Composition for mitochondrial autophagy induction and application thereof

Abstract

A polypeptide having a mitochondrial targeting sequence (MTS), an endonuclease sequence, and a destabilizing domain sequence; a nucleic acid encoding the polypeptide; the use of such polypeptides and nucleic acids to induce mitochondrial autophagy, increase mitochondrial turnover and / or induce double-strand breakage of mitochondrial DNA in a cell; and the aforementioned therapeutic applications, for example in the treatment of mitochondrial diseases and conditions.

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 426,424, filed on November 18, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0003] 2. Sequence Listing

[0004] This application contains a sequence listing submitted electronically in XML format and is hereby 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 Art

[0005] Mitochondrial dysfunction is caused by a variety of factors, including mutations in genes encoding mitochondrial proteins, tRNA and rRNA, accumulation of mutations in the mitochondrial genome, poor management of mitochondrial proteins, poor management of intracellular organelles such as the endoplasmic reticulum and lysosomes, and defects in mitochondrial protein quality control. Mitochondrial protein quality control defects can be caused by a variety of factors, such as poor management of intracellular organelles such as the endoplasmic reticulum and lysosomes. Among them, the quality control and fusion / fission dynamics of mitochondrial biogenesis and autophagy (mitochondrial autophagy) are the focus of various mitochondrial dysfunctions. Mitochondrial quality control defects associated with various diseases have been 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 cancer immunodeficiency, infectious diseases and autoimmune diseases.

[0006] Small molecule compounds have been developed to enhance biogenesis, induce mitophagy, and inhibit excessive fission, etc. For example, coenzyme Q10, idebenone, and metformin have been shown to induce mitophagy.

[0007] However, no compounds with a clear mode of action have reached the clinic. Thus, there remains a need for compositions and methods for ameliorating mitochondrial dysfunction. 2. Summary of the Invention

[0009] The present disclosure provides polypeptides capable of inducing double-strand breaks (DSBs) in mitochondria to achieve a temporary and partial reduction in the number of mitochondria in cells. Compared with the nucleus, mitochondria have a poor gene repair mechanism. In response to the stress of DSBs, mitochondria strongly transmit signals to the nucleus to promote the replication of the mitochondrial genome and increase the production of mitochondrial component proteins. Without being bound by theory, it is believed that DSBs induced in mitochondrial DNA can be used to effectively promote mitochondrial turnover, thereby improving mitochondrial dysfunction by generating new mitochondria. DSBs can be introduced into mitochondrial DNA using a polypeptide comprising a mitochondrial targeting sequence (MTS) fused to an endonuclease. However, a potential problem of this method is excessive endonuclease activity. In order to provide a sensitive on / off control of an endonuclease, the present disclosure provides a polypeptide having 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 retain structure and endonuclease activity; in the absence of a stabilizer, the destabilization domain loses stability, causing the polypeptide to be degraded by the proteasome.

[0010] Accordingly, in one aspect, the present disclosure provides polypeptides comprising a mitochondrial targeting sequence (MTS), an endonuclease sequence (e.g., XbaIR), and a destabilization domain sequence. The inclusion of the MTS can be used to direct the polypeptide to the desired endonuclease active site, i.e., the mitochondrial genome. The inclusion of a destabilization domain sequence (which is stabilized by a stabilizer) enables sensitive on / off control of the endonuclease. For example, a cell can be contacted with the polypeptide in the presence of a stabilizer for a period of time (during which the polypeptide is active) and, subsequently, the stabilizer can be removed, resulting in destabilization and degradation of the polypeptide.

[0011] Exemplary features of the polypeptides of the present disclosure are described below in Section 4.2 and in Specific Embodiments 1 to 84.

[0012] On the other hand, the present disclosure provides nucleic acids encoding polypeptides of the present disclosure, particles comprising nucleic acids (such as viral particles), and host cells comprising nucleic acids 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 to 96.

[0013] On the other hand, the present disclosure provides the following methods: by contacting cells with polypeptides, nucleic acids or particles of the present disclosure and stabilizers, (a) inducing mitophagy in cells, and / or (b) increasing mitochondrial turnover in cells, and / or (c) increasing mitochondrial mass, and / or (d) inducing double-strand breaks in mitochondrial DNA and / or (e) inducing epigenomic modifications in cells. For example, cells can be transfected with nucleic acids encoding polypeptides, transduced with viral particles containing nucleic acids encoding polypeptides, or injected with polypeptides, and cultured in the presence of stabilizers. After a period of time (e.g., 6 hours to 5 days), the stabilizer can be removed to allow destabilization and degradation of the polypeptide.

[0014] In another aspect, the present disclosure provides cells and cell populations obtained or obtainable by the methods described herein: (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 epigenomic modifications in a cell. Unless the context requires otherwise, reference to "cell" herein encompasses single cells as well as cell populations.

[0015] In another aspect, the present disclosure provides methods for treating a subject using the cells and cell populations of the present disclosure. For example, the subject can be a subject suffering from an age-related disease, a mitochondrial disease or disorder, a neurodegenerative disease, an eye disease (e.g., a retinal disease), diabetes, a hearing impairment, a genetic disease, heart failure, an immunodeficiency, a cancer, or an infectious disease.

[0016] Further exemplary features of the methods and cells of the present disclosure are described below in Section 4.4 and in Specific Embodiments 97 to 195.

[0017] In a further aspect, the present disclosure provides a pharmaceutical composition comprising a polypeptide, nucleic acid, particle or cell (including a cell population) of the present disclosure. Such a pharmaceutical composition can be used, for example, in the therapeutic methods described herein.

[0018] In another aspect, the present disclosure provides a kit comprising a polypeptide, nucleic acid or particle of the present disclosure and a stabilizer. The kit can be used, for example, in the method of the present disclosure.

[0019] Further exemplary features of pharmaceutical compositions and kits are described below in Section 4.5 and in specific embodiments 196 to 200. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A map of a retroviral vector harboring the MTS-XbaIR-DHFR polypeptide is shown (Example 1).

[0021] Figure 2The molecular model of the MTS-XbaIR-DHFR polypeptide of Example 1 is shown.

[0022] Figure 3 A map of a retroviral vector harboring the EFGR-DHFR polypeptide is shown (Example 1).

[0023] Figure 4 The molecular model of the EGFR-DHFR polypeptide of Example 1 is shown.

[0024] Figure 5 Shown are fluorescence microscopy images of HeLa cells transduced with the EGFP-DHFR retroviral vector (Example 1).

[0025] Figure 6 Shown are FACS data of HeLa cells transduced with an EGFP-DHFR retroviral vector, demonstrating EGFP expression when cultured in the presence of TMP (Example 1).

[0026] Figure 7 Shown are the relative EGFP mRNA expression levels of Hela cells transduced with an EGFP-DHFR retroviral vector when cultured with TMP (Example 1).

[0027] Figures 8A-8D The figure shows Hela cells transduced with EGFP-DHFR retroviral vector and cultured in a medium containing TMP for two days, followed by TMP washout, and the fluorescence intensity detected by fluorescence microscopy and FACS at 0, 1, 2, 4, 6, 8, 24 and 48 hours after TMP washout (Example 1). Figure 8A : Time course; Figure 8B : Fluorescence image; Figure 8C : FACS analysis, Figure 8D : Mean fluorescence intensity (MFI) over time in FACS analysis.

[0028] Figures 9A-9D Shown are the fluorescence intensities detected by fluorescence microscopy and FACS after Hela cells transduced with EGFP-DHFR retroviral vector were cultured in a medium containing TMP for several time periods (0, 1, 2, 4, 6, 8, 24, and 48 hours). Figure 9A : Time course; Figure 9B : Fluorescence image; Figure 9C : FACS analysis; Figure 9D : MFI changes over time in FACS analysis.

[0029] Figures 10A-10CShows the expression of XbaI in HeLa cells (Hela MXD sc20) transfected with a retroviral vector encoding the MTS-XbaIR-DHFR polypeptide ( Figure 10A ) and mtDNA copy number (CN) ( Figure 10B ), and the cells were cultured for two days in the absence or presence of 0.5 μM TMP, followed by TMP washout ( Figure 10C )(Example 1).

[0030] Figures 11A-11D Shown are HeLa MXD sc20 cells incubated with TMP for three different time periods (16, 20, and 48 hours) and then cultured over time. Figure 11D ), the MFI of Mitogreen staining ( Figure 11A ), MFI of TMRM staining as an indicator of global mitochondrial membrane potential (mtMP) ( Figure 11B ), and the relative TMRM / Mitogreen ratio as an indicator of mtMP per mass unit ( Figure 11C ).

[0031] Figures 12A-12C Shown are HeLa MXD sc20 cells cultured for two days in the absence or presence of 0.5 μM TMP followed by TMP washout ( Figure 12C ) of cells ( Figure 12A ) and cell viability ( Figure 12B ). (Example 1).

[0032] Figures 13A-13B The mitochondrial autophagy index of Hela MDX sc20 cells analyzed by FACS is shown ( Figure 13A ) and mtDNA CN estimated by qPCR ( Figure 13B HeLa MDX sc20 cells were transduced with retroviral vectors encoding mtKeimaRed and PARK2 and cultured for several time periods in the absence or presence of TMP. 10 μM CCCP was used as a positive control for mitophagy (Example 1).

[0033] Figures 14A-14D Figure 5. Changes in mitochondrial biogenesis in HeLa-GiM cells stably expressing a genetically induced mitophagy (GiM) unit in the presence and absence of TMP. Mitochondrial ROS (mtROS) were assessed over time using FACS. Figure 14A )、PGC1α( Figure 14B )、NRF1( Figure 14C ) and TFAM( Figure 14D ) (Example 2).

[0034] Figures 15A-15F The expression of several mitochondrial proteins in HeLa_GiM cells over time in the presence and absence of TMP is shown. Figure 15A The Western blot images of mitochondrial proteins on days 2, 4, 6 and 8 are shown. Figures 15B-15F The quantified levels of the same proteins are shown. (Example 2).

[0035] Figures 16A-16B The phase contrast (PhC) and fluorescence microscopy images of HeLa_GiM cells expressing mtKeimaRed in the presence and absence of TMP are shown Figure 16A ), as well as the percentage of cells positive for mitophagy Figure 16B ) (Example 2).

[0036] Figures 17A-17B 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 is shown Figure 17A ), as well as the quantified values of the area corresponding to autophagosomes Figure 17B ) (Example 3).

[0037] Figures 18A-18B The LC3-II expression levels in HeLa_GiM cells in the absence or presence of TMP or TMP+BafA1 are shown. Figure 18A The Western blot images are shown, Figure 18B Graphical representation of the quantification in triplicate is shown. (Example 3).

[0038] Figures 19A-19C The results of the respirometry assay showing the change in OXPHOS and glycolysis over time Figure 19A ), the two-dimensional expansion of the OXPHOS and glycolysis relationship Figure 19B ), and the change in ATP production, basal respiration, proton leak and spare capacity over time Figure 19C ) are shown. (Example 4).

[0039] Figures 20A-20G The assay setup used in Example 5 is shown Figure 20A ), the FACS results of untreated Alzheimer’s disease (AD) fibroblasts and control NHDF cells Figure 20B ) and the FACS results of AD fibroblasts at 7, 14 and 21 days after genetic induction of mitochondrial mitophagy (GiM) Figure 20C ) are shown. The untreated cells are Figure 20D and 20E ) and mtMass and mtMP levels in the control group ( Figure 20F and 20G ), mtMass and mtMP levels in AD fibroblasts 7 and 14 days after GiM, which are further described in Example 5. 4. Detailed Description of the Invention

[0041] The present disclosure provides polypeptides capable of inducing double-strand breaks (DSBs) in mitochondria to achieve a temporary and partial reduction in the number of mitochondria in cells.

