Method for improving mitochondrial function

Abstract

The present invention addresses the problem of elucidating the function of human Mhr1 and providing a method for improving mitochondrial function. Provided is a method for promoting mitochondrial DNA homoplasmy formation or for improving mitochondrial function, the method including introducing a protein having 80% or higher sequence identity with the amino acid sequence represented by SEQ ID NO: 1 or DNA or mRNA encoding a protein having 80% or higher sequence identity with the amino acid sequence represented by SEQ ID NO: 1 into a cell.

Description

Methods for improving mitochondrial function

[0001] The present invention relates to a method for promoting homoplasmy, a method for improving mitochondrial function, an agent for promoting homoplasmy, an agent for improving mitochondrial function, and a therapeutic agent for diseases associated with heteroplasmy of mitochondrial DNA.

[0002] Mitochondria in eukaryotes are organelles that produce over 90% of the energy (ATP) required for cellular activity through oxidative phosphorylation reactions via oxygen respiration, and are involved in a variety of cellular functions. Mitochondria contain hundreds to thousands of units of unique genomic DNA, known as mitochondrial DNA (mtDNA). Mitochondrial DNA (mtDNA) encodes factors essential for cellular respiration.

[0003] Healthy human mtDNA is born with a single genotype in each cell that makes up the entire body; in other words, all cells have the same base sequence (a state known as homoplasmy). However, because mtDNA is more prone to mutation than nuclear chromosomal DNA, in patients with mitochondrial disease and in adult human cells, mutant mtDNA coexists with normal mtDNA within the cell (a state known as heteroplasmy). Mutations and deletions in somatic mtDNA can expand clonally to reach harmful concentrations within the cell, and when the ratio of mutant mtDNA exceeds a certain threshold, symptoms such as carcinogenesis, diabetes, Parkinson's disease, Alzheimer's disease, and various mitochondrial diseases can develop. For example, large deletions have been reported at position 3243 of mtDNA in MELAS and cardiomyopathy patients, at position 8344 in MERRF patients, and in CPEO and Pearson's disease (Non-Patent Document 1). Furthermore, the A3243G substitution mutation in mtDNA is known to cause diabetes.

[0004] Furthermore, induced pluripotent stem cells (iPSCs) can proliferate indefinitely and differentiate into any type of cell in the body, but because the reprogramming process to establish iPSCs does not reset mtDNA, the mtDNA in iPSCs remains heteroplasmic (Non-Patent Document 2). For this reason, preventing mtDNA mutations and removing mutated mtDNA are also important in the field of regenerative medicine.

[0005] As a method for promoting mtDNA homogeneity, the present inventors have developed a method for changing the proportion of mutant mtDNA in eukaryotic cells by contacting them with reactive oxygen species or chemical species that generate reactive oxygen species within the cells (Patent Document 1).The present inventors have also proposed a method for promoting mitochondrial DNA homogeneity, which involves expressing the Mhr1 gene product of budding yeast in yeast cells (Patent Document 2).

[0006] Furthermore, Patent Document 3 discloses a method for treating mitochondrial disease, in which the endogenous mtDNA copy number is reduced, exogenous mitochondria from a healthy donor are transferred to produce mitochondrial replacement cells, and the cells are administered to a subject in need of mitochondrial replacement (e.g., a patient suffering from mitochondrial disease).

[0007] Japanese Patent Application Laid-Open No. 2011-125248 Japanese Patent Application Laid-Open No. 2005-87150 Special Publication No. 2021-533827

[0008] Wallace, Annu Rev Genet. , 39, 359-407, 2005Cherry, A. B. et al. , Stem Cells, 31, 1287-1297.

[0009] As mentioned above, the present inventors have demonstrated that Mhr1, a mitochondrial homologous DNA synthase, promotes homoplasmy in budding yeast. However, the role of human Mhr1 remains unclear, and elucidation of its function has been eagerly awaited.

[0010] An objective of the present invention is to elucidate the function of human Mhr1 and provide a method for improving mitochondrial function.A further objective of the present invention is to provide an agent for promoting homoplasmy, an agent for improving mitochondrial function, and an agent for treating diseases associated with heteroplasmy of mitochondrial DNA.

[0011] Examples of specific embodiments of the present invention are given below.

[0012] [1] A method for improving mitochondrial function, comprising introducing into a cell a protein having 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1. [2] The method for improving mitochondrial function according to [1], wherein the protein is human Mhr1 protein. [3] The method for improving mitochondrial function according to [1] or [2], wherein the cell is a human-derived cell. [4] The method for improving mitochondrial function according to any of [1] to [3], wherein the cell is an iPS cell. [5] A method for promoting mitochondrial DNA homoplasmy, comprising introducing into a cell a protein having 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1. [6] The method for promoting homoplasmy according to [5], wherein the protein is human Mhr1 protein. [7] The method for promoting homoplasmy according to [5] or [6], wherein the cell is a human-derived cell. [8] The method for promoting homoplasmy establishment according to any one of [5] to [7], wherein the cells are iPS cells. [9] An agent for improving mitochondrial function, comprising a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[10] The agent for improving mitochondrial function according to [9], wherein the protein is human Mhr1 protein.

