduction or partial reduction of endogenous mtDNA Agents or depletion that cause the complete depletion of all endogenous mtDNA (i.e., ρ0 cells) It is understood that the term "increase" as used herein does not necessarily mean Typically, an increase of at least 5%, for example, at least about 10%, or at least about 20%, or less at least about 30%, or at least about 40%, or at least about 50%, or at least about 60% , or at least about 70%, or at least about 80%, or at least about 90%, or more than 90% It means an increase.

[0190] As used herein, the term "endogenous" means originating from within or originating from within. For example, endogenous mitochondria are those that are native to the cell. These are mitochondria.

[0191] As used herein, the term "exogenous" refers to a substance that is not native to the host. refers to cellular material (e.g., mitochondria or mtDNA) that is not derived from the cell, e.g., externally derived cellular material. "External" usually means from a different source, e.g., from the host where the mitochondrial genome resides. Mitochondria are classified as mitochondrial mitochondria when they originate from a different cell type or a different species than the host cell or host mitochondria. The rearrangement genome is exogenous to the host cell or host mitochondria. "Sexuality" refers to mitochondria that are taken out of mitochondria, manipulated, and then put back into the same mitochondria. It can also refer to the chondrial genome.

[0192] As used herein, the term "sufficient period of time" refers to an amount that produces a desired result. A sufficient period of time may vary depending on, but is not limited to, the temperature, the amount of reagents used, and the cell type. It is understood that the protocol will vary depending on the experimental conditions, including the amount of time required to complete the assay. These are provided throughout as "intermediate" guidelines and will be readily apparent to those skilled in the art without undue experimentation. It may be possible to specify a period of time that is sufficient.

[0193] As used herein, the term "majority" means a quantity that is greater than or equal to the quantity being compared. It is also intended to mean the greatest amount. The percentage of the total population is about 50% or more, about 60% or more, about 70% or more, about 80% or more, including any integers therebetween. % or more, or about 90% or more, or any integer greater than about 95% or more. The percentage depends on the total population being compared and is less than 50% when three or more groups are compared. It is understood that the amount may be any amount.

[0194] As used herein, the term "non-invasively" refers to the introduction of exogenous material. When used in conjunction with an invasive device (e.g., nanoblade or electroporation) using chemicals such as centrifugation, physical forces (e.g., centrifugation), or harmful culture conditions (e.g., heat shock). In a preferred embodiment, the non-invasive transfer procedure is , which involves the co-incubation of recipient cells and donor mitochondria.

[0195] As used herein, the term "subject in need of mitochondrial replacement" refers to a The term refers to a condition in which a person has dysfunctional mitochondria or is predisposed to have dysfunctional mitochondria. A subject in need of mitochondrial replacement is intended to mean a subject who is asymptomatic. Patients in need of mitochondrial replacement may be in a state of The elephant may also be asymptomatic and in need of treatment. The subject in need of mitochondrial replacement may have an age-related disease or a mitochondrial disease or disorder. Having dysfunctional mitochondria is not the result of harm.

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

[0197] As used herein, the term "effective amount" refers to an amount of a compound that inhibits heteroplasmy and / or for regulating, treating, or ameliorating any disease or disorder associated with dysfunctional mitochondria An effective amount refers to an amount of the composition of the present invention that is effective. Thus, an effective amount is, for example, a therapeutically effective amount. or a biologically effective amount, which refers to an amount effective for a biological effect. The terms "therapeutically effective amount" and "effective amount" refer to an amount that improves overall treatment or reduces symptoms or reduces or prevents the cause of a disease or disorder or increases the therapeutic efficacy of another therapeutic agent The amount of a given composition corresponding to such an amount can be, for example, For example, a given composition, pharmaceutical formulation, route of administration, type of disease, disorder, or disorder, treatment, Although this will depend on various factors, such as the identity of the subject or host being used, it is nonetheless well known to those skilled in the art. As defined herein, the activity of an agent can be determined routinely by A therapeutically effective amount can be readily determined by one of ordinary skill in the art by routine methods known in the art. It can be done.

[0198] As used herein, the term "age-related disease" refers to any condition that results from aging. These diseases include, but are not limited to, osteoporosis, bone loss, and arthritis. , joint stiffness, cataracts, macular degeneration, metabolic diseases including diabetes mellitus, Alzheimer's disease and Neurodegenerative diseases including Parkinson's disease, immunosenescence, and atherosclerosis and lipids The term "age-related diseases" includes neurodegenerative diseases, e.g. For example, Alzheimer's disease and related disorders, ALS, Huntington's disease, Parkinson's disease, and It further includes cancer.

[0199] As used herein, the term "autoimmune disease" refers to an autoimmune disease that affects an individual's own tissues, organs, or tissues. A disease or disorder resulting from, or manifestation of, or arising as a result of, an immune response to Autoimmune diseases are intended to mean conditions in which the immune system is affected by an autoimmune antigen or its epitope. Refers to a condition caused by or exacerbated by the production of autoantibodies that react with the antibody. Autoimmune diseases can be tissue or organ specific, or they can be global. Systemic autoimmune diseases include connective tissue diseases (CTDs) ), such as systemic lupus erythematosus (SLE), mixed connective tissue disease, systemic sclerosis, and Examples include myofasciitis (PM), dermatomyositis (DM), and Sjogren's syndrome (SS). Common autoimmune diseases include rheumatoid arthritis and antineutrophil cytoplasmic antibody (ANCA) polyangiitis. Further examples include:

[0200] As used herein, the term "genetic disease" refers to an abnormality in the nuclear genome, e.g., For example, it refers to a disease caused by a mutation. Exemplary genetic diseases include Hatch Johnson-Gilford-Progeria syndrome, Werner syndrome, and Huntington's disease These include, but are not limited to:

[0201] As used herein, the term "cancer" includes solid cancers and hematogenous cancers. The terms "cancer" and "cancerous" refer to uncontrolled cell growth, typically but not limited to: The term "condition" refers to or describes a physiological state in mammals characterized by:

[0202] As used herein, the terms "mitochondrial disease or disorder" and "mitochondrial The terms "disorder" and "mitochondrial dysfunction" are interchangeable and refer to a mitochondrial disorder that causes an energy deficiency in an area of ​​the body. Mitochondrial diseases are a group of diseases caused by genetic or acquired damage to the mitochondrial system. Exemplary organs affected by disease or disorder include organs that consume large amounts of energy. Organs, such as the liver, muscles, brain, eyes, ears, and heart, can be affected. Weakness, fatigue, and problems with the heart, ears, and various other systems are common.

[0203] As used herein, the term "mitochondrial DNA abnormality" refers to a gene whose product is a mitochondrial DNA abnormality. Mitochondrial genetic alterations that are localized in mitochondria and are not observed in cells of healthy subjects Exemplary diseases associated with mitochondrial DNA abnormalities include: , chronic progressive external ophthalmoplegia (CPEO), Pearson syndrome, Kearns-Sayre syndrome (KSS), diabetes Diarrhea and Deafness (DAD), Leber's Hereditary Optic Neuropathy (LHON), LHON-plus, Neuropathy , ataxia, and retinitis pigmentosa syndrome (NARP), caused by mutant mtDNA Maternally inherited Leigh syndrome (MILS), also known as Leigh syndrome, mitochondrial encephalomyopathy, Lactic acidosis and stroke-like episodes (MELAS), myoclonic epilepsy and ragged-red fibers Disease (MERRF), Familial Bilateral Striatal Necrosis / Striatonigral Degeneration (FBSN), Luft's Disease, Aminoglycosides These include mitochondrial DNA multiple deletion syndrome (MDNA multiple deletion syndrome), and mitochondrial DNA multiple deletion syndrome (MDD).

[0204] As used herein, "nuclear DNA" in the context of a mitochondrial disease or disorder The term "abnormality" refers to a mutation or coding region 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 disorder, sensory ataxic neuropathy, dysarthria, ophthalmoplegia (SAN) Leukoencephalopathy with brainstem and spinal cord involvement and elevated lactate (LBSL), coenzyme Q10 deficiency, Leukemia syndrome (caused by nuclear mutations), mitochondrial complex abnormalities, fumarase deficiency α-Ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl-CoA ligase pyruvate dehydrogenase complex deficiency (PDHC), pyruvate carboxylate Carnitine palmitoyltransferase deficiency (PCD), carnitine palmitoyltransferase I (CPT I) deficiency, carnitine Carnitine palmitoyltransferase II (CPT II) deficiency, carnitine acyl-carnitine (C ACT) deficiency, autosomal dominant / autosomal recessive progressive external ophthalmoplegia (ad- / ar-PEO), infantile-onset spinal cord injury Myocerebellar atrophy (IOSCA), mitochondrial myopathy (MM), spinal muscular atrophy (SMA), growth retardation, Aminoaciduria, cholestasis, iron overload, and early death (GRACILE) and Charcot-Marie syndrome One example is Cox's Tongue-Mouth disease type 2A (CMT2A).

[0205] As used herein, the term "dysfunctional mitochondria" refers to a group of mitochondria that lack functional mitochondria. Mitochondria are the opposite of chondria. Exemplary dysfunctional mitochondria include: Mitochondria that are unable to synthesize ATP by oxidative phosphorylation or synthesize insufficient amounts of ATP As used herein, the term "functional mitochondria" includes The term mitochondria refers to the cells that consume oxygen and produce ATP.

[0206] As used herein, the term "mutation" refers to a variant ("mutant"). Refers to any change in genetic structure that results in a morphology (also called a mutation). Mutations can be single base changes in DNA or deletions or insertions of larger segments of genes or chromosomes. In some embodiments, the mutation may be can affect the function or the resulting protein. A single nucleotide mutation (i.e., point mutation) in the coding region of a gene can lead to different This can result in a codon encoding an amino acid other than the amino acid (i.e., a missense mutation). The different amino acids can change the structure of the protein, and In certain circumstances, as described in the specification, the function of organelles such as mitochondria can be controlled. It is understood that this can be varied.

[0207] As used herein, the terms "heteroplasmy" and "heteroplasmic" The term refers to the occurrence of multiple types of mitochondrial DNA genomes in an individual or sample. Varying degrees of heteroplasmy are associated with varying degrees of physiological conditions as described herein. Heteroplasmy can be identified by means known in the art, particularly The severity of a physiological condition associated with a given nucleotide allele depends on the distribution of such associated alleles within an individual. It is thought that this will vary depending on the percentage of

[0208] As used herein, "wild" when used in reference to mitochondrial DNA. The term "type" refers to the typical morphological genotype of a species as it occurs in nature. An exemplary reference genome for the human mtDNA genome is the Cambridge Reference Sequence (CRS). can be.

[0209] As used herein, the terms "old" or "older" refer to the structure of mtDNA. The source is from a subject that is older than the recipient cells or Compared to human cells, they have undergone a greater number of population doublings (i.e., a greater number of population doublings) since their in vitro culture. , PDL), meaning that they originate from cells within a population of cells that have doubled that population. It is intended that

[0210] As used herein, the terms "young" or "younger" refer to the origin of the mtDNA. or derived from a subject younger than the recipient cells, or Compared to the blastocysts, they have undergone fewer population doublings (i.e., population doubling level, PDL) since their in vitro culture. ), meaning that it originated from a cell within a population of cells that doubled that population. is intended.

[0211] As used herein, "isolated" when used in reference to mitochondria The term "phytoplasmic" refers to a cell that has been physically separated or removed from other cellular components of its natural biological environment. This refers to mitochondria that are involved in the synthesis of proteins.

[0212] As used herein, the terms "intact" and "intact mitochondria" refer to It contains the outer and inner membranes, the intermembrane space, the cristae (formed by the inner membrane), and the matrix. An exemplary intact mitochondrion contains mtDNA. In certain embodiments, the intact mitochondria are functional mitochondria. However, it is understood that intact and dysfunctional mitochondria can also be used in the present invention. .

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

[0214] As used herein, the term "allogeneic" refers to biologically derived The biological composition is intended to be a target of a genotype different from that of the subject receiving the biological composition. It is meant to be tasted.

[0215] As used herein, the term "animal cell" refers to any cell of eukaryotic origin. Animal cells are intended to mean cells from mammalian and non-mammalian species, e.g., amphibians. , fish, insects (e.g., Drosophila), and helminths (e.g., Caenorhabditis elegans It is understood that the term "group" can include "groups" and "groups".

[0216] As used herein, the term "fusion protein" does not necessarily mean However, it primarily refers to a sequence of amino acids connected to each other by peptide bonds, where A portion of a sequence may be derived from one source (natural or synthetic) (i.e., it may have sequence similarity to a sequence and another portion of the sequence is derived from one or more other sources. The entire fusion protein is encoded so that essentially all bonds are peptide bonds (both (e.g., encoding the mitochondrial targeting sequence and endonuclease) Fusions can be prepared by constructing vectors. Chemical conjugation by using any of the known methods used for gating It is also understood that the ion exchange membrane can be produced by gating.

[0217] As used herein, "mitochondrial-targeted sequence" refers to a sequence that is a sequence of a mitochondrial targeting molecule. "Mitochondrial targeting sequence (MTS)" and "mitochondrial targeting sequence (MTS)" The terms "(MTS)" and "(MTS)" are interchangeable and refer to any enzyme, peptide, sequence, or compound attached thereto. It refers to any amino acid sequence that results in the transport of a substance into mitochondria. In another embodiment, the MTS is human MTS. In another embodiment, the MTS is from another species. A non-limiting example of such a sequence is cytochrome c oxidase subunit X (COX10 )MTS [ka] and cytochrome c oxidase subunit VIII (COX8) MTS [ka] Another non-limiting example of an MTS sequence is one that is encoded by nuclear DNA and translated in the cytoplasm ( Each individual mitochondrial protein is produced and transported into the mitochondria, as well as citrate synthase (cs), lipoamide dehydrogenase (LAD), and C6ORF66 (ORF) The various MTSs are interchangeable for each mitochondrial enzyme in them. Each possibility represents a separate embodiment of a fusion protein for use in the present invention. vinegar.

[0218] As used herein, the term "small molecule" refers to a molecule that has an effect on a biological process. An exemplary small molecule is a compound having a molecular weight of about 300 Daltons or less. It has a molecular weight of about 700 daltons.

[0219] As used herein, when used in conjunction with numbers, the terms "about" or "approximately" are used interchangeably. The term "immediately" refers to any number within 1, 5, 10, 15, or 20% of the number mentioned. vinegar.

[0220] As used herein, the term "somatic cells" includes stem cells, progenitor cells, as well as living cells. Germline cells (i.e., oogonia and spermatogonia) and cells derived therefrom (e.g., oocytes, spermatogonia, and refers to any differentiated cell that forms the body of an organism, apart from a mother cell, sperm, etc. For example, an internal organ The body's cells, skin, bones, blood, and connective tissue are all made up of somatic cells. Preferably, the cells are obtained from a human subject and cultured according to standard cell culture protocols available to those skilled in the art. and cultivated.

[0221] As used herein, the term "endocytic pathway" refers to the pathway through which a cell The endocytic pathway refers to the cellular process of taking in molecules from the environment. They form "clathrin" vesicles, which require the recruitment of clathrin to help them organize into vesicles that absorb molecules. These include those that are "clathrin-dependent" or "clathrin-independent" that do not require clathrin recruitment. Exemplary types of clathrin-independent endocytosis include Examples of such a pathway include macropinocytosis. The term "activator of endocytosis" refers to a compound that increases the endocytic pathway, e.g. The term "endocytic pathway" refers to an agent that induces or activates an endocytic pathway or process, such as a cytoplasmic receptor. An exemplary "activator of endocytosis" is a protein that transports mitochondria from the extracellular environment. Increases uptake.

[0222] As used herein, the term "macropinocytosis" refers to the transport of solute molecules, Clathrin-independent forms of endocytic cells that mediate nonselective uptake of nutrients and antigens This refers to the system.

[0223] As used herein, the term "compound" refers to a compound that provides a desired biological function. This term refers to compounds that can be synthesized by DNA, RNA, proteins, polypeptides, and other compounds. These include, but are not limited to, long-acting factors, cytokines, hormones, or other compounds, including small molecules. It will not be done.

[0224] As used herein, the term "peptide" means a peptide, a polypeptide, or a The terms "de" and "protein" are constrained (i.e., for example, β-turn or the presence of amino acids that cause β-pleated sheets, or e.g., disulfides (having some elements of a structure cyclized by the presence of a peptide-linked Cys residue) or or an unconstrained (e.g., linear or unstructured) amino acid sequence. The amino acids that make up a polypeptide are used interchangeably and in their broadest sense. The acid may be naturally occurring or synthetic. The peptide can be purified from a biological sample. Tides include modified polypeptides, proteins, and peptides, e.g., glycopolypeptides. , glycoprotein, or glycopeptide; or lipopolypeptide, lipoprotein, or also encompasses lipopeptides.

[0225] As used herein, "modulate," "modulation," "modulator," and " The term "modulate" refers to a change in the nature or composition of the basic homeostatic state. Exemplary modulation includes homeostatic regulation, such as a significant decrease in cellular metabolism. The term "modulator" refers to an alteration of cellular metabolism through disruption of insulin action. Inhibitors include inhibitors and activators. Inhibitors can be used to inhibit, for example, a desired protein, pathway, or or inhibit the expression or modification of a process, or bind to, or partially or completely inhibit, stimulation Completely block, reduce, prevent, delay activation of the target protein described, Agents that inactivate, desensitize, or down-regulate the activity of a pathway or process In some embodiments, an inhibitor inhibits a target protein, pathway, or process. Activators are, for example, antagonists of the target proteins described herein. Induce or activate, or bind to, inhibit the expression or modification of pathways or processes Stimulates, increases, initiates, activates, promotes, enhances, and is described as The activity of the target protein (or encoding polynucleotide), pathway, or process In some embodiments, the activator is an agent that sensitizes or upregulates sex. Modulators are agonists of the target protein, pathway, or process. The present invention relates to natural and synthetic ligands, antagonists, and agonists (e.g., agonists or These include small chemical molecules, antibodies, etc., that act as either antagonists or inhibitors. It is further understood that the initiator can be biological (eg, an antibody) or chemical.

[0226] As used herein, the term "before" means before the intended event begins. The desired result (e.g., antibiotic selection) or effect is maintained without complete dissipation of the desired result or effect. is the duration of an event that is long enough to achieve and sustain an effect (e.g., a biological effect). It is intended to mean the period of time preceding the onset. For example, in the exemplary situation, Modulating cellular metabolism prior to the transfer of exogenous mitochondria and / or exogenous mtDNA can be achieved, e.g. homeostasis before the transfer of exogenous mitochondria and / or exogenous mtDNA causes biological effects The desired biological effect is achieved without reverting to stasis (e.g., S6 kinase activity). It is understood that the period of time during which the treatment is continued includes a period of time sufficient to induce the formation of a cytotoxic effect (e.g., an increase in phosphorylation).

[0227] As used herein, the term "nutritional stress" refers to the upregulation of autophagy, AMPK, Disruption of cellular homeostasis, such as signal transduction and / or induction of the mTOR signaling pathway Nutritional stress refers to a state of nutrient deficiency or starvation sufficient to cause These include serum starvation, deprivation of essential amino acids, and / or disruption of metabolic pathways.

[0228] The terms "nucleic acid" and "polynucleotide" refer to two naturally occurring nucleic acids. capable of hybridizing in a sequence-specific manner to naturally occurring nucleic acids, e.g., Watson et al. Nucleotides capable of participating in click-type base pairing interactions, e.g., deoxynucleotides Polymers of any length composed of silibonucleotides or ribonucleotides, or are used interchangeably herein to describe compounds that are produced synthetically. As used herein in the context of polynucleotide sequences, "bases (or The term "nucleotides" (or "nucleotides") is a mistranslation of "nucleotides" (or "nucleotides"). is synonymous with "nucleotide," i.e., the monomeric subunit of a polynucleotide. The abbreviation "A" when used in reference to nucleotides means adenine (A). The abbreviation "G" when used in reference to a nucleotide is intended to mean guanine. "C" when used in connection with a nucleotide is intended to mean a nucleotide (G). The abbreviation α, β ... The abbreviation "T" when used herein is intended to mean thymine (T).

[0229] The term "pharmaceutically acceptable" when used in reference to a carrier means a carrier, diluent, the agent or excipient is compatible with the other ingredients of the formulation and not harmful to the recipient thereof. is intended to mean that the

[0230] Implementation of the embodiments provided herein is within the skill of one of ordinary skill in the art unless otherwise indicated. The present invention utilizes conventional techniques of molecular biology, microbiology, and immunology, such as: , which are fully explained in the literature. Examples of particularly suitable reference texts include: Sambrook et al., Molecular Cloning: A Labora- tory Manual Laboratory Manual), 3rd ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al. References, Current Protocols in Molecular Biology, John W Iley and Sons, Baltimore, MD (1999); Glover (ed.), DNA Cloning, Vol. I Vol. II and Vol. II (1985); Gait, ed., Oligonucleotide Synthesis (19 84); Hames and Higgins, eds., Nucleic Acid Hybridization ( 1984); Hames and Higgins, eds., Transcription and Translation (1984); Fr Maeshney (ed.), Animal Cell Culture: Immobilized Cells and Enzymes and Enzymes) (IRL Press, 1986); Kallen et al., Plant Molecular Biology - A Laboratory Manual (Plant Molecular Biology - A Laboratory Manual) (edited by Melody S. Clark; Springer-Verl ag, 1997; Immunochemical Methods in Cells and Molecular Biology and Molecular Biology) (Academic Press, London); Scopes, Protein Purification: Protein Purification: Principles and Practice (Springer Verlag, NY ., 2nd ed., 1987); and Weir and Blackwell, eds., Handbook of Experimental Immunology Experimental Immunology, Vols. I to IV (1986).