[0042] The mitochondrial genome encodes respiratory chain proteins, which are tightly regulated in translational balance with nuclear-encoded respiratory proteins. Therefore, partial loss of the mitochondrial genome is directly associated with insufficient proton uptake, leading to depolarization of the mitochondrial membrane potential. The mitochondrial membrane potential is depolarized in dysfunctional mitochondrial compartments and is a key regulator of mitophagy. Without being bound by theory, it is believed that upon the introduction of a DSB, the more depolarized compartment may preferentially undergo mitophagy.

[0043] In response to the stress of DSB, mitochondria strongly transmit signals to the nucleus (for example, as part of the mitochondrial unfolded protein response (UPRmt)) to promote the replication of the mitochondrial genome and increase the production of mitochondrial component proteins. In addition to the signal of UPRmt, the metabolic changes caused by the reduction of the mitochondrial genome can affect the epigenomic state of the cell. For example, the reduction can reduce some intermediates of the TCA cycle used for acetylation and methylation of the nuclear genome and histones. Without being bound by theory, it is believed that DSBs inducing mitochondrial DNA can be used to effectively promote mitochondrial turnover, thereby improving mitochondrial dysfunction by generating new mitochondria. Mitochondrial DNA may have some modifications, such as 8-oxo-7,8-dihydroguanine (8-OXOG), which is an oxidized form of guanine. As age increases, destructive modifications accumulate. Since the newly generated mitochondrial genome lacks these modifications, mitochondrial generation can promote the recovery of healthy mitochondrial function.

[0044] DSBs can be introduced into mitochondrial DNA by using a polypeptide comprising a mitochondrial targeting sequence (MTS) fused to an endonuclease. However, a potential problem with this approach is excessive endonuclease activity. In order to provide sensitive on / off control of the endonuclease, the present disclosure provides polypeptides having a destabilizing domain in addition to the mitochondrial targeting sequence and the endonuclease sequence. When stabilized by a stabilizer, the destabilizing domain allows the polypeptide to retain structure and endonuclease activity; in the absence of a stabilizer, the destabilizing domain loses stability, causing the polypeptide to be degraded by the proteasome.

[0045] 4.1. Definitions

[0046] 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 in this specification.

[0047] As used herein, the following terms are intended to have the following meanings:

[0048] 1. A kind of: As used herein, the terms "a," "an," "the," and similar terms used in the context of this disclosure should be construed to include both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0049] and / or : The term "and / or" means that each or two or all of the components or features in the list are possible variations, especially two or more thereof in an alternative or cumulative manner.

[0050] Destabilization domain (DD)The term destabilizing domain refers to a polypeptide domain that, when fused to a second polypeptide domain such as an endonuclease, causes degradation of the polypeptide in the absence of a stabilizing agent that otherwise prevents or inhibits degradation initiated by the destabilizing domain. Exemplary destabilizing domains include a dihydrofolate reductase (DHFR) destabilizing domain (which can be stabilized by the exemplary stabilizing agent trimethoprim), an FK506 binding protein (FKBP) destabilizing domain (which can be stabilized by the exemplary stabilizing agents Shield-1 (Shldl), rapamycin, and FK506), and a PDE5 destabilizing domain (which can be stabilized by the exemplary stabilizing agents sildenafil, vardenafil, tadalafil, avanafil, lodenafil, mirodenafil, udenafil, benzamidenafil, dansantafil, 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 US 9,487,787; exemplary FKBP destabilizing domains are described in Banaszynski et al., 2006, Cell 126(5):995-1104, and US 9,487,787; and exemplary PDE5 destabilizing domains are described in WO 2018 / 237323, the contents of each of which are incorporated by reference in their entirety.

[0051] effective dose The term "effective amount" or "therapeutically effective amount" means the amount of an agent or composition that is sufficient to elicit a needed or desired response, or in other words, an amount that is sufficient to elicit a significant biological response when administered to a subject. The amount preferably relates to an amount that is therapeutically effective, or in a broader sense, also prophylactically effective, against the progression of a disease or disorder disclosed herein. It will be appreciated that an "effective amount" or "therapeutically effective amount" can vary from subject to subject, due to variation in the metabolism of the agent, age, body weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.

[0052] Endonucleases:The term "endonuclease" refers to an enzyme that cuts a polynucleotide chain by separating nucleotides other than the nucleotides at the 5' or 3' ends. Endonucleases are different from exonucleases, which cut nucleotides from the 5' or 3' ends of a polynucleotide chain. Exemplary endonucleases include restriction endonucleases that can cut double-stranded DNA at or near the specific recognition site to form double-strand breaks (DSBs) in DNA. Exemplary restriction endonucleases include XbaIR, EcoRI, SmaI, AflII, BamHI, BclI, EcoRI, HaeIII, HindII, HindIII, NdeI, PvuII, PstI, and SpeI. Exemplary endonuclease amino acid sequences are described in publicly available databases such as UniProt. For example, the exemplary XbaIR amino acid sequence has UniProt Accession No. 068567; the exemplary EcoRI amino acid sequence has UniProt Accession No. P00642; the exemplary SmaI amino acid sequence has UniProt Accession No. P14229; the exemplary AflII amino acid sequence has UniProt Accession No. E3VX87; the exemplary BamHI amino acid sequence has UniProt Accession No. P23940; the exemplary BclI amino acid sequence has UniProt Accession No. E5LGB8; the exemplary The HaeIII amino acid sequence has UniProt Accession No. 068584; the exemplary HindIII amino acid sequence has UniProt Accession No. P44413; the exemplary HindIII amino acid sequence has UniProt Accession No. P43870; the exemplary PvuII amino acid sequence has UniProt Accession No. A0A4R7BM34; the exemplary PstI amino acid sequence has UniProt Accession No. P00640; and the exemplary SpeI amino acid sequence has UniProt Accession No. F1KM35.

[0053] Mitochondrial targeting sequence (MTS) The term "mitochondrial targeting sequence" refers to an amino acid sequence that directs the transport of a polypeptide containing the sequence to the mitochondria. An MTS is typically 10-70 amino acids in length. An MTS typically comprises an alternating pattern of hydrophobic and positively charged amino acids to form an amphipathic helix.

[0054] or : Unless otherwise indicated, the conjunction "or" is intended to be used in its proper sense as a Boolean logic operator, encompassing both feature selection in alternatives (A or B, where the selection of A is mutually exclusive with B) and feature selection in conjunctions (A or B, where both A and B are selected). In some places herein, the term "and / or" is used for the same purpose, which should not be interpreted as implying that "or" is used to refer to mutually exclusive alternatives.

[0055] Peptides, proteins and polypeptides : The terms peptide, protein, and polypeptide are used interchangeably to refer to natural or synthetic molecules comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha-amino group of another amino acid. Amino acids can be natural or synthetic and can contain chemical modifications such as disulfide bonds, replacement of radioisotopes, phosphorylation, substrate chelation (e.g., chelation of iron or copper atoms), glycosylation, acetylation, formylation, amidation, biotinylation, and a wide range of other modifications. There is no explicit requirement that a polypeptide must contain the intended function; a polypeptide can be functional, non-functional, function for an unexpected / unintended purpose, or have an unknown function. Polypeptides are composed of approximately twenty standard naturally occurring amino acids, although natural and synthetic amino acids that are not members of the standard twenty amino acids can also be used. The standard twenty 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" is an alphabetical representation of a polypeptide molecule.

[0056] Percent identity The percent identity between two amino acid sequences is by multiplying the number of matches between a pair of alignments by 100, and calculating divided by the length of the alignment region.The identity score is only calculated to match completely, does not consider the similarity of amino acids to each other, does not consider replacement or disappearance as coupling.The comparison that is used to determine the purpose of sequence identity percent identity can be realized in the many ways within the scope of this art, for example, by manual comparison or use publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.Those skilled in the art can determine to be used to realize the appropriate parameters of maximum comparison.

[0057] Subjects : As used herein, the term "subject" means a human.

[0058] Treat, treat, treatAs used herein, in one embodiment, the terms "treat," "treating," or "treatment" of any disease or condition refer to ameliorating the disease or condition (e.g., slowing or inhibiting or reducing the development of the disease or at least one clinical symptom or pathological feature thereof). In another embodiment, "treat," "treating," or "treatment" refers to alleviating or ameliorating at least one physical parameter or pathological feature of the disease, e.g., including those that may not be discernible to the subject. In yet another embodiment, "treat," "treating," or "treatment" refers to regulating the disease or condition 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 or development or progression of a disease or condition or at least one symptom or pathological feature associated therewith. In yet another embodiment, "treat," "treating," or "treatment" refers to preventing or delaying the progression of a disease to a more advanced stage or a more serious condition. The benefit to the patient being treated is statistically significant or at least perceptible to the patient or physician. However, it should be understood that when a drug is administered to a patient to treat a disease, the result may not always be effective treatment.

[0059] 4.2. Peptides

[0060] In one aspect, the present disclosure provides polypeptides comprising a mitochondrial targeting sequence (MTS), an endonuclease sequence, and a destabilization domain (DD) sequence. Exemplary features of mitochondrial targeting sequences, endonucleases, and destabilization domains that can be included in the polypeptides of the present disclosure are described in Sections 4.2.1, 4.2.2, and 4.2.3, respectively.

[0061] The MTS, endonuclease sequence, and DD sequence can be positioned in any suitable N-terminal to C-terminal order. For example, the MTS can be located at the N-terminus or C-terminus of the polypeptide. In some embodiments, the MTS is located at the N-terminus of the polypeptide. The endonuclease sequence can be located at the N-terminus or C-terminus of the DD. In some embodiments, the polypeptide comprises the MTS, endonuclease sequence, and DD sequence in N-terminal to C-terminal order. The MTS, endonuclease sequence, and DD sequence can be directly linked or separated by a spacer sequence (e.g., a short amino acid sequence, such as one, two, three, four, or more amino acids).

[0062] 4.2.1. Mitochondrial targeting sequence

[0063] Mitochondria have about 1500 proteins encoded by the nuclear genome. They are translated in the cytosol and, depending on the MTS, are imported into the inner or outer mitochondrial membrane, the intermembrane space, or the matrix. The polypeptides disclosed herein may include a full-length MTS or a variant of a wild-type MTS of a mitochondrial protein (e.g., a truncated version of the full-length MTS and / or an MTS having one or more amino acid substitutions (e.g., one or more conservative amino acid substitutions) compared to the wild-type sequence).

[0064] The polypeptides of the present disclosure may include human MTS or non-human MTS (e.g., rodents such as mice or rats or non-human primates such as cynomolgus monkeys). For example, the MTS of the polypeptides of the present disclosure may include TCA cycle-related enzymes, chaperone proteins, mitochondrial genome replication proteins, proteases, mRNA processing proteins, mitochondrial RNA degradation proteins, deoxynucleotide triphosphate synthesis-related proteins, mitochondrial ribosomal proteins, phospholipid metabolism-related proteins, proteins involved in the metabolism of toxic compounds, disulfide bond relay system-related proteins, iron-sulfur protein assembly proteins, tRNA modification proteins, aminoacyl-tRNA synthetases, release factors, or elongation factors.

[0065] In some embodiments, the MTS comprises an MTS of a cytochrome c oxidase subunit (e.g., a full-length MTS or a truncated version thereof that retains 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).

[0066] In some embodiments, the MTS comprises the MTS of frataxin (FXN).

[0067] In some embodiments, the MTS comprises an MTS of a TCA cycle-related enzyme, e.g., an MTS of pyruvate dehydrogenase, citrate synthase, aconitase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinyl-CoA synthetase, succinate dehydrogenase, fumarase, malate dehydrogenase, or pyruvate carboxylase.