[11] The agent for improving mitochondrial function according to [9] or

[10] , wherein the agent is a pharmaceutical product, a food or beverage, or a cosmetic product.

[12] An agent for promoting mitochondrial DNA homoplasmy establishment, comprising a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[13] The agent for promoting homoplasmy establishment according to

[12] , wherein the protein is human Mhr1 protein.

[14] The agent for promoting homoplasmy establishment according to

[12] or

[13] , wherein the protein is human Mhr1 protein.

[15] A therapeutic agent for a disease associated with mitochondrial DNA heteroplasmy, comprising a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[16] The therapeutic agent according to

[15] , wherein the protein is human Mhr1 protein.

[0013] [A] A protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, for use in treatment for promoting homoplasmy. [B] Use of a protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, for the manufacture of an agent for promoting homoplasmy. [C] A protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, for use in treatment for improving mitochondrial function. [D] Use of a protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, for the manufacture of an agent for improving mitochondrial function. [E] A protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, for use in treating a disease associated with heteroplasmy. [F] Use of a protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, for the manufacture of a therapeutic agent for a disease associated with heteroplasmy. [G] A method for treating a disease associated with heteroplasmy, comprising introducing into a cell DNA or mRNA encoding a protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1.[H] A method for treating a disease associated with heteroplasmy, comprising administering to an animal a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0014] The present invention provides a method for promoting homoplasmy using human Mhr1 and a method for improving mitochondrial function. The present invention also provides a homoplasmy promoter, a mitochondrial function improver, and a therapeutic agent for diseases associated with mitochondrial DNA heteroplasmy, each containing human Mhr1.

[0015] The results of the production and purification of His-tagged human Mhr1 in silkworms are shown below. The left panel shows the results of SDS-PAGE analysis and silver staining of partially purified human Mhr1. The right panel shows the results of Western blotting using an antibody against the His-tag. Human Mhr1 was confirmed to promote the formation of triplex DNA strand exchangers in vitro. When partially purified human Mhr1 was mixed with negatively supercoiled circular DNA and single-stranded DNA with a homologous base sequence, it was unable to form DNA strand exchangers resistant to restriction enzyme cleavage (Fig. 2A). On the other hand, when partially purified human Mhr1 was mixed with double-stranded DNA and single-stranded DNA with a homologous base sequence, it formed DNA strand exchangers resistant to restriction enzyme cleavage (Fig. 2B). The addition of an anti-His tag antibody that specifically binds to C-terminal His-tagged human Mhr1 reduced the amount of DNA strand exchangers formed and inhibited the homologous DNA pairing promotion reaction of human Mhr1 (Fig. 2B). Human Mhr1 was overexpressed in budding yeast, and the subcellular localization of the expressed human Mhr1 is shown. Western blotting using a known localization protein as a marker revealed that human Mhr1 was present in the mitochondrial fraction (Figure 3B). This figure illustrates that human Mhr1 partially complements the mhr1-1 mutation, which causes defects in mitochondrial DNA recombination (gene exchange type) at the Omega and Endo.SceI loci. This figure illustrates that human Mhr1 partially complements the mhr1-1 mutation, which causes defects in mitochondrial DNA recombination (gene exchange type) at the Omega and Endo.SceI loci. This figure illustrates that human Mhr1 partially complements the mhr1-1 mutation, which causes defects in mitochondrial DNA recombination (gene exchange type) at the Omega and Endo.SceI loci. This figure shows the distribution of the m.3243 A>G mutation upon knockdown of the human Mhr1 gene in MELAS cells. Knockdown of human Mhr1 in MELAS cells prevented the bimodal distribution observed with H2O2 treatment, maintaining heteroplasmy (Figure 6d). This shows the percentage of cells with mtDNA deletion mutations (mitochondrial common deletions) when the human Mhr1 gene was knocked down in Tig3 cells.It was shown that knockdown of human Mhr1 increased the proportion of cells with typical mtDNA deletion mutations (mitochondrial common deletions) and promoted heteroplasmy. This shows that expression of the human Mhr1 gene in Tig3 cells improves mitochondrial function. A higher OCR / ECAR value indicates improved mitochondrial function. This shows that expression of the human Mhr1 gene in BJ-P cells improves mitochondrial function.

[0016] The present invention will be described in detail below. The following description may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0017] In the present specification, the 20 types of amino acid residues in an amino acid sequence may be represented by single-letter abbreviations, such as G for glycine (Gly), A for alanine (Ala), V for valine (Val), L for leucine (Leu), I for isoleucine (Ile), F for phenylalanine (Phe), Y for tyrosine (Tyr), W for tryptophan (Trp), S for serine (Ser), T for threonine (Thr), C for cysteine ​​(Cys), M for methionine (Met), D for aspartic acid (Asp), E for glutamic acid (Glu), N for asparagine (Asn), Q for glutamine (Gln), K for lysine (Lys), R for arginine (Arg), H for histidine (His), and P for proline (Pro). In addition, in the amino acid sequences shown herein, the left end is the N-terminus and the right end is the C-terminus.

