Methods for detection of macro-heteroplasmy and micro-heteroplasmy in mitochondrial DNA

Abstract

To provide methods for detecting macro-heteroplasmy and / or micro-heteroplasmy in mitochondrial DNA.SOLUTION: The methods can include detecting or monitoring the presence of heteroplasmy, and / or identifying a threshold level of heteroplasmy. In addition, the methods can be used for diagnosing a mitochondrial related disease or disorder, as well as for monitoring the efficacy of a therapy affecting mitochondrial DNA (mtDNA) in a subject having or suspected of having heteroplasmy.SELECTED DRAWING: Figure 1-1

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 925,677, filed October 24, 2019. No. 6,239,999, the entire contents of which are incorporated herein by reference.

[0002] (2. Sequence Listing) This application contains a Sequence Listing which was submitted in ASCII format via EFS-Web and is incorporated by reference in its entirety. 145 It is called 95-002-228_Sequence_Listing.txt and is 6,264 bytes in size.

[0003] 3. FIELD OF THE INVENTION The present invention relates to the treatment of macro-heteroplasmy and / or macro-heteroplasmy in mitochondrial DNA. Also provided is a method for detecting microheteroplasmy. [Background technology]

[0004] 4. BACKGROUND OF THE INVENTION Mitochondrial diseases are genetically heterogeneous disorders characterized by mitochondrial dysfunction. Currently, there are no treatment options for patients with mitochondrial diseases, Therefore, patients are only given temporary treatments to alleviate their symptoms (Gorman, GS et al. "Mitochondrial diseases." Nat Rev Dis Primers 2, 160 80, doi:10.1038 / nrdp.2016.80 (2016)).

[0005] Mitochondrial precursors and their interactions with eukaryotes, α-proteobacteria, and archaea For symbiosis, many mitochondrial genes were transferred to the nuclear genome and evolved at the same time. The number of genes has expanded by 200,000 fold (Lane, N. and Martin, W., "Genome The energetics of genome complexity. Nature 467, 929-9 34, doi:10.1038 / nature09486 (2010)).

[0006] Mitochondrial diseases involve mitochondrial structural proteins or mitochondrial function. Genes in nuclear DNA (nDNA) and / or mitochondrial DNA (mtDNA) that encode proteins It can manifest from mutations in any organ, can occur at any age, and can be transmitted through any may indicate severity (Lightowlers, RN, Taylor, RW and Turnbull, DM, " Mutations causing mitochondrial diseases: what is new and what remains to be explored? tations causing mitochondrial disease: What is new and what challenges remain?) " Science 349, 1494-1499 (2015)). Phenotypic variation resulting from pathogenic mutations in mtDNA This can result in multiple copies of mtDNA. Each cell contains multiple copies of the mitochondrial genome. range from 100,000 in unfertilized oocytes to approximately 100 in sperm (Stew Art, JB and Chinnery, PF, "Drivens of Mitochondrial DNA Heteroplasmy" The dynamics of mitochondrial DNA heteroplasmy: implications for human health and disease applications for human health and disease.) Nat Rev Genet 16, 530-542, doi:10.1038 / nrg3966 (2015)). Women with nuclear mutations that cause mitochondrial disease should be encouraged to have children. There are several options for obtaining such information, such as prenatal and preimplantation genetic testing. These options are not a solution for all women. Patients with causative mutations inherit the disease due to genetic bottlenecks and relaxed mtDNA replication. The transmission patterns are extremely complex, and many different challenges are encountered (Wai, T., Teoli, D and Shoubridge, EA, "Mitochondrial DNA genetic bottlenecks in the genome" The mitochondrial DNA genetic bottleneck results from eplication of a subpopulation of genomes.)” Nature Genetics 40, 1484-1488, doi: 10.1038 / ng.258 (2008); Chinnery, PF and Samuels, DC, "Relaxed mtDNA Relaxed replication of mtDNA: a model with implicated disease lications for the expression of disease.)” The American Journal of Human Genetic s 64, 1158-1165 (1999)).

[0007] The mixture of mutant and wild-type genomes is termed heteroplasmy (Holt, I. J., Harding, AE, and Morgan-Hughes, JA, "Mitochondrial Myopathy" Deletions of muscle mitochondrial DNA in patients with A in patients with mitochondrial myopathies.) Nature 331, 717-719, doi:10.1038 / 3 31717a0 (1988)). The severity of mitochondrial disease depends on the mutation of the protein-coding gene. It has been observed that the presence of genomic DNA often correlates with the level of heteroplasmy. Although there are widespread examples of a small proportion of mitochondrial genomes with genetic mutations, There appears to be a typical 60-80% threshold for the manifestation of psychological dysfunction (Stewart, J.B. and and Chinnery, P.F., "Dynamics of mitochondrial DNA heteroplasmy: implications for human health." The dynamics of mitochondrial DNA heteroplasmy: implications for health and disease for human health and disease.)” Nat Rev Genet 16, 530-542, doi:10.1038 / nrg3966 ( For example, heteroplasmy of less than 70% is commonly caused by mutations in MT-ATP6. Clinical presentation of NARP (neurogenic muscular weakness, ataxia, and retinitis pigmentosa), a mitochondrial disease caused by In contrast, when heteroplasmy ranges from 70% to 90%, NARP does not exhibit a floor phenotype. Symptoms may appear and remain stable into adulthood. Extreme heteroplasmy (e.g., >90% heteroplasmy) aberrant mtDNA) leads to Leigh syndrome (Tatuch, Y. et al., "Percentage of abnormal mtDNA When the level is high, a heteroplasmic mtDNA mutation (T----G) at 8993 causes Leigh disease. Heteroplasmic mtDNA mutation (T----G) at 8993 can cause Leigh disease when the percentage of abnormal mtDNA is high.)”American journal of human gene tics 50, 852-858 (1992)).

[0008] However, heteroplasmy does not explain all variability in mitochondrial diseases. For example, mtDNA mutations in tRNA genes show high clinical variability. , which cannot be explained by heteroplasmy (Nunnari, J. and Suomalaine n, A., "Mitochondria: in sickness and health" in health,” Cell 148, 1145-1159, doi:10.1016 / j.cell.2012.02.035 (2012). The ordinal method for mitochondrial heteroplasmy involves the identification of mtDNA. A cell-to-cell isogenic population consisting of cells with similar intracellular heterogeneity and a cell-to-cell heterogeneity population consisting of cells with different intracellular heterogeneity. To identify cell-to-cell heterogeneous populations consisting of cells with a ratio of mutant mtDNA I can't do that.

[0009] Therefore, we aimed to develop an improved method for determining heteroplasmy in single cells. There is a huge unmet need. Summary of the Invention

[0010] (5. SUMMARY OF THE INVENTION) In one aspect, the presence of mitochondrial DNA (mtDNA) heteroplasmy is used herein. 1. A method for detecting or monitoring a disorder comprising: (a) obtaining or having obtained a biological sample containing one or more single cells; (b) determining the sequence of intracellular mtDNA in the one or more single cells; (c) wild-type and mutant forms of the intracellular mtDNA sequence in the one or more single cells. determining the ratio of; and (d) Cells in the arrangement of intracellular mtDNA between the one or more single cells and within the one or more single cells The inter- and / or intracellular variation is calculated, thereby determining the heteroplasmy of mtDNA in the sample. determining the presence or absence of the marker. In another aspect, provided herein is a method for determining mitochondrial-associated 1. A method for use in diagnosing a disease or disorder, comprising: (a) obtaining or having obtained from the subject a biological sample comprising one or more single cells; ; (b) determining the sequence of intracellular mitochondrial DNA (mtDNA) in the one or more single cells. ; (c) wild-type and mutant forms of the intracellular mtDNA sequence in the one or more single cells. determining the ratio of; (d) Cells in the arrangement of intracellular mtDNA between the one or more single cells and within the one or more single cells The inter- and / or intracellular variation is calculated, thereby determining the heteroplasmy of mtDNA in the sample. To decide; and (e) if mtDNA heteroplasmy is present in the sample, a mitochondrial-related disease or diagnosing the subject as having or suspected of having the disorder. Provide the law.

[0011] In yet another aspect, provided herein is a method for treating a patient with a mitochondrial-related disease or disorder, or Efficacy of treatments affecting mitochondrial DNA (mtDNA) in subjects suspected of having HIV 1. A method for monitoring: (a) administering to the subject a treatment that affects mtDNA; (b) obtaining a biological sample from the subject comprising one or more single cells; (c) determining the sequence of intracellular mtDNA in the one or more single cells; (d) wild-type and mutant forms of the intracellular mtDNA sequence in the one or more single cells. determining the ratio of; (e) determining whether the sequence of intracellular mtDNA between the one or more single cells and within the one or more single cells is identical to that of the sequence of intracellular mtDNA between the one or more single cells and within the one or more single cells; The amount of intercellular and / or intracellular variation is calculated, thereby determining the heteroplasmy of mtDNA in the sample. determining the level of; and (f) determining the level of mtDNA heteroplasmy in the sample by comparing the level of mtDNA heteroplasmy obtained from a reference sample; and comparing the level of heteroplasmy of the mtDNA to the level of heteroplasmy of the mtDNA. wherein the comparison indicates the effectiveness of the treatment in the subject. To provide.

[0012] In some embodiments, the treatment affecting mtDNA is mitochondrial replacement therapy. In some embodiments, the treatment that affects mtDNA is mitochondrial cross-linking. In a specific embodiment, the treatment affecting mtDNA is mitochondrial replacement therapy. In some embodiments, the reference sample comprises administering a reference replacement cell (MirC). It is taken from the same subject before administering the treatment to the elephant.