[0068] In other embodiments, the MTS comprises the MTS of a chaperone protein, eg, the MTS of mtHSP10, mtHSP60, mtHSP70, or mtHSP90.

[0069] In other embodiments, the MTS comprises the MTS of a mitochondrial genome replication protein, e.g., the MTS of TFAM, Twinkle, PolG, TFB2M, TEFM, or MTERF1.

[0070] In other embodiments, the MTS comprises the MTS of a protease, e.g., the MTS of MPP, CLPXP, LON ATPase, or PreP.

[0071] In other embodiments, the MTS comprises the MTS of a mRNA processing protein, e.g., the MTS of LRPPRC, TACOl, ELAC2, PNPT1, HSD17B10, MTPAP, or PTCD1.

[0072] In other embodiments, the MTS comprises the MTS of a mitochondrial RNA degradation protein, e.g., the MTS of PNPasse, REX02, or SUV3.

[0073] In other embodiments, the MTS comprises the MTS of a deoxynucleotide triphosphate synthesis related protein, e.g., the MTS of DGUOK, TK2, TYMP, MGME1, SUCLG1, SUCLA2, RNASEH1, or C10orf2.

[0074] In other embodiments, the MTS comprises the MTS of a mitochondrial ribosomal protein, e.g., the MTS of MRPS16, MRPS22, MRPL3, MRP12, or MRPL44.

[0075] In other embodiments, the MTS comprises the MTS of a phospholipid metabolism related protein, e.g., the MTS of AGK, SERAC1, or TAZ.

[0076] In other embodiments, the MTS comprises the MTS of a protein involved in toxic compound metabolism, e.g., the MTS of HIBCH, ECHS1, ETHE1, or MPV17.

[0077] In other embodiments, the MTS comprises the MTS of a disulfide bond relay system related protein, e.g., the MTS of GFER.

[0078] In other embodiments, the MTS comprises the MTS of an iron-sulfur protein assembly protein, e.g., the MTS of ISCU, BOLA3, NFU1, or IBA57.

[0079] In other embodiments, the MTS comprises the MTS of a tRNA modification protein, e.g., the MTS of MTOl, GTP3BP, TRMU, PUS1, MTFMT, TRIT1, TRNT1, or TRMT5.

[0080] In other embodiments, the MTS comprises an MTS of an aminoacyl tRNA synthetase, e.g., an MTS of AARS2, DARS2, EARS2, RARS2, YARS2, FARS2, HARS2, LARS2, VARS2, TARS2, IARS2, CARS2, PARS2, NARS2, KARS, GARS, SARS2, or MARS2.

[0081] In other embodiments, the MTS comprises an MTS of an elongation factor, e.g., an MTS of TUFM, TSFM, or GFM1.

[0082] Exemplary mitochondrial targeting sequences are listed in Table 1.

[0083]

[0084] A polypeptide of the present disclosure can include an MTS identified in Table 1 or a variant thereof (e.g., an MTS having one or more conservative amino acid substitutions and / or truncations). The truncation can be a truncation of the C-terminal sequence (e.g., the MTS can correspond to the sequence listed in Table 1, but with one or more amino acids, e.g., a C-terminal truncation of one, two, three, four, five, or more than five amino acids). In some embodiments, the MTS comprises at least 15 N-terminal amino acids of an MTS sequence listed in Table 1. A variant MTS can include, e.g., an MTS that is at least 80%, at least 95%, at least 90%, or at least 95% identical to an MTS listed in Table 1.

[0085] Those of skill in the art will appreciate that additional mitochondrial targeting sequences can be used in addition to those identified in this section. A variety of tools for predicting MTSs can be used to identify additional mitochondrial targeting sequences, including 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).

[0086] 4.2.2. Endonucleases

[0087] A variety of endonucleases can be used in the polypeptides of the present disclosure. For example, the endonuclease can be a restriction endonuclease, an RNA-guided endonuclease (e.g., Cas9 or Cas12), a zinc finger nuclease, or a transcription activator-like effector nuclease (TALEN). The endonuclease can include a catalytic domain (e.g., from a wild-type or engineered endonuclease) and optionally one or more additional domains, such as all domains present in a full-length wild-type or engineered endonuclease.

[0088] The endonuclease may be of bacterial origin. Many restriction endonucleases are known in the art, including, for example, XbaIR, EcoRI, SmaI, AflII, BamHI, BclI, HaeIII, HindII, HindIII, NdeI, PvuII, PstI, and SpeI.

[0089] In some embodiments, the endonuclease is XbaIR. An exemplary XbaIR sequence is set forth in SEQ ID NO: 16: MTTLEKIKLLADGYADRLKLAIDGRVLEMQGDDVSHYLIYRVLGVAQEEGRLIDVYQNKGRFLYKYAGSFLEAATKLCFKEAFPDSASLRLPNTQGQRPRTVEIDCLVGNDALEIKWKDATTDGDHITKEHTRIKVISDAGYKPIRIMFYYPHRTQAIRIQETLETLYNGVHGEYHYGEAAWDYVLQRTSVNLKVALEQIADSRTNEAA (SEQ ID NO: 16).

[0090] In some embodiments, the endonuclease comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, greater than 95%, or 100% identical to SEQ ID NO: 16.

[0091] In some embodiments, the endonuclease is EcoRI.

[0092] In some embodiments, the endonuclease is SmaI.

[0093] In some embodiments, the endonuclease is AflII.

[0094] In some embodiments, the endonuclease is BamHI.

[0095] In some embodiments, the endonuclease is BclI.

[0096] In some embodiments, the endonuclease is HaeIII.

[0097] In some embodiments, the endonuclease is HindII.

[0098] In some embodiments, the endonuclease is HindIII.

[0099] In some embodiments, the endonuclease is NdeI.

[0100] In some embodiments, the endonuclease is PvuII.

[0101] In some embodiments, the endonuclease is PstI.

[0102] In some embodiments, the endonuclease is SpeI.

[0103] 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.

[0104] Exemplary RNA-guided endonucleases such as Cas9 and Cas12 are described in US 11,001,863 B2, WO 2014 / 093661, and 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.

[0105] 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 entirety.

[0106] Exemplary TALEN nucleases are described in WO 2014 / 134412, WO 2015 / 013583, and WO 2013 / 163628, the contents of which are herein incorporated by reference in their entirety.

[0107] 4.2.3. Destabilization domain

[0108] The polypeptides of the present disclosure include destabilization domains (DDs) that allow on / off control of endonucleases. Exemplary DDs include DHFR, FKBP, and PDE5 DDs.

[0109] Exemplary DHFR DDs are described in US 9,487,787, the contents of which are incorporated herein in their entirety. The amino acid sequence of wild-type E. coli DHFR is set forth below:

[0110] MISLIAALAVDHVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR (SEQ ID NO: 17).

[0111] DHFR DDs can comprise wild-type DHFR sequences or can comprise one or more amino acid substitutions and / or truncations at the N and / or C-terminus. For example, a DHFR DD sequence can 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 a DHFR DD include Y100I, G121V, N18T / A19V, F103L, H12Y / Y100I, H12L / Y100I, R98H / F103S, M42T / H114R, I61F / T68S. Combinations of the foregoing substitutions can also be used. In some embodiments, a DHFR comprises the same amino acid sequence as SEQ ID NO: 17, except for a Y100I, G121V, N18T / A19V, F103L, H12Y / Y100I, H12L / Y100I, R98H / F103S, M42T / H114R, or I61F / T68S substitution or combination thereof. In some embodiments, a DHFR DD lacks the N-terminal methionine. For example, in some embodiments, a DHFR comprises the same amino acid sequence as SEQ ID NO: 17, except for a Y100I, G121V, N18T / A19V, F103L, H12Y / Y100I, H12L / Y100I, R98H / F103S, M42T / H114R, or I61F / T68S substitution or combination thereof, and lacks the N-terminal methionine.

[0112] In some embodiments, the DHFR DD has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to ISLIAALAVDHVIGMETVMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR (SEQ ID NO: 18). An exemplary nucleotide sequence encoding SEQ ID NO: 18 is shown below:

[0113] atcagtctgattgcggcgttagcggtagatcacgttatcggcatggaaaccgtcatgccgtggaacctgcctgccgatctcgcctggtttaaacgcaacaccttaaataaacccgtgat tatgggccgccatacctgggaatcaatcggtcgtccgttgccaggacgcaaaaatattatcctcagcagtcaaccgagtacggacgatcgcgtaacgtgggtgaagtcggtggatgaag ccatcgcggcgtgtggtgacgtaccagaaatcatggttattggcggcggtcgcgtttatgaacagttcttgccaaaagcgcaaaaactgtatctgacgcatatcgacgcagaagtggaa ggcgacacccatttcccggattacgagccggatgactgggaatcggtattcagcgaattccacgatgctgatgcgcagaactctcacagctattgctttgagattctggagcggcgataa (SEQ ID NO: 19).

[0114] An exemplary stabilizer for DHFR DD is trimethoprim.

[0115] An exemplary FKBP DD is described in US 9,487,787, the contents of which are incorporated herein in their entirety. The amino acid sequence of an exemplary FKBPDD (having an F36V substitution compared to the wild-type sequence) is shown below:

[0116] GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 20).

[0117] The FKBP DD may comprise the wild-type FKBP sequence or may comprise 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 may be included in the FKBP DD include F15S, V24A, H25R, E60G, L106P, D100G, M66T, R71G, D100N, E102G, and K105I. Combinations of the foregoing substitutions may also be used. In some embodiments, the DD comprises the same amino acid sequence as SEQ ID NO: 20, except for the substitutions F15S, V24A, H25R, E60G, L106P, D100G, M66T, R71G, D100N, E102G, or K105I, or a combination thereof.

[0118] Exemplary FKBP DD stabilizers include Shield-1 (Shld1), rapamycin, and FK506.

[0119] Exemplary PDE5 DDs are described in WO 2018 / 237323, the contents of which are incorporated herein in their entirety. PDE5 DDs may 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 common sequence of 823 amino acids.

[0120] All PDE5A isoforms contain a catalytic domain located near the C-terminus of the protein and relatively selective for cGMP as a substrate at physiological levels. This substrate binding site also serves as the binding site for several known PDE5 inhibitors, such as sildenafil, which have been used to treat cardiovascular disease and erectile dysfunction. At the N-terminus, two homologous GAF domains are present. One of the GAF domains, GAF-A, contains a high-affinity binding site for cGMP. Occupancy of this domain by cGMP is known to activate the catalytic domain. Furthermore, the affinity of this site for cGMP is increased by phosphorylation of serine 92 mediated by cGMP-dependent protein kinases. In another embodiment, the PDE5A DD may comprise the catalytic domain of PDE5A, spanning amino acid positions 535 to 860 of UniProt ID: 076074 (SEQ ID NO: 21), as shown in SEQ ID NO: 24. In addition to the catalytic domain, the PDE5A DD may also comprise 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 positions 466 to 875 or positions 420 to 875 of SEQ ID NO: 21. Exemplary PDE5 DD sequences are listed in Table 2.

[0121]

[0122]

[0123]

[0124] Exemplary amino acid substitutions that can be included in PDE5 DD include one or more amino acid substitutions selected from the group consisting of: 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, H678R, D687A, T711A, In some embodiments, the PDE5 DD sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-35 of WO 2018 / 237323 and SEQ ID NOs: 66-69 of WO 2018 / 237323.

[0125] Exemplary stabilizers for PDE5 DD include sildenafil, vardenafil, tadalafil, avanafil, lodenafil, milonafil, udenafil, benzenafil, daxenafil, and beminafil.