[0231] 5.2 Method for producing mitochondrial exchange cells (MirC) The present invention provides endogenous mitochondrial DNA (mtDNA)-reducing agents, including agents that reduce endogenous mitochondrial DNA. Any agent that reduces chondrial function may inhibit the non-invasive import of exogenous mitochondria. However, the internal structure of the ρ(0) cell is not fully understood. Complete depletion of endogenous mtDNA prevents this enhancement. Invasive import is energy-dependent, and complete depletion of endogenous mtDNA is a non-invasive import process. This significantly limits the energy available to promote exogenous metabolism. Non-invasive transfer of mitochondrial DNA is possible if mitochondrial function and / or mtDNA are not disrupted. For example, mitochondria may simply be co-incubated (i.e., "added") or centrifuged. Separate addition is also inefficient.

[0232] Thus, provided herein are methods for generating mitochondrial exchange cells (MirCs). (a) injecting recipient cells with an agent or mitochondria that reduces endogenous mtDNA copy number; (b) contacting the recipient cells with an agent that reduces mitochondrial function; The agent partially reduces the endogenous mtDNA copy number in the recipient cells, respectively. for a period of time sufficient to completely or partially reduce said endogenous mitochondrial function; and (c)(1) incubating the endogenous mtDNA or the endogenous mitochondrial DNA, respectively. (b) recipient cells derived from a healthy donor with partially reduced apoptotic function; and (2) recipient cells derived from a healthy donor. Non-invasively transferring exogenous mitochondria into the recipient cells The cells are then co-incubated for a period of time sufficient to produce mitochondria-exchanged cells. Also provided herein are methods for detecting mitochondrial dysfunction, which may include: A method for producing a replacement cell, comprising carrying out the steps (a) and (b) above, and and (c)(1) the endogenous mtDNA or the endogenous mitochondrial function is partially reduced, respectively. (b) recipient cells derived from the exogenous mtDNA of a healthy donor; and (2) exogenous mtDNA derived from a healthy donor. Co-incubate for a period of time sufficient to non-invasively transfer the target mtDNA into the recipient cells. thereby producing mitochondria-exchanged cells. In embodiments, the exogenous mtDNA is imported by exogenous mitochondria.

[0233] The generation of MirC can be a useful strategy for a variety of applications. The transfer of exogenous mitochondria, exogenous mtDNA, or a combination thereof into a cell can result in, for example, functional Endogenous mitochondria that are defective and / or comprise mutant mtDNA are functionally mitochondria, e.g., mitochondria composed of wild-type mtDNA, In certain embodiments, the methods provided herein can be used to treat dysfunctional It is performed in recipient cells that have endogenous mtDNA encoding mitochondria. In certain embodiments, the endogenous mtDNA is mutant mtDNA. The endogenous mtDNA is heteroplasmic and contains both wild-type and mutant mtDNA. It is composed of two parts.

[0234] As noted above, in some applications, exogenous mitochondria, exogenous mtDNA, or combinations thereof may be used. The introduction of a mutant mtDNA may involve, for example, the transfer of an endogenous mtDNA that is dysfunctional or composed of mutant mtDNA. This involves the transfer of functional mitochondria or wild-type mtDNA to replace mitochondria. Thus, in some embodiments, the exogenous mtDNA is wild-type mtDNA. In other embodiments, the endogenous mitochondria of the recipient cells contain wild-type mtDNA and Have dysfunctional endogenous mitochondria, e.g., recipients with wild-type mtDNA An exemplary dysfunctional mitochondrion in a cell is a sudden gene encoding a mitochondrial protein. Mutant nuclear DNA or dysfunctional mitochondria caused by the side effects of aging or disease It may contain doria.

[0235] Therefore, they may be dysfunctional, consist of mutant mtDNA, or a combination thereof. Certain endogenous mitochondria can be replaced using the methods described herein. Mitochondrial dysfunction can occur as a result of many factors, including but not limited to: Examples include diseases (e.g., age-related diseases, mitochondrial diseases or disorders, neurodegenerative diseases, etc.) mitochondrial disease due to glaucoma, retinal disease, genetic disorders, diabetes, hearing loss, or any combination of these Mitochondrial dysfunction is seen in over 5%, over 10%, over 20%, and over 3% of patients. Endogenous mitochondria that are reduced by more than 0%, 40%, 50%, 60%, 70%, 80%, or 90% Thus, in some embodiments, endogenous Mitochondria function at approximately 5%, 10%, 15%, 20%, 25%, 30%, and 40% , about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% reduced mitochondria Includes.

[0236] The methods provided herein are directed to the detection of both homoplasmic and heteroplasmic mtDNA. In a specific embodiment, the endogenous mtDNA is a single type of mtDNA. (i.e., the endogenous mtDNA is homoplasmic). In this study, endogenous mtDNA contains multiple types of mtDNA (i.e., endogenous mtDNA is heterologous). In some embodiments, the heteroplasmic mtDNA is It contains both normal and mutant mtDNA. The proportion of mutant mtDNA usually depends on the severity of the phenotype. These factors can determine the severity of the disease and influence the extent to which mitochondrial function declines. In some embodiments, the heteroplasmic mtDNA is 5% mutant mtDNA and 9% 5% wild-type mtDNA and mitochondrial function is reduced by 5%. The heteroplasmic mtDNA was 55% mutant mtDNA and 45% wild-type mtDNA. Mitochondrial function is reduced by 55%. However, the percentage of mutant mtDNA It is understood that the amount of mitochondrial function does not necessarily correlate with mitochondrial function.

[0237] Dysfunctional mitochondria result in a loss of efficiency in the electron transport chain and adenosine Decreased synthesis of high-energy molecules such as ATP-5'-triphosphate and leakage of harmful reactive oxygen species (ROS) Those skilled in the art will understand how mitochondrial function is affected by mitochondrial deficiency and / or disruption of cellular respiration. For example, you will understand how to evaluate the baseline data in a single experiment. Seahorse Biopsy was performed to determine oxygen consumption, glycolytic rate, ATP production, and respiratory capacity. Using cell-based assays such as the Bioscience XF Extracellular Flux Analyzer, Chondriac dysfunction can be assessed using the Oroboros 02K respiratory activity monitor. The above assay examples can also be used to establish quantitative functional mitochondrial diagnostics. are exemplary and not all-inclusive of methods for assessing mitochondrial function. It is understood that there is no

[0238] In some embodiments, functional mitochondria have an intact outer membrane. In some embodiments, the functional mitochondria are intact mitochondria. In embodiments, functional mitochondria consume oxygen at a rate that increases over time. In another embodiment, mitochondrial functionality is measured by oxygen consumption. In one embodiment, mitochondrial oxygen consumption can be measured using, but not limited to, the MitoXpress fluorescent probe. It can be measured by any method known in the art, such as Robe (Luxcel). In some embodiments, functional mitochondria contain, but are not limited to, ADP and glutamate. It exhibits an increased rate of oxygen consumption in the presence of substrates such as thamate, malate, or succinate. Each possibility represents a separate embodiment of the present invention. In this context, functional mitochondria are mitochondria that produce ATP.

[0239] The methods provided herein include the treatment of dysfunctional mitochondria, mutant mitochondria, and These may be useful in producing MirC from recipient cells containing tDNA, or combinations thereof. However, MirC production does not need to be performed in recipient cells with dysfunctional mitochondria. In some embodiments, MirC is a functional endogenous mitochondrial protein. The mitochondrial DNA fragments were generated using recipient cells expressing exogenous mitochondrial DNA (mtDNA), wild-type mtDNA, or a combination thereof. The mtDNA may also be functional, contain wild-type mtDNA, or a combination thereof. Endogenous wild-type mtDNA can be reduced using the methods provided herein, and healthy individuals can be Using exogenous mtDNA derived from healthy donor cells (e.g., young cells with relatively low PDL) "Aged" recipient cells (e.g., cells from an aged subject or cells with a relatively high population doubling rate) Recipes incorporating exogenous wild-type mtDNA, such as mitochondrial replacement in cells with PDL (Primary Defective Lymphocytes). Thus, in one embodiment, exogenous mtDNA can be introduced into an endogenous cell. The donor cells are healthy donor cells, e.g., donor cells that are younger than the recipient cells. In one embodiment, the donor and recipient cells are about 1.5 times larger than the donor and recipient cells. , about 2-fold, about 2.5-fold, about 3-fold, about 4-fold, about 5-fold, or more than 5-fold difference in PDL. In the above, the donor and recipient cells are multiplied by about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 4 times, They are derived from subjects separated by approximately or more than five times the age of the donor cells. It is understood that an age difference between the cells and the recipient cells is not a requirement. In this study, donor and recipient cells were of the same age, and the donor cells were transplanted from healthy cells. It is a cell.

[0240] In yet other embodiments, the production of MirC involves the production of functional endogenous mitochondria, e.g., It is performed in recipient cells with wild-type endogenous mtDNA and in recipients where the exogenous mtDNA is mutant. or encodes a dysfunctional mitochondrion, or the exogenous mitochondrion is dysfunctional. In other embodiments, exogenous mitochondria, exogenous mtDNA, or a combination thereof. A, or a combination thereof, is derived from donor cells that are older than the recipient cells. For example, in some embodiments, the model for a mitochondrial disease or disorder is Functional mitochondria and mutant and / or dysfunctional mitochondria in patient cells It can be generated by exchanging exogenous mtDNA from donor cells that encodes the endogenous The examples described herein are illustrative and not all combinations involving mtDNA exchange. It is understood that this does not include.

[0241] As provided herein, methods for producing MirC have the effect of reducing endogenous mtDNA. The method is performed using either a factor or an agent that reduces endogenous mitochondrial function. In some circumstances, a combination of two agents can be used. Agents capable of reducing mitochondrial function are well known in the art and are well known in the art. Exemplary agents include ADP or uncouplers, e.g., Inhibitors of complex III (e.g., myxothiazole), inhibitors of complex IV (e.g., , sodium azide, potassium cyanide (KCN)), or inhibitors of complex V (e.g. , oligomycin); which abolishes the burst of oxygen consumption after the addition of ADP but does not Inhibitors of phosphorylation have no effect on drug-stimulated respiration; intact mitochondria Uncoupling agents (e.g., ATP) that abolish the forced coupling between the respiratory chain and the phosphorylation system observed in endogenous e.g., dinitrophenol, CCCP, FCCP); ATP efflux or crossing the inner mitochondrial membrane ATP / ADP transport inhibitors, such as adenine nucleotides, which block the influx of either atractyloside translocase inhibitors (e.g., atractyloside); Ionophores that make cells permeable to compounds that cannot be permeated (e.g., valinomycin nigericin); or Krebs cycle enzymes that block one or more TCA cycle enzymes or their associated reactions Blocks respiration in the presence of inhibitors (e.g., arsenite, aminooxyacetate) Inhibitors of the mitochondrial respiratory chain are examples of inhibitors that reduce the mitochondrial functions mentioned above. The agents that can be used are not limiting, and those skilled in the art will appreciate that they can be used by techniques known in the art. The method can be used to readily identify suitable agents capable of reducing mitochondrial function. It is understood that it is possible to

[0242] In a specific embodiment, the agent that reduces endogenous mitochondrial function is In another embodiment, the compound induces transient reduction of endogenous mitochondrial function. Agents that reduce endogenous mitochondrial function permanently reduce endogenous mitochondrial function. In a preferred embodiment, the agent that reduces endogenous mitochondrial function is Partially reduces mitochondrial function.

[0243] A variety of agents can be used to reduce mtDNA. The tDNA degradation agent binds the mitochondrial targeting sequence (MTS) and the endonuclease. a nucleic acid encoding a fusion protein, an endonuclease, or a small molecule comprising In some 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 that degrades mtDNA encodes an endonuclease. In a preferred embodiment, the function of the plasmid DNA expression vector cassette is The factor is a plasmid DNA expression vector cassette encoding the endonuclease together with MTS. A variety of expression vector cassettes can be used, and those skilled in the art will be able to identify the host cells. Necessary considerations required to allow successful expression of the endonuclease according to the cell For example, the cytomegalovirus (CMV) promoter, the SV40 promoter, Mammalian expression vectors, such as vectors with a CAG promoter, can be used in non-mammalian It would be preferable to express the endonuclease in mammalian, but not animal, cells. It is understood that viral expression vectors can also be used, and those skilled in the art will understand how to do so. The viral expression vector is transferred in tandem with the helper plasmid ( It is understood that the plasmid may require additional components (i.e., envelope and packaging plasmids). In other embodiments, the agent encodes an endonuclease. In another preferred embodiment, the agent is an endonuclease with MTS. In yet a further embodiment, the agent is a recombinant mRNA encoding the ATPase. In other embodiments, the agent is a protein, e.g., an endonuclease. For example, small molecules that disrupt mtDNA synthesis. Techniques for doing so are known to those of skill in the art and are readily performed without undue experimentation. In a preferred embodiment, the agent is suitable for clinical use.

[0244] In specific embodiments, the endonuclease is selected from, for example, the following DNA sequence: [ka] restriction enzymes that cut the DNA double helix at specific sites to fragment it, such as XbaI, which cuts As the endonuclease, for example, a restriction enzyme other than XbaI, such as EcoRI, BamHI, HindIII, or PstI may also be mentioned, all of which bind to mtD at multiple sites. Endonuclease has a specific recognition site, which allows it to digest mtDNA. For example, the sensitivity of restriction enzymes such as XbaI, EcoRI, and SmaI can be predicted. A given recognition site is specific for a given nucleic acid sequence. In this study, the degradation of endogenous mtDNA was mediated by zinc finger proteins that are coupled to DNA nucleases. and transcription activator-like effectors (TALEs). Two types of DNA-binding proteins have been shown to have specificity for novel DNA sequences of interest. Similarly, clustered regularly spaced repeats of short palindromic sequences can be modified as follows. CRISPR / Cas9 proteins are introduced into cells by adding the corresponding coding genes. Thus, in some embodiments, the endonuclease may programmable nucleases, such as RNA-guided DNA endonucleases (e.g., Cas 9), zinc finger nucleases (ZFNs), or transcription activator-like effector nucleases The above nucleases are not intended to be limiting. and those skilled in the art can easily obtain suitable endonucleases using techniques known in the art. It is understood that the sequences can be identified using, for example, the Cambridge Reference Sequence or similar codes. Using the consensus sequence, suitable endonucleases that recognize mtDNA sequences can be identified, e.g., In a specific embodiment, the endonuclease can be identified by in silico analysis. In other embodiments, the endonuclease cleaves the wild-type sequence of mtDNA. The enzyme cleaves the mutant sequence in mtDNA. The agent that reduces endogenous mtDNA is odor. agents that inhibit mtDNA biosynthesis, such as ethidium chloride, It is not necessary to have any mtDNA that can be reduced. It is also understood that, for example, endogenous mitochondrial Inducing autophagy to promote selective degradation, mediated by urolithin A or small molecules p62 First, the action factors such as mitophagy inducers (PMIs) (i.e., mitophagy agonists) The present invention relates to a method for treating mtDNA by using nucleoside reverse transcriptase inhibitors (NRTIs). It can also be carried out using the compound as an agent that causes the reaction.

[0245] Furthermore, in some embodiments, the expression vector cassette is containing one or more antibiotic resistance genes to allow selection of a population of cells expressing the For example, in some embodiments, the expression vector may be a Streptomyces cerevisiae vector. The puromycin N-acetyltransferase gene (p) from Streptomyces a c) and the cells can be selected with puromycin. In situations where selection is performed using a substance, e.g., puromycin, selection may be performed using a drug The time period can be short (e.g., 24 to 48 hours) to limit prolonged exposure. However, it should be understood that the examples provided above are merely illustrative and that the expression vector cassette The kit may contain, for example, bsr, bls, or BSD genes for selection with blasticidin, or hybrids. It is possible that the nucleotide sequence may contain other antibiotic resistance genes, such as the hph gene for selection with gromycin B. It is understood that the concentration of antibiotic used for selection will depend on the type of antibiotic and the type of cell. and are generally readily available to those skilled in the art without undue experimentation. It is understood that selection can be effected by any means known in the art, It is further understood that antibiotic resistance need not be involved. For example, in some embodiments In this case, the selection of cells can be performed, for example, by fluorescence activated cell sorting (FACS) or expression of cell surface markers. This can be achieved by expression of a fluorescent protein encoded by In some embodiments, selection can be performed according to the phenotype of the cells. In some embodiments, the mutant endogenous mt in cells with heteroplasmy The DNA can result in a selectable phenotypic response, such as cell survival.

[0246] Thus, in some embodiments, the cells contain endonucleases that degrade mtDNA. In some embodiments, the expression vector cassette containing the enzyme is introduced and then selected. In this process, cells obtain a homogenous cell population that expresses an endonuclease that degrades mtDNA. In a specific embodiment, the cells are selected to express endonucleases that degrade mtDNA. After transfection with an expression vector cassette containing the ATPase, a homogeneous stable cell line was generated by selection. In other embodiments, the cells express an endonuclease that degrades mtDNA. As mentioned above, this enrichment by selection is achieved by the selection of the antibody. The cells that are enriched may be enriched by varying the degree and / or type of selection pressure. Depending on the expression, the endonuclease can be stably expressed or the endonuclease can be transiently expressed. The enriched population does not need to be homogeneous, and the mtDNA can be degraded. The enriched cell population expressing the endonuclease involved was compared with the unselected cell population. Compared to the endonuclease-containing cells, It is understood that the cells may also contain some cells that do not express the nuclease.

[0247] In other embodiments, the cells contain an expression vector containing an endonuclease that degrades mtDNA. In a specific embodiment, the cells are not selected for after introducing the vector. The expression vector containing the endonuclease that breaks down the endonuclease is not selected for after the introduction of the vector. The nuclease is transiently expressed.

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

[0249] When the agent that degrades endogenous mitochondria includes an endonuclease, the endonuclease Expression of the endonuclease can be achieved by introducing mRNA encoding the endonuclease or by recombinant transcription. This may involve the introduction of the endonuclease as a protein. The MaxCyte electroporator meets the standards of Good Manufacturing Practice and Good Clinical Practice, among others. This can be used for mRNA transfection in clinical settings. The electroporation was performed using a MaxCyte electroporator according to the manufacturer's protocol. The above methods are merely exemplary and may be used to identify mRNA and / or recombinant proteins. It is further understood that any means of introducing the protein can be used.

[0250] Specific targeting of endonucleases to mitochondria is a promising strategy for targeting mitochondria. The endonuclease coding sequence is flanked by mitochondria, resulting in a fusion protein that This can be achieved by incorporating a targeting sequence (MTS). which, when fused to its N-terminus, can target proteins to specific compartments. It has been shown that the mitochondrial targeting sequence can be expressed by the mitochondrial targeting sequence. MTS is well known to those skilled in the art (see, for example, U.S. Pat. No. 6,223,699, which is incorporated herein by reference in its entirety). See Patent No. 8,039,587B2. For example, cytochrome c oxidase subunit IV (COX 4), MTS, a targeting peptide derived from subunit VIII (COX 8) or subunit X (COX 10) In principle, any MTS for the mitochondrial matrix can be used. Any target sequence or sequences derived from any nuclear-encoded mitochondrial matrix or inner membrane enzyme. The fusion protein was converted to a mitochondrial import protein (hydrophobic moment > 5.5, At least two basic residues, amphipathic α-helical conformation; see e.g., Bedwell et al. Mol Cell Biol. 9(3)(1989), 1014-1025) can be used in the present invention. It is useful for the purpose of

[0251] In one embodiment, the MTS is human MTS. In another embodiment, the MTS is human MTS. Non-limiting examples of such sequences are those derived from cytochrome c oxidase. Subunit X (COX 10) MTS [ka] and cytochrome c oxidase subunit VIII (COX 8) MTS [ka] Further non-limiting examples of MTS sequences are those encoded by nuclear DNA and translated in the cytoplasm. Each individual mitochondrial protein that is produced and transported into the mitochondria, and citrate synthase (cs), lipoamide dehydrogenase (LAD), and C6ORF66 (ORF) The various MTSs are interchangeable for each mitochondrial enzyme in them. Thus, in some embodiments, MTS may be a mitochondrial matrix protein. In a specific embodiment, the target is a mitochondrial matrix protein. The enzyme is subunit VIII of human cytochrome c oxidase. Each of these represents a separate embodiment of the fusion protein for use in the present invention.

[0252] Recipient cells are transfected with an agent that reduces endogenous mtDNA copy number or endogenous mitochondria. When contacted with an agent that reduces donor function, the recipient cells react with the agent to: partially reduce or decrease the endogenous mtDNA copy number in recipient cells, respectively. for a period sufficient to partially reduce endogenous mitochondrial function in recipient cells, The agent partially reduces the endogenous mtDNA copy number or Identifying a "sufficient period" that allows for partial reduction of endogenous mitochondrial function It is within the skill of one of ordinary skill in the art to determine a sufficient or appropriate period of time, including but not limited to, The specific type of cells, the amount of starting material (e.g., the number and / or reduced number of recipient cells) the amount of mtDNA to be transfected), the amount and type of agent, the plasmid promoter regulator, and In various embodiments, the recipe may vary depending on various factors, including the culture conditions. Approximately 1 day is sufficient to allow for a partial reduction in endogenous mtDNA copy number in human cells. , 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 to 2 weeks, about In a preferred embodiment, the sufficient period is obtained Recipient cells are transformed with exogenous mtDNA and / or exogenous mitochondria. Before incubation with α-glucan, most of the endogenous mtDNA copy number was reduced or most of the endogenous mitochondria was removed. The agent that reduces the function of mitochondria and reduces endogenous mtDNA or endogenous mitochondria. It is also long enough to be substantially free of agents that reduce mitochondrial function.