[0018] (Method for promoting homoplasmy / method for improving mitochondrial function) A first embodiment of the present invention relates to a method for promoting homoplasmy of mitochondrial DNA, comprising introducing into cells a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1. However, the method for promoting homoplasmy of the first embodiment may exclude methods for treating humans. In this specification, the "method for promoting homoplasmy" refers to a method for restoring heteroplasmic cells to normal homoplasmic cells.

[0019] The first embodiment may also relate to a method for promoting homoplasmy, comprising introducing into a cell in vitro a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0020] A second embodiment of the present invention relates to a method for improving mitochondrial function, which includes introducing into cells a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding the protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1. However, the method for improving mitochondrial function of the second embodiment may exclude methods for treating humans. In this specification, the "method for improving mitochondrial function" refers, for example, to a method for increasing the OCR / ECAR value of a cell into which a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 has been introduced.

[0021] Furthermore, the second embodiment may relate to a method for improving mitochondrial function, which includes introducing into cells in vitro a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0022] Homoplasmy refers to a state in which the mtDNA base sequence is uniform at the cellular or individual level. In contrast, heteroplasmy refers to a state in which the mtDNA base sequence is not uniform at the cellular or individual level. For example, if the mtDNA base sequence contained in a cell (or individual) is uniform in all mitochondrial genome copies, the cell (or individual) is a "homoplasmic cell (or individual)." If a cell (or individual) contains both mutated mtDNA and normal mtDNA, the cell (or individual) is a "heteroplasmic cell (or individual)." Note that even if a mutation occurs in mtDNA, if the base sequence at the mutated site is uniform at the cellular or individual level, the state is homoplasmic with respect to the specific mutation. In this specification, the term "homoplasmy" simply refers to "normal homoplasmy," and the term "mutation" is added when referring to "mutated homoplasmy."

[0023] As used herein, "mutated mtDNA" refers to mtDNA with a sequence that differs from the normal mtDNA sequence. The number of mutations present per mtDNA may be one or more, and may be one to three, or even only one. Furthermore, the "mutation" in the mutant mtDNA is not particularly limited and includes all mutations that differ from the normal mtDNA sequence, and may result in a mutation that causes some disease. Examples of such "mutations" include substitution mutations at position 3243 (the causative mutation of MELAS) and position 8344 (the causative mutation of MERRF) in the mtDNA (see Non-Patent Document 1, etc.).

[0024] Reducing the proportion of mutant mtDNA present in cells as much as possible enables the preparation of homoplasmic cells. In this specification, it is preferred that all mtDNA present in homoplasmic cells has the same base sequence and is normal mtDNA, but homoplasmic cells may also contain small amounts of mutant mtDNA. Specifically, the proportion of normal mtDNA present in homoplasmic cells (intracellular proportion) is preferably 80% or more (mutant mtDNA is 20% or less), more preferably 90% or more (mutant mtDNA is 10% or less), even more preferably 95% or more (mutant mtDNA is 5% or less), and particularly preferably 99% or more (mutant mtDNA is 1% or less). The proportion of mutant mtDNA present in cells can be easily determined, for example, using restriction enzymes whose cleavage sensitivity differs depending on the presence or absence of a mutation (see, for example, Goto et al., Nature, 348, 651-653, 1990). Other methods that can be used include allele-specific PCR, quantitative PCR, the Invader method, and analytical methods using next-generation sequencers. As used herein, homoplasmy refers to increasing the proportion of normal mtDNA in cells, and in individuals, it means increasing the proportion of homoplasmic cells.

[0025] Restoring heteroplasmic cells to normal homoplasmic cells is expected to protect mitochondria, improve mitochondrial damage, and improve mitochondrial function in humans and other organisms, including invertebrates, birds, fish, amphibians, reptiles, and vertebrates such as mammals.

[0026] A method for promoting homoplasmy in a first embodiment includes introducing into a cell a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1. A method for improving mitochondrial function in a second embodiment includes introducing into a cell a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1. The sequence identity between the amino acid sequence of the protein and the amino acid sequence shown in SEQ ID NO: 1 may be 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more. As used herein, "sequence identity" refers to the percentage (%) of identical bases or amino acids relative to the total overlapping base sequence or total amino acid sequence in optimal alignment when two base sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm can take into account the introduction of gaps into one or both of the sequences for optimal alignment). The "sequence identity" of a base sequence or amino acid sequence can be easily confirmed by those skilled in the art. For example, NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) can be used.

[0027] The protein having the amino acid sequence shown in SEQ ID NO: 1 is preferably human Mhr1 protein. Proteins having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 are proteins that exhibit functions equivalent to human Mhr1 protein. The amino acid sequence of such a protein may differ partially from the reference amino acid sequence (the amino acid sequence shown in SEQ ID NO: 1). Here, "partially different from the reference amino acid sequence" refers to, for example, deletion or substitution of one or more amino acids among the amino acids constituting the amino acid sequence, addition or insertion of one or more amino acids to the amino acid sequence, or any combination thereof. Partial differences in the amino acid sequence are permissible as long as the function as human Mhr1 protein is maintained. Furthermore, the position of the difference in the amino acid sequence is not particularly limited as long as the function as human Mhr1 protein is maintained. Furthermore, differences in the amino acid sequence may occur at multiple locations (positions).

