CN120769907A - Composition for inducing mitochondrial autophagy and use thereof - Google Patents

Abstract

Polypeptides having a Mitochondrial Targeting Sequence (MTS), an endonuclease sequence and a destabilizing domain sequence, nucleic acids encoding such polypeptides, use of such polypeptides and nucleic acids in inducing mitochondrial autophagy, increasing mitochondrial turnover and/or inducing double strand breaks in mitochondrial DNA in cells, and therapeutic uses as described above, for example, in the treatment of mitochondrial diseases and conditions.

Description

Composition for inducing mitochondrial autophagy and use thereof

1. Cross-reference to related applications

The present application claims the benefit of priority from U.S. provisional application No. 63/426,424 filed on 11/18 of 2022, the contents of which are hereby incorporated by reference in their entirety.

2. Sequence listing

The present application contains a sequence listing submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML sequence table was created at 11/8 of 2023 and named RMG-004WO_SL.xml, size 42,441 bytes.

Background

Mitochondrial dysfunction is caused by a number of factors, including mutations in genes encoding mitochondrial proteins, trnas and rrnas, 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 defective quality control of mitochondrial proteins. Defects in mitochondrial protein quality control can be caused by a variety of factors, such as poor management of intracellular organelles such as the endoplasmic reticulum and lysosomes. Among them, quality control of mitochondrial biogenesis and autophagy (mitochondrial autophagy) and kinetics of fusion/division are a focus of various mitochondrial dysfunctions. Mitochondrial quality control defects associated with a variety of diseases have been intensively studied as targets for disease treatment. The diseases of interest 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.

Small molecule compounds have been developed to enhance biogenesis, induce mitochondrial autophagy, inhibit excessive division, and the like. For example, coenzyme Q10, idebenone and metformin have been shown to induce mitochondrial autophagy.

However, no compounds with a clear mode of action have arrived at the clinic. Thus, there remains a need for compositions and methods for improving mitochondrial dysfunction.

2. Summary of the invention

The present disclosure provides polypeptides capable of inducing Double Strand Breaks (DSBs) in mitochondria to achieve temporary and partial reduction of mitochondrial numbers in cells. Mitochondria have a poorer mechanism of gene repair than the nucleus. In response to DSB stress, mitochondria strongly transmit signals to the nucleus to promote replication of the mitochondrial genome and increase production of mitochondrial component proteins. Without being bound by theory, it is believed that DSBs that induce 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 by using polypeptides comprising a Mitochondrial Targeting Sequence (MTS) fused to an endonuclease. However, one potential problem with this approach is excessive endonuclease activity. In order to provide sensitive ON/OFF (ON/OFF) control of endonucleases, the present disclosure provides polypeptides having a destabilizing domain in addition to a mitochondrial targeting sequence and an endonuclease sequence. The destabilizing domain allows the polypeptide to retain structure and endonuclease activity when stabilized by a stabilizer, and destabilizes in the absence of a stabilizer, resulting in degradation of the polypeptide by the proteasome.

Accordingly, in one aspect, the present disclosure provides polypeptides comprising a Mitochondrial Targeting Sequence (MTS), an endonuclease sequence (e.g., xbaIR), and a destabilizing domain sequence. Inclusion of MTS can be used to direct the polypeptide to a desired endonuclease active site, i.e., the mitochondrial genome. Inclusion of a destabilizing domain sequence (which is stabilized by a stabilizer) enables sensitive on/off control of the endonuclease. For example, a cell may be contacted with a polypeptide in the presence of a stabilizing agent for a period of time during which the polypeptide is active and, subsequently, the stabilizing agent may be removed, resulting in destabilization and degradation of the polypeptide.

Exemplary features of the polypeptides of the present disclosure are described in section 4.2 and embodiments 1 through 84 below.

In another aspect, the disclosure provides nucleic acids encoding the polypeptides of the disclosure, particles (e.g., viral particles) comprising the nucleic acids, and host cells comprising the nucleic acids of the disclosure. Exemplary nucleic acids include vectors such as viral (e.g., retrovirus) 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 and embodiments 85 to 96 below.

In another aspect, the present disclosure provides methods of (a) inducing mitochondrial autophagy 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 modification in a cell, by contacting a cell with a polypeptide, nucleic acid, or particle of the present disclosure and a stabilizer. For example, the cells can be transfected with a nucleic acid encoding the polypeptide, transduced with a viral particle containing the nucleic acid encoding the polypeptide or injected with the polypeptide, and cultured in the presence of a stabilizer. After a period of time (e.g., 6 hours to 5 days), the stabilizing agent may be removed to allow destabilization and degradation of the polypeptide.

In another aspect, the present disclosure provides cells and cell populations obtained or obtainable by the methods described herein (a) inducing mitochondrial autophagy 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 modification in cells. Unless the context requires otherwise, references herein to "cells" encompass single cells as well as cell populations.

In another aspect, the present disclosure provides methods of treating a subject with the cells and cell populations of the present disclosure. For example, the subject may be a subject having an age-related disease, a mitochondrial disease or disorder, a neurodegenerative disease, an ocular disease (e.g., retinal disease), diabetes, a hearing disorder, a genetic disease, heart failure, an immunodeficiency, a cancer, or an infectious disease.

Further exemplary features of the methods and cells of the present disclosure are described in section 4.4 and embodiments 97-195 below.

In a further aspect, the present disclosure provides a pharmaceutical composition comprising a polypeptide, nucleic acid, particle, or cell (including a population of cells) of the present disclosure. Such pharmaceutical compositions may be used, for example, in the methods of treatment described herein.

In yet another aspect, the present disclosure provides a kit comprising a polypeptide, nucleic acid or particle of the present disclosure and a stabilizer. Kits can be used, for example, in the methods of the present disclosure.

Further exemplary features of pharmaceutical compositions and kits are described in section 4.5 and embodiments 196-200 below.

Drawings

FIG. 1 shows a retroviral vector map with MTS-XbaIR-DHFR polypeptides (example 1).

FIG. 2 shows a molecular model of the MTS-XbaIR-DHFR polypeptide of example 1.

FIG. 3 shows a retroviral vector map with an EFGR-DHFR polypeptide (example 1).

FIG. 4 shows a molecular model of EGFR-DHFR polypeptide of example 1.

FIG. 5 shows a fluorescence microscope image of Hela cells transduced with EGFP-DHFR retroviral vector (example 1).

FIG. 6 shows FACS data of Hela cells transduced with EGFP-DHFR retroviral vector, showing EGFP expression when cultured in the presence of TMP (example 1).

FIG. 7 shows relative EGFP mRNA expression levels of Hela cells transduced with EGFP-DHFR retroviral vector when cultured with TMP (example 1).

FIGS. 8A-8D show fluorescence intensities detected by fluorescence microscopy and FACS at 0,1, 2, 4, 6, 8, 24 and 48 hours after TMP elution following two days of culture of Hela cells transduced with EGFP-DHFR retroviral vector in TMP-containing medium (example 1). FIG. 8A time course, FIG. 8B fluorescence image, FIG. 8C FACS analysis, FIG. 8D Mean Fluorescence Intensity (MFI) over time in FACS analysis.

FIGS. 9A-9D show the fluorescence intensities detected by fluorescence microscopy and FACS after culturing Hela cells transduced with EGFP-DHFR retroviral vector in TMP-containing medium for several time periods (0, 1, 2, 4, 6, 8, 24 and 48 hours). FIG. 9A time course, FIG. 9B fluorescence image, FIG. 9C FACS analysis, FIG. 9D MFI over time in FACS analysis.

FIGS. 10A-10C show XbaI expression (FIG. 10A) and mtDNA Copy Number (CN) of HeLa cells transfected with a retroviral vector encoding a MTS-XbaIR-DHFR polypeptide (Hela MXD sc 20) (FIG. 10B), and the cells were cultured in the absence or presence of 0.5 μm TMP over time for two days, followed by TMP elution (FIG. 10C) (example 1).

FIGS. 11A-11D show Mitogreen-stained MFI (FIG. 11A), an index of mitochondrial mass (mtMass), TMRM-stained MFI (FIG. 11B), an index of mitochondrial membrane potential (mtMP) as a whole, and a relative TMRM/Mitogreen ratio (FIG. 11C), an index of mtMP per mass unit, in Hela MXD sc20 (FIG. 11D) incubated with TMP for 3 different time periods (16, 20, and 48 hours), followed by cell culture over time.

FIGS. 12A-12C show cell numbers (FIG. 12A) and cell viability (FIG. 12B) of Hela MXD sc20 cells (FIG. 12C) incubated for two days in the absence or presence of 0.5 μm TMP followed by TMP elution. (example 1).

FIGS. 13A-13B show mitochondrial autophagy index by FACS analysis (FIG. 13A) and mtDNA CN estimated by qPCR (FIG. 13B) of Hela MDX sc20 cells transduced with retroviral vectors encoding MTKEIMARED and PARK2 and cultured for several time periods without or with TMP. 10. mu.M CCCP was used as a positive control for mitochondrial autophagy. (example 1).

FIGS. 14A-14D show changes in mitochondrial biogenesis in Hela_ GiM cells stably expressing a genetically induced mitochondrial autophagy (GiM) unit in the presence and absence of TMP. Mitochondrial ROS (mtROS) (fig. 14A), pgc1α (fig. 14B), NRF1 (fig. 14C), and TFAM (fig. 14D) were evaluated over time using FACS. (example 2).

FIGS. 15A-15F show the time-dependent expression of several mitochondrial proteins in Hela_ GiM cells in the presence and absence of TMP. Fig. 15A shows western blot images of mitochondrial proteins at days 2, 4, 6 and 8. FIGS. 15B-15F show quantitative levels of the same protein. (example 2).

FIGS. 16A-16B show phase contrast (PhC) and fluorescence microscopy images of MTKEIMARED expressing HeLa-GiM cells in the presence and absence of TMP (FIG. 16A) and the percentage of cells positive for mitochondrial autophagy (FIG. 16B). (example 2).

FIGS. 17A-17B show the co-localization of autophagosome membrane marker LC3, mitochondrial marker TOM20 and nuclear stain DAPI in Hela_ GiM cells in the presence or absence of TMP and BafA1 (FIG. 17A) and quantitative values for the region corresponding to autophagosome (FIG. 17B) (example 3).

FIGS. 18A-18B show the level of LC3-II expression in HeLa-GiM cells in the absence or presence of TMP or TMP+ BafA 1. Fig. 18A shows western blot images and fig. 18B is a graphical presentation of triplicate quantification. (example 3).

FIGS. 19A-19C show the results of a breath assay showing the changes over time in OXPHOS and glycolysis (FIG. 19A), a two-dimensional expansion of the relationship of OXPHOS and glycolysis (FIG. 19B), and changes over time in ATP production, basal respiration, proton leak and sparing capacity (FIG. 19C). (example 4).

Fig. 20A-20G show the assay setup used in example 5 (fig. 20A), FACS results for untreated Alzheimer's Disease (AD) fibroblasts and control NHDF cells (fig. 20B) and FACS results for AD fibroblasts 7, 14 and 21 days after genetically induced mitochondrial autophagy (GiM) (fig. 20C), mtMass and mtMP levels in untreated cells (fig. 20D and 20E, respectively), and mtMass and mtMP levels in AD fibroblasts 7 and 14 days after GiM relative to control (fig. 20F and 20G, respectively), as further described in example 5.

4. Detailed description of the invention

The present disclosure provides polypeptides capable of inducing Double Strand Breaks (DSBs) in mitochondria to achieve temporary and partial reduction of mitochondrial numbers in cells.