[0253] An important and novel aspect of the present invention is that the mitochondrial import efficiency is comparable to the completeness of endogenous mitochondria. The endogenous mtDNA copy number is severely reduced in cells with complete depletion (i.e., (ρ)0 cells), whereas the endogenous mtDNA copy number is significantly reduced in cells with complete depletion (i.e., (ρ)0 cells). - When the number of bacteria is declining but not completely depleted (i.e., (ρ) -cells), greatly improved Furthermore, the present invention has been found to be feasible without a simple addition or centrifugation protocol. It also shows that it is inefficient without partial reduction of endogenous mtDNA copy number. Therefore, in a preferred embodiment, the endogenous mtDNA copy number in the recipient cells The reduction in endogenous mtDNA is less than 100% depletion. The endogenous mtDNA copy number in the resident cells is reduced by approximately 5% to approximately 99%. In embodiments, the agent that reduces the endogenous mtDNA copy number reduces the endogenous mtDNA copy number by about In another embodiment, the endogenous mtDNA copy number is reduced by 30% to about 70%. The active factor is approximately 50% or more, approximately 60% or more, approximately 70% or more, or approximately 80% or more of the endogenous mtDNA copy number. or by about 90% or more, or by about 95% or more. Factors that reduce endogenous mtDNA copy number reduce endogenous mtDNA copy number by approximately 60% to 90%. In some embodiments, an agent that reduces endogenous mtDNA copy number It is also understood that reduces mitochondrial clumping.

[0254] In certain embodiments, the exogenous mtDNA is isolated exogenous mitochondria from donor cells. Mitochondrial isolation can be performed using methods described herein and in the cited references, including, but not limited to, the methods described herein and the cited references. This can be accomplished by any of several well-known techniques, including those described in the In one embodiment, exogenous mitochondria for use in mitochondrial transfer can be used. The kit includes, for example, the Qproteum mitochondrial isolation kit (Qiagen, USA), the MITOISO2 mitochondrial isolation kit (Qiagen, USA), and the Mitochondria isolation kit (Sigma, USA) or mitochondrial isolation kit for cultured cells (Thermo Science) In another embodiment, mitochondria are isolated using commercially available kits such as the ntific. Exogenous mitochondria for use in transfection are manually isolated. The manual isolation of mitochondria involves pelleting donor cells and extracting approximately 100% mitochondria grown in culture. 10 9 Wash 1-2 mL of cell pellet obtained from 100 cells and swell the cells in hypotonic buffer. Dounce or Potter-Elvehjem homogenizer with a tensioned, tight-fitting pestle By rupturing the cells with HCl and isolating the mitochondria by differential centrifugation, This involves isolating mitochondria from donor cells. Manual isolation can be performed, for example, by using a microscope. It may also include density gradient ultracentrifugation, or free-flow electrophoresis. Without being bound by theory, the kits and manual methods described herein The methods are exemplary and any mitochondrial isolation method can be used; It is understood that this is within the skill of one of ordinary skill in the art.

[0255] In some embodiments, the isolated donor mitochondria are isolated from other organelles. In other embodiments, the isolated mitochondria are substantially pure from impurities. For example, in some embodiments, In the above, the isolated mitochondria were about 90% pure, about 80% pure, about 70% pure, about 60% pure, , pure, about 50% pure, or any integer between these. Generally, isolated donor molecules Any impurities contained in the mitochondria are transferred to the recipient cells during mitochondrial transfer. It is understood that the exogenous ATP does not affect viability or function. The transfer of mitochondria, exogenous mtDNA, or a combination thereof can be used to induce the transfer of non-mitochondrial organelles. This does not involve the introduction of

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

[0257] In some embodiments, the isolated mitochondria are functional mitochondria. In a further embodiment, the isolated mitochondria are characterized by dysfunctional mitochondria. In some embodiments, mitochondrial function is measured in the donor cells prior to isolation. In other embodiments, mitochondrial function can be assessed using isolated mitochondria. It can be assayed from chondria.

[0258] Preserving the integrity of the mitochondrial membrane is another important factor during mitochondrial isolation. In some embodiments, the mtDNA used in the methods provided herein is intact. In a specific embodiment, the isolated mitochondrial About 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% of A, or More than 90% are intact. Mitochondrial membrane integrity is defined as, but not limited to, the integrity of the mitochondrial membrane as used herein. This may be accomplished by any of several well-known techniques, including those described in the cited references. For example, TMRM, Rhod123, JC-1, and DiOC6 can inhibit the mitochondrial membrane potential. Typical probes for measuring ATP (see, for example, Perry et al., Biotechniques, 2011). b;50(2):98-115). JC-1 is a novel antibody for measuring the inner membrane potential of isolated mitochondria. A widely used dye that reacts with the electrochemical proton gradient across the inner mitochondrial membrane .

[0259] In certain embodiments of the methods provided herein, a method is provided comprising the steps of: A recipient cell is then non-invasively transferred with exogenous mitochondria. Co-incubated with exogenous mitochondria from healthy donors for a period sufficient to In another embodiment, the mitochondrial replacement cells are prepared by replacing a portion of the endogenous mtDNA. Recipients with partial reductions in mitochondria were treated by non-invasively injecting exogenous mitochondria into the recipient cells. The cells were co-incubated with exogenous mtDNA from a healthy donor for a period sufficient to allow for the rapid transfer of mtDNA. , thereby producing mitochondrial replacement cells. Identifying a "sufficient period" for non-invasive transfer of endogenous mtDNA into recipient cells The period of time that is sufficient or appropriate depends on, but is not limited to, the characteristics of the cells. the type of cell type, the amount of starting material (e.g., the number of recipient cells and / or the endogenous amount of exogenous mtDNA), amount of donor material (e.g., quantity, quality, and / or purity of exogenous mtDNA), and In various embodiments, the exogenous factors may vary depending on the cell type and / or the culture conditions. sufficient to non-invasively transfer endogenous mitochondria and / or exogenous mtDNA into recipient cells. The available periods are 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, and about 10 days. In some embodiments, the ink is At the end of incubation, the recipient cells contain most of the exogenous mtDNA and optionally Substantially free of exogenous mitochondrial organelles.

[0260] Another feature of the present invention is that the total mtDNA copy number in MirC is increased compared to the original recipient cell. In contrast, other less efficient methods The method involves the transfer of mitochondria to the recipient cells without conditioning them prior to the co-incubation step. Centrifugation was used without adding acetone or conditioning the recipient cells before centrifugation. Attempts have been made to import exogenous mitochondria using inefficient methods. The resulting cell populations tend to have a large increase in total mtDNA copy number. In one embodiment, the mitochondrial replacement cells have the effect of reducing endogenous mtDNA copy number. Approximately 1.1-fold and 1.2-fold higher than the total mtDNA copy number of recipient cells before exposure to the factor , about 1.3 times, about 1.4 times, about 1.5 times, or more of the total mtDNA copy number.

[0261] The use of non-invasive transfer is another unique aspect of the present invention. Previous methods have required the use of invasive instruments. In this method, exogenous mitochondria are injected and then separated by centrifugation or other methods that are effective for recipient cells. The same harsh conditions that are harmful have physically forced mitochondria into cells. In clinical practice, especially in the case of hematopoietic stem cells or T cells, the number of recipient cells is limited. Harsh cell manipulation is undesirable when cells are in a state where they are not properly grafted. Therefore, the use of non-invasive transfer is of great importance in clinical practice. It is a beneficial feature of the present invention that it lends itself to field use.

[0262] As provided herein, exogenous mitochondria, exogenous mtDNA, or a combination thereof The cells can be autologous or allogeneic to the recipient cells. In embodiments, the exogenous mtDNA is allogeneic to the recipient cells. For example, the exogenous mtDNA can be obtained from the same species as the recipient cells, In another embodiment, the exogenous mitochondrial cells have a genotype different from that of the host cells. The exogenous mtDNA, the exogenous mtDNA, or a combination thereof may be autologous. The intrinsic mtDNA may be derived from healthy donor cells, e.g., "young" donor cells, e.g., umbilical cord blood The recipient cells may contain mtDNA derived from the same subject. and "old" recipient cells, where "young" and "old" The term "has been" refers to the total number of cell doublings in a population or the age of the subject from which the cells are taken. Another exemplary example of autologous exogenous mtDNA is, for example, mtDNA derived from the same subject as the recipient cell. The donor mtDNA is isolated from the recipient cells and modified before being exchanged with the recipient cells. In some embodiments, only the mtDNA and / or mitochondria can be allogeneic. and the recipient cells require exogenous mtDNA and / or exogenous mitochondria. It is something that belongs to the subject.

[0263] In one embodiment, the exchange of mtDNA in the recipient cell comprises a hypervariable region of mtDNA. (HVR), for example, the DNA sequence of HV1 and / or HV2 of the D-loop is sequenced and By comparing the sequences of both the donor mitochondria and the recipient cells, In a specific embodiment, the recipient cell and the donor mitochondrial sequence Differences can be identified by single nucleotide polymorphism assays. For example, Amplified sequences of mtDNA from recipient cells and donor mitochondria were used as quantitative standards. It can be cloned into a plasmid for use in

[0264] In some embodiments, the cells (i.e., donor cells and recipient cells) are: The cell is an animal cell or a plant cell. In a specific embodiment, the cell is mammalian. In some embodiments, the cells are human, horse, dog, cat, mouse, rat, bovine, and sheep. In some embodiments, the cells are human cells. In some embodiments, the cells are cultured in The cells are mammalian cells, either prepared in situ or purchased from a commercial cell source. (preferably human) directly, or from commercial sources, or from tissue, or e.g., cultured The cells may be obtained in the form of cells, and the like. In some embodiments, the cells are primary cells. (i.e., cells obtained directly from living tissue, e.g., biopsy material). to any organ, including the blood or lymphatic system, but not to the muscles, any organ, gland, or skin In some embodiments, the cells are somatic cells. In embodiments, the cells are epithelial cells, neuronal cells, epithelial cells, keratinocytes, hematopoietic cells. (e.g., bone marrow cells), melanocytes, chondrocytes, hepatocytes, B cells, T cells, red blood cells, macrophages Phages, monocytes, fibroblasts, muscle cells, vascular smooth muscle cells, hepatocytes, spleen cells, and pancreatic beta cells.

[0265] As provided herein, in specific embodiments, the donor cells are selected from the group consisting of: The donor cells are commercially available cells cultured under current good manufacturing practice (cGMP). For example, the donor cells are from Waisman Biosciences. cell repositories, such as manufacturing, or similar commercial sources, e.g., cGMP-compliant cells; In some embodiments, donor cells can be obtained from commercially available sources. are cGMP manufactured bone marrow-derived mesenchymal stromal cells (BM-MSCs). In other embodiments, the cells are , cGMP-grade human hepatocytes. Therefore, donor cells are used for isolating mitochondria. It is also understood that the cells may be frozen and thawed prior to use. The doria do not need to be isolated after freezing the cells, but can be isolated from fresh cells and immediately In some embodiments, mitochondria can be isolated and used for , which can then be frozen before being transferred into recipient cells.

[0266] In some embodiments, the cells are cancer cells. Typically, the cancer cells are breast cancer, Prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain tumor, Brain tumors, 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, Gastric cancer, colon cancer, prostate cancer, genitourinary cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, Renal cancer, renal cell carcinoma, endometrial cancer, adrenocortical carcinoma, malignant pancreatic insulinoma, malignant carcinoid cancer , choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic Myeloid leukemia, chronic lymphocytic leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, acute myeloid leukemia Myeloid leukemia, chronic myeloid leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, thyroid cancer, and thyroid cancer. Polycythemia vera, essential thrombocythemia, Hodgkin's disease, non-Hodgkin's lymphoma, soft tissue sarcoma, osteosarcoma , primary macroglobulinemia, and retinoblastoma. do.

[0267] In some embodiments, the cell is a stem cell. The term "stem cell" refers to an undifferentiated cell that can be induced to proliferate. Cells can be self-sustaining or self-renewing, which means that with each cell division, one This means that the daughter cells can also become stem cells. Stem cells can be found in embryonic, postnatal, juvenile, or adult tissues. Stem cells are pluripotent or multipotent. As used herein, the term "progenitor cell" refers to a cell derived from a stem cell and The term "progenitor cells" refers to undifferentiated cells that are not stem cells themselves. Some progenitor cells differentiate into multiple cell types. Stem cells can give rise to offspring that can give rise to the body's tissue lineages: mesoderm, endoderm, and These include pluripotent stem cells that can form either ectoderm or ectoderm cells. Thus, for example, stem cells include human embryonic stem (ES) cells; human inner cell mass (ICM) / epithelial cells; human primitive ectodermal cells, human primitive endoderm cells, human primitive mesodermal cells, and human primitive Stem cells can be selected from germ cells (EG) cells. Stem cells can be cells that make up an entire tissue or multiple tissues. Multipotent stem cells that can form multiple cell lineages, e.g., Stem cells include, but are not limited to, hematopoietic stem cells or neural progenitor cells. Stem cells are the cells that give rise to the entire organism. In some embodiments, the stem cells are mesenchymal stem cells. The term "mesenchymal stem cells" or "MSCs" refers to stem cells that are not terminally differentiated or can divide to give rise to any stem cell, or produce cytokines, etc. Depending on the various influences from which bioactive factors, cells of mesenchymal lineage, e.g., adipose, bone, cartilage, elastic, and fibrous connective tissues, myoblasts), and tissues derived from the embryonic mesoderm For adult cells that irreversibly differentiate to give rise to tissues other than the nucleus (e.g., nerve cells), In some embodiments, stem cells are partially differentiated or differentiated. In some embodiments, the stem cells are reprogrammed. The cells are induced pluripotent stem cells (iPSCs) that have been differentiated or dedifferentiated. In some embodiments, the recipient cells are iPSCs. The stem cells are hematopoietic stem cells (HSCs) or MSCs. Stem cells can be derived from embryonic, fetal, or adult tissues. can be obtained from

[0268] In other embodiments, the cells are immune cells. In some embodiments, the immune cells are T cells, phagocytic cells, or the like. The cells are selected from the group consisting of cells, microglial cells, and macrophages. In one embodiment, the T cells are CD4+ T cells. In another embodiment, 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 those derived from a state of or associated with T cell dysfunction. These are exhausted or nearly exhausted T cells.

[0269] 5.3 Methods for enhancing mitochondrial import Also provided herein are methods for treating rheumatoid arthritis in combination with any of the methods described in Section 5.2. Methods for mtDNA and / or mitochondrial transfer involving the use of a second active agent Mitochondrial import occurs via the endocytic pathway, an ATP-dependent process. For example, under certain cell culture conditions, mitochondria It has been observed that they are engulfed by macropinocytosis (e.g., Kitani et al. J Cell Mol Med., 2014, 18(8):1694-1703). The second step before co-incubating the patient cells with exogenous mitochondria and / or exogenous mtDNA US201301226633A1 - Use of active agents to promote uptake of exogenous mitochondria and / or exogenous mtDNA - Google Patents This also relates to the novel discovery that it is possible to

[0270] Various types of agents can be used to induce the uptake of exogenous mitochondria and / or exogenous mtDNA. In some embodiments, the second active agent can promote the growth of large a second active agent selected from the group consisting of a molecule, a small molecule, or a cell therapy; Syn, NR (nicotinamide riboside), bezafibrate, idebenone, cysteamine tartar Hydrogen benzoate (RP103), elamipretide (MTP131), omaveloxolone (RTA408), KH176, batiki Non (Epi743), thioctic acid, A0001 (α-tocopherol quinone), mitochondrial CoQ10 (Mi toQ), SkQ1 (bisomitin), resveratrol, curcumin, ketogenic diet treatment, hypoxia, and activators of endocytosis. In a similar manner, activators of endocytosis are modulators of cellular metabolism. Cellular metabolism can be modulated using a variety of methods known to those skilled in the art. In B. cerevisiae, modulation of cellular metabolism involves nutrient starvation, chemical inhibitors, or small molecules.

[0271] As mentioned above, the import of intact mitochondria occurs via the endocytic pathway. For example, exogenous mitochondria and / or exogenous mtDNA have been reported to Imported by uptake of intact mitochondria via the endocytic pathway. Cytosis pathways are divided into four categories: 1) clathrin-mediated endocytosis; 2) wall endocytosis; ola, 3) macropinocytosis, and 4) phagocytosis Clathrin-mediated endocytosis is primarily associated with the cytosolic protein clathrin. They are small (approximately 1 mm in diameter) with a distinctive morphological envelope composed of a complex of proteins. 1000 nm) vesicles. Thus, in one embodiment, mitochondria The endocytic pathway for import is clathrin-dependent. In other embodiments, the endocytic pathway for mitochondrial import is In a specific embodiment, the endocytic pathway is a raslin-independent pathway. Macropinocytosis.

[0272] Macropinocytosis is suggested to be an important process in nutrient-deficient environments. As a result, pathways or pathways that are activated by cellular nutrient deficiency or sufficient nutrient intake are Inhibition of target molecules, e.g., mTOR, leads to cellular engulfment of intact mitochondria into the cytosol. Specifically, the methods provided herein are hypothesized to be a strategy to enhance Thus, it was 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 is an important site of protein, lipid, and nucleotide synthesis involved in extracellular nutrient uptake Thus, in some embodiments, the methods provided herein The researchers then transformed recipient cells into small compounds that can increase macropinocytosis. In particular embodiments, the method further comprises contacting the cell with a substance, peptide, or protein. Therefore, the methods provided herein include the transfer of exogenous mitochondria and / or exogenous mtDNA. In one embodiment, the method further comprises modulating the cellular metabolism of the recipient cells prior to the transplantation. The same microenvironmental pathways that regulate cell metabolism can increase macropinocytosis. The method is carried out using small compounds, peptides, or proteins.

[0273] Regulating cellular metabolism includes, but is not limited to, the use of compounds described herein and in the cited references. This can be achieved by any of several well-known techniques, including those described in For example, in some embodiments, modulating cellular metabolism is achieved by nutrient starvation or nutrient deprivation. In another embodiment, modulating cellular metabolism is performed by chemical inhibitors. In a specific embodiment, the inhibitor is a chemical inhibitor or a small molecule. The small molecule is an mTOR inhibitor.

[0274] Rapamycin, also known as sirolimus (CAS number 53123-88-9; C 51 H 79 NO 13 ) and rapamycin Various compounds, including derivatives (e.g., rapamycin analogs, also known as "rapalogs"), have been shown to inhibit mT It is known that OR is inhibited. Examples of rapamycin derivatives include temsirolimus. (CAS No. 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). Therefore, In some embodiments, the methods of mitochondrial import provided herein involve the use of rapamycin. before the transfer of exogenous mitochondria and / or exogenous mtDNA using .beta.-amycin or its derivatives The method further comprises regulating the cellular metabolism of the recipient cells. The above embodiments are non-limiting and that modulating cellular metabolism may be achieved by chemical compounds or It is understood that small molecules need not be involved.

[0275] Thus, in some embodiments, rapamycin containing clinically approved drugs is used. Utilizing isin or a derivative thereof as a stand-alone method or in the methods provided herein , e.g., any method involving partial reduction of endogenous mitochondria in recipient cells. In combination with other methods, the efficiency of import of exogenous mitochondria can be increased.

[0276] Those skilled in the art will appreciate that additional delivery methods may be used to deliver exogenous mitochondria and / or exogenous m tDNA can be introduced, and macropinocytosis is an exemplary pathway. In some embodiments, the mtDNA is a clathrin-dependent delivery by clathrin-dependent or clathrin-independent endocytosis In a specific embodiment, the clathrin-independent pathway is, for example, CLIC / GEEC Endocytic pathway, Arf6-dependent endocytosis, flotillin-dependent endocytosis cytosis, macropinocytosis, circular doral ruffles, It can be phagocytosis or trans-endocytosis. Delivery of mitochondrial and / or exogenous mtDNA may be achieved by any compound that stimulates mitochondrial delivery. This can be enhanced by the use of substances such as activators of endocytosis. Non-limiting exemplary compounds suitable for activating endocytosis include, for example: Phorbol-12-myristate-13-acetate (PMA) (C 36 H 56 O8), 12-O-tetradecanoyl Rubor 13-acetate (TPA) (C 36 H 56 O8), Tanshinone IIA sodium sulfonate (TSN-SS) (C 19 H 17 O6S.Na), and phorbol-12,13-dibutyrate, or their derivatives. It is further understood that the method for bypassing endocytosis and / or cell fusion is also Non-endocytosis-mediated transfer of mtDNA and / or mitochondria, including It is also understood that this is possible.

[0277] (5.4 Treatment method) Provided herein are methods for treating mitochondrial dysfunction associated with mutant mtDNA and / or dysfunctional mitochondria. Various methods for treating diseases associated with mutant mtDNA and / or dysfunctional mitochondria Use of the composition for the treatment of diseases associated with mutant mtDNA and / or dysfunction The use of the composition in the manufacture of a medicament for the treatment of a mitochondrial-associated disease. Also provided are exogenous mitochondrial pathways for restoring or enhancing endogenous mitochondrial function. Use of chondria and / or exogenous mtDNA to restore or enhance endogenous mitochondrial function and the use of the composition for treating a subject in need of mitochondrial replacement. and a method of treatment involving the use of the composition in the manufacture of a medicament. In some embodiments, the treatment , accompanied by prevention of mitochondrial dysfunction.

