Compositions for inducing mitophagy and uses thereof

Abstract

Polypeptides having a mitochondrial targeting sequence (MTS), an endonuclease sequence, and a destabilization domain sequence; nucleic acids encoding the same; uses of the polypeptides and nucleic acids to induce mitophagy, increase mitochondrial turnover, and / or induce double-strand breaks in mitochondrial DNA in cells; and the aforementioned therapeutic uses, for example, in the treatment of mitochondrial diseases and disorders.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 426,424, filed November 18, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] 2. Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated by reference in its entirety. The XML Sequence Listing, created on November 8, 2023, is named RMG-004WO_SL.xml and is 42,441 bytes in size. [Background technology]

[0003] 1. Background Mitochondrial dysfunction is caused by various factors, including mutations in genes encoding mitochondrial proteins, tRNAs, and rRNAs, accumulation of mutations in the mitochondrial genome, improper management of mitochondrial proteins, improper management of intracellular organelles such as the endoplasmic reticulum and lysosomes, and defective mitochondrial protein quality control. Defects in mitochondrial protein quality control are caused by various factors, including improper management of intracellular organelles such as the endoplasmic reticulum and lysosomes. Among these, quality control through mitochondrial biogenesis and autophagy (mitophagy) and the dynamics of fusion / fission are key contributors to various mitochondrial dysfunctions. Mitochondrial quality control defects associated with various diseases continue to be intensively studied as targets for disease treatment. Target diseases include not only mitochondrial diseases but also neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and ALS, heart failure, diabetes, and immune deficiencies against cancer, infectious diseases, and autoimmune diseases. Small molecule compounds are being developed to enhance biogenesis, induce mitophagy, and suppress excessive fission. For example, coenzyme Q10, idebenone, and metformin have been shown to induce mitophagy.

[0004] However, no compound with a clear mechanism of action has yet reached the clinic. Thus, there remains a need for compositions and methods for ameliorating mitochondrial dysfunction. Summary of the Invention

[0005] 2. Overview The present disclosure provides polypeptides that can induce double-strand breaks (DSBs) in mitochondria, thereby enabling a temporary and partial reduction in the number of mitochondria in cells. Compared to the nucleus, mitochondria have poor gene repair mechanisms. In response to DSB stress, mitochondria potently transmit signals to the nucleus to promote mitochondrial genome replication and increase the production of mitochondrial component proteins. Without being bound by theory, it is believed that introducing DSBs into mitochondrial DNA can efficiently promote mitochondrial turnover and thereby be used to ameliorate mitochondrial dysfunction by generating new mitochondria. DSBs can be introduced into mitochondrial DNA by using a polypeptide containing a mitochondrial targeting sequence (MTS) fused to an endonuclease. However, one potential problem with this approach is excessive endonuclease activity. To provide sensitive ON / OFF control of the endonuclease, the present disclosure provides polypeptides that contain a destabilization domain in addition to the mitochondrial targeting sequence and endonuclease sequence. When stabilized by a stabilizer, the destabilization domain allows the polypeptide to maintain structure and endonuclease activity; in the absence of the stabilizer, the destabilization domain loses stability, which causes degradation of the polypeptide by the proteasome.

[0006] Thus, in one aspect, the present disclosure provides a polypeptide comprising a mitochondrial targeting sequence (MTS), an endonuclease sequence (e.g., XbaIR), and a destabilization domain sequence. The inclusion of an MTS is useful for targeting the polypeptide to the desired site of endonuclease activity, i.e., the mitochondrial genome. The inclusion of a destabilization domain sequence stabilized by a stabilizer enables sensitive ON / OFF control of the endonuclease. For example, cells can be contacted with the polypeptide in the presence of a stabilizer for a certain period of time (the period during which the polypeptide is active), and then the stabilizer can be removed, causing destabilization and degradation of the polypeptide.

[0007] Exemplary features of the polypeptides of the present disclosure are described below in Section 4.2 and in specific embodiments 1-84.

[0008] In another aspect, the present disclosure provides a nucleic acid encoding a polypeptide of the present disclosure, a particle containing the nucleic acid, such as a viral particle, and a host cell containing the nucleic acid of the present disclosure. Exemplary nucleic acids include vectors, such as viral (e.g., retroviral) genomes, plasmids, and mRNA molecules. Exemplary particles include viral particles (e.g., retroviral particles). Further exemplary features of the nucleic acids, particles, and host cells of the present disclosure are described in Section 4.3 below and in specific embodiments 85-96.

[0009] In another aspect, the present disclosure provides a method for (a) inducing mitophagy in cells, (b) increasing mitochondrial turnover in cells, (c) increasing mitochondrial mass, (d) inducing double-strand breaks in mitochondrial DNA, and / or (e) inducing epigenetic modifications in cells by contacting cells with a polypeptide, nucleic acid, or particle of the present disclosure and a stabilizer. For example, cells can be transfected with a nucleic acid encoding the polypeptide, transduced with a viral particle containing a nucleic acid encoding the polypeptide, or injected with a polypeptide and cultured in the presence of a stabilizer. After a certain period of time (e.g., 6 hours to 5 days), the stabilizer can be removed to destabilize and degrade the polypeptide.

[0010] In another aspect, the present disclosure provides cells and cell populations obtained or obtainable by the methods described herein of (a) inducing mitophagy in a cell, and / or (b) increasing mitochondrial turnover in a cell, and / or (c) increasing mitochondrial mass, and / or (d) inducing double-strand breaks in mitochondrial DNA, and / or (e) inducing epigenetic modifications in a cell. Unless otherwise required by context, references herein to "cells" encompass single cells as well as cell populations.

[0011] In another aspect, the present disclosure provides the method for treating subject with the cell and cell population of the present disclosure.For example, subject can be the subject with aging-related disease, mitochondrial disease or disorder, neurodegenerative disease, eye disease (for example, retinal disease), diabetes, hearing impairment, genetic disease, heart failure, immunodeficiency, cancer or infectious disease.

[0012] Further exemplary features of the methods and cells of the present disclosure are described in Section 4.4 below and in specific embodiments 97-195.

[0013] In a further aspect, the present disclosure provides pharmaceutical compositions comprising the polypeptides, nucleic acids, particles, or cells (including cell populations) of the present disclosure. Such pharmaceutical compositions can be used, for example, in the treatment methods described herein.

[0014] In yet another aspect, the present disclosure provides a kit comprising a polypeptide, nucleic acid, or particle of the present disclosure and a stabilizing agent. The kit can be used, for example, in a method of the present disclosure.

[0015] Further exemplary features of the pharmaceutical compositions and kits are described below in Section 4.5 and in specific embodiments 196-200. [Brief explanation of the drawings]

[0016] [Figure 1]FIG. 1 shows a retroviral vector map using MTS-XbaIR-DHFR polypeptide (Example 1). [Figure 2] FIG. 1 shows a molecular model of the MTS-XbaIR-DHFR polypeptide of Example 1. [Figure 3] FIG. 1 shows a retroviral vector map based on EFGR-DHFR polypeptides (Example 1). [Figure 4] FIG. 1 shows a molecular model of the EGFR-DHFR polypeptide of Example 1. [Figure 5] FIG. 1 shows fluorescence microscopy images of HeLa cells transduced with an EGFP-DHFR retroviral vector (Example 1). [Figure 6] FIG. 1 shows FACS data of Hela cells transduced with an EGFP-DHFR retroviral vector showing EGFP expression when cultured in the presence of TMP (Example 1). [Figure 7] FIG. 1 shows the relative EGFP mRNA expression levels in Hela cells transduced with an EGFP-DHFR retroviral vector and cultured with TMP (Example 1). [Figure 8-1] 8A-8D show the fluorescence intensity of Hela cells transduced with EGFP-DHFR retroviral vectors by fluorescence microscopy and FACS at 0, 1, 2, 4, 6, 8, 24, and 48 hours after culturing in medium with TMP for 2 days and then washing (Example 1). Figure 8A: Time course; Figure 8B: Fluorescence images; Figure 8C: FACS analysis; Figure 8D: Mean fluorescence intensity (MFI) in FACS analysis over time. [Figure 8-2] 8A-8D show the fluorescence intensity of Hela cells transduced with EGFP-DHFR retroviral vectors by fluorescence microscopy and FACS at 0, 1, 2, 4, 6, 8, 24, and 48 hours after culturing in medium with TMP for 2 days and then washing (Example 1). Figure 8A: Time course; Figure 8B: Fluorescence images; Figure 8C: FACS analysis; Figure 8D: Mean fluorescence intensity (MFI) in FACS analysis over time. [Figure 9-1]9A and 9B show the fluorescence intensity of Hela cells transduced with EGFP-DHFR retroviral vectors by fluorescence microscopy and FACS after culturing in medium with TMP for several periods (0, 1, 2, 4, 6, 8, 24, and 48 hours). Figure 9A shows time course; Figure 9B shows fluorescence images; Figure 9C shows FACS analysis; Figure 9D shows MFI in FACS analysis over time. [Figure 9-2] 9A and 9B show the fluorescence intensity of Hela cells transduced with EGFP-DHFR retroviral vectors by fluorescence microscopy and FACS after culturing in medium with TMP for several periods (0, 1, 2, 4, 6, 8, 24, and 48 hours). Figure 9A shows time course; Figure 9B shows fluorescence images; Figure 9C shows FACS analysis; Figure 9D shows MFI in FACS analysis over time. [Figure 10-1] Figure 10A shows XbaI expression (Figure 10A) and mtDNA copy number (CN) (Figure 10B) for Hela cells (Hela MXD sc20) transduced with a retroviral vector encoding the MTS-XbaIR-DHFR polypeptide and cultured for 2 days in the absence or presence of 0.5 μM TMP, after which the TMP was washed out (Figure 10C) (Example 1). [Figure 10-2] Figure 10A shows XbaI expression (Figure 10A) and mtDNA copy number (CN) (Figure 10B) for Hela cells (Hela MXD sc20) transduced with a retroviral vector encoding the MTS-XbaIR-DHFR polypeptide and cultured for 2 days in the absence or presence of 0.5 μM TMP, after which the TMP was washed out (Figure 10C) (Example 1). [Figure 11-1] Figure 11A shows the MFI of Mitogreen staining, an indicator of mitochondrial mass (mtMass), (Figure 11A), the MFI of TMRM staining, an indicator of total mitochondrial membrane potential (mtMP), (Figure 11B), and the relative TMRM / Mitogreen ratio, an indicator of mtMP per mass unit, (Figure 11C) in Hela MXD sc20 cells incubated with TMP for three different periods (16, 20, and 48 hours) following cell culture over time (Figure 11D). [Figure 11-2] Figure 11A shows the MFI of Mitogreen staining, an indicator of mitochondrial mass (mtMass), (Figure 11A), the MFI of TMRM staining, an indicator of total mitochondrial membrane potential (mtMP), (Figure 11B), and the relative TMRM / Mitogreen ratio, an indicator of mtMP per mass unit, (Figure 11C) in Hela MXD sc20 cells incubated with TMP for three different periods (16, 20, and 48 hours) following cell culture over time (Figure 11D). [Figure 12] 12A and 12B show the cell count (FIG. 12A) and cell viability (FIG. 12B) of Hela MXD sc20 cells cultured for 2 days in the absence or presence of 0.5 μm TMP, and washing with TMP (FIG. 12C) (Example 1). [Figure 13-1] Figure 13A shows the mitophagy index by FACS analysis (Figure 13A) and mtDNA CN estimated by qPCR (Figure 13B) of Hela MDX sc20 cells transduced with retroviral vectors encoding mtKeimaRed and PARK2 and cultured with or without TMP for several time periods. CCCP is used as a positive control for mitophagy at 10 μM (Example 1). [Figure 13-2] Figure 13A shows the mitophagy index by FACS analysis (Figure 13A) and mtDNA CN estimated by qPCR (Figure 13B) of Hela MDX sc20 cells transduced with retroviral vectors encoding mtKeimaRed and PARK2 and cultured with or without TMP for several time periods. CCCP is used as a positive control for mitophagy at 10 μM (Example 1). [Figure 14-1] Figure 14 shows the changes in mitochondrial biogenesis in Hela_GiM cells stably expressing the genetically induced mitophagy (GiM) unit in the presence and absence of TMP. Mitochondrial ROS (mtROS) (Figure 14A), PGC1α (Figure 14B), NRF1 (Figure 14C), and TFAM (Figure 14D) were assessed over time using FACS (Example 2). [Figure 14-2] Figure 14 shows the changes in mitochondrial biogenesis in Hela_GiM cells stably expressing the genetically induced mitophagy (GiM) unit in the presence and absence of TMP. Mitochondrial ROS (mtROS) (Figure 14A), PGC1α (Figure 14B), NRF1 (Figure 14C), and TFAM (Figure 14D) were assessed over time using FACS (Example 2). [Figure 15-1] Figure 15 shows the expression of several mitochondrial proteins over time in Hela_GiM cells in the presence and absence of TMP. Figure 15A shows Western blot images of mitochondrial proteins on days 2, 4, 6, and 8. Figures 15B-15F show the quantitative levels of the same proteins (Example 2). [Figure 15-2] Figure 15 shows the expression of several mitochondrial proteins over time in Hela_GiM cells in the presence and absence of TMP. Figure 15A shows Western blot images of mitochondrial proteins on days 2, 4, 6, and 8. Figures 15B-15F show the quantitative levels of the same proteins (Example 2). [Figure 15-3] Figure 15 shows the expression of several mitochondrial proteins over time in Hela_GiM cells in the presence and absence of TMP. Figure 15A shows Western blot images of mitochondrial proteins on days 2, 4, 6, and 8. Figures 15B-15F show the quantitative levels of the same proteins (Example 2). [Figure 15-4] Figure 15 shows the expression of several mitochondrial proteins over time in Hela_GiM cells in the presence and absence of TMP. Figure 15A shows Western blot images of mitochondrial proteins on days 2, 4, 6, and 8. Figures 15B-15F show the quantitative levels of the same proteins (Example 2). [Figure 16-1]16A and 16B show phase contrast (PhC) and fluorescence microscopy images of mtKeimaRed-expressing HeLa_GiM cells in the presence and absence of TMP, and the percentage of cells that are mitophagy-positive (FIG. 16B) (Example 2). [Figure 16-2] 16A and 16B show phase contrast (PhC) and fluorescence microscopy images of mtKeimaRed-expressing HeLa_GiM cells in the presence and absence of TMP, and the percentage of cells that are mitophagy-positive (FIG. 16B) (Example 2). [Figure 17-1] Figure 17A shows the colocalization of the autophagosomal membrane marker LC3, the mitochondrial marker TOM20, and the nuclear stain DAPI in Hela_GiM cells in the presence or absence of TMP and BafA1, as well as quantification in the area corresponding to autophagosomes (Figure 17B) (Example 3). [Figure 17-2] Figure 17A shows the colocalization of the autophagosomal membrane marker LC3, the mitochondrial marker TOM20, and the nuclear stain DAPI in Hela_GiM cells in the presence or absence of TMP and BafA1, as well as quantification in the area corresponding to autophagosomes (Figure 17B) (Example 3). [Figure 18] Figure 18 shows LC3-II expression levels in HeLa_GiM cells in the absence or presence of TMP or TMP + BafA1 (Figure 18A shows a Western blot image, and Figure 18B is a graphical representation of triplicate quantification (Example 3)). [Figure 19-1] Figure 19 shows the results of respirometry depicting the changes in OXPHOS and glycolysis over time (Figure 19A), a two-dimensional development of the relationship between OXPHOS and glycolysis (Figure 19B), and the changes in ATP production, basal respiration, proton leak, and spare respiratory capacity over time (Figure 19C) (Example 4). [Figure 19-2]Figure 19 shows the results of respirometry depicting the changes in OXPHOS and glycolysis over time (Figure 19A), a two-dimensional development of the relationship between OXPHOS and glycolysis (Figure 19B), and the changes in ATP production, basal respiration, proton leak, and spare respiratory capacity over time (Figure 19C) (Example 4). [Figure 19-3] Figure 19 shows the results of respirometry depicting the changes in OXPHOS and glycolysis over time (Figure 19A), a two-dimensional development of the relationship between OXPHOS and glycolysis (Figure 19B), and the changes in ATP production, basal respiration, proton leak, and spare respiratory capacity over time (Figure 19C) (Example 4). [Figure 20-1] 20A and 20B show the assay setup used in Example 5, as further described in Example 5 (FIG. 20A), FACS results of untreated Alzheimer's disease (AD) fibroblasts and control NHDF cells (FIG. 20B), and AD fibroblasts after 7, 14, and 21 days of gene-induced mitophagy (GiM) (FIG. 20C), mtMass and mtMP levels in untreated (FIGS. 20D and 20E, respectively), and AD fibroblasts at 7 and 14 days after GiM compared to controls (FIGS. 20F and 20G, respectively). [Figure 20-2] 20A and 20B show the assay setup used in Example 5, as further described in Example 5 (FIG. 20A), FACS results of untreated Alzheimer's disease (AD) fibroblasts and control NHDF cells (FIG. 20B), and AD fibroblasts after 7, 14, and 21 days of gene-induced mitophagy (GiM) (FIG. 20C), mtMass and mtMP levels in untreated (FIGS. 20D and 20E, respectively), and AD fibroblasts at 7 and 14 days after GiM compared to controls (FIGS. 20F and 20G, respectively). [Figure 20-3]20A and 20B show the assay setup used in Example 5, as further described in Example 5 (FIG. 20A), FACS results of untreated Alzheimer's disease (AD) fibroblasts and control NHDF cells (FIG. 20B), and AD fibroblasts after 7, 14, and 21 days of gene-induced mitophagy (GiM) (FIG. 20C), mtMass and mtMP levels in untreated (FIGS. 20D and 20E, respectively), and AD fibroblasts at 7 and 14 days after GiM compared to controls (FIGS. 20F and 20G, respectively). [Figure 20-4] 20A and 20B show the assay setup used in Example 5, as further described in Example 5 (FIG. 20A), FACS results of untreated Alzheimer's disease (AD) fibroblasts and control NHDF cells (FIG. 20B), and AD fibroblasts after 7, 14, and 21 days of gene-induced mitophagy (GiM) (FIG. 20C), mtMass and mtMP levels in untreated (FIGS. 20D and 20E, respectively), and AD fibroblasts at 7 and 14 days after GiM compared to controls (FIGS. 20F and 20G, respectively). [Figure 20-5] 20A and 20B show the assay setup used in Example 5, as further described in Example 5 (FIG. 20A), FACS results of untreated Alzheimer's disease (AD) fibroblasts and control NHDF cells (FIG. 20B), and AD fibroblasts after 7, 14, and 21 days of gene-induced mitophagy (GiM) (FIG. 20C), mtMass and mtMP levels in untreated (FIGS. 20D and 20E, respectively), and AD fibroblasts at 7 and 14 days after GiM compared to controls (FIGS. 20F and 20G, respectively). DETAILED DESCRIPTION OF THE INVENTION

