Methods for detecting macroheteroplasmy and microheteroplasmy in mitochondrial DNA.

Abstract

The present disclosure provides methods for detecting macroheteroplasmy and / or microheteroplasmy in mitochondrial DNA. The methods may include detecting or monitoring the presence of heteroplasmy and / or identifying a threshold level of heteroplasmy. Furthermore, the methods can be used to diagnose mitochondrial-related diseases or disorders in subjects with or suspected of having heteroplasmy, as well as to monitor the effectiveness of treatments that affect mitochondrial DNA (mtDNA).

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, the entire contents of which are incorporated herein by reference.

[0002] (2. Sequence Listing) This application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created on October 21, 2020, is named 14595-002-228_Sequence_Listing.txt and is 6,264 bytes in size.

[0003] 3. FIELD OF THE INVENTION The present invention provides methods for detecting macro- and / or micro-heteroplasmy in mitochondrial DNA. [Background technology]

[0004] 4. BACKGROUND OF THE INVENTION Mitochondrial diseases are a group of genetically heterogeneous disorders characterized by mitochondrial dysfunction. Currently, there are no treatment options for patients with mitochondrial diseases, leaving them with only palliative care to alleviate their symptoms (Gorman, GS et al., "Mitochondrial diseases." Nat Rev Dis Primers 2, 16080, doi:10.1038 / nrdp.2016.80 (2016)).

[0005] Due to the symbiosis of mitochondrial precursors with eukaryotes, alphaproteobacteria, and archaea, respectively, numerous mitochondrial genes have been transferred to the nuclear genome and evolved simultaneously, resulting in a 200,000-fold expansion in gene number (Lane, N. and Martin, W., "The energetics of genome complexity." Nature 467, 929-934, doi:10.1038 / nature09486 (2010)).

[0006] Mitochondrial diseases can manifest from mutations in genes in nuclear DNA (nDNA) and / or mitochondrial DNA (mtDNA) that encode mitochondrial structural proteins or proteins involved in mitochondrial function, can affect any organ, can occur at any age, and can range in severity (Lightowlers, RN, Taylor, RW, and Turnbull, DM, "Mutations causing mitochondrial disease: What is new and what challenges remain?" Science 349, 1494-1499 (2015)). Phenotypic variation resulting from pathogenic mutations in mtDNA can be attributed to multiple copies of mtDNA. Each cell has multiple copies of the mitochondrial genome, ranging from 100,000 in unfertilized oocytes to approximately 100 in sperm (Stewart, JB and Chinnery, PF, “The dynamics of mitochondrial DNA heteroplasmy: implications for human health and disease.” Nat Rev Genet 16, 530–542, doi:10.1038 / nrg3966 (2015)). Women with nuclear mutations that cause mitochondrial disease have several options for having children, including prenatal and preimplantation genetic testing, but these options are not a solution for all women.Furthermore, individuals with mitochondrial DNA pathogenic mutations face many different challenges because genetic bottlenecks and relaxed mtDNA replication result in highly complex inheritance patterns (Wai, T., Teoli, D., and Shoubridge, EA, "The mitochondrial DNA genetic bottleneck results from replication of a subpopulation of genomes." Nature Genetics 40, 1484-1488, doi:10.1038 / ng.258 (2008); Chinnery, PF, and Samuels, DC, "Relaxed replication of mtDNA: a model with implications for the expression of disease." The American Journal of Human Genetics 64, 1158-1165 (1999)).

[0007] The mixing of mutant and wild-type genomes is termed heteroplasmy (Holt, IJ, Harding, AE, and Morgan-Hughes, JA, "Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies." Nature 331, 717-719, doi:10.1038 / 331717a0 (1988)). It has been observed that the severity of mitochondrial disease often correlates with the level of heteroplasmy when protein-coding genes are mutated. Although low proportions of the mitochondrial genome carrying pathogenic mutations are widespread, there appears to be a typical 60-80% threshold for the manifestation of biochemical dysfunction (Stewart, JB, and Chinnery, PF, "The dynamics of mitochondrial DNA heteroplasmy: implications for human health and disease." for human health and disease.” Nat Rev Genet 16, 530–542, doi:10.1038 / nrg3966 (2015)). For example, heteroplasmy below 70% typically does not result in the clinical phenotype of the mitochondrial disease NARP (neurogenic muscular weakness, ataxia, and retinitis pigmentosa), which is caused by mutations in MT-ATP6. In contrast, when heteroplasmy ranges from 70% to 90%, NARP becomes symptomatic and can remain stable into adulthood.Extreme heteroplasmy (e.g., greater than 90% heteroplasmy) results in Leigh syndrome (Tatuch, Y. et al., "Heteroplasmic mtDNA mutation (T----G) at 8993 can cause Leigh disease when the percentage of abnormal mtDNA is high." American Journal of Human Genetics 50, 852-858 (1992)).

[0008] However, heteroplasmy cannot explain the variability of all mitochondrial diseases. For example, mtDNA mutations in tRNA genes show high clinical variability, which cannot be explained by heteroplasmy (Nunnari, J. and Suomalainen, A., "Mitochondria: in sickness and in health." Cell 148, 1145-1159, doi:10.1016 / j.cell.2012.02.035 (2012)). Furthermore, conventional methods for mitochondrial heteroplasmy cannot distinguish between cell-to-cell homogeneous populations, which are composed of cells with similar intracellular heterogeneity for mtDNA, and cell-to-cell heterogeneous populations, which are composed of cells with different ratios of mutant mtDNA.