[0028] Proteins having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 may be part of a larger protein (eg, a fusion protein).

[0029] DNA encoding the amino acid sequence shown in SEQ ID NO: 1 has the nucleotide sequence shown in SEQ ID NO: 2. That is, a first embodiment may relate to a method for promoting homoplasmy, comprising introducing DNA having the nucleotide sequence shown in SEQ ID NO: 2 or mRNA transcribed from the DNA into a cell. Furthermore, a second embodiment may relate to a method for improving mitochondrial function, comprising introducing DNA having the nucleotide sequence shown in SEQ ID NO: 2 or mRNA transcribed from the DNA into a cell. The sequence identity of the DNA introduced into a cell with the nucleotide sequence shown in SEQ ID NO: 2 is not particularly limited as long as it can encode a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1. However, the sequence identity of the DNA with the nucleotide sequence shown in SEQ ID NO: 2 is, for example, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more.

[0030] DNA encoding a protein having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 may be inserted into an expression vector. That is, the first embodiment may relate to a method for promoting homoplasmy, comprising introducing into a cell an expression vector containing DNA encoding a protein having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. Any expression vector can be used as long as it is capable of expressing human Mhr1 protein in a host cell. Specifically, to express in a host cell DNA encoding a protein having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, the gene is inserted downstream of a promoter in the expression vector, and then this recombinant expression vector is introduced into a host cell (preferably a human-derived cell) compatible with the expression vector.

[0031] The expression vector may include, for example, an expression vector comprising a yeast promoter such as the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock protein promoter, MFα1 promoter, or CUP1 promoter. Methods for introducing a recombinant expression vector into a host include, for example, the calcium phosphate method, protoplast method, electroporation method, spheroblast method, lithium acetate method, and lipofection method, and can be appropriately selected depending on the type of host cell.

[0032] A protein having 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, may be supported on a carrier. For example, the protein, DNA, or mRNA may be encapsulated or supported in nanoparticles having a lipid membrane. Known methods can be appropriately used to introduce such nanoparticles into cells.

[0033] A cell into which a protein having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, is introduced is a eukaryotic cell. The eukaryotic cells used in this embodiment are not particularly limited, but include, for example, animal cells, particularly mammalian cells, and especially cells derived from humans and non-human organisms. Examples of non-human cells include cells derived from mice, rats, cows, horses, pigs, sheep, monkeys, dogs, cats, birds, etc., but the eukaryotic cells are preferably human-derived cells. Furthermore, the eukaryotic cells used in the method for promoting homoplasmy are preferably human-derived cells that have become heteroplasmic.

[0034] Preferably, the cell into which a protein having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or DNA or mRNA encoding a protein having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 is introduced is an iPS cell (iPSC). iPS cells are cells that have acquired pluripotency equivalent to that of ES cells by introducing several types of transcription factor genes that confer pluripotency, transcription factor proteins, and compounds that induce pluripotency (collectively referred to as pluripotency factors) into somatic cells. Many factors have already been reported as pluripotency factors, and examples include, but are not limited to, the Oct family (e.g., Oct3 / 4), the SOX family (e.g., SOX2, SOX1, SOX3, SOX15, and SOX17), the Klf family (e.g., Klf4 and Klf2), the MYC family (e.g., c-MYC, N-MYC, L-MYC), NANOG, and LIN28. Although iPS cells can proliferate indefinitely and differentiate into any type of cell in the body, the mtDNA remains heteroplasmic in iPS cells because the reprogramming used to establish iPS cells does not reset the mtDNA. Therefore, introducing a protein having 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, into iPS cells can make them homoplasmic, which is also useful in the field of regenerative medicine.

[0035] A protein having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, is introduced into cells, and the concentration of the protein increases within the cells, thereby promoting a shift from glycolysis to mitochondrial respiration. The shift from glycolysis to mitochondrial respiration indicates improved mitochondrial function. More specifically, the OCR / ECAR value is measured, and an increase in this value indicates improved mitochondrial function. Here, OCR is the oxygen consumption rate in the process of ATP production by mitochondrial oxidative phosphorylation, and ECAR is the extracellular acidification rate, which is an indicator of glycolysis.

[0036] The present inventors have found that knockdown of the human Mhr1 gene in human-derived cells results in the H gene, which promotes homoplasmy. ２ O ２ It was confirmed that homoplasmy did not occur even after treatment, suggesting that mitochondrial function was not improved. Based on these findings, it is believed that the protein having the amino acid sequence shown in SEQ ID NO: 1 (human Mhr1 protein) improves mitochondrial function by suppressing mtDNA deletion mutations and promoting mtDNA homoplasmy in cells.