[0013] In another aspect, the present invention relates to a method for treating a mitochondrial disease or disorder, or Pathogenic mitochondrial DNA ( 1. A method for identifying a threshold level of heteroplasmy for a mutation in mtDNA (mtDNA), comprising: (a) obtaining or having obtained a biological sample containing one or more single cells from a subject; (b) determining the sequence of intracellular mtDNA in the one or more single cells; (c) wild-type and mutant forms of the intracellular mtDNA sequence in the one or more single cells. determining the ratio of; (d) Cells in the arrangement of intracellular mtDNA between the one or more single cells and within the one or more single cells The inter- and / or intracellular variation is calculated, thereby determining the heteroplasmy of mtDNA in the sample. Determining the level; and (e) Identifying the minimum level of heteroplasmy that positively correlates with mitochondrial disease or disorder. and thereby the heteroplasmy that is manifested in the mitochondrial-related disease or disorder. determining a threshold level of

[0014] In some embodiments of any of the methods provided herein, the method further comprises administering to said one or more The method includes calculating the amount of cell-to-cell variation in the sequence of the intracellular mtDNA between the above single cells.

[0015] In some embodiments of any of the methods provided herein, the method further comprises administering to said one or more The method includes calculating the amount of intracellular variation in the sequence of the intracellular mtDNA within the single cell.

[0016] In some embodiments of any of the methods provided herein, the method further comprises administering to said one or more Inter- and intracellular variations in the sequence of the intracellular mtDNA between or within single cells on the This includes calculating the quantity.

[0017] In one embodiment, determining the sequence of the intracellular mtDNA in the one or more single cells. In certain embodiments, the one or more single cells are assayed in a single assay. determining the sequence of the intracellular mtDNA in the one or more single cells; and Determining the ratio of wild-type and mutant forms of a sequence is carried out in a single assay will be done.

[0018] In some embodiments, determining the sequence of the intracellular mtDNA is performed using quantitative polymerase chain reaction (PCR). In a specific embodiment, the quantitative PCR assay is a deoxyribonucleotide-binding protein (DNA) assay. In some embodiments, the assay is a digital droplet PCR (ddPCR) assay. The quantitative PCR assay includes TaqMan polymerase.

[0019] In some embodiments, the one or more single cells have uniform intercellular mtDNA. In some embodiments, the one or more single cells have heterologous intracellular mtDNA. In the method, the one or more single cells have heterologous intracellular mtDNA. [Brief explanation of the drawings]

[0020] (6. Brief description of the drawings) [Figure 1]The strategy used for heteroplasmy analysis of fibroblasts (BK01, BK02, and BK04) obtained from patients with primary mitochondrial disease harboring a single mitochondrial DNA mutation, and the design of primers used in SNP genotyping assays are shown. SEQ ID NOs: 1 to 15 are shown.

[0021] [Figure 2] The total heteroplasmy of three fibroblasts obtained from patients with primary mitochondrial disease was shown to be 99.8%, 96.9%, and 99.7% for BK01, BK02, and BK04, respectively.

[0022] [Figure 3] A concentration of 1 x 105 cells per milliliter was shown to be the optimal dilution to obtain a cell suspension that allowed ddPCR at the single cell level.

[0023] [Figure 4] A threshold line was obtained based on the proportional relationship between the number of cells with a positive signal and the number of cells loaded.

[0024] [Figure 5]The results of ddPCR analysis at the single-cell level are shown. The healthy signal is plotted on the Y axis, and the mutant signal is plotted on the X axis. Quadrant analysis showed that cells with only mutant mtDNA were shown in the lower right quadrant, cells with both mutant and healthy mtDNA were shown in the upper right quadrant, and cells with only healthy mtDNA were shown in the upper left quadrant. The lower left quadrant represents a droplet containing no cells. Quadrant analysis of BK01 showed that the majority of cells (96.56%) were homoplasmic for mutant mtDNA (plotted in the lower right), while a minority of cells had both mutant and healthy mtDNA (plotted in the upper right). This is a state of intracellular heteroplasmy (Figure 5, upper panel). Furthermore, a population of cells composed only of healthy mtDNA was present at the same ratio as cells with intracellular heteroplasmy, 1.72%. BK02 contained a population of cells with both mutant and healthy mtDNA, plotted at the top right with a ratio of 4.76% (Fig. 5, middle panel). BK04 differed from the other patients in that there was only a single fraction of cells with only mutant mtDNA (Fig. 5, bottom panel). Both BK02 and BK04 did not contain a population of cells with only healthy mtDNA.

[0025] [Figure 6] Cell cycle analysis of fibroblasts from three patients with primary mitochondrial disease (BK01, BK02, and BK04) versus NHDF cells was performed. The analysis revealed that the S-phase fraction in these cells was less than half that of NHDFs. The sum of G2 / M and S-phase fractions ranged from 10 to 20% in affected fibroblasts.

[0026] [Figure 7] The human T-cell D-loop region used for sequencing mtDNA is shown.

[0027] [Figure 8]The sequences of HVR1 mtDNA in human T cells and EPC100 cells are shown in SEQ ID NOs: 16 and 17.

[0028] [Figure 9] 1 shows human T cell versus EPC100 SNP assay probes / primers. SEQ ID NOs: 18 to 24 are shown.

[0029] [Figure 10] 1 shows the experimental design for the production of MirC and the timeline for the heteroplasmy assay.

[0030] [Figure 11] PCR amplification products obtained from human T cells and EPC100 cells are shown.

[0031] [Figure 12] Quantification of TaqMan qPCR SNP genotyping assays for the total T cell population with and without mitochondrial replacement shows that exogenous mtDNA accounted for half of the total T cell population within 2 days and approximately 70% within 7 days post-transfer.

[0032] [Figure 13] Quadrant analysis of mitochondrial-exchanged human primary T cells is shown.

[0033] [Figure 14] Figure 1 shows the total heteroplasmy of mononuclear cells from patients with MELAS compared to control GJ mononuclear cells as determined by TaqMan qPCR.

[0034] [Figure 15] 1 shows exemplary results of sc-ddPCR analysis by FACs on control GJ mononuclear cells.

[0035] [Figure 16] 1 shows exemplary results of sc-ddPCR analysis by FACs on control MELAS mononuclear cells.

[0036] [Figure 17] 1 shows exemplary results of sc-ddPCR analysis by FACs on control MELAS CD3+ T cells.

[0037] [Figure 18] 1 shows exemplary results of sc-ddPCR analysis by FACs on control MELAS CD11b+ macrophage-monocyte lineage cells.

[0038] [Figure 19] 1 shows a comparison of the heteroplasmy of mononuclear cells, CD3+ T cells, and CD11b+ macrophage-monocyte lineage cells from patients with MELAS compared to control GJ mononuclear cells. DETAILED DESCRIPTION OF THE INVENTION

[0039] (7. Detailed Description of the Invention) (7.1 definition) Unless otherwise defined, all terms, including technical and scientific terms, used in this application are The terms "a," "b," and "c" have the same meaning as commonly understood by a person skilled in the art to which this invention pertains. In general, the nomenclature used in this specification and the experimental procedures described below is consistent with that of the relevant art. It is widely known and commonly used in

[0040] As used herein, the term "mitochondrial exchange cell" or MirC refers to endogenous mitochondrial Cells in which mitochondria and / or mtDNA have been replaced with foreign mitochondria and / or mtDNA For example, the exemplary mitochondrial exchange cell (MirC) has a function Endogenous mtDNA encoding defective mitochondria, e.g., those with mitochondrial diseases or disorders mtDNA originating from the target organism is replaced with foreign mtDNA encoding functional mitochondria, e.g. For example, it involves replacing mtDNA with mtDNA originating from a healthy subject. However, there are cells in which endogenous mitochondria are replaced by foreign mitochondria. However, replacement of endogenous mitochondria and / or mtDNA may occur in some cells, e.g., older cells. Functional endogenous mtDNA from the cells is transferred to different cells, e.g., healthier cells from younger subjects. It will be understood that this may also include those in which functional foreign mtDNA has been replaced. In this case, healthy endogenous mitochondria and / or mtDNA may be expressed in a manner that mimics, for example, a mitochondrial disease or disorder. Replace dysfunctional mitochondria and / or mtDNA with foreign mitochondria, for example, to mimic the It will further be appreciated that the

[0041] As used herein, the terms "treat," "treating," and "treatment" refer to a disease. Reduction in the severity, progression, spread, and / or frequency of symptoms; elimination of symptoms and / or underlying causes; "Treatment" refers to the prevention of the occurrence of a condition and / or its underlying cause, as well as the amelioration or correction of damage. "Medical treatment" is meant to include therapeutic treatment as well as preventative or suppressive measures for disease, illness, or disorder.

[0042] As used herein, the term "agent" is used in reference to the removal or reduction of mtDNA. In this case, it refers to an enzyme or compound that can reduce mtDNA. Restriction enzymes that cut DNA without causing toxicity in recipient cells include XbaI. However, some drugs inhibit mtDNA synthesis or selectively promote mitochondrial degradation. The enzyme or compound may also be included.

[0043] As used herein, the terms "decreasing" or "reducing" mean that the term Generally, a decrease of at least 5% compared to a reference level, e.g., at least At least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or means a reduction of at least about 90%, or any reduction between 5% and 99%. Partial reduction, or agents that partially reduce endogenous mtDNA, or reduction, does not reduce all endogenous mtDNA. It will be appreciated that this does not result in complete removal of tDNA (i.e., ρ0 cells). The term "increase" as used generally refers to an increase of at least 5%, for example at least about 10%. , or at least about 20%, or at least about 30%, or at least about 40%, or at least About 50%, or at least about 60%, or at least about 70%, or at least about 80%, or less Both mean an increase of about 90% or more than 90%.

[0044] As used herein, the term "intrinsic" means having or originating from within. For example, endogenous mitochondria are the mitochondria that are naturally present in cells.

[0045] As used herein, the term "foreign" refers to cellular material that is not native to the host (e.g., "Externally" refers to cellular material that originates from outside the body, such as mitochondria or mtDNA. For example, the mitochondrial genome is derived from a different source than the host cell. or originating from a different cell type or species than the host mitochondria, The genome is foreign to the host cell or host mitochondria. This refers to mitochondria that have been removed from mitochondria, manipulated, and then returned to the same mitochondria. It may also refer to the mitochondrial genome.