[0009] Therefore, there is a great unmet need to develop improved methods for determining heteroplasmy in single cells. Summary of the Invention

[0010] (5. SUMMARY OF THE INVENTION) In one aspect, provided herein is a method for detecting or monitoring the presence of mitochondrial DNA (mtDNA) heteroplasmy, 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) determining the ratio of wild-type and mutant forms of the intracellular mtDNA sequence in the one or more single cells; and (d) calculating the amount of cell-to-cell and / or intracellular variation in the sequences of intracellular mtDNA between the one or more single cells and within the one or more single cells, thereby determining mtDNA heteroplasmy in the sample. In another aspect, provided herein is a method for use in diagnosing a mitochondrial-related disease or disorder in a subject based on the method, 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) determining the ratio of wild-type and mutant forms of the intracellular mtDNA sequence in the one or more single cells; (d) calculating the amount of cell-to-cell and / or intracellular variation in the sequence of the cellular mtDNA between the one or more single cells and within the one or more single cells, thereby determining the heteroplasmy of mtDNA in the sample; and (e) diagnosing the subject as having or suspected of having a mitochondrial-related disease or disorder if mtDNA heteroplasmy is present in the sample.

[0011] In yet another aspect, provided herein is a method for monitoring the effectiveness of a treatment affecting mitochondrial DNA (mtDNA) in a subject having or suspected of having a mitochondrial-associated disease or disorder, comprising: (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) determining the ratio of wild-type and mutant forms of the intracellular mtDNA sequence in the one or more single cells; (e) calculating the amount of cell-to-cell and / or intracellular variation in the sequence of intracellular mtDNA between the one or more single cells and within the one or more single cells, thereby determining the level of mtDNA heteroplasmy in the sample; and (f) comparing the level of mtDNA heteroplasmy in the sample with the level of mtDNA heteroplasmy obtained from a reference sample, wherein a change in the level of mtDNA heteroplasmy indicates the effectiveness of the treatment in the subject.

[0012] In some embodiments, the treatment that affects mtDNA is mitochondrial replacement therapy.In some embodiments, the treatment that affects mtDNA is mitochondrial replacement therapy.In specific embodiments, the treatment that affects mtDNA comprises administering mitochondrial replacement cells (MirC).In some embodiments, the reference sample is obtained from the same subject before the subject is administered treatment.

[0013] In another aspect, provided herein is a method for identifying a threshold level of heteroplasmy for a pathogenic mitochondrial DNA (mtDNA) mutation for use in stratifying a patient population having or suspected of having a mitochondrial-related disease or disorder, 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) determining the ratio of wild-type and mutant forms of the intracellular mtDNA sequence in the one or more single cells; (d) calculating the amount of cell-to-cell and / or intracellular variation in the sequence of the cellular mtDNA between the one or more single cells and within the one or more single cells, thereby determining the level of mtDNA heteroplasmy in the sample; and (e) identifying a minimum level of heteroplasmy that positively correlates with a mitochondrial disease or disorder, thereby determining a threshold level of said heteroplasmy that becomes manifest in said mitochondrial-associated disease or disorder.

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

[0015] In some embodiments of any of the methods provided herein, the method comprises calculating the amount of intracellular variability in the sequence of the intracellular mtDNA within the one or more single cells.

[0016] In some embodiments of any of the methods provided herein, the method includes calculating the amount of cell-to-cell and intracellular variability in the sequence of the intracellular mtDNA between or within the one or more single cells.

[0017] In certain embodiments, determining the sequence of the intracellular mtDNA in the one or more single cells is performed in a single assay. In certain embodiments, 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 sequence of the intracellular mtDNA in the one or more single cells are performed in a single assay.

[0018] In some embodiments, determining the sequence of the intracellular mtDNA comprises quantitative polymerase chain reaction (PCR) assay. In specific embodiments, the quantitative PCR assay is digital droplet PCR (ddPCR) assay. In some embodiments, the quantitative PCR assay comprises TaqMan polymerase.

[0019] In some embodiments, the one or more single cells have uniform intercellular mtDNA. In other embodiments, the one or more single cells have heterogeneous intracellular mtDNA. In some embodiments, the one or more single cells have heterogeneous 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 herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used in this specification and in the experimental procedures described below is widely known and commonly used in the relevant field.

[0040] As used herein, the term "mitochondrial replacement cells" or MirCs is intended to refer to cells in which endogenous mitochondria and / or mtDNA have been replaced with exogenous mitochondria and / or mtDNA. For example, exemplary mitochondrial replacement cells (MirCs) involve replacing endogenous mtDNA encoding dysfunctional mitochondria, e.g., mtDNA originating from a subject with a mitochondrial disease or disorder, with exogenous mtDNA encoding functional mitochondria, e.g., mtDNA originating from a healthy subject. Also included among exemplary MirCs are cells in which endogenous mitochondria have been replaced with exogenous mitochondria. However, it will be understood that replacement of endogenous mitochondria and / or mtDNA can also include, for example, replacing functional endogenous mtDNA from one cell, e.g., an older cell, with functional exogenous mtDNA from a different cell, e.g., a healthier cell obtained from a younger subject. It will further be understood that healthy endogenous mitochondria and / or mtDNA may be replaced with dysfunctional exogenous mitochondria and / or exogenous mtDNA, e.g., to mimic a mitochondrial disease or disorder.

[0041] As used herein, the terms "treat," "treating," and "treatment" refer to reducing the severity, progression, spread, and / or frequency of symptoms, eliminating symptoms and / or their underlying causes, preventing the onset of symptoms and / or their underlying causes, and ameliorating or correcting damage. "Treatment" is meant to include therapeutic treatment as well as preventative or suppressive measures for a disease, disorder, or disorder.

[0042] As used herein, the term "agent," when used in reference to the removal or reduction of mtDNA, refers to an enzyme or compound that can reduce mtDNA. Preferred agents include restriction enzymes, such as XbaI, that cleave mtDNA at one or more sites and do not cause toxicity in recipient cells. However, agents can also include enzymes or compounds that inhibit mtDNA synthesis or selectively promote mitochondrial degradation.