Mitochondrial genomes encode respiratory chain proteins, which are tightly regulated by translational balance with nuclear-encoded respiratory proteins. Thus, partial loss of the mitochondrial genome is directly related to insufficient proton uptake, resulting in depolarization of the mitochondrial membrane potential. Mitochondrial membrane potential is depolarized in dysfunctional mitochondrial compartments and is a key regulator of mitochondrial autophagy. Without being bound by theory, it is believed that upon introduction of a DSB, the more depolarized portion may preferentially undergo mitochondrial autophagy.

In response to stress by DSBs, mitochondria strongly transmit signals to the nucleus (e.g., as part of a mitochondrial unfolded protein response (UPRmt)) to promote replication of the mitochondrial genome and increase production of mitochondrial component proteins. In addition to the UPRmt signal, metabolic changes caused by mitochondrial genome depletion can affect the apparent genomic state of the cell. For example, this reduction may result in a reduction of some intermediates of the TCA cycle for acetylation and methylation of nuclear genomes and histones. Without being bound by theory, it is believed that DSBs that induce 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. With age, destructive modifications can accumulate. Mitochondrial genesis promotes the restoration of healthy mitochondrial function due to the lack of these modifications in the newly generated mitochondrial genome.

DSBs can be introduced into mitochondrial DNA by using polypeptides comprising a Mitochondrial Targeting Sequence (MTS) fused to an endonuclease. However, one potential problem with this approach is excessive endonuclease activity. In order to provide sensitive on/off control of endonucleases, the present disclosure provides polypeptides having a destabilizing domain in addition to a mitochondrial targeting sequence and an endonuclease sequence. The destabilizing domain allows the polypeptide to retain structure and endonuclease activity when stabilized by a stabilizer, and destabilizes in the absence of a stabilizer, resulting in degradation of the polypeptide by the proteasome.

4.1. Definition of the definition

Unless defined otherwise, 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.

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

1. the use of the terms "a," "an," "the," and similar terms in the context of this disclosure should be construed to include both the singular and the plural, unless the context clearly dictates otherwise or is contradicted by context. Thus, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

And/or the term "and/or" means that each or two or all of the components or features in the list are possible variants, in particular two or more of their alternatives or cumulatively.

Destabilizing 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 caused by the destabilizing domain. Exemplary destabilizing domains include a dihydrofolate reductase (DHFR) destabilizing domain (which may be stabilized by the exemplary stabilizer trimethoprim), an FK506 binding protein (FKBP) destabilizing domain (which may be stabilized by the exemplary stabilizers Shield-1 (Shield 1), rapamycin, and FK 506), and a PDE5 destabilizing domain (which may be stabilized by the exemplary stabilizers sildenafil, vardenafil, tadalafil (tadalafil), avanafil (avanafil), lodinafil (lodenafil), mi Luona non (mirodenafil), udenafil (udenafil), beninafil (benzamidenafil), darifenacin (dasantafil), and bemilnafil (beminafil). Exemplary DHFR destabilization 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 destabilization domains are described in Banaszynski et al, 2006, cell 126 (5): 995-1104 and US 9,487,787, and exemplary PDE5 destabilization domains are described in WO 2018/237323, the contents of each of which are incorporated herein by reference in their entirety.

Effective amount the term "effective amount" or "therapeutically effective amount" means an amount or quantity of an agent or composition sufficient to elicit a necessary or desired response, or in other words, an amount 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 progression of the diseases or conditions disclosed herein. It will be appreciated that an "effective amount" or "therapeutically effective amount" can vary from subject to subject due to the metabolism of the agent, the age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the discretion of the prescribing physician.

Endonuclease the term "endonuclease" refers to an enzyme that cleaves a polynucleotide strand by separation of nucleotides other than the 5 'or 3' terminal nucleotides. Endonucleases are distinct from exonucleases, which cleave nucleotides from the 5 'or 3' ends of a polynucleotide strand. Exemplary endonucleases include restriction endonucleases capable of cleaving double-stranded DNA at or near a specific recognition site to form a double-strand break (DSB) in the 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 the UniProt accession number O68567, the exemplary EcoRI amino acid sequence has the UniProt accession number P00642, the exemplary SmaI amino acid sequence has the UniProt accession number P14229, the exemplary afli amino acid sequence has the UniProt accession number E3VX87, the exemplary BamHI amino acid sequence has the UniProt accession number P23940, the exemplary BclI amino acid sequence has the UniProt accession number E5LGB8, the exemplary haeii amino acid sequence has the UniProt accession number O68584, the exemplary HindII amino acid sequence has the UniProt accession number P44413, the exemplary HindIII amino acid sequence has the UniProt accession number P43870, the exemplary PvuII amino acid sequence has the UniProt accession number A0A4R7BM34, the exemplary PstI amino acid sequence has the UniProt accession number P00640, and the exemplary SpeI amino acid sequence has the UniProt accession number F1KM35.

Mitochondrial Targeting Sequence (MTS): the term "mitochondrial targeting sequence" refers to an amino acid sequence capable of directing the transport of a polypeptide containing this sequence to the mitochondria. MTS is typically 10-70 amino acids in length. MTS typically comprises an alternating pattern of hydrophobic and positively charged amino acids to form an amphipathic helix.

Or "conjunctions are intended to be used in their proper sense as boolean logic operators, covering feature choices in alternative choices (a or B, where a's choice and B's choice are mutually exclusive) and feature choices in conjunctions (a or B, where a and B are both chosen), unless otherwise indicated. In some places herein, the term "and/or" is used for the same purpose, which should not be construed as implying "or" as referring to mutually exclusive alternatives.

Peptides, proteins and polypeptides the terms peptide, protein and polypeptide are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked to the alpha amino group of one amino acid by the carboxyl group of another amino acid. Amino acids may be natural or synthetic and may contain chemical modifications such as disulfide bonds, substitution of radioisotopes, phosphorylation, substrate chelation (e.g., chelation of iron or copper atoms), glycosylation, acetylation, formylation, amidation, biotinylation, and a wide variety of other modifications. There is no explicit requirement that the polypeptide must contain the intended function, and the polypeptide may be functional, nonfunctional, functional for unexpected/unintended purposes, 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 twenty standard amino acids may also be used. The twenty standard amino acids include alanine (Ala, a), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (gin, 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.

Percent identity the percent identity between two amino acid sequences is calculated by multiplying the number of matches between a pair of aligned sequences by 100 and dividing by the length of the aligned region. The identity score only calculates a perfect match, irrespective of the degree of similarity of amino acids to each other, and irrespective of substitution or deletion as a match. Alignment for the purpose of determining percent sequence identity can be accomplished in a variety of ways within the skill of the art, for example, by manual alignment or using publicly available computer software such as BLAST, BLAST-2, ALIGN-2 or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for achieving maximum alignment.

Subject as used herein, the term "subject" means a human.

Treatment, treatment as used herein, in one embodiment, the terms "treatment" or "treatment" refer to any disease or condition that ameliorates a disease or condition (e.g., slows or inhibits or reduces the progression of a disease or at least one clinical symptom or pathological feature thereof). In another embodiment, "treatment" or "treatment" refers to alleviating or ameliorating at least one physical parameter or pathological feature of a disease, including, for example, those that a subject may not be able to distinguish. In yet another embodiment, "treatment" or "treatment" refers to modulating a disease or disorder on the body (e.g., stabilization of at least one discernible or non-discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. In yet another embodiment, "treatment" or "treatment" refers to preventing or delaying the onset or development or progression of a disease or disorder or at least one symptom or pathological feature associated therewith. In yet another embodiment, "treatment" or "treatment" refers to preventing or delaying the progression of a disease to a more advanced stage or a more severe condition. The benefit to the patient to be treated is statistically significant, or at least perceptible to the patient or to the physician. However, it should be appreciated that when a drug is administered to a patient to treat a disease, the result may not always be effective treatment.

4.2. Polypeptides

In one aspect, the present disclosure provides polypeptides comprising a Mitochondrial Targeting Sequence (MTS), an endonuclease sequence, and a Destabilizing Domain (DD) sequence. Exemplary features of mitochondrial targeting sequences, endonucleases, and destabilizing domains that may be included in the polypeptides of the present disclosure are described in sections 4.2.1, 4.2.2, and 4.2.3, respectively.

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

4.2.1. Mitochondrial targeting sequences

Mitochondria have about 1500 proteins encoded by the nuclear genome. They are translated in the cytosol and introduced into the mitochondrial inner or outer membrane, the inter-membrane space or the matrix, depending on the MTS. The polypeptides of the present disclosure may include variants of a full-length MTS or a wild-type MTS of a mitochondrial protein (e.g., truncated versions of a full-length MTS and/or MTS having one or more amino acid substitutions (e.g., one or more conservative amino acid substitutions) as compared to the wild-type sequence).

The polypeptides of the present disclosure can include a human MTS or a 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 comprise TCA cycle-related enzymes, chaperones, 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 toxic compound metabolism, disulfide relay system-related proteins, iron-sulfur protein assembly proteins, tRNA modification proteins, aminoacyl tRNA synthetases, factor-releasing or elongation factor MTS.

In some embodiments, the MTS comprises a 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 a MTS of cytochrome c oxidase subunit VIII (COX 8), cytochrome c oxidase subunit X (COX 10), or cytochrome c oxidase subunit IV (COX 4).

In some embodiments, the MTS comprises an ataxin (FXN) MTS.

In some embodiments, the MTS comprises a TCA cycle related enzyme MTS, e.g., a pyruvate dehydrogenase, citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinyl CoA synthase, succinate dehydrogenase, fumarate, malate dehydrogenase, or pyruvate carboxylase MTS.

In other embodiments, the MTS comprises an MTS that is a chaperone protein, e.g., mtHSP10, mtHSP60, mtHSP70, or mtHSP 90.

In other embodiments, the MTS comprises a mitochondrial genome replication protein MTS, e.g., TFAM, twinkle, polG, TFB, 2, M, TEFM, or MTERF1 MTS.

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

In other embodiments, the MTS comprises an MTS of an mRNA processing protein, e.g., LRPPRC, TACO1, ELAC2, PNPT1, HSD17B10, MTPAP, or an MTS of PTCD 1.

In other embodiments, the MTS comprises a MTS of a mitochondrial RNA degradation protein, e.g., PNPasse, REX02, or a MTS of SUV 3.

In other embodiments, the MTS comprises a deoxynucleotide triphosphate synthesis-related protein MTS, e.g., DGUOK, TK2, TYMP, MGME1, sullg 1, sulla 2, RNASEH1, or C10orf2 MTS.

In other embodiments, the MTS comprises a MTS of a mitochondrial ribosomal protein, e.g., a MTS of MRPS16, MRPS22, MRPL3, MRP12, or MRPL 44.

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

In other embodiments, the MTS comprises a MTS of a protein involved in the metabolism of a toxic compound, e.g., a MTS of HIBCH, ECHS1, ETHE1, or MPV 17.

In other embodiments, the MTS comprises a disulfide relay system related protein MTS, e.g., GFER MTS.

In other embodiments, the MTS comprises an MTS of a ferrosulfur assembly protein, e.g., an MTS of ISCU, BOLA3, NFU1, or IBA 57.

In other embodiments, the MTS comprises a MTS of a tRNA modified protein, e.g., MTO1, GTP3BP, TRMU, PUS1, MTFMT, TRIT1, TRNT1, or TRMT 5.

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

In other embodiments, the MTS comprises an elongation factor MTS, e.g., a TUFM, TSFM, or GFM1 MTS.