[0046] As used herein, the term "majority" means the largest amount relative to another amount being compared. An exemplary majority when comparing two groups is approximately 50% of the total population. or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 95% or more The majority is the total number of people being compared. It is understood that this may vary depending on the group and may be less than 50% when three or more groups are compared. Let's do it.

[0047] As used herein, the term "subject" is intended to mean a mammal. The subject may be a human or non-human mammal, such as a dog, cat, bovid, horse, mouse, or lamb. A "subject" may be a "patient", e.g., a rat, a rabbit, or a transgenic species thereof. It will be understood that the term may also refer to, for example, a human patient.

[0048] As used herein, the term "effective amount" refers to an amount of an amount of a compound that inhibits heteroplasmy and / or mitochondrion. Effective in regulating, treating, or ameliorating any disease or disorder associated with dysfunction of the Thus, an effective amount can include, for example, a therapeutically effective amount, and this use The term refers to a therapeutically effective amount or a biologically effective amount, and this biologically effective amount is a biological The terms "therapeutically effective amount" and "effective amount" refer to an amount effective to improve overall treatment. improve, reduce or avoid the symptoms or cause of the disease or disorder, or prevent the release of another therapeutic agent The amount of a given composition that corresponds to such an amount may vary. The composition may vary depending on various factors, such as the type of composition, pharmaceutical formulation, route of administration, type of disease, disorder or injury being treated, Although the nature of the target or host may vary, it is nevertheless possible for one skilled in the art to determine The therapeutically effective amount of a drug as defined herein can be determined by methods known in the art. This can be readily determined by those skilled in the art using known routine methods.

[0049] As used herein, the term "age-related disease" refers to any of a number of diseases that are attributable to aging. These diseases include, but are not limited to, osteoporosis, bone loss, arthritis, and arthrosis. , cataracts, macular degeneration, metabolic diseases including diabetes, Alzheimer's disease and Parkinson's disease neurodegenerative diseases, including immunosenescence, and heart disease, including atherosclerosis, and lipid The term "age-related diseases" includes neurodegenerative diseases such as Alzheimer's disease and and related disorders, ALS, Huntington's disease, Parkinson's disease, and cancer.

[0050] As used herein, the term "autoimmune disease" refers to an immune response to an individual's own tissues and organs. Diseases or disorders resulting from a directed immune response, or their manifestations or Autoimmune disease is intended to mean a disease resulting from an autoimmune Caused by or exacerbated by the production of autoantibodies that react with an antigen or its epitope Autoimmune diseases can be tissue-specific or organ-specific, or can refer to systemic autoimmune diseases. The systemic autoimmune disease may be a connective tissue disease (CTD), such as systemic lupus erythematosus (SLE). Malignant fibrosis (lupus, SLE), mixed connective tissue disease, systemic sclerosis, polymyositis (PM), skin Myositis (DM), and Sjogren's syndrome (SS). Others include rheumatoid arthritis and antineutrophil cytoplasmic antibody (ANCA) polyangiitis.

[0051] As used herein, the term "genetic disease" refers to an abnormality, such as a mutation, in the nuclear genome. Exemplary genetic disorders include, but are not limited to, Hatch disease, These include Johnson-Gilford progeria syndrome, Werner syndrome, and Huntington's disease.

[0052] As used herein, the term "cancer" includes, but is not limited to, solid tumors and hematological tumors. The terms "cancer" and "cancerous" refer to a type of cancer typically characterized by unregulated cell growth. refers to or describes the physiological state of a mammal in which

[0053] As used herein, the terms "mitochondrial disease or disorder" and "mitochondrial disorder" The terms are used interchangeably and refer to the mitochondria that cause energy deficiency in these body regions. Mitochondrial diseases or disorders refer to a group of diseases resulting from inherited or acquired damage to the mitochondrial Exemplary organs affected include organs that consume large amounts of energy, such as the liver. The effects include liver failure, muscle weakness, fatigue, and heart failure. Problems with the liver, eyes, and various other systems are common.

[0054] As used herein, the term "mitochondrial DNA abnormality" refers to a condition in which the product is a mitochondrial Mutations in mitochondrial genes localized in the mitochondrial membrane and not observed in cells of healthy subjects Exemplary diseases associated with mitochondrial DNA abnormalities include, for example, chronic progressive extraocular muscle Paralysis (CPEO), Pearson syndrome, Kearns-Sayre syndrome (KSS), diabetes and hearing loss (DAD), Leber's hereditary optic neuropathy (LHON), LHON and neuropathy, ataxia, and retinitis pigmentosa Maternally inherited NARP syndrome, also known as Leigh syndrome, caused by a mutant form of mtDNA Mitochondrial Leigh syndrome (MILS), mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes Myoclonic epilepsy with ragged red fibers (MERRF), familial bilateral striatal striatum (MELAS) Corpus necrosis / striatonigral degeneration (FBSN), Luft's disease, aminoglycoside-induced hearing loss (AID), and Multiple mitochondrial DNA deletion syndrome.

[0055] As used herein, the term "nuclear DNA abnormality" in the context of a mitochondrial disease or disorder The term refers to a mutation or change in the coding sequence of a nuclear gene whose product is localized to the mitochondria. Exemplary mitochondrial diseases or disorders associated with nuclear mutations include mitochondrial DNA depletion syndrome 4A, mitochondrial recessive ataxia syndrome (MIRAS), mitochondrial neurogastrointestinal Myocardial encephalopathy (MNGIE), mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase gamma (POLG)-related disorder Sensory ataxia neuropathy dysarthria ophthalmoplegia (SANDO), white matter with brainstem and spinal cord lesions Encephalopathy and elevated lactate level (LBSL), coenzyme Q10 deficiency, Leigh syndrome (caused by nuclear mutations), myelopathy Mitochondrial complex disorders, fumarase deficiency, α-ketoglutarate dehydrogenase complex KGDHC deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenase complex deficiency PDHC, pyruvate carboxylase deficiency (PCD), carnitine palmitoyltransferase Carnitine palmitoyltransferase I (CPT I) deficiency, Carnitine palmitoyltransferase II (CPT II) deficiency Carnitine-acyl-carnitine (CACT) deficiency, autosomal dominant / autosomal recessive progressive External ophthalmoplegia (ad- / ar-PEO), infantile-onset spinocerebellar atrophy (IOSCA), mitochondrial myopathy ( MM), spinal muscular atrophy (SMA), developmental delay, aminoaciduria, cholestasis, iron overload, early death (GRA CILE), and Charcot-Marie-Tooth disease type 2A (CMT2A).

[0056] As used herein, the term "dysfunctional mitochondria" refers to dysfunctional mitochondria. Exemplary dysfunctional mitochondria include oxidative phosphorylation Mitochondria that are unable to synthesize ATP by oxidation or that synthesize insufficient amounts of ATP As used herein, the term "functional mitochondria" refers to mitochondria that consume oxygen and This refers to mitochondria, which produce ATP.

[0057] As used herein, the term "mutation" refers to any change in the structure of a gene. Mutations in genes result in the formation of variant (also called "mutant") forms. changes in the gene, resulting from deletions, insertions, or rearrangements of larger parts of genes or chromosomes. In some embodiments, the mutations affect the function or the resulting protein. For example, the quality of a single nucleotide of DNA in a protein coding region can be affected. Mutations in the codon (i.e., point mutations) result in codons that code for different amino acids. This different amino acid can change the structure of the protein (i.e., a missense mutation). In some circumstances, as described herein, organelles, e.g., mitochondria, may be involved. It will be appreciated that chondrial function may be altered.

[0058] As used herein, the terms "heteroplasmy" and "heteroplasmic" refer to This refers to the occurrence of two or more types of mitochondrial DNA genomes in an individual or sample. Degrees of heteroplasmy are associated with varying degrees of physiological conditions as described herein. Heteroplasmy can be identified by means known in the art, and specific nucleic acids can be identified by The severity of the physiological condition associated with a leiotide allele depends on the relative abundance of the associated allele within such an individual. It is expected to vary with the percentage of genes.

[0059] As used herein, the term "wild-type" is used in the context of mitochondrial DNA. When used in this context, it refers to the genotype that is typical of the species as it occurs in nature. Exemplary reference genomes for the human mtDNA genome include the Cambridge Reference Sequence (CRS).

[0060] As used herein, the terms "about" or "approximately" are used in conjunction with numbers. In this case, it refers to any number within 1, 5, 10, 15, or 20% of the referenced number.

[0061] Practice of the embodiments provided herein is within the skill of those of ordinary skill in the art unless otherwise indicated. Conventional techniques in molecular biology, microbiology, and immunology shall be utilized. Such techniques shall The literature provides a thorough explanation. Examples of textbooks that are particularly suitable for reference are: See mbrook et al., "Molecular Cloning: A Laboratory Manual" ory Manual), 3rd ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al. Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); Glover, ed., DNA Cloning, Vols. I and II (1985); Gait Eds., Oligonucleotide Synthesis (1984); Hames and Higgi ns, ed., Nucleic Acid Hybridization (1984); Hames and Higgins, eds., Transcription and Translation (1984); Freshney "Animal Cell Culture: Immobilized Cells and Enzymes" Plant Molecular Biology: Laboratory Manual (IRL Press, 1986); Kallen et al. Plant Molecular Biology - A Laboratory Manual (Melody S. Clark, ed.; Springer, 1999) Inger-Verlag, 1997; "Immunochemical Methods in Cell and Molecular Biology" s in Cell and Molecular Biology (Academic Press, London); Scopes's article, Protein Purification: Principles and Practice (Spr Inger Verlag, NY, 2nd ed., 1987); and Weir and Blackwell, eds., Handbook of Experimental Immunology. Handbook of Experimental Immunology, Vols. I-IV (1986).