[0278] 5.4.1 Methods of Treating Age-Related Disorders In some embodiments, provided herein are methods for treating a patient with an age-related disease or who is at an age 5. A method of treating a subject suspected of having a related disorder, comprising administering to a subject a compound according to any one of Sections 5.2 and / or 5.3. In some embodiments, the method comprises any of the methods described in the section. Provided herein are methods for treating or preventing age-related disorders in patients with or suspected of having an age-related disorder. A method of treating a subject receiving a gene encoding ... or contacting the cells with an agent that reduces endogenous mitochondrial function, Partially reduce mtDNA copy number in piñon cells or endogenous mitochondria Incubating the recipient cells for a period of time sufficient to partially reduce function. and (1) recipients with partial loss of endogenous mtDNA or endogenous mitochondrial function. cells and (2) exogenous mitochondria and / or exogenous mtDNA from a healthy donor. Co-incubation is continued for a period of time sufficient to non-invasively transfer mitochondrial material into the recipient cells. By transferring the mitochondria to the cells, mitochondria exchange cells can be generated ex vivo or in vitro. By doing this, mitochondria exchange cells were prepared, and then the mitochondria exchange recipe was A therapeutically effective amount of peritoneal cells is administered to a subject having or suspected of having an age-related disorder. administering to the subject.

[0279] In some embodiments, the age-related disease includes an autoimmune disease, a metabolic disease, a genetic disease, or Diseases include cancer, neurodegenerative diseases, and immunosenescence. Metabolic diseases include diabetes. Neurodegenerative diseases that can be treated by the methods provided herein Non-limiting examples of include Alzheimer's disease or Parkinson's disease. Treatable genetic disorders include Hutchinson-Gilford progeria Syndrome, Werner's syndrome, and Huntington's disease. Dysfunctional mitochondria Further age-related diseases are also contemplated, including:

[0280] In certain embodiments, a patient with or suspected of having an age-related disorder is The method of treating a subject suffering from the disease includes producing MirC, wherein for producing the MirC, The recipient cells used in this study are T cells or hematopoietic stem cells (HSCs). Endogenous mtDNA, endogenous mitochondria, or a combination thereof in T cells or hematopoietic stem cells (HSCs) The cells can be exchanged for rejuvenation. The exchange provides for the treatment of affected patients with human T cells and / or hematopoietic stem cells. Therefore, in some embodiments, the present invention Using the methods provided herein, isolated exogenous mitochondria derived from healthy, non-senescent cells were obtained. Non-invasive transfer of chondria into senescent and senescent cells delays senescence and / or extend the lifespan of the cells to rejuvenate the recipient cells, and then The rejuvenated MirC is administered to patients with or suspected of having an age-related disease. It can be administered to a patient.

[0281] As shown herein, rejuvenation of senescent T cells can be achieved using the present invention in an age-related manner. In one possible embodiment, a subject having a related disease, such as cancer, can be treated. Examples include aging-associated secretions consisting of inflammatory cytokines, growth factors, and proteases. Phenotype (SASP), slowed and / or delayed population doubling rate, telomere shortening, DNA damage response Aged T cells exhibiting an increased DDR (Discrete Differential), or a combination thereof, can be identified using the methods provided herein. For example, young, healthy T cells that are autologous to a subject with an age-related disease, e.g., cancer. It is possible to rejuvenate cells by non-invasively transferring isolated mitochondria from cells. Subsequently, T cell-derived MirC, which has characteristics of young, non-senescent cells, has been shown to be a key regulator of age-related diseases. The compound can be administered to a subject for treatment of a disease.

[0282] Thus, in a specific embodiment, a person having or suffering from an age-related disease The method of treating a subject suspected of having MirC comprises producing MirC, wherein the recipient The cells are T cells. T cell fate is regulated by metabolic pathways, such as glycolysis or oxidative phosphate depletion. Either OXPHOS or OXPHOS is involved in providing most of the energy to T cells. Glucose-dominant T cells choose to differentiate into effector T cells, whereas OXPHOS-dominant T The cells are intended for memory T cells. Therefore, exogenous mitochondria and / or mtDNA can be used to regulate T cell fate. For example, in the case of allergies, exogenous Using mitochondria and / or mtDNA to calm over-activated T cells In other situations, such as cancer immunotherapy, exogenous mitochondria and / or mtDNA may be Empowering anti-tumor T cells to allow them to persist for a long time or to promote T cell lytic capacity; Furthermore, chimeric antigens can be used to treat or reduce tumor burden. A new treatment using receptor T cells (CAR T) uses autologous T cells. These CAR T cells Fatigue symptoms due to aging or malnutrition such as cachexia, which are frequently seen in severe pathological stages of cancer. Mitochondrial replacement technology can provide more ATP by increasing C ART can be activated and rejuvenated, leading to better outcomes.

[0283] Thus, in one embodiment, the method of treating a subject includes administering to a recipient T cell The T cells can be CD4+ T cells, CD8+ T cells, or CAR T cells. In a specific embodiment, mitochondrial replacement in recipient T cells is achieved by administering to recipient T cells a mitochondrial replacement therapy that is life-span-competent. Generates extended T cell lifespan, e.g., about 1.5 times, about 2 times, about 3 times, about 4 times, about 5 times In a specific embodiment, the recipient T Mitochondrial exchange in T cells was associated with a decrease in the number of T cells with mitochondrial exchange compared to T cells without mitochondrial exchange. Inhibit or delay senescence of recipient T cells. As described in Section 5.2, Exogenous mitochondria and / or exogenous mtDNA from donor cells that are younger than the recipient cells By performing mtDNA exchange using In the above, the donor and recipient cells are multiplied by about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 4 times, In other embodiments, the donor and recipient have a PDL difference of about 5-fold or more than 5-fold. The human cells may be derived from subjects separated in age by about 5 years, about 10 years, about 15 years, about 20 years, or more than 20 years. In another specific embodiment, mitochondria in recipient T cells are expressed. Mitochondrial exchange generates T cells with increased lytic capacity compared to T cells without mitochondrial exchange. In still further embodiments, mitochondrial replacement in T cells is This results in a reduction in tumor burden.

[0284] In one embodiment, T cells with exogenous mitochondria and / or exogenous mtDNA are Plasmid-based gene transfection can be used to generate However, in other embodiments, mRNA transfection can be used. The use of transfection reduces the chances of the RNA sequence being integrated into the host genome. and have minimal long-term gene expression that causes endogenous mtDNA degradation. can.

[0285] In certain embodiments, the MaxCyte electroporator is manufactured in accordance with, among other things, Good Manufacturing Practices and It meets the standards of clinical practice and is suitable for use in mRNA transfection in clinical settings. Transfections can be performed using a MaxCyte Electrophoresis Kit according to the manufacturer's protocol. This can be done using a microporator.

[0286] Methods of treating a subject having or suspected of having an age-related disorder The method can also include producing MirC using the methods provided herein, wherein the recipe The primary stem cells are hematopoietic stem cells (HSCs). HSCs are responsible for not only blood cells but also other cells, e.g. By transdifferentiation, they also provide endothelium that replaces damaged resident cells in distant organs. Furthermore, it has been reported that dysfunction of HSCs is involved in systemic aging. Therefore, HSC-derived Mi It is envisioned that rC can be used as a method of treatment for any age-related disease.

[0287] Furthermore, allogeneic HSC transplantation can lead to graft rejection or even graft-versus-host disease. Autologous HSC transplantation is often a safer and more practical means of disease intervention. For example, autologous HSC transplantation is typically preceded by immunosuppressive agents such as radiation and chemicals. Therefore, the healthy, young cells in the autologous HSCs that are then reintroduced into the patient are not affected. In vitro or ex vivo production of MirC using exogenous mtDNA from intact mitochondria was demonstrated. It is envisaged using the methods provided in the specification.

[0288] In one embodiment, the HSCs are in need of mitochondrial and / or mtDNA replacement. The mtDNA is autologous to the subject, and the exogenous mtDNA is allogeneic. As presented, mtDNA exchange in HSCs leads to differentiated cells with functional mitochondria and and / or may result in differentiated cells with improved function. The methods provided herein can be used in the context of HSC transplantation.

[0289] Aging alters biological processes, leading to conditions such as Alzheimer's disease, atherosclerosis, Osteoporosis, type 2 diabetes mellitus, and tissue disorders that contribute to chronic kidney disease and chronic obstructive pulmonary disease Mitochondria affect the aging process, leading to the development of degenerative disorders such as fibrosis. may play a role in aging via reactive oxygen species generated by mitochondria, which may Mitochondrial dysfunction in aging can be attributed to nicotinamide phosphorylation. Downregulation of NAMPT and poly(ADP-ribose) polymerase 1 (PAR Nicotinamide adenine dinucleotide (NAD) is a ubiquitous nucleotide that is caused by overactivation of P1. + ) deficiency of NAD +Nutrient sensing leads to inhibition of the sirtuin-dependent deacetylase sirtuin 1 (SIRT1) This then leads to a vicious cycle of deregulation of PGC1α, which is triggered by the acetylation-dependent inactivation of PGC1α. As a result, NAD + This results in a decrease in mitochondrial biogenesis, which increases the availability of Low activity of PGC1α not only affects the expression of nuclear-encoded mitochondrial proteins, but also the expression of mitochondria. It also down-regulates the expression of the mitochondrial transcription factor TFAM, which is located around the mitochondrial DNA. .

[0290] In addition to two core senescence-regulating pathways, including p53 and p16 / Rb, IL-1, IL-6 / VEGF, and IL-8 Many inflammatory cytokines, chemokines, and proteases, such as CXCL9 / MMP, are released. The senescence-associated secretory phenotype (SASP) is one of the best-characterized phenomena in aging. The transcription factor GATA4 mediates selective autophagy under normal conditions. The DNA damage response (DDR) kinase ATM (ataxia) is degraded upon association with the adaptor p62. Ataxia telangiectasia (mutated) and ATR (ataxia telangiectasia and Rad3-related) receptor senescence The activation signal promotes the dissociation of GATA4 and p62, stabilizing GATA4 and subsequently TRAF3IP2 (a tumor catalyzer). Activates NF-kB through death factor receptor-associated factor-interacting protein 2 and IL1A, resulting in SASP SASP supports this finding. cells) is completely blocked. Mitochondria in oocytes derived from older ones in experimental IVF The exchange of embryos definitely increases the success rate of fertilization, embryo development and implantation, and offspring birth. I did.

[0291] Impairment of protein homeostasis is another hallmark of aging. The integrity of protein homeostasis depends on translational regulation, protein folding chaperones, and ubiquitin. It is strictly maintained by the ATP-proteasome system (UPS) and the autophagy-lysosome system. Because chaperones are ATP-dependent, the age-related decline in bioenergetics may be due to the need for accurate timing. Compromises functions related to protein folding, including UPS and mitophagy. Both phagocytic and lysosomal systems decline over time. They form aggregates in the cytosol that are not recycled, leading to degenerative damage. In Ricks, the accumulation of abnormal proteins triggers this system to degrade them. In addition, the mitochondrial unfolded protein response (UPR) mt ) called the nuclear and communal All of the above pathways provide an opportunity to restore mitochondrial function. , including mitochondria. If mitochondria are replaced in somatic cells, harmful It can disrupt the aging cycle, slow down the aging process, and even rejuvenate cells.

[0292] Thus, the methods provided herein are directed to treating endogenous dysfunctional mitochondria, e.g., Endogenous mitochondria with mutant mtDNA were cultured either of autologous or allogeneic origin. By replacing mitochondria with young and / or healthy mitochondria that may have For treating teloplasmy and / or various diseases, such as diseases associated with aging This provides a clinically viable method for

[0293] In some embodiments, the methods of mitochondrial replacement provided herein involve the use of mitochondria. It can be used to treat mitochondrial diseases or disorders, as well as aging, cancer, and immune system deficiencies. Cut.

[0294] 5.4.2 Methods of Treating Mitochondrial Diseases or Disorders Also provided herein are any of the methods described in Section 5.2 and / or Section 5.3. Therefore, the patient may be diagnosed with a mitochondrial disease or disorder or may be diagnosed with a mitochondrial disease or disorder. is a method of treating a subject suspected of having a disorder. , having a mitochondrial disease or disorder or The method for treating a subject suspected of having a rheumatoid arthritis may be any of the methods described in Sections 5.2 and / or 5.3. Produce MirC according to either of the following methods, and then treat the mitochondria exchange recipient cells. An effective amount is administered to a subject having a mitochondrial disease or disorder or a subject with a mitochondrial disease or disorder. This includes administering to a subject suspected of having the disorder.

[0295] A variety of mitochondrial diseases or disorders are known, all of which can be treated with the methods provided herein. For example, mycobacterium tuberculosis can be treated using the methods provided herein. The mitochondrial disease or disorder can be complex I deficiency (OMIM:252010). I deficiency is caused by a mutation in one of its subunits In another embodiment, the complex I deficiency can be caused by NDUFV1 (OMIM:161015), NDUFV2 (OMIM:161016), or M:600532), NDUFS1(OMIM:157655), NDUFS2(OMIM:602985), NDUFS3(OMIM:603846), NDUFS4 (OMIM:602694), NDUFS6(OMIM:603848), NDUFS7(OMIM:601825), NDUFS8(OMIM:602141), and and NDUFA2 (OMIM:602137). can be.

[0296] Additionally, mitochondrial diseases that can be treated using the methods provided herein Or the disorder can be complex IV deficiency (cytochrome c oxidase; OMIM:220110). Complex IV deficiency is caused by a mutation in one of its subunits. In some circumstances, complex IV deficiency can be attributed to MTCO1 (OMIM:516030), MTCO 2(OMIM:516040), MTCO3(OMIM:516050), COX10(OMIM:602125), COX6B1(OMIM:124089), SCO 1 (OMIM: 603644), FASTKD2 (OMIM: 612322), and SCO2 (OMIM: 604272). It is caused by a mutation in a gene that

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

[0298] Specific types of mitochondria that can be treated using the methods provided herein Non-limiting examples of diseases or disorders include ornithine transcarbamylase deficiency ( Hyperammonemia (OTCD), Carnitine O-palmitoyltransferase II deficiency (CPT 2) Fumarase deficiency, Leigh syndrome-associated cytochrome c oxidase deficiency, MAPE Syrup urine disease (MSUD), medium-chain acyl-CoA dehydrogenase deficiency (MCAD), very long-chain acyl-CoA Mitochondrial DNA deletions, mitochondrial oA dehydrogenase deficiency (LCAD), trifunctional protein deficiency Progressive external ophthalmoplegia (POLG), DGUOK, TK2, pyruvate decarboxylase deficiency, and In another embodiment, the mitochondrial disease or disorder is: Alpers disease; Barth syndrome; β-oxidation defect; Carnitine acyl-carnitine deficiency; Carnitine Coenzyme Q10 deficiency; complex II deficiency (OMIM:252011), complex III deficiency (OMIM:12400 0), Complex V deficiency (OMIM:604273), LHON-Leber hereditary optic neuropathy; MM-Mitochondrial Myopathy; LIMM - lethal infantile mitochondrial myopathy; MMC - maternally inherited myopathy and cardiomyopathy; NARP - neurogenic muscle weakness, ataxia, and retinitis pigmentosa; Leigh's disease; FICP - fatal Infantile cardiomyopathy plus MELAS-associated cardiomyopathy; MELAS-associated cardiomyopathy with lactic acidosis and stroke-like episodes Mitochondrial encephalomyopathy; LDYT-Leber's hereditary optic neuropathy and dystonia; MERRF-Myoc Rhonus epilepsy and ragged red muscle fibers; MHCM-Maternally inherited hypertrophic cardiomyopathy; CPEO-Chronic progressive External ophthalmoplegia; KSS-Kerns-Sayre syndrome; DM-Diabetes mellitus; DMDF-Diabetes mellitus + hearing loss; CIPO-chronic intestinal pseudo-obstruction with myopathy and ophthalmoplegia; DEAF-maternally inherited hearing loss; PEM-progression SNHL-Sensorineural Hearing Loss; Encephalomyopathy; Mitochondrial Cytopathy; DEMCHO-Dementia and Myopathy AMDF-Ataxia, myoclonus; ESOC-Epilepsy; Optic atrophy; FBSN-Familial dyspraxia lateral striatal necrosis; FSGS focal segmental glomerulosclerosis; LIMM lethal infantile mitochondrial myopathy MDM Myopathy and diabetes mellitus; MEPR Myoclonic epilepsy and psychomotor Cardiomyopathy; MERME MERRF / MELAS overlap disorder; MHCM maternally inherited hypertrophic cardiomyopathy; MICM maternally inherited Mitochondrial cardiomyopathy; MILS Maternally inherited Leigh syndrome; Mitochondrial encephalocardiopathy; Multisystemic mitochondrial Disorders (myopathy, encephalopathy, blindness, hearing loss, peripheral neuropathy); NAION Non-arteritic anterior ischemia PEM progressive encephalopathy; PME progressive myoclonic epilepsy; RTT Rett syndrome SIDS, sudden infant death syndrome; and MIDD, a group consisting of maternally inherited diabetes and hearing loss. are selected.

[0299] The methods for treating mitochondrial diseases or disorders provided herein include In an embodiment, a mitochondrial disease caused by a mitochondrial DNA abnormality or disorders, wherein the mitochondrial DNA abnormality is chronic progressive external ophthalmoplegia. palsy (CPEO), Pearson syndrome, Kearns-Sayre syndrome (KSS), diabetes, and deafness (DAD), Leber's hereditary optic neuropathy (LHON), LHON-plus, neuropathy, ataxia, and Retinitis Pigmentosa Syndrome (NARP), Maternally Inherited Leigh Syndrome (MILS), Mitochondrial Encephalomyopathy, Breast Acidosis and stroke-like episodes (MELAS), myoclonic epilepsy and ragged-red fibers Disease (MERRF), Familial Bilateral Striatal Necrosis / Striatonigral Degeneration (FBSN), Luft's Disease, Aminoglycosides mitochondrial DNA multiple deletion syndrome (MDD) and mitochondrial DNA multiple deletion syndrome (MDD). do.

[0300] Mutations in mtDNA are thought to be associated with a number of clinical disorders. These include neurological disorders (e.g., migraine, stroke, epilepsy, dementia, myopathies, Qi, peripheral neuropathy, diplopia, ataxia, speech disorder, and sensorineural hearing loss), gastrointestinal disorders ( constipation, irritable bowel, and swallowing problems), heart disease (e.g., heart failure, heart block, and and cardiomyopathy), respiratory diseases (e.g., respiratory failure, nocturnal hypoventilation, recurrent aspiration, and pneumonia), endocrine endocrine disorders (e.g., diabetes, thyroid disease, parathyroid disease, and ovarian failure), ophthalmological disorders (e.g., Symptoms include optic atrophy, cataracts, ophthalmoplegia, and ptosis. Disorders thought to be associated with NA mutations include neurological diseases (e.g., epilepsy, myeloma, and encephalopathy). opiates, psychomotor retardation, ataxia, spasticity, dystonia, and sensorineural hearing loss), gastrointestinal diseases (e.g., vomiting, failure to thrive, and swallowing disorders), heart diseases (e.g., biventricular hypertrophic cardiomyopathy and and rhythm abnormalities), respiratory disorders (e.g., central hypoventilation and apnea), hematological disorders (e.g., anemia and pancytopenia), kidney disease (e.g., tubular abnormalities), liver disease (e.g., liver failure), These include urinary disorders (e.g., diabetes and adrenal insufficiency), and ophthalmological disorders (e.g., optic atrophy). Thus, the methods and compositions provided herein are directed to the detection of mtDNA mutations associated with Use in the treatment or prevention of diseases and disorders is envisaged.

[0301] In other specific embodiments, the methods provided herein are directed to a patient with a mitochondrial disease or The present invention allows treating disorders, wherein the mitochondrial disease or disorder is caused by a nuclear DNA mutation. The nuclear DNA abnormality is commonly called mitochondrial DNA depletion syndrome-4A, or mitochondrial DNA depletion syndrome. Mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), mitochondrial Chondriac DNA depletion syndrome (MTDPS), DNA polymerase gamma (POLG)-related disorder, sensory ataxia Neuropathic dysarthria-ophthalmoplegia (SANDO), associated with brainstem and spinal cord involvement and elevated lactate Leukoencephalopathy (LBSL), coenzyme Q10 deficiency, Leigh syndrome, mitochondrial complex disorder, Humahr α-Ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl-CoA Gauze deficiency, pyruvate dehydrogenase complex deficiency (PDHC), pyruvate carboxylase Carnitine palmitoyltransferase 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 IOSCA (Interosteoarthritis of the Cerebellum), Mitochondrial Myopathy (MM), Spinal Muscular Atrophy (SMA), Growth Retardation Diarrhea, amino aciduria, cholestasis, iron overload, early death (GRACILE), and Charcot-Marie syndrome - Tooth disease type 2A (CMT2A).