[0017] 4. Detailed Description The present disclosure provides polypeptides that can induce double-strand breaks (DSBs) in mitochondria, thereby enabling a transient and partial reduction in the number of mitochondria in a cell.

[0018] The mitochondrial genome encodes respiratory chain proteins, which are under tight regulation and balanced with the translation of nuclear-encoded respiratory proteins. Therefore, partial loss of the mitochondrial genome is directly related to insufficient proton uptake, resulting in depolarization of the mitochondrial membrane potential. Mitochondrial membrane proteins are depolarized in dysfunctional mitochondrial compartments and are key regulators of mitophagy. Without being bound by theory, it is thought that more depolarized proteins may preferentially undergo mitophagy after the introduction of DSBs.

[0019] In response to the stress of DSBs, mitochondria potently transmit signals to the nucleus to promote mitochondrial genome replication and increase the production of mitochondrial proteins (e.g., as part of the mitochondrial unfolded protein response (UPRmt)). In addition to UPRmt signals, metabolic changes resulting from mitochondrial genome reduction can also affect the epigenomic state of the cell. For example, this reduction may decrease some TCA cycle intermediates that are utilized for acetylation and methylation in the nuclear genome and histones. Without being bound by theory, the introduction of DSBs into mitochondrial DNA can be used to efficiently promote mitochondrial turnover, thereby improving mitochondrial dysfunction through the generation of new mitochondria. Mitochondrial DNA can contain several modifications, such as 8-oxo-7,8-dihydroguanine (8-OXOG), an oxidized form of guanine. Damaging modifications accumulate with aging. Because newly generated mitochondrial genomes lack these modifications, mitochondrial biogenesis can promote the restoration of healthy mitochondrial function.

[0020] DSB can be introduced into mitochondrial DNA by using a polypeptide containing a mitochondrial targeting sequence (MTS) fused to an endonuclease. However, one potential problem with this approach is excessive endonuclease activity. To provide sensitive ON / OFF control of the endonuclease, the present disclosure provides a polypeptide that has a destabilization domain in addition to the mitochondrial targeting sequence and the endonuclease sequence. When stabilized by a stabilizer, the destabilization domain allows the polypeptide to maintain its structure and endonuclease activity; in the absence of the stabilizer, the destabilization domain loses stability, which causes the polypeptide to be degraded by proteasomes.

[0021] 4.1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following definitions are provided for a full understanding of the terms used herein.

[0022] As used herein, the following terms are intended to have the following meanings:

[0023] "A," "An," "The": As used herein, the terms "a," "an," "the," and similar terms used in the context of this specification (especially in the context of the claims) are intended to include both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably.

[0024] And / or: The term "and / or" means that each one or both or all of the components or functions in a list are possible variations, especially two or more of them, in an alternative or cumulative manner.

[0025] Destabilization domain (DD): The term destabilization domain refers to a polypeptide domain that, when fused with a second polypeptide domain such as endonuclease, causes the degradation of the polypeptide in the absence of a stabilizer that prevents or inhibits the degradation caused by the destabilization domain.Exemplary destabilization domains include dihydrofolate reductase (DHFR) destabilization domain (can be stabilized by the exemplary stabilizer trimethoprim), FK506 binding protein (FKBP) destabilization domain (can be stabilized by the exemplary stabilizer Shield-1 (Shld1), rapamycin, FK506), and PDE5 destabilization domain (can be stabilized by the exemplary stabilizer sildenafil, vardenafil, tadalafil, avanafil, lodenafil, mirodenafil, udenafil, benzamidenafil, dasantafil and beminafil). Exemplary DHFR destabilizing domains are described in Iwamoto et al., 2010, Chem Biol. 17(9):981-8, Liu et al., 2014 Int. J. Parasitol. 44(10):729-735, and U.S. Pat. No. 9,487,787; exemplary FKBP destabilizing domains are described in Banaszynski et al., 2006, Cell 126(5):995-1104 and U.S. Pat. No. 9,487,787; and exemplary PDE5 destabilizing domains are described in WO 2018 / 237323, the contents of each of which are incorporated herein by reference in their entireties.

[0026] Effective amount: The term "effective amount" or "therapeutically effective amount" refers to an amount or quantity of an agent or composition sufficient to elicit the necessary or desired response, in other words, an amount sufficient to elicit an appreciable biological response when administered to a subject. The amount preferably relates to an amount that is therapeutically, or more broadly, prophylactically, effective against the progression of a disease or disorder as disclosed herein. An "effective amount" or "therapeutically effective amount" may vary from subject to subject, depending on differences in drug metabolism, age, weight, the subject's general condition, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.

[0027] Endonuclease: The term "endonuclease" refers to an enzyme that cleaves a polynucleotide chain by separating nucleotides other than those at the 5' or 3' end. Endonucleases are distinct from exonucleases, which cleave nucleotides from the 5' or 3' end of a polynucleotide chain. Exemplary endonucleases include restriction endonucleases that can cleave double-stranded DNA at or near specific recognition sites in DNA to form double-stranded breaks (DSBs). Exemplary restriction endonucleases include XbaIR, EcoRI, SmaI, AflII, BamHI, BcII, EcoRI, HaeIII, HindII, HindIII, NdeI, PvuII, PstI, and SpeI. Exemplary endonuclease amino acid sequences are listed in publicly available databases, such as UniProt. For example, an exemplary XbaIR amino acid sequence has UniProt accession number 068567; an exemplary EcoRI amino acid sequence has UniProt accession number P00642; an exemplary SmaI amino acid sequence has UniProt accession number P14229; an exemplary AflII amino acid sequence has UniProt accession number E3VX87; an exemplary BamHI amino acid sequence has UniProt accession number P23940; an exemplary BclI amino acid sequence has UniProt accession number E5LGB8; an exemplary An exemplary HaeIII amino acid sequence has UniProt accession number O68584; an exemplary HindII amino acid sequence has UniProt accession number P44413; an exemplary HindIII amino acid sequence has UniProt accession number P43870; an exemplary PvuII amino acid sequence has UniProt accession number A0A4R7BM34; an exemplary PstI amino acid sequence has UniProt accession number P00640; and an exemplary SpeI amino acid sequence has UniProt accession number F1KM35.

[0028] Mitochondrial Targeting Sequence (MTS): The term "mitochondrial targeting sequence" refers to an amino acid sequence that can target the transport of a polypeptide containing that amino acid sequence to mitochondria. MTSs are typically 10-70 amino acids in length. MTSs often contain an alternating pattern of hydrophobic and positively charged amino acids that form an amphipathic helix.

[0029] Or: Unless otherwise specified, the conjunction "or" is intended to be used in its proper sense as a Boolean logic operator and encompasses both alternative feature selections (A or B, where selection of A is mutually exclusive with B) and joint feature selections (A or B, where both A and B are selected). In several places in the text, the term "and / or" is used for the same purpose, and this should not be interpreted to mean that "or" is used to refer to mutually exclusive alternatives.

[0030] Peptides, proteins, and polypeptides: The terms peptide, protein, and polypeptide are used interchangeably to refer to natural or synthetic molecules containing two or more amino acids linked by the carboxyl group of one amino acid to the alpha-amino group of another. The amino acids can be natural or synthetic and can include chemical modifications such as disulfide bonds, radioisotope substitution, phosphorylation, substrate chelation (e.g., iron or copper chelation), glycosylation, acetylation, formylation, amidation, biotinylation, and various other modifications. There is no explicit requirement that a polypeptide must contain its intended function; a polypeptide can be functional, non-functional, function for an unexpected / unintended purpose, or have unknown function. Polypeptides are composed of approximately 20 naturally occurring amino acids, although natural and synthetic amino acids that are not members of the standard 20 amino acids can also be used. The 20 standard amino acids include alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). The term "polypeptide sequence" or "amino acid sequence" refers to the alphabetical representation of a polypeptide molecule.

[0031] Percentage of identity: The percentage of identity between two amino acid sequences is calculated by multiplying the number of matches between the pair of aligned sequences by 100 and dividing by the length of the aligned region.Scored identity only counts perfect matches, and does not consider the similarity between amino acids, nor does it consider substitution or deletion as a match.The alignment for determining the percentage of sequence identity can be achieved in various ways within the skill of the art, such as by manual alignment or by using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.Those skilled in the art can determine the appropriate parameters to achieve maximum alignment.

[0032] Subject: As used herein, the term "subject" means a human.

[0033] Treat, treating, treatment: As used herein, the terms "treat," "treating," or "treatment" of any disease or disorder refer, in one embodiment, to ameliorating the disease or disorder (e.g., slowing, inhibiting, or alleviating the progression of the disease or at least one of its clinical symptoms or pathological features). In another embodiment, "treat," "treating," or "treatment" refers to alleviating or ameliorating at least one physical parameter or pathological feature of the disease, including, for example, those that may not be discernible by the subject. In yet another embodiment, "treat," "treating," or "treatment" refers to modulating the disease or disorder physically (e.g., stabilization of at least one discernible or indiscernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. In yet another embodiment, "treat," "treating," or "treatment" refers to preventing or delaying the onset, development, or progression of the disease or disorder, or at least one symptom or pathological feature associated therewith. In yet another embodiment, "treat," "treating," or "treatment" means preventing or slowing the progression of a disease to a more advanced or more severe state. The benefit to the treated patient will be statistically significant, or at least discernible by the patient or the physician. However, it will be understood that when a pharmaceutical agent is administered to a patient to treat a disease, the result will not always be an effective treatment.

[0034] Polypeptides In one aspect, the present disclosure provides a polypeptide comprising a mitochondrial targeting sequence (MTS), an endonuclease sequence, and a destabilization domain (DD) sequence. Exemplary features of mitochondrial targeting sequences, endonuclease sequences, and destabilization domains that can be included in polypeptides of the present disclosure are described in Sections 4.2.1, 4.2.2, and 4.2.3, respectively.

[0035] The MTS, endonuclease sequence, and DD can be positioned in any suitable order from N-terminus to C-terminus. For example, the MTS can be positioned at the N-terminus or C-terminus of the polypeptide. In some embodiments, the MTS is positioned at the N-terminus of the polypeptide. The endonuclease sequence can be positioned N-terminal to the DD or C-terminal to the DD. In some embodiments, the polypeptide comprises the MTS, endonuclease sequence, and DD sequence in order from N-terminus to C-terminus. The MTS, endonuclease sequence, and DD sequence can be directly linked or separated by a spacer sequence, e.g., a short amino acid sequence, e.g., one, two, three, four, or more amino acids.