Exemplary mitochondrial targeting sequences are listed in table 1.

The polypeptides of the present disclosure may include MTS identified in table 1 or variants thereof (e.g., MTS having one or more conservative amino acid substitutions and/or truncations). The truncation may be a truncation of the C-terminal sequence (e.g., the MTS may correspond to the sequences listed in table 1, but have 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 15N-terminal amino acids of the MTS sequences listed in table 1. Variant MTS may include, for example, MTS at least 80%, at least 95%, at least 90%, or at least 95% identical to the MTS listed in table 1.

Those skilled in the art will appreciate that additional mitochondrial targeting sequences other than those identified in this section may be used. A variety of tools for predicting MTS 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).

4.2.2. Endonuclease enzyme

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 Cas 12), a zinc finger nuclease, or a transcription activator-like effector nuclease (TALEN). The endonuclease may comprise 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.

The endonuclease may be of bacterial origin. Many restriction enzymes are known in the art, including, for example XbaIR, ecoRI, smaI, aflII, bamHI, bclI, haeIII, hindII, hindIII, ndeI, pvuII, pstI and SpeI.

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

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

In some embodiments, the endonuclease is EcoRI.

In some embodiments, the endonuclease is SmaI.

In some embodiments, the endonuclease is AflII.

In some embodiments, the endonuclease is BamHI.

In some embodiments, the endonuclease is BclI.

In some embodiments, the endonuclease is HaeIII.

In some embodiments, the endonuclease is HindII.

In some embodiments, the endonuclease is HindIII.

In some embodiments, the endonuclease is NdeI.

In some embodiments, the endonuclease is PvuII.

In some embodiments, the endonuclease is PstI.

In some embodiments, the endonuclease is SpeI.

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 Unit Prot accession O68567, unit Prot accession P00642, unit Prot accession P14229, unit Prot accession E3VX87, unit Prot accession P23940, unit Prot accession E5LGB8, unit Prot accession O68584, unit Prot accession P44413, unit Prot accession P43870, unit Prot accession A0A4R7BM34, unit Prot accession P00640, or Unit Prot accession F1KM35.

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 may be used in combination with one or more guide RNA molecules that target mitochondrial DNA.

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.

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

4.2.3. Destabilizing domains

The polypeptides of the present disclosure include a Destabilizing Domain (DD) that allows for on/off control of endonucleases. Exemplary DDs include DHFR, FKBP, and PDE5 DD.

An exemplary DHFR DD is described in US 9,487,787, the contents of which are incorporated herein in their entirety. The amino acid sequence of wild E.coli DHFR is shown below:

MISLIAALAVDHVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR (SEQ ID NO: 17).

The DHFR DD may comprise a wild-type DHFR sequence or may comprise one or more amino acid substitutions and/or truncations at the N-and/or C-terminus. For example, the 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 may be included in the DHFR DD include Y100I, G121V, N T/A19V, F103L, H Y/Y100I, H L/Y100I, R H/F103S, M T/H114R, I F/T68S. Combinations of the foregoing substitutions may also be used. In some embodiments, the DHFR comprises the same amino acid sequence as SEQ ID NO. 17 except for the Y100I, G121V, N T/A19V, F103L, H Y/Y100I, H L/Y100I, R H/F103S, M T/H114R or I61F/T68S substitution or a combination thereof. In some embodiments, the DHFR DD lacks an N-terminal methionine. For example, in some embodiments, the DHFR comprises the same amino acid sequence as SEQ ID NO. 17 except for the Y100I, G121V, N T/A19V, F103L, H Y/Y100I, H L/Y100I, R H/F103S, M T/H114R or I61F/T68S substitution or combination thereof and lacks an N-terminal methionine.

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:

atcagtctgattgcggcgttagcggtagatcacgttatcggcatggaaaccgtcatgccgtggaacctgcctgccgatctcgcctggtttaaacgcaacaccttaaataaacccgtgattatgggccgccatacctgggaatcaatcggtcgtccgttgccaggacgcaaaaatattatcctcagcagtcaaccgagtacggacgatcgcgtaacgtgggtgaagtcggtggatgaagccatcgcggcgtgtggtgacgtaccagaaatcatggttattggcggcggtcgcgtttatgaacagttcttgccaaaagcgcaaaaactgtatctgacgcatatcgacgcagaagtggaaggcgacacccatttcccggattacgagccggatgactgggaatcggtattcagcgaattccacgatgctgatgcgcagaactctcacagctattgctttgagattctggagcggcgataa (SEQ ID NO: 19).

an exemplary stabilizer for DHFR DD is trimethoprim.

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

GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 20).

The FKBP DD may comprise a 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, R G, D100N, E G and K105I. Combinations of the foregoing substitutions may also be used. In some embodiments, DD comprises the same amino acid sequence as SEQ ID NO. 20 except that the F15S, V24A, H25R, E60G, L106P, D100G, M66T, R71G, D100N, E G or K105I substitution or a combination thereof.

Exemplary FKBP DD stabilizers include Shield-1 (Shield 1), rapamycin, and FK506.

An exemplary PDE5 DD is described in WO 2018/237323, the contents of which are incorporated herein in their entirety. PDE5 DD 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 region and have a unique first exon followed by a 823 amino acid common sequence.

All PDE5A isoforms contain a catalytic domain, which is located near the C-terminus of the protein and is relatively selective at physiological levels for cGMP as a substrate. The substrate binding site is also a binding site for several known PDE5 inhibitors (e.g. sildenafil) which have been used in the treatment of cardiovascular diseases and erectile dysfunction. At the N-terminus, there are two homologous GAF domains. One of the GAF domains, GAF-A, contains A high affinity binding site for cGMP. cGMP is known to occupy this domain leading to activation of the catalytic domain. Furthermore, the affinity of this site for cGMP is increased by cGMP-dependent protein kinase mediated phosphorylation of serine 92. In another embodiment, PDE5A DD may comprise the catalytic domain of PDE5A, which spans amino acid positions 535 to 860 of UniProt ID: O76074 (SEQ ID NO: 21) as shown in SEQ ID NO: 24. In addition to the catalytic domain, the PDE5A DD may further comprise one or more GAF domains and/or C-terminal portions extending beyond the catalytic domain. In one embodiment, the PDE 5A-derived DD comprises amino acids from position 535 to position 875 of SEQ ID NO. 21. In another embodiment, 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.

Exemplary amino acid substitutions that may be included in PDE5 DD include one or more amino acid substitutions ：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、T712S、D724N、L738H、N742S、F744L、L746S、F755L、A762S、D764V、D764N、D764G、S766F、K795E、L797F、I799T、L804P、T802P、S815C、M816A、M816T、I824T、C839S、F840S and K852E selected from the group consisting of. PDE5 DD may also contain additional substitutions, such as Q589R. 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.

Exemplary stabilizers for PDE5 DD include sildenafil, vardenafil, tadalafil, avanafil, lobanaf, milonafil, ubendinafil, a benzene amide nafil, darifenacin and beminafil.

4.3. Nucleic acids, particles and host cells

In another aspect, the disclosure provides nucleic acids encoding the polypeptides of the disclosure (e.g., polypeptides as described in section 4.2). The nucleic acid may be, for example, a vector, such as a viral genome or plasmid, or an mRNA molecule.

Exemplary vectors include viral expression vectors (e.g., viral vectors based on vaccinia virus, polio virus, adenovirus (see, e.g., ,Li et al., 1994, Invest Opthalmol Vis Sci 35:2543-2549; Borras et al, 1999, Gene Ther 6:515-524; Li and Davidson, 1995, PNAS 92:7700-7704; Sakamoto et al., 1999, H Gene Ther 5:1088-1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655), adeno-associated virus (AAV) (see, e.g., ,Ali et al., 1998, Hum Gene Ther 9:81 86; Flannery et al., 1997, PNAS 94:6916-6921; Bennett et al., 1997, Invest Opthalmol Vis Sci 38:2857-2863; Jomary et al., 1997, Gene Ther 4:683 690; Rolling et al., 1999, Hum Gene Ther 10:641-648; Ali et al., 1996, Hum Mol Genet 5:591-594; WO 93/09239);SV40; herpes simplex virus, human immunodeficiency virus (see, e.g., miyoshi et al, 1997, PNAS 94:10319-23; TAKAHASHI ET al, 1999, J Virol 73:7812-7816), retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as rous sarcoma virus, hawk sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and papilloma virus), and the like).

In some embodiments, the vector comprises a retroviral genome. Nucleic acids, such as retroviral genomes, may be provided in the form of particles, such as in the form of viral particles (e.g., retroviral particles).

The nucleic acid encoding a polypeptide of the present disclosure may further comprise one or more regulatory sequences, such as a promoter, e.g., an SV40, CMV or CAG promoter. Exemplary SV40 promoter sequences are shown below:

GTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAA (SEQ ID NO: 25).

in another aspect, the present disclosure provides a host cell comprising a nucleic acid of the present disclosure. The host cell may be prokaryotic (e.g., a bacterium such as e.coli) or eukaryotic (e.g., a human cell line 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.

4.4. Methods of inducing mitochondrial autophagy and methods of treatment

In a further aspect, the present disclosure provides methods of using the polypeptides, nucleic acids and particles of the present disclosure, e.g., as described in sections 4.2 and 4.3, to induce mitochondrial autophagy in a cell, and/or to increase mitochondrial turnover in a cell, and/or to increase mitochondrial mass, and/or to induce double strand breaks in mitochondrial DNA and/or (e) to induce epigenomic modifications in a cell.

The method generally comprises contacting the cell with a polypeptide, nucleic acid or particle, and a stabilizing agent capable of stabilizing the DD. The polypeptide may 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 a polypeptide to a cell. The nucleic acid may be introduced into the cell by transfection, electroporation, injection, carrier, or any other means known in the art for delivering nucleic acids to cells. The viral particles may be introduced into the cells by transduction.

The cells may be contacted with a stabilizing agent, for example, by culturing the cells in a medium comprising the stabilizing agent. The cells may be cultured in a medium with a stabilizing agent for a period of time to allow the endonuclease to introduce the DSB into the mitochondrial DNA. In some embodiments, the cells are cultured in a medium with a stabilizing agent for at least 8 hours (e.g., at least 12 hours, at least 1 day, at least 2 days, or longer) and/or up to 5 days (e.g., up to 4 days, up to 3 days, or up to 2 days). The stabilizer may then be removed, for example, by culturing the cells in a medium that does not contain the stabilizer. Once the stabilizing agent is removed, the polypeptide will be destabilized, resulting in degradation of the polypeptide.

After removal of the stabilizer, the cells may be cultured in the absence of the stabilizer for a period of time during which the cells may produce new mitochondria. In some embodiments, the cells are cultured in a medium that does not include a 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).

In some embodiments, the methods of the 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 a 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 DSB of mitochondrial DNA in the cell. In some embodiments, the method results in an epigenomic modification in the cell, e.g., induced by mitochondrial depletion. It has been previously reported that rho0 cells with complete depletion of mitochondrial genomes 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 may be useful for inducing epigenomic modifications. In some embodiments, the methods of the present disclosure result in the appearance in a cell of one, two, three, four, or all five of (a) induction of mitochondrial autophagy, (b) increased mitochondrial turnover, (c) increased mitochondrial mass, (d) DSB of mitochondrial DNA, and (e) epigenomic modification.

Exemplary cells useful in the method include mammalian cells, preferably human cells, more preferably human somatic cells. Cell types that may 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 offspring, may be used.