[0302] Many individuals with mtDNA mutations are diagnosed with Kearns-Sayre syndrome (KSS), a chronic progressive disorder characterized by Mitochondrial encephalomyopathy (MELAS) with external ophthalmoplegia (CPEO), lactic acidosis, and stroke-like episodes ), myoclonic epilepsy with ragged-red fibers (MERRF), ataxia, and retinitis pigmentosa Clinically classified into distinct clinical syndromes, such as Neural Asthenia with Recurrent Attack (NARP), or Leigh syndrome (LS). However, considerable clinical variability exists, and many individuals exhibit overlapping Mitochondrial diseases or disorders have emerged as the primary causes of various disease phenotypes (nuclear genetic Mitochondrial recessive ataxia syndrome (MIRAS) caused by mutations in POLG It doesn't fit neatly into one particular category that can be easily explained.

[0303] Exemplary diseases in which mitochondrial disorders are known to play an important role include: Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis, These include, but are not limited to, the pathogenesis of many neurodegenerative diseases. A genetic disease or disorder is subdivided into several syndromes according to symptoms, not 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 Methods of Treating a Subject in Need of Mitochondrial Replacement Also provided herein are methods for treating a cancer, comprising administering to a patient a cancer-causing agent, comprising administering to the patient a cancer-causing agent, a method for treating a cancer-causing agent, or ... a method for treating a cancer-ca and methods of treating a subject in need of mitochondrial replacement according to any of the preceding claims. In some embodiments, the method of treating a subject in need of mitochondrial replacement comprises: MirC is produced according to any of the methods described in Section 5.2 and / or Section 5.3. Thereafter, a therapeutically effective amount of mitochondrial exchange recipient cells is administered to the recipient cells in need of said mitochondrial exchange. This includes administering the compound to a subject.

[0305] A subject in need of mitochondrial replacement may be any subject with dysfunctional mitochondria. In one embodiment, the subject in need of mitochondrial replacement is a Age-related diseases, mitochondrial diseases or disorders, neurodegenerative diseases, retinal diseases, diabetes, hearing have a genetic disorder, a genetic disorder, or a combination thereof, who may benefit from mitochondrial replacement Neurodegenerative diseases that can occur include amyotrophic lateral sclerosis (ALS), Huntington's disease, and Alzheimer's disease. Marger's disease, Parkinson's disease, Friedreich's ataxia, Charcot-Marie-Tooth disease Retinal diseases include, but are not limited to, retinal dystrophies, retinal dysplasias, and leukodystrophies. or dry age-related macular degeneration, macular edema, or glaucoma. Other exemplary diseases, such as mitochondrial diseases or disorders, are described in Sections 5.4.1 and 5.4.2. This is described in more detail in section 5.4.2.

[0306] Subjects in need of mitochondrial replacement are predisposed to mitochondrial dysfunction For example, the subject may have mutant mtDNA. However, there are no signs of, for example, mitochondrial disease. This is because the disease may be an adult-onset disease or Therefore, the methods provided herein can be used to treat mitochondrial replacement and preventing any of the diseases described herein by treating a subject suffering from the disease. It can also be done as follows.

[0307] 5.5 Methods for Producing iPSCs The present invention provides a method for producing or enhancing the production of induced pluripotent stem cells (iPSCs) from non-pluripotent cells. The methods described in Sections 5.2 and 5.3 for the generation of iPSCs are also provided. Produced from non-pluripotent cells using exogenous expression of stemness factors such as α, Sox2, and c-Myc. Furthermore, it has been shown that a small number of mitochondrial DNA (mtDNA) copies are present in undifferentiated ESCs. This number increases with differentiation and the level of mitochondrial maturity. (Facucho-Oliveira JM et al., J Cell Sci 2007;120(Pt 22):4025-4034). Thus, the present invention provides a method for transforming non-pluripotent recipient cells into endogenous cells using the methods provided herein. The non-pluripotent cells are contacted with an agent that reduces endogenous mtDNA, and the agent reduces the endogenous mtDNA of the non-pluripotent cells. Incubating the non-pluripotent recipient cells for a period of time sufficient to partially reduce This reduces endogenous mtDNA in non-pluripotent embryos, followed by the expression of Oct3 / 4, Klf4, and S Generation of iPSCs by introducing one or more expression cassettes for the expression of ox2 and c-Myc In one embodiment, the exogenous mtDNA and / or Alternatively, exogenous mitochondria are non-invasively transferred into recipient cells.

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

[0309] In one embodiment, the method comprises incorporating exogenous mitochondria and / or exogenous mtDNA into a recombinant plant. Transfect recipient cells with exogenous mitochondria for a period of time sufficient to non-invasively transfer the mitochondria into recipient cells. The method further includes incubating the cells with ATP and / or exogenous mtDNA. In an embodiment, the method comprises administering the recipient a sufficient amount of the endogenous mtDNA for a period of time sufficient to replace a large portion of the endogenous mtDNA. Incubating the cells with exogenous mitochondria and / or exogenous mtDNA Further, the present invention includes exogenous mitochondria and / or exogenous mtDNA and / or exogenous mitochondria. A method for producing iPSCs from non-pluripotent cells, including transfecting pluripotent stem cells, is described in Section 5.3. The present invention may also include any of the embodiments described above.

[0310] A small number of mitochondrial DNA (mtDNA) copies have been detected in undifferentiated embryonic stem cells (ESCs). and promoting pluripotency of non-pluripotent stem cells and generating iPSCs using the methods provided herein. It is also possible to reduce the number of exogenous genes required to produce a gene. For example, In embodiments, the methods provided herein are used to transform non-pluripotent recipient cells into The non-pluripotent cells are contacted with an agent that reduces endogenous mtDNA, and the agent reduces the endogenous mtDNA of the non-pluripotent cells. Incubate non-pluripotent recipient cells for a period of time sufficient to partially reduce NA. This reduces endogenous mtDNA in non-pluripotent cells, followed by the expression of Oct3 / 4 and Klf4. , Sox2, and c-Myc into non-pluripotent cells, thereby generating pluripotent stem cells. The cells can also be generated to generate iPSCs. iPSCs can even be generated without exogenous factors, using only small molecule agents.

[0311] In certain embodiments, the iPSCs can contain mutant mtDNA. Mutant mtDNA contains point mutations, such as point mutations in tRNA (e.g., MELAS). Mutant mtDNA can also include mtDNA with long mtDNA deletions. In other embodiments, non-pluripotent cells for use in generating iPSCs can be heterologous. The integration of mutant mtDNA can facilitate the creation of disease models, for example. It can be made into.

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

[0313] Culture conditions, identification, and establishment of iPSCs are within the skill of one of ordinary skill in the art. For example, methods include: U.S. Patent Nos. 8,058,065 and 8,278,104, which are incorporated herein by reference in their entirety. This includes the methods provided in

[0314] 5.6 Assays for Measuring Heteroplasmy As previously disclosed, mutant mtDNA and / or heteroplasmy may result in dysfunctional Therefore, the method proposed herein for mtDNA exchange can be used to generate mitochondria. To assess mitochondrial function and / or mtDNA mutations in relation to the methods provided Assays useful for determining or predicting the functionality of mitochondrial and / or mtDNA mutations are The present invention includes any assay known to those skilled in the art that can be used to determine whether a particular

[0315] By way of example, assays for determining mitochondrial function include, for example, the following: secreted factors associated with inflammatory processes (e.g., proinflammatory cytokines, proteases, and proliferation and and angiogenic factors, e.g., IL-1, IL-6 / VEGF, IL-8, and CXCL9 / MMP); Mitochondrial function by using Keima-Red; mitophagy by using Keima-Red; Chondriac permeability; Mitochondrial membrane potential; Cytochrome c levels; Reactive oxygen species; Cellular respiration; Activity Enhanced innate immunity, overactivation of overactive glycolysis, alleviation of ER stress, and mTOR-S6 pathway Transcriptomics and proteomics for measuring inhibition of ATP and cell cycle; Mitochondrial DNA is observed by microscopy and quantified by specialized software. dynamics, e.g., measurement of any one of fission and fusion; or mitochondrial function This includes any assay known in the art that measures

[0316] Various sequencing methods may be used in combination with any of the methods provided herein. (1) detect mutant mtDNA, (2) quantify heteroplasmy, and / or (3) detect exogenous mtDNA. The transfer of mitochondrial and / or exogenous mtDNA can be assessed or confirmed. A stretch of approximately 1,100 nucleotides does not contain a gene, but contains a D-loop, a displacement The D-loop is the region where the mutation occurs and is called the control region. It contains two regions that accumulate more frequently than elsewhere in the The variable regions are referred to as HV1 and HV2. Thus, in some embodiments, the mtDNA mutation Mutations were identified by sequencing the hypervariable regions (HV) (i.e., HV1 and / or HV2) of the D-loop of mtDNA. mtDNs can be identified in the context of the methods provided herein by performing mtDN analysis. A. Sequencing is performed using any sequencing method known in the art. In a specific embodiment, the sequencing method is a single nucleotide polynucleotide sequence. 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 are methods for producing a medicament for use in any of the methods described in Sections 5.2 through 5.5. In one embodiment, provided herein is a composition of cells obtained by (a) contacting recipient cells with an agent that reduces endogenous mtDNA copy number; (b) The agent is sufficient to partially reduce the endogenous mtDNA copy number in the recipient cells. (c) (1) incubating the recipient cells for a period of time during which the endogenous mtDNA is partially reduced; (2) transferring exogenous mitochondria from the recipient cells obtained in step (b) to the exogenous mitochondria; Co-incubation is continued for a period of time sufficient to non-invasively transfer chondria into the recipient cells. and then subjecting the resulting mitochondria to a transfection, thereby producing a mitochondria-exchanged cell. 1. A composition comprising mitochondrial replacement cells, wherein the mitochondrial replacement cells comprise more than 5% exogenous In another aspect, provided herein is a composition comprising: (a) a recipe for a recombinant mtDNA; (b) contacting the patient cells with an agent that reduces the endogenous mtDNA copy number; and (b) determining whether the agent reduces the endogenous mtDNA copy number. for a period of time sufficient to partially reduce the endogenous mtDNA copy number in recipient cells. incubating the recipient cells; and (c)(1) the endogenous mtDNA is partially depleted. The recipient cells from step (b) and (2) exogenous mtDNA from a healthy donor are combined to form exogenous mtDNA. and co-incubating the cells for a period of time sufficient to non-invasively transfer the Produce mitochondria-exchanged cells by: A composition comprising mitochondrial replacement cells, wherein the mitochondrial replacement cells contain more than 5% exogenous mtDNA. Well, it is a composition.

[0318] The composition comprises contacting a cell with an agent that reduces mitochondrial function, and then The drug partially reduces endogenous mitochondrial function in the recipient cells. and incubating the recipient cells for a sufficient period of time. In some embodiments, endogenous mitochondrial function is then partially restored. The recipient cells, which are depleted in the presence of either exogenous mitochondria derived from a healthy donor, were then transplanted into the recipient. and for a period of time sufficient to non-invasively transfer exogenous mitochondria into the recipient cells. , and co-incubate, thereby producing mitochondria-exchanged cells. In another embodiment, the endogenous mitochondrial function is then partially reduced. The recipient cells were transfected with either exogenous mtDNA or exogenous mitochondria from a healthy donor. and co-incubating the cells for a period of time sufficient to non-invasively transfer the In some embodiments, mitochondria-exchanged cells can be produced by The mitochondrial replacement cells produced by the above method contain more than 5% exogenous mtDNA. .

[0319] As noted above, exogenous mitochondria can be composed of exogenous mtDNA. Therefore, in some embodiments, both exogenous mitochondria and exogenous mtDNA are MirC is transferred to recipient cells and expresses both exogenous mitochondria and exogenous mtDNA. In other embodiments, the exogenous mtDNA is transduced via exogenous mitochondria into the genome. The exogenous mtDNA is then delivered to endogenous mitochondria. In some situations, exogenous mitochondria may be involved in the transfer of exogenous mtDNA to endogenous mitochondria. After reaching the target site, MirC is removed from the cell. , with exogenous mtDNA and no exogenous mitochondria.

[0320] The endogenous mtDNA of the recipient cells is partially degraded, so exogenous mitochondria and exogenous MirC containing exogenous mtDNA, endogenous mtDNA, or a combination thereof may contain both exogenous and endogenous mtDNA. Similarly, in a scenario where exogenous mitochondria are transferred into recipient cells, In this study, MirC was found to be contained in both exogenous and endogenous mitochondria. Thus, in specific embodiments, the compounds obtained by the methods provided herein are The composition of one or more mitochondrial replacement cells to be used may comprise endogenous mitochondria and exogenous mitochondria. In other embodiments, the method provided herein comprises the step of: The composition of one or more mitochondrial replacement cells obtained by the method comprises a combination of endogenous mtDNA and exogenous mtDNA. In yet a further embodiment, the mtDNA is heteroplasmic. These mitochondrial replacement cells were contacted with an agent that reduces the endogenous mtDNA copy number. Compared to the total mtDNA copy number of the recipient cells before transplantation, the number of copies was approximately 1.1 times, 1.2 times, 1.3 times, and 1.4 times higher. The total mtDNA copy number is no more than 1.4 times, about 1.5 times, or more.

[0321] The present invention relates to an agent that reduces endogenous mtDNA or an agent that reduces mitochondrial function. and a second activating agent for use in the method of producing a mitochondrial exchanger. In certain embodiments, the composition comprises exogenous mitochondria, one or more or a combination thereof. In the above, the composition may further comprise exogenous mtDNA.

[0322] As described in Section 5.3, various second active agents may act on one or more mitochondria. For example, in some embodiments, The second active agent may comprise a macromolecule, a small molecule, or a cell therapy, and the second active agent may comprise a The agents used are rapamycin, NR (nicotinamide riboside), bezafibrate, and idebenone. , cysteamine hydrogen tartrate (RP103), elamipretide (MTP131), omaveloxolone (RTA4 08), KH176, Batchiquinone (Epi743), Thioctic Acid, A0001 (α-Tocopherolquinone), Mito Chondria CoQ10 (MitoQ), SkQ1 (Bisomitin), resveratrol, curcumin, ketogenic diet The therapeutic agent is optionally selected from hypoxia and an activator of endocytosis.

[0323] The use of endocytic activators was demonstrated in cells treated with the MTS-XbaIR plasmid. It has been shown to enhance the uptake of exogenous mitochondria in the vesicles, but the "additional" or "mimic" There was no uptake-enhancing effect in mock-transfected cells, which resulted in exogenous mitochondria being This mechanism of chondrial migration has been shown to be unique to the methods provided herein. Non-limiting exemplary compounds suitable for activating endocytosis include: , e.g., phorbol-12-myristate-13-acetate (PMA) (C 36 H 56 O8), 12-O-tetradecane Canoylphorbol 13-acetate (TPA) (C 36 H 56 O8), Tanshinone IIA Sodium Sulfonate (TSN-SS) (C 19 H 17 O6S.Na), and phorbol-12,13-dibutyrate, or their derivatives In some embodiments, the activator of endocytosis is Includes modulators of cellular metabolism.

[0324] Regulating cellular metabolism includes, but is not limited to, the use of compounds described herein and in the cited references. This can be achieved by any of several well-known techniques, including, for example, In some embodiments, modulating cellular metabolism is a process that reduces the risk of death due to nutrient starvation or nutrient deprivation. In another embodiment, modulating cellular metabolism is performed by chemical inhibitors. In a specific embodiment, the inhibitor is a chemical inhibitor or small molecule. The compound is an mTOR inhibitor.

[0325] Rapamycin, also known as sirolimus (CAS number 53123-88-9; C 51 H 79 NO 13 ) and rapamycin A variety of compounds, including derivatives (e.g., rapamycin analogs, also known as "rapalogs"), have been shown to inhibit mT It is known that OR is inhibited. Examples of rapamycin derivatives include temsirolimus. (CAS No. 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). In some embodiments, the compositions provided herein contain rapamycin or a derivative thereof. The above embodiments for modulating cellular metabolism are not limiting and include any of the above. This does not require chemical compounds or small molecules and involves modulation of other pathways beyond mTOR. It is understood that the composition can contain an activator of endocytosis. It is also understood that it may be optionally included and that it is not a required component. Furthermore, in some embodiments, the inventions provided herein can be used to treat, for example, non- Involvement of non-endocytosis-mediated transfer of mtDNA and / or mitochondria in clinical settings can be done.

[0326] As described in Section 5.5, the present invention provides, in certain embodiments, a method for reducing endogenous mtDNA. one or more expression cassettes for expression of the down-regulating factors Oct3 / 4, Klf4, Sox2, and c-Myc and generating induced pluripotent stem cells (iPSCs) from non-pluripotent cells, including recipient cells. Also provided are compositions for use in the method, wherein the recipient cells are non-pluripotent cells. wherein the agent that reduces endogenous mtDNA is an agent that reduces endogenous mtDNA. Compared to non-pluripotent cells that were not treated with IFN-γ, induced pluripotent stem cells (IPS) were generated from non-pluripotent cells. In some embodiments, the IL-16 receptor is present in an amount effective to increase the efficiency of producing a PSC. , an agent that reduces endogenous mtDNA is treated with an agent that reduces endogenous mtDNA. Generate induced pluripotent stem cells (iPSCs) from non-pluripotent cells compared to non-pluripotent cells without The compound is present in an amount effective to increase the efficiency of the pluripotent cells to increase mtDNA copy number. In a specific embodiment, the method for producing iPSCs is based on the observation that iPSCs have a decreased number of The composition for use in further comprises exogenous mitochondria and / or exogenous mtDNA.

[0327] The present invention relates to an age-related disease, a mitochondrial disease or disorder, as described in Section 5.4, Neurodegenerative diseases, diabetes, genetic disorders, or any condition requiring mitochondrial replacement In certain embodiments, the present invention also includes pharmaceutical compositions for use in the treatment of elephants. Provided are isolated mitochondrial cells with exogenous mitochondria from healthy donors. A pharmaceutical composition comprising a population of erythrocyte-exchanged cells, the cells being described herein, e.g., in Section 5.2. In other embodiments, the pharmaceutical composition is obtained by the method described in Sections 5.1 to 5.3. isolated with exogenous mitochondria and / or exogenous mtDNA from healthy donors The present invention relates to a population of mitochondrial exchanged cells, and the cells may be any of the cells described herein, e.g., in Sections 5.2 to 5.9. For example, in some embodiments, the exogenous Mitochondria-exchanged cells with endogenous mtDNA may optionally further contain exogenous mitochondria. In other embodiments, exogenous mtDNA can be delivered to endogenous mitochondria. They are imported into cells via exogenous mitochondria, which are then transported to the cell via , are removed from the recipient cells.

[0328] The present disclosure provides an isolated mitochondrial cell line with exogenous mitochondria from healthy donors. Also provided is a pharmaceutical composition comprising a population of mitochondrial exchange cells, wherein the cells are mitochondrial exchange cells. In another embodiment, the cells are obtained by any of the methods provided herein for obtaining the cells. In this disclosure, isolated mitochondria with exogenous mtDNA from healthy donors are used. A pharmaceutical composition is provided comprising a population of replacement cells, wherein the cells are mitochondrial replacement cells. In some embodiments, the method is obtained by any of the methods provided herein for obtaining In mice, isolated mitochondrial exchange cells with exogenous mtDNA from healthy donors were used. The pharmaceutical composition comprising the population of cells further comprises exogenous mitochondria.

[0329] For example, in some embodiments, exogenous mitochondria from healthy donors are included. The pharmaceutical composition comprises contacting a cell with an agent that reduces mtDNA copy number, followed by administration of a recipe. The agent partially reduces the endogenous mtDNA copy number of the recipient cells. and incubating for a period of time sufficient to cause the In some embodiments, the gene is then transformed with a partially reduced endogenous mtDNA copy number. The recipient cells are then non-invasively transferred with exogenous mitochondria into the recipient cells. Co-incubate with either exogenous mitochondria from a healthy donor for a period sufficient to This allows the production of mitochondrial-exchanged cells. and then transfecting recipient cells with a partially reduced endogenous mtDNA copy number. The recipient cells are maintained in a healthy state for a period of time sufficient to non-invasively transfer exogenous mitochondria into the recipient cells. The cells were then co-incubated with exogenous mtDNA from either donor, thereby allowing the mitochondrial In some embodiments, the above-described methods can be used to generate replacement cells. The mitochondrial replacement cells produced by this method contain more than 5% exogenous mtDNA.

[0330] In other embodiments, the cells are contacted with an agent that reduces mitochondrial function. and then the agent ameliorates endogenous mitochondrial function in the recipient cells. and incubating the recipient cells for a period of time sufficient to effectively reduce the In some embodiments, the partially reduced endogenous Recipient cells with endogenous mitochondrial function are then cultured by transferring exogenous mitochondria to the recipient cells. The patient was given either exogenous myeloid leukemia virus (MLL) derived from a healthy donor for a period sufficient to non-invasively transfer the patient's cells to the donor. Co-incubation with mitochondrial cells, thereby creating mitochondrial-exchanged cells. In another embodiment, the partially reduced endogenous mitochondria are then The recipient cells with mitochondria function were then transfected with exogenous mitochondria noninvasively. Co-injected with any exogenous mtDNA from a healthy donor for a period sufficient to rapidly transfer Incubate the cells, thereby creating mitochondrial replacement cells. In an embodiment, the mitochondria-exchanged cells produced by the above method have more than 5% Factors that reduce mitochondrial function, including exogenous mtDNA, can affect mitochondrial function. The agent can transiently or permanently reduce mitochondrial function. The inhibitors may be transient (e.g., reversible inhibitors) or permanent (e.g., It is within the skill of one of ordinary skill in the art to be able to determine the level of a specific inhibitor (irreversible inhibitor).