[0036] 4.2.1. Mitochondrial targeting sequence Mitochondria have approximately 1,500 proteins encoded by the nuclear genome. They are translated in the cytosol and imported into the inner or outer mitochondrial membrane, the intermembrane space, or the matrix depending on the MTS. The polypeptides of the present disclosure may include the full-length mitochondrial protein MTS, or a variant of the wild-type MTS (e.g., a truncated version of the full-length MTS and / or an MTS with one or more amino acid substitutions (e.g., one or more conservative amino acid substitutions) compared to the wild-type sequence).

[0037] The polypeptides of the present disclosure may comprise a human MTS or a non-human MTS (e.g., a rodent such as a mouse or a rat, or a non-human primate such as a cynomolgus monkey). For example, the MTS of a polypeptide of the present disclosure may comprise an MTS of a TCA cycle-related enzyme, a chaperone protein, a mitochondrial genome replication protein, a protease, an mRNA processing protein, a mitochondrial RNA degradation protein, a deoxynucleotide triphosphate synthesis-related protein, a mitoribosomal protein, a phospholipid metabolism-related protein, a protein involved in the metabolism of toxic compounds, a disulfide relay system-related protein, an iron-sulfur protein assembly protein, a tRNA-modifying protein, an aminoacyl-tRNA synthetase, a release factor, or an elongation factor.

[0038] In some embodiments, the MTS comprises a cytochrome c oxidase subunit (e.g., a full-length MTS or a truncated form thereof that maintains mitochondrial targeting activity), such as an MTS of cytochrome c oxidase subunit VIII (COX8), cytochrome c oxidase subunit X (COX10), or cytochrome c oxidase subunit IV (COX4).

[0039] In some embodiments, the MTS comprises an MTS of a frataxin (FXN) protein.

[0040] In some embodiments, the MTS comprises an MTS for a TCA cycle-related enzyme, such as pyruvate dehydrogenase, citrate synthase, aconitase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, malate dehydrogenase, or pyruvate carboxylase.

[0041] In other embodiments, the MTS comprises an MTS of a chaperone protein, eg, mtHSP10, mtHSP60, mtHSP70, or mtHSP90.

[0042] In other embodiments, the MTS comprises the MTS of a mitochondrial genome replication protein, such as TFAM, Twinkle, PolG, TFB2M, TEFM, or MTERF1.

[0043] In other embodiments, the MTS comprises the MTS of a protease, eg, MPP, CLPXP, LONATPase, or PreP.

[0044] In other embodiments, the MTS comprises an MTS for an mRNA processing protein, for example, LRPPRC, TACO1, ELAC2, PNPT1, HSD17B10, MTPAP, or PTCD1.

[0045] In other embodiments, the MTS comprises the MTS of a mitochondrial RNA degradation protein, eg, PNPasse, REX02, or SUV3.

[0046] In other embodiments, the MTS comprises an MTS of a deoxynucleotide triphosphate synthesis-related protein, for example, DGUOK, TK2, TYMP, MGME1, SUCLG1, SUCLA2, RNASEH1, or C10orf2.

[0047] In other embodiments, the MTS comprises the MTS of a mitoribosomal protein, eg, MRPS16, MRPS22, MRPL3, MRP12, or MRPL44.

[0048] In other embodiments, the MTS comprises the MTS of a phospholipid metabolism-related protein, eg, AGK, SERAC1, or TAZ.

[0049] In other embodiments, the MTS comprises the MTS of a protein involved in the metabolism of toxic compounds, for example, HIBCH, ECHS1, ETHE1, or MPV17.

[0050] In other embodiments, the MTS comprises a disulfide relay system-associated protein, eg, the MTS of GFER.

[0051] In other embodiments, the MTS comprises the MTS of an iron-sulfur protein assembly protein, eg, ISCU, BOLA3, NFU1, or IBA57.

[0052] In other embodiments, the MTS comprises an MTS of a tRNA modifying protein, for example, MTO1, GTP3BP, TRMU, PUS1, MTFMT, TRIT1, TRNT1, or TRMT5.

[0053] In other embodiments, the MTS comprises an aminoacyl-tRNA synthetase, e.g., an MTS for AARS2, DARS2, EARS2, RARS2, YARS2, FARS2, HARS2, LARS2, VARS2, TARS2, IARS2, CARS2, PARS2, NARS2, KARS, GARS, SARS2, or MARS2.

[0054] In other embodiments, the MTS comprises the MTS of an elongation factor, eg, TUFM, TSFM, or GFM1.

[0055] Exemplary mitochondrial targeting sequences are listed in Table 1.

[0056] [Table 1]

[0057] Polypeptides of the present disclosure can include an MTS identified in Table 1 or a variant thereof (e.g., an MTS with one or more conservative amino acid substitutions and / or truncations). The truncation can be a truncation of the C-terminal sequence (e.g., an MTS can correspond to a sequence set forth in Table 1, but with a C-terminal truncation of one or more amino acids, e.g., 1, 2, 3, 4, 5, or 6 or more amino acids). In some embodiments, an MTS comprises at least 15 N-terminal amino acids of an MTS sequence set forth in Table 1. A variant MTS can include, for example, an MTS that is at least 80%, at least 95%, at least 90%, or at least 95% identical to an MTS set forth in Table 1.

[0058] Those skilled in the art will understand that additional mitochondrial targeting sequences other than those identified in this section can also be used. Additional mitochondrial targeting sequences can be identified using various tools for predicting MTS, such as SignalP (Bendtsen et al., 2004, J. Mol. Biol. 340:783-795; Teufel et al., 2022 Nat Biotechnol. doi.org / 10.1038 / s41587-021-01156-3), MitoFates (Fukasawa et al., 2015 Mol Cell Proteomics 14(4):1113-1126), and MitoProt (Claros, 1995, Comput Apl Biosci. 11(4):441-7).

[0059] 4.2.2 Endonucleases Various endonucleases can be used in the polypeptide of the present disclosure.For example, endonucleases can be restriction endonucleases, RNA-guided endonucleases (for example, Cas9 or Cas12), zinc finger nucleases, or transcription activator-like effector nucleases (TALENs).Endonucleases can include catalytic domains (for example, from wild-type or engineered endonucleases), and optionally one or more additional domains, such as all domains present in full-length wild-type or engineered endonucleases.

[0060] The endonuclease may be of bacterial origin. Many restriction enzymes are known in the art, including, for example, XbaIR, EcoRI, SmaI, AflII, BamHI, BclI, HaeIII, HindII, HindIII, NdeI, PvuII, PstI, and SpeI.

[0061] In some embodiments, the endonuclease is XbaIR. An exemplary XbaIR sequence is set forth in SEQ ID NO: 16:

[0062] [ka]

[0063] In some embodiments, the endonuclease comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, more than 95%, or 100% identity to SEQ ID NO:16.

[0064] In some embodiments, the endonuclease is EcoRI.

[0065] In some embodiments, the endonuclease is SmaI.

[0066] In some embodiments, the endonuclease is AflII.

[0067] In some embodiments, the endonuclease is BamHI.

[0068] In some embodiments, the endonuclease is BclI.

[0069] In some embodiments, the endonuclease is HaeIII.

[0070] In some embodiments, the endonuclease is HindII.

[0071] In some embodiments, the endonuclease is HindIII.

[0072] In some embodiments, the endonuclease is NdeI.

[0073] In some embodiments, the endonuclease is PvuII.

[0074] In some embodiments, the endonuclease is PstI.

[0075] In some embodiments, the endonuclease is SpeI.

[0076] In some embodiments, the endonuclease sequence is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of UniProt Accession No. O68567, UniProt Accession No. P00642, UniProt Accession No. P14229, UniProt Accession No. E3VX87, UniProt Accession No. P23940, UniProt Accession No. E5LGB8, UniProt Accession No. O68584, UniProt Accession No. P44413, UniProt Accession No. P43870, UniProt Accession No. A0A4R7BM34, UniProt Accession No. P00640, or UniProt Accession No. F1KM35.

[0077] Exemplary RNA-guided endonucleases, such as Cas9 and Cas12, are described in U.S. Patent No. 11,001,863 (B2), International Publication No. WO 2014 / 093661, and International Publication No. WO 2019 / 233990, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the endonuclease is SaCas9 or SpCas9. When an RNA-guided endonuclease is used, the polypeptide can be used in combination with one or more guide RNA molecules that target mitochondrial DNA.

[0078] Exemplary zinc finger nucleases are described in WO 2001 / 025255 and WO 2003 / 066828, the contents of which are incorporated herein by reference in their entireties.

[0079] Exemplary TALEN nucleases are described in WO 2014 / 134412, WO 2015 / 013583, and WO 2013 / 163628, the contents of which are incorporated by reference in their entireties.

[0080] 4.2.3. Destabilization Domains The polypeptides of the present disclosure contain a destabilization domain (DD) that allows for on / off control of the endonuclease. Exemplary DDs include DHFR, FKBP, and PDE5 DDs.

[0081] An exemplary DHFR DD is described in U.S. Patent No. 9,487,787, the contents of which are incorporated herein in their entirety. The amino acid sequence of wild-type E. coli DHFR is as follows:

[0082] [ka]

[0083] The DHFR DD may contain a wild-type DHFR sequence or may contain one or more amino acid substitutions and / or truncations at the N- and / or C-terminus. For example, the DHFR DD sequence may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 17. Exemplary amino acid substitutions and combinations that can be included in the DHFR DD include Y100I, G121V, N18T / A19V, F103L, H12Y / Y100I, H12L / Y100I, R98H / F103S, M42T / H114R, and I61F / T68S. Combinations of the foregoing substitutions may also be used. In some embodiments, DHFR comprises an amino acid sequence identical to SEQ ID NO: 17 except for Y100I, G121V, N18T / A19V, F103L, H12Y / Y100I, H12L / Y100I, R98H / F103S, M42T / H114R, or I61F / T68S substitutions, or a combination thereof. In some embodiments, the DHFR DD lacks an N-terminal methionine. For example, in some embodiments, DHFR comprises an amino acid sequence identical to SEQ ID NO: 17 except for Y100I, G121V, N18T / A19V, F103L, H12Y / Y100I, H12L / Y100I, R98H / F103S, M42T / H114R, or I61F / T68S substitutions, or a combination thereof, and the lack of an N-terminal methionine.

[0084] In some embodiments, the DHFR DD is

[0085] [ka] An exemplary nucleotide sequence encoding SEQ ID NO: 18 is as follows:

[0086] [ka]

[0087] An exemplary stabilizer for DHFR DD is trimethoprim.

[0088] An exemplary FKBP DD is described in U.S. Patent No. 9,487,787, the contents of which are incorporated herein in their entirety. The amino acid sequence of an exemplary FKBP DD (having an F36V substitution compared to the wild-type sequence) is as follows: GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 20)

[0089] The FKBP DD may contain a wild-type FKBP sequence or may contain one or more amino acid substitutions. For example, the FKBP DD sequence may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 20. Exemplary amino acid substitutions that can be included in the FKBP DD include F15S, V24A, H25R, E60G, L106P, D100G, M66T, R71G, D100N, E102G, and K105I. Combinations of the aforementioned substitutions may also be used. In some embodiments, DD comprises an amino acid sequence identical to SEQ ID NO: 20 except for an F15S, V24A, H25R, E60G, L106P, D100G, M66T, R71G, D100N, E102G, or K105I substitution, or a combination thereof.

[0090] Exemplary FKBP DD stabilizers include Shield-1 (Shld1), rapamycin, and FK506.

[0091] Exemplary PDE5 DDs are described in International Publication No. 2018 / 237323, the contents of which are incorporated herein in their entirety. PDE5 DDs can be derived from PDE5A isoform 1 (SEQ ID NO: 21); PDE5A isoform 2 (SEQ ID NO: 22), and / or PDE5A isoform 3 (SEQ ID NO: 23). These isoforms differ in their N-terminal regions and have a unique first exon followed by a consensus sequence of 823 amino acids.

[0092] All PDE5A isoforms contain a catalytic domain located near the C-terminus of the protein, which is relatively selective for cGMP as a substrate at physiological levels. The substrate-binding site is also the binding site for several known PDE5 inhibitors, such as sildenafil, which are used to treat cardiovascular disease and erectile dysfunction. Two homologous GAF domains are located toward the N-terminus. One of the GAF domains, GAF-A, contains a high-affinity binding site for cGMP. Occupation of this domain by cGMP is known to cause activation of the catalytic domain. Furthermore, the affinity of this site for cGMP increases with cGMP-dependent protein kinase-mediated phosphorylation of serine 92. In another embodiment, the PDE5A DD may contain the catalytic domain of PDE5A, spanning from amino acid position 535 to position 860 of UniProt ID: O76074 (SEQ ID NO: 21), as shown in SEQ ID NO: 24. In addition to the catalytic domain, the PDE5A DD may also contain one or more GAF domains and / or a C-terminal portion extending beyond the catalytic domain. In one embodiment, the PDE5A-derived DD comprises amino acids from position 535 to position 875 of SEQ ID NO: 21. In another embodiment, the PDE5 DD comprises amino acids from position 466 to 875 or from position 420 to 875 of SEQ ID NO: 21. Exemplary PDE5 DD sequences are set forth in Table 2.

[0093] [Table 2-1]

[0094] [Table 2-2]

[0095] Exemplary amino acid substitutions that can be included in the PDE5 DD include E535D, E536G, Q541R, K555R, F559L, S560G, F561L, F564L, F564S, V585A, N587S, K591E, I599V, K604E, K608E, N609H, K630R, K633E, N636S, I648V, N661S, S663P, L675P, Y676D, Y676N, C677R, H678 PDE5 DD can comprise one or more amino acid substitutions selected from R, D687A, T711A, T712S, D724N, L738H, N742S, F744L, L746S, F755L, A762S, D764V, D764N, D764G, S766F, K795E, L797F, I799T, L804P, T802P, S815C, M816A, M816T, I824T, C839S, F840S and K852E.PDE5 DD can also comprise additional substitutions such as Q589R. In some embodiments, the PDE5 DD sequence comprises a sequence selected from the group of amino acid sequences identified by SEQ ID NOs: 19-35 of WO 2018 / 237323 and SEQ ID NOs: 66-69 of WO 2018 / 237323.

[0096] Exemplary stabilizers of PDE5 DD include sildenafil, vardenafil, tadalafil, avanafil, lodenafil, mirodenafil, udenafil, benzamidenafil, dasantafil, and beminafil.

[0097] 4.3. Nucleic Acids, Particles, and Host Cells In another aspect, the disclosure provides a nucleic acid encoding a polypeptide of the disclosure, e.g., as described in Section 4.2. The nucleic acid can be, for example, a vector, such as a viral genome or a plasmid, or an mRNA molecule.