[0043] The terms "lower" or "reduce," as those terms are defined herein, generally refer to a decrease of at least 5% compared to a reference level, e.g., 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 at least about 90%, or any decrease between 5% and 99%. As used herein, it is understood that a partial decrease, or an agent that partially reduces endogenous mtDNA, or a decrease, does not result in complete elimination of all endogenous mtDNA (i.e., ρ0 cells). The term "increase," as used herein, generally refers to an increase of at least 5%, e.g., 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 at least about 90%, or an increase of more than 90%.

[0044] As used herein, the term "endogenous" refers to having or originating from within. For example, endogenous mitochondria are mitochondria that are native to a cell.

[0045] As used herein, the term "foreign" refers to cellular material (e.g., mitochondria or mtDNA) that is not native to the host, e.g., cellular material that originates from an external source. "Exogenous" typically means from a different source. For example, a mitochondrial genome is foreign to a host cell or host mitochondrion if it originates from a different cell type or a different species than the host cell or host mitochondrion. Additionally, "foreign" can refer to a mitochondrial genome that has been removed from a mitochondrion, manipulated, and returned to the same mitochondrion.

[0046] As used herein, the term "majority" is intended to mean the largest amount relative to other amounts being compared. When comparing two groups, an exemplary majority is an amount greater than about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more, or about 95% or more of the total population, including any integer therebetween. It will be understood that the majority will vary depending on the total population being compared, and may be an amount less than 50% when more than two groups are being compared.

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

[0048] As used herein, the term "effective amount" refers to an amount of a composition of the present invention effective to regulate, treat, or ameliorate any disease or disorder associated with heteroplasmy and / or mitochondrial dysfunction. Thus, an effective amount can include, for example, a therapeutically effective amount, which refers to a therapeutically effective amount or a biologically effective amount, which refers to an amount effective for a biological effect. The terms "therapeutically effective amount" and "effective amount" can encompass an amount that improves overall treatment, reduces or avoids the symptoms or causes of a disease or disorder, or enhances the therapeutic effect of another therapeutic agent. The amount of a given composition that corresponds to such an amount will vary depending on various factors, such as the given composition, pharmaceutical formulation, route of administration, type of disease, disorder, or disorder, and the identity of the subject or host being treated, but can nevertheless be routinely determined by one of ordinary skill in the art. Therapeutically effective amounts of agents as defined herein can be readily determined by one of ordinary skill in the art using routine methods known in the art.

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

[0050] As used herein, the term "autoimmune disease" is intended to mean a disease or disorder resulting from an immune response directed against an individual's own tissues or organs, or a disease manifesting or resulting therefrom. Autoimmune disease may refer to a disease caused by or exacerbated by the production of autoantibodies reactive with autoimmune antigens or epitopes thereof. Autoimmune diseases may be tissue-specific or organ-specific, or may be systemic. Systemic autoimmune diseases include connective tissue diseases (CTDs), such as systemic lupus erythematosus (lupus, SLE), mixed connective tissue disease, systemic sclerosis, polymyositis (PM), dermatomyositis (DM), and Sjögren's syndrome (SS). Further exemplary autoimmune diseases include rheumatoid arthritis and antineutrophil cytoplasmic antibody (ANCA) polyangiitis.

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

[0052] The term "cancer" as used herein includes, but is not limited to, solid tumors and hematological cancers. The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0053] As used herein, the terms "mitochondrial disease or disorder" and "mitochondrial disorder" are used interchangeably and refer to a group of diseases resulting from inherited or acquired damage to mitochondria, which causes energy deficiency in these body regions. Exemplary organs affected by mitochondrial disease or disorder include organs that consume large amounts of energy, such as the liver, muscles, brain, eyes, ears, and heart. The results are often liver failure, muscle weakness, fatigue, and problems with the heart, eyes, and various other systems.

[0054] As used herein, the term "mitochondrial DNA abnormality" refers to a mutation in a mitochondrial gene whose product is localized in mitochondria and is not observed in cells of healthy subjects. Exemplary diseases associated with mitochondrial DNA abnormalities include chronic progressive external ophthalmoplegia (CPEO), Pearson syndrome, Kearns-Sayre syndrome (KSS), diabetes and deafness (DAD), Leber hereditary optic neuropathy (LHON), LHON with neuropathy, ataxia, and retinitis pigmentosa syndrome (NARP), maternally inherited Leigh syndrome (MILS), also known as Leigh syndrome due to mutant mtDNA, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), and myoclonic epilepsy with ragged-red fibers (MERRF), familial bilateral striatal necrosis / striatonigral degeneration (FBSN), Luft disease, aminoglycoside-induced hearing loss (AID), and multiple mitochondrial DNA deletion syndromes.

[0055] As used herein, the term "nuclear DNA abnormality" in the context of a mitochondrial disease or disorder refers to a mutation or alteration 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 encephalomyopathy (MNGIE), mitochondrial DNA depletion syndrome (MTDPS), DNA polymerase gamma (POLG)-associated disorders, sensory ataxia neuropathy dysarthria ophthalmoplegia (SANDO), leukoencephalopathy with brainstem and spinal cord involvement, and elevated lactate level (LBSL), coenzyme Q10 deficiency, Leigh syndrome (caused by nuclear mutations), mitochondrial complex disorders, fumarase deficiency, α-ketoglutarate dehydrogenase complex (KGDHC) deficiency, succinyl-CoA ligase deficiency, pyruvate dehydrogenase complex deficiency (PDHC), pyruvate carboxylase deficiency (PCD), carnitine palmitoyltransferase I (CPT I) deficiency, carnitine palmitoyltransferase II (CPT II) deficiency, and others. 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 (GRACILE), and Charcot-Marie-Tooth disease type 2A (CMT2A).

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

[0057] As used herein, the term "mutation" refers to any change in the structure of a gene, resulting in a variant (also referred to as a "mutant") form. A genetic mutation can be caused by a single base change in DNA, or by the deletion, insertion, or rearrangement of larger portions of a gene or chromosome. In some embodiments, a mutation can affect the function or the resulting protein. For example, a mutation of a single nucleotide in DNA in the coding region of a protein (i.e., a point mutation) can result in a codon that codes for a different amino acid (i.e., a missense mutation). It will be understood that this different amino acid can alter the structure of the protein and, in certain circumstances described herein, can alter the function of an organelle, such as mitochondria.