[0126] 4.3. Nucleic Acids, Particles, and Host Cells

[0127] In another aspect, the present disclosure provides nucleic acids encoding a polypeptide of the present disclosure (e.g., a polypeptide as described in Section 4.2). The nucleic acid can be, for example, a vector, such as a viral genome or plasmid, or an mRNA molecule.

[0128] Exemplary vectors include viral expression vectors (e.g., viral vectors based on vaccinia virus; 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, 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); al., 1998, Hum Gene Ther 9:81-86; Flannery et al., 1997, PNAS 94:6916-6921; Bennett et al., 1997, Invest Opthalmol VisSci 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 10:10319-23). 73:7812-7816); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); etc. In some cases, the recombinant expression vector of the present disclosure is a recombinant lentiviral vector. In some cases, the recombinant expression vector of the present disclosure is a recombinant retroviral vector.

[0129] In some embodiments, the vector comprises a retroviral genome.The nucleic acid, such as a retroviral genome, can be provided in the form of a particle, such as a viral particle (eg, a retroviral particle).

[0130] The nucleic acids encoding the polypeptides of the present disclosure may further include one or more regulatory sequences, such as promoters, such as SV40, CMV or CAG promoters. An exemplary SV40 promoter sequence is shown below:

[0131] GTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCG CCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAA (SEQ ID NO: 25).

[0132] In another aspect, the present disclosure provides host cells comprising nucleic acids of the present disclosure. The host cells can be prokaryotic (e.g., bacteria such as E. coli) or eukaryotic (e.g., human cell lines such as HEK293 or 293T). The host cells can be used, for example, to propagate nucleic acids such as retroviral genomes or plasmids, or to propagate and package particles, such as retroviral particles.

[0133] 4.4. Methods and treatments for inducing mitophagy

[0134] In a further aspect, the present disclosure provides methods of inducing mitophagy in a cell, and / or increasing mitochondrial turnover in a cell, and / or increasing mitochondrial mass, and / or inducing double-strand breaks in mitochondrial DNA and / or (e) inducing epigenomic modifications in a cell using the polypeptides, nucleic acids, and particles of the present disclosure, e.g., the polypeptides, nucleic acids, and particles described in Sections 4.2 and 4.3.

[0135] The method generally comprises contacting the cell with a polypeptide, nucleic acid, or particle and a stabilizer capable of stabilizing DD. The polypeptide can be introduced into the cell by electroporation, injection, or a carrier (e.g., a lipid-based carrier such as a liposome), or any other means known in the art for delivering polypeptides to cells. The nucleic acid can be introduced into the cell 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 the cell by transduction.

[0136] The cells can be contacted with the stabilizer, for example, by culturing the cells in a culture medium comprising the stabilizer. The cells can be cultured in the culture medium with the stabilizer for a period of time to allow the endonuclease to introduce a DSB into the mitochondrial DNA. In some embodiments, the cells are cultured in the culture medium with the stabilizer 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). Subsequently, the stabilizer can be removed, for example, by culturing the cells in a culture medium without the stabilizer. Once the stabilizer is removed, the polypeptide will be destabilized, leading to degradation of the polypeptide.

[0137] After the stabilizer is removed, the cells can be cultured without the stabilizer for a period of time during which the cells can produce new mitochondria. In some embodiments, the cells are cultured in a culture medium that does not include the stabilizer 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).

[0138] In some embodiments, the methods of the present disclosure result in induction of mitochondrial autophagy in the cell. In some embodiments, the methods of the present disclosure result in an increase in mitochondrial turnover in the cell. In some embodiments, the methods of the present disclosure result in an increase in mitochondrial mass in the cell. In some embodiments, the method results in induction of a DSB in mitochondrial DNA in the cell. In some embodiments, the method results in epigenomic modifications in the cell, for example, induced by mitochondrial depletion. It has been previously reported that rho0 cells, which have a complete depletion of the mitochondrial genome, show significant levels of epigenomic changes (see, e.g., Hertzog Santos, 2021 Free Radic Biol Med. 170:69-69). Thus, it is believed that the compositions of the present disclosure can be used to induce epigenomic modifications. In some embodiments, the methods of the present disclosure result in one, two, three, four, or all five of the following in the cell: (a) induction of mitochondrial autophagy, (b) increased mitochondrial turnover, (c) increased mitochondrial mass, (d) a DSB in mitochondrial DNA, and (e) epigenomic modifications.

[0139] Exemplary cells that can be used in the methods include mammalian cells, preferably human cells, more preferably human somatic cells. Cell types 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 CD4+ T cells. Primary cells obtained from a subject, as well as their progeny, can be used.

[0140] The cells are normal cells (e.g., from a healthy donor) or have dysfunctional mitochondria (e.g., from a subject suffering from a disease or disorder). For example, the cells can be from a subject suffering from an age-related disease or disorder, such as an autoimmune disease, a metabolic disease, a genetic disease, cancer, a neurodegenerative disease, or immunosenescence.

[0141] As another example, the cell can be from a subject with a mitochondrial disease or disorder, such as chronic progressive external ophthalmoplegia (CPEO), Pearson syndrome, Kearns-Sell syndrome (KSS), diabetes mellitus and deafness (DAD), mitochondrial diabetes, Leber hereditary optic neuropathy (LHON), LHON-plus, neuropathy, ataxia, retinitis pigmentosa syndrome (NARP), maternally inherited Leigh syndrome (MILS), mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), myoclonus epilepsy with ragged red myofibers (MERRF), familial bilateral striatal necrosis / striatonigral degeneration (FBSN), Rafter disease, aminoglycoside-induced deafness (AID), or mitochondrial DNA multiple deletion syndrome. Additional mitochondrial diseases and disorders include mitochondrial DNA depletion syndrome 4A, mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase gamma (POLG)-related disorders, sensory ataxic neuropathy with dysarthria and ophthalmoplegia (SANDO), leukoencephalopathy involving the brainstem and spinal cord with elevated lactate (LBSL), coenzyme Q10 deficiency, Leigh syndrome, mitochondrial complex abnormalities, fumarase deficiency, alpha-ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenase complex deficiency (PDHC), pyruvate carboxylase deficiency (PCD), carnitine palmitoyltransferase I (CPT I) deficiency, carnitine palmitoyltransferase II (CPT II) deficiency, and pyruvate carboxylase deficiency (PCD). IT) deficiency, carnitine-acylcarnitine (CACT) deficiency, autosomal dominant / autosomal recessive progressive external ophthalmoplegia (ad- / ar-PEO), infantile-onset spinocerebellar atrophy (IOSCA), mitochondrial myopathy (MM), spinal muscular atrophy (SMA), growth retardation, aminoaciduria, cholestasis, iron overload, premature death (GRACILE), and Charcot-Marie-Tooth disease type 2A (CMT2A).

[0142] As another example, the cells can be from a subject having 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. In some embodiments, the cells are from a subject having Alzheimer’s disease, e.g., a subject having an APOE4 allele, e.g., an E3 / E4 or E4 / E4 genotype.

[0143] As yet another example, the cells can be from a subject having an eye disease (e.g., a retinal disease) such as age-related macular degeneration, macular edema, or glaucoma.

[0144] In further examples, the cells can be from a subject having diabetes, a hearing disorder, a genetic disease (such as Hutchinson-Gilford Progeria Syndrome, Wohrman Syndrome, or Huntington’s disease), heart failure, an immunodeficiency, a cancer, or an infectious disease.

[0145] The cells obtained or obtainable by the methods described herein can be administered to a subject, e.g., to the subject from which the cells were derived, or to a different subject in the case that the cells are from a healthy donor.

[0146] Accordingly, in another aspect, the disclosure provides methods of treating a subject having an age-related disease, a mitochondrial disease or disorder, a neurodegenerative disease, a retinal disease, diabetes, a hearing disorder, a genetic disease, heart failure, an immunodeficiency, a 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.

[0147] 4.5. Pharmaceutical Compositions and Kits

[0148] In another aspect, the present disclosure provides pharmaceutical compositions comprising a polypeptide of the disclosure (e.g., as described in Section 4.2), a nucleic acid of the disclosure (e.g., as described in Section 4.3), a particle of the disclosure (e.g., as described in Section 4.3), or a cell of the disclosure (e.g., obtained by the method described in 4.4) and a pharmaceutically acceptable excipient. For example, pharmaceutical compositions can be prepared by mixing the polypeptide, nucleic acid, particle or cell with one or more physiologically acceptable carriers, excipients or stabilizers, for example, 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).

[0149] In another aspect, 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, when the DD sequence of the polypeptide is a DHFR DD sequence, the kit may include trimethoprim (TMP), when the DD sequence of the polypeptide is an FKBP DD sequence, the kit may include Shield-1, rapamycin, or FK506, or when the DD sequence of the polypeptide is a PDE5 DD sequence, the kit may include sildenafil, vardenafil, tadalafil, avanafil, lodenafil, milonafil, udenafil, benzamidine, dasafil, and beminafil. Example

[0150] 5.1. Example 1: Polypeptides for inducing mitophagy, biogenesis, and accelerating mitochondrial turnover

[0151] This example describes compositions and methods for simultaneously activating not only mitophagy but also mitophagy biogenesis, and for accelerating mitochondrial turnover by eliminating dysfunctional mitochondria and generating new mitochondria.

[0152] 5.1.1. Transgenic design

[0153] XbaIR is selected to induce DSB in the mitochondrial genome. XbaIR has five cleavage sites in the mitochondrial genome consensus sequence. In order to guide the XbaIR endonuclease to mitochondria, the Cox8a post-mitochondrial signal is placed on the N-terminal side of XbaIR. Inducing DSB in the mitochondrial genome leads to strong temporary energy depletion, and, in order to control endonuclease activity, the DHFR destabilization domain (Liu et al., 2014 Int. J. Parasitol. 44(10):729-735) is fused to the C-terminus of XbaIR to allow sensitive on / off control of the endonuclease. The DHFR destabilization domain is stabilized by the antibiotic trimethoprim (TMP).

[0154] Construction of a retroviral vector with the MTS-XbaIR-DHFR coding sequence ( Figure 1 ). The nucleotide sequence of the vector is shown in Table 3.

[0155]

[0156]

[0157]

[0158]

[0159]

[0160] Molecular modeling was used to confirm that the three functional domains of the polypeptide construct (MTS, XbaI and DHFR) adopted a three-dimensional structure and did not interfere with each other's structure ( Figure 2 In addition, a retroviral vector with EGFP-DHFR as a transgene was created to evaluate the responsiveness of the DHFR / TMP system ( Figure 3 and Figure 4 ). The nucleotide sequence of the vector is shown in Table 4.

[0161]

[0162]

[0163]

[0164]

[0165]

[0166] 5.1.2. Responsiveness of the DHFR / TMP System

[0167] EGFP-DHFR retroviral vector was transduced into Hela cells. Infection efficiency was high and without enrichment of infected cells, TMP exposure was performed for two days at various concentrations, followed by detection of EGFP fluorescence expression using fluorescence microscopy and FACS Figure 5 and Figure 6 ). Transcription and presence of EGFP mRNA was observed Figure 7 ), but no protein was observed Figure 5 and Figure 6 ) in the absence of TMP.

[0168] To confirm OFF control of the construct, medium washout was performed after two days of TMP exposure and fluorescence intensity was checked over time by fluorescence microscopy and FACS Figure 8A ). EGFP fluorescence dropped abruptly one hour after removal of TMP in the medium, then fluorescence dissipated after four hours Figure 8B and Figure 8C ). In addition, no expression leakage of the transgene was observed Figure 8D ) within 48 hours after TMP OFF.

[0169] To confirm ON control of the construct, addition of TMP was performed and fluorescence intensity was checked over time by fluorescence microscopy and FACS Figure 9A ). EGFP fluorescence turned on abruptly one hour after addition of TMP in the medium, then fluorescence reached more than 80% of maximum intensity after six hours Figure 9B and Figure 9C ). From eight hours, intensity tended to stabilize until the 48 hours time point Figure 9D ).