[0098] Exemplary vectors include viral expression vectors (e.g., vaccinia virus-based viral vectors); poliovirus; adenovirus (see, e.g., Li et al., 1994, Invest Opthalmol Vis Sci 35:2543-2549; Borras et al., 1999, Gene Ther 6:515-524; Li and Davidson, 1995, PNAS 92:7700-7704; Sakamoto et al., 1999, H Gene Ther 5:1088-1097, 1999; WO 94 / 12649, WO 93 / 03769, WO 93 / 19191, WO 94 / 28938, WO 95 / 11984, and WO 95 / 00655); adeno-associated virus (AAV) (see, e.g., Ali et al., 1998, Hum Gene Ther 9:81 86; Flannery et al., 1997, PNAS 94:6916-6921; Bennett et al., 1997, Invest Opthalmol Vis Sci 38:2857-2863; Jomary et al., 1997, Gene Ther 4:683 690; Rolling et al., 1999, Hum Gene Ther 10:641-648; Ali et al., 1996, Hum Mol Genet 5:591-594; WO 93 / 09239); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., 1997, PNAS 94:10319-23; Takahashi et al., 1999, J Virol 73:7812-7816); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and retroviral-derived vectors such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. In some cases, the recombinant expression vector of the present disclosure is a recombinant lentiviral vector.In some cases, the recombinant expression vectors of the present disclosure are recombinant retroviral vectors.

[0099] In some embodiments, the vector comprises a retroviral genome. Nucleic acids, such as retroviral genomes, can be provided in the form of particles, for example, viral particles (e.g., retroviral particles).

[0100] Nucleic acids encoding polypeptides of the present disclosure can further comprise one or more regulatory sequences, e.g., a promoter, e.g., an SV40, CMV, or CAG promoter. An exemplary SV40 promoter sequence is as follows:

[0101] [ka]

[0102] In another aspect, the present disclosure provides a host cell comprising the nucleic acid of the present disclosure.The host cell can be a prokaryotic cell (for example, a bacterium such as E. coli) or a eukaryotic cell (for example, a human cell line such as HEK293 or 293T).The host cell can be used to propagate nucleic acid, such as a retroviral genome or a plasmid, or to propagate and package particles, such as retroviral particles.

[0103] 4.4. Methods of Inducing Mitophagy and Treatment Methods In further aspects, the present disclosure provides methods of using the polypeptides, nucleic acids, and particles of the present disclosure, such as the polypeptides, nucleic acids, and particles described in Sections 4.2 and 4.3, to induce mitophagy in a cell, and / or increase mitochondrial turnover in a cell, and / or increase mitochondrial mass, and / or induce double-strand breaks in mitochondrial DNA, and / or (e) induce epigenomic modifications in a cell.

[0104] This method typically includes contacting cells with a polypeptide, nucleic acid, or particle and a stabilizing agent that can stabilize the DD.Polypeptides can be introduced into cells by electroporation, injection, or a carrier (e.g., a lipid-based carrier such as liposome), or any other means known in the art for delivering polypeptides to cells.Nucleic acids can be introduced into cells by transfection, electroporation, injection, a carrier, or any other means known in the art for delivering nucleic acids to cells.Viral particles can be introduced into cells by transduction.

[0105] The cells can be contacted with the stabilizing agent, for example, by culturing the cells in a medium containing the stabilizing agent. The cells can be cultured in the medium with the stabilizing agent for a certain period of time, allowing the endonuclease to introduce DSB into the mitochondrial DNA. In some embodiments, the cells are cultured in the medium with the stabilizing agent for at least 8 hours (e.g., at least 12 hours, at least 1 day, at least 2 days, or more) and / or up to 5 days (e.g., up to 4 days, up to 3 days, or up to 2 days). Then, the stabilizing agent can be removed, for example, by culturing the cells in a culture medium that does not contain the stabilizing agent. After the stabilizing agent is removed, the polypeptide is destabilized, which causes the polypeptide to be degraded.

[0106] After removal of the stabilizing agent, the cells can be cultured without the stabilizing agent for a period of time during which the cells can produce new mitochondria. In some embodiments, the cells are cultured in stabilizing agent-free medium for at least 6 hours (e.g., at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days) and / or up to 10 days (e.g., up to 8 days, up to 6 days, or up to 4 days).

[0107] In some embodiments, the methods of the present disclosure result in the induction of mitophagy in cells. In some embodiments, the methods of the present disclosure result in an increase in mitochondrial turnover in cells. In some embodiments, the methods of the present disclosure result in an increase in mitochondrial mass in cells. In some embodiments, the methods result in the induction of DSBs in mitochondrial DNA in cells. In some embodiments, the methods result in epigenetic modifications in cells, for example, induced by mitochondrial depletion. It has previously been reported that rho0 cells, in which the mitochondrial genome is completely depleted, exhibit significant levels of epigenetic changes (see, e.g., Hertzog Santos, 2021 Free Radic Biol Med. 170:69-69). Therefore, it is believed that the compositions of the present disclosure can be used to induce epigenetic modifications. In some embodiments, the methods of the present disclosure result in one, two, three, four, or all five of the following in a cell: (a) induction of mitophagy, (b) increased mitochondrial turnover, (c) increased mitochondrial mass, (d) DSBs in mitochondrial DNA, and (e) epigenomic modifications.

[0108] Exemplary cells that can be used in this method include mammalian cells, preferably human cells, more preferably human somatic cells. Types of cells that can be used include bone marrow cells, stem cells, such as hematopoietic stem cells (HSCs) or mesenchymal stem cells (MSCs), immune cells, such as T cells, phagocytes, microglia, and macrophages. In some embodiments, the cells are T cells, such as CD4+ and / or CD8+ T cells. Primary cells obtained from a subject, as well as their progeny, can be used.

[0109] The cells may be normal (e.g., from a healthy donor) or may have dysfunctional mitochondria (e.g., from a subject with a disease or disorder). For example, the cells may be derived from a subject with an age-related disease or disorder, such as an autoimmune disease, a metabolic disease, a genetic disease, cancer, a neurodegenerative disease, or immunosenescence.

[0110] As another example, the cells may be derived from a subject with a mitochondrial disease or disorder, such as chronic progressive external ophthalmoplegia (CPEO), Pearson syndrome, Kearns-Sayre syndrome (KSS), diabetes and deafness (DAD), mitochondrial diabetes, Leber's hereditary optic neuropathy (LHON), LHON-plus, neuropathy, ataxia, retinitis pigmentosa syndrome (NARP), maternally inherited Leigh syndrome (MILS), mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), myoclonic epilepsy and ragged-red fibrosis (MERRF), familial bilateral striatal necrosis / striatonigral degeneration (FBSN), Luft's disease, aminoglycoside-induced hearing loss (AID), or multiple mitochondrial DNA deletion syndrome. Additional mitochondrial diseases and disorders include mitochondrial DNA depletion syndrome 4A, mitochondrial recessive ataxia syndrome (MIRAS), neurogastrointestinal mitochondrial encephalomyopathy (MNGIE), mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase gamma (POLG)-related disorders, sensory ataxia neuropathy dysarthria ophthalmoplegia (SANDO), leukoencephalopathy with brainstem and spinal cord involvement and elevated lactate (LBSL), coenzyme Q10 deficiency, Leigh syndrome, mitochondrial complex disorders, fumarase deficiency, α-ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenase complex deficiency (PDHC), pyruvate carboxylase deficiency (PCD), carnitine palmitoyltransferase I (CPT I) deficiency, carnitine palmitoyltransferase II (CPT II) deficiency, and coenzyme Q10 deficiency. These include IIT (Integrated Tissue-Regulating Protein) Deficiency, Carnitine Acylcarnitine (CACT) Deficiency, Autosomal Dominant / Autosomal Recessive Progressive External Ophthalmoplegia (ad- / ar-PEO), Infantile-Onset Spinal Cerebellar Atrophy (IOSCA), Mitochondrial Myopathy (MM), Spinal Muscular Atrophy (SMA), Growth Retardation, Amino Aciduria, Cholestasis, Iron Overload, Early Death (GRACILE), and Charcot-Marie-Tooth Disease Type 2A (CMT2A).

[0111] As another example, the cells can be derived from a subject with a neurodegenerative disease, such as amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, Friedreich's ataxia, Charcot-Marie-Tooth disease, or leukodystrophy, etc. In some embodiments, the cells are derived from a subject with Alzheimer's disease, e.g., a subject with an APOE4 allele, e.g., an E3 / E4 or E4 / E4 genotype.

[0112] As yet another example, the cells can be derived from a subject with an ocular disease (eg, a retinal disease), such as age-related macular degeneration, macular edema, or glaucoma.

[0113] In further examples, the cells may be derived from a subject with diabetes, hearing impairment, a genetic disease (such as Hutchinson-Gilford Progeria Syndrome, Werner Syndrome, or Huntington's disease), heart failure, an immune deficiency, cancer, or an infectious disease.

[0114] Cells obtained or obtainable by the methods described herein can be administered to a subject, e.g., the subject from which the cells were derived, or, if derived from a healthy donor, can be administered to a different subject.

[0115] Thus, in another aspect, the present disclosure provides methods of treating a subject having an age-related disease, a mitochondrial disease or disorder, a neurodegenerative disease, a retinal disease, diabetes, hearing impairment, a genetic disease, heart failure, an immune deficiency, cancer, or an infectious disease by administering a therapeutically effective amount of cells obtained or obtainable by the methods described herein. For example, the subject can have a disease or disorder described in this section.

[0116] 4.5. Pharmaceutical Compositions and Kits In another aspect, the present disclosure provides a pharmaceutical composition comprising a polypeptide of the present disclosure (e.g., as described in Section 4.2), a nucleic acid of the present disclosure (e.g., as described in Section 4.3), a particle of the present disclosure (e.g., as described in Section 4.3), or a cell of the present disclosure (e.g., a cell obtained by a method described in Section 4.4) and a pharmaceutically acceptable excipient. For example, pharmaceutical compositions can be prepared by mixing polypeptides, nucleic acids, particles, or cells with one or more physiologically acceptable carriers, excipients, or stabilizers, e.g., in the form of an aqueous solution or suspension (see, e.g., Hardman et al., 2001, Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro, 2000, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Weiner and Kotkoskie, 2000, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0117] In another embodiment, the present disclosure provides a kit comprising a polypeptide of the present disclosure (e.g., as described in Section 4.2), a nucleic acid of the present disclosure (e.g., as described in Section 4.3), or a particle of the present disclosure and a stabilizer. For example, the kit can include trimethoprim (TMP) when the DD sequence of the polypeptide is a DHFR DD sequence, Shield-1, rapamycin, or FK506 when the DD sequence of the polypeptide is an FKBP DD sequence, or sildenafil, vardenafil, tadalafil, avanafil, lodenafil, mirodenafil, udenafil, benzamidenafil, dasantaf[iota]fil, or beminafil when the DD sequence of the polypeptide is a PDE5 DD sequence. [Example]

[0118] 5. Working Example

[0119] [Example 1] 5.1. Example 1: Polypeptides that induce mitophagy, biogenesis, and promotion of mitochondrial turnover This example describes compositions and methods for simultaneously activating mitophagy as well as biogenesis, and for promoting mitochondrial turnover by eliminating dysfunctional mitochondria and generating new mitochondria.

[0120] 5.1.1. Transgene Design XbaIR was selected to induce DSBs in the mitochondrial genome. XbaIR has five cleavage sites in the mitochondrial genome consensus sequence. To target the XbaIR endonuclease to mitochondria, a Cox8a postmitochondrial signal was placed at the N-terminus of XbaIR. Induction of DSBs in the mitochondrial genome causes a strong, transient energy depletion. To control endonuclease activity, a DHFR destabilization domain (Liu et al., 2014 Int. J. Parasitol. 44(10):729-735) was fused to the C-terminus of XbaIR, enabling sensitive on / off control of the endonuclease. The DHFR destabilization domain is stabilized by the antibiotic trimethoprim (TMP).

[0121] A retroviral vector carrying the MTS-XbaIR-DHFR coding sequence was constructed (Figure 1). The nucleotide sequence of the vector is shown in Table 3.

[0122] [Table 3-1]

[0123] [Table 3-2]

[0124] [Table 3-3]

[0125] Using molecular simulations, we confirmed that the three functional domains of the polypeptide construct (MTS, XbaI, and DHFR) adopt a three-dimensional structure without interfering with each other's structure (Figure 2). Furthermore, to estimate the responsiveness of the DHFR / TMP system, we constructed a retroviral vector carrying EGFP-DHFR as a transgene (Figures 3 and 4). The nucleotide sequence of the vector is shown in Table 4.

[0126] [Table 4-1]

[0127] [Table 4-2]

[0128] [Table 4-3]

[0129] [Table 4-4]

[0130] 5.1.2. Response of the DHFR / TMP system HeLa cells were transduced with the EGFP-DHFR retroviral vector. High infection efficiency was achieved by exposing infected cells to various concentrations of TMP for 2 days without concentrating them. Subsequently, fluorescent expression of EGFP was observed using a fluorescent microscope and FACS (Figures 5 and 6). The presence of transcribed EGFP mRNA was observed (Figure 7), but no protein was observed in the absence of TMP (Figures 5 and 6).

[0131] To confirm the OFF-regulation of the construct, the medium was washed after 2 days of exposure to TMP, and the fluorescence intensity over time was examined by fluorescence microscopy and FACS (Figure 8A). One hour after removing TMP from the culture medium, EGFP fluorescence rapidly decreased, and after 4 hours, the fluorescence disappeared (Figure 8B and Figure 8C). Furthermore, no leakage of transgene expression was observed 48 hours after TMP OFF (Figure 8D).

[0132] To confirm the ON control of the construct, TMP was added, and the fluorescence intensity over time was confirmed by fluorescence microscopy and FACS (Figure 9A). After adding TMP to the culture medium, EGFP fluorescence rapidly turned on within 1 hour, and after 6 hours, the fluorescence reached over 80% of its maximum intensity (Figures 9B and 9C). After 8 hours, the intensity remained stable until 48 hours (Figure 9D).

[0133] 5.1.3. Characteristics of mitochondria after genetically induced mitophagy Hela cells were transfected with the MTS-XbaIR-DHFR vector and exposed to 0.5 μM TMP for 2 days, followed by washout. XbaRI RNA expression was measured after TMP exposure. The transgene transcription level did not change significantly (Fig. 10A). By day 2, CN had decreased to less than half of its initial value (Fig. 10B).

[0134] Hela transfectants containing MTS-XbaIR-DHFR were cloned by limiting dilution and designated Hela-MXD sc20. Hela-MXD sc20 was exposed to 0.5 μM TMP for several periods (16, 20, and 48 hours) to measure mitochondrial mass (mtMass) measured by MitoGreen staining, total mitochondrial membrane potential (mtMP) measured by TMRM staining, and mtMP corrected for mtMass, calculated as the ratio of mtMP to mtMass (Figures 11A-11C). Regardless of the duration of TMP exposure, mtMass transiently increased approximately twofold compared to the resting state and then returned to its initial value 10 hours after TMP ON, suggesting a transient and potent activation of mitochondrial biogenesis (Figure 11A). Both total and corrected mtMP showed a rapid decrease followed by an increase, indicating insufficient hydrogen ion pumping by either respiratory chain complexes I, III, and IV or counterclockwise rotated complex V (Figures 6B and 6C).