The cells are normal cells (e.g., from a healthy donor) or mitochondria with dysfunction (e.g., from a subject with a disease or disorder). For example, the cells may be from a subject suffering from an age-related disease or disorder, such as an autoimmune disease, metabolic disease, genetic disease, cancer, neurodegenerative disease, or immune aging.

As another example, the cells may be from a subject having a mitochondrial disease or disorder, such as chronic progressive extraocular muscle paralysis (CPEO), pearson syndrome, kanns-ser syndrome (KSS), diabetes and deafness (DAD), mitochondrial diabetes, leber Hereditary Optic Neuropathy (LHON), LHON-plus, neuropathy, ataxia, retinitis pigmentosa syndrome (NARP), maternal hereditary Leigh syndrome (MILS), mitochondrial encephalomyopathy with lactic acidosis and stroke-like attacks (MELAS), sarcotic epilepsy with red ragged Myofibrosis (MERRF), familial bilateral striatal necrosis/striatal substantia degeneration (FBSN), lamivudine, aminoglycoside-induced deafness (AID), or mitochondrial DNA multiple deficiency syndrome. Additional mitochondrial diseases and conditions include mitochondrial DNA depletion syndrome 4A, mitochondrial recessive ataxia syndrome (mils), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase γ (POLG) -related diseases, sensory ataxia neuropathy buffer block (SANDO), brain leukosis (LBSL) involving brain stem and spinal cord with elevated lactic acid, 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 palmitoyl transferase I (CPT I) deficiency, carnitine palmitoyl transferase II (CPT IT) deficiency, carnitine-acyl carnitine (CACT) deficiency, autosomal paralysis/autosomal recessive progressive extra ocular muscle ad (amar), mitochondrial atrophy (glad), advanced prompter-atrophy, advanced muscle atrophy (CMT 2), choiceps (achalar), advanced muscle atrophy (achalasia), advanced muscle atrophy (CMT 2), choiceps (CMT), advanced muscle atrophy (achalasia), and CMT2 (CMT).

As another example, the cells may be from a subject suffering from a neurodegenerative disease such as Amyotrophic Lateral Sclerosis (ALS), huntington's disease, alzheimer's disease, parkinson's disease, friedreich's ataxia, fibular muscular atrophy, or white matter dystrophy. In some embodiments, the cells are from a subject with alzheimer's disease, e.g., a subject with an APOE4 allele, e.g., E3/E4 or E4/E4 genotype.

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

In further examples, the cells may be from a subject suffering from diabetes, a hearing disorder, a genetic disorder (e.g., hakinsen-Ji Erfu de early-senescence syndrome, wanner syndrome, or huntington's disease), heart failure, immunodeficiency, cancer, or an infectious disease.

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

Accordingly, in another aspect, the present disclosure provides a method of treating a subject suffering from an age-related disease, mitochondrial disease or disorder, neurodegenerative disease, retinal disease, diabetes, hearing impairment, genetic disease, heart failure, immunodeficiency, cancer, or infectious disease by administering a therapeutically effective amount of cells obtained or obtainable by the methods described herein. For example, the subject may have a disease or disorder described in this section.

4.5. Pharmaceutical compositions and kits

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

In another aspect, the disclosure provides a kit 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), or a particle of the 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, lodinafil, milonafil, ubenafil, benzomidafil, dabtafil, and beminafil.

Examples

5.1. Example 1 Polypeptides for inducing mitochondrial autophagy, biogenesis and accelerating mitochondrial turnover

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

5.1.1. Design of transgenes

XbaIR was selected to induce DSB in the mitochondrial genome. XbaIR have five cleavage sites in the mitochondrial genome consensus sequence. To direct XbaIR endonucleases to mitochondria, the Cox8a postmitochondrial signal was placed on the N-terminal side of XbaIR. Induction of DSBs of the mitochondrial genome results in strong temporary energy depletion and, in order to control endonuclease activity, DHFR destabilizing domains (Liu et al, 2014 Int. J. Parasitol. 44 (10): 729-735) are fused to the C-terminus of XbaIR to allow sensitive on/off control of the endonuclease. The DHFR destabilizing domain is stabilized by the antibiotic Trimethoprim (TMP).

Retroviral vectors having the MTS-XbaIR-DHFR coding sequence were constructed (FIG. 1). The nucleotide sequences of the vectors are shown in table 3.

Molecular modeling was used to confirm that the three functional domains of the polypeptide construct (MTS, xbaI, and DHFR) adopt three-dimensional structures that do not interfere with each other (fig. 2). In addition, retroviral vectors with EGFP-DHFR as transgene were created to assess the responsiveness of the DHFR/TMP system (FIGS. 3 and 4). The nucleotide sequences of the vectors are shown in table 4.

Responsiveness of DHFR/TMP System

EGFP-DHFR retroviral vectors were transduced into HeLa cells. Infection was efficient and TMP exposure was performed at various concentrations for two days without enrichment of infected cells, and fluorescence expression of EGFP was then detected using fluorescence microscopy and FACS (fig. 5 and 6). Transcription and presence of EGFP mRNA was observed (fig. 7), but no protein was observed without TMP (fig. 5 and 6).

To confirm the OFF control of the construct, medium elution was performed two days after TMP exposure and the fluorescence intensity over time was checked by fluorescence microscopy and FACS (fig. 8A). EGFP fluorescence suddenly decreased one hour after TMP was removed from the medium, and then after four hours, fluorescence was dissipated (FIG. 8B and FIG. 8C). In addition, no leakage of expression of the transgene product was observed thereafter within 48 hours after TMP was turned off (fig. 8D).

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

5.1.3. Mitochondrial characterization after genetically induced mitochondrial autophagy

HeLa cells were transfected with MTS-XbaIR-DHFR vector and exposed to 0.5. Mu.M TMP for two days, followed by elution. XbaRI RNA expression was measured after TMP exposure. The transcript levels of the transgenes were not significantly altered (fig. 10A). On day 2, CN was reduced to less than half of the initial value (fig. 10B).

Hela transfectants with MTS-XbaIR-DHFR, designated Hela MXD sc20, were cloned by limiting dilution. Hela MXD sc20 was subjected to 0.5 μM TMP exposure for several time periods (16, 20, and 48 hours) to examine mitochondrial quality (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) (FIGS. 11A-11C). Regardless of the duration of TMP exposure, mtMass temporarily increased to approximately twice as compared to resting state, and then recovered to the original volume 10 hours after TMP was turned on, indicating that mitochondrial biogenesis was temporarily and strongly activated (fig. 11A). Both overall and corrected mtMP showed a sharp drop and then increase, indicating poor pumping of hydrogen ions through respiratory chain complexes I, III and IV or counter-clockwise rotating complex V (fig. 11B and 11C).

The introduced transgene appears to cause DSB of the mitochondrial genome and reduce CN, and in addition, although proteins from the nucleus are required, the increase in MM shows a high response to this stress. Based on these two factors, the density of the respiratory chain complex is thought to decrease and MMP, which is a phenotype, is thought to decrease. Mitochondrial genome appeared to return to normal on day 6 with a slight increase in MM. Without being bound by theory, it is believed that this suggests that mitochondrial biogenesis is enhanced in the MTS-XbaIR-DHFR/TMP system and that mitochondrial capacity is increased due to the abundance of mitochondria.

Next, it was investigated whether changes in cell proliferation and viability occurred in the system and affected the above changes. In this system with or without TMP, no significant difference in cell number or viability was observed (fig. 12A-12B). Thus, the major benefit of this system was determined to be the intervention of the MTS-XbaIR-DHFR polypeptide on mitochondria.

Effects on mitochondrial autophagy after MTS-XbaIR-DHFR/TMP

MtKeima-Red transfected Hela MXD sc20 cells with PARK2 overexpression were generated to quantify mitochondrial autophagy in a finer manner. TMP was examined for exposure to transfectants of varying duration to measure mitochondrial autophagy (16, 20, 24, 40, 44 and 48 hours). As a positive control, the mitochondrial autophagy inducer carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used. As the duration of TMP exposure increased, more mitochondrial autophagy was observed (fig. 13A). At 40 hours of TMP exposure, mitochondrial autophagy reached stationary phase (FIG. 13A). At the same time, it was observed that the measured mtDNA CN was restored to the initial value regardless of the duration of TMP exposure (fig. 13B). In summary, the results indicate that mitochondrial turnover is accelerated by MTS-XbaIR-DHFR/TMP.

5.2. Example 2 mitochondrial function and biogenesis changes due to genetically induced mitochondrial autophagy

This example describes mitochondrial biogenesis and functional changes associated with genetically induced mitochondrial autophagy (GiM).

5.2.1. Materials and methods

After gene transfer using retrovirus, MTS-XbaI-ecDHFR was cloned into Hela cells and stable transfectants (Hela_ GiM) were generated that constitutively expressed GiM units. During 2 days of treatment of hela_ GiM cells with Trimethoprim (TMP), the endonuclease XbaI was transiently present in the mitochondrial matrix. Since TMP was dissolved in DMSO, the control group was treated with the same amount of DMSO.

Reactive Oxygen Species (ROS) over time were assessed by staining cells with mitoSox and measuring fluorescence intensity with FACS, setting a threshold line compared to unstressed cells, and measuring the percentage of positive cells. The peroxisome proliferator-activated receptor gamma co-activator 1- α (pgc1α) involved in mitochondrial biogenesis, nuclear factor receptor 1 (NRF 1) involved in mitochondrial biogenesis and mitochondrial Transcription Factor A (TFAM) which forms a nuclear class with mtDNA and is involved in mtDNA transcription, replication and maintenance deeply were evaluated by qPCR. The expression levels of several nuclear-encoded and mtDNA-encoded proteins were quantified via western blotting.

5.2.2. Results

Throughout the evaluation, ROS levels were initially increased in the TMP treated group, but were essentially unchanged in the control group. On day 2, the percentage of mitoSox positive cells in the TMP treated group was higher than in the control group. The percentage of mitoSox positive cells was comparable for both groups on and after day 4 (fig. 14A). At all observations, it was also found that transcription factors pgc1α and NRF1, which promote mitochondrial production, were elevated in the TMP-treated group relative to the control group (fig. 14B and 14C), and TFAM remained essentially unchanged at days 2,4 and 6 in the TMP-treated group (fig. 14D).

Without being bound by theory, it is believed that these results suggest that ROS production is temporarily enhanced by transient mitochondrial genome reduction, whereby mitochondrial biogenesis is caused by amplification of transcription factors such as pgc1α and NRF 1. Again without being bound by theory, it is further believed that the maintenance of elevated transcript levels of TFAM (nuclear-like major constituent protein) suggests that mitochondrial biogenesis may continue for a period of time after mitochondrial genomic depletion is triggered.

Next, the effect of GiM on the expression of nuclear-encoded and mtDNA-encoded proteins was assessed. As the mitochondrial genome decreased, the expression level of ATP5A (respiratory chain complex encoded in the nucleus) was not substantially altered (fig. 15A and 15B). Expression of outer membrane translocator (TOM 20), also encoded in the nucleus, decreased immediately after mitochondrial genome depletion, but recovered by day 6 to levels comparable to those observed in DMSO-treated groups (fig. 15A and 15C). On the other hand, in the TMP treated group, ATP6 (which is encoded by mtDNA) was greatly reduced immediately after the mitochondrial genome was reduced, then increased on day 4, and then restored to a level comparable to that of the control group (fig. 15A and 15D). This trend was also observed in terms of ATP6/ATP5A ratio (fig. 15A and 15E). These results indicate that GiM causes a transient surge in mitochondrial proteins derived from the mitochondrial genome (rather than the nuclear genome).