[0170] 5.1.3. Mitochondrial features after genetic induction of mitophagy

[0171] Hela cells were transfected with MTS-XbaIR-DHFR vector and exposed to 0.5 μΜ TMP for two days, followed by washout. XbaRI RNA expression was measured after TMP exposure. Transcriptional level of the transgene did not significantly change Figure 10A ). CN decreased to less than half of the initial value at day 2 Figure 10B ).

[0172] Hela transfectants with MTS-XbaIR-DHFR were cloned by limiting dilution, named Hela MXD sc20. Hela MXD sc20 was subjected to 0.5 mM TMP exposure for several time periods (16, 20 and 48 hours) to examine mitochondrial mass (mtMass), which was measured by Mito Green staining, overall mitochondrial membrane potential (mtMP), which was measured by TMRM staining, and mtMP corrected with mtMass, which was calculated as the ratio of mtMP to mtMass Figures 11A-11C ) Regardless of the duration of TMP exposure, mtMass temporarily increased to about twice compared to the resting state, and then returned to the initial volume 10 hours after TMP was turned on, indicating that mitochondrial biogenesis was temporarily and strongly activated Figure 11A ) Both overall and corrected mtMP showed a sharp decrease and then an increase, indicating poor pumping of hydrogen ions through respiratory chain complexes I, III and IV or counterclockwise rotating complex V Figure 11B and Figure 11C ).

[0173] The introduced transgene seems to cause DSB of the mitochondrial genome and decrease CN, in addition, although proteins from the nucleus are required, the increase in MM shows a high responsiveness to this stress. Based on these two factors, it is considered that the density of the respiratory chain complex will decrease, and it is considered that as a phenotype MMP will decrease. The mitochondrial genome seems to return to normal at day 6, with a slight increase in MM. Without being bound by theory, it is considered that this indicates that mitochondrial biogenesis is enhanced in the MTS-XbaIR-DHFR / TMP system, and that mitochondrial capacity increases due to the abundance of mitochondria.

[0174] Next, whether changes in cell proliferation and viability occur in this system and affect the above changes were investigated. In this system with or without TMP, no significant difference in cell number or viability was observed Figures 12A-12B ) Therefore, it was determined that the main benefit of this system is the intervention of the MTS-XbaIR-DHFR polypeptide on mitochondria.

[0175] 5.1.4. Effect on mitochondrial autophagy after MTS-XbaIR-DHFR / TMP

[0176] HeLa MXD sc20 cells transfected with mtKeima-Red were generated to overexpress PARK2 in order to quantify mitophagy in a more refined manner. Transfectants exposed to TMP for different durations were examined to measure mitophagy (16, 20, 24, 40, 44, and 48 hours). The mitophagy inducer carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as a positive control. With increasing duration of TMP exposure, more mitophagy was observed ( Figure 13A After 40 hours of TMP exposure, mitochondrial autophagy reached a stable phase ( Figure 13A ). At the same time, it was observed that the measured mtDNA CN returned to its initial value regardless of the duration of TMP exposure ( Figure 13B Taken together, the results indicate that mitochondrial turnover is accelerated by MTS-XbaIR-DHFR / TMP.

[0177] 5.2. Example 2: Changes in mitochondrial function and biogenesis due to genetically induced mitophagy

[0178] This example describes the changes in mitochondrial biogenesis and function associated with genetically induced mitophagy (GiM).

[0179] Materials and methods

[0180] After using retroviral gene transfer, MTS-XbaI-ecDHFR was cloned into Hela cells and a stable transfectant (Hela_GiM) constitutively expressing the GiM unit was generated. During the 2-day treatment of Hela_GiM cells with trimethoprim (TMP), the endonuclease XbaI was transiently present in the mitochondrial matrix. Since TMP is dissolved in DMSO, the control group was treated with the same amount of DMSO.

[0181] By staining cells with mitoSox and measuring fluorescence intensity by FACS, setting a threshold line compared to unstressed cells, and measuring the percentage of positive cells, reactive oxygen species (ROS) that change over time were evaluated. qPCR was used to evaluate the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) associated with mitochondrial biogenesis, nuclear factor receptor 1 (NRF1) involved in mitochondrial biogenesis, and mitochondrial transcription factor A (TFAM) that forms a nucleoid with mtDNA and is deeply involved in mtDNA transcription, replication, and maintenance. The expression levels of several nuclear-encoded and mtDNA-encoded proteins were quantified by Western blotting.

[0182] 5.2.2. Results

[0183] Throughout the evaluation, the levels of ROS initially increased in the TMP-treated group, but were essentially unchanged in the control group. On day 2, the percentage of mitoSox-positive cells was higher in the TMP-treated group than in the control group. On day 4 and onwards, the percentage of mitoSox-positive cells was comparable in both groups Figure 14A ). At all observation points, the transcription factors promoting mitochondrial biogenesis, PGC1a and NRF1, were also found to be elevated in the TMP-treated group relative to the control group Figure 14B and 14C , and TFAM remained essentially unchanged in the TMP-treated group on days 2, 4 and 6 Figure 14D .

[0184] Without being bound by theory, it is believed that these results indicate that ROS production is temporarily enhanced by the transient mitochondrial genome reduction, whereby mitochondrial biogenesis is induced by the amplification of transcription factors such as PGC1a and NRF1. Also without being bound by theory, it is further believed that the maintenance of elevated transcriptional levels of TFAM, the major component protein of the nucleoid, indicates that mitochondrial biogenesis continues for a period of time after the mitochondrial genome reduction is triggered.

[0185] Next, the effect of GiM on the expression of nuclear-encoded and mtDNA-encoded proteins was evaluated. The expression level of ATP5A, a respiratory chain complex encoded in the nucleus, was essentially unchanged with mitochondrial genome reduction Figure 15A and 15B ). The expression of TOM20, an outer membrane translocase also encoded in the nucleus, decreased immediately after mitochondrial genome reduction, but recovered to levels comparable to those observed in the DMSO-treated group by day 6 Figure 15A and 15C ). On the other hand, in the TMP-treated group, ATP6, which is encoded by mtDNA, decreased substantially immediately after mitochondrial genome reduction, then increased on day 4, and thereafter recovered to levels comparable to the control group Figure 15A and 15D ). This trend was also observed in terms of the ATP6 / ATP5A ratio Figure 15A and 15E ). These results indicate that GiM induces a transient surge of mitochondrial proteins derived from the mitochondrial genome, but not the nuclear genome.

[0186] Changes in the levels of AMPK-activated form (p-AMPK) were used to evaluate changes in mitochondrial energy production after mitochondrial genome reduction. Mitochondrial energy production was confirmed by the increase in p-AMPK by day 4 Figure 15A and 15F). Transient mitochondrial genome reduction was associated with a decrease in respiratory chain complex capacity at day 2, and this effect was directly related to cellular energy depletion, leading to a significant increase in p-AMPK at day 4.

[0187] 5.3. Example 3: Autophagy associated with GiM-induced transient mitochondrial genome reduction

[0188] Mitophagy occurs when mitochondria are incorporated into phagophores and become autolysosomes by fusing with lysosomes. This example describes compositions and methods for detecting autolysosome formation and autophagy following GiM-induced transient mitochondrial genome reduction.

[0189] 5.3.1. Materials and methods

[0190] Autolysosomes have a lower pH than mitochondria that do not fuse with lysosomes. Autolysosome formation was assessed in cells following 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, and has a transit signal that allows it to be transported into mitochondria. Cells stably expressing mKeimaRed emit red light when the environmental pH is below 5. To quantify autolysosomes following GiM-induced transient mitochondrial genome reduction, HeIa_GiM cells described in section 5.2.1 were retrovirally engineered with the sequence encoding mKeimaRed. The percentage of cells undergoing mitophagy was quantified every two days for two weeks using fluorescence microscopy and phase-contrast microscopy.

[0191] The last step of the autophagic flux depends on lysosomal V-ATPase activity. Therefore, the last step of the autophagic flux was inhibited using the lysosomal V-ATPase inhibitor Bafilomycin Al (BafAl) following GiM-induced transient mitochondrial genome reduction to assess the autophagic flux targeting mitochondria. Antibody staining of LC3 (MAP1LC3: microtubule-associated protein 1 light chain 3), a representative marker of autophagosome formation, and TOM20 staining as a mitochondrial membrane marker were used to quantify autophagosomes. Given that LC3 exists as LC3-II with PE on the autophagosome membrane, LC3-II was quantified by western blot in HeIa_GiM cells treated with BafAl, where proteins were extracted from cells at day 8 following exposure to TMP for 48 h.

[0192] 5.3.2. Results

[0193] The peak of mtKeimaRed signal associated with GiM-induced autolysosome formation was at day 8 ( Figure 16A ), mitochondrial autophagy occurs in approximately 20% of cells ( Figure 16B Time course analysis showed that the kinetics of mitophagy increased on day 6 and decreased to control levels by day 14, with only a small percentage of cells undergoing mitophagy ( Figure 16B This transient surge in induced mitophagy was not associated with cell death or decreased 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.

[0194] Autophagosomes formed after fusion of mitochondria and lysosomes were detected as spots with overlapping LC3 and TOM20 staining ( Figure 17A In the absence of TMP, the size of the spots with overlapping LC3 and TOM20 staining was approximately 80 μm 2 , and was not significantly affected by the use of BafA1. On the other hand, in the TMP-exposed group, the area of ​​puncta with overlapping LC3 and TOM20 staining increased significantly to approximately 120 μm with the addition of BafA1. 2 ( Figure 17B Although LC3-II was found to be significantly increased by GiM, this increase was more pronounced and significant when BafA1 was used ( Figure 18A and 18B Taken together, these results indicate that GiM significantly promotes autophagy.

[0195] 5.4. Example 4: Metabolic effects of mitochondrial genome reduction

[0196] Using Hela_GiM cells as described in Section 5.2.1, mitochondrial genome reduction was performed after 2 days of TMP exposure. Respiration was performed over time using SeaHorse to assess oxidative phosphorylation (OXPHOS) and glycolysis. Figure 19A , left panel) decreased until day 4 and gradually increased from day 6 until approaching the starting level on day 10. On the other hand, glycolysis increased until day 8 and then decreased on day 10 ( Figure 19A , right). A two-dimensional diagram depicting the relationship between OXPHOS and glycolysis shows cyclic changes, indicating that metabolic changes are transient ( Figure 19B Separate assessments of ATP production, basal respiration, proton leak, and spare capacity further support the transient nature of GiM-associated metabolic changes ( Figure 19C Even with respect to oxygen consumption, the metabolic effects of GiM suggest that transient mitochondrial genome reduction is a reversible change.

[0197] 5.5. Example 5: Genetically Induced Mitophagy in AD Fibroblasts

[0198] Alzheimer's disease (AD) is associated with mitochondrial dysfunction. This example describes how transient mitochondrial genome reduction by gene transfer transforms cell phenotype by enhancing mitochondrial turnover in fibroblasts obtained from patients with AD.

[0199] 5.5.1. Materials and methods

[0200] Normal human skin fibroblasts (NHDF) and fibroblasts obtained from a forearm skin sample of a patient with Alzheimer's disease, whose APOE genotype was E3 / E45 (AD fibroblasts), were used as target cells. Transient mitochondrial genome reduction was performed by plasmid transfer, which carries the gene encoding endonuclease XbaIR downstream of the mitochondrial transfer signal derived from human Cox8, and expresses puromycin resistance as a selection marker under different promoters (pCAGGS-MTS-XbaIR). Electroporation was used as gene transfer method, and enrichment was performed by exposing the transfected target cells to puromycin at a concentration of 3 pg / mL for 24 hours at day 2 after electroporation. This condition was set using a plasmid with the recombinant GFP gene instead of XbaIR, following the criteria of 70-80% GFP expression and more than 90% viability. Copy number of mitochondrial genome was assessed at days 7, 14 and 21 after gene transfer to confirm genome reduction and subsequent biogenesis Figure 20A ). In addition, mitochondrial phenotype was assessed by measuring mitochondrial volume (mtMass) and mitochondrial membrane potential (mtMP) using MitoTracker Green and TMRM, respectively.