[0135] The introduced transgene appears to induce DSBs in the mitochondrial genome, reduce CN, and increase MM, despite the need for nuclear proteins. Based on these two factors, the density of respiratory chain complexes is thought to decrease, leading to a decrease in MMP, as well as a phenotype. The mitochondrial genome returned to normal by day 6, and MM appeared to increase slightly. Without being bound by theory, this suggests that mitochondrial biogenesis was enhanced in the MTS-XbaIR-DHFR / TMP system, resulting in an increase in mitochondrial capacity, as measured by mitochondrial abundance.

[0136] Next, we investigated whether this system would alter cell proliferation and viability, and whether it would affect the changes described above. No significant differences in cell number or viability were observed in this system, regardless of the presence or absence of TMP (Figures 12A-12B). Therefore, the main advantage of this system was identified as the intervention of the MTS-XbaIR-DHFR polypeptide into mitochondria.

[0137] 5.1.4. Effect on mitophagy after MTS-XbaIR-DHFR / TMP To quantify mitophagy in a more refined manner, we generated mtKeima-Red-transfected Hela MXD sc20 cells with PARK2 overexpression. To measure mitophagy, we examined TMP exposure in transfectants for various periods (16, 20, 24, 40, 44, and 48 hours). The mitophagy inducer carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as a positive control. As TMP exposure increased, more mitophagy was observed (Figure 13A). Mitophagy reached a plateau at 40 hours of TMP exposure (Figure 13A). At the same time, the measured mtDNA CN was observed to return to its initial value regardless of the duration of TMP exposure (Figure 13B). Overall, the results indicate that mitochondrial turnover was promoted by MTS-XbaIR-DHFR / TMP.

[0138] [Example 2] 5.2. Example 2: Altered mitochondrial function and biogenesis due to genetically induced mitophagy This example describes the changes in mitochondrial biogenesis and function associated with genetically induced mitophagy (GiM).

[0139] 5.2.1 Materials and Methods After gene transfer, we used retroviruses to clone MTS-XbaI-ecDHFR into Hela cells, generating stable transfectants constitutively expressing the GiM unit (Hela_GiM). The endonuclease, XbaI, was transiently present within the mitochondrial matrix during treatment of Hela_GiM cells with trimethoprim (TMP) for 2 days. Because TMP is dissolved in DMSO, the control group also received the same amount of DMSO.

[0140] Reactive oxygen species (ROS) over time were assessed by staining cells with mitoSox and measuring the fluorescence intensity by FACS. A threshold was set compared to unstressed cells, and the percentage of positive cells was measured. Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α), which is involved in mitochondrial biogenesis; nuclear factor receptor 1 (NRF1), which is involved in mitochondrial biogenesis; and mitochondrial transcription factor A (TFAM), which forms nucleoids with mtDNA and is deeply involved in mtDNA transcription, replication, and maintenance, were assessed by qPCR. The expression levels of several nuclear-encoded and mtDNA-encoded proteins were quantified by Western blotting.

[0141] 5.2.2.Results Initially, ROS levels increased in the TMP-treated group but remained essentially unchanged in the control group throughout the evaluation. By day 2, the percentage of mitoSox-positive cells in the TMP-treated group was higher than in the control group. From day 4 onward, the percentage of mitoSox-positive cells was comparable in the two groups (Figure 14A). We also found that the mitochondrial biogenesis-promoting transcription factors PGC1α and NRF1 were elevated in the TMP-treated group compared with the control group at all observation time points (Figures 14B and 14C), and TFAM was largely maintained in the TMP-treated group on days 2, 4, and 6 (Figure 14D).

[0142] Without being bound by theory, these results suggest that ROS production is temporarily enhanced by transient mitochondrial genome reduction, which then triggers mitochondrial biogenesis through the amplification of transcription factors such as PGC1α and NRF1. Furthermore, without being bound by theory, the maintenance of high transcription levels of TFAM, a major component of nucleoids, suggests that mitochondrial biogenesis continues for some time after mitochondrial genome reduction has been triggered.

[0143] Next, we evaluated the effect of GiM on the expression of nuclear- and mtDNA-encoded proteins. The expression level of ATP5A, a nuclear-encoded respiratory chain complex, was not substantially altered by mitochondrial genome reduction (Figures 15A and 15B). Expression of the nuclear-encoded outer membrane translocase (TOM20) also decreased immediately after mitochondrial genome reduction but returned to levels comparable to those observed in the DMSO-treated group by day 6 (Figures 15A and 15C). On the other hand, mtDNA-encoded ATP6 significantly decreased in the TMP-treated group immediately after mitochondrial genome reduction, then increased by day 4 and subsequently returned to levels comparable to those in the control group (Figures 15A and 15D). This trend was also observed in the ATP6 / ATP5A ratio (Figures 15A and 15E). These results indicate that GiM induces a transient surge in mitochondrial genome-derived mitochondrial proteins, but not nuclear genome-derived mitochondrial proteins.

[0144] Changes in the levels of p-AMPK, the activated form of AMPK, were used to estimate changes in mitochondrial energy production after mitochondrial genome reduction. Mitochondrial energy production was confirmed by an increase in p-AMPK on day 4 (Figures 15A and 15F). Transient mitochondrial genome reduction was associated with a decrease in the capacity of respiratory chain complexes on day 2, an effect directly related to cellular energy depletion, which led to a significant increase in p-AMPK on day 4.

[0145] [Example 3] 5.3. Example 3: Autophagy Associated with Transient Mitochondrial Genome Reduction by GiM Induction Mitophagy occurs when mitochondria are incorporated into phagophores and fuse with lysosomes to form autolysosomes. This example describes compositions and methods for detecting autolysosome formation and autophagy after transient mitochondrial genome reduction by GiM induction.

[0146] Materials and Methods Autolysosomes have a lower pH than mitochondria that have not fused with lysosomes. Autolysosome formation was assessed in cells after GiM-induced transient mitochondrial genome reduction using the pH-sensitive mitochondrial reporter mtKeimaRed. mtKeimaRed emits fluorescence with a peak at 440 nm (green) at pH > 6 and a peak at 620 nm (red) at pH < 5, providing a transport signal that enables its transport into mitochondria. Cells stably expressing mKeimaRed emit red light when the environmental pH is below 5. To quantify autolysosomes in GiM-induced transient mitochondrial genome reduction, Hela_GiM cells described in Section 5.2.1 were retrovirally engineered with a sequence encoding mKeimaRed. The percentage of cells undergoing mitophagy was quantified every two days over a two-week period using fluorescence and phase-contrast microscopy.

[0147] The final step of autophagic flux depends on lysosomal V-ATPase activity. Therefore, to assess mitochondria-targeted autophagic flux, we suppressed the final step of flux using bafilomycin A1 (BafA1), a lysosomal V-ATPase inhibitor, after transient mitochondrial genome reduction induced by GiM. We quantified autophagosomes using antibody staining for LC3 (MAP1LC3: microtubule-associated protein 1 light chain 3), a representative marker for autophagosome formation, along with TOM20 staining as a mitochondrial membrane marker. Assuming that LC3 exists on the autophagosome membrane as LC3-II with PE, LC3-II was quantified by Western blotting in Hela_GiM cells treated with BafA1. In this case, proteins were extracted from cells on day 8 after 48 hours of exposure to TMP.

[0148] 5.3.2.Results The peak of mtKeimaRed signal, associated with GiM-induced autolysosome formation, occurred on day 8 (Figure 16A), when mitophagy occurred in approximately 20% of cells (Figure 16B). Time course analysis suggested that mitophagy kinetics increased on day 6 and decreased to control levels by day 14, when only a small percentage of cells underwent mitophagy (Figure 16B). This transient increase in induced mitophagy was not associated with cell death or reduced viability. Without being bound by theory, these results suggest that the adverse effects of excessive mitophagy, such as mitophagy-induced cell death, can be suppressed by controlling GiM.

[0149] The autophagosomes formed during the fusion of mitochondria and lysosomes were detected as overlapping spots of LC3 and TOM20 staining (Fig. 17A). In the absence of TMP, the size of the overlapping spots of LC3 and TOM20 staining was approximately 80 μm.2 On the other hand, in the TMP-exposed group, the area of ​​the overlapping spots of LC3 staining and TOM20 staining increased by approximately 120 μm with the addition of BafA1. 2 LC3-II was significantly increased by GiM, and this increase was more pronounced and significant when BafA1 was used (Figures 18A and 18B). Taken together, these results suggest that GiM significantly promotes autophagy.

[0150] [Example 4] 5.4. Example 4: Metabolic Effects of Mitochondrial Genome Reduction Mitochondrial genome reduction was performed using Hela_GiM cells as described in Section 5.2.1 after 2 days of TMP exposure. Time-course respiration measurements were performed using SeaHorse to estimate oxidative phosphorylation (OXPHOS) and glycolysis. OXPHOS (Figure 19A, left panel) decreased until day 4, gradually increased from day 6, and approached the starting level by day 10. Meanwhile, glycolysis increased until day 8 and then decreased by day 10 (Figure 19A, right panel). A two-dimensional plot depicting the relationship between OXPHOS and glycolysis showed a circular change, indicating that the metabolic changes were transient (Figure 19B). Separate assessments of ATP production, basal respiration, proton leak, and spare respiratory capacity further supported the transient nature of GiM-associated metabolic changes (Figure 19C). The metabolic effects of GiM, including oxygen consumption, also indicate that the temporary mitochondrial genome reduction is a reversible change.

[0151] [Example 5] 5.5. Example 5: Genetically Induced Mitophagy in AD Fibroblasts Alzheimer's disease (AD) is associated with mitochondrial dysfunction. This example describes how transient mitochondrial genome reduction by gene transfer can transform cellular phenotype by enhancing mitochondrial turnover in fibroblasts derived from patients with AD.

[0152] 5.5.1 Materials and Methods Normal human dermal fibroblasts (NHDFs) and fibroblasts (AD fibroblasts) collected from forearm skin samples of patients with Alzheimer's disease (APOE E3 / E45 genotype) were used as target cells. Transient mitochondrial genome reduction was performed by introducing a plasmid carrying the gene encoding the endonuclease XbaIR downstream of the human Cox8-derived mitochondrial transport signal and expressing puromycin resistance as a selectable marker under a different promoter (pCAGGS-MTS-XbaIR). Electroporation was used as the gene transfer method, and transfected target cells were enriched by exposing them to 3 μg / mL puromycin for 24 hours two days after electroporation. The conditions were set using a plasmid carrying a recombinant GFP gene in place of XbaIR, with the criteria of 70-80% GFP expression and >90% viability. After gene transfer, mitochondrial genome copy number was assessed at days 7, 14, and 21 to confirm genome reduction and subsequent biogenesis (Figure 20A). Furthermore, mitochondrial phenotype was estimated by measuring mitochondrial mass (mtMass) and mitochondrial membrane potential (mtMP) using MitoTracker Green and TMRM, respectively.

[0153] 5.5.2.Results In untreated AD fibroblasts, mtMP was significantly reduced compared to untreated NHDF (Figures 20B and 20E). Similarly, mtMass was reduced in untreated AD fibroblasts compared to untreated NHDF (Figures 20B and 20D). Quadrant analysis was performed to plot the two fluorescent signals in two dimensions and set the threshold line using NHDF as a positive control. The fraction ratio was used as a biomarker of senescence in lymphocytes to detect mitochondrial dysfunction. The double-positive rate was found to be approximately half that of NHDF. After gene-induced mitophagy (GiM), the double-positive rate did not change significantly on day 7, but increased to 60.3% on day 14 and further increased to 87.3% on day 21, becoming comparable to that of control NHDF (Figure 20C).

[0154] Both mtMass and mtMP were quantified using mean fluorescence intensity (MFI) as an index of fluorescence intensity. Both mtMass and mtMP were lower in untreated AD fibroblasts compared to NHDF cells. In AD fibroblasts, mtMass and mtMP levels further decreased on day 7 after GIM induction, but increased to levels comparable to those in NHDF on day 14 (Figures 20F-20G), suggesting that GiM restored both mitochondrial mass and mitochondrial membrane potential in AD fibroblasts to the same levels as in healthy NHDF cells.

[0155] These results indicate that GiM-mediated promotion of mitochondrial turnover is qualitatively favorable for newly generated mitochondria. Given that the accumulation of dysfunctional mitochondria is a common phenomenon in various neurodegenerative diseases and aging, GiM can be used as a therapeutic strategy for treating neurodegenerative diseases and restoring normal function in aging cells.