[0058] As used herein, the terms "heteroplasmy" and "heteroplasmic" refer to the occurrence of two or more types of mitochondrial DNA genomes in an individual or sample. Varying degrees of heteroplasmy are associated with varying degrees of physiological conditions described herein. Heteroplasmy can be identified by means known in the art, and the severity of a physiological condition associated with a particular nucleotide allele is expected to vary with the percentage of the associated allele within such an individual.

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

[0060] As used herein, the terms "about" or "approximately," when used in conjunction with a number, refer to any number within 1, 5, 10, 15, or 20% of the referenced number.

[0061] The practice of the embodiments provided herein will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, and immunology that are within the skill of those in the art, and such techniques are fully explained in the literature.Examples of particularly suitable textbooks for reference are: Sambrook et al., Molecular Cloning: A Laboratory 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, Volumes I and II (1985); Gait, ed., Oligonucleotide Synthesis (1984); Hames and Higgins, eds., Nucleic Acid Hybridization (1984); Hames and Higgins, eds., Transcription and Translation (1984); Freshney, ed., Animal Cell Culture: Immobilized Cells and Enzymes (IRL Press, 1986); Kallen et al., Plant Molecular Biology - A Laboratory Manual (Melody S. Clark, ed.; Springer-Verlag, 1997); Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, Protein Purification: Principles and Practice (Springer Verlag, NY, 2nd ed., 1987); and Weir and Blackwell, eds., Handbook of Experimental Immunology, vols. I-IV (1986).

[0062] (7.2 Institutional Insights) Heteroplasmy in dividing or non-dividing cells changes over time through various mechanisms, which influences the onset and progression of disease. One proposed mechanism for altering heteroplasmy is asexual segregation, in which proliferating mitochondria are randomly and unevenly distributed among daughter cells, and the ratio of wild-type mtDNA to mutant mtDNA is sometimes biased toward the former or the latter due to random genetic drift (Birky, CW, Maruyama, T., and Fuerst, P., "An Approach To Population And Evolutionary Genetic Theory For Genes In Mitochondria And Chloroplasts, And Some Results." Genetics 103, 513 (1983)). Another is relaxed replication. In this model, mitochondria are randomly selected, replicate, and destroy independently of the host cell cycle, and their genomes are tightly regulated to replicate in sync with cell division (see Birky Jr., C., "Relaxed and stringent genomes: why cytoplasmic genes don't obey Mendel's laws." Journal of Heredity 85, 355-365 (1994)). These neutral genetic drifts are subject to positive or negative selection pressures.Long deletion mutations in mtDNA that result in pathogenic consequences exhibit a replicative advantage and outnumber wild-type genomes (Clark, KA et al., "Selfish little circles: transmission bias and evolution of large deletion-bearing mitochondrial DNA in Caenorhabditis briggsae nematodes." PLoS One 7, e41433, doi:10.1371 / journal.pone.0041433 (2012)). Given that wild-type mtDNA is quantitatively regulated in individual cells, the occurrence of mutations in mtDNA promotes mitochondrial proliferation, regardless of the presence or absence of mutations in mtDNA. This has been termed the wild-type maintenance theory (Durham, SE, Brown, DT, Turnbull, DM and Chinnery, PF, "Progressive depletion of mtDNA in mitochondrial myopathy." Neurology 67, 502-504 (2006)), where positive selection enhances the replicative advantage of mutations.Conversely, undesirable biochemical features of pathogenic mutations reduce the viability of hematopoietic stem or progenitor cells and lead to the loss of mtDNA mutations in the blood (Rajasimha, HK, Chinnery, PF, and Samuels, DC, "Selection against pathogenic mtDNA mutations in a stem cell population leads 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] In extreme cases of mitochondrial disease, mtDNA maintenance defects caused by mutations in 20 nuclear genes result in a number of diseases based on the resulting mtDNA depletion or multiple mtDNA deletions (El-Hattab, AW, Craigen, WJ, and Scaglia, F., "Mitochondrial DNA maintenance defects." Biochim Biophys Acta Mol Basis Dis 1863, 1539-1555, doi:10.1016 / j.bbadis.2017.02.017 (2017)). Targets involved in specific, post-differentiated cells, such as neurons and skeletal muscle, are not proliferating cells, which have up to 1,000-fold higher nucleotide pools in their cytoplasm than, for example, hematopoietic cells and fibroblasts (Gorman, GS et al., "Mitochondrial diseases." Nat Rev Dis Primers 2, 16080, doi:10.1038 / nrdp.2016.80 (2016)). Considering the treatment of mitochondrial diseases, specific molecularly targeted anti-cancer drugs will require the development of companion diagnostics to assist in determining indications (Jorgensen, JT, "Companion and complementary diagnostics: clinical and regulatory perspectives." Trends in cancer 2, 706-712 (2016)).

[0064] Recent advances in mitochondrial biomarkers have led to the development of fibroblast growth factor 21 (FGF21), which has a sensitivity and specificity of 92% (Suomalainen, A. et al., "FGF-21 as a biomarker for muscle-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., "Growth differentiation factor 15 as a useful biomarker for mitochondrial disorders." Ann Neurol 78, While the efficacy of mitochondrial heteroplasmy (MtDNA heteroplasmy) has been established, the reacquisition of wild-type mtDNA and the elimination of mutation burden in affected cells should be the optimal indicators of therapeutic success. Heteroplasmy should be useful not only for diagnosing mitochondrial diseases but also for estimating the effectiveness of therapeutic processes.