[0201] 5.5.2. Results

[0202] In untreated AD fibroblasts, mtMP was significantly reduced relative to untreated NHDF Figure 20B and 20E ). Similarly, mtMass was reduced in untreated AD fibroblasts relative to untreated NHDF Figure 20B and 20D ). Both fluorescence signals were two-dimensionally amplified and quadrant analysis was performed using NHDF as a positive control to set the threshold line. The score ratio was used as a biomarker to detect senescence of lymphocytes with mitochondrial dysfunction. This double-positive rate was found to be about half of NHDF. The percentage of double-positive did not change significantly at day 7 after genetic induction of mitochondrial autophagy (GiM), but increased to 60.3% at day 14 and further to 87.3% at day 21, which was comparable to control NHDF Figure 20C ).

[0203] Both mtMass and mtMP were quantified using mean fluorescence intensity (MFI) as an indicator of fluorescence intensity. Both mtMass and mtMP were lower in untreated AD fibroblasts relative to NHDF cells. In AD fibroblasts, mtMass and mtMP levels were even lower on day 7 after GIM induction, but increased to levels comparable to NHDF on day 14 ( Figures 20F-20G ), indicating that both mitochondrial volume and mitochondrial membrane potential in AD fibroblasts were restored to the same level as those in healthy NHDF cells by GiM.

[0204] These results suggest that GiM-promoted mitochondrial turnover is qualitatively beneficial for newly generated mitochondria. Given that the accumulation of dysfunctional mitochondria is a common phenomenon in various neurodegenerative diseases and aging, GiM may be used as a therapeutic strategy for treating degenerative diseases and may also be used to restore normal function in aging cells.

[0205] 6. Specific implementation plan

[0206] The present disclosure is exemplified by the following specific embodiments.

[0207] 1. A polypeptide comprising:

[0208] (a) Mitochondrial targeting sequence (MTS);

[0209] (b) an endonuclease sequence; and

[0210] (c) Destabilization domain sequence.

[0211] 2. The polypeptide of embodiment 1, wherein the MTS comprises human MTS.

[0212] 3. The polypeptide of embodiment 1, wherein the MTS comprises a non-human MTS.

[0213] 4. The polypeptide of any one of embodiments 1 to 3, wherein the MTS comprises the MTS of a mitochondrial protein.

[0214] 5. The polypeptide of any one of embodiments 1 to 4, wherein the MTS comprises TCA cycle-related enzymes, chaperone proteins, mitochondrial genome replication proteins, proteases, mRNA processing proteins, mitochondrial RNA degradation proteins, deoxynucleotide triphosphate synthesis-related proteins, mitochondrial ribosomal proteins, phospholipid metabolism-related proteins, proteins involved in the metabolism of toxic compounds, disulfide bond relay system-related proteins, iron-sulfur protein assembly proteins, tRNA modification proteins, aminoacyl-tRNA synthetases, release factors or elongation factors.

[0215] 6. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises the MTS of a cytochrome c oxidase subunit.

[0216] 7. The polypeptide of embodiment 6, wherein the MTS comprises the MTS of cytochrome c oxidase subunit VIII (COX8).

[0217] 8. The polypeptide of embodiment 6, wherein the MTS comprises the MTS of cytochrome c oxidase subunit X (COX10).

[0218] 9. The polypeptide of embodiment 6, wherein the MTS comprises the MTS of cytochrome c oxidase subunit IV (COX4).

[0219] 10. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises the MTS of frataxin (FXN).

[0220] 11. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises the MTS of a TCA cycle-associated enzyme, optionally the TCA cycle-associated enzyme is pyruvate dehydrogenase, citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinyl CoA synthetase, succinate dehydrogenase, fumarase, malate dehydrogenase, or pyruvate carboxylase.

[0221] 12. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises the MTS of a chaperone, optionally the chaperone is mtHSP10, mtHSP60, mtHSP70, or mtHSP90.

[0222] 13. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises the MTS of a mitochondrial genome replication protein, optionally the mitochondrial genome replication protein is TFAM, Twinkle, PolG, TFB2M, TEFM, or MTERF1.

[0223] 14. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises the MTS of a protease, optionally the protease is MPP, CLPXP, LON ATPase, or PreP.

[0224] 15. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises the MTS of an mRNA processing protein, optionally the mRNA processing protein is LRPPRC, TACOl, ELAC2, PNPT1, HSD17B10, MTPAP, or PTCD1.

[0225] 16. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises an MTS of a mitochondrial RNA degradation protein, optionally the mitochondrial RNA degradation protein is PNPasse, REX02, or SUV3.

[0226] 17. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises an MTS of a deoxynucleotide triphosphate synthesis-related protein, optionally wherein the deoxynucleotide triphosphate synthesis-related protein is DGUOK, TK2, TYMP, MGME1, SUCLG1, SUCLA2, RNASEH1 or C10orf2.

[0227] 18. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises the MTS of a mitochondrial ribosomal protein, optionally the mitochondrial ribosomal protein is MRPS16, MRPS22, MRPL3, MRP12 or MRPL44.

[0228] 19. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises the MTS of a phospholipid metabolism-related protein, optionally the phospholipid metabolism-related protein is AGK, SERAC1 or TAZ.

[0229] 20. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises the MTS of a protein involved in the metabolism of a toxic compound, optionally the protein involved in the metabolism of a toxic compound is HIBCH, ECHS1, ETHE1 or MPV17.

[0230] 21. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises the MTS of a disulfide-relay system-associated protein, optionally wherein the disulfide-relay system-associated protein is GFER.

[0231] 22. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises the MTS of an iron-sulfur protein assembly protein, optionally the iron-sulfur protein assembly protein is ISCU, BOLA3, NFU1 or IBA57.

[0232] 23. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises an MTS of a tRNA-modifying protein, optionally the tRNA-modifying protein is MTO1, GTP3BP, TRMU, PUS1, MTFMT, TRIT1, TRNT1, or TRMT5.

[0233] 24. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises an MTS of an aminoacyl-tRNA synthetase, optionally the aminoacyl-tRNA synthetase is AARS2, DARS2, EARS2, RARS2, YARS2, FARS2, HARS2, LARS2, VARS2, TARS2, IARS2, CARS2, PARS2, NARS2, KARS, GARS, SARS2, or MARS2.

[0234] 25. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises an MTS that releases a factor, optionally the release factor is C12orf65.

[0235] 26. The polypeptide of any one of embodiments 1 to 5, wherein the MTS comprises the MTS of an elongation factor, optionally the elongation factor is TUFM, TSFM or GFM1.

[0236] 27. The polypeptide of embodiment 1, wherein the MTS comprises a sequence at least 80% identical to msvltplllrgltgsarr (SEQ ID NO: 1).

[0237] 28. The polypeptide of embodiment 27, wherein the MTS comprises a sequence at least 85% identical to msvltplllrgltgsarr (SEQ ID NO: 1).

[0238] 29. The polypeptide of embodiment 27, wherein the MTS comprises a sequence at least 90% identical to msvltplllrgltgsarr (SEQ ID NO: 1).

[0239] 30. The polypeptide of embodiment 27, wherein the MTS comprises a sequence at least 95% identical to msvltplllrgltgsarr (SEQ ID NO: 1).

[0240] 31. The polypeptide of embodiment 27, wherein the MTS comprises a sequence that is 100% identical to msvltplllrgltgsarr (SEQ ID NO: 1).

[0241] 32. The polypeptide of embodiment 1, wherein the MTS comprises a sequence at least 80% identical to MSVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO: 2).

[0242] 33. The polypeptide of embodiment 32, wherein the MTS comprises a sequence at least 85% identical to MSVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO: 2).

[0243] 34. The polypeptide of embodiment 32, wherein the MTS comprises a sequence at least 90% identical to MSVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO: 2).

[0244] 35. The polypeptide of embodiment 32, wherein the MTS comprises a sequence at least 95% identical to MSVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO: 2).

[0245] 36. The polypeptide of embodiment 32, wherein the MTS comprises a sequence 100% identical to MSVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO: 2)

[0246] 37. The polypeptide of embodiment 1, wherein the MTS comprises a sequence at least 80% identical to MSVLTPLLLRSLTGSARRLMVPRA (SEQ ID NO: 3).

[0247] 38. The polypeptide of embodiment 37, wherein the MTS comprises a sequence at least 85% identical to MSVLTPLLLRSLTGSARRLMVPRA (SEQ ID NO: 3).

[0248] 39. The polypeptide of embodiment 37, wherein the MTS comprises a sequence at least 90% identical to MSVLTPLLLRSLTGSARRLMVPRA (SEQ ID NO: 3).

[0249] 40. The polypeptide of embodiment 37, wherein the MTS comprises a sequence at least 95% identical to MSVLTPLLLRSLTGSARRLMVPRA (SEQ ID NO: 3).

[0250] 41. The polypeptide of embodiment 37, wherein the MTS comprises a sequence that is 100% identical to MSVLTPLLLRSLTGSARRLMVPRA (SEQ ID NO: 3).

[0251] 42. The polypeptide of embodiment 1, wherein the MTS comprises a sequence at least 80% identical to MAASPHTLSSRLLTGCVGGSVWYLERRT (SEQ ID NO: 4).

[0252] 43. The polypeptide of embodiment 42, wherein the MTS comprises a sequence at least 85% identical to MAASPHTLSSRLLTGCVGGSVWYLERRT (SEQ ID NO: 4).

[0253] 44. The polypeptide of embodiment 42, wherein the MTS comprises a sequence at least 90% identical to MAASPHTLSSRLLTGCVGGSVWYLERRT (SEQ ID NO: 4).

[0254] 45. The polypeptide of embodiment 42, wherein the MTS comprises a sequence at least 95% identical to MAASPHTLSSRLLTGCVGGSVWYLERRT (SEQ ID NO: 4).

[0255] 46. ​​The polypeptide of embodiment 42, wherein the MTS comprises a sequence that is 100% identical to MAASPHTLSSRLLTGCVGGSVWYLERRT (SEQ ID NO: 4).

[0256] 47. The polypeptide of embodiment 1, wherein the MTS comprises a sequence at least 80% identical to MWTLGRRAVAGLLASPSPAQ (SEQ ID NO: 5).

[0257] 48. The polypeptide of embodiment 47, wherein the MTS comprises a sequence at least 85% identical to MWTLGRRAVAGLLASPSPAQ (SEQ ID NO: 5).

[0258] 49. The polypeptide of embodiment 47, wherein the MTS comprises a sequence at least 90% identical to MWTLGRRAVAGLLASPSPAQ (SEQ ID NO: 5).

[0259] 50. The polypeptide of embodiment 47, wherein the MTS comprises a sequence at least 95% identical to MWTLGRRAVAGLLASPSPAQ (SEQ ID NO: 5).

[0260] 51. The polypeptide of embodiment 47, wherein the MTS comprises a sequence that is 100% identical to MWTLGRRAVAGLLASPSPAQ (SEQ ID NO: 5).

[0261] 52. 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).

[0262] 53. 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).

[0263] 54. 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).

[0264] 55. 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).

[0265] 56. 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).

[0266] 57. 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).

[0267] 58. 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).