[0156] 6. Specific Embodiments The present disclosure is illustrated by the following specific embodiments. Embodiment 1 (a) mitochondrial targeting sequence (MTS) and; (b) endonuclease sequence and; (c) Destabilization domain sequence and A polypeptide comprising: Embodiment 2 2. The polypeptide of embodiment 1, wherein the MTS comprises human MTS. Embodiment 3 2. The polypeptide of embodiment 1, wherein the MTS comprises a non-human MTS. Embodiment 4 4. The polypeptide according to any one of embodiments 1 to 3, wherein the MTS comprises a mitochondrial protein MTS. Embodiment 5 5. The polypeptide of any one of embodiments 1 to 4, wherein the MTS comprises an MTS of a TCA cycle-related enzyme, a chaperone protein, a mitochondrial genome replication protein, a protease, an mRNA processing protein, a mitochondrial RNA degradation protein, a deoxynucleotide triphosphate synthesis-related protein, a mitoribosomal protein, a phospholipid metabolism-related protein, a protein involved in the metabolism of toxic compounds, a disulfide relay system-related protein, an iron-sulfur protein assembly protein, a tRNA-modifying protein, an aminoacyl-tRNA synthetase, a release factor, or an elongation factor. Embodiment 6 6. The polypeptide according to any one of embodiments 1 to 5, wherein the MTS comprises an MTS of a cytochrome c oxidase subunit. Embodiment 7 7. The polypeptide of embodiment 6, wherein the MTS comprises an MTS of cytochrome c oxidase subunit VIII (COX8). Embodiment 8 7. The polypeptide of embodiment 6, wherein the MTS comprises an MTS of cytochrome c oxidase subunit X (COX10). Embodiment 9 7. The polypeptide of embodiment 6, wherein the MTS comprises an MTS of cytochrome c oxidase subunit IV (COX4). Embodiment 10 6. The polypeptide according to any one of embodiments 1 to 5, wherein the MTS comprises the MTS of the frataxin (FXN) protein. Embodiment 11 6. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises an MTS for a TCA cycle-related enzyme that is, optionally, pyruvate dehydrogenase, citrate synthase, aconitase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, malate dehydrogenase, or pyruvate carboxylase. Embodiment 12 6. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises an MTS of a chaperone protein, optionally mtHSP10, mtHSP60, mtHSP70, or mtHSP90. Embodiment 13 6. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises an MTS of a mitochondrial genome replication protein, which is optionally TFAM, Twinkle, PolG, TFB2M, TEFM, or MTERF1. Embodiment 14 6. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises an MTS of a protease, which is optionally MPP, CLPXP, LON ATPase, or PreP. Embodiment 15 6. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises an MTS for an mRNA processing protein, which MTS is optionally LRPPRC, TACO1, ELAC2, PNPT1, HSD17B10, MTPAP, or PTCD1. Embodiment 16 6. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises an MTS for a mitochondrial RNA degradation protein, optionally PNPasse, REX02, or SUV3. Embodiment 17 6. The polypeptide of any one of embodiments 1 to 5, comprising an MTS for a deoxynucleotide triphosphate synthesis-related protein, where the MTS is optionally DGUOK, TK2, TYMP, MGME1, SUCLG1, SUCLA2, RNASEH1, or C10orf2. Embodiment 18 6. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises an MTS of a mitoribosomal protein, which is optionally MRPS16, MRPS22, MRPL3, MRP12, or MRPL44. Embodiment 19 6. The polypeptide of any one of embodiments 1 to 5, comprising an MTS of a phospholipid metabolism-related protein, wherein the MTS is, optionally, AGK, SERAC1, or TAZ. Embodiment 20 6. The polypeptide of any one of embodiments 1 to 5, comprising an MTS of a protein involved in the metabolism of toxic compounds, wherein the MTS is optionally HIBCH, ECHS1, ETHE1, or MPV17. Embodiment 21 6. The polypeptide of any one of embodiments 1 to 5, comprising an MTS of a disulfide relay system-associated protein, optionally GFER. Embodiment 22 6. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises an MTS of an iron-sulfur protein assembly protein, optionally ISCU, BOLA3, NFU1, or IBA57. Embodiment 23 6. The polypeptide of any one of embodiments 1 to 5, comprising an MTS for a tRNA modifying protein, wherein the MTS is optionally MTO1, GTP3BP, TRMU, PUS1, MTFMT, TRIT1, TRNT1, or TRMT5. Embodiment 24 6. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises an MTS of an aminoacyl-tRNA synthetase that is, optionally, AARS2, DARS2, EARS2, RARS2, YARS2, FARS2, HARS2, LARS2, VARS2, TARS2, IARS2, CARS2, PARS2, NARS2, KARS, GARS, SARS2, or MARS2. Embodiment 25 6. The polypeptide of any one of embodiments 1 to 5, comprising an MTS of a release factor, wherein the MTS is optionally C12orf65. Embodiment 26 6. The polypeptide of any one of embodiments 1 to 5, comprising an MTS of an elongation factor, wherein the MTS is optionally TUFM, TSFM, or GFM1. Embodiment 27 2. The polypeptide of embodiment 1, wherein the MTS comprises a sequence that is at least 80% identical to MSVLTPLLLRGLTGSARR (SEQ ID NO: 1). Embodiment 28 28. The polypeptide of embodiment 27, wherein the MTS comprises a sequence that is at least 85% identical to MSVLTPLLLRGLTGSARR (SEQ ID NO: 1). Embodiment 29 28. The polypeptide of embodiment 27, wherein the MTS comprises a sequence that is at least 90% identical to MSVLTPLLLRGLTGSARR (SEQ ID NO: 1). Embodiment 30 28. The polypeptide of embodiment 27, wherein the MTS comprises a sequence that is at least 95% identical to MSVLTPLLLRGLTGSARR (SEQ ID NO: 1). Embodiment 31 28. The polypeptide of embodiment 27, wherein the MTS comprises a sequence that is 100% identical to MSVLTPLLLRGLTGSARR (SEQ ID NO: 1). Embodiment 32 2. The polypeptide of embodiment 1, wherein the MTS comprises a sequence that is at least 80% identical to MSVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO: 2). Embodiment 33 33. The polypeptide of embodiment 32, wherein the MTS comprises a sequence that is at least 85% identical to MSVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO: 2). Embodiment 34 33. The polypeptide of embodiment 32, wherein the MTS comprises a sequence that is at least 90% identical to MSVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO: 2). Embodiment 35 33. The polypeptide of embodiment 32, wherein the MTS comprises a sequence that is at least 95% identical to MSVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO: 2). Embodiment 36 33. The polypeptide of embodiment 32, wherein the MTS comprises a sequence that is 100% identical to MSVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO: 2). Embodiment 37 2. The polypeptide of embodiment 1, wherein the MTS comprises a sequence that is at least 80% identical to MSVLTPLLLRSLTGSARRLMVPRA (SEQ ID NO: 3). Embodiment 38 38. The polypeptide of embodiment 37, wherein the MTS comprises a sequence that is at least 85% identical to MSVLTPLLLRSLTGSARRLMVPRA (SEQ ID NO: 3). Embodiment 39 38. The polypeptide of embodiment 37, wherein the MTS comprises a sequence that is at least 90% identical to MSVLTPLLLRSLTGSARRLMVPRA (SEQ ID NO: 3). Embodiment 40 38. The polypeptide of embodiment 37, wherein the MTS comprises a sequence that is at least 95% identical to MSVLTPLLLRSLTGSARRLMVPRA (SEQ ID NO: 3). Embodiment 41 38. The polypeptide of embodiment 37, wherein the MTS comprises a sequence that is 100% identical to MSVLTPLLLRSLTGSARRLMVPRA (SEQ ID NO: 3). Embodiment 42 2. The polypeptide of embodiment 1, wherein the MTS comprises a sequence that is at least 80% identical to MAASPHTLSSRLLTGCVGGSVWYLERRT (SEQ ID NO: 4). Embodiment 43 43. The polypeptide of embodiment 42, wherein the MTS comprises a sequence that is at least 85% identical to MAASPHTLSSRLLTGCVGGSVWYLERRT (SEQ ID NO: 4). Embodiment 44 43. The polypeptide of embodiment 42, wherein the MTS comprises a sequence that is at least 90% identical to MAASPHTLSSRLLTGCVGGSVWYLERRT (SEQ ID NO: 4). Embodiment 45 43. The polypeptide of embodiment 42, wherein the MTS comprises a sequence that is at least 95% identical to MAASPHTLSSRLLTGCVGGSVWYLERRT (SEQ ID NO: 4). Embodiment 46 43. The polypeptide of embodiment 42, wherein the MTS comprises a sequence that is 100% identical to MAASPHTLSSRLLTGCVGGSVWYLERRT (SEQ ID NO: 4). Embodiment 47 2. The polypeptide of embodiment 1, wherein the MTS comprises a sequence that is at least 80% identical to MWTLGRRAVAGLLASPSPAQ (SEQ ID NO: 5). Embodiment 48 48. The polypeptide of embodiment 47, wherein the MTS comprises a sequence that is at least 85% identical to MWTLGRRAVAGLLASPSPAQ (SEQ ID NO: 5). Embodiment 49 48. The polypeptide of embodiment 47, wherein the MTS comprises a sequence that is at least 90% identical to MWTLGRRAVAGLLASPSPAQ (SEQ ID NO: 5). Embodiment 50 48. The polypeptide of embodiment 47, wherein the MTS comprises a sequence that is at least 95% identical to MWTLGRRAVAGLLASPSPAQ (SEQ ID NO: 5). Embodiment 51 48. The polypeptide of embodiment 47, wherein the MTS comprises a sequence that is 100% identical to MWTLGRRAVAGLLASPSPAQ (SEQ ID NO: 5). Embodiment 52 2. The polypeptide of embodiment 1, wherein the MTS comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to MAPYSLLVTRLQKALG (SEQ ID NO: 6). Embodiment 53 2. The polypeptide of embodiment 1, wherein the MTS comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to MALLTAAARLLGTKNASCLVLAARHASA (SEQ ID NO: 7). EMBODIMENT 54 2. The polypeptide of embodiment 1, wherein the MTS comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to MVKQIESKTAFQEALDAAGDKLVVVDFSATWC (SEQ ID NO: 8). Embodiment 55 2. The polypeptide of embodiment 1, wherein the MTS comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to MATNWGSLLQDKQQLEELARQAVDRALAEGVLLRTSQ (SEQ ID NO: 9). Embodiment 56 2. The polypeptide of embodiment 1, wherein the MTS comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to MAFLRSMWGVLSALGRSGA (SEQ ID NO: 10). Embodiment 57 2. The polypeptide of embodiment 1, wherein the MTS comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to MWVLLRSGYPLRILLPLRG (SEQ ID NO: 11). Embodiment 58 2. The polypeptide of embodiment 1, wherein the MTS comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to MSRLLWRKVAGATVGPGPVPAPG (SEQ ID NO: 12). Embodiment 59 2. The polypeptide of embodiment 1, wherein the MTS comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to MKRNTLVELLTFWKNWHFRLL (SEQ ID NO: 13). Embodiment 60 2. The polypeptide of embodiment 1, wherein the MTS comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to MISASRAAAARLVGAAASRGPTAA (SEQ ID NO: 14). Embodiment 61 2. The polypeptide of embodiment 1, wherein the MTS comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to MEALIPVINKLQDVFNTVGA (SEQ ID NO: 15). Embodiment 62 62. The polypeptide of any one of embodiments 1 to 61, wherein the endonuclease is a restriction endonuclease, an RNA-guided endonuclease (e.g., Cas9 or Cas12), a zinc finger nuclease, or a transcription activator-like effector nuclease (TALEN). Embodiment 63 63. The polypeptide of embodiment 62, wherein the endonuclease is a restriction endonuclease. EMBODIMENT 64 64. The polypeptide of embodiment 63, wherein the restriction endonuclease is XbaIR, EcoRI, SmaI, AflII, BamHI, BclI, HaeIII, HindII, HindIII, NdeI, PvuII, PstI, or SpeI endonuclease. Embodiment 65 65. The polypeptide of embodiment 64, wherein the restriction endonuclease is XbaIR endonuclease. Embodiment 66 The endonuclease is

[0157] [ka] 64. The polypeptide of any one of embodiments 1 to 63, comprising a sequence that is at least 80% identical to Embodiment 67 The endonuclease sequence is

[0158] [ka] 67. The polypeptide of embodiment 66, comprising a sequence that is at least 85% identical to Embodiment 68 The endonuclease sequence is

[0159] [ka] 67. The polypeptide of embodiment 66, comprising a sequence that is at least 90% identical to Embodiment 69 The endonuclease sequence is

[0160] [ka] 67. The polypeptide of embodiment 66, comprising a sequence that is at least 95% identical to Embodiment 70 The endonuclease sequence is

[0161] [ka] 67. The polypeptide of embodiment 66, comprising a sequence that is 100% identical to Embodiment 71 71. The polypeptide of any one of embodiments 1 to 70, wherein the destabilization domain sequence is a DHFR, FKBP, or PDE5 destabilization domain sequence. Embodiment 72 72. The polypeptide of embodiment 71, wherein the destabilization domain sequence is a DHFR destabilization domain sequence. Embodiment 73 73. The polypeptide of embodiment 72, wherein the destabilization domain sequence is an Escherichia coli (E. coli) DHFR (ecDHFR) destabilization domain sequence. EMBODIMENT 74 The destabilization domain sequence is

[0162] [ka] 74. The polypeptide of any one of embodiments 1 to 73, comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to Embodiment 75 The destabilization domain sequence is