[0062] (7.2 Mechanistic insight) Heteroplasmy in dividing or non-dividing cells changes over time through various mechanisms. This has implications for disease onset and progression. The mechanism by which this occurs is asexual segregation, and in this model, the proliferated mitochondria The ratio of wild-type mtDNA to mutant mtDNA is randomly and unevenly distributed in daughter cells. Random genetic drift sometimes biases the former or the latter (Birky, C.W., Maruyama, T., and F 1999, pp. 1317-1323, 1999. See Uerst, P., "Population and evolutionary changes in genes in mitochondria and chloroplasts." An Approach to the Theory of Population and Evolution and Some Results lutionary Genetic Theory For Genes In Mitochondria And Chloroplasts, And Some Re results.) Genetics 103, 513 (1983). The other is relaxed replication. In this model, Mitochondria are randomly selected, replicated, destroyed, and then destroyed independently of the host cell cycle. The genome of the human genome is tightly regulated to replicate in sync with cell division (Birky Jr., C., ed.). "Relaxed and Rigid Genomes: Why Cytoplasmic Genes Do Not Follow Mendelian Laws" d and stringent genomes: why cytoplasmic genes don't obey Mendel's laws.)” Jo (Journal of Heredity 85, 355-365 (1994)). These neutral genetic drifts are caused by either positive or negative selection. Long deletion mutations in mtDNA that result in pathogenic consequences may offer a replicative advantage. and exceed the number of wild-type genomes (Clark, KA et al., "Selfish Little Circles: Evolution of mitochondrial DNA with transmission bias and large deletions in rhizal nematodes (Se) lfish little circles: transmission bias and evolution of large deletion-bearing mitochondrial DNA in Caenorhabditis briggsae nematodes.)” PLoS One 7, e41433, do i:10.1371 / journal.pone.0041433 (2012)). Wild-type mtDNA is quantitatively regulated in individual cells. Considering that mutations in mtDNA are present in the genome, the occurrence of mutations in mtDNA may lead to the This is called the wild-type maintenance theory. (Durham, SE, Brown, DT, Turnbull, DM and Chinnery, PF, "Mitco Progressive depletion of mtDNA in myopathies mitochondrial myopathy.) Neurology 67, 502-504 (2006)) in the replication of mutations Positive selection promotes beneficial effects, whereas undesirable effects in pathogenic mutations The main features of this disease are reduced survival of hematopoietic stem or progenitor cells and loss of mtDNA mutations in the blood. (Rajasimha, HK, Chinnery, PF and Samuels, DC, "Stem Cell Selection against pathogenic mtDNA mutations in the population has led to the loss of the 3243A-->G mutation in blood. (Selection against pathogenic mtDNA mutations in a stem cell population lead s to the loss of the 3243A-->G mutation in blood.)” Am J Hum Genet 82, 333-343, doi:10.1016 / j.ajhg.2007.10.007 (2008)).

[0063] Extreme cases of mitochondrial disease are caused by mutations in 20 nuclear genes. The mtDNA maintenance defect, which is caused by the resulting mtDNA depletion or multiple mtDNA deletions, (El-Hattab, AW, Craigen, WJ and Scaglia, F., "Minor Mitochondrial DNA maintenance defects. Biochim Biop Hys Acta Mol Basis Dis 1863, 1539-1555, doi:10.1016 / j.bbadis.2017.02.017 (2017)) The pool of nucleotides in the cytoplasm is up to 1,000 times higher than in hematopoietic cells and fibroblasts, for example. It is involved in cells that have completed specific differentiation, such as nerve cells and skeletal muscles, rather than proliferating cells with Targets (Gorman, GS et al., "Mitochondrial diseases." Na t Rev Dis Primers 2, 16080, doi:10.1038 / nrdp.2016.80 (2016)). Mitochondrial disease Considering treatment, anti-cancer drugs that target specific molecules can help determine indications. Companion diagnostics to assist in the diagnosis of rheumatoid arthritis should be developed (see Jorgensen, JT, "Companion Diagnostics"). Companion and complementary diagnostics: clinical and regulatory perspectives stics: clinical and regulatory perspectives.)” Trends in cancer 2, 706-712 (201 6).

[0064] Recent advances in mitochondrial biomarkers have led to a line with a sensitivity and specificity of 92%. Fibroblast growth factor 21 (FGF21) (Suomalainen, A. et al., "Muscle-Elicited Mitochondrial Respiration") FGF-21 as a biomarker for muscle chain defects: a diagnostic study -manifesting mitochondrial respiratory chain deficiencies: a diagnostic study.) ” The Lancet Neurology 10, 806-818, doi:10.1016 / s1474-4422(11)70155-7 (2011)) and Growth / differentiation factor 15 (GDF15), which has a sensitivity of 98% and a specificity of 86% (Yatsuga, S. et al., "Malignant Pathogenesis"). Growth differentiation factor 15 (GDIF) as a useful biomarker for mitochondrial disorders ion factor 15 as a useful biomarker for mitochondrial disorders.) Ann Neurol 7 8, 814-823, doi:10.1002 / ana.24506 (2015)) has been established, but The reacquisition of wild-type mtDNA in cells and the elimination of the mutation burden are the optimal indicators of therapeutic success. Heteroplasmy is not only important for the diagnosis of mitochondrial diseases, but also for the treatment process. It should also be useful for estimating efficacy.

[0065] 7.3 Intercellular vs. intracellular heteroplasmy Mitochondrial genotype heterogeneity is due to differences in mtDNA copy number in individual cells as well as mtD Microheteroplasmy and intercellular mtDNA mutations in single cells Mutations are redefined as macroheteroplasmy occurring between putatively identical cells (Ary Aman, J., Johnston, I.G., and Jones, N.S., "Mitochondrial Heterogeneity" Front Genet 9, 718, doi:10.3389 / fgene.2018.00718 (2018)). Microheteroplasmy can lead to macroheteroplasmy. On the other hand, macroheteroplasmy is characterized by the occurrence of mtDNA with wild-type or mutant genomes. This can result in the differentiation of distinct cell populations.

[0066] Microheteroplasmy occurs due to replication errors rather than oxidative damage (Kau ppila, JH & Stewart, JB "Radical generation without free radical-driven mutations" Mitochondrial DNA: Radically free of free-radical driven mutations Biochim Biophys Acta 1847, 1354-1361, doi:10.1016 / j.bbabio.2015.06.00 1 (2015)). Sequencing of single mitochondria in neurons revealed microheteroplasmic While it was speculated that there may be a mechanism of negative selection that neutralizes this, some mutations It has been shown that β-glucan dominates over 90% of single cells, suggesting the existence of a mechanism to bypass negative selection. It has been suggested that there is a single mitochondrial sequence (Morris, J. et al., "Single mitochondrial sequence revealed"). Heteroplasmic single nucleotide variants permeating the revealed mitochondria -(Pervasive within-Mitochondrion Single-Nucleotide Variant Heteroplasmy as Reve Cell Rep 21, 2706-2713, doi:10.1016 / j.celrep.2017.11.031 (2017)). The wide range of mutations that can be achieved and the Whether this structure can result in pathological outcomes remains an open question.

[0067] On the other hand, macroheteroplasmy can arise through neutral genetic drift (Wonnapinij, P., Chinnery, PF and Samuels, DC, "Mitochondrial dynamics due to random genetic drift." The distribution of mitochondrial DNA heteroplasmy Am J Hum Genet 83, 582-593, doi:10.1016 / j.ajh g.2008.10.007 (2008)), which shows high mutation levels involved in disease (Rossignol, R. et al. "Mitochondrial threshold effects." The Bioch emical journal 370, 751-762, doi:10.1042 / BJ20021594 (2003)). The variation in macroheteroplasmy due to mitophagy can be mathematically correlated with time, mitophagy rate, and network structure. It is predicted that the risk of catastrophic brain damage will increase with increasing fragmentation (Aryaman, J., Bowles, C., Jones, N S. and Johnston, I.G., "Fragmentation of the mitochondrial network is an absolute disruption" Mitochondrial network fragment regulates the accumulation of mutant mtDNA independently of fusion rate ation modulates mutant mtDNA accumulation independently of absolute fission-fusi on rates.)” bioRxiv, 409128, doi:10.1101 / 409128 (2018)), overall copy number of mtDNA (Chinnery, PF and Samuels, DC, "Relaxed Relaxed replication of mtDNA: a model with implications for disease development h implications for the expression of disease.)” The American Journal of Human Geology Netics 64, 1158-1165 (1999)). During development, mitochondria are formed, characterized by a decrease in mtDNA copy number. Doria bottleneck increases heteroplasmy fluctuations and mutation load above a threshold Cells with mitochondrial DNA fragments are eliminated (Cree, LM et al., "Mitochondrial DNA Fragmentation During Embryogenesis"). A reduction of mitochondrial DNA molecules explains the rapid segregation of genotypes. Nat Gene t 40, 249-254, doi:10.1038 / ng.2007.63 (2008)). High degree of pathogenic heteroplasmy variation Children with mitochondrial disease or disorders may develop, but at later life stages There is no direct correlation between the mtDNA and mitochondrial dysfunction. The accumulation of mutations is not only responsible for neurodegenerative diseases, such as Parkinson's disease, but also for the physiological aging process. This may be observed even in people with aging (Bender, A. et al., "Aging and Parkinson's Disease"). High levels of mitochondrial DNA deletion in substantia nigra neurons NA deletions in substantia nigra neurons in aging and Parkinson disease.) Nat Genet 38, 515-517, doi:10.1038 / ng1769 (2006)).