[0268] 59. 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).

[0269] 60. 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).

[0270] 61. 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).

[0271] 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).

[0272] 63. The polypeptide of embodiment 62, wherein the endonuclease is a restriction endonuclease.

[0273] 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.

[0274] 65. The polypeptide of embodiment 64, wherein the restriction endonuclease is XbaIR endonuclease.

[0275] 66. The polypeptide of any one of embodiments 1 to 63, wherein the endonuclease comprises a sequence at least 80% identical to MTTLEKIKLLADGYADRLKLAIDGRVLEMQGDDVSHYLIYRVLGVAQEEGRLIDVYQNKGRFLYKYAGSFLEAATKLCFKEAFPDSASLRLPNTQGQRPRTVEIDCLVGNDALEIKWKDATTDGDHITKEHTRIKVISDAGYKPIRIMFYYPHRTQAIRIQETLETLYNGVHGEYHYGEAAWDYVLQRTSVNLKVALEQIADSRTNEAA (SEQ ID NO: 16).

[0276] 67. The polypeptide of embodiment 66, wherein the endonuclease sequence comprises a sequence that is at least 85% identical to MTTLEKIKLLADGYADRLKLAIDGRVLEMQGDDVSHYLIYRVLGVAQEEGRLIDVYQNKGRFLYKYAGSFLEAATKLCFKEAFPDSASLRLPNTQGQRPRTVEIDCLVGNDALEIKWKDATTDGDHITKEHTRIKVISDAGYKPIRIMFYYPHRTQAIRIQETLETLYNGVHGEYHYGEAAWDYVLQRTSVNLKVALEQIADSRTNEAA (SEQ ID NO: 16).

[0277] 68. The polypeptide of embodiment 66, wherein the endonuclease sequence comprises a sequence that is at least 90% identical to MTTLEKIKLLADGYADRLKLAIDGRVLEMQGDDVSHYLIYRVLGVAQEEGRLIDVYQNKGRFLYKYAGSFLEAATKLCFKEAFPDSASLRLPNTQGQRPRTVEIDCLVGNDALEIKWKDATTDGDHITKEHTRIKVISDAGYKPIRIMFYYPHRTQAIRIQETLETLYNGVHGEYHYGEAAWDYVLQRTSVNLKVALEQIADSRTNEAA (SEQ ID NO: 16).

[0278] 69. The polypeptide of embodiment 66, wherein the endonuclease sequence comprises a sequence that is at least 95% identical to MTTLEKIKLLADGYADRLKLAIDGRVLEMQGDDVSHYLIYRVLGVAQEEGRLIDVYQNKGRFLYKYAGSFLEAATKLCFKEAFPDSASLRLPNTQGQRPRTVEIDCLVGNDALEIKWKDATTDGDHITKEHTRIKVISDAGYKPIRIMFYYPHRTQAIRIQETLETLYNGVHGEYHYGEAAWDYVLQRTSVNLKVALEQIADSRTNEAA (SEQ ID NO: 16).

[0279] 70. The polypeptide of embodiment 66, wherein the endonuclease sequence comprises a sequence that is 100% identical to MTTLEKIKLLADGYADRLKLAIDGRVLEMQGDDVSHYLIYRVLGVAQEEGRLIDVYQNKGRFLYKYAGSFLEAATKLCFKEAFPDSASLRLPNTQGQRPRTVEIDCLVGNDALEIKWKDATTDGDHITKEHTRIKVISDAGYKPIRIMFYYPHRTQAIRIQETLETLYNGVHGEYHYGEAAWDYVLQRTSVNLKVALEQIADSRTNEAA (SEQ ID NO: 16).

[0280] 71. The polypeptide of any one of embodiments 1-70, wherein the destabilization domain sequence is a DHFR, FKBP, or PDE5 destabilization domain sequence.

[0281] 72. The polypeptide of embodiment 71, wherein the destabilization domain sequence is a DHFR destabilization domain sequence.

[0282] 73. The polypeptide of embodiment 72, wherein the destabilization domain sequence is an E. coli DHFR (ecDHFR) destabilization domain sequence.

[0283] 74. The polypeptide of any one of embodiments 1-73, 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 MISLIAALAVDHVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR (SEQ ID NO: 17).

[0284] 75. The polypeptide of any one of embodiments 1-73, 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 ISLIAALAVDHVIGMETVMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR (SEQ ID NO: 18).

[0285] 76. 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.

[0286] 77. The polypeptide of any one of embodiments 1-71, 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 GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 20).

[0287] 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.

[0288] 79. The polypeptide of any one of embodiments 1-71, 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.

[0289] 80. The polypeptide of any one of embodiments 1-79, wherein the MTS is N-terminal to the endonuclease sequence and the destabilization domain sequence.

[0290] 81. The polypeptide of any one of embodiments 1-79, wherein the MTS is located C-terminal to the endonuclease sequence and the destabilization domain sequence.

[0291] 82. The polypeptide of any one of embodiments 1-81, wherein the endonuclease sequence is located N-terminal to the destabilization domain sequence.

[0292] 83. The polypeptide of any one of embodiments 1-81, wherein the endonuclease sequence is located C-terminal to the destabilization domain sequence.

[0293] 84. The polypeptide of any one of embodiments 1-79, wherein the MTS is located N-terminal to the endonuclease sequence and the endonuclease sequence is located N-terminal to the destabilization domain sequence.

[0294] 85. A nucleic acid encoding the polypeptide of any one of embodiments 1-84.

[0295] 86. The nucleic acid of embodiment 85, comprising a promoter operably linked to the nucleotide sequence encoding the polypeptide.

[0296] 87. The nucleic acid of embodiment 86, wherein the promoter is an SV40 promoter, a CMV promoter, or a CAG promoter.

[0297] 88. The nucleic acid of embodiment 87, wherein the promoter is an SV40 promoter.

[0298] 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.

[0299] 90. The nucleic acid of any one of embodiments 85-89, which is a vector.

[0300] 91. The nucleic acid of embodiment 90, wherein the vector is a retroviral genome.

[0301] 92. The nucleic acid of embodiment 91, wherein the vector is a mouse retroviral genome.

[0302] 93. The nucleic acid of embodiment 90, wherein the vector is a plasmid.

[0303] 94. The nucleic acid of embodiment 85, which is an mRNA molecule.

[0304] 95. A particle comprising the nucleic acid of any one of embodiments 85-94, optionally wherein the particle is a retroviral particle.

[0305] 96. A host cell comprising the nucleic acid of any one of embodiments 85 to 94.

[0306] 97. 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 epigenomic modifications in a cell, the method comprising contacting the cell with (i) the polypeptide of any one of embodiments 1 to 84, the nucleic acid of any one of embodiments 85 to 94, or the particle of embodiment 95, and (ii) a stabilizer.

[0307] 98. The method of embodiment 97, wherein when the destabilization domain sequence is a DHFR destabilization domain sequence, the stabilizer is trimethoprim (TMP).

[0308] 99. The method of embodiment 97, wherein when the destabilization domain sequence is a FKBP destabilization domain sequence, the stabilizer is Shield-1, rapamycin, or FK506.

[0309] 100. The method of any one of embodiments 97 to 99, wherein contacting the cell with the stabilizer comprises culturing the cell in a medium comprising the stabilizer.

[0310] 101. The method of embodiment 100, comprising culturing the cell in a medium comprising the stabilizer for at least 8 hours.

[0311] 102. The method of embodiment 100, comprising culturing the cell in a medium comprising the stabilizer for at least 12 hours.

[0312] 103. The method of embodiment 100, comprising culturing the cell in a medium comprising the stabilizer for at least 1 day.

[0313] 104. The method of embodiment 100, comprising culturing the cell in a medium comprising the stabilizer for at least 2 days.

[0314] 105. The method of any one of embodiments 100 to 104, comprising culturing the cell in a medium comprising the stabilizer for up to 5 days.

[0315] 106. The method of any one of embodiments 100 to 104, comprising culturing the cell in a medium comprising the stabilizer for up to 4 days.

[0316] 107. The method of any one of embodiments 100 to 104, comprising culturing the cells in a culture medium comprising the stabilizer for up to 3 days.

[0317] 108. The method of any one of embodiments 100 to 104, comprising culturing the cells in a culture medium comprising the stabilizer for up to 2 days.

[0318] 109. The method of any one of embodiments 97 to 108, further comprising removing the stabilizer from the cell after contacting the cell with the stabilizer.

[0319] 110. The method of embodiment 109, wherein removing the stabilizer from the cells comprises culturing the cells in a culture medium that does not contain the stabilizer.

[0320] 111. The method of embodiment 110, comprising culturing the cells in a culture medium that does not contain the stabilizer for at least 3 hours.

[0321] 112. The method of embodiment 110, comprising culturing the cells in a culture medium that does not contain the stabilizer for at least 6 hours.

[0322] 113. The method of embodiment 110, comprising culturing the cells in a culture medium that does not contain the stabilizer for at least 12 hours.

[0323] 114. The method of embodiment 110, comprising culturing the cells in a culture medium that does not contain the stabilizer for at least 1 day.

[0324] 115. The method of embodiment 110, comprising culturing the cells in a culture medium that does not contain the stabilizer for at least 2 days.

[0325] 116. The method of embodiment 110, comprising culturing the cells in a culture medium that does not contain the stabilizer for at least 3 days.

[0326] 117. The method of embodiment 110, comprising culturing the cells in a culture medium that does not contain the stabilizer for at least 4 days.

[0327] 118. The method of any one of embodiments 110 to 117, comprising culturing the cells in a culture medium that does not comprise the stabilizer for up to 10 days.

[0328] 119. The method of any one of embodiments 100 to 117, comprising culturing the cells in a culture medium that does not contain the stabilizer for up to 8 days.

[0329] 120. The method of any one of embodiments 100 to 117, comprising culturing the cells in a culture medium that does not contain the stabilizer for up to 6 days.

[0330] 121. The method of any one of embodiments 100 to 117, comprising culturing the cells in a culture medium that does not contain the stabilizer for up to 4 days.

[0331] 122. The method of any one of embodiments 97 to 121, comprising contacting the cell with the polypeptide of any one of embodiments 1 to 84.

[0332] 123. The method of embodiment 122, wherein the contacting comprises introducing the polypeptide into the cell via electroporation, injection, or a carrier.

[0333] 124. The method of any one of embodiments 97 to 121, comprising contacting the cell with the nucleic acid of any one of embodiments 85 to 94.

[0334] 125. The method of embodiment 124, wherein the contacting comprises introducing the nucleic acid into the cell via transfection, electroporation, injection, or a carrier.

[0335] 126. The method of embodiment 124 or embodiment 125, wherein the polypeptide is transiently expressed in the cell.

[0336] 127. The method of any one of embodiments 97 to 126, further comprising administering the cells to a subject.

[0337] 128. The method of any one of embodiments 97 to 127, which induces mitophagy in the cell.

[0338] 129. The method of any one of embodiments 97 to 128, which increases mitochondrial turnover in the cell.

[0339] 130. The method of any one of embodiments 97 to 129, which increases mitochondrial mass.

[0340] 131. The method of any one of embodiments 97 to 130, which induces double-strand breaks in mitochondrial DNA.

[0341] 132. The method of any one of embodiments 97 to 131, which induces epigenomic modification in the cell.

[0342] 133. A cell obtained or obtainable by the method of any one of embodiments 97 to 132.

[0343] 134. A cell comprising the polypeptide of any one of embodiments 1 to 84, the nucleic acid of any one of embodiments 85 to 94, or the particle of embodiment 95.

[0344] 135. The cell of embodiment 134, further comprising a stabilizer.

[0345] 136. The cell of embodiment 135, wherein when the destabilization domain sequence is a DHFR destabilization domain sequence, the stabilizer is trimethoprim (TMP).