[0163] [ka] 74. The polypeptide of any one of embodiments 1 to 73, comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to Embodiment 76 75. The polypeptide of embodiment 74, wherein the destabilization domain sequence has one or more of the following amino acid substitutions: N18T / A19V, F103L, Y100I, G121V, H12Y / Y100I, H12L / Y100I, R98H / F103S, M42T / H114R, and I61F / T68S. Embodiment 77 The destabilization domain sequence is GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 20) 72. The polypeptide of any one of embodiments 1 to 71, comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to Embodiment 78 78. The polypeptide of embodiment 77, wherein the destabilization domain sequence has one or more of the following amino acid substitutions: F15S, V24A, H25R, E60G, L106P, D100G, M66T, R71G, D100N, E102G, and K105I. Embodiment 79 72. The polypeptide of any one of embodiments 1 to 71, wherein the destabilization domain sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOs: 19-35 and 66-69 of WO 2018 / 237323. Embodiment 80 80. The polypeptide of any one of embodiments 1-79, wherein the MTS is positioned N-terminal to the endonuclease sequence and the destabilization domain sequence. Embodiment 81 80. The polypeptide of any one of embodiments 1-79, wherein the MTS is positioned C-terminal to the endonuclease sequence and the destabilization domain sequence. Embodiment 82 82. The polypeptide of any one of embodiments 1 to 81, wherein the endonuclease sequence is positioned N-terminal to the destabilization domain sequence. Embodiment 83 82. The polypeptide of any one of embodiments 1 to 81, wherein the endonuclease sequence is positioned C-terminal to the destabilization domain sequence. Embodiment 84 80. The polypeptide of any one of embodiments 1 to 79, wherein the MTS is positioned N-terminal to the endonuclease sequence, and the endonuclease sequence is positioned N-terminal to the destabilization domain sequence. Embodiment 85 A nucleic acid encoding the polypeptide of any one of embodiments 1 to 84. Embodiment 86 86. The nucleic acid of embodiment 85, comprising a promoter operably linked to a nucleotide sequence encoding a polypeptide. Embodiment 87 87. The nucleic acid of embodiment 86, wherein the promoter is an SV40 promoter, a CMV promoter, or a CAG promoter. Embodiment 88 88. The nucleic acid of embodiment 87, wherein the promoter is an SV40 promoter. Embodiment 89 89. The nucleic acid of embodiment 88, wherein the SV40 promoter comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:25. Embodiment 90 90. The nucleic acid of any one of embodiments 85 to 89, which is a vector. Embodiment 91 91. The nucleic acid of embodiment 90, wherein the vector is a retroviral genome. Embodiment 92 92. The nucleic acid of embodiment 91, wherein the vector is a murine retroviral genome. Embodiment 93 91. The nucleic acid of embodiment 90, wherein the vector is a plasmid. Embodiment 94 86. The nucleic acid of embodiment 85, which is an mRNA molecule. Embodiment 95 95. The nucleic acid-containing particle of any one of embodiments 85 to 94, wherein the particle is a retroviral particle. Embodiment 96 A host cell comprising a nucleic acid according to any one of embodiments 85 to 94. Embodiment 97 1. A method for (a) inducing mitophagy in a cell, and / or (b) increasing mitochondrial turnover in a cell, and / or (c) increasing mitochondrial mass, and / or (d) inducing double-strand breaks in mitochondrial DNA, and / or (e) inducing epigenetic modifications in a cell, the method comprising contacting the cell with (i) a polypeptide described in any one of embodiments 1 to 84, a nucleic acid described in any one of embodiments 85 to 94, or a particle described in embodiment 95, and (ii) a stabilizing agent. Embodiment 98 98. The method of embodiment 97, wherein when the destabilization domain sequence is a DHFR destabilization domain sequence, the stabilizer is trimethoprim (TMP). Embodiment 99 98. The method of embodiment 97, wherein when the destabilization domain sequence is an FKBP destabilization domain sequence, the stabilizer is Shield-1, rapamycin, or FK506. Embodiment 100 100. The method of any one of embodiments 97 to 99, wherein contacting the cells with the stabilizing agent comprises culturing the cells in a medium comprising the stabilizing agent. Embodiment 101 101. The method of embodiment 100, comprising culturing the cells in a medium containing the stabilizing agent for at least 8 hours. Embodiment 102 101. The method of embodiment 100, comprising culturing the cells in a medium containing the stabilizing agent for at least 12 hours. Embodiment 103 101. The method of embodiment 100, comprising culturing the cells in a medium comprising a stabilizing agent for at least 1 day. Embodiment 104 101. The method of embodiment 100, comprising culturing the cells in a medium containing a stabilizing agent for at least 2 days. Embodiment 105 105. The method of any one of embodiments 100 to 104, comprising culturing the cells in a medium containing a stabilizing agent for up to 5 days. Embodiment 106 105. The method of any one of embodiments 100 to 104, comprising culturing the cells in a medium containing a stabilizing agent for up to 4 days. Embodiment 107 105. The method of any one of embodiments 100 to 104, comprising culturing the cells in a medium containing a stabilizing agent for up to 3 days. Embodiment 108 105. The method of any one of embodiments 100 to 104, comprising culturing the cells in a medium containing a stabilizing agent for up to 2 days. Embodiment 109 109. The method of any one of embodiments 97-108, further comprising the step of removing the stabilizing agent from the cells after contacting the cells with the stabilizing agent. Embodiment 110 110. The method of embodiment 109, wherein removing the stabilizing agent from the cells comprises culturing the cells in medium that does not contain the stabilizing agent. Embodiment 111 111. The method of embodiment 110, comprising culturing the cells in a medium that does not contain a stabilizing agent for at least 3 hours. Embodiment 112 111. The method of embodiment 110, comprising culturing the cells in a medium that does not contain a stabilizing agent for at least 6 hours. Embodiment 113 111. The method of embodiment 110, comprising culturing the cells in a medium that does not contain a stabilizing agent for at least 12 hours. Embodiment 114 111. The method of embodiment 110, comprising culturing the cells in a medium that does not contain a stabilizing agent for at least 1 day. Embodiment 115 111. The method of embodiment 110, comprising culturing the cells in a stabilizing agent-free medium for at least 2 days. Embodiment 116 111. The method of embodiment 110, comprising culturing the cells in a stabilizing agent-free medium for at least 3 days. Embodiment 117 111. The method of embodiment 110, comprising culturing the cells in a stabilizing agent-free medium for at least 4 days. Embodiment 118 118. The method of any one of embodiments 110 to 117, comprising culturing the cells in a stabilizing agent-free medium for up to 10 days. Embodiment 119 118. The method of any one of embodiments 100 to 117, comprising culturing the cells in a stabilizing agent-free medium for up to 8 days. Embodiment 120 118. The method of any one of embodiments 100 to 117, comprising culturing the cells in a stabilizing agent-free medium for up to 6 days. Embodiment 121 118. The method of any one of embodiments 100 to 117, comprising culturing the cells in a stabilizing agent-free medium for up to 4 days. Embodiment 122 The method of any one of embodiments 97 to 121, comprising contacting a cell with a polypeptide of any one of embodiments 1 to 84. Embodiment 123 123. The method of embodiment 122, wherein the contacting step comprises introducing the polypeptide into the cell by electroporation, injection, or a carrier. Embodiment 124 The method of any one of embodiments 97 to 121, comprising contacting a cell with a nucleic acid of any one of embodiments 85 to 94. Embodiment 125 125. The method of embodiment 124, wherein the contacting step comprises introducing the nucleic acid into the cell by transfection, electroporation, injection, or a carrier. Embodiment 126 126. The method of embodiment 124 or embodiment 125, wherein the polypeptide is transiently expressed in the cell. Embodiment 127 127. The method of any one of embodiments 97-126, further comprising administering the cells to a subject. Embodiment 128 The method of any one of embodiments 97 to 127, wherein mitophagy is induced in the cell. Embodiment 129 129. The method of any one of embodiments 97-128, wherein the method increases mitochondrial turnover in the cell. Embodiment 130 130. The method of any one of embodiments 97-129, wherein the mitochondrial mass is increased. Embodiment 131 The method of any one of embodiments 97 to 130, wherein double-strand breaks are induced in mitochondrial DNA. Embodiment 132 The method of any one of embodiments 97 to 131, wherein epigenetic modifications are induced in cells. Embodiment 133 A cell obtained or obtainable by a method according to any one of embodiments 97 to 132. Embodiment 134 A cell comprising a polypeptide according to any one of embodiments 1 to 84, a nucleic acid according to any one of embodiments 85 to 94, or a particle according to embodiment 95. Embodiment 135 135. The cell of embodiment 134, further comprising a stabilizing agent. Embodiment 136 136. The cell of embodiment 135, wherein when the destabilization domain sequence is a DHFR destabilization domain sequence, the stabilizer is trimethoprim (TMP). Embodiment 137 136. The cell of embodiment 135, wherein when the destabilization domain sequence is an FKBP destabilization domain sequence, the stabilizer is Shield-1, rapamycin, or FK506. Embodiment 138 The method according to any one of embodiments 97 to 132 or the cell according to any one of embodiments 133 to 137, wherein the cell is a mammalian cell. Embodiment 139 139. The method or cell of embodiment 138, wherein the cell is a human cell. Embodiment 140 140. The method or cell according to any one of embodiments 138 to 139, wherein the cell is a somatic cell. Embodiment 141 141. The method or cell according to any one of embodiments 138 to 140, wherein the cell is a bone marrow cell. Embodiment 142 142. The method or cell according to any one of embodiments 138 to 141, wherein the cell is a hematopoietic stem cell (HSC) or a mesenchymal stem cell (MSC). Embodiment 143 141. The method or cell according to any one of embodiments 138 to 140, wherein the cell is an immune cell. Embodiment 144 144. The method or cell of embodiment 143, wherein the cell is a T cell, a phagocyte, a microglia cell, or a macrophage. Embodiment 145 145. The method or cell of embodiment 144, wherein the cell is a CD4+ T cell. Embodiment 146 146. The method or cell of embodiment 144 or embodiment 145, wherein the cell is a CD8+ T cell. Embodiment 147 147. The method or cell according to any one of embodiments 138 to 146, wherein the cell is a primary cell. Embodiment 148 147. The method or cell of any one of embodiments 138 to 146, wherein the cell is a progeny of a primary cell. Embodiment 149 149. The method or cell of any one of embodiments 138 to 148, wherein the cell has dysfunctional mitochondria. Embodiment 150 150. The method or cell of any one of embodiments 138 to 149, wherein the cell is derived from a subject with an age-related disease. Embodiment 151 151. The method or cell of embodiment 150, wherein the age-related disease is an autoimmune disease, a metabolic disease, a genetic disease, cancer, a neurodegenerative disease, or immunosenescence. Embodiment 152 152. The method or cell of any one of embodiments 138-151, wherein the cell is derived from a subject with a mitochondrial disease or disorder. Embodiment 153 153. The method or cell of embodiment 152, wherein the mitochondrial disease or disorder is caused by a mitochondrial DNA abnormality, a nuclear DNA abnormality, or both. Embodiment 154 154. The method or cell of embodiment 152 or embodiment 153, wherein the mitochondrial disease or disorder is chronic progressive external ophthalmoplegia (CPEO), Pearson syndrome, Kearns-Sayre syndrome (KSS), diabetes and deafness (DAD), mitochondrial diabetes, Leber's hereditary optic neuropathy (LHON), LHON-plus, neuropathy, ataxia, retinitis pigmentosa syndrome (NARP), maternally inherited Leigh's syndrome (MILS), mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), myoclonic epilepsy and ragged-red fibrosis (MERRF), familial bilateral striatal necrosis / striatonigral degeneration (FBSN), Luft's disease, aminoglycoside-induced hearing loss (AID), or multiple mitochondrial DNA deletion syndrome. Embodiment 155 Mitochondrial diseases or disorders include mitochondrial DNA depletion syndrome 4A, mitochondrial recessive ataxia syndrome (MIRAS), neurogastrointestinal mitochondrial encephalomyopathy (MNGIE), mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase gamma (POLG)-related disorders, sensory ataxia neuropathy dysarthria ophthalmoplegia (SANDO), leukoencephalopathy with brainstem and spinal cord involvement and elevated lactate (LBSL), coenzyme Q10 deficiency, Leigh syndrome, mitochondrial complex disorders, fumarase deficiency, α-ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenase complex deficiency (PDHC), pyruvate carboxylase deficiency (PCD), carnitine palmitoyltransferase I (CPT I) deficiency, carnitine palmitoyltransferase II (CPT II) deficiency, and coenzyme Q10 deficiency. 154. The method or cell of embodiment 152 or embodiment 153, wherein the patient is a patient with progressive external ophthalmoplegia (PEO), progressive cerebellar atrophy (IOSCA), mitochondrial myopathy (MM), spinal muscular atrophy (SMA), growth retardation, aminoaciduria, cholestasis, iron overload, premature death (GRACILE), or Charcot-Marie-Tooth disease type 2A (CMT2A). Embodiment 156 156. The method or cell of any one of embodiments 138 to 155, wherein the cell is derived from a subject with a neurodegenerative disease. Embodiment 157 157. The method or cell of embodiment 156, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, Friedreich's ataxia, Charcot-Marie-Tooth disease, or leukodystrophy. Embodiment 158 157. The method or cell of embodiment 156, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS). Embodiment 159 157. The method or cell of embodiment 156, wherein the neurodegenerative disease is Huntington's disease. Embodiment 160 157. The method or cell of embodiment 156, wherein the neurodegenerative disease is Alzheimer's disease, and optionally the cell is derived from a subject having an APOE4 allele, for example an E3 / E4 or E4 / E4 genotype. Embodiment 161 157. The method or cell of embodiment 156, wherein the neurodegenerative disease is Parkinson's disease. Embodiment 162 157. The method or cell of embodiment 156, wherein the neurodegenerative disease is Friedreich's ataxia, Charcot-Marie-Tooth disease. Embodiment 163 157. The method or cell of embodiment 156, wherein the neurodegenerative disease is a leukodystrophy. Embodiment 164 164. The method or cell of any one of embodiments 138 to 163, wherein the cell is derived from a subject with a retinal disease. Embodiment 165 165. The method or cell of embodiment 164, wherein the retinal disease is age-related macular degeneration, macular edema, or glaucoma. Embodiment 166 166. The method or cell according to any one of embodiments 138 to 165, wherein the cell is derived from a subject with diabetes. Embodiment 167 167. The method or cell according to any one of embodiments 138 to 166, wherein the cell is derived from a subject with hearing impairment. Embodiment 168 168. The method or cell of any one of embodiments 138 to 167, wherein the cell is derived from a subject with a genetic disease. Embodiment 169 169. The method or cell of embodiment 168, wherein the genetic disease is Hutchinson-Gilford Progeria Syndrome, Werner Syndrome, or Huntington's Disease. Embodiment 170 169. The method or cell of any one of embodiments 138 to 169, wherein the cell is derived from a subject with heart failure. Embodiment 171 171. The method or cell of any one of embodiments 138 to 170, wherein the cell is derived from a subject with an immunodeficiency. Embodiment 172 172. The method or cell of any one of embodiments 138 to 171, wherein the cell is derived from a subject with cancer. Embodiment 173 173. The method or cell according to any one of embodiments 138 to 172, wherein the cell is derived from a subject having an infection. Embodiment 174 149. The method or cell according to any one of embodiments 138 to 148, wherein the cell is derived from a healthy donor. Embodiment 175 175. The method or cell according to any one of embodiments 138 to 174, which is an ex vivo cell. Embodiment 176 176. The method of embodiment 175, further comprising administering the cells to a subject, optionally the subject being the same subject from which the cells were derived. Embodiment 177 A method of treating a subject having an age-related disease, a mitochondrial disease or disorder, a neurodegenerative disease, a retinal disease, diabetes, hearing impairment, a genetic disease, heart failure, an immune deficiency, cancer, or an infectious disease, comprising administering to the subject a therapeutically effective amount of a cell described in any one of embodiments 133 to 175. Embodiment 178 178. The method of embodiment 177, wherein the subject has an age-related disease. Embodiment 179 179. The method of embodiment 178, wherein the age-related disease is an autoimmune disease, a metabolic disease, a genetic disease, cancer, a neurodegenerative disease, or immunosenescence. Embodiment 180 178. The method of embodiment 177, wherein the subject has a mitochondrial disease or disorder. Embodiment 181 181. The method of embodiment 180, wherein the mitochondrial disease or disorder is caused by a mitochondrial DNA abnormality, a nuclear DNA abnormality, or both. Embodiment 182 The method of embodiment 180 or embodiment 181, wherein the mitochondrial disease or disorder is chronic progressive external ophthalmoplegia (CPEO), Pearson syndrome, Kearns-Sayre syndrome (KSS), diabetes and deafness (DAD), mitochondrial diabetes, Leber's hereditary optic neuropathy (LHON), LHON-plus, neuropathy, ataxia, and retinitis pigmentosa syndrome (NARP), maternally inherited Leigh syndrome (MILS), mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), myoclonic epilepsy and ragged-red fibrosis (MERRF), familial bilateral striatal necrosis / striatonigral degeneration (FBSN), Luft's disease, aminoglycoside-induced hearing loss (AID), and multiple mitochondrial DNA deletion syndrome. Embodiment 183 Mitochondrial diseases or disorders include mitochondrial DNA depletion syndrome 4A, mitochondrial recessive ataxia syndrome (MIRAS), neurogastrointestinal mitochondrial encephalomyopathy (MNGIE), mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase gamma (POLG)-related disorders, sensory ataxia neuropathy dysarthria ophthalmoplegia (SANDO), leukoencephalopathy with brainstem and spinal cord involvement and elevated lactate (LBSL), coenzyme Q10 deficiency, Leigh syndrome, mitochondrial complex disorders, fumarase deficiency, α-ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenase complex deficiency (PDHC), pyruvate carboxylase deficiency (PCD), carnitine palmitoyltransferase I (CPT I) deficiency, carnitine palmitoyltransferase II (CPT II) deficiency, and coenzyme Q10 deficiency. 182. The method of embodiment 180 or embodiment 181, wherein the genetic disorder is progressive external ophthalmoplegia (PEO), progressive cerebellar atrophy (IOSCA), mitochondrial myopathy (MM), spinal muscular atrophy (SMA), growth retardation, aminoaciduria, cholestasis, iron overload, early death (GRACILE), or Charcot-Marie-Tooth disease type 2A (CMT2A). Embodiment 184 178. The method of embodiment 177, wherein the subject has a neurodegenerative disease. Embodiment 185 185. The method of embodiment 184, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, Friedreich's ataxia, Charcot-Marie-Tooth disease, or leukodystrophy. Embodiment 186 178. The method of embodiment 177, wherein the subject has a retinal disease. Embodiment 187 187. The method of embodiment 186, wherein the retinal disease is age-related macular degeneration, macular edema, or glaucoma. Embodiment 188 178. The method of embodiment 177, wherein the subject has diabetes. Embodiment 189 178. The method of embodiment 177, wherein the subject has a hearing impairment. Embodiment 190 178. The method of embodiment 177, wherein the subject has a genetic disease. Embodiment 191 191. The method of embodiment 190, wherein the genetic disease is Hutchinson-Gilford Progeria Syndrome, Werner Syndrome, or Huntington's Disease. Embodiment 192 The method of embodiment 177, wherein the subject has heart failure. Embodiment 193 178. The method of embodiment 177, wherein the subject has an immunodeficiency. Embodiment 194 178. The method of embodiment 177, wherein the subject has cancer. Embodiment 195 The method of embodiment 177, wherein the subject has an infection. Embodiment 196 A pharmaceutical composition comprising a polypeptide according to any one of embodiments 1 to 84, a nucleic acid according to any one of embodiments 85 to 94, a particle according to embodiment 95, or a cell according to any one of embodiments 133 to 175, and a pharmaceutically acceptable excipient. Embodiment 197 A kit comprising: (a) a polypeptide according to any one of embodiments 1 to 84, a nucleic acid according to any one of embodiments 85 to 94, or a particle according to embodiment 95; and (b) a stabilizer. Embodiment 198 198. The kit of embodiment 197, wherein when the destabilization domain sequence is a DHFR destabilization domain sequence, the stabilizer is trimethoprim (TMP). Embodiment 199 198. The kit of embodiment 197, wherein when the destabilization domain sequence is an FKBP destabilization domain sequence, the stabilizer is Shield-1, rapamycin, or FK506. Embodiment 200 The kit of embodiment 197, wherein when the destabilization domain sequence is a PDE5 destabilization domain sequence, the stabilizer is sildenafil, vardenafil, tadalafil, avanafil, lodenafil, mirodenafil, udenafil, benzamidenafil, dasantafil, or beminafil.