[0068] Mitophagy is a key regulator of various mitochondrial stresses associated with mutational burden, e.g. It is activated by nutrient deprivation, hypoxia, and oxidative stress (Wei, H., Liu, L., and Che n, Q., "Selective removal of mitochondria via mitophagy: Different mitochondria Selective removal of mitochondria via mitophagy: distinct pathways for different mitochondrial stresses.)” Biochim Biophys Acta 1 853, 2784-2790, doi:10.1016 / j.bbamcr.2015.03.013 (2015)). Mitochondrial dynamics , fusion and fission play essential roles in maintaining their function and quality (Sebastian, D., Palacin, M. and Zorzano, A., "Mitochondrial dynamics: Mitochondrial Dynamics: Coupling Mitochondrial Fitness ess with Healthy Aging.)” Trends Mol Med 23, 201-215, doi:10.1016 / j.molmed.2017. 01.003 (2017)). Fragmented mitochondria have a depolarized membrane potential, which allows mitochondria to separate. preferentially degraded by phasing (Twig, G. et al., "Fission and selective fusion in mitochondrial cells"). Fission and selective fusion The EMBO Jour nal 27, 433-446, doi:10.1038 / sj.emboj.7601963 (2008)). Mitochondrial Disease and Aging Mitophagy promotes mtDNA turnover and alters heteroplasmy in yeast It increases and maintains the negative pressure of mutation load without increasing it (Aryaman , J., Johnston, IG and Jones, NS, "Mitochondrial heterogeneity" Front Genet 9, 718, doi:10.3389 / fgene.2018.00718 (2018 )) So far, mutation burden estimation has been performed using bulk cell samples, e.g., peripheral blood and These measurements have been performed on skeletal muscle biopsies. It is not possible to distinguish heteroplasmy. Understanding the cellular events that result from mutation burden This will require single-cell analysis of various biological processes that involve mutational burden.

[0069] 7.4 Detection Systems including single nucleotide polymorphisms (SNPs) used in the diagnosis of cancer, genetic disorders, and infectious diseases. Therefore, there are many methods for detecting and genotyping DNA sequence variations (Angulo, B., Lopez- Rios, F. and Gonzalez, D., "New Generation Companion Diagnostics: cobas BRAF, KRAS, and E GFR mutation detection test (A new generation of companion diagnostics: cobas BRAF, K RAS and EGFR mutation detection tests.)”Expert Review of Molecular Diagnostics 14, 517-524, doi:10.1586 / 14737159.2014.910120 (2014); Urata, M. et al., "Conflict By combining gene-specific PCR and peptide nucleic acid-directed PCR clamping High-sensitivity detection of the A3243G mutation in mitochondrial DNA A3243G mutation of mitochondrial DNA by a combination of allele-specific PCR and peptide nucleic acid-directed PCR clamping.)” Clin Chem 50, 2045-2051, doi:10.1 373 / clinchem.2004.033761 (2004); Payungporn, S., Tangkijvanich, P., Jantaradsame e, P., Theamboonlers, A. and Poovorawan, Y., "Real-time PCR and Melting Curves." Simultaneous quantification and genotyping of hepatitis B virus by assay n and genotyping of hepatitis B virus by real-time PCR and melting curve analysis s.)” J Virol Methods 120, 131-140, doi:10.1016 / j.jviromet.2004.04.012 (2004)). Hard-to-amplify mutagenesis based on oligonucleotides with mismatched 3'-residues to the template The ARMS system does not efficiently extend PCR strands (Newton, CR et al., "Any DNA fragment"). Analysis of any point mutation in the refractory amplification mutation system (ARMS) DNA.The amplification refractory mutation system (ARMS). Nucleic Acids Resear ch 17, 2503-2516, doi:10.1093 / nar / 17.7.2503 (1989)). ARMS requires a 3'-matrix. Only primers designed to contain a 3'- or 3'-mismatched end are isotopic, No restriction enzymes, sequencing reactions, or special machinery are required. Specificity of ARMS primers is dependent on the template sequence (Huang, M.-M., Arnheim, N., and Goodman, M.F., "Taq DNA Extension of base mispairs by A polymerase: Implications for single nucleotide discrimination in PCR (E xtension of base mispairs by Taq DNA polymerase: implications for single nucleot ide discrimination in PCR.)” Nucleic Acids Research 20, 4567-4573, doi:10.1093 / n ar / 20.17.4567 (1992)), so some modifications include an additional mismatch upstream of the 3' end. There are some methods that improve specificity without compromising the simplicity of the method. Quenching, defined as the reduction of signal intensity, is a key factor in various PCR-based SNP assays. (Gibson, NJ, "Real-Time Peptide Analysis for DNA Sequence Variation Analysis") Use of real-time PCR methods in DNA sequence variation analysis. ” Clin Chim Acta 363, 32-47, doi:10.1016 / j.cccn.2005.06.022 (2006)).

[0070] Taq-Man quantitative PCR was applied in combination with sequence-specific probes to detect DNA sequence variations. (Holland, PM, Abramson, RD, Watson, R. and Gelfand, DH, "Thermal 5'→3' exonuclease of Thermus aquaticus DNA polymerase Detection of specific polymerase chain reaction products by using ATPase activity ic polymerase chain reaction product by utilizing the 5' -> 3' exonuclease act of Thermus aquaticus DNA polymerase.)” Proceedings of the National Academy of Sciences 88, 7276, doi:10.1073 / pnas.88.16.7276 (1991)). and a quencher, and exhibits little fluorescence in the aqueous phase. The nucleotide probe is then introduced into the 5' exonuclease of TaqMan polymerase during the PCR extension process. The enzyme activity digests the fragments, releasing the fluorescence from the dissociated fluorescent substance. By using this method, multiplex detection of several mutations can be performed in a single reaction tube. It is feasible (Nurmi, J., Ylikoski, A., Soukka, T., Karp, M. and Lovgren, T. "A novel labeling technique for the detection of specific polymerase chain reaction products in sealed tubes" (A new label technology for the detection of specific polymerase chain reaction (products in a closed tube.)” Nucleic Acids Research 28, e28-00 (2000)). To detect rare variants, a number of modifications, e.g., CataCleave (Harvey, J.J. "Characterization and application of CataCleave probes in real-time detection assays," (Characterization and applications of CataCleave probe in real-time detection as Anal Biochem 333, 246-255, doi:10.1016 / j.ab.2004.05.037 (2004)), Scorpio n-ARMS (Whitcombe, D., Theaker, J., Guy, S.P., Brown, T., and Little, S., " Detection of PCR products using self-probing amplicons and fluorescence ducts using self-probing amplicons and fluorescence.)”Nature Biotechnology 17, 804-807, doi:10.1038 / 11751 (1999)), and "Peptide Nucleic Acid-Locked Nucleic Acid Polymerase" Dual PNA Clamping Mediated PCR (PNA-LNA PCR) Clamp (Zhang, S. et al., "Dual PNA Clamping Mediated PCR" EGF in plasma samples obtained from patients with non-small cell lung cancer using an LNA-PNA PCR clamp Ultrasensitive and quantitative detection of mutations in R EGFR mutations in plasma samples from patients with non-small-cell lung cancer using a dual PNA clamping-mediated LNA-PNA PCR clamp.)” Analyst 144, 1718-1724 ( 2019)) (the sensitivity of each method is 5%, 1%, and 1%, respectively) in combination with primers and probes. Double-stranded DNA fragments have a specific melting temperature, which is used as a probe. This method is applied to fluorescent melting curve analysis based on the fluorophore and quencher at different ends. The probe is used to generate a temperature-dependent negative differential signal of the probe-template hybrid. The fluorescence melting curves are plotted against the nucleotide sequence, even when a single nucleotide is determined. Sequence-dependent identification peaks are obtained as a result of the analysis (Huang, Q. et al., "Dual Labeling, Self-Encapsulating Multiplex Fluorescence Melting Curve Analysis for Mutation Detection Using Fluorescent Probes ex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes.)” PLoS One 6, e19206, doi:10.1371 / journal.pone.0019206 (2 Although the above methodologies have improved in specificity and sensitivity, single-cell biology still requires the use of single-cell-based The development of a microwell chip for detecting target sequences of 1 is required. Third generation PCR, based on water-in-oil and microfluidic technologies, and digital PCR, based on Poisson distribution. The digital MIQE guidelines have emerged along with the analysis systems (Huggett, JF et al., "Digital MIQE Guidelines"). The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments Information for Publication of Quantitative Digital PCR Experiments.)”Clinical Chemistry 59, 892, doi:10.1373 / clinchem.2013.206375 (2013)). Water-in-oil droplets Droplet digital PCR (ddPCR) using dPCR technology detects rare mutations with a sensitivity of 0.001%. showed higher sensitivity in identifying markers (Watanabe, M. et al., "Droplet Digital PCR" Pretreatment EGFR T790M in patients with non-small cell lung cancer harboring EGFR activating mutations Ultra-Sensitive Detection of Pretreatment EGFR T790M Mutations Mutation in Non-Small Cell Lung Cancer Patients with an EGFR-Activating Mutation n Using Droplet Digital PCR.)” Clinical Cancer Research 21, 3552, doi:10.1158 / 1078-0432.CCR-14-2151 (2015)).

[0071] Digital droplet PCR is a TaqMan qPCR-based assay that uses microfluidic technology. High sensitivity and specificity for accurate quantification of target nucleotides, e.g., rare variants It is possible to detect SNPs / SNPs (Mazaika, E. and Homsy, J. (2014) "Digital Droplet Digital Droplet PCR: CNV Analysis and Other Applications ications." Curr Protoc Hum Genet 82, 7 24 21-13). introduced ddPCR as a general detection method to identify heteroplasmy in single cells. This analysis revealed that heteroplasmy based on current conventional methods was macroheteroplasmy. The intercellular hybridization, in which each cell has a different homoplasmic mtDNA, is called plasmolysis. Heteroplasmy is when each cell is of a different type, termed teloplasmy or microheteroplasmy. It is clear whether the mtDNA-containing cells are heteroplasmic (Aryaman, J., Johnston, IG and Jones, NS, "Mitochondrial Heterogeneity" geneity.)” Front Genet 9, 718, doi:10.3389 / fgene.2018.00718 (2018)).

[0072] The detection systems provided herein can be used to detect heterogeneity within cells, between cells, or both. The presence of mitochondrial dysfunction can be detected or monitored. Diagnosis of a leukemia-related disease or disorder and having or suspected of having heteroplasmy Applicability for use in monitoring the efficacy of mitochondrial replacement therapy in patients Furthermore, the detection system will be able to identify patient populations that are being treated with mitochondrial replacement cells (MirCs). of heteroplasmy for use in stratifying patients as likely to respond to It can be used in methods to identify threshold levels.