[0065] 7.3 Intercellular vs. intracellular heteroplasmy Mitochondrial genotypic heterogeneity can be further defined as microheteroplasmy, where intracellular mutations in mtDNA exist within a single cell, in addition to differences in mtDNA copy number between individual cells, and macroheteroplasmy, where intercellular mutations in mtDNA exist between putatively identical cells (Aryaman, J., Johnston, I.G., and Jones, N.S., "Mitochondrial Heterogeneity." Front Genet 9, 718, doi:10.3389 / fgene.2018.00718 (2018)). Microheteroplasmy can give rise to macroheteroplasmy, while macroheteroplasmy can result in the differentiation of distinct cell populations that are homogeneous with respect to mtDNA carrying wild-type or mutant genomes.

[0066] Microheteroplasmy arises from replication errors rather than oxidative damage (Kauppila, JH & Stewart, JB "Mitochondrial DNA: Radically free of free-radical driven mutations." Biochim Biophys Acta 1847, 1354-1361, doi:10.1016 / j.bbabio.2015.06.001 (2015)). Although sequencing of single mitochondria in neurons has suggested that there may be mechanisms of negative selection that neutralize microheteroplasmy, some mutations have been shown to dominate over 90% of single cells, suggesting the existence of mechanisms to circumvent negative selection (Morris, J. et al., "Pervasive intra-Mitochondrion Single-Nucleotide Variant Heteroplasmy as Revealed by Single-Mitochondrion Sequencing." Cell Rep 21, 2706-2713, doi:10.1016 / j.celrep.2017.11.031 (2017)). The extent to which widespread mutations can be achieved and whether these mechanisms can result in pathological outcomes remain open questions.

[0067] On the other hand, macroheteroplasmy can arise through neutral genetic drift (Wonnapinij, P., Chinnery, PF, and Samuels, DC, "The distribution of mitochondrial DNA heteroplasmy due to random genetic drift." Am J Hum Genet 83, 582-593, doi:10.1016 / j.ajhg.2008.10.007 (2008)) and represents a high level of mutations associated with disease (Rossignol, R. et al., "Mitochondrial threshold effects." The Biochemical journal 370, 751-762, doi:10.1042 / BJ20021594 (2003)).Although variation in macroheteroplasmy due to neutral genetic drift is mathematically predicted to increase with time, mitophagy rate, and network fragmentation (Aryaman, J., Bowles, C., Jones, NS, and Johnston, IG, "Mitochondrial network fragmentation modulates mutant mtDNA accumulation independently of absolute fission-fusion rates." bioRxiv, 409128, doi:10.1101 / 409128 (2018)), overall mtDNA copy number is predicted to decrease (Chinnery, PF, and Samuels, DC, "Relaxed replication of mtDNA: a model with implications for the expression of disease." The American Journal of Human Genetics 64, 1158-1165). (1999)). During development, a mitochondrial bottleneck characterized by a reduction in mtDNA copy number increases heteroplasmy fluctuations, and cells with mutational burdens above a threshold are eliminated (Cree, LM et al., "A reduction of mitochondrial DNA molecules during embryogenesis explains the rapid segregation of genotypes." Nat Genet 40, 249-254, doi:10.1038 / ng.2007.63 (2008)). Children with high pathogenic heteroplasmy fluctuations may develop mitochondrial diseases or disorders, but there does not appear to be a direct correlation with mitochondrial dysfunction later in life.The accumulation of mutations in mtDNA can be observed not only in neurodegenerative diseases, Parkinson's disease, but also in the physiological aging process (Bender, A. et al., "High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson's disease." Nat Genet 38, 515-517, doi:10.1038 / ng1769 (2006)).

[0068] Mitophagy is activated by various mitochondrial stresses related to mutational burden, such as nutrient deprivation, hypoxia, and oxidative stress (Wei, H., Liu, L., and Chen, Q., "Selective removal of mitochondria via mitophagy: distinct pathways for different mitochondrial stresses." Biochim Biophys Acta 1853, 2784-2790, doi:10.1016 / j.bbamcr.2015.03.013 (2015)). Mitochondrial dynamics, fusion, and fission play essential roles in maintaining mitochondrial function and quality (Sebastian, D., Palacin, M., and Zorzano, A., "Mitochondrial Dynamics: Coupling Mitochondrial Fitness 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 and are preferentially degraded by mitophagy (Twig, G. et al., "Fission and selective fusion govern mitochondrial segregation and elimination by autophagy." The EMBO Journal 27, 433-446, doi:10.1038 / sj.emboj.7601963 (2008)).In mitochondrial diseases and aging, mitophagy promotes mtDNA turnover, increasing and maintaining fluctuations in heteroplasmy, without increasing the negative pressure caused by mutational load (Aryaman, J., Johnston, I.G., and Jones, N.S., "Mitochondrial Heterogeneity." Front Genet 9, 718, doi:10.3389 / fgene.2018.00718 (2018)). Estimation of mutational load has thus far been performed on bulk cell samples, such as peripheral blood and skeletal muscle biopsies. Such measurements cannot distinguish between microheteroplasmy and macroheteroplasmy. Understanding the cellular events associated with mutational load requires single-cell analysis of various biological processes that involve mutational load.