[0037] The Mhr1 protein of budding yeast is a homologous DNA recombinase that is known to pair double-stranded DNA and single-stranded DNA having the same base sequence to form an intermolecular double-stranded (heteroduplex) under ATP-free conditions. Here, the sequence identity between the human Mhr1 protein having the amino acid sequence set forth in SEQ ID NO: 1 and the Mhr1 protein in budding yeast is less than 40%, so the sequence identity is not very high. However, the present invention has revealed that the human Mhr1 protein, like the Mhr1 protein in budding yeast, is a homologous DNA recombinase and exhibits similar functions. Thus, the present invention is useful in that it has identified and isolated the human Mhr1 protein and further elucidated its function. Furthermore, the human Mhr1 protein is expected to be used for the treatment and prevention of diseases caused by mtDNA heterogeneity.

[0038] (Homoplasmy promoter / mitochondrial function improver) A third embodiment of the present invention relates to an agent for promoting homoplasmy of mitochondrial DNA, comprising a protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1.

[0039] Furthermore, a fourth embodiment of the present invention relates to an agent for improving mitochondrial function, comprising a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0040] The protein, DNA, and mRNA used in the third and fourth embodiments are the same as those used in the first embodiment. Of these, the protein having the amino acid sequence shown in SEQ ID NO: 1 is preferably human Mhr1 protein.

[0041] In the third and fourth embodiments, the homoplasmy establishment promoter and mitochondrial function improver comprise a protein having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. The homoplasmy establishment promoter and mitochondrial function improver may contain other components in addition to the above protein, DNA, or mRNA, or may consist solely of the above protein, DNA, or mRNA. Furthermore, the protein, DNA, and mRNA used in the third and fourth embodiments may be supported on a carrier. For example, the protein, DNA, or mRNA may be encapsulated in or supported by nanoparticles having a lipid membrane.

[0042] The form of the homoplasmy promoter and the mitochondrial function improver is not particularly limited, and may be granules, powder, solid, liquid, or the like.

[0043] The homoplasmy promoter and the mitochondrial function improver may be a pharmaceutical product (including quasi-drugs), a food or beverage product, a cosmetic product, or the like. Pharmaceutical products are not particularly limited, but specific examples include oral pharmaceutical products such as tablets, pills, powders, fine granules, granules, capsules (including hard capsules and soft capsules), troches, chewable tablets, extracts (including soft extracts and dry extracts), jellies, syrups, spirits, elixirs, and liposome preparations; topical pharmaceutical products such as ointments, creams, lotions, gels, sprays, patches, emulsions, suspensions, poultices, liniments, aerosols, ointments, packs, inhalants, and suppositories; and injections. Examples of foods and beverages include general foods and beverages, as well as foods with health claims (including foods for specified health uses, foods with nutrient functions, and foods with functional claims), and foods for patients. The form of these foods and drinks is not particularly limited, but specific examples include beverages such as tea drinks, energy drinks, fruit juice drinks, carbonated drinks, and lactic acid drinks; supplements such as capsules (soft capsules and hard capsules), tablets, granules, powders, jellies, and liposome preparations; and luxury goods such as gummies, candies, and jellies. Specific examples of cosmetics include creams, lotions, gels, emulsions, liquids, ointments, and packs.

[0044] Among these, the homoplasmy promoter and mitochondrial function improver are preferably pharmaceuticals, and are particularly preferably therapeutic agents for diseases associated with mitochondrial DNA heteroplasmy. This embodiment may relate to a therapeutic agent for diseases associated with mitochondrial DNA heteroplasmy, comprising a protein having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. Diseases associated with mitochondrial DNA heteroplasmy include mitochondrial diseases. Mitochondrial diseases are a general term for pathological conditions in which symptoms appear due to impaired mitochondrial function. The homoplasmy promoter and mitochondrial function improver of the present invention can improve various symptoms associated with mitochondrial diseases. For example, various symptoms associated with mitochondrial diseases can be improved by administering the homoplasmy promoter and mitochondrial function improver of the present invention to animals (e.g., mammals).

[0045] The homoplasmy promoter, mitochondrial function improver, and therapeutic agent for diseases associated with heteroplasmy of mitochondrial DNA may be further combined with other components or pharmaceuticals to the extent that the effects of the present invention are not impaired. For example, other components include reactive oxygen species or chemical species that generate reactive oxygen species within cells. Examples of reactive oxygen species or chemical species that generate reactive oxygen species within cells include superoxide anion radical, hydroxyl radical, hydrogen peroxide, singlet oxygen, nitric oxide, nitrogen dioxide, ozone, lipid peroxide, hypoxanthine, hydrogen peroxide, and iron(II) chloride (FeCl). ２ ), alloxan, antimycin A, oligomycin, etc. Furthermore, the homoplasmy promoter, mitochondrial function improver, and therapeutic agent for diseases involving heteroplasmy of mitochondrial DNA may contain a nucleic acid derivative or an organic compound having a nucleoside structure. Examples of nucleic acid derivatives include compounds that generate hydrogen peroxide intracellularly (e.g., hypoxanthine, etc.). Examples of organic compounds having a nucleoside structure include inosine, etc.