[0073] Detection of heteroplasmy at the single-cell level identifies cells with uniform intercellular mtDNA It is also possible to identify single cells that contain heterologous mtDNA. Comparison of single-cell mtDNA sequences allows identification of mtDNA within different cells. , yielding a mitochondrial disease or disorder phenotype from a comparison of multiple single-cell mtDNA sequences. Identifying threshold levels of heteroplasmy and / or mitochondrial replacement cells (MirCs) in patient populations ) and stratification into those likely to respond to treatment.

[0074] Throughout this application, various publications are referenced. The disclosures of these publications are incorporated herein by reference in their entireties. The present application is hereby incorporated by reference in order to more fully describe the state of the art to which the present invention pertains. The present invention has been described with reference to the examples provided above. It will be understood that various modifications can be made without departing from the spirit and scope of the invention. Hello. [Example]

[0075] 8. Working Example Example I (Single-cell digital droplet PCR) This example demonstrates the ability to identify mutant mt to assess mtDNA heteroplasmy in single cells in the presence or absence of DNA Demonstrate the method.

[0076] Normal human dermal fibroblasts (NHDFs) were obtained from Lonza (Walkersville, MD, USA). Chondriac disease patient-derived skin fibroblasts (BK01 / 02 / 04) were provided by the Department of Regenerative Medicine, Kyoto Prefectural University of Medicine, Supporting research into mitochondrial disease with the consent of both Kyoto, Japan and the Koinobori Ethics Committee These were kindly provided by Koinobori, a non-profit organization (NPO). The clinical characteristics of the primary cells are summarized in Table 1. Table 1 [Table 1]

[0077] NHDF was prepared using 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin, and 1% ethanol. Dulbecco's modified Eagle's medium (Thermo Fisher Scientific) supplemented with phosphate / streptomycin BK01 was maintained in FGM™-2 SingleQuots™ (hFGF-B, insulin, FBS FBM™ fibroblast basal medium supplemented with cephalosporin, cephalosporin, and gentamicin / amphotericin-B (LONZA) BK02 and BK04 were cultured in 10% fetal bovine serum (Thermo Fisher Scientific) and 1% Low glucose Dulbecco's modified Eagle's medium (DMEA) supplemented with penicillin / streptomycin ( All cells were cultured in a humidified 5% CO2 incubator (Thermo Fisher Scientific). The mixture was incubated at 37°C.

[0078] The cells were trypsinized, suspended in culture medium, and then centrifuged (1000 rpm, 5 min) The cells were pelleted and resuspended in PBS. The resuspended cells were then resuspended in 4% paraformaldehyde (Fujifilm) The cells were then fixed in a 500 ml solution of 1000 ml of PBS (Wako Pure Chemical Industries, Ltd., Osaka, Japan) at room temperature for at least 15 minutes. The cells were centrifuged (1500 rpm, 5 min) and resuspended in 50 μg / ml propidium iodide, 0.1 mg / ml RNase A, 0 The cells were resuspended in a propidium iodide solution consisting of 0.05% Triton X-100 and PBS, and incubated at 37°C for 40 min. After washing with PBS, the cells were pelleted (1500 rpm, 5 min) and washed with PBS. The samples were immediately analyzed by flow cytometry. Fabric was determined using Flowjo software.

[0079] Mitochondrial DNA heteroplasmy was determined by TaqMan SNP genotyping assays. Wild-type and mutant allele-specific TaqMan probes and primers were designed. The probes were prepared by Thermo Fisher Scientific. The two probes were fluorescently labeled with different fluorescent materials (FAM and The genomic DNA was labeled with NucleoSpi and VIC, and a quencher was attached to the other end. Extraction from cells was performed using n® Tissue (Takara Bio, Tokyo, Japan). The extracted genomic DNA (100 ng) was used with forward and reverse primers, probes, and The mixture was mixed with TaqMan genotyping master mix (Thermo Fisher Scientific) under the following conditions: Conditions: Initial denaturation (95°C for 10 minutes) followed by 40 cycles of PCR (95°C for 15 seconds and 60°C for 1 minute). The wild-type or mutant strains were used for quantitative PCR on a CFX connect real-time system (BioRad). Copy number determined plasmid containing the targeted mtDNA fragment amplified for the mutant sequence A calibration curve was generated by quantitative PCR using the primers listed above. They are listed in Figure 1.

[0080] The sc-ddPCR system begins with encapsulating a single cell in a single oil droplet, followed by 5' → Primers and fluorescent probes using TaqMan polymerase with 3' exonuclease activity The probe set is used in the PCR step, during which fluorescent material is released from the probe. The PCR reaction mixture is: 1.25 x10 5 4 μl resuspended cells at a concentration of 100 cells / ml; 10 μl 2x ddPCR supermix (Bio-Rad); 0.25 μM Concentration of wild-type and mutant allele-specific TaqMan probes; 0.9μ for target genes The primer mix is ​​added to a concentration of 1 μM, and nuclease-free water is added up to 20 μl. Droplets were analyzed using a Bio-Rad QX200 system (Bio-Rad) according to the manufacturer's instructions. The reaction mixture was prepared by PCR reaction using a thermal cycler (Bio-Rad) under the following conditions: The mixture was transferred to a 96-well plate (Eppendorf, Hamburg, Germany) for amplification at 2.0°C / sec. The standard ramp rate was 95°C for 10 minutes, followed by 94°C for 30 seconds and 53°C for 1 minute. The cycle was performed 40 times. A final enzyme inactivation step was performed at 98°C for 10 minutes. The droplets were transferred to a QX200 droplet reader (Bio-Rad) and the number of fluorescent-positive droplets was analyzed. Each droplet was analyzed using a two-color detection system (configured to detect FAM and VIC). Fluorescent droplets were counted and analyzed individually. Using the io-Rad, absolute quantification of target mtDNA in digital form is provided. added various numbers of target cells to the PCR reaction mix to ensure single-cell encapsulation. 500 cells per sample were successfully prepared as droplets. The cells were encapsulated in the vesicles, and single cell encapsulation was observed.

[0081] Three types of cells derived from patients with mitochondrial diseases were examined in this study. The characteristics of these cells are summarized in Table 1. These primary fibroblasts were isolated from skin biopsies of patients. The Ethics Committee of Koinobori General Incorporated Association, a Japanese NPO for mitochondrial disease, The cells were established as cultured cells with the consent of the Kyoto Prefectural University of Medicine Research Institute Ethics Committee. BK01 is the m3243 tRNA for leucine. Mitochondrial myopathy, encephalopathy, and lactic acidosis resulting from an A to G mutation in β-lactamase inhibitors The patient was a 30-year-old female patient with a history of stroke-like episodes (MELAS). The other two fibroblasts were generated from female patients with Leigh syndrome, aged 6 and 1 year. One of the syndrome cell lines expresses 37 nuclear-encoded genes that are part of respiratory chain complex I. and 7 mitochondrially encoded subunits. Modified NADH dehydrogenase 3 (MT-ND3) (also known as NADH dehydrogenase (ubiquinone) The other Leigh syndrome cell line contains a T to C mutation at m10158 located in the nucleus of the leukemia virus (LSV). is a subunit of the F1F0 ATPase (also known as complex V) and is one of 14 nuclear-encoded The ATP synthase F0 subunit is composed of two mitochondrially encoded subunits. Mitochondrial-encoded ATP synthase membrane subunit 6 (MT-ATP6) The proband with BK01 has a T to C mutation at m9185 located in unit 6. Her mother The mutation in BK02 was de novo. The inheritance of BK04 was not determined. There wasn't.

[0082] TaqMan single nucleotide polymorphism (SNP) assays are amenable to ddPCR, so from the perspective of simplicity, Select the primer set to determine heteroplasmy in the mtDNA of the target cells. , targeting the region encompassing the SNP, and The PCR product is designed to amplify a 151 bp sequence that matches the Cambridge Reference Sequence (CRS). To separately identify and quantify healthy and mutant mtDNA, FAM was added to the 5' end. Or VIC fluorescent dye and two TaqMan probes with non-fluorescent quenchers at the 3' end were used. Designed in combination with groove binding (MGB) to maximize the difference in melting temperature of various fibroblasts The amplified sequences were subcloned into a plasmid and used in a standard assay to quantify the target sequence. The lines are shown in Figure 1. The total heteroplasmy of the three fibroblasts was 0.01 for BK01, BK02, and BK04. The efficiencies were 99.8%, 96.9%, and 99.7%, respectively (Figure 2). Table 2 [Table 2]

[0083] Droplets were divided into various groups so that they contained a single cell or no cells. The concentration of the cell suspension was made by using 1 x 10 cells per milliliter. 5 concentration of individuals The cells were optimal (Fig. 3). The mtDNA was sequenced, and MT-ND3, MT-ATP6, and normal human skin, which is certain to have a sequence matching the CRS in the tRNA for leucine. Human dermal fibroblasts (NHDF) were used as a control. All three primer sets and probe sets were predicted between the probe and template. The PCR reaction was designed based on melting temperature. After initial denaturation (95°C for 10 minutes), 40 cycles were performed. Optimized to include a final heating step (98°C for 10 minutes) in addition to the PCR (94°C for 30 seconds and 56°C for 2 minutes). Following optimization of the PCR reaction, the correlation between the number of cells with a positive signal and the number of cells loaded was Based on the proportional relationship, the threshold line became constant (Figure 4).