Changes in the level of the activated form of AMPK (p-AMPK) were used to assess changes in mitochondrial energy production following mitochondrial genome depletion. Mitochondrial energy production was confirmed by an increase in p-AMPK on day 4 (fig. 15A and 15F). Transient mitochondrial genome decrease is associated with reduced ability of the respiratory chain complex on day 2, and this effect is directly related to cell energy depletion, resulting in a significant increase in p-AMPK on day 4.

5.3. Example 3 autophagy associated with GiM-induced transient mitochondrial genome reduction

Mitochondrial autophagy occurs when mitochondria are incorporated into phagosome and become autophagic lysosomes by fusion with lysosomes. This example describes compositions and methods for detecting autophagy lysosomal formation and autophagy following GiM-induced transient mitochondrial genome reduction.

5.3.1. Materials and methods

Autophagic lysosomes have a lower pH than mitochondria that do not fuse with lysosomes. Autophagic lysosomal formation was assessed in cells following GiM-induced transient mitochondrial genome reduction using a pH-sensitive mitochondrial reporter MTKEIMARED. MTKEIMARED fluoresces at pH >6 at 440 nm (green), at pH <5 at 620 nm (red) and has a transport signal that allows it to be transported into the mitochondria. Cells stably expressing MKEIMARED will emit red light when the environmental pH is below 5. To quantify the GiM-induced autophagic lysosomes following transient mitochondrial genome depletion, hela_ GiM cells described in section 5.2.1 were retrovirus engineered with a sequence encoding MKEIMARED. The percentage of cells undergoing mitochondrial autophagy was quantified every two days using fluorescence microscopy and phase contrast microscopy for two weeks.

The last step in autophagy flow depends on lysosomal V-ATPase activity. Thus, the last step of inhibiting autophagy flow following GiM-induced transient mitochondrial genome reduction was performed using the lysosomal V-ATPase inhibitor bafilomycin A1 (BafA 1) to assess autophagy flow targeted to mitochondria. Antibody staining of LC3 (MAP 1LC3: microtubule-associated protein 1 light chain 3) (a representative marker for autophagosome formation), and TOM20 staining as a mitochondrial membrane marker were used to quantify autophagosomes. Considering that LC3 exists on autophagosome membranes as LC3-II with PE, LC3-II was quantified by western blotting in hela_ GiM cells treated with BafA1, where protein was extracted from the cells on day 8 after exposure to TMP 48 h.

5.3.2. Results

Peaks in MTKEIMARED signal associated with GiM-induced autophagy lysosomal formation occurred at day 8 (fig. 16A) and mitochondrial autophagy occurred in about 20% of the cells (fig. 16B). Time course analysis showed that the kinetics of mitochondrial autophagy increased on day 6, decreased to control levels by day 14, and only a small percentage of cells underwent mitochondrial autophagy (fig. 16B). This transient increase in induced mitochondrial autophagy is not associated with cell death or reduced viability. Without being bound by theory, these results indicate that adverse reactions such as mitochondrial autophagy-induced cell death can be inhibited by controlling GiM.

Autophagosomes formed after mitochondrial and lysosomal fusion were detected as spots overlapping LC3 staining and TOM20 staining (fig. 17A). In the absence of TMP, the spot size with overlapping LC3 and TOM20 staining was about 80 μm² and was not significantly affected by the use of BafA 1. On the other hand, in the TMP exposed group, with the addition of BafA a, the spot area with overlapping LC3 and TOM20 staining increased significantly to about 120 μm² (fig. 17B). Although a significant increase in LC3-II through GiM was found, this increase was more pronounced and significant when BafA1 was used (fig. 18A and 18B). Taken together, these results indicate GiM significantly promote autophagy.

5.4. Example 4 reduced Metabolic Effect of mitochondrial genome

Using Hela GiM cells described in section 5.2.1, TMP was exposed for 2 days for mitochondrial genome reduction. Respiratory assays were performed using SeaHorse over time to assess oxidative phosphorylation (OXPHOS) and glycolysis. OXPHOS (fig. 19A, left panel) decreased until day 4 and gradually increased from day 6 until near the starting level on day 10. On the other hand, glycolysis increased until day 8, and then decreased by day 10 (fig. 19A, right panel). A two-dimensional plot depicting the relationship of OXPHOS and glycolysis, showing cyclic changes, indicating that metabolic changes are transient (fig. 19B). Separate assessment of ATP production, basal respiration, proton leakage and sparing capacity further supports GiM the transient nature of the associated metabolic changes (fig. 19C). Even in terms of oxygen consumption, the metabolic effects of GiM indicate that transient mitochondrial genome depletion is a reversible change.

5.5. EXAMPLE 5 genetically induced mitochondrial autophagy in AD fibroblasts

Alzheimer's Disease (AD) is associated with mitochondrial dysfunction. This example describes how transient mitochondrial genome reduction by gene transfer converts cell phenotypes by enhancing mitochondrial turnover in fibroblasts obtained from patients with AD.

5.5.1. Materials and methods

Normal human skin fibroblasts (NHDF) and fibroblasts (AD fibroblasts) obtained from a forearm skin sample of a patient suffering from alzheimer's disease, whose APOE genotype is E3/E45, were used as target cells. Transient mitochondrial genome reduction was performed by transferring a plasmid carrying the gene encoding endonuclease XbaIR downstream of the mitochondrial transfer signal derived from human Cox8 and expressing puromycin resistance (pCAGGS-MTS-XbaIR) as a selectable marker at a different promoter. Using electroporation as a gene transfer method, transfected target cells were enriched by exposing them to puromycin at a concentration of 3. Mu.g/mL for 24 hours on day 2 post electroporation. The conditions were set using a plasmid with recombinant GFP gene instead of XbaIR, according to the criteria of 70-80% GFP expression and viability exceeding 90%. Following gene transfer, the copy number of mitochondrial genomes was assessed on days 7, 14 and 21 to confirm genomic reduction and subsequent biogenesis (fig. 20A). In addition, mitochondrial phenotypes were assessed by measuring mitochondrial volume (mtMass) and mitochondrial membrane potential (mtMP) using MitoTracker Green and TMRM, respectively.

5.5.2. Results

MtMP was significantly reduced in untreated AD fibroblasts relative to untreated NHDF (fig. 20B and 20E). Similarly, mtMass was reduced in untreated AD fibroblasts relative to untreated NHDF (fig. 20B and 20D). Two fluorescent signals were amplified in two dimensions and quadrant analysis was performed using NHDF as positive control to set the threshold line. The fractional ratio is used as a biomarker for detecting the senescence of mitochondrially dysfunctional lymphocytes. The double positive rate was found to be about half that of NHDF. The percentage of double positives did not change significantly on day 7 after genetically induced mitochondrial autophagy (GiM), but increased to 60.3% on day 14 and further to 87.3% on day 21, comparable to control NHDF (fig. 20C).

MtMass and mtMP were both quantified using the Mean Fluorescence Intensity (MFI) as an indicator of fluorescence intensity. mtMass and mtMP were lower in untreated AD fibroblasts relative to NHDF cells. In AD fibroblasts, mtMass and mtMP levels were even lower at day 7 after GIM induction, but increased to levels comparable to NHDF at day 14 (fig. 20F-20G), indicating that both mitochondrial volume and mitochondrial membrane potential in AD fibroblasts were restored to the same levels as healthy NHDF cells by GiM.

These results indicate that mitochondrial turnover facilitated by GiM is qualitatively beneficial to newly generated mitochondria. Given that accumulation of dysfunctional mitochondria is a common phenomenon in a variety of neurodegenerative diseases and aging, giM can be used as a therapeutic strategy for treating degenerative diseases and also for the restoration of normal function in aging cells.

6. Detailed description of the preferred embodiments

The present disclosure is exemplified by the following specific embodiments.

1. A polypeptide comprising:

(a) Mitochondrial Targeting Sequences (MTS);

(b) Endonuclease sequences, and

(C) Destabilizing domain sequences.

2. The polypeptide of embodiment 1, wherein said MTS comprises human MTS.

3. The polypeptide of embodiment 1, wherein said MTS comprises a non-human MTS.

4. The polypeptide of any one of embodiments 1 to 3, wherein said MTS comprises a MTS of a mitochondrial protein.

5. The polypeptide of any one of embodiments 1 to 4, wherein said MTS comprises a TCA cycle associated enzyme, chaperone protein, mitochondrial genome replication protein, protease, mRNA processing protein, mitochondrial RNA degradation protein, deoxynucleotide triphosphate synthesis associated protein, mitochondrial ribosomal protein, phospholipid metabolism associated protein, proteins involved in toxic compound metabolism, disulfide relay system associated protein, iron-sulfur protein assembly protein, tRNA modification protein, aminoacyl tRNA synthetase, MTS releasing factor or elongation factor.

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

7. The polypeptide of embodiment 6, wherein said MTS comprises the MTS of cytochrome c oxidase subunit VIII (COX 8).

8. The polypeptide of embodiment 6, wherein said MTS comprises the MTS of cytochrome c oxidase subunit X (COX 10).

9. The polypeptide of embodiment 6, wherein said MTS comprises a MTS of cytochrome c oxidase subunit IV (COX 4).

10. The polypeptide of any one of embodiments 1 to 5, wherein said MTS comprises an MTS of ataxin (FXN).

11. The polypeptide of any one of embodiments 1 to 5, wherein said MTS comprises a MTS of a TCA cycle related enzyme, optionally said TCA cycle related enzyme is a pyruvate dehydrogenase, a citrate synthase, a aconitase, an isocitrate dehydrogenase, an alpha-ketoglutarate dehydrogenase, a succinyl CoA synthase, a succinate dehydrogenase, a fumarate, a malate dehydrogenase, or a pyruvate carboxylase.

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

13. The polypeptide of any one of embodiments 1 to 5, wherein said MTS comprises a mitochondrial genome replication protein, optionally said mitochondrial genome replication protein is TFAM, twinkle, polG, TFB, M, TEFM or MTERF1.

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

15. The polypeptide of any one of embodiments 1 to 5, wherein said MTS comprises an MTS of an mRNA processing protein, optionally said mRNA processing protein is LRPPRC, TACO1, ELAC, PNPT1, HSD17B10, MTPAP, or PTCD1.

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

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

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

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

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

21. The polypeptide of any one of embodiments 1 to 5, wherein said MTS comprises a MTS of a disulfide relay related protein, optionally said disulfide relay related protein is GFER.

22. The polypeptide of any one of embodiments 1 to 5, wherein said MTS comprises an MTS of a ferrosulfur assembly protein, optionally said ferrosulfur assembly protein is ISCU, BOLA3, NFU1, or IBA57.

23. The polypeptide of any one of embodiments 1 to 5, wherein said MTS comprises a MTS of a tRNA modification protein, optionally said tRNA modification protein is MTO1, GTP3BP, TRMU, PUS1, MTFMT, TRIT1, TRNT1, or TRMT.

24. The polypeptide of any one of embodiments 1 to 5, wherein said MTS comprises an aminoacyl tRNA synthetase, optionally said aminoacyl tRNA synthetase is AARS2, DARS2, EARS2, RARS2, YARS2, far 2, HARS2, LARS2, VARS2, TARS2, IARS2, CARS2, PARS2, NARS2, KARS, GARS, SARS2, or MARS2.

25. The polypeptide of any one of embodiments 1 to 5, wherein said MTS comprises a release factor MTS, optionally said release factor is C12orf65.

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

27. The polypeptide of embodiment 1, wherein said MTS comprises a sequence at least 80% identical to MSVLTPLLLRGLTGSARR (SEQ ID NO: 1).