[0046] (Homoplasmic Cells) This embodiment may relate to homoplasmic cells prepared using the above-described method for promoting homoplasmy or method for improving mitochondrial function. The prepared homoplasmic cells are preferably cells having normal mitochondrial DNA. This embodiment may also relate to homoplasmic iPS cells (iPSCs) prepared using the above-described method for promoting homoplasmy or method for improving mitochondrial function. Many publications have been published regarding methods for establishing iPS cells, and these can be used as reference (e.g., Takahashi et al., Cell 2006, 126:663-676; Okita et al., Nature 2007, 448:313-317; Wernig et al., Nature 2007, 448:318-324; Maherali et al., Cell Stem Cell 2007, 1:55-70; Park et al., Nature 2007, 451:141-146; Nakagawa et al., Nat Biotechnol 2008, 26:101-106; Wernig et al., Cell Stem Cell 2008, 10:10-12; Yu et al., Science 2007, 318:1917-1920; Takahashi et al., Cell 2007, 131:861-872; Stadtfeld et al., Science 2008 322:945-949.) For example, mitochondrial diseases and the like can be effectively treated by transplanting homoplasmic cells or homoplasmic iPS cells into individuals.

[0047] (Treatment of Diseases Associated with Heteroplasmy) This embodiment may relate to a protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, for use in treating diseases associated with heteroplasmy. This embodiment may also relate to the use of a protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, for the manufacture of a therapeutic agent for diseases associated with heteroplasmy.

[0048] Furthermore, this embodiment may relate to a method for treating a disease associated with heteroplasmy, comprising introducing into a cell a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or a method for treating a disease associated with heteroplasmy, comprising administering to an animal (e.g., a mammal) a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0049] Diseases involving heteroplasmy of mitochondrial DNA include mitochondrial diseases. Mitochondrial diseases are a general term for pathological conditions in which symptoms appear due to a decrease in mitochondrial function. By using the therapeutic agent and treatment method of the present invention, the homoplasmy promoter and mitochondrial function improver are expected to improve various symptoms caused by mitochondrial diseases.

[0050] The features of the present invention will be explained in more detail below with reference to examples and comparative examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

[0051] <Cloning of the human Mhr1 gene> The human Mhr1 gene was amplified by polymerase chain reaction (PCR) using KOD DNA polymerase (Toyobo) and total DNA prepared from HeLa cells as the template DNA. Based on the nucleotide sequence of the human Mhr1 open reading frame (ORF), the following primers containing the restriction enzymes HindIII and XhoI were designed and used in the PCR reaction: 5'-HindIII-FLJ: 5'-caagcttatgtcctatttgagtggcag ca-3' (SEQ ID NO: 3) 3'-XhoI-FLJ: 5'-cctcgagttaaattctgaagacagggaggtgtg-3' (SEQ ID NO: 4)

[0052] The PCR-amplified human Mhr1 DNA fragment was digested with the restriction enzymes HindIII and XhoI and then inserted into the HindIII / XhoI site of the budding yeast expression vector pYES2 and the HindIII / XhoI site of the mammalian cell expression vector pCDNA3, respectively, to construct pYES-humanMhr1 and pCDNA3-humanMhr1, which are vectors used for overexpression of human Mhr1 in budding yeast and human cells. A standard yeast transformation method was used to introduce pYES-humanMhr1 into yeast, and Lipofectamine P3000 (Thermo Fisher Scientific) was used as the transfection reagent to transfect human cells with pCDNA3-humanMhr1.

[0053] Human Mhr1 was expressed in insects and partially purified (Figure 1). Specifically, His-tagged human Mhr1 was expressed in silkworms, and then 30 mL of the supernatant of the pupal homogenate was diluted with a solution containing 50 mM Tri-HCl, 300 mM NaCL, 20 mM imidazole, and 10% glycerol (pH 8.0) to prepare a purification sample of approximately 150 mL. This sample was then purified using a nickel column. The wash buffer (40 mL, pH 8.0) consisted of 50 mL Tri-HCl, 300 mM NaCL, 20 mM imidazole, and 0% glycerol. Elution was then performed in three steps. Elu1 (lanes 7 and 8 in Figure 1) was eluted with 19 mL of an elution solution containing Tri-HCl, 300 mM NaCL, 50 mM imidazole, and 10% glycerol (pH 8.0). Elu2 (lanes 9 and 10) was eluted with 15 mL of elution buffer containing Tri-HCl, 300 mM NaCL, 80 mM imidazole, and 10% glycerol (pH 8.0). Elu3 was eluted with 16.5 mL of elution buffer containing Tri-HCl, 300 mM NaCL, 250 mM imidazole, and 10% glycerol (pH 8.0). Lanes 2 and 4 represent the fractions that flowed freely through the nickel column after loading the crude protein homogenate onto the column. Lanes 5 and 6 represent the fractions washed with a wash buffer containing 20 mM imidazole. Western blotting and silver staining combined revealed partially purified human Mhr1 in fraction 5 (lane 5).