[0069] 7.4 Detection Systems There are many methods for detecting and genotyping DNA sequence variations, including single nucleotide polymorphisms (SNPs), which are utilized in the diagnostic fields of cancer, genetic disorders, and infectious diseases (Angulo, B., Lopez-Rios, F., and Gonzalez, D., "A new generation of companion diagnostics: cobas BRAF, KRAS, and EGFR mutation detection tests." Expert Review of Molecular Diagnostics 14, 517-524, doi:10.1586 / 14737159.2014.910120 (2014); Urata, M. et al., "High-sensitivity detection of the A3243G mutation of mitochondrial DNA by a combination of allele-specific PCR and peptide nucleic acid-directed PCR clamping." Payungporn, S., Tangkijvanich, P., Jantaradsamee, P., Theamboonlers, A., and Poovorawan, Y., "Simultaneous quantitation and genotyping of hepatitis B virus by real-time PCR and melting curve analysis." J Virol Methods 120, 131-140, doi:10.1016 / j.jviromet.2004.04.012 (2004).The refractory amplification mutation system (ARMS), based on oligonucleotides with mismatched 3'-residues relative to the template, does not efficiently extend PCR strands (Newton, C.R. et al., "Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS)." Nucleic Acids Research 17, 2503-2516, doi:10.1093 / nar / 17.7.2503 (1989)). ARMS requires only primers designed to contain 3'-matched or 3'-mismatched ends; it does not require isotopes, restriction enzymes, sequencing reactions, or special machinery. The specificity of ARMS primers depends on the sequence of the template (Huang, M.-M., Arnheim, N., and Goodman, M.F., "Extension of base mispairs by Taq DNA polymerase: Implications for single-nucleotide discrimination in PCR"). (Gibson, NJ, "The 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, "Detection of specific polymerase chain reaction product by utilizing the 5' -> 3' exonuclease activity of Thermus aquaticus DNA polymerase." Proceedings of the National Academy of Sciences 88, 7276, doi:10.1073 / pnas.88.16.7276 (1991)). Oligonucleotide probes that have both a fluorophore and a quencher at different ends and exhibit little fluorescence in the aqueous phase are digested by the 5' exonuclease activity of TaqMan polymerase during the PCR extension process, releasing fluorescence from the dissociated fluorophore. The use of different fluorophores enables multiplex detection of several types of mutations in a single reaction tube (Nurmi, J., Ylikoski, A., Soukka, T., Karp, M., and Lovgren, T., "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 extremely rare variants, a number of modifications have been made, such as CataCleave (Harvey, JJ et al., "Characterization and applications of CataCleave probe in real-time detection assays." Anal Biochem 333, 246-255, doi:10.1016 / j.ab.2004.05.037 (2004)), Scorpion-ARMS (Whitcombe, D., Theaker, J., Guy, SP, Brown, T., and Little, S., "Detection of PCR products using self-probing amplicons and fluorescence." Nature Biotechnology 17, 804-807, doi:10.1038 / 11751 (1999)) and "peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp" (Zhang, S. et al., "Ultrasensitive and quantitative detection of 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) have been applied to primer and probe combination methods. Double-stranded DNA fragments have unique melting temperatures, which are applicable to probe-based fluorescence melting curve analysis.Using probes with fluorophores and quenchers at different ends, the negative differential signal of the probe-template hybrid is plotted against temperature, yielding sequence-dependent discrimination peaks as fluorescence melting curve analysis, even when a single nucleotide is being determined (Huang, Q. et al., "Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes." PLoS One 6, e19206, doi:10.1371 / journal.pone.0019206 (2011)). While the above methodologies have improved in specificity and sensitivity, the development of single-cell-based technologies is necessary for single-cell biology. Third-generation PCR, digital PCR, based on microwell chip, water-in-oil, and microfluidic technologies, has emerged with analysis systems based on Poisson distribution for detecting target sequences (Huggett, JF et al., "The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments." Clinical Chemistry 59, 892, doi:10.1373 / clinchem.2013.206375 (2013)).Droplet digital PCR (ddPCR) using water-in-oil droplet technology demonstrated higher sensitivity for identifying rare mutations, with a sensitivity of 0.001% (Watanabe, M. et al., "Ultra-Sensitive Detection of the Pretreatment EGFR T790M Mutation in Non-Small Cell Lung Cancer Patients with an EGFR-Activating Mutation 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 using microfluidic technology that enables accurate quantification of target nucleotides with high sensitivity and specificity, e.g., detection of rare variants / SNPs (Mazaika, E. and Homsy, J. (2014) "Digital Droplet PCR: CNV Analysis and Other Applications." Curr Protoc Hum Genet 82, 7 24 21-13). In this invention, the inventors have introduced ddPCR as a general detection method for identifying heteroplasmy in single cells. This analysis reveals whether heteroplasmy based on current conventional methods is intercellular heteroplasmy, in which each cell has a different homoplasmic mtDNA, termed macroheteroplasmy, or intracellular heteroplasmy, in which each cell has a different type of mtDNA, termed microheteroplasmy (Aryaman, J., Johnston, I.G., and Jones, N.S., "Mitochondrial Heterogeneity." Front Genet 9, 718, doi:10.3389 / fgene.2018.00718 (2018)).

[0072] The detection system provided herein can be used to detect or monitor the presence of intracellular, intercellular, or both heteroplasmy. This has application for use in diagnosing mitochondrial-related diseases or disorders and monitoring the effectiveness of mitochondrial replacement therapy in patients with or suspected of having heteroplasmy. Furthermore, the detection system can be used in methods to identify threshold levels of heteroplasmy for use in stratifying patient populations as likely to respond to treatment with mitochondrial replacement cells (MirCs).

[0073] Detection of heteroplasmy at the single-cell level allows for the identification of cells with uniform intercellular mtDNA as well as single cells with heterogeneous intercellular mtDNA. Furthermore, comparison of multiple single-cell mtDNA sequences allows for the identification of heterogeneous intracellular mtDNA. Furthermore, as described above, comparison of multiple single-cell mtDNA sequences allows for the identification of threshold levels of heteroplasmy that result in mitochondrial disease or disease phenotypes and / or the stratification of patient populations as more likely to respond to treatment with mitochondrial replacement cells (MirCs).

[0074] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. While the 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 of the invention. [Example]

[0075] 8. Working Example Example I (Single-cell digital droplet PCR) This example demonstrates a method for assessing mtDNA heteroplasmy in single cells in the presence or absence of mutant mtDNA without the need for laser capture or other sorting assays.