28. The polypeptide of embodiment 27, wherein said MTS comprises a sequence at least 85% identical to MSVLTPLLLRGLTGSARR (SEQ ID NO: 1).

29. The polypeptide of embodiment 27, wherein said MTS comprises a sequence at least 90% identical to MSVLTPLLLRGLTGSARR (SEQ ID NO: 1).

30. The polypeptide of embodiment 27, wherein said MTS comprises a sequence at least 95% identical to MSVLTPLLLRGLTGSARR (SEQ ID NO: 1).

31. The polypeptide of embodiment 27, wherein said MTS comprises a sequence 100% identical to MSVLTPLLLRGLTGSARR (SEQ ID NO: 1).

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

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

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

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

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

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

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

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

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

41. The polypeptide of embodiment 37, wherein said MTS comprises a sequence 100% identical to MSVLTPLLLRSLTGSARRLMVPRA (SEQ ID NO: 3).

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

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

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

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

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

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

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

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

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

51. The polypeptide of embodiment 47, wherein said MTS comprises a sequence 100% identical to MWTLGRRAVAGLLASPSPAQ (SEQ ID NO: 5).

52. The polypeptide of embodiment 1, wherein said MTS comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to MAPYSLLVTRLQKALG (SEQ ID NO: 6).

53. The polypeptide of embodiment 1, wherein said MTS comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to MALLTAAARLLGTKNASCLVLAARHASA (SEQ ID NO: 7).

54. The polypeptide of embodiment 1, wherein said MTS comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to MVKQIESKTAFQEALDAAGDKLVVVDFSATWC (SEQ ID NO: 8).

55. The polypeptide of embodiment 1, wherein said MTS comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to MATNWGSLLQDKQQLEELARQAVDRALAEGVLLRTSQ (SEQ ID NO: 9).

56. The polypeptide of embodiment 1, wherein said MTS comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to MAFLRSMWGVLSALGRSGA (SEQ ID NO: 10).

57. The polypeptide of embodiment 1, wherein said MTS comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to MWVLLRSGYPLRILLPLRG (SEQ ID NO: 11).

58. The polypeptide of embodiment 1, wherein said MTS comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to MSRLLWRKVAGATVGPGPVPAPG (SEQ ID NO: 12).

59. The polypeptide of embodiment 1, wherein said MTS comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to MKRNTLVELLTFWKNWHFRLL (SEQ ID NO: 13).

60. The polypeptide of embodiment 1, wherein said MTS comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to MISASRAAAARLVGAAASRGPTAA (SEQ ID NO: 14).

61. The polypeptide of embodiment 1, wherein said MTS comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to MEALIPVINKLQDVFNTVGA (SEQ ID NO: 15).

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 Cas 12), a zinc finger nuclease, or a transcription activator-like effector nuclease (TALEN).

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

64. The polypeptide of embodiment 63, wherein the restriction endonuclease is XbaIR, ecoRI, smaI, aflII, bamHI, bclI, haeIII, hindII, hindIII, ndeI, pvuII, pstI or a SpeI endonuclease.

65. The polypeptide of embodiment 64, wherein the restriction endonuclease is a XbaIR endonuclease.

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

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).

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).

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).

70. The polypeptide of embodiment 66, wherein the endonuclease sequence comprises a sequence that is the same as MTTLEKIKLLADGYADRLKLAIDGRVLEMQGDDVSHYLIYRVLGVAQEEGRLIDVYQNKGRFLYKYAGSFLEAATKLCFKEAFPDSASLRLPNTQGQRPRTVEIDCLVGNDALEIKWKDATTDGDHITKEHTRIKVISDAGYKPIRIMFYYPHRTQAIRIQETLETLYNGVHGEYHYGEAAWDYVLQRTSVNLKVALEQIADSRTNEAA(SEQ ID NO: 16) 100%.

71. The polypeptide of any one of embodiments 1 to 70, wherein the destabilizing domain sequence is a DHFR, FKBP or PDE5 destabilizing domain sequence.

72. The polypeptide of embodiment 71, wherein the destabilizing domain sequence is a DHFR destabilizing domain sequence.

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

74. The polypeptide of any one of embodiments 1 to 73, wherein the destabilizing domain sequence comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to MISLIAALAVDHVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR(SEQ ID NO: 17).

75. The polypeptide of any one of embodiments 1 to 73, wherein the destabilizing domain sequence comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to ISLIAALAVDHVIGMETVMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR(SEQ ID NO: 18).

76. The polypeptide of embodiment 74 wherein the destabilizing domain sequence has one or more of the following amino acid substitutions N18T/A19V, F103L, Y100I, G121V, H Y/Y100I, H L/Y100I, R98H/F103S, M T/H114R and I61F/T68S.

77. The polypeptide of any one of embodiments 1 to 71, wherein the destabilizing domain sequence comprises a sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE(SEQ ID NO: 20).

78. The polypeptide of embodiment 77, wherein the destabilizing domain sequence has one or more of the following amino acid substitutions F15S, V24A, H25R, E60G, L106P, D G, M3266T, R G, D100N, E G and K105I.

79. The polypeptide of any one of embodiments 1 to 71, wherein the destabilizing 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.

80. The polypeptide of any one of embodiments 1 to 79, wherein said MTS is located N-terminal to said endonuclease sequence and said destabilizing domain sequence.

81. The polypeptide of any one of embodiments 1 to 79, wherein said MTS is located C-terminal to said endonuclease sequence and said destabilizing domain sequence.

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

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

84. The polypeptide of any one of embodiments 1 to 79, wherein said MTS is located N-terminal to said endonuclease sequence and said endonuclease sequence is located N-terminal to said destabilizing domain sequence.

85. A nucleic acid encoding the polypeptide of any one of embodiments 1 to 84.

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

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

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

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.

90. The nucleic acid of any one of embodiments 85 to 89, which is a vector.

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

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

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

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

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

96. A host cell comprising the nucleic acid of any one of embodiments 85-94.

97. A method of (a) inducing mitochondrial autophagy in a cell, and/or (b) increasing mitochondrial turnover in a cell, and/or (c) increasing mitochondrial mass, and/or (d) inducing a double strand break in mitochondrial DNA, and/or (e) inducing an epigenomic modification in a cell, the method comprising contacting the cell with (i) the polypeptide of any one of embodiments 1-84, the nucleic acid of any one of embodiments 85-94, or the particle of embodiment 95, and (ii) a stabilizer.

98. The method of embodiment 97, wherein when the destabilizing domain sequence is a DHFR destabilizing domain sequence, the stabilizer is Trimethoprim (TMP).

99. The method of embodiment 97, wherein when the destabilizing domain sequence is an FKBP destabilizing domain sequence, the stabilizer is Shield-1, rapamycin or FK506.

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.

101. The method of embodiment 100, comprising culturing the cells in a medium comprising the stabilizing agent for at least 8 hours.

102. The method of embodiment 100, comprising culturing the cells in a medium comprising the stabilizing agent for at least 12 hours.

103. The method of embodiment 100, comprising culturing the cells in a medium comprising the stabilizing agent for at least 1 day.

104. The method of embodiment 100, comprising culturing the cells in a medium comprising the stabilizing agent for at least 2 days.

105. The method of any one of embodiments 100 to 104, comprising culturing the cells in a medium comprising the stabilizing agent for up to 5 days.

106. The method of any one of embodiments 100 to 104, comprising culturing the cells in a medium comprising the stabilizing agent for up to 4 days.

107. The method of any one of embodiments 100 to 104, comprising culturing the cells in a medium comprising the stabilizing agent for up to 3 days.

108. The method of any one of embodiments 100 to 104, comprising culturing the cells in a medium comprising the stabilizing agent for up to 2 days.

109. The method of any one of embodiments 97-108, further comprising, after contacting the cell with the stabilizing agent, removing the stabilizing agent from the cell.

110. The method of embodiment 109, wherein removing the stabilizing agent from the cells comprises culturing the cells in a medium that does not comprise the stabilizing agent.

111. The method of embodiment 110, comprising culturing the cells in a medium that does not comprise the stabilizing agent for at least 3 hours.

112. The method of embodiment 110, comprising culturing the cells in a medium that does not comprise the stabilizing agent for at least 6 hours.

113. The method of embodiment 110, comprising culturing the cells in a medium that does not comprise the stabilizing agent for at least 12 hours.

114. The method of embodiment 110, comprising culturing the cells in a medium that does not comprise the stabilizing agent for at least 1 day.

115. The method of embodiment 110, comprising culturing the cells in a medium that does not comprise the stabilizing agent for at least 2 days.

116. The method of embodiment 110, comprising culturing the cells in a medium that does not comprise the stabilizing agent for at least 3 days.

117. The method of embodiment 110, comprising culturing the cells in a medium that does not comprise the stabilizing agent for at least 4 days.

118. The method of any one of embodiments 110 to 117, comprising culturing the cells in a medium that does not comprise the stabilizing agent for up to 10 days.

119. The method of any one of embodiments 100 to 117, comprising culturing the cells in a medium that does not comprise the stabilizing agent for up to 8 days.

120. The method of any one of embodiments 100 to 117, comprising culturing the cells in a medium that does not comprise the stabilizing agent for up to 6 days.

121. The method of any one of embodiments 100 to 117, comprising culturing the cells in a medium that does not comprise the stabilizing agent for up to 4 days.

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.

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

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.

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

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

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

128. The method of any one of embodiments 97 to 127, which induces mitochondrial autophagy in the cell.

129. The method of any one of embodiments 97-128, which increases mitochondrial turnover in the cell.

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

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

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

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

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.

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

136. The cell of embodiment 135, wherein when the destabilizing domain sequence is a DHFR destabilizing domain sequence, the stabilizer is Trimethoprim (TMP).

137. The cell of embodiment 135, wherein when the destabilizing domain sequence is an FKBP destabilizing domain sequence, the stabilizer is Shield-1, rapamycin or FK506.

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.

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

140. The method or cell of any one of embodiments 138-139, wherein the cell is a somatic cell.

141. The method or cell of any one of embodiments 138-140, wherein the cell is a bone marrow cell.

142. The method or cell of any one of embodiments 138-141, wherein the cell is a Hematopoietic Stem Cell (HSC) or a Mesenchymal Stem Cell (MSC).

143. The method or cell of any one of embodiments 138-140, wherein the cell is an immune cell.

144. The method or cell of embodiment 143, wherein the cell is a T cell, a phagocytic cell, a microglial cell, or a macrophage.

145. The method or cell of embodiment 144, wherein the cell is a cd4+ T cell.

146. The method or cell of embodiment 144 or embodiment 145, wherein the cell is a cd8+ T cell.

147. The method or cell of any one of embodiments 138-146, wherein the cell is a primary cell.

148. The method or cell of any one of embodiments 138-146, wherein the cell is a progeny of a primary cell.

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

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

151. The method or cell of embodiment 150, wherein the age-related disorder is an autoimmune disorder, a metabolic disorder, a genetic disorder, cancer, a neurodegenerative disorder, or immune aging.

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.

153. The method or cell of embodiment 152, wherein the mitochondrial disease or disorder is caused by mitochondrial DNA abnormalities, nuclear DNA abnormalities, or both.