[0054] <Molecular Function of Human Mhr1 in Vitro> In vitro, homologous pairing of budding yeast Mhr1 with RecA-type proteins (RecA, Rad51, and Dmc1) essential for DNA recombination in the cell nucleus has been shown to produce not only D-loops but also triplex DNA strand exchangers. The partially purified human Mhr1 described above was also confirmed to promote the formation of triplex DNA strand exchangers in vitro (Figure 2). In Figure 2A, when partially purified human Mhr1 was mixed with negatively supercoiled circular DNA and single-stranded DNA with a homologous base sequence, no DNA strand exchangers resistant to restriction enzyme cleavage were detected. However, in Figure 2B, when partially purified human Mhr1 was mixed with double-stranded DNA and single-stranded DNA with a homologous base sequence, DNA strand exchangers resistant to restriction enzyme cleavage were detected. The addition of an anti-His tag antibody that specifically binds to human Mhr1 with a His tag at the C-terminus reduced the amount of DNA strand exchange complexes formed and inhibited the homologous DNA pairing promotion reaction mediated by human Mhr1. These results suggest that human Mhr1 has a molecular function in vitro similar to that of budding yeast Mhr1.

[0055] <Localization of human Mhr1 in budding yeast> Human Mhr1 was overexpressed in budding yeast, and the intracellular localization of the expressed human Mhr1 was investigated. Budding yeast overexpressing human Mhr1 was cultured in a medium containing raffinose and glucose as carbon sources, and the budding yeast in which human Mhr1 expression was induced was fractionated. Using a protein known to be localized as a marker, the localization of human Mhr1 was detected by Western blotting. The results showed that it was present in the mitochondrial fraction (Figure 3B). In Figures 3 to 9, "hsMhr1," "HsMHR1," and "hsMHR1" refer to "human Mhr1."

[0056] As shown in Figures 4 and 5, human Mhr1 partially complemented the mhr1-1 mutation, which causes defects in mitochondrial DNA recombination (gene exchange type) at the Omega and Endo.SceI loci. These results suggest that human Mhr1 prevents the formation of mtDNA deletion mutations and possesses the molecular function of budding yeast Mhr1. As shown in Figure 4, omega endonuclease introduces a double-strand break at the omega locus, inducing site-specific gene conversion homologous recombination. Here, omega plus inserts into omega minus sites almost 100% of the time. This type of homologous recombination is also known as omega homing. The mhr1-1 mutation reduces 100% omega homing to approximately 37%, but overexpression of human Mhr1 restores it to approximately 60%. As shown in Figure 5, End.SceI endonuclease introduces a double-strand break at the End.SceI locus, inducing site-specific gene conversion homologous recombination at approximately 52.6% of sites. Overexpression of Mhr1 increased the rate of gene conversion recombination to 83.2%. Overexpression of mutant Mhr1 (mhr1-1) increased the rate of gene conversion recombination at the End.SceI locus to 82.5%, even with the mutation. Furthermore, overexpression of human Mhr1 increased the rate of gene conversion recombination at the End.SceI locus to 87.2%. Overexpression of mutant human Mhr1 (four-base deletion) increased the rate of gene conversion recombination at the End.SceI locus to 76.9%.

[0057] <Suppression of human Mhr1 gene expression in MELAS cells> In MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes) cells derived from a patient with a mitochondrial DNA point mutation (m.3243 A>G (A at position 3243 in mitochondrial DNA is changed to G)), wild-type and m.3243 A>G mutant mtDNA are known to mix and not separate (Fig. 6a). However, treatment of MELAS cells with hydrogen peroxide (H2O2) activated rolling-circle replication in human cells, resulting in a bimodal distribution of cells with different proportions of m.3243 A>G mutation, and the wild-type and m.3243 A>G mutant mtDNA segregated, leading to homoplasmy (Fig. 6b). However, knockdown of human Mhr1 in MELAS cells prevented the bimodal distribution observed with H2O2 treatment (Fig. 6d). These results demonstrate that knockdown of human Mhr1 in MELAS cells maintains heteroplasmy and prevents the formation of homoplasmy. Therefore, human Mhr1 is required for mitochondrial allele segregation. siRNA duplexes targeting human Mhr1 were designed using existing methods and synthesized by Dharmacon RNAi Technologies (USA). The sense strand of the siRNA duplex was 5'-auucaaggcacuaaaggaguggaug-3 (SEQ ID NO: 5), and the antisense strand was 5'-cauccacuccuuuagugccuugaa-3' (SEQ ID NO: 6). Lipofectamine™ RNAiMAX (Thermo Fisher Scientific) was used as the transfection reagent for transfection of MELAS cells with siRNA targeting human Mhr1. The efficiency of human Mhr1 knockdown was confirmed by measuring mRNA levels by real-time RT-PCR.Figure 6c shows the results of transfection of non-targeting control siRNA (siCtrl) followed by H2O2 treatment. In this case, wild-type and m.3243 A>G mutant mtDNA separated, promoting homoplasmy.

[0058] <Suppression of human Mhr1 gene expression in Tig3 cells> Tig3 cells were transfected with siRNA targeting human Mhr1 or a non-targeting control siRNA (siCtrl) using the same method as described above. As a result, knockdown of human Mhr1 increased the proportion of cells with typical mtDNA deletion mutations (mitochondrial common deletions) (Figure 7). This indicates that mtDNA deletion mutations promote heteroplasmy.