[0084] The inventors plotted the results as healthy signals on the Y axis and mutant signals on the X axis. The quadrant analysis shows that cells with only mutant mtDNA cells with both mutant and healthy mtDNA are in the lower right quadrant, cells with both mutant and healthy mtDNA are in the upper left quadrant, and healthy Cells with only mtDNA are shown in the upper left quadrant. The lower left quadrant is a droplet containing no cells. The quadrant analysis of BK01 showed that the majority of cells, 95.56%, were homozygous for mutant mtDNA. Plasmonic plasminogen activator (PLA) is shown in the bottom left, but a small proportion of cells are mutant and healthy. The two types of mtDNA are plotted in the upper right corner. This is the state of Smy (upper panel of Figure 5). Furthermore, a cell population consisting only of healthy mtDNA is However, the ratio was 1.72%, the same as that of cells with intracellular heteroplasmy. The cell population with both mutant and healthy mtDNA is plotted at the top right with a ratio of .76%. BK04 contained a single mtDNA fragment from cells with only mutant mtDNA (Fig. 5, middle panel). The presence of only the BK02 fraction differed from that of other patients (Figure 5, lower panel). Neither BK04 nor BK04 contained a cell population with only healthy mtDNA.

[0085] In the quadrant analysis, there were two distinct fractions in the upper left or lower right quadrant. The small size of the events made it difficult to determine whether the two groups were in the upper right quadrant. Cell cycle analysis revealed that mitochondrial markers were expressed in three cell lines derived from patients with mitochondrial diseases. The S-phase fraction in the NHDF was less than half of that in the NHDF. The sum of G2 / M and S phases ranged from 10 to 20% of affected fibroblasts (Fig. 6). The ratio of mtDNA fragments to mtDNA fragments was almost identical, suggesting that the duplicated mtDNA fragments could occupy half of the cell cycle. was done.

[0086] In this study, we investigated the heterozygote structure of mtDNA in single cells in the presence or absence of mutant mtDNA. A method for assessing heteroplasmy in mitochondrial diseases is provided. Conventional methods uniformly target cell populations, such as mononuclear cells and biopsied skeletal muscle. Therefore, the output was intracellular heteroplasmy (microheteroplasmy) and intercellular heteroplasmy. No heteroplasmy (macroheteroplasmy) could be identified. The Leah disease phenotype appears when the heteroplasmy of mutant mtDNA reaches 60-70% or more. Although the threshold theory is based on clinical samples, it is difficult to predict whether all cells are heterozygous. have homoplasmy containing normal or mutant mtDNA Are the cells mixed in a heteroplasmic ratio, or a mixture of the former and the latter? Furthermore, it is unclear whether the threshold theory can be justified at the single-cell level. Whether this will be the case remains to be investigated.

[0087] Furthermore, single-cell biology of mtDNA heteroplasmy is not limited to mitochondrial diseases. rather, they shed light on the path to a fuller understanding of neurodegenerative diseases, cancer, and aging. .

[0088] Example II Heterogenesis of Mitochondrial Exchanged T Cells (MirT) Using Single-Cell Digital Droplet qPCR Analysis of loplasmic In this example, we used single-cell digital droplet qPCR to measure mitochondrial exchange. We present a method for analyzing heteroplasmy of T cells (MirT).

[0089] Human peripheral blood was collected from healthy volunteers and purified by Ficoll density gradient method. Human primary T cells (hereinafter referred to as GT cells) were separated into nuclear fractions. Grown in the presence of IL-7 and IL-15 at concentrations of 20 μg / ml and 10 μg / ml, respectively, and treated with anti-CD3 and anti-CD28 The mitochondria-exchanged T cells were derived from EPC100 and transferred to antibody-coated plates. It was produced using donor mitochondria.

[0090] The sequence of hypervariable region 1 (HVR1) in mitochondrial DNA (mtDNA) (Fig. 7) was used to distinguish between the two mtDNA species. The positions of mtDNA124 and mtDNA130 were C and C in GT cells, whereas the positions of mtDNA124 and mtDNA130 were C and C in EPC10 cells. 0, the results were T and T (Figure 8). These differences were used to analyze TaqMan qPCR single nucleotide polymorphism (SNP) genetic Probes specific to GT cells and EPC100 in the genomic typing assay encompass these points. Each has a fluorescent FAM. [ka] and fluorescent VIC [ka] The lowercase letters in the sequences represent the differences between the two types of cells. A primer set containing two PCR fragments specific to GT cells or EPC100 is also designed. The recombinant plasmid was prepared, and the recombinant plasmid was analyzed for mtDNA copy number by TaqMan qPCR. A standard curve was provided for quantification of the numbers.

[0091] MirT cells were generated according to the following protocol. On day 0, GT cells were electroporated. The mRNA encoding XbaIR was introduced by a transfection method (ATX, MaxCyte). GT cells, which are called ρ(-) cells, were treated with uridine and pyruvate. The cells were then maintained under modified conditions of the initial expansion culture by being placed in a separate container for one week. Isolated mitochondria were co-cultured with ρ(-)GT cells under growth conditions. qMan qPCR SNP genotyping assays were performed on days 9 and 14, i.e., post-mitochondrial transfer. Single-cell digital droplet PCR (sc-ddPCR) was performed on days 2 and 7, while single-cell digital droplet PCR (sc-ddPCR) was performed on days 14 and 16. That is, the experiment was carried out 7 days after the introduction of mitochondria (FIG. 10).

[0092] The SNP assay for the whole population (Figure 11) was performed using the hmtDNA D-loop (hmtD_loop-F: [ka] ; hmtHV1-R: [ka] PCR was carried out under the following conditions: [Table 3]

[0093] After initial denaturation (94°C for 2 min), the PCR reaction was subjected to 35 cycles of PCR (94°C for 30 s, 59°C for 30 s, and 68°C for 1 minute) and a final extension at 68°C for 2 minutes. The results showed that the number of sc- infected individuals accounted for half within two days of introduction and approximately 70% within seven days of introduction (Fig. 12). For ddPCR, the SNP assay is compatible with population-wide SNP assays for endogenous and exogenous mtDNA. The ratio of α-ddPCR to α-ddPCR was shown, demonstrating the robustness of the sc-ddPCR analysis (Figure 13).

[0094] Importantly, quadrant analysis revealed that nearly all cells contained endogenous or exogenous mtDNA. It was shown that the heteroplasmy was intracellular, i.e., microheteroplasmy. The results of the sc-ddPCR by the present inventors showed that the mitochondrial We found that the mitochondrial exchange cell (MirC) can bring about almost complete mtDNA exchange at the single-cell level. This clearly shows that:

[0095] Taken together, these results suggest that identifying mtDNA content at the single-cell level is possible because mtDNA is nearly We demonstrate that this technique can be used to identify completely exchanged MirC. This method can be used not only for T cells but also for stem cells, which is currently This could result in the eradication of mitochondrial diseases, for which current medical science only provides temporary treatments.

[0096] Example III (Application of sc-ddPCR to patient-derived peripheral blood) In this example, heteroplasmy in MELAS cells was detected using sc-ddPCR and FACS analysis. Show how to analyze.

[0097] A 23-year-old female MELAS patient carrying the mitochondrial A3243G mutation and a healthy donor (GJ and Peripheral blood obtained from the patients (indicated) was examined by single-cell ddPCR protocol. Following isolation of mononuclear cells, the cells were labeled with surface markers of T cell and macrophage-monocyte lineages, respectively. The cells were further sorted into CD3+ cells or CD11b+ cells.

[0098] Conventional single nucleotide polymorphism (SNP) genotyping assays using TaqMan polymerase were performed on cells The A3243G antibody was applied to the entire population, which was divided into three samples. The heteroplasmy of the mutation was 27%, while the level of A3243G in healthy controls was It was negligible (Figure 14).

[0099] Next, the sorted lineage cells and total mononuclear cells were subjected to sc-ddPCR to prevent bias in the results. The samples were divided into four groups. To improve the visibility of the sc-ddPCR results, digital data was used. was imported into FlowJo, a standard FACS instrument application software. The results, after appropriate smoothing, are plotted as quadrant contours with mutant sequences on the x-axis and healthy sequences on the y-axis. Shown as line plots (see, for example, Figures 15-18).

[0100] Control samples showed negligible A3243G heteroplasmy and no sample-to-sample variation. This is consistent with conventional SNP genotyping assays (see, e.g., Figure 15). Blood samples from MELAS patients showed that the entire mononuclear population was homoplasmic for the A3243G mutation. , healthy homoplasmy, and intracellular A3243G heteroplasmy were approximately The results show that the incidence rates of MELAS patients were 18.4%, 72.4%, and 9.1% (see, e.g., Figure 16). T cells have a lower rate of mutant A3243G homoplasmy (approximately 6.9%) compared to the overall population. ) and showed a higher ratio of healthy homoplasmy (about 88.2%) (see, e.g., Figure 17 In T cells of MELAS patients, cells with intracellular A3243G heteroplasmy were rare. (See, e.g., Figure 17) On the other hand, cells of the macrophage-monocyte lineage are present in the mononuclear population. Compared with the overall population, there was a higher rate of mutant A3243G homoplasmy (approximately 25%) and a lower rate of healthy controls. The cells showed a normal homoplasmy (approximately 62.4%) (see, for example, Figure 18). Rasmi was much higher in the macrophage-monocyte lineage compared to the overall T cell population. (See, for example, FIG. 18).

[0101] These results suggest that the A3243G heteroplasmy between cells is due to the mutant A324 These results indicated that there was a difference in the expression of 3G. Cells with mutant A3243G homoplasmy was predominant in each lineage compared to intracellular A3243G heteroplasmy, which Intracellular A3243G heteroplasmy is unstable in contrast to the stability of homoplasmy. This suggests that (Figure 19).