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

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

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

[0079] Mitochondrial DNA heteroplasmy was determined by TaqMan SNP genotyping assay. Wild-type and mutant allele-specific TaqMan probes and primers were designed and produced by Thermo Fisher Scientific. Two probes were labeled with different fluorophores (FAM and VIC) and a quencher was attached to the other end. Genomic DNA was extracted from cells using NucleoSpin® Tissue (Takara Bio, Tokyo, Japan). The extracted genomic DNA (100 ng) was mixed with forward and reverse primers, probes, and TaqMan Genotyping Master Mix (Thermo Fisher Scientific) and used for quantitative PCR on a CFX connect real-time system (BioRad) under the following 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). Calibration curves were generated by the above quantitative PCR using the plasmids containing the targeted mtDNA fragments amplified for the wild-type or mutant sequences, whose copy numbers were determined. The primers used in this experiment are listed in FIG.

[0080] The sc-ddPCR system begins with encapsulating a single cell in an oil droplet, followed by PCR using a set of primers and a fluorescent probe using TaqMan polymerase with 5' to 3' exonuclease activity, which releases fluorescent material from the probe and then detects the fluorescent signal in the droplet. The PCR reaction mixture contains: 1.25 x 10 5The mixture consisted of 4 μl of resuspended cells at a concentration of 1000 cells / ml, 10 μl of 2x ddPCR supermix (Bio-Rad), wild-type and mutant allele-specific TaqMan probes at a concentration of 0.25 μM, a primer mix for the target gene at a concentration of 0.9 μM, and nuclease-free water added to a volume of 20 μl. Droplets were generated using a Bio-Rad QX200 system (Bio-Rad) according to the manufacturer's instructions. The reaction mixture was transferred to a 96-well plate (Eppendorf, Hamburg, Germany) for PCR using a thermal cycler (Bio-Rad) under the following conditions: amplification was performed at 95°C for 10 min with a standard ramp rate of 2.0°C / s, followed by 40 cycles of 94°C for 30 s and 53°C for 1 min. A final enzyme inactivation step was performed at 98°C for 10 min. The 96-well plate was transferred to a QX200 droplet reader (Bio-Rad) and the number of fluorescently positive droplets was analyzed. Each droplet was individually analyzed using a two-color detection system (configured to detect FAM and VIC). Counting the fluorescent droplets provided absolute quantification of the target mtDNA in digital form using QuantaSoft software (Bio-Rad). To ensure single-cell encapsulation, we added various numbers of target cells to the PCR reaction mix and generated droplets. 500 cells per sample were successfully encapsulated in droplets, and single-cell encapsulation was observed.

[0081] Three types of cells derived from patients with mitochondrial disease were examined in this study. The characteristics of these cells are summarized in Table 1. These primary fibroblasts were isolated from the patient's skin biopsy and established as cultured cells under the approval of the Ethics Committee of the Koinobori General Incorporated Association, a Japanese nonprofit organization for mitochondrial disease. They were also donated for research under the approval of the Kyoto Prefectural University of Medicine Research Institute Ethics Committee. BK01 was derived from a 30-year-old female patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) resulting from an A to G mutation at m3243 of tRNA for leucine. The other two fibroblasts were generated from female patients with Leigh syndrome, aged 6 and 1 year. One of the Leigh syndrome cell lines contains a T-to-C mutation at m10158, located in mitochondrially encoded NADH dehydrogenase 3 (MT-ND3) (also known as NADH dehydrogenase (ubiquinone)), which is part of respiratory chain complex I and consists of 37 nuclear-encoded and 7 mitochondrially encoded subunits. The other Leigh syndrome cell line contains a T-to-C mutation at m9185, located in mitochondrially encoded ATP synthase membrane subunit 6, which encodes ATP synthase F0 subunit 6 (MT-ATP6), a subunit of the F1F0 ATPase (also known as complex V) and consists of 14 nuclear-encoded and 2 mitochondrially encoded subunits. The proband in BK01 was identified in her mother, and the mutation in BK02 was de novo. The inheritance of BK04 was not determined.

[0082] The TaqMan single nucleotide polymorphism (SNP) assay was chosen for its simplicity and ease of use in ddPCR to determine heteroplasmy in the mtDNA of target cells. A set of primers was designed to target the region encompassing the SNP and amplify 83-bp, 100-bp, and 151-bp sequences for BK01, BK02, and BK04, respectively. To separately identify and quantify healthy genotype and mutant mtDNA that match the Cambridge Reference Sequence (CRS), two TaqMan probes were designed with a FAM or VIC fluorescent dye at the 5' end and a non-fluorescent quencher at the 3' end, combined with minor groove binding (MGB) to maximize the difference in melting temperature between the various fibroblast types. The amplified sequences were subcloned into a plasmid, and a standard line for quantifying the target sequence is shown (Figure 1). The total heteroplasmy of the three fibroblast types was 99.8%, 96.9%, and 99.7% for BK01, BK02, and BK04, respectively (Fig. 2 ). Table 2 [Table 2]

[0083] Droplets were created by using cell suspensions of various concentrations, such that they contained either a single cell or no cells: 1 x 10 per milliliter. 5A cell concentration of 1000kJ / ml was optimal (Figure 3). Normal human dermal fibroblasts (NHDFs), whose mtDNA had been sequenced and confirmed to have CRS sequences matching those in MT-ND3, MT-ATP6, and the tRNA for leucine, were used as a control. Three primer and probe sets for the mutations in m3243, m10158, and m9185 were all designed based on predicted melting temperatures between the probe and template. The PCR reaction was optimized to include an initial denaturation (95°C for 10 minutes), followed by 40 cycles of PCR (94°C for 30 seconds and 56°C for 2 minutes), with a final heating step (98°C for 10 minutes). Following PCR reaction optimization, a constant threshold line was achieved based on the proportional relationship between the number of cells with a positive signal and the number of cells loaded (Figure 4).