[0346] 137. The cell of embodiment 135, wherein when the destabilization domain sequence is a FKBP destabilization domain sequence, the stabilizer is Shield-1, rapamycin, or FK506.

[0347] 138. The method of any one of embodiments 97 to 132 or the cell of any one of embodiments 133 to 137, wherein the cell is a mammalian cell.

[0348] 139. The method or cell of embodiment 138, wherein the cell is a human cell.

[0349] 140. The method or cell of any one of embodiments 138 to 139, wherein the cell is a somatic cell.

[0350] 141. The method or cell of any one of embodiments 138 to 140, wherein the cell is a bone marrow cell.

[0351] 142. The method or cell of any one of embodiments 138 to 141, wherein the cell is a hematopoietic stem cell (HSC) or a mesenchymal stem cell (MSC).

[0352] 143. The method or cell of any one of embodiments 138 to 140, wherein the cell is an immune cell.

[0353] 144. The method or cell of embodiment 143, wherein the cell is a T cell, a phagocyte, a microglia, or a macrophage.

[0354] 145. The method or cell of embodiment 144, wherein the cell is a CD4+ T cell.

[0355] 146. The method or cell of embodiment 144 or embodiment 145, wherein the cell is a CD8+ T cell.

[0356] 147. The method or cell of any one of embodiments 138 to 146, wherein the cell is a primary cell.

[0357] 148. The method or cell of any one of embodiments 138-146, wherein the cell is a descendant of a primary cell.

[0358] 149. The method or cell of any one of embodiments 138-148, wherein the cell has dysfunctional mitochondria.

[0359] 150. The method or cell of any one of embodiments 138-149, wherein the cell is from a subject having an age-related disease.

[0360] 151. The method or cell of embodiment 150, wherein the age-related disease is an autoimmune disease, a metabolic disease, a genetic disease, a cancer, a neurodegenerative disease, or immunosenescence.

[0361] 152. The method or cell of any one of embodiments 138-151, wherein the cell is from a subject having a mitochondrial disease or disorder.

[0362] 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.

[0363] 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 mellitus 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 fiber syndrome (MERRF), familial bilateral striatal necrosis / striatonigral degeneration (FBSN), Lafferty disease, aminoglycoside-induced deafness (AID), or mitochondrial DNA multiple deletions syndrome.

[0364] 155. The method or cell of embodiment 152 or embodiment 153, wherein the mitochondrial disease or disorder is mitochondrial DNA depletion syndrome 4A, mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase gamma (POLG)-related disease, sensory ataxic neuropathy dysarthria ophthalmoplegia (SANDO), leukoencephalopathy involving the brainstem and spinal cord with elevated lactate (LBSL), coenzyme Q10 deficiency, Leigh syndrome, mitochondrial complex abnormalities, fumarase deficiency, alpha-ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenase complex deficiency (PDHC), pyruvate carboxylase deficiency (PCD), carnitine palmitoyltransferase I (CPT) deficiency. I) deficiency, carnitine palmitoyltransferase II (CPTIT) deficiency, carnitine-acylcarnitine (CACT) deficiency, autosomal dominant / autosomal recessive progressive external ophthalmoplegia (ad- / ar-PEO), infantile-onset spinocerebellar atrophy (IOSCA), mitochondrial myopathy (MM), spinal muscular atrophy (SMA), growth retardation, aminoaciduria, bile obstruction, iron overload, premature death (GRACILE), or Charcot-Marie-Tooth disease type 2A (CMT2A).

[0365] 156. The method or cell of any one of embodiments 138 to 155, wherein the cell is from a subject suffering from a neurodegenerative disease.

[0366] 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.

[0367] 158. The method or cell of embodiment 156, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

[0368] 159. The method or cell of embodiment 156, wherein the neurodegenerative disease is Huntington's disease.

[0369] 160. The method or cell of embodiment 156, wherein the neurodegenerative disease is Alzheimer's disease, optionally wherein the cell is from a subject having an APOE4 allele, such as an E3 / E4 or E4 / E4 genotype.

[0370] 161. The method or cell of embodiment 156, wherein the neurodegenerative disease is Parkinson's disease.

[0371] 162. The method or cell of embodiment 156, wherein the neurodegenerative disease is Friedreich's ataxia or Charcot-Marie-Tooth disease.

[0372] 163. The method or cell of embodiment 156, wherein the neurodegenerative disease is a leukodystrophy.

[0373] 164. The method or cell of any one of embodiments 138 to 163, wherein the cell is from a subject having a retinal disease.

[0374] 165. The method or cell of embodiment 164, wherein the retinal disease is age-related macular degeneration, macular edema, or glaucoma.

[0375] 166. The method or cell of any one of embodiments 138 to 165, wherein the cell is from a subject having diabetes.

[0376] 167. The method or cell of any one of embodiments 138 to 166, wherein the cell is from a subject with a hearing impairment.

[0377] 168. The method or cell of any one of embodiments 138 to 167, wherein the cell is from a subject having a genetic disease.

[0378] 169. The method or cell of embodiment 168, wherein the genetic disease is Hutchinson-Gilford Progeria Syndrome, Werner Syndrome, or Huntington's disease.

[0379] 170. The method or cell of any one of embodiments 138 to 169, wherein the cell is from a subject suffering from heart failure.

[0380] 171. The method or cell of any one of embodiments 138 to 170, wherein the cell is from a subject with an immunodeficiency.

[0381] 172. The method or cell of any one of embodiments 138 to 171, wherein the cell is from a subject having cancer.

[0382] 173. The method or cell of any one of embodiments 138 to 172, wherein the cell is from a subject having an infectious disease.

[0383] 174. The method or cell of any one of embodiments 138 to 148, wherein the cell is from a healthy donor.

[0384] 175. The method or cell of any one of embodiments 138 to 174, which is an ex vivo cell.

[0385] 176. The method of embodiment 175, further comprising administering the cells to a subject, optionally wherein the subject is the same subject from which the cells were derived.

[0386] 177. A method of treating a subject having an age-related disease, a mitochondrial disease or disorder, a neurodegenerative disease, a retinal disease, diabetes, a hearing disorder, a genetic disease, heart failure, an immunodeficiency, cancer, or an infectious disease, the method comprising administering to the subject a therapeutically effective amount of the cells according to any one of embodiments 133-175.

[0387] 178. The method of embodiment 177, wherein the subject has an age-related disease.

[0388] 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.

[0389] 180. The method of embodiment 177, wherein the subject has a mitochondrial disease or disorder.

[0390] 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.

[0391] 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 with ragged-red fibers (MERRF), familial bilateral striatal necrosis / pallidonigral degeneration (FBSN), Leigh’s disease, aminoglycoside-induced deafness (AID), and mitochondrial DNA multiple deletions syndrome.

[0392] 183. The method of embodiment 180 or embodiment 181, wherein the mitochondrial disease or disorder is mitochondrial DNA depletion syndrome 4A, mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase gamma (POLG)-related disease, sensory ataxic neuropathy dysarthria ophthalmoplegia (SANDO), leukoencephalopathy involving the brainstem and spinal cord with elevated lactate (LBSL), coenzyme Q10 deficiency, Leigh syndrome, mitochondrial complex abnormalities, fumarase deficiency, alpha-ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenase complex deficiency (PDHC), pyruvate carboxylase deficiency (PCD), carnitine palmitoyltransferase I (CPT I) deficiency, carnitine palmitoyltransferase II (CPT II) deficiency. IT) deficiency, carnitine-acylcarnitine (CACT) deficiency, autosomal dominant / autosomal recessive progressive external ophthalmoplegia (ad- / ar-PEO), infantile-onset spinocerebellar atrophy (IOSCA), mitochondrial myopathy (MM), spinal muscular atrophy (SMA), growth retardation, aminoaciduria, bile obstruction, iron overload, premature death (GRACILE), or Charcot-Marie-Tooth disease type 2A (CMT2A).

[0393] 184. The method of embodiment 177, wherein the subject suffers from a neurodegenerative disease.

[0394] 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.

[0395] 186. The method of embodiment 177, wherein the subject has a retinal disease.

[0396] 187. The method of embodiment 186, wherein the retinal disease is age-related macular degeneration, macular edema, or glaucoma.

[0397] 188. The method of embodiment 177, wherein the subject has diabetes.

[0398] 189. The method of embodiment 177, wherein the subject suffers from a hearing impairment.

[0399] 190. The method of embodiment 177, wherein the subject has a genetic disease.

[0400] 191. The method of embodiment 190, wherein the genetic disease is Hutchinson-Gilford Progeria Syndrome, Werner Syndrome, or Huntington's disease.

[0401] 192. The method of embodiment 177, wherein the subject suffers from heart failure.

[0402] 193. The method of embodiment 177, wherein the subject has an immunodeficiency.

[0403] 194. The method of embodiment 177, wherein the subject has cancer.

[0404] 195. The method of embodiment 177, wherein the subject has an infectious disease.

[0405] 196. A pharmaceutical composition comprising the polypeptide of any one of embodiments 1 to 84, the nucleic acid of any one of embodiments 85 to 94, the particle of embodiment 95, or the cell of any one of embodiments 133 to 175, and a pharmaceutically acceptable excipient.

[0406] 197. A kit comprising (a) the polypeptide of any one of embodiments 1 to 84, the nucleic acid of any one of embodiments 85 to 94, or the particle of embodiment 95, and (b) a stabilizer.

[0407] 198. The kit of embodiment 197, wherein when the destabilization domain sequence is a DHFR destabilization domain sequence, the stabilizer is trimethoprim (TMP).

[0408] 199. The kit of embodiment 197, wherein when the destabilization domain sequence is a FKBP destabilization domain sequence, the stabilizer is Shield-1, rapamycin, or FK506.

[0409] 200. The kit of embodiment 197, wherein when the destabilizing domain sequence is a PDE5 destabilizing domain sequence, the stabilizer is sildenafil, vardenafil, tadalafil, avanafil, lodenafil, mildenafil, udenafil, benzamide, dasafil, and beminafil.

[0410] 7. Citation of references

[0411] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes. In the event of an inconsistency between the teachings of the one or more references incorporated herein and the present disclosure, the teachings of the present specification are intended to prevail.

Claims

1. A polypeptide comprising: (a) Mitochondrial targeting sequence (MTS); (b) an endonuclease sequence; and (c) Destabilization domain sequence.

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

5. The polypeptide of any one of claims 1 to 4, wherein the MTS comprises 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 mitochondrial ribosomal protein, a phospholipid metabolism-related protein, a protein involved in the metabolism of toxic compounds, a disulfide bond relay system-related protein, an iron-sulfur protein assembly protein, a tRNA modification protein, an aminoacyl-tRNA synthetase, a release factor, or an elongation factor.

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

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

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

9. The polypeptide of claim 1, wherein the MTS comprises a sequence 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. 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. 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. 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 at least 80% identical to SEQ ID NO:

16.

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

16.

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

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

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

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

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

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

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

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

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

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

31. 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 epigenomic modifications in a cell, the method comprising contacting the cell with (i) the polypeptide of any one of claims 1 to 27, the nucleic acid of claim 28, or the particle of claim 29, and (ii) a stabilizer.

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

33. 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, after contacting the cell with the stabilizing agent, removing the stabilizing agent from the cell.

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

36. A cell comprising the polypeptide of any one of claims 1-27, the nucleic acid of claim 28, or the particle of 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. The method or cell of claim 37, wherein the cell is a human cell.

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

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

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

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

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

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 a leukodystrophy.

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

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

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

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

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

52. A kit comprising (a) the polypeptide of any one of claims 1 to 27, the nucleic acid of claim 28, or the particle of claim 29 and (b) a stabilizer.

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