154. The method or cell of embodiment 152 or embodiment 153, wherein the mitochondrial disease or disorder is chronic progressive extraocular myoparalysis (CPEO), pearson syndrome, kanns-ser syndrome (KSS), diabetes and deafness (DAD), mitochondrial diabetes, leber Hereditary Optic Neuropathy (LHON), LHON-plus, neuropathy, ataxia, retinitis pigmentosa syndrome (NARP), maternal hereditary Leigh syndrome (MILS), mitochondrial encephalomyopathy with lactic acidosis and stroke-like attacks (MELAS), muscle-aspiration epilepsy with bilateral red ragged Myofibrosis (MERRF), familial striatal necrosis/striatal melanin degeneration (FBSN), raffint disease, aminoglycoside-induced deafness (AID), or mitochondrial DNA multiple deficiency syndrome.

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 (milas), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase γ (POLG) -related disease, sensory ataxia neuropathy, dystonia (SANDO), leukoencephalopathy (LBSL) involving brain stem and spinal cord with elevated lactate, coenzyme Q10 deficiency, leigh syndrome, mitochondrial complex abnormality, fumarase deficiency, alpha-ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl-coa ligase deficiency, pyruvate dehydrogenase complex deficiency (PDHC), pyruvate Carboxylase Deficiency (PCD), carnitine palmitoyl transferase I (CPT I) deficiency, carnitine palmitoyl transferase II (CPT) deficiency, carnitine-acyl carnitine (CACT), autosomal/autosomal progressive, mitochondrial-dominant-phase-muscle atrophy (mmc), advanced-atrophy (mmc), or amyotrophic lateral atrophy (CMT 2), mitochondrial-dominant-phase-deficiency (mmc), or amyotrophic lateral-motor-deficiency (mmc), gastric-muscle-deficiency (mmc), or (mmc-2).

156. The method or cell of any one of embodiments 138-155, wherein the cell is from a subject having a neurodegenerative disease.

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, fibula muscular atrophy, or white matter dystrophy.

158. The method or cell of embodiment 156, wherein the neurodegenerative disease is Amyotrophic Lateral Sclerosis (ALS).

159. The method or cell of embodiment 156, wherein the neurodegenerative disease is huntington's disease.

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, e.g., E3/E4 or E4/E4 genotype.

161. The method or cell of embodiment 156, wherein the neurodegenerative disease is parkinson's disease.

162. The method or cell of embodiment 156, wherein the neurodegenerative disease is friedreich ataxia. Fibular muscular atrophy.

163. The method or cell of embodiment 156, wherein the neurodegenerative disease is white matter dystrophy.

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

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

166. The method or cell of any one of embodiments 138-165, wherein the cell is from a subject with diabetes.

167. The method or cell of any of embodiments 138-166, wherein the cell is from a subject having a hearing disorder.

168. The method or cell of any one of embodiments 138-167, wherein the cell is from a subject with a genetic disorder.

169. The method or cell of embodiment 168, wherein the genetic disorder is hakinsen-Ji Erfu de early senescence syndrome, wona syndrome, or huntington's disease.

170. The method or cell of any one of embodiments 138-169, wherein the cell is from a subject having heart failure.

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

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

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

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

175. The method or cell of any one of embodiments 138-174, which is an ex vivo cell.

176. The method of embodiment 175, further comprising administering the cell to a subject, optionally wherein the subject is the same subject from which the cell originated.

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

178. The method of embodiment 177, wherein the subject has an age-related disorder.

179. The method of embodiment 178, wherein the age-related disorder is an autoimmune disorder, a metabolic disorder, a genetic disorder, cancer, a neurodegenerative disorder, or immune aging.

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

181. The method of embodiment 180, wherein the mitochondrial disease or disorder is caused by mitochondrial DNA abnormalities, nuclear DNA abnormalities, or both.

182. The method of embodiment 180 or embodiment 181, wherein the mitochondrial disease or disorder is chronic progressive extraocular myoparalysis (CPEO), pearson syndrome, kanns-ser syndrome (KSS), diabetes and deafness (DAD), mitochondrial diabetes, leber Hereditary Optic Neuropathy (LHON), LHON-plus, neuropathy, ataxia, and retinitis pigmentosa syndrome (NARP), maternal hereditary Leigh syndrome (MILS), mitochondrial encephalomyopathy with lactic acidosis and stroke-like attacks (MELAS), sarcoidosis with red ragged Myofibrosis (MERRF), familial bilateral striatal necrosis/striatal melanin degeneration (FBSN), raffint disease, aminoglycoside-induced deafness (AID), and mitochondrial DNA multiple deficiency syndrome.

183. Embodiment 180 or embodiment 181, wherein the mitochondrial disease or disorder is mitochondrial DNA depletion syndrome 4A, mitochondrial recessive ataxia syndrome (milas), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase γ (POLG) -related disease, sensory ataxia neuropathy, buffer in the eye (SANDO), leukoencephalopathy (LBSL) involving brain stem and spinal cord with elevated lactic acid, coenzyme Q10 deficiency, leigh syndrome, mitochondrial complex abnormality, fumarase deficiency, alpha-ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl-coa ligase deficiency, pyruvate dehydrogenase complex deficiency (PDHC), pyruvate Carboxylase Deficiency (PCD), carnitine palmitoyl transferase I (CPT I) deficiency, carnitine palmitoyl transferase II (CPT IT) deficiency, carnitine-acyl carnitine (CACT) deficiency, autosomal/normothermic-type, mitochondrial phase-advanced-muscle atrophy (tsunabated), tsubular-muscle atrophy (glabrosis), tsunabated-type (CMT), tsunabated muscle atrophy (glabrosis), or (CMT 2-type, myotonic muscle atrophy (myopic muscle atrophy).

184. The method of embodiment 177, wherein the subject has a neurodegenerative disease.

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, fibula muscular atrophy, or white matter dystrophy.

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

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

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

189. The method of embodiment 177, wherein the subject has a hearing disorder.

190. The method of embodiment 177, wherein the subject has a genetic disorder.

191. The method of embodiment 190, wherein the genetic disorder is hakinsen-Ji Erfu de early senescence syndrome, wona syndrome, or huntington's disease.

192. The method of embodiment 177, wherein the subject has heart failure.

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

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

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

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.

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 stabilizing agent.

198. The kit of embodiment 197, wherein when the destabilizing domain sequence is a DHFR destabilizing domain sequence, the stabilizer is Trimethoprim (TMP).

199. The kit of embodiment 197, wherein when the destabilizing domain sequence is an FKBP destabilizing domain sequence, the stabilizer is Shield-1, rapamycin or FK506.

200. The kit of embodiment 197, wherein when the destabilizing domain sequence is a PDE5 destabilizing domain sequence, the stabilizing agent is sildenafil, vardenafil, tadalafil, avanafil, lodinafil, milonafil, nadinafil, phenylamidinafil, darifenacin and beminafil.

7. Citation of reference

All publications, patents, patent applications, and other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent application, or other document was individually indicated to be incorporated by reference for all purposes. If there is an inconsistency between the teachings of one or more of the references incorporated herein and the present disclosure, it is intended that the teachings of the present specification shall control.

Claims (52)