[0102] The above embodiments are intended to be merely exemplary, and those skilled in the art will appreciate that specific The inventors have recognized numerous equivalents of conventional compounds, materials, and procedures, or have been able to use no more than routine experimentation. All such equivalents are considered to be within the scope of this invention. , are encompassed by the appended claims. The present application provides the following aspects of the invention. (Aspect 1) To detect or monitor the presence of mitochondrial DNA (mtDNA) heteroplasmy 1. A method of: (a) obtaining or having obtained a biological sample containing one or more single cells; (b) determining the sequence of intracellular mtDNA in the one or more single cells; (c) wild-type and mutant forms of the intracellular mtDNA sequence in the one or more single cells. determining the ratio of; and (d) Cells in the arrangement of intracellular mtDNA between the one or more single cells and within the one or more single cells The inter- and / or intracellular variation is calculated, thereby determining the heteroplasmy of mtDNA in the sample. determining. (Aspect 2) Use in diagnosing a mitochondrial-related disease or disorder in a subject based on the method 1. A method for: (a) obtaining or having obtained from the subject a biological sample comprising one or more single cells; ; (b) determining the sequence of intracellular mitochondrial DNA (mtDNA) in the one or more single cells. ; (c) wild-type and mutant forms of the intracellular mtDNA sequence in the one or more single cells. determining the ratio of; (d) Cells in the arrangement of intracellular mtDNA between the one or more single cells and within the one or more single cells The inter- and / or intracellular variation is calculated, thereby determining the heteroplasmy of mtDNA in the sample. To decide; and (e) if mtDNA heteroplasmy is present in the sample, a mitochondrial-related disease or diagnosing the subject as having or suspected of having the disorder. Law. (Aspect 3) Mitochondrial disease or disorder in subjects with or suspected of having the disease or disorder A method for monitoring the effectiveness of a treatment affecting mtDNA. : (a) administering to the subject a treatment that affects mtDNA; (b) obtaining a biological sample from the subject comprising one or more single cells; (c) determining the sequence of intracellular mtDNA in the one or more single cells; (d) wild-type and mutant forms of the intracellular mtDNA sequence in the one or more single cells. determining the ratio of; (e) Cells in the arrangement of intracellular mtDNA between the one or more single cells and within the one or more single cells The inter- and / or intracellular variation is calculated, thereby determining the heteroplasmy of mtDNA in the sample. Determining the level; and (f) determining the level of mtDNA heteroplasmy in the sample by comparing the level of mtDNA heteroplasmy obtained from a reference sample; and comparing the level of heteroplasmy of the mtDNA to the level of heteroplasmy of the mtDNA. wherein said comparing indicates the effectiveness of said treatment in said subject. (Aspect 4) The method of embodiment 3, wherein the mtDNA-affecting treatment is a cell therapy. (Aspect 5) The method of embodiment 3, wherein the mtDNA-affecting treatment is mitochondrial replacement therapy. (Aspect 6) The mtDNA-affecting treatment includes administering mitochondrial replacement cells (MirCs). 4. The method of embodiment 3. (Aspect 7) Aspects 3-6, wherein the reference sample is obtained from the same subject prior to administering the treatment to the subject. The method according to any one of the preceding claims. (Aspect 8) Stratification of patient populations with or suspected of having a mitochondrial-related disease or disorder Heteroplasmy of pathogenic mitochondrial DNA (mtDNA) mutations for use in 1. A method for identifying a threshold level of: (a) obtaining or having obtained a biological sample containing one or more single cells from a subject; (b) determining the sequence of intracellular mtDNA in the one or more single cells; (c) wild-type and mutant forms of the intracellular mtDNA sequence in the one or more single cells. determining the ratio of; (d) intercellular and / or intracellular mtDNA sequences between the one or more single cells and within the single cells; Alternatively, the amount of variation within the cells is calculated, thereby determining the level of mtDNA heteroplasmy in the sample. determining the (e) Identifying the minimum level of heteroplasmy that positively correlates with mitochondrial disease or disorder. and thereby the heteroplasmy that is manifested in the mitochondrial-related disease or disorder. determining a threshold level of (Aspect 9) calculating the amount of cell-to-cell variation in the sequence of the intracellular mtDNA between the one or more single cells; The method according to any one of embodiments 1 to 8, comprising: (Aspect 10) Calculating the amount of intracellular variation in the sequence of the intracellular mtDNA within the one or more single cells. The method according to any one of embodiments 1 to 8, comprising: (Aspect 11) Intercellular and intracellular mtDNA sequences between and within said one or more single cells The method according to any one of aspects 1 to 8, comprising calculating intracellular variation. (Aspect 12) Determining the sequence of the intracellular mtDNA in the one or more single cells is a single assay. The method according to any one of aspects 1 to 11, wherein the method is carried out in step (a). (Aspect 13) determining the sequence of the intracellular mtDNA in the one or more single cells and Determining the ratio of wild-type and mutant forms of the intracellular mtDNA sequence in a single cell 12. The method of any one of embodiments 1 to 11, wherein said determining said number of individuals is performed in a single assay. (Aspect 14) Determining the sequence of the intracellular mtDNA is performed using a quantitative polymerase chain reaction (PCR) assay. 14. The method according to any one of aspects 1 to 13, comprising: (Aspect 15) 15. The method of claim 14, wherein the quantitative PCR assay is a digital droplet PCR (ddPCR) assay. How to post. (Aspect 16) 16. The method of embodiment 14 or 15, wherein the quantitative PCR assay comprises TaqMan polymerase. (Aspect 17) 17. The method of any one of embodiments 1 to 16, wherein the one or more single cells comprise heterologous intercellular mtDNA. . (Aspect 18) 17. The method of any one of embodiments 1 to 16, wherein the one or more single cells harbor heterologous intracellular mtDNA. .

Claims

1. To detect or monitor the presence of mitochondrial DNA (mtDNA) heteroplasmy 1. A method of: (a) To determine the intracellular mtDNA sequences of multiple single cells contained in a biological sample. Thus, determining the sequences of the intracellular mtDNA in the plurality of single cells is referred to as single cell digitalization. and performing a single droplet PCR (ddPCR) on each of the plurality of single cells in a single assay. determining that each of the nucleotides is encapsulated in a single droplet of ddPCR; (b) determining the wild-type and mutant forms of the intracellular mtDNA sequence in the plurality of single cells; Determining the ratio; and (c) calculating intracellular variation in the sequence of intracellular mtDNA within the plurality of single cells; and Calculating the amount of cell-to-cell variation in the sequence of intracellular mtDNA among the plurality of single cells, thereby determining the heteroplasmy of mtDNA in the sample.

2. Method for obtaining data for use in diagnosing a mitochondrial-related disease or disorder in a subject The law is: (a) determining whether a single cell in a biological sample obtained from the subject is a single cell; determining the sequence of intracellular mtDNA (mtDNA) in the plurality of single cells; Sequencing can be performed using single-cell digital droplet PCR (ddPCR) in a single assay. wherein each of the plurality of single cells is encapsulated in a single droplet of ddPCR. determining that: (b) determining the wild-type and mutant forms of the intracellular mtDNA sequence in the plurality of single cells; Determining the ratio; and (c) calculating intracellular variation in the sequence of intracellular mtDNA within the plurality of single cells; and Calculating the amount of cell-to-cell variation in the sequence of intracellular mtDNA among the plurality of single cells, thereby determining the heteroplasmy of mtDNA in the sample; If mtDNA heteroplasmy is present in the sample, the subject is diagnosed with a mitochondrial-associated disease. The method further comprises the step of determining whether the individual has or is suspected of having a disease or disorder.

3. Mitochondrial disease or disorder in subjects with or suspected of having the disease or disorder Obtaining data to monitor the effectiveness of treatments affecting mtDNA 10. A method comprising: (a) the sequences of intracellular mtDNA in a plurality of single cells contained in a biological sample obtained from the subject; wherein the subject is undergoing a treatment that affects mtDNA, and the plurality of Determining the sequence of intracellular mtDNA in single cells is a promising approach for single-cell digital droplet detection. and performing a single-cell PCR (ddPCR) in a single assay, wherein each of the plurality of single cells is subjected to ddPCR. determining that the compound is encapsulated in a single droplet of the compound; (b) determining the wild-type and mutant forms of the intracellular mtDNA sequence in the plurality of single cells; Determining the ratio; and (c) calculating intracellular variation in the sequence of intracellular mtDNA within the plurality of single cells; and Calculating the amount of cell-to-cell variation in the sequence of intracellular mtDNA among the plurality of single cells, thereby determining the level of mtDNA heteroplasmy in the sample; The level of mtDNA heteroplasmy in the sample was determined by comparing the mtDNA heteroplasmy obtained from a reference sample. A change in the level of mtDNA heteroplasmy compared to the level of mtDNA heteroplasmy is The method further comprises demonstrating the effectiveness of said treatment in

4. The method of claim 3, wherein the mtDNA-affecting treatment is a cell therapy.

5. The method of claim 3, wherein the treatment affecting mtDNA is mitochondrial replacement therapy.

6. The mtDNA-affecting treatment includes administering mitochondrial replacement cells (MirCs). The method of claim 3.

7. 3. The method of claim 2, wherein the reference sample is obtained from the same subject prior to administering the treatment to the subject. The method described in any one of items 1 to 6.

8. Stratification of patient populations with or suspected of having a mitochondrial-related disease or disorder Heteroplasmy of pathogenic mitochondrial DNA (mtDNA) mutations for use in 1. A method for identifying a threshold level of: (a) the sequences of intracellular mtDNA in a plurality of single cells contained in a biological sample obtained from the subject; determining the sequence of intracellular mtDNA in the plurality of single cells. The present invention relates to a method for detecting single-cell digital droplet PCR (ddPCR) in a single assay. Each of the plurality of single cells is encapsulated in a single droplet of ddPCR. and; (b) determining the wild-type and mutant forms of the intracellular mtDNA sequence in the plurality of single cells; Determining ratios; (c) calculating intracellular variation in the sequence of intracellular mtDNA within the plurality of single cells; and Calculating the amount of cell-to-cell variation in the sequence of intracellular mtDNA among the plurality of single cells, thereby Determining the level of mtDNA heteroplasmy in the sample; and (d) The minimum level of mtDNA heteroplasmy that positively correlates with a mitochondrial disease or disorder. and identifying mtDNA heterogeneity that is manifested in the mitochondrial-related disease or disorder. determining a threshold level of plasminogen activator.

9. The method of any one of claims 1 to 8, wherein the ddPCR assay comprises TaqMan polymerase. Law.

10. The method of any one of claims 1 to 9, wherein the plurality of single cells comprises heterologous intercellular mtDNA. Law.

11. The method of any one of claims 1 to 9, wherein the plurality of single cells has heterologous intracellular mtDNA. Law.

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