[0331] In certain embodiments of the pharmaceutical compositions provided herein, the cells are recipient cells. recipient cells before co-incubating with exogenous mitochondria and / or exogenous mtDNA. and a method further comprising contacting the mouse cell with a second active agent. In some embodiments, the second active agent is a large molecule, a small molecule, or a cell. and the second active agent is selected from the group consisting of rapamycin, NR (nicotine), amide riboside), bezafibrate, idebenone, cysteamine bitartrate (RP103) , elamipretide (MTP131), omaveloxolone (RTA408), KH176, vatiquinone (Epi743), Thioctic acid, A0001 (α-tocopherol quinone), mitochondrial CoQ10 (MitoQ), SkQ1 (Bi somitin), resveratrol, curcumin, ketogenic diet, hypoxia, and endothelial In a specific embodiment, the activator of ATPase activity is selected from the group consisting of: Activators of endocytosis are modulators of cellular metabolism. In this manner, modulators of cellular metabolism may be nutrient starvation, chemical inhibitors, or small molecules. In a further embodiment, the chemical inhibitor or small molecule is an mTOR inhibitor. In still further embodiments, the mTOR inhibitor is rapamycin or a derivative thereof. Includes conductors.

[0332] As described in Section 5.2 above, various cell types can be used as recipient cells and recipient cells. For example, the present disclosure provides methods for treating mammalian cells, such as mammalian recipient cells. However, any cell with mitochondria It is also understood that the recipient cell can be a recipient cell. Thus, the recipient cell is , or plant cells.

[0333] In some embodiments, the animal cell is mammalian. In a further embodiment, the cell is a somatic cell. In a further embodiment, the somatic cell is an epithelial cell. In still further embodiments, the epithelial cells are thymic epithelial cells (TECs).

[0334] The present disclosure also provides compositions in which the somatic cells are immune cells. wherein the immune cells are T cells, e.g., exhausted T cells. In some embodiments, the composition comprises exogenous mitochondria and / or exogenous mtDNA. For example, senescent or senescent T cells (e.g., immune-mediated T cells) may be used. These cells (those that have undergone senescence) can serve as recipient cells, and T cell-derived MirC for generating T cells with healthy exogenous mitochondria and / or exogenous mtDNA They can be made using the methods provided herein. In a specific embodiment, 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. For example, In some embodiments, the present disclosure provides CAR-T cells that are effective in killing cancer cells. MirC-derived CART can increase immune surveillance and enhance cancer cell killing. In other embodiments, the immune cells are phagocytes and have long-term survival. be.

[0335] As described above, the compositions provided herein may be used to delay senescence and / or promote longevity in cells. It may also include compositions for use in life extension. The compositions may include endogenous mitochondrial Isolated extracellular matrix from senescent and senescent-prone cells, non-senescent cells, and These may include factors that reduce endogenous mitochondrial and endogenous mtDNA copy number. The composition is useful for senescent and senescent-prone cells that have endogenous mitochondria. Isolated exogenous mitochondria from cells that do not have reduced mitochondrial function The compound may also include an agent that

[0336] Also provided herein are one or more recipient 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 contain mitochondria. Mitochondrial disease, age-related disease, or mitochondrial disease, age-related disease Suspected or otherwise in need of mitochondrial replacement and exogenous mitochondria The mitochondria can be isolated from subjects whose endogenous mitochondria have been replaced with mitochondrial cells. The HSC or MSC-derived MirCs are then transplanted back into the subject in need of the mitochondrial replacement. In yet a further embodiment, the recipient cells are iPS cells. The composition can be used in a clinical setting and is useful for treating age-related diseases, mitochondrial disorders, may be effective in treating diseases or disorders, neurodegenerative diseases, diabetes, or genetic disorders For example, in some embodiments, iPSCs are cultured in a manner consistent with the art before being administered back to the subject. The cells can be differentiated into specific cell types using methods known in the art.

[0337] In other embodiments, provided herein are vectors having reduced amounts of endogenous mtDNA. a pharmaceutical composition comprising a population of isolated pluripotent cells comprising a pluripotent cell selected from the group consisting of: In a specific embodiment, the isolated The resulting population of pluripotent cells are iPS cells.

[0338] The administration of the cells or compounds described herein is not limited to the methods commonly used to deliver drugs. The pharmaceutical compositions of the present invention may comprise a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" refers to a compound that is pharmaceutically acceptable for animals, such as Specifically, it is not approved by any federal or state regulatory agency for use in humans. or listed in the United States Pharmacopoeia or other generally recognized foreign pharmacopoeias. The term "carrier" refers to a diluent, agent, or substance with which the therapeutic agent is administered. A pharmaceutically acceptable carrier refers to a substance, excipient, or vehicle that is used in the administration of a particular determined in part by the composition and by the method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention (e.g., (See Remington's Pharmaceutical Sciences, 17th ed., 1985).

[0339] Formulations suitable for administration may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic. Aqueous and non-aqueous solutions, isotonic sterile solutions, and suspending agents, solubilizing agents, Examples of suitable suspensions include aqueous and non-aqueous sterile suspensions which may contain anti-inflammatory agents, thickening agents, stabilizers, and preservatives. In the practice of the invention, compositions can be administered, for example, orally, intranasally, topically, intravenously, intraperitoneally, or intravenously. The compound can be administered intrathecally, intrathecally, or intraocularly (e.g., by eye drops or injection). The formulations may be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials. Solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described. It can be manufactured.

[0340] The dose administered to a patient in the context of the present invention is determined to induce a beneficial response in the subject over time. induces, i.e., is sufficient to prevent, ameliorate, or reverse the disease in a subject; The optimal dosage level for any patient will depend on the particular modulator utilized. The efficacy of the drug, the patient's age, weight, physical activity, and diet, as well as possible interactions with other drugs, The size of the dose will also depend on a variety of factors, including the combination of The existence, nature, and extent of any adverse side effects that accompany the administration of the compounds or vectors. 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 may be made in accordance with the foregoing description and accompanying drawings. Such modifications are intended to be within the scope of the appended claims. It is intended to enter.

[0342] All patents, applications, published applications, and other publications cited herein are incorporated by reference in their entirety. Any explanation of a term described is incorporated herein by reference. Any explanation of a term used herein, if inconsistent with any document incorporated herein, shall take precedence.

[0343] Throughout this application, various publications are referenced. No. 6,299,625, filed Dec. 1, 2003, in which: The present invention has been described with reference to the examples provided above, but the present invention It should be understood that various changes may be made without departing from the spirit of the present invention. be. [Example]

[0344] 6. Working Example The examples in this section are offered by way of illustration and not by way of limitation. These are presented as exemplary embodiments of the invention and should not be construed as limiting the broad scope of the invention. should not be seen as

[0345] Example I: Optimization of the MirC protocol demonstrates that XbaI degrades mtDNA in vitro and cleaves the MTS expression vector. revealed that catalyzes the targeting of mitochondria A scheme of the method used to generate mitochondrial exchange cells (MirC) is shown in Figure 1A. First, the MTS-XbaI sequence was inserted into pCAGGS using standard techniques known in the art. Xba fused to a mitochondrial targeting sequence (MTS) by cloning into a vector The mammalian expression vector used to express the I restriction enzyme was engineered (Figure 1B Among the reported mitochondrial import signals (MTS), the present inventors have identified the ND4 signal. The resulting expression vector also contained a puromycin sequence to allow selection. It contained the mycin resistance gene (Fig. 1B).

[0346] XbaIR is one of the most potent endonucleases and is involved in the regulation of the human mitochondrial genome. The standard sequences of mtDNA, named according to the Cambridge Reference Sequence (CRS) of the genome, are The isolated mtDNA has as many as five recognition sites targeted by the Xb clease (Figure 1D). In vitro endonuclease co-incubation leads to digestion at multiple sites by aIR In contrast, NotI digestion of mtDNA resulted in the formation of mitochondrial DNA fragments (Fig. 1C). The DNA fragments showed a single fragment, as predicted by the cross-bridge reference sequence (CRS) (Fig. 1C).

[0347] The protocol for transfecting cells with plasmid DNA is described in Nucleofector electroporation. By using a transcription-based transfection method, enhanced green fluorescent protein (E Optimization was performed using normal human dermal fibroblast (NHDF) cells expressing GFP. After 1 day of mycin exposure, >90% efficacy and >90% survival were achieved (Figure 1E).

[0348] To specifically evaluate the effectiveness of the MTS targeting sequence, a plasmid carrying MTS fused to EGFP was prepared. The smid was generated by subcloning the EGFP gene in place of the XbaIR gene. The pCAGGS-MTS-EGFP-PuroR plasmid was generated using the GFP-PuroR gene (Figure 1F). ) was transfected with an MTS-EGFP expression vector, and the cells were incubated in an active medium with an intact membrane potential. TMRM (tetramethylrhodamine, methyl methyl rhodamine), a cell-permeable dye that accumulates in mitochondria The cells were counterstained with esters (Figure 1G).

[0349] Taken together, these results demonstrate that XbaI can be used to digest mitochondrial DNA. This allows cells to be efficiently transfected without affecting cell viability. Therefore, we demonstrated that MTS-containing expression vectors can effectively target mitochondria. showed.

[0350] Example II: Endonuclease MTS-XbaIR treatment improves mtDNA degradation compared to conventional EtBr method (Indicates The efficiency and effectiveness of the MTS-XbaIR expression vector compared to the conventional method using ethidium bromide (EtBr) The efficacy was evaluated according to the scheme shown in Figure 2A. Placental vein endothelial-derived cell line EPC100 was cultured in 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (PSC). The cells were cultured in pyruvate-free DMEM (Wako cat# 044-29765) containing phospholipase S (P / S), and on day 0 Then, cells were either untreated ("normal") or transfected with the MTS-XbaIR expression vector ("MTS-XbaIR"). On day 1, cells were either transfected or treated with 50 ng / mL EtBr. DM containing 10% FBS and 1% P / S supplemented with 0 μg / mL pyruvate and 50 μg / mL uridine Cultured in EM. Measure mtDNA relative to the housekeeping gene β-actin (Actb). To determine the percentage of α-glucan-1-phosphate dehydrogenase (α-glucan-1-phosphate dehydrogenase), quantitative polymerase chain reaction (qPCR) was performed at 3 and 5 days according to methods known in the art. These results demonstrate that XbaIR reduces the mtDNA copy number to 2715.8141. In contrast, EtBr treatment resulted in a DNA copy number of 5169.125, similar to 6189.6867 in untreated cells. The endonuclease-treated group showed that the mtDNA copy number was only reduced to 8 (Figure 2B). The reduction in mtDNA in the group treated with conventional methods was superior to that in the group treated with conventional methods. In this case, the mtDNA was not completely deleted, but about 30% of the endogenous mtDNA remained (Fig. 2B). Cells with a decrease were designated ρ(-) cells.

[0351] Enhanced degradation of endogenous mtDNA in the MTS-XbaIR-treated group compared with the EtBr group was observed using DsRed-labeled mitochondria. This was further confirmed by microscopic examination of chondria (Fig. 2C). This is reflected in the estimated remaining healthy mitochondrial volume, which is Furthermore, FACS analysis of NHDF cells revealed that XbaIR-treated NHDF cells increased the expression of IL-1, which was significantly lower than that of the control group (Fig. 2C). A decrease in TMRM was observed after treatment ( Figure 2 D).

[0352] The dynamics of XbaIR expression after plasmid transfection was examined by qPCR. On day 3, expression was , peaked, and then decreased to 0 on day 7 (Fig. 2E). For example, GFP) showed the same kinetics as XbaIR (Fig. 2E). Transfection with the MTS-EGFP-PuroR plasmid after puromycin selection compared with before transfection The enrichment of GFP in transfected cells was confirmed (Fig. 2F and Fig. 2G). The fraction of GFP-positive cells was , and a 1-day exposure to puromycin significantly increased the expression level to almost 100% (Fig. 2 F).

[0353] These results suggest that the reduction in mtDNA copy number in the XbaI endonuclease-treated group is due to the This is superior to the group treated with the conventional EtBr method, and completely deletes all endogenous mtDNA. Furthermore, short-term selection with puromycin resulted in the formation of MTS. A significant enrichment of cells expressing the construct was achieved.

[0354] Example III: Expression of endogenous mitochondria using the MTS-XbaIR construct in recipient cells Partial degradation of mitochondrial DNA allowed for mitochondrial exchange from exogenous donor cells. Exogenous mitochondria from healthy donor cells were subjected to XbaI-mediated mtDNA deletion. Therefore, to evaluate whether the NHDF cells could be transfected into recipient cells, The cells were transfected with MTS-GFP or MTS-XbaIR plasmid, and then treated with puromycin 48 hours later. Six days after transfection, DsRed-labeled uterine endothelial derived cells were selected. Isolated mitochondria from a human cell line (designated EPC100) were transferred to donor cells. A scheme of the protocol is shown in Figure 3A.

[0355] After transfection with MTS-GFP or MTS-XbaI and selection with puromycin, Mitochondrial content was assessed by TMRM staining. As shown in Figure 3B, MTS-GFP transfectants were Transfected cells showed strong staining for TMRM, indicating high levels of mitochondria in NHDF cells. In contrast, MTS-XbaI transfected cells (ρ-) showed no visible cytoplasmic changes by TMRM staining. When induced, they showed a decrease in mitochondrial volume (Fig. 3B).

[0356] Mitochondrial DNA coding was determined by qPCR of 12S-rRNA after adjusting for nuclear β-actin (Actb). The reduction of mitochondrial DNA was further confirmed by quantifying the number of peaks (Figure 3C). In contrast to MTS-GFP transfected NHDF control cells, MTS-XbaI transfected NHDF cells A significant decrease in mitochondrial DNA from mitochondrial cells (ρ-) was observed (Fig. 3C). The significant reduction in mitochondrial DNA in the sera persisted for the duration of the assay, and this ceased at day 12. Specifically, the copy number dropped to about one-third of the original copy number on day 6, and On day 12, the number of ρ-cells decreased to approximately one-fourth (Fig. 3C).

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

[0358] Consistent with the results shown in Figures 2C and 2D, ρ(-) cells showed a significantly higher cellular phenotype than ρ(-) cells, as measured by TMRM visualization. As expected, the mitochondrial content was reduced after MTS-XbaI transfection. Notably, the reduction in mitochondria was due to the uptake of DsRed-labeled isolated mitochondria. As indicated by the ρ(-) cells contacted with isolated exogenous mitochondria. In contrast, the DsRed-marked cells were rescued by and isolated mitochondria and NHDF control cells or mock transfectants MTS-EG Co-culture with either NHDF cells transfected with an FP expression vector resulted in the expression of exogenous It was found that mitochondria gather at the periphery of cells and form aggregates, but are not internalized. (Fig. 3D, lower panel). Although a small proportion of mitochondria were engulfed, The majority of DsRe are outside the cells with intact endogenous mitochondria, and during this period, The intensity of DsRed was maintained. The DsRed aggregates became smaller and less abundant, and the intensity of DsRed was The concentration of mitochondria decreases, and exogenous mitochondria are collected on the cell membrane, after which they are engulfed and the mitochondria are This suggests that the membrane portion of mitochondrial membranes is rapidly digested.

[0359] Comparison with existing methods shows that the endonuclease method of the present invention is effective for the treatment of mitochondrial It was shown that this method is more effective in generating mitochondria-exchanged cells (Figure 3F). For example, the endonuclease method of the present invention can be implemented by (1) adding the additional nucleotides described in our previous work. Mitochondrial transfer method (e.g., Kitani, T. et al., J Cell Mol Med (2014) 18, 1694) (see reference) or (2) spinoculation of isolated mitochondria with metabolically healthy cells. Recently reported methods utilizing this method (e.g., Kim, MJ et al., Sci Rep 8, 3330, (2018) (See Figure 3F) compared with a previously reported method (i.e., mitochondrial addition; "Mt Both "addition" and "spinoculation at 800 x g or 1500 x g" were performed on DsRed-labeled exosomes. Significant import of exogenous mitochondria as measured by FACS analysis of endogenous mitochondria On the other hand, the MTS-XbaI-mediated endogenous mitochondrial fraction did not show any significant changes (Fig. 3F). Degradation followed by non-invasive transfer or the use of exogenous mitochondria (Mt EPC100) The novel method provided herein allows for significant DsRed positivity after the transfer of exogenous mitochondria. The sex ratio and increased mean fluorescence intensity were shown (Figure 3F, upper right graph, rightmost line).

[0360] Previously established methods used in mitochondrial biology have been used to isolate intact mitochondria. ρ(0) cells, cells with a specific deletion, were utilized (see, e.g., filed September 23, 2010, and published as US 2011-0008778 A1, which is incorporated herein by reference. (See Patent Application No. 12 / 747,771.) However, ρ(0) cells do not contain exogenous mitochondria. Based on these results presented herein, , ρ(0) cells lack the energy required to undergo macropinocytosis Therefore, it was hypothesized that exogenous mitochondria could not be engulfed. To confirm this theory, we examined the effects of exposure to antimycin, which induces mitophagy. We engineered genetically modified cells to generate ρ(0) cells by adjusting the mitochondrial import level. The results showed that engulfment of exogenous mitochondria was sufficient to induce cell death in cells with a complete lack of mitochondria. These results suggest that complete deletion of the nucleus does not cause cytotoxicity in the nucleus (Fig. 3G-I). The partial deletion of existing mtDNA is not responsible for the macropinocytic differentiation of exogenous and extracellular mitochondria. These results suggest that it is an important factor in cis.

[0361] Furthermore, the uptake of DsRed-labeled exogenous mitochondria was investigated using a cytosol containing exogenous mitochondria. Treated or untreated ρ(-) cells, untransfected cells (added Mt) The fluorescence intensity of DsRed was monitored in cells treated with either the GFP or mock MTS-GFP plasmid. The intensity was quantified every 24 hours using NIH image software. The bar graphs show relative values ​​to the initial intensity. This quantification demonstrates the effectiveness of simple additional mitochondrial co-incubation. Co-incubation of mitochondria with mock transfectants resulted in the formation of isolated mitochondria. The intensity increased at the same rate due to mitochondrial aggregation, and the Ds-red labeled mitochondria In contrast, isolated exogenous mitochondria showed accumulation, rather than engulfment, of the mitochondria. The intensity of ρ(-) cells co-incubated with Doria gradually decreased with time, This suggests that the incorporated mitochondria are degraded.

[0362] These results suggest that the MTS-XbaI expression vector expresses ρ, which has a partial deletion of endogenous mitochondria. (-) cells and that the mitochondrial content is solely derived from the donor cells. can be rescued by transferring exogenous isolated mitochondria As described herein, the method of the present invention can be implemented by combining the previously described methods, For example, methods performed in combination with centrifugation or involving partial reduction of endogenous mtDNA. Improved efficiency of mitochondrial import compared to simple "addition" of mitochondria without However, mitochondrial import does not result in complete degradation of endogenous mitochondria. This is because the exogenous mitochondria cannot be used in cells with ρ(0) mitochondrial It was shown that uptake likely requires energy.

[0363] Example IV: Isolated exogenous mitochondria fuse with endogenous mitochondria to form donor cells. -transfer of mtDNA) To further elucidate how mitochondrial import of intact mitochondria occurs, To clarify the fate of mitochondria imported into cells, we investigated the outer and inner membranes and nucleoids separately. In some situations, transient mitochondrial fusion events have been observed, and this In this case, two mitochondria are closely aligned and soluble intermembrane space proteins are released. and matrix proteins, then separate again, retaining their original morphology (e.g., Liu X et al., EMBO J. 2009;28(20):3074-3089; Huang X et al., Proc Natl Acad Sci US A. 2013;110(8):2846-2851). Therefore, transient intermitochondrial fusion events were investigated in this study. The analysis was carried out under the conditions described in the specification.

[0364] Isolated mitochondria from EPC100 donor cells were labeled with DsRed and marked with EGFP. Recipient cells with grafted mitochondria were used. A schematic is shown in Figure 4A. Microscopic image of transient contact between donor and endogenous mitochondria. It was revealed that extensive mitochondrial fusion was not observed (Fig. 4B and Fig. 4C). Most of the donor mitochondria were separated from the endogenous mitochondria. A slight transient fusion image was observed, after which the donor mitochondria merged with the donor mitochondria. They appeared to run away before disappearing into the background (Fig. 4C).

[0365] Mitochondrial transfer was performed according to the protocol shown in Figure 4F. The mitochondria of the recipient NHDF cells were marked with DsRed. (Figure 4D) and mitochondria from donor EPC100 cells were transfected with mitochondria that bind to mtDNA and The recipient NHDF cells were marked with TFAM, which allows tracking of the cells (Figure 4E). The cells were transfected with the -XbaIR-P2A-PuroR expression vector and then incubated with puromycin for 24 hours. On day 6, mitochondria from TFAM-GFP-labeled mitochondria derived from EPC100 donor cells were analyzed. Mitochondria transfer was performed. Then, on day 8, the cells were imaged. Microscopic examination of post-implantation revealed that donor nucleoids were deposited into the pre-existing mitochondrial matrix. It was revealed that exogenous mitochondria were involved in the migration of recipient mitochondria (Fig. 4G). The TFAM-containing mitochondrial nucleoids transiently contact the mitochondrial membrane and then transfer to the existing membrane. This suggests that it is imported into mitochondria.

[0366] These results suggest that donor mitochondria are integrated into the mitochondrial matrix of recipient cells. This indicates that the mitochondria are transferred to the nucleus and become dominant following the decline of existing mitochondria. Furthermore, these experiments showed that almost all isolated mitochondria were engulfed. On the other hand, the additional mitochondrial transfer transfectants and mock transfectants , rather than showing strict engulfment, we instead deposited the majority of these exogenous mitochondria on the cell surface. It was agglomerated.