[0059] Overexpression of human Mhr1 in Tig3 cells. Primary Tig3 cells (Tig3-P) cultured in Dulbecco's Modified Eagle Medium (DMEM) were transfected with pCDNA3 or pCDNA3-human Mhr1. Lipofectamine P3000 (Thermo Fisher Scientific) was used as the transfection reagent. Because pCDNA3 contains a neomycin resistance gene, after 4 days, the cells were subcultured in DMEM containing 250 μg / mL neomycin. The medium was changed every 2 days. After 1 week, the oxygen consumption rate (OCR) during mitochondrial oxidative phosphorylation (ATP production), the extracellular acidification rate (ECAR) as an indicator of glycolysis, and the OCR / ECAR ratio, which indicates a shift from glycolysis to mitochondrial respiration, were calculated using an extracellular flux analyzer (Agilent Technologies). A higher OCR / ECAR value indicates improved mitochondrial function. As a result, high expression of human Mhr1 in human cells (Tig3 cells) improved mitochondrial function in human cells (Figure 8).

[0060] Overexpression of human Mhr1 in BJ-P cells. BJ-P cells were transfected with pCDNA3 or pCDNA3-human Mhr1 using the same method as described above. After 4 days, the oxygen consumption rate (OCR) during mitochondrial oxidative phosphorylation (ATP production), the extracellular acidification rate (ECAR) as an indicator of glycolysis, and the OCR / ECAR ratio, which indicates a shift from glycolysis to mitochondrial respiration, were calculated using an extracellular flux analyzer (Agilent Technologies). Overexpression of human Mhr1 in BJ-P cells improved mitochondrial function (Figure 9).

[0061] These results suggest that human Mhr1 plays an important role in preventing heteroplasmy caused by mtDNA deletion mutations.

[0062] <SEQ ID NO: 1> Homo sapiens (human) Mhr1 MSYLSGSSASPARRLGVVKVVPRPCLQWLENPGGCLGPSGQREAGSSSPGDCGHIGACLGLEGQVTSPATLPSLLWGPHFRATLPEAHASIHSFSALNLIHKQPPPFPSPSHSVDVILPPPVSILRQASKEALTLLPKWCFLKNTITTLGAIFSHLPVFRI

[0063] <SEQ ID NO: 2> Homo sapiens (human) human Mhr1 atgtcctatt tgagtggcag cagcgccagc ccagcaagaa ggctgggggt tgtcaaggtt gtccccagac cttgcttgca gtggttggag aacccagggg gctgccttgg gccctctggc cagagggaag cgggcagctc tagccctgga gattgtggtc acattggggc ttgtttagga ttggagggcc aggtcacctc cccagccacc ctcccttctc tcctctgggg tccccacttt agggcgactt tgccccgaggc ccacgcatcc atccactcct ttagtgcctt gaatcttatt cacaagcagc cccctccctt cccctcccct tctcactctg ttgatgtaat cctcccacc ccagtgtcca tcctaagaca ggcatcaaaa gaggccctaa ctttacttcc caaatggtgc tttttaaaaa acaccatcac tacattaggg gcaatttttt cacacctccc tgtcttcaga atttaa

[0064] <SEQ ID NO: 3> Primer (5'-HindIII-FLJ) caagcttatgtcctatttgagtggcag ca

[0065] <SEQ ID NO: 4> Primer (3'-XhoI-FLJ) cctcgagttaaattctgaagacagggaggtgtg

[0066] <SEQ ID NO: 5> Sense strand of siRNA duplex auucaaggcacuaaaggaguggaug

[0067] <SEQ ID NO: 6> Antisense strand of siRNA duplex cauccacuccuuuagugccuugaa

Claims

1. A method for improving mitochondrial function, comprising introducing into a cell a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

2. The method for improving mitochondrial function according to claim 1, wherein the protein is human Mhr1 protein.

3. A method for improving mitochondrial function according to claim 1 or 2, wherein the cells are human-derived cells.

4. A method for improving mitochondrial function as described in claim 1 or 2, wherein the cells are iPS cells.

5. A method for promoting homoplasmy of mitochondrial DNA, comprising introducing into a cell a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

6. The method for promoting homoplasmy according to claim 5, wherein the protein is human Mhr1 protein.

7. The method for promoting homoplasmy described in claim 5 or 6, wherein the cells are human-derived cells.

8. A method for promoting homoplasmy as described in claim 5 or 6, wherein the cells are iPS cells.

9. A mitochondrial function improving agent comprising a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

10. The mitochondrial function improving agent according to claim 9, wherein the protein is human Mhr1 protein.

11. The mitochondrial function improving agent according to claim 9 or 10, which is a pharmaceutical product, a food or beverage product, or a cosmetic product.

12. A mitochondrial DNA homoplasmy promoter comprising a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

13. The homoplasmy promoter of claim 12, wherein the protein is human Mhr1 protein.

14. The homoplasmy promoter according to claim 12 or 13, which is a pharmaceutical, food or beverage, or cosmetic.

15. A therapeutic agent for a disease associated with heteroplasmy of mitochondrial DNA, comprising a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1, or DNA or mRNA encoding a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

16. The therapeutic agent according to claim 15, wherein the protein is human Mhr1 protein.

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