[0164] 7. Citation of References All publications, patents, patent applications, and other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes. In the event of a conflict between the teachings of one or more references incorporated herein and the present disclosure, the teachings of the present disclosure are intended to control.

Claims

1. (a) a mitochondrial targeting sequence (MTS); (b) an endonuclease sequence; (c) a destabilization domain sequence; A polypeptide comprising:

2. The polypeptide of claim 1 , wherein the MTS comprises human MTS.

3. The polypeptide of claim 1 , wherein the MTS comprises a non-human MTS.

4. The polypeptide according to any one of claims 1 to 3, wherein the MTS comprises a mitochondrial protein MTS.

5. The polypeptide of any one of claims 1 to 4, wherein the MTS comprises an MTS of a TCA cycle-related enzyme, a chaperone protein, a mitochondrial genome replication protein, a protease, an mRNA processing protein, a mitochondrial RNA degradation protein, a deoxynucleotide triphosphate synthesis-related protein, a mitoribosomal protein, a phospholipid metabolism-related protein, a protein involved in the metabolism of toxic compounds, a disulfide relay system-related protein, an iron-sulfur protein assembly protein, a tRNA-modifying protein, an aminoacyl-tRNA synthetase, a release factor, or an elongation factor.

6. The polypeptide according to any one of claims 1 to 5, wherein the MTS comprises an MTS of a cytochrome c oxidase subunit.

7. The polypeptide of claim 6, wherein the MTS comprises an MTS of cytochrome c oxidase subunit VIII (COX8), cytochrome c oxidase subunit X (COX10), or cytochrome c oxidase subunit IV (COX4).

8. The MTS is (a) MTS of frataxin (FXN) protein; (b) an MTS of a TCA cycle-related enzyme that is optionally pyruvate dehydrogenase, citrate synthase, aconitase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, malate dehydrogenase, or pyruvate carboxylase; (c) an MTS of a chaperone protein, optionally mtHSP10, mtHSP60, mtHSP70, or mtHSP90; (d) a mitochondrial genome replication protein MTS, which is optionally TFAM, Twinkle, PolG, TFB2M, TEFM, or MTERF1; (e) an MTS of a protease, which is optionally an MPP, a CLPXP, a LON ATPase, or a PreP; (f) an MTS for mRNA processing protein, which is optionally LRPPRC, TACO1, ELAC2, PNPT1, HSD17B10, MTPAP, or PTCD1; (g) a mitochondrial RNA degradation protein MTS, which is optionally PNPasse, REX02, or SUV3; (h) a deoxynucleotide triphosphate synthesis-related protein MTS, which is optionally DGUOK, TK2, TYMP, MGME1, SUCLG1, SUCLA2, RNASEH1, or C10orf2; (i) a mitoribosomal protein MTS, which is optionally MRPS16, MRPS22, MRPL3, MRP12, or MRPL44; (j) a phospholipid metabolism related protein MTS, which is optionally AGK, SERAC1, or TAZ; (k) an MTS of a protein involved in the metabolism of toxic compounds, which is optionally HIBCH, ECHS1, ETHE1, or MPV17; (l) MTS, a disulfide relay system associated protein, optionally GFER; (m) an iron-sulfur protein assembly protein MTS, which is optionally ISCU, BOLA3, NFU1, or IBA57; (n) a tRNA-modifying protein MTS, which is optionally MTO1, GTP3BP, TRMU, PUS1, MTFMT, TRIT1, TRNT1, or TRMT5; (o) an aminoacyl-tRNA synthetase MTS that is optionally AARS2, DARS2, EARS2, RARS2, YARS2, FARS2, HARS2, LARS2, VARS2, TARS2, IARS2, CARS2, PARS2, NARS2, KARS, GARS, SARS2, or MARS2; (p) a release factor MTS, optionally C12orf65; or (q) an elongation factor MTS, which is optionally TUFM, TSFM, or GFM1; The polypeptide according to any one of claims 1 to 5, comprising:

9. The polypeptide of claim 1, wherein the MTS comprises a sequence that is at least 80% identical to any one of SEQ ID NOs: 1-15.

10. The polypeptide of claim 9, wherein the MTS comprises a sequence that is 100% identical to SEQ ID NO:

1.

11. The polypeptide of claim 9, wherein the MTS comprises a sequence that is 100% identical to SEQ ID NO:

2.

12. 12. The polypeptide of any one of claims 1 to 11, wherein the endonuclease is a restriction endonuclease, an RNA-guided endonuclease (e.g., Cas9 or Cas12), a zinc finger nuclease, or a transcription activator-like effector nuclease (TALEN).

13. The polypeptide of claim 12 , wherein the endonuclease is a restriction endonuclease.

14. 14. The polypeptide of claim 13, wherein the restriction endonuclease is XbaIR, EcoRI, SmaI, AflII, BamHI, BclI, HaeIII, HindII, HindIII, NdeI, PvuII, PstI, or SpeI endonuclease.

15. 15. The polypeptide of claim 14, wherein the restriction endonuclease is XbaIR endonuclease.

16. The polypeptide of any one of claims 1 to 13, wherein the endonuclease comprises a sequence that is at least 80% identical to SEQ ID NO:

16.

17. 17. The polypeptide of claim 16, wherein the endonuclease sequence comprises a sequence that is 100% identical to SEQ ID NO:

16.

18. 18. The polypeptide of any one of claims 1 to 17, wherein the destabilization domain sequence is a DHFR, FKBP, or PDE5 destabilization domain sequence.

19. 19. The polypeptide of claim 18, wherein the destabilization domain sequence is a DHFR destabilization domain sequence.

20. 20. The polypeptide of claim 19, wherein the destabilization domain sequence is an E. coli DHFR (ecDHFR) destabilization domain sequence.

21. 21. The polypeptide of any one of claims 1 to 20, wherein the destabilization domain sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:

18.

22. 19. The polypeptide of any one of claims 1 to 18, wherein the destabilization domain sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:

20.

23. 23. The polypeptide of any one of claims 1 to 22, wherein the MTS is positioned N-terminal to the endonuclease sequence and the destabilization domain sequence.

24. 23. The polypeptide of any one of claims 1 to 22, wherein the MTS is positioned C-terminal to the endonuclease sequence and the destabilization domain sequence.

25. 25. The polypeptide of any one of claims 1 to 24, wherein the endonuclease sequence is positioned N-terminal to the destabilization domain sequence.

26. 25. The polypeptide of any one of claims 1 to 24, wherein the endonuclease sequence is positioned C-terminal to the destabilization domain sequence.

27. 23. The polypeptide of any one of claims 1 to 22, wherein the MTS is positioned N-terminal to the endonuclease sequence, and the endonuclease sequence is positioned N-terminal to the destabilization domain sequence.

28. A nucleic acid encoding the polypeptide of any one of claims 1 to 27.

29. 29. A particle comprising the nucleic acid of claim 28, optionally a retroviral particle.

30. 29. A host cell comprising the nucleic acid of claim 28.

31. 30. A method of (a) inducing mitophagy in a cell, and / or (b) increasing mitochondrial turnover in a cell, and / or (c) increasing mitochondrial mass, and / or (d) inducing double-strand breaks in mitochondrial DNA, and / or (e) inducing epigenetic modifications in a cell, the method comprising contacting the cell with (i) a polypeptide according to any one of claims 1 to 27, a nucleic acid according to claim 28, or a particle according to claim 29, and (ii) a stabilizing agent.

32. 32. The method of claim 31, wherein the destabilization domain sequence is a DHFR destabilization domain sequence, and the stabilizer is trimethoprim (TMP).

33. 32. The method of claim 31, wherein when the destabilization domain sequence is an FKBP destabilization domain sequence, the stabilizer is Shield-1, rapamycin, or FK506.

34. The method of any one of claims 31 to 33, further comprising the step of removing the stabilizing agent from the cells after contacting the cells with the stabilizing agent.

35. A cell obtained or obtainable by the method according to any one of claims 31 to 34.

36. A cell comprising a polypeptide according to any one of claims 1 to 27, a nucleic acid according to claim 28, or a particle according to claim 29.

37. The method of any one of claims 31 to 34 or the cell of claim 35 or claim 36, wherein the cell is a mammalian cell.

38. 38. The method or cell of claim 37, wherein the cell is a human cell.

39. 39. The method or cell of claim 37 or claim 38, wherein the cell is a somatic cell, a bone marrow cell, a hematopoietic stem cell (HSC) or a mesenchymal stem cell (MSC), or an immune cell.

40. 40. The method or cell of any one of claims 37 to 39, wherein the cell is derived from a subject with an age-related disease.

41. 41. The method or cell of claim 40, wherein the age-related disease is an autoimmune disease, a metabolic disease, a genetic disease, cancer, a neurodegenerative disease, or immunosenescence.

42. 42. The method or cell of any one of claims 37 to 41, wherein the cell is derived from a subject with a mitochondrial disease or disorder.

43. 43. The method or cell of claim 42, wherein the mitochondrial disease or disorder is chronic progressive external ophthalmoplegia (CPEO), Pearson syndrome, Kearns-Sayre syndrome (KSS), diabetes and deafness (DAD), mitochondrial diabetes, Leber's hereditary optic neuropathy (LHON), LHON-plus, neuropathy, ataxia, retinitis pigmentosa syndrome (NARP), maternally inherited Leigh's syndrome (MILS), mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), myoclonic epilepsy and ragged-red fibrosis (MERRF), familial bilateral striatal necrosis / striatonigral degeneration (FBSN), Luft's disease, aminoglycoside-induced hearing loss (AID), or multiple mitochondrial DNA deletion syndrome.

44. The mitochondrial diseases or disorders include mitochondrial DNA depletion syndrome 4A, mitochondrial recessive ataxia syndrome (MIRAS), neurogastrointestinal mitochondrial encephalomyopathy (MNGIE), mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase gamma (POLG)-associated disorders, sensory ataxia neuropathy dysarthria ophthalmoplegia (SANDO), leukoencephalopathy with brainstem and spinal cord involvement and elevated lactate (LBSL), coenzyme Q10 deficiency, Leigh's syndrome, mitochondrial complex disorders, fumarase deficiency, α-ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenase complex deficiency (PDHC), pyruvate carboxylase deficiency (PCD), carnitine palmitoyltransferase I (CPT I) deficiency, carnitine palmitoyltransferase II (CPT 43. The method or cell of claim 42, wherein the disease is a progressive external ophthalmoplegia (AD- / ar-PEO) deficiency, carnitine acylcarnitine (CACT) deficiency, autosomal dominant / autosomal recessive progressive external ophthalmoplegia (ad- / ar-PEO), infantile-onset spinal cerebellar atrophy (IOSCA), mitochondrial myopathy (MM), spinal muscular atrophy (SMA), growth retardation, amino aciduria, cholestasis, iron overload, premature death (GRACILE), or Charcot-Marie-Tooth disease type 2A (CMT2A).

45. 45. The method or cell of any one of claims 37 to 44, wherein the cell is derived from a subject with a neurodegenerative disease.

46. 46. ​​The method or cell of claim 45, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, Friedreich's ataxia, Charcot-Marie-Tooth disease, or leukodystrophy.

47. 47. The method or cell of any one of claims 37 to 46, wherein the cell is derived from a subject with a retinal disease, diabetes, hearing impairment, a genetic disease, heart failure, an immune deficiency, cancer, or an infectious disease.

48. 48. The method or cell of any one of claims 37 to 47, wherein the cell is an ex vivo cell.

49. 49. The method of claim 48, further comprising administering the cells to a subject, optionally the subject being the same subject from which the cells were derived.

50. 50. A method of treating a subject having an age-related disease, a mitochondrial disease or disorder, a neurodegenerative disease, a retinal disease, diabetes, hearing impairment, a genetic disease, heart failure, an immune deficiency, cancer, or an infectious disease, comprising administering to the subject a therapeutically effective amount of the cells of any one of claims 35-48.

51. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 27, a nucleic acid according to claim 28, a particle according to claim 29, or a cell according to any one of claims 35 to 48, and a pharmaceutically acceptable excipient.

52. A kit comprising: (a) a polypeptide according to any one of claims 1 to 27, a nucleic acid according to claim 28, or a particle according to claim 29; and (b) a stabilizing agent.

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