Translated fromChinese

1.多肽，其包含：1. A polypeptide comprising:(a)线粒体靶向序列(MTS)；(a) Mitochondrial targeting sequence (MTS);(b)核酸内切酶序列；和(b) an endonuclease sequence; and(c)去稳定化结构域序列。(c) Destabilization domain sequence.2.权利要求1所述的多肽，其中所述MTS包含人MTS。2. The polypeptide of claim 1, wherein the MTS comprises human MTS.3.权利要求1所述的多肽，其中所述MTS包含非人MTS。3. The polypeptide of claim 1, wherein the MTS comprises a non-human MTS.4.权利要求1至3中任一项所述的多肽，其中所述MTS包含线粒体蛋白的MTS。4. The polypeptide of any one of claims 1 to 3, wherein the MTS comprises the MTS of a mitochondrial protein.5.权利要求1至4中任一项所述的多肽，其中所述MTS包含TCA循环相关酶、伴侣蛋白、线粒体基因组复制蛋白、蛋白酶、mRNA加工蛋白、线粒体RNA降解蛋白、脱氧核苷酸三磷酸合成相关蛋白、线粒体核糖体蛋白、磷脂代谢相关蛋白、参与毒性化合物代谢的蛋白、二硫键中继系统相关蛋白、铁硫蛋白组装蛋白、tRNA修饰蛋白、氨酰基tRNA合成酶、释放因子或延伸因子的MTS。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.权利要求1至5中任一项所述的多肽，其中所述MTS包含细胞色素c氧化酶亚基的MTS。6. The polypeptide of any one of claims 1 to 5, wherein the MTS comprises the MTS of a cytochrome c oxidase subunit.7.权利要求6所述的多肽，其中所述MTS包含细胞色素c氧化酶亚基VIII(COX8)、细胞色素c氧化酶亚基X(COX10)、细胞色素c氧化酶亚基IV(COX4)的MTS。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.权利要求1至5中任一项所述的多肽，其中所述MTS包含：8. The polypeptide of any one of claims 1 to 5, wherein the MTS comprises:(a)共济蛋白(FXN)的MTS；(a) MTS of frataxin (FXN);(b)TCA循环相关酶的MTS，任选地所述TCA循环相关酶是丙酮酸脱氢酶、柠檬酸合酶、顺乌头酸酶、异柠檬酸脱氢酶、α-酮戊二酸脱氢酶、琥珀酰CoA合成酶、琥珀酸脱氢酶、延胡索酸酶、苹果酸脱氢酶或丙酮酸羧化酶；(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)伴侣蛋白的MTS，任选地所述伴侣蛋白是mtHSP10、mtHSP60、mtHSP70或mtHSP90；(c) an MTS of a chaperone protein, optionally wherein the chaperone protein is mtHSP10, mtHSP60, mtHSP70 or mtHSP90;(d)线粒体基因组复制蛋白的MTS，任选地所述线粒体基因组复制蛋白是TFAM、Twinkle、PolG、TFB2M、TEFM或MTERF1；(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)蛋白酶的MTS，任选地所述蛋白酶是MPP、CLPXP、LON ATPase或PreP；(e) an MTS of a protease, optionally wherein the protease is MPP, CLPXP, LON ATPase or PreP;(f)mRNA加工蛋白的MTS，任选地所述mRNA加工蛋白是LRPPRC、TACO1、ELAC2、PNPT1、HSD17B10、MTPAP或PTCD1；(f) an MTS of an mRNA processing protein, optionally wherein the mRNA processing protein is LRPPRC, TACO1, ELAC2, PNPT1, HSD17B10, MTPAP or PTCD1;(g)线粒体RNA降解蛋白的MTS，任选地所述线粒体RNA降解蛋白是PNPasse、REX02或SUV3；(g) an MTS of a mitochondrial RNA degradation protein, optionally wherein the mitochondrial RNA degradation protein is PNPasse, REX02 or SUV3;(h)脱氧核苷酸三磷酸合成相关蛋白的MTS，任选地所述脱氧核苷酸三磷酸合成相关蛋白是DGUOK、TK2、TYMP、MGME1、SUCLG1、SUCLA2、RNASEH1或C10orf2；(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)线粒体核糖体蛋白的MTS，任选地所述线粒体核糖体蛋白是MRPS16、MRPS22、MRPL3、MRP12或MRPL44；(i) an MTS of a mitochondrial ribosomal protein, optionally wherein the mitochondrial ribosomal protein is MRPS16, MRPS22, MRPL3, MRP12 or MRPL44;(j)磷脂代谢相关蛋白的MTS，任选地所述磷脂代谢相关蛋白是AGK、SERAC1或TAZ；(j) an MTS of a phospholipid metabolism-related protein, optionally wherein the phospholipid metabolism-related protein is AGK, SERAC1 or TAZ;(k)参与毒性化合物代谢的蛋白的MTS，任选地所述参与毒性化合物代谢的蛋白是HIBCH、ECHS1、ETHE1或MPV17；(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;(l)二硫键中继系统相关蛋白的MTS，任选地所述二硫键中继系统相关蛋白是GFER；(1) an MTS of a disulfide bond relay system-associated protein, optionally wherein the disulfide bond relay system-associated protein is GFER;(m)铁硫蛋白组装蛋白的MTS，任选地所述铁硫蛋白组装蛋白是ISCU、BOLA3、NFU1或IBA57；(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)tRNA修饰蛋白的MTS，任选地所述tRNA修饰蛋白是MTO1、GTP3BP、TRMU、PUS1、MTFMT、TRIT1、TRNT1或TRMT5；(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)氨酰基tRNA合成酶的MTS，任选地所述氨酰基tRNA合成酶是AARS2、DARS2、EARS2、RARS2、YARS2、FARS2、HARS2、LARS2、VARS2、TARS2、IARS2、CARS2、PARS2、NARS2、KARS、GARS、SARS2或MARS2；(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)释放因子的MTS，任选地所述释放因子是C12orf65；或(p) an MTS that releases a factor, optionally the release factor is C12orf65; or(q)延伸因子的MTS，任选地所述延伸因子是TUFM、TSFM或GFM1。(q) an MTS of an elongation factor, optionally wherein the elongation factor is TUFM, TSFM or GFM1.9.权利要求1所述的多肽，其中所述MTS包含与SEQ ID NO: 1-15中的任一个至少80%相同的序列。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.权利要求9所述的多肽，其中所述MTS包含与SEQ ID NO: 1 100%相同的序列。10. The polypeptide of claim 9, wherein the MTS comprises a sequence that is 100% identical to SEQ ID NO: 1.11.权利要求9所述的多肽，其中所述MTS包含与SEQ ID NO: 2 100%相同的序列。11. The polypeptide of claim 9, wherein the MTS comprises a sequence that is 100% identical to SEQ ID NO: 2.12.权利要求1至11中任一项所述的多肽，其中所述核酸内切酶是限制性核酸内切酶、RNA引导的核酸内切酶(例如，Cas9或Cas12)、锌指核酸酶或转录激活因子样效应物核酸酶(TALEN)。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.权利要求12所述的多肽，其中所述核酸内切酶是限制性核酸内切酶。13. The polypeptide of claim 12, wherein the endonuclease is a restriction endonuclease.14.权利要求13所述的多肽，其中所述限制性核酸内切酶是XbaIR、EcoRI、SmaI、AflII、BamHI、BclI、HaeIII、HindII、HindIII、NdeI、PvuII、PstI或SpeI核酸内切酶。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.权利要求14所述的多肽，其中所述限制性核酸内切酶是XbaIR核酸内切酶。15. The polypeptide of claim 14, wherein the restriction endonuclease is XbaIR endonuclease.16.权利要求1至13中任一项所述的多肽，其中所述核酸内切酶包含与SEQ ID NO: 16至少80%相同的序列。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.权利要求16所述的多肽，其中所述核酸内切酶序列包含与SEQ ID NO: 16 100%相同的序列。17. The polypeptide of claim 16, wherein the endonuclease sequence comprises a sequence that is 100% identical to SEQ ID NO: 16.18.权利要求1至17中任一项所述的多肽，其中所述去稳定化结构域序列是DHFR、FKBP或PDE5去稳定化结构域序列。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.权利要求18所述的多肽，其中所述去稳定化结构域序列是DHFR去稳定化结构域序列。19. The polypeptide of claim 18, wherein the destabilization domain sequence is a DHFR destabilization domain sequence.20.权利要求19所述的多肽，其中所述去稳定化结构域序列是大肠杆菌DHFR(ecDHFR)去稳定化结构域序列。20. The polypeptide of claim 19, wherein the destabilization domain sequence is an Escherichia coli DHFR (ecDHFR) destabilization domain sequence.21.权利要求1至20中任一项所述的多肽，其中所述去稳定化结构域序列包含与SEQ IDNO: 18至少80%、至少85%、至少90%、至少95%或100%相同的序列。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.权利要求1至18中任一项所述的多肽，其中所述去稳定化结构域序列包含与SEQ IDNO: 20至少80%、至少85%、至少90%、至少95%或100%相同的序列。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.权利要求1至22中任一项所述的多肽，其中所述MTS位于所述核酸内切酶序列和所述去稳定化结构域序列的N端。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.权利要求1至22中任一项所述的多肽，其中所述MTS位于所述核酸内切酶序列和所述去稳定化结构域序列的C端。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.权利要求1至24中任一项所述的多肽，其中所述核酸内切酶序列位于所述去稳定化结构域序列的N端。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.权利要求1至24中任一项所述的多肽，其中所述核酸内切酶序列位于所述去稳定化结构域序列的C端。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.权利要求1至22中任一项所述的多肽，其中所述MTS位于所述核酸内切酶序列的N端，并且所述核酸内切酶序列位于所述去稳定化结构域序列的N端。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.核酸，其编码权利要求1至27中任一项所述的多肽。28. A nucleic acid encoding the polypeptide of any one of claims 1 to 27.29.颗粒，其包含权利要求28所述的核酸，任选地其中所述颗粒是逆转录病毒颗粒。29. A particle comprising the nucleic acid of claim 28, optionally wherein the particle is a retroviral particle.30.宿主细胞，其包含权利要求28所述的核酸。30. A host cell comprising the nucleic acid of claim 28.31.(a)诱导细胞中的线粒体自噬，和/或(b)增加细胞中的线粒体周转，和/或(c)增加线粒体质量，和/或(d)诱导线粒体DNA中的双链断裂和/或(e)诱导细胞中的表观基因组修饰的方法，所述方法包括使所述细胞与(i)权利要求1至27中任一项所述的多肽、权利要求28所述的核酸或权利要求29所述的颗粒和(ii)稳定剂接触。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.权利要求31所述的方法，其中当所述去稳定化结构域序列是DHFR去稳定化结构域序列时，所述稳定剂是甲氧苄啶(TMP)。32. The method of claim 31, wherein when the destabilization domain sequence is a DHFR destabilization domain sequence, the stabilizer is trimethoprim (TMP).33.权利要求31所述的方法，其中当所述去稳定化结构域序列是FKBP去稳定化结构域序列时，所述稳定剂是Shield-1、雷帕霉素或FK506。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.权利要求31至33中任一项所述的方法，其进一步包括，在使所述细胞与所述稳定剂接触之后，从所述细胞中去除所述稳定剂。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.细胞，其通过权利要求31至34中任一项所述的方法获得或可获得。35. A cell obtained or obtainable by the method of any one of claims 31 to 34.36.细胞，其包含权利要求1-27中任一项所述的多肽、权利要求28所述的核酸或权利要求29所述的颗粒。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.权利要求31至34中任一项所述的方法或权利要求35或权利要求36所述的细胞，其中所述细胞是哺乳动物细胞。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.权利要求37所述的方法或细胞，其中所述细胞是人细胞。38. The method or cell of claim 37, wherein the cell is a human cell.39.权利要求37或权利要求38所述的方法或细胞，其中所述细胞是体细胞、骨髓细胞、造血干细胞(HSC)或间充质干细胞(MSC)或免疫细胞。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.权利要求37至39中任一项所述的方法或细胞，其中所述细胞来自患有年龄相关疾病的受试者。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.权利要求40所述的方法或细胞，其中所述年龄相关疾病是自身免疫性疾病、代谢性疾病、遗传性疾病、癌症、神经退行性疾病或免疫衰老。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.权利要求37至41中任一项所述的方法或细胞，其中所述细胞来自患有线粒体疾病或病症的受试者。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.权利要求42所述的方法或细胞，其中所述线粒体疾病或病症是慢性进行性眼外肌麻痹(CPEO)、皮尔逊综合征、卡恩斯-塞尔综合征(KSS)、糖尿病和耳聋(DAD)、线粒体糖尿病、Leber遗传性视神经病变(LHON)、LHON-plus、神经病变、共济失调、视网膜色素变性综合征(NARP)、母系遗传性Leigh综合征(MILS)、线粒体脑肌病伴乳酸酸中毒和卒中样发作(MELAS)、肌抽跃癫痫合并红色褴褛肌纤维症(MERRF)、家族性双侧纹状体坏死/纹状体黑质变性(FBSN)、拉夫特病、氨基糖苷类诱导的耳聋(AID)或线粒体DNA多重缺失综合征。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.权利要求42所述的方法或细胞，其中所述线粒体疾病或病症是线粒体DNA耗竭综合征4A、线粒体隐性共济失调综合征(MIRAS)、线粒体神经胃肠型脑肌病(MNGIE)、线粒体DNA耗竭综合征(MTDPS)、DNA聚合酶γ(POLG)相关疾病、感觉性共济失调神经病变构音障碍眼肌麻痹(SANDO)、累及脑干和脊髓并伴有乳酸升高的脑白质病(LBSL)、辅酶Q10缺乏症、Leigh综合征、线粒体复合体异常、延胡索酸酶缺乏症、α-酮戊二酸脱氢酶复合物(KGDHC)缺乏症、琥珀酰辅酶A连接酶缺乏症、丙酮酸脱氢酶复合物缺乏症(PDHC)、丙酮酸羧化酶缺乏症(PCD)、肉碱棕榈酰转移酶I(CPT I)缺乏症、肉碱棕榈酰转移酶II(CPT IT)缺乏症、肉碱-酰基肉碱(CACT)缺乏症、常染色体显性/常染色体隐性进行性外眼肌麻痹(ad-/ar-PEO)、婴儿型脊髓性小脑萎缩(IOSCA)、线粒体肌病(MM)脊髓性肌萎缩症(SMA)、生长迟缓、氨基酸尿、胆汁阻塞、铁过载、早逝(GRACILE)或腓骨肌萎缩症2A型(CMT2A)。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.权利要求37至44中任一项所述的方法或细胞，其中所述细胞来自患有神经退行性疾病的受试者。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.权利要求45所述的方法或细胞，其中所述神经退行性疾病是肌萎缩性侧索硬化(ALS)、亨廷顿病、阿尔茨海默病、帕金森病、弗里德赖希共济失调、腓骨肌萎缩症或脑白质营养不良。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.权利要求37至46中任一项所述的方法或细胞，其中所述细胞来自患有视网膜疾病、糖尿病、听力障碍、遗传性疾病、心力衰竭、免疫缺陷、癌症或感染性疾病的受试者。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.权利要求37至47中任一项所述的方法或细胞，其中所述细胞是离体细胞。48. The method or cell of any one of claims 37 to 47, wherein the cell is an ex vivo cell.49.权利要求48所述的方法，其进一步包括向受试者施用所述细胞，任选地其中所述受试者是所述细胞所源自的同一受试者。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.治疗患有年龄相关疾病、线粒体疾病或病症、神经退行性疾病、视网膜疾病、糖尿病、听力障碍、遗传疾病、心力衰竭、免疫缺陷、癌症或感染性疾病的受试者的方法，所述方法包括向所述受试者施用治疗有效量的根据权利要求35至48中任一项的细胞。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.药物组合物，其包含权利要求1至27中任一项所述的多肽、权利要求28所述的核酸、权利要求29所述的颗粒或权利要求35至48中任一项所述的细胞，以及药学上可接受的赋形剂。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.试剂盒，其包含(a)权利要求1至27中任一项所述的多肽、权利要求28所述的核酸或权利要求29所述的颗粒和(b)稳定剂。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.