[0367] Taken together, the results of Examples III and IV demonstrate that ρ(-) cells separate engulfed mitochondria. (Fig. 3J) and that exogenous mitochondria transiently contacted existing mitochondria. On the other hand, when exogenous mtDNA containing TFAM is present in existing mitochondria, This shows that (Figure 4G).

[0368] Therefore, exogenous mitochondria briefly interact with endogenous mitochondria, and this It is hypothesized that mtDNA can be transferred during brief contact with the disassembles exogenous mitochondrial membrane complexes in the cytosol, allowing for mitochondrial remodeling. Recipients that accept exogenous mitochondria can provide the building blocks for Mitochondria in mouse cells can gradually reconstruct mitochondrial membrane complexes, allowing them to function. It shows potential recovery.

[0369] Example V: SNP assay reveals exogenous mitochondria after transfer of isolated exogenous mitochondria (An increase in chondria was detected) To assess the origin of mtDNA after mtDNA exchange, we sequenced hypervariable regions 1 and 2. The differential nucleotides identified between NHDF and EPC100 by B). NHDF retains A at position 16362 of the CRS, while EPC100 introduces a change from A to G. Importantly, mitochondrial exchange in ρ(-) cells was significantly reduced in the ρ(-) cells, which harbored mutations at the same positions as the ρ(-) cells (Fig. 5B). From the evaluation of the cells (NHDFρ(-)Mt), the original nucleotides in the minor wave and the exogenous nucleotides in the major wave were The presence of both leukocyte and protease G was demonstrated, demonstrating that the cells were heteroplasmic. was shown (Fig. 5B, lower panel).

[0370] Heteroplasmy in mitochondrial exchange NHDF between recipient NHDF and donor EPC1 This was further evaluated by single nucleotide polymorphism assay to detect differences in the 00 (Figure 5C). hmt16318-F primer [ka] and hmt16414-R primer [ka] Amplify the HV1 region using a NHDF-specific probe. [ka] and EPC100-specific probe [ka] SNPs were detected using the method (Figure 5C). The SNP assay results showed that the ratio of EPC100 to NHDF was significantly correlated with mtDNA exchange. On day 12, the cellular efficiencies reached 66.6% (Figure 5D). This result was unexpected from the previous method. This is a significant improvement, as a relatively small fraction of extracellular mitochondria is derived from human endometrial glands. This results in the cells being engulfed by mesenchymal cells, resulting in little to no heteroplasmy levels. had no effect (see, e.g., Kitani, T. et al., Journal of Cellular and Molecular Med icine, 18, 1694-1703(2014)).

[0371] These results suggest that the exchange of mtDNA with exogenous mitochondria and / or exogenous mtDNA may be responsible for the The methods provided herein are entirely novel and represent an improvement over existing technology. As mentioned above, the method provided demonstrates that MTS-XbaI mediated The import of mitochondria after degradation of endogenous mitochondria results in the exogenous mitochondria This indicates that mtDNA can become the dominant mtDNA.

[0372] (Example VI: Replaced mitochondria produce energy and MirC is as efficient as normal control cells) (showing recovery of the phenotype) The manufacturer will test whether the replaced mitochondria function to produce energy. The negative control cells, ρ(-) cells, and mitochondria-exchanged cells, and then respiration Flux and control ratios were calculated (Figures 6A and 6B). Basal respiration, maximum capacity of the electron transport chain, and ATP All of the production (free routine activity) showed similar kinetics, and these indices were significantly higher in ρ(-) cells. (Figure 6B, top row). Importantly, these indices were significantly reduced in mitochondrial In the replaced cells, the levels returned to the original values ​​(Fig. 6A and Fig. 6B). Non-mitochondrial ATP production (ROX) was upregulated and the coupling ratio was downregulated in ρ(-) cells (Fig. 6B, bottom row). The energy supply mechanism shifts from mitochondrial ATP generation to glycolysis, and these changes are mediated by mtD After NA exchange, the original state was restored (Fig. 6B, upper right).

[0373] Furthermore, the phenotypic recovery of mitochondria-exchanged cells (MirC) was demonstrated by their proliferation ability ( Specifically, ρ(-) cells showed low proliferation potential, whereas MirC increased proliferation by 6 to 12 days. , the proliferation capacity of the control cells was restored to a level close to that of the control cells (Fig. 6C, right).

[0374] These results demonstrate that this method provides mtDNA exchange with clinically applicable material and is useful for mitochondrial DNA replication. Cells with functional mitochondria allow for phenotypic recovery of mitochondria-exchanged cells (MirC). It has been shown to produce

[0375] Example VII: Inhibition of mTOR by rapamycin reduces exogenous mitochondrial (enhances macropinocytosis) To determine how to increase the ability of cells to receive MirC, exogenous mitochondrial We investigated the mechanisms regulating macropinocytosis. ρ(-) cells have a mitochondrial As a result of the decrease in ATP, the intracellular energy state of ρ(-) cells becomes starved. To this effect, two molecular pathways were investigated: the rapamycin complex 1 ( We investigated the mammalian target of ATPase (mTORC1) and AMP-activated protein kinase (AMPK). essential sensors of amino acids, energy, oxygen, and growth factors, and proteins AMPK is a key regulator of lipid and nucleotide synthesis. It is a sensor of AMP levels. Its activation also inhibits autophagy, mitochondrial biogenesis, glycolysis, and lipolysis. Both pathways are involved in the uptake of extracellular nutrients.

[0376] As shown in Figure 6D, the mechanism of macropinocytosis in ρ(-) cells. To investigate this, we used starvation to stimulate AMPK / mTORC1 while administering the drug palmitoyltransferase. We used rapamycin and rapamycin to specifically stimulate mTORC1 activation and mTORC1 activation, respectively. Rapamycin was added to the culture medium at a concentration of 50 ng / ml for 24 hours, and serum-free Starvation was stimulated by exposing the cells to a glucose- and essential amino acid-free medium for 1 hour. Palmitic acid (PA) has been reported to activate mTORC1 in vivo at a concentration of 200 μM. However, titration of PA on cultured fibroblasts showed that at a concentration of 50 μM and a duration of 24 hours The ratio of phosphorylated AMPK to AMPK and the ratio of phospho-AMPK to AMPK were shown to be optimal based on cell viability. The ratio of phosphorylated p70 S6 kinase to p70 S6 kinase (which is a downstream target of mTORC1) was calculated as kinase activity. The samples were examined by capillary electrophoresis using Wes™ (Protein Simple).

[0377] Treatment with PA or rapamycin did not significantly activate the AMPK pathway in ρ(-) cells. Although the mTORC1 pathway was not affected by starvation and rapamycin, as measured by pS6 / S6 (Figures 6G and 6H), the mTORC1 pathway was not affected by starvation and rapamycin, as measured by pS6 / S6. The results showed that the expression of α-glucan in ρ(-) cells was dramatically suppressed in ρ(-) cells at a level similar to that of α-glucan in ρ(-) cells (Fig. 6E-F). Our results suggest that mTORC1 plays a key role in mitochondrial macropinocytosis in ρ(-) cells. This indicates that the target is an important one.

[0378] Next, the present inventors investigated whether rapamycin or palmitic acid could be used to induce cell proliferation in mitochondria co-culture. Simultaneous treatment with rapamycin and palmitate inhibits mitochondrial engulfment The effect of NHDF was investigated. The protocol is shown in Figure 6I. Recipient cells were transfected with the MTS-XbaI expression vector and treated with or without rapamycin. The MTS-XbaI-expressing strains were cultured without or with palmitic acid (PA). Puromycin selection of (-) cells was performed after 48 hours. On day 6, DsRe derived from EPC100 cells was The transfer of isolated mitochondria marked with d was performed. On day 8, FACS analysis was performed. To confirm donor mitogenesis, we performed a transfection assay using NHDF recipient cells by measuring DsRed expression. Chondria was detected.

[0379] As shown in Figures 6I-6L, rapamycin treatment increased the DsRed-labeled isolated extracellular matrix. The engulfment of mitochondrial cells was significantly enhanced by palmitic acid, whereas palmitic acid significantly enhanced the engulfment of mitochondrial cells. These experiments were repeated four times, and the positive rates were calculated. A statistically significant difference was observed between rapamycin and palmitic acid (Fig. 6I and Fig. 6K). There was no significant difference between mock transfection and added mitochondrial import. Furthermore, these results suggest that the effect of modulating mTORC1 activity is related to the mitochondria in ρ(-) cells. Affects only transfection and has no effect on "additional" or mock-transfected cells Thus, this mechanism for importing exogenous mitochondria is provided herein. It has been shown that the invention is unique to the invention being developed.

[0380] These results suggest that activation of mTORC1 by rapamycin during mitochondrial import is essential for the development of mitochondrial Furthermore, it has been shown that the macropinocytosis of endogenous ATP can be enhanced. These methods use the clinically available drug rapamycin to generate MirC. These results indicate that the efficiency of macropinocytosis can be increased by activating ATP.

[0381] Example VIII: Reverse Heteroplasmy in Fibroblasts Derived from Patients with Leigh Syndrome (mtDNA exchange with translocation) Mitochondrial disease cells were corrected using in vitro mtDNA replacement techniques. To investigate whether or not this could be achieved, a patient was diagnosed with Leigh syndrome with the mtDNA T10158C mutation. Primary fibroblasts (7SP) derived from a patient were used as recipient cells (Figure 7A). The same protocol described for F cells was applied to 7SP fibroblasts. DNA sequencing of mtDNA from EPC100 donor mitochondria in the rat confirmed that it was T. In contrast to the 7SP fibroblasts (Fig. 7B, top), the 7SP fibroblasts were heteroplasmic, showing a major wave of T and non-T cells. It had a mosaic of major wave C ( Fig. 7B , bottom).

[0382] The dynamics of mtDNA content in 7S fibroblasts are similar to those in NHDFs after mitochondrial exchange (Fig. 7C and Fig. 7J). Time-lapse observations showed that ρ(-)7SP fibroblasts were significantly different from ρ(-)NHDF cells. In particular, the exogenous mitochondrial membranes on the surface of ρ(-) cells showed the same behavior as those of ρ(-) cells. The accumulated aggregates of endonucleases became smaller and less numerous with time, and the cytoplasmic zone The intensity of DsRed in the cells rapidly decreased, indicating efficient engulfment and cytosolic degradation. This suggests a digestion during digestion, which is consistent with the results obtained using ρ(-) NHDF cells.

[0383] Importantly, the number of mtDNA copies after mitochondrial exchange was significantly higher than that of the original 7S fiber at day 12. The number of mtDNA copies in 7SP fibroblasts recovered to the same value as that in the fibroblasts (Fig. 7D). Transfectants (additional mitochondrial import) were isolated from mitochondria under the same conditions. Despite being co-cultured with the rhesus monkeys, the number of mtDNA copies could not be increased, and the exogenous factors The cells showed low import of mitochondrial mitochondrial markers (Fig. 7D, light gray bars).

[0384] By sequencing a mitochondrial genome fragment containing 10,158 nucleotides, We investigated whether the mitochondria of 7S fibroblasts contained exogenous and healthy mtDNA. As shown in Figure 7E, the mtDNA sequences of 7SP cells were similar to those of the recipe after mitochondrial exchange. In ent 7SPρ(-) cells, the majority of mutant heteroplasias were located at nucleotide position 10158. Those with Smy (large wave of C and small wave of T) to those with mostly wild-type mtDNA (large waves in T and small waves in C) (Fig. 7E, bottom).

[0385] To provide quantitative information, single nucleotide polymorphism (SNP) assays were performed to assess this The resulting heteroplasmy was estimated using the technique. hmt10085-F primer [ka] and hmt10184-R primer [ka] and EPC100-specific probe [ka] or 7SP-specific probe [ka] The ND3 region of mitochondrial DNA was amplified using 7SP fibroblasts (Figure 7F). The original hmt10158 heteroplasmy level in cells was approximately 90% mutant mtDNA. The heterozygote in 7SP cells that received mitochondrial import (7SP ρ(-)Mt) showed the following (Fig. 7G). plasmy showed only 10% heteroplasmy levels 12 days after exchange. (Figure 7G), mock transfectants (additional mitochondrial import) showed heteroplasmy. These results did not change significantly and remained at approximately the same ratio or above 90% (Figures 7H and 7I). The mitochondrial exchange technique provided herein is based on previously reported additional mitochondria. The ρ(-) cells treated with endonuclease showed superiority over ρ(-) cells. , which increased heteroplasmy to approximately 75%, accompanied by an approximately 80% reduction in mtDNA copy number. .

[0386] Taken together, these results suggest that MTS-XbaI can partially deplete endogenous mitochondria. The method for producing the mitochondria-exchanged cells described herein is carried out by Effectively used in cells derived from subjects with a disease or disorder to improve heteroplasmy levels and demonstrate that the amount of mutant mtDNA can be reduced.

[0387] Example IX: mtDNA exchange in fibroblasts derived from patients with Leigh syndrome is associated with cell life span and improves cellular metabolism) The functional activity of mitochondrial-exchanged 7SP fibroblasts was assessed. As shown in Fig. 1, the proliferation of mitochondrial-exchanged 7SP fibroblasts (ρ(-)Mt) cells was significantly improved around day 12. The levels of 7SP in fibroblasts were restored to levels comparable to those in fibroblasts.

[0388] Furthermore, mitochondrial exchange in 7SP fibroblasts (ρ(-)Mt) cells occurred at approximately 63 population doublings (P While the lifespan was dramatically extended until the growth arrest threshold of 120 h, the doubling time was significantly longer. (Figure 8C). Cells that received mtDNA exchange and reconstituted with healthy mtDNA were identified after approximately 8 PDL. The induced cells can continue to divide beyond 55 PDL, which is the threshold for cell division arrest. This is thought to be the number of times a normal human cell population divides before it can be differentiated (i.e., the Hyflick limit). In contrast, untreated 7S fibroblasts entered a senescent state after 25 PDL (Fig. 8C). Therefore, this experiment demonstrated that mtDNA exchange significantly affects the proliferation and lifespan of cells with mitochondrial disease. It has been shown that senescence increases with age and cancer cells have a significant impact on mitochondrial function. Considering that this is often accompanied by a lack of function, this method offers a crucial clue for rejuvenation. This may provide a novel approach to cancer therapy and therapy for other age-related diseases. This may provide the basis for a strategy.

[0389] Function of mitochondrial import in 7S fibroblasts by measuring cell size The effect of the 7S fiber in the coding sequence of the ND4 gene of complex I in the respiratory chain was further evaluated (Fig. 8D). Mutations in blastocytes cause protons to be pumped from the matrix into the intermembrane space This, combined with the impairment of the electron-transporting complex I, resulted in glycolysis. , which predominates over and damages mitochondrial ATP production in 7S fibroblasts. Despite their lower function, cells are larger in size and contain more mitochondria. This results in a greater compensatory adaptation (Fig. 8D). Compared with PDL 15 (solid black line), PDL 25 The diameter of 7S fibroblasts was approximately 1.5 times larger than that of NHDFs, and the increase in cell size was due to the PDL 3 5, and eventually their size increased until they were approximately 3-8 times larger (Figure 8D, left ).

[0390] Consistent with the functional recovery of 7SP cells after mtDNA exchange, the 7SP fibroblasts showed a significant increase in the PDL at early stages. The significant increase in cell size observed was inhibited after mitochondrial replacement (Fig. 8D, right). Furthermore, the size of mitochondrial-exchanged 7SP cells receiving exogenous mitochondria at PDL 8 was This was maintained up to the 50th PDL (Fig. 8D, right). By the 10th PDL, the CS concentration was 2-fold higher in 7SP fibroblasts than in NHDF cells. This is consistent with the increased size of 7SP fibroblasts (data not shown).

[0391] The observed improvement in cell function after mitochondrial replacement is not due to contamination with other cell types. To ensure that cells of various origins are not A tandem repeat (STR) assay was performed (Figure 8E). Importantly, the mitochondria at different time points The STR pattern in the chondriac-exchanged cells was similar to that of the original 7SP fibroblasts. The results were completely identical (Fig. 8E), indicating the absence of contamination. The transfer of exogenous mitochondria from cells expressing telomerase and E6 resulted in the development of primary lineages. It was found that fibroblasts did not transform into cancer cells (Fig. 8F).

[0392] Taken together, these results demonstrate that the expression of wild-type m Mitochondrial transfer of exogenously isolated mitochondria carrying tDNA extends the lifespan of 7SP cells. Importantly, this transfer was shown to increase mitochondrial function and improve cell function. The doria-exchanged 7SP cells did not transform into cancer cells.

[0393] Example X: Transfer of exogenous mitochondria into fibroblasts derived from patients with Leigh syndrome (Introducing functional mitochondria) By using Oroboros O2k, we analyzed the respiratory function of cells and 7SP lines. The functional effects of mitochondrial exchange in fibroblasts were further evaluated (Figure 9A). Therefore, basal respiration and ATP production (free routine activities) were suppressed after the preparation of mitochondria-exchanged cells. , which continued to decrease from the 10th PDL to the 20th PDL, and the maximum capacity of the electron transport chain was After the transfer of exogenous mitochondria, the original level of mitochondrial activity was maintained (Fig. 9B). By the time of the PDL, all three indices of respiratory function (normal, ETS, and free routine activity) were elevated. These results suggest that after mtDNA exchange, electron transfer There is a slight delay in reconstituting the delivery system with healthy, unmutated complex I. Proton leak exhibited the same kinetics as non-mitochondrial ATP production, which , which steadily improved from the initial stage (Figure 9B).

[0394] These results suggest that mitochondrial disease may be a contributing factor to the development of mitochondrial fibroblasts derived from patients with mitochondrial diseases or disorders. We demonstrated that the transfer of exogenous mitochondria can generate functional mitochondria. Ta.

[0395] Example XI: Import of exogenous mitochondria abolishes chronic and persistent reactive oxygen species (ROS) generation can be lost) Reperfusion and Starvation Models in Culture Conditions to Characterize the Properties of 7SP Fibroblast-Derived MirC Both were used for 7SP fibroblast-derived MirC, the original 7SP fibroblasts, and NHDF as a control. These stress conditions induced apoptosis in cultured cells, the extent of which was Annexin V as an early marker and propidium iodide (PI) as a late marker Among environmental insults, reperfusion injury is the most common cause of mitochondrial dysfunction. Cells that are predisposed to mitochondrial dysfunction due to mtDNA mutations contribute primarily to , are more vulnerable to reperfusion injury than healthy cells.

[0396] 1 x 10 cells per well 5 The cells were seeded in a 6-well plate. The next day, 600 μM H2O2 (FUJ IFILM Wako Pure Chemical) was added to the cells for the reperfusion model, or serum-free DMEM (FUJIFILM Wako Pure Chemical) containing no essential amino acids (-EAA) was used as the medium for the starvation model. After 3 hours of H2O2 treatment or 48 hours of starvation, the cells were washed with PBS and centrifuged. The cells were collected in a centrifuge tube. Annexin V-FITC and PI solution were added to the cells, and the cells were then placed in a chamber in the dark. The cells were then incubated at room temperature for 30 minutes. Afterwards, the cells were analyzed using 488 and 561 nm laser lines. The samples were immediately subjected to FCM analysis. Fluorescence data were collected using SH800 (Sony). FlowJo software Flow cytometry files were analyzed using software (TreeStar).

[0397] These results suggest that 7SP cells derived from subjects with Leigh syndrome can tolerate both forms of stress ( The results shown in Figures 10A-10D indicate that the cells are highly susceptible to H2O2 and starvation. As shown in Fig. 1, 7SP cells treated with H2O2 showed both early and late apoptosis. In the reperfusion model (H2O2), NHDF showed a significant increase in Annexin V and PI staining. Based on the results, no significant damage was observed in the apoptotic process (Fig. 10B-D). However, this mild reperfusion stress did induce apoptosis in 7SP fibroblasts. In contrast, the positive rates of both Annexin V and PI in 7SP fibroblast-derived MirCs were This was significantly lower than in parental 7SP fibroblasts and closer to the levels in NHDF cells (Figures 10B-10D). Interestingly, there was no significant difference between 7SP fibroblast-derived MirCs and NHDFs, and MirCs were the only cells that survived this mild reperfusion. This suggests that the body regains the ability to withstand injury.

[0398] The same tendency as in the reperfusion model was observed using the starvation model (Fig. 10E-H). More apoptosis was observed in 7SP fibroblasts than in 7SP fibroblasts. The derived MirCs showed basal apoptosis levels similar to those of NHDFs, which is consistent with the original 7SP These results were significantly lower than those of fibroblasts (Fig. 10F to 10H). This further confirms that the chondrial exchange method improves the functional recovery of recipient cells. There are.

[0399] These results suggest that exogenous mitochondria from healthy cells to cells with mutant mtDNA We demonstrated that transfection of ribosomal RNA can improve the function of recipient cells.

[0400] Example XII: Transfer of exogenous mitochondria into recipient cells inhibits early-stage senescence-associated processes reversed the secretory phenotype (SASP) This example demonstrates that the transfer of exogenous mitochondria into recipient cells inhibits early-stage senescence-associated These results suggest that the secretory phenotype (SASP) of inflammatory cytokines, growth factors, and The SASP, which consists of proteases, is a characteristic feature of senescent cells.