[0084] The inventors presented the results in a quadrant format, with the healthy signal on the Y axis and the mutant signal on the X axis. In the quadrant analysis, cells with only mutant mtDNA are shown in the lower right quadrant, cells with both mutant and healthy mtDNA are shown in the upper left quadrant, and cells with only healthy mtDNA are shown in the upper left quadrant. The lower left quadrant represents a droplet containing no cells (Figure 5). Quadrant analysis of BK01 showed that the majority of cells, 95.56%, were homoplasmic for mutant mtDNA, as plotted in the lower left, while a minority of cells contained both mutant and healthy mtDNA, as plotted in the upper right. This represents intracellular heteroplasmy (upper panel of Figure 5). Furthermore, a cell population composed exclusively 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.

[0085] Quadrant analysis revealed two distinct fractions in the upper left or lower right quadrant, but the small size of the events made it difficult to determine whether the two populations were in the upper right quadrant. Cell cycle analysis revealed that the S-phase fraction in three cell lines derived from patients with mitochondrial disease was less than half that in NHDFs. The sum of G2 / M and S phases ranged from 10 to 20% in affected fibroblasts (Figure 6). The ratio of the two fractions was nearly identical, suggesting that overlapping mtDNA contents may occupy half of the cell cycle.

[0086] This study provides a method for assessing mtDNA heteroplasmy in single cells in the presence or absence of mutant mtDNA. Previous methods for heteroplasmy in mitochondrial disease uniformly target cell populations, such as mononuclear cells and biopsied skeletal muscle, and therefore the output cannot distinguish between intracellular heteroplasmy (microheteroplasmy) and intercellular heteroplasmy (macroheteroplasmy). The threshold theory, which states that mitochondrial disease phenotypes emerge when mutant mtDNA heteroplasmy reaches 60–70% or more, is based on clinical samples. However, it is unclear whether all cells have similar heteroplasmy, whether cells with homoplasmy containing healthy or mutant mtDNA are mixed in the same ratio, or whether the former and the latter are mixed. Furthermore, whether the threshold theory can be justified at the single-cell level remains to be investigated.

[0087] Furthermore, the single-cell biology of mtDNA heteroplasmy sheds light on the way to a fuller understanding of not only mitochondrial diseases, but also neurodegenerative diseases, cancer, and aging.

[0088] Example II Analysis of Mitochondrial Exchange T Cell (MirT) Heteroplasmy Using Single-Cell Digital Droplet qPCR This example demonstrates how single-cell digital droplet qPCR can be used to analyze heteroplasmy in mitochondrial-exchanged T cells (MirTs).

[0089] Human peripheral blood was collected from healthy volunteers and separated into mononuclear fractions using a Ficoll density gradient. Human primary T cells (hereafter referred to as GT cells) were grown in the presence of IL-7 and IL-15 at concentrations of 20 μg / ml and 10 μg / ml, respectively, and transferred to plates coated with anti-CD3 and anti-CD28 antibodies. Mitochondria-exchanged T cells were generated using donor mitochondria derived from EPC100.

[0090] The sequence of hypervariable region 1 (HVR1) in mitochondrial DNA (mtDNA) (Figure 7) was examined to distinguish the two mtDNA species. Positions mtDNA124 and mtDNA130 were C and C in GT cells, whereas they were T and T in EPC100 (Figure 8). Taking advantage of these differences, probes specific to GT cells and EPC100 in TaqMan qPCR single nucleotide polymorphism (SNP) genotyping assays were designed to encompass these points, each carrying a fluorescent FAM. [ka] and fluorescent VIC [ka] The sequences were analyzed using the same primer sets (Figure 9). Lowercase letters in the sequences indicate differences between the two cell types. Primer sets containing probe regions were also designed. Two recombinant plasmids carrying PCR fragments specific to GT cells or EPC100 were constructed, and these recombinant plasmids provided a standard curve for quantification of mtDNA copy number by TaqMan qPCR.

[0091] MirT cells were generated according to the following protocol. On day 0, GT cells were transfected with mRNA encoding XbaIR by electroporation (ATX, MaxCyte). GT cells, designated ρ(-) cells, which exhibited significant mitochondrial DNA depletion, were maintained under modified initial growth culture conditions by adding uridine and pyruvate for one week. Mitochondria isolated from donors were co-cultured with ρ(-) GT cells under growth culture conditions. Whole-cell TaqMan qPCR SNP genotyping assays were performed on days 9 and 14, i.e., 2 and 7 days after mitochondrial introduction, while single-cell digital droplet PCR (sc-ddPCR) was performed on day 14, i.e., 7 days after mitochondrial introduction (Figure 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 included 35 cycles of PCR (94°C for 30 s, 59°C for 30 s, and 68°C for 1 min), with a final extension at 68°C for 2 min. The results showed that the exogenous mtDNA accounted for half of the total mtDNA at 2 days and approximately 70% at 7 days (Figure 12). For sc-ddPCR, the SNP assay showed a ratio of endogenous and exogenous mtDNA that was compatible with the population-wide SNP assay, demonstrating the robustness of the sc-ddPCR analysis (Figure 13).

[0094] Importantly, quadrant analysis showed that nearly all cells were homoplasmic, harboring either endogenous or exogenous mtDNA, but a small number of cells had intracellular heteroplasmy, or microheteroplasmy. Our sc-ddPCR results clearly demonstrate that mitochondrial exchange cells (MirC) can result in nearly complete mtDNA exchange at the single-cell level.

[0095] "Together, these results demonstrate that characterization of mtDNA content at the single-cell level can be used to identify MirCs that have undergone nearly complete mtDNA exchange. This technology can be applied not only to T cells but also to stem cells, potentially leading to the eradication of mitochondrial diseases for which current medical treatments only provide temporary cures."

[0096] Example III (Application of sc-ddPCR to patient-derived peripheral blood) This example demonstrates a method for analyzing heteroplasmy in MELAS cells using sc-ddPCR and FACS analysis.

[0097] Peripheral blood samples from a 23-year-old female MELAS patient carrying