[0401] We investigated whether the transfer of exogenous mitochondria into senescent cells can reverse the SASP. To determine whether the SASP cytokines IL-6 and IL-8, the chemokine CXCL-1, and The expression level of growth factor ICAM1 was approximately 15-20% in NHDF and 7SP fibroblasts, whose PDL levels were similar. The expression of MirC in 7SP fibroblast-derived cells was quantitatively measured at the transcriptional level (Fig. 11). L-6 was significantly higher in 7SP fibroblasts than in NHDF and 7SP fibroblast-derived MirC, whereas The other three factors did not show significant differences between these cells. did not show typical SASP, but showed higher IL-6 expression, indicating an early stage of senescence. Importantly, 7SP fibroblast-derived MirC inhibited the early stage of senescence in this PDL. I was able to turn Seth around.

[0402] Taken together, these data suggest that mitochondrial exchange leads to mitochondrial remodeling accompanied by mtDNA mutations. In addition to treating endocrine disorders, it is also used to treat neurodegenerative, cardiovascular, metabolic, and autoimmune diseases, as well as It can also rejuvenate aging cells, such as those involved in various diseases, including cancer. This shows that it is possible.

[0403] Example XIII: iPS cells generated from mtDNA-exchanged fibroblasts Induced pluripotent stem cells (iPSCs) were generated using cells derived from patients with long mtDNA deletions. To determine whether it is possible to create a NHDF, we performed well. A standard transfection method using Sendai virus carrying Oct3 / 4, Klf4, Sox2, and c-Myc (OKSM) was used. We attempted to generate iPSCs from 7SP fibroblasts using a novel method. The formula is shown in Figure 12A.

[0404] Alkaline phosphatase staining (AP staining) detects early stage iPSC colonies. While the 7SP fibroblast-derived colonies appear to have a ramshackle appearance, the mitochondrial Endothelial-exchanged 7SP fibroblast-derived colonies were shown to be robust at day 21 (Figure 12B). In contrast, ρ(-)7SP fibroblasts that had not received mtDNA exchange did not generate colonies. As measured by AP staining, some lines of iPS cells exhibited mitochondrial cross-linking. iPSC clones could be generated from recombinant 7S fibroblasts (Fig. 12C and Fig. 12D). The colonies were stable and showed similar morphology among independent colonies (Fig. 12E). Human pluripotency was enhanced on mitochondrial exchange 7SP fibroblast-derived colonies overexpressing OKSM. Expression of reproductive stem cell markers SOX2, OCT3 / 4, NANOG, SSEA4, TRA1-81, and TRA1-60 was confirmed. This was confirmed (Figure 12F).

[0405] iPSCs generated by the methods described herein were used to generate commercially available KYOU-DXR0109B human inducible iPSCs. Further comparison was made with pluripotent stem (IPS) cells [201B7]. Importantly, the mitochondrial exchange 7SP line Fibroblasts showed the same level of efficiency in iPS generation as healthy fibroblasts. Furthermore, consistent with previous studies, qPCR of 12S-rRNA normalized to nuclear β-actin The iPS cells generated by mitochondrial exchange with 7SP fibroblasts were half as viable as the control. The mtDNA content of the 201B7 iPSCs was shown to be similar to that of the 201B7 iPSC standard. (Figure 12G).

[0406] Furthermore, hmt10158 heteroplasmy levels were less than 10% in the generated iPSCs (Figure 1 2H). Quantification of absolute mtDNA copy number revealed reduced levels of mtDNA and mutant mtDNA. A decrease was confirmed (Figure 12I).

[0407] These results demonstrate that iPSCs were generated using the mitochondrial replacement technique provided herein. and the entire procedure uses only clinically applicable materials, This indicates that it may be applicable in the clinical field.

[0408] Example XIV: Mitochondrial exchange of donor cell-derived mitochondria with recipient cell (changes lifespan cells) This example demonstrates that mtDNA exchange can alter the lifespan of recipient cells. We investigated the hypothesis that mitochondrial replacement can rejuvenate aging cells. To demonstrate this, two models were used to characterize cell cycle competence, including doubling time and PDL at growth arrest. The rule was estimated.

[0409] Early PDL (approximately 5-10, referred to as "young") and late PDL (approximately 40-45, referred to as "old") Models were designed using NHDF and TIG1 embryonic lung cells with O2. The young cells were replaced with mitochondria from the old cells, named "Y." Another model, named "Y2O," involved replacing mitochondria with those from younger cells. accompanied by aged cells (Fig. 13A).

[0410] The extent of mtDNA exchange is A and G at position 16145 between NHDF and TIG1, respectively. TaqMan SNP genotyping assays based on single nucleotide differences in mtDNA NHDF-derived MirC showed that over 90% of the endogenous mtDNA (hmt16145-A) was derived from TIG1 (Fig. 13B). The results clearly showed that the mtDNA was exchanged with the original mtDNA (hmt16145-G) (Fig. 13C). The small percentage of hmt16145-A detected was considered to be background error. (Figure 13C).

[0411] Furthermore, the Y2O model clearly demonstrated a restoration of the lifespan of aged cells to approximately 65 PDL (Figure 13D). Aged control cells and mock transfectants showed growth arrest at 55 PDL. On the other hand, O2Y is consistent with the low lifespan of young cells at approximately 45 PDL. The difference of about 10 PDL in both models could be attributed to exogenous mtDNA. These results suggest that the transfer of exogenous mitochondria from young cells to old cells can improve cell It shows that it can be rejuvenating.

[0412] Example XV: Mitochondrial Exchange from Human Primary T Cells Using mRNA Transfection Cell (MirC) optimization) This example demonstrates the generation of myeloma derived from human primary T cells by using mRNA transfection. We describe the generation of Mitochondrial Exchange Cells (MirCs).

[0413] Prior to the experiment, the use of human primary T cells was approved by our institutional review board. Peripheral blood was collected from healthy volunteers and purified at a specific gravity of 1.077 by pulsed PBS at 400g for 35 minutes at 20°C. Lymphocytes were isolated by centrifugation using ethanol. 6 Cell isolation The lymphocytes were plated onto a 96-well flat plate coated with anti-CD3 and anti-CD28 antibodies. Plates were incubated overnight with 5 μg / ml anti-CD3 and 1 μg / ml anti-CD28. The cells were prepared by the following procedure and pre-warmed at 37°C for 2 hours before seeding. The day after seeding, IL-7 and IL-15 were added to the cells. The medium was then added at the same concentrations as the first addition. The media was replaced every 3 days with IL-7 and IL-15 at concentrations of 0.05%.

[0414] A MaxCyte electroporator that meets GMP / GCP standards was used according to the manufacturer's protocol. Transfections were performed using mMESSAGE mMACHINE T7 U with slight modifications. mRNA was produced according to the manufacturer's protocol using the Ultra kit (Thermo Fisher). To minimize the chance of messing up RNases, The template is then digested with an endonuclease and the fragments are purified without further purification, resulting in a plasmid carrying the DNA sequence. It was prepared from sumidi (Figure 14A).

[0415] The results showed that the unpurified DNA template for EGFP mRNA production was sufficient for gene transfection. The transfection efficiency was nearly 100%, with high expression and high viability after 24 hours. Because of the high transfection efficiency, this method was used When MTS-XbaIR was used, antibiotic selection was not required. This leads to a decrease in mitochondrial membrane potential, which is attributed to a decrease in endogenous mtDNA. There was a possibility (Figure 14D).

[0416] Optimal protocol for timing of co-incubation of isolated mitochondria GFP mRNA by electroporation using MaxCyte ATX to determine colony Fluorescence images of human primary T cells receiving IgG were taken over an 8-day period as indicated. (Figure 14E). Electroporated cells transfected with GFP plasmid. The fluorescence image of the control cells (Fig. 14F, upper panel) is identical to that seen in the fluorescence image of the fibroblasts. Expression peaked on day 2 and disappeared by day 8. In contrast, MTS- Cells that received GFP mRNA (Fig. 14F, lower panel) were found in the plasmid-transfected cells. The expression was higher within 4 hours after electroporation and disappeared earlier on day 6 than that of the control. showed.

[0417] GFP protein expression in cells receiving MTS-GFP mRNA was measured using capillary electrophoresis. The results were evaluated by Western blot analysis using electrophoresis. Peak expression occurred on day 4, and expression continued until day 6, as quantified in Figure 14H) and Figure 14G. The dynamics of XbaIR transcript levels were quantified by qPCR, which revealed that The expression of the nuclease transcript was found to be fully maximal 4 hours after transfection. XbaIR transcript levels decreased rapidly by day 2 and were negligible by day 6 (Figure 14I). The mitochondrial content was estimated by quantifying 12S rRNA (Fig. 14J), mitochondria were reduced to approximately 30% by day 2 and remained at 20% throughout the experimental period. It was shown that the α-glucan concentration was maintained at less than 1%.

[0418] Taken together, these results suggest that the mRNA transcripts of endonucleases such as XbaI fused to MTSs are highly conserved. Transfection can efficiently degrade host mtDNA, and mitochondria derived from human primary T cells We have shown that it can be used to generate mitochondrial replacement cells (MirCs).

[0419] Example XVI: Mitochondrial Exchange from Human Primary T Cells Using mRNA Transfection Preparation of recombinant cells (MirC) The optimal time point for performing mitochondrial transfer in human primary T cells was determined in Example XV. After the transfection, the mitochondrial DNA was removed to prevent digestion of the exogenous mtDNA by residual endonucleases. Rearrangement and co-incubation were performed on day 7. MirC protocol for human primary T cells The scheme is shown in Figure 15A.

[0420] mtDNA heteroplasmy in recipient human primary T cells after mitochondrial exchange To determine the differences in mtDNA between donor mitochondria and recipient cells, TaqMan SN The P genotyping assay was performed on normal human primary T cells and EPC100 (mitochondrial Sequencing of the D-loop of mtDNA in the donor cells revealed two nucleotides Differences in mtDNA positions (nucleotides 218 and 224) were noted, which were associated with T cells and EPC100 cells. The genotypes of the cells were C / C and T / T, respectively (Fig. 15B). To construct a standard curve for seq, the 218 and 224 nucleotides of mtDNA were included. The variable region fragments were subcloned into pBluescript SK(-). The desired region of the D-loop was then mapped onto the Cambridge reference sequence and targeted for amplification. Primers and probes were designed to: (Fig. 15C). Using TaqMan polymerase with 5' exonuclease activity, qPCR was performed to determine the threshold cycle (Ct value), which was then compared with several different plasmids listed above. The different copy numbers for each sequence were used to generate a standard curve. After chondrial exchange, the EPC100 mtDNA origin predominated in human T cells at both days 7 and 12. While the β-actin receptor occupies the same position as MirC, it accepts electroporation without genetic material and follows the same process as MirC. Mock transfectants co-incubated with isolated mitochondria in vitro showed exogenous mtDNA origin at less than 10% on day 7 and at background levels on day 12 ( (Fig. 15D), demonstrating that MTS-XbaIR mRNA promotes efficient mitochondrial import in human primary T cells. It was shown that

[0421] Next, to assess the effect of mitochondrial import on the function of MirC human T cells, To investigate this, we performed a respiration measurement experiment using Oroboros O2k. The results showed that the respiration of human T cell-derived Mi ATP production and coupling efficiency in rC were restored by electroporation of XbaIR. ρ(-) human T cells generated by mRNA transfer showed a loss of ATP production throughout the experiment. Representative raw data using the Coupling Control Protocol (CCP) is shown in Figure 15 F and G, which demonstrate that MirC T cells can restore mitochondrial respiration. It shows that it can be done.

[0422] These results demonstrate that human primary T cells are electroporated using an electroporator produced by MaxCyte. Mitochondria were transfected to produce MirC using a GMP-grade electroporator such as It was shown that it is possible to exchange

[0423] Example XVII: Mitochondria from Mouse Primary T Cells Using mRNA Transfection Preparation of replacement cells (MirC) Further characterization of T cell-derived MirC was performed on mouse T cells. EasySep Mouse Isolation Kit (STEM CELL) provides highly purified T cell populations by selection Using a spleen immunoassay (SIL-1000) and a spleen immunoassay (SIL-1000) ...), mouse T cells were isolated from the suspension obtained from the spleen. Isolated mouse T cells (1 × 10 6 cells / ml) at a bead-to-cell ratio of 1:1 and a combination of 30 U / ml Recombinant IL-2 was used in 96-well plates with Dynabeads mouse T-activator CD3 / CD28 (Invitrogen). The culture medium for mouse T cells was used to evaluate cell proliferation and CD3 expression. The results showed that RPMI1640 was superior to TexMACS (Figure 16A). The rate and total cell number were greater for cells cultured in RPMI1640 compared to TexMACS. The medium was changed every 3 or 4 days.

[0424] Next, we electroporated mouse T cells using the Nucleofector device and mRNA. The kinetics of GFP expression after mRNA transfection was similar to that in human T cells (Fig. 16B). Six hours after electroporation of TS-GFP mRNA, almost all cells strongly expressed GFP. This indicates that MTS-GFP was transfected with high efficiency. The GFP intensity decreased rapidly over time and eventually disappeared after electroporation. The lesions disappeared 6 days after administration (Fig. 16B).

[0425] Transfection of MTS-XbaI mRNA resulted in mouse T cells showing a higher showed a more moderate decrease in XbaIR transcript expression, which persisted at low levels on day 6. Quantification of 12S rRNA levels as a surrogate marker for mtDNA revealed that mouse mtDNA It was shown that the Ds-Red labeling persisted at approximately 40% of the control level even on day 6 (Fig. 16D). Co-incubation of exogenous mitochondria with ρ(-) mouse T cells was performed (Figure 16E). Longer persistence of XbaIR and lower levels of endogenous mtDNA degradation compared to human T cells Nevertheless, the drinking water after 48 hours of co-incubation with isolated mitochondria FACS analysis of embedded fluorescently labeled mitochondria confirmed the presence of exogenous mitochondria. A significant positive rate (9.73%) of T cells was revealed (Figure 16F). The percentage of positive cells was even higher than in the fibroblast experiments. Therefore, this mouse T cell protocol may be optimal for generating T cell-derived MirC. showed.

[0426] Example XVIII: Transfer of exogenous mitochondria into T cells reversed aging. This example demonstrates that mitochondrial replacement is successful in mouse T cells and rejuvenates senescent T cells. This shows that...

[0427] Exogenous mitochondria can be successfully transferred to generate mouse T cell-derived MirC. To evaluate whether mtDNA heteroplasmy levels were measured in BL6 (recipient) cells and The donor cells were selected from NZB cells. Two consecutive polymorphisms at 2766 and 2767 mtDNA of ND1 were identified. Specifically, BL6 mitochondria contained AT at positions 2766 and 2767 of the mtDNA. NZB mitochondria contained GC at the same position, whereas NZB mitochondria contained GC at the same position. To identify polymorphisms, a primer set and two In addition, two probes were designed to express the GC and AT polymorphisms, respectively. to generate separate plasmids for MirC and facilitate quantitative estimation of heteroplasmy in MirC. To do this, a standard curve was constructed.

[0428] Co-transfected with MTS-XbaI and isolated mitochondria from NZB mice Quantification of mitochondrial exchange (XbaIR Mt) in incubated BL6 cells revealed that exogenous The overwhelming predominance of the endonuclease mtDNA was demonstrated, whereas in the absence of the endonuclease mRNA Mock co-incubated with isolated mitochondria after electroporation of Transfection did not result in the engulfment of exogenous mtDNA (Fig. 17C). We demonstrated that cells tolerate mitochondrial exchange.

[0429] The results described herein demonstrate that fibroblast-derived MirC undergoes rejuvenation in vitro. Since it was shown that this is possible (Figure 13), we investigated the possibility of rejuvenation of T cell-derived Mir...

Claims

1. Mitochondrial replacement primary cells show phenotypic recovery of mitochondrial function equivalent to that of normal cells 1. An ex vivo or in vitro method of producing cells, comprising: (a) Primary cells were cultured in a fusion medium containing an endonuclease with a mitochondrial targeting sequence (MTS). contacting the recombinant protein ex vivo or in vitro with a polynucleotide encoding the recombinant protein; Achieving partial depletion of endogenous mitochondrial DNA (mtDNA) of 99% or less; and (b) the primary cells are substantially free of a 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; As a result, the mitochondrial function of the mitochondrial cells is restored to a phenotype equivalent to that of normal cells. Creating doria-exchanged primary cells The method comprising:

2. The exogenous mitochondria: (i) Functional mitochondria that consume oxygen and produce ATP; (ii) containing wild-type mtDNA; (iii) isolated mitochondria; and / or (iv) the method of claim 1 is allogeneic.

3. 3. The method of claim 2, wherein the isolated mitochondria are intact mitochondria. 。

4. (i) the MTS targets a mitochondrial matrix protein; (ii) the endonuclease is XbaI, EcoRI, BamHI, HindIII, PstI, Cas9, zinc Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TA LEN); or (iii) The polynucleotide is a messenger ribonucleic acid (mRNA) or a deoxyribonucleic acid ( The method of any one of claims 1 to 3, wherein the nucleic acid sequence is a DNA sequence.

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

6. The mitochondrial matrix protein is a cytochrome c oxidase subunit Cytochrome c oxidase subunit IV, cytochrome c oxidase subunit VIII, and cytochrome c oxidase subunit 5. The method of claim 4, wherein the knit is selected from the group consisting of knit X.

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

8. Between about 5% and about 99%, between about 30% and about 70%, between about 50% and about 95%, or between ... or between about 30% and about 70%, or between about 50% and about 95%, or between about 5% and about 99%, or between about 30 8. The method of any one of claims 1 to 7, wherein the IL-16 expression level is reduced by about 60% to about 90%.

9. The endogenous mtDNA comprises: (i) Encoding dysfunctional mitochondria; (ii) containing mutant mtDNA; (iii) contains mtDNA associated with a mitochondrial disease or disorder; (iv) is heteroplasmic; and / or (v) the method of any one of claims 1 to 8, comprising wild-type mtDNA.

10. The mitochondrial exchange primary cells contain a polynucleotide encoding the fusion protein. The total mtDNA copy number of the primary cells was about 1.1 times, about 1.2 times, or about 1.3 times that of the total mtDNA copy number of the primary cells before contact with the 10. Any of claims 1 to 9, wherein the total mtDNA copy number is not more than about 1.5 times, about 1.4 times, or about 1.5 times The method described in claim 1.

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

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

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

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

15. 14. The method of claim 13, wherein the exogenous mtDNA alters heteroplasmy in the primary cells. The method described.

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

17. 17. The method of claim 16, wherein the mTOR inhibitor comprises rapamycin or a derivative thereof.

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

19. One or more mitochondrial exchange initials obtained by the method according to any one of claims 1 to 18. a composition comprising mitochondrial replacement primary cells, wherein the mitochondrial replacement primary cells contain more than 50% exogenous mtDNA; The composition comprising:

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

21. (i) The neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, disease, Parkinson's disease, Friedreich's ataxia, Charcot-Marie-Tooth disease, and and leukodystrophies; (ii) the retinal disease is selected from the group consisting of age-related macular degeneration, macular edema, and glaucoma. ru; (iii) The age-related disease is an autoimmune disease, a metabolic disease, a genetic disease, cancer, or a neurodegenerative disease. disease, and immunosenescence; (iv) The genetic disease is Hutchinson-Gilford Progeria Syndrome, Werner's syndrome, or - syndrome, and Huntington's disease; (v) The mitochondrial disease or disorder is a mitochondrial DNA abnormality, a nuclear DNA abnormality, or both. caused by (vi) The mitochondrial disease or disorder is caused by a mitochondrial DNA abnormality. and chronic progressive external ophthalmoplegia (CPEO), Pearson syndrome, Kearns-Sayre syndrome (KSS) ), diabetes and hearing loss (DAD), mitochondrial diabetes, Leber's hereditary optic neuropathy (LHON), LHON-plus, neuropathy, ataxia, and retinitis pigmentosa syndrome (NARP), maternal inheritance Hereditary Leigh syndrome (MILS), mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MEL AS), Myoclonic Epilepsy and Ragged Red Fiber Disease (MERRF), Familial Bilateral Striatal Necrosis / Striatal Syndrome Striatonigral degeneration (FBSN), Luft's disease, aminoglycoside-induced hearing loss (AID), mitochondrial D NA multiple deletion syndrome; or (vii) The mitochondrial disease or disorder is caused by a nuclear DNA abnormality, and Mitochondrial DNA depletion syndrome-4A, mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial Neurogenic gastrointestinal encephalomyopathy (MNGIE), mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase Protease gamma (POLG)-related disorders, sensory ataxic neuropathy with dysarthria and ophthalmoplegia (SANDO), brain Leukoencephalopathy with trunk and spinal cord involvement and elevated lactate (LBSL), coenzyme Q10 deficiency, Leigh syndrome , mitochondrial complex disorder, fumarase deficiency, α-ketoglutarate dehydrogenase Complex (KGDHC) deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenase complex Pyruvate carboxylase deficiency (PDHC), pyruvate carboxylase deficiency (PCD), carnitine palmitoyl 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 atrophy (IOSCA), mitochondrial myopathy Myopathy (MM), spinal muscular atrophy (SMA), growth retardation, amino aciduria, cholestasis, iron overload, From the group consisting of early death (GRACILE), and Charcot-Marie-Tooth disease type 2A (CMT2A) 21. The mitochondrial exchanged primary cells for use according to claim 20, selected.

22. The metabolic disease is diabetes, and the neurodegenerative disease is Alzheimer's disease or pulmonary embolism.

22. The mitochondrial replacement primary cells for use according to claim 21, wherein the primary cell is Parkinson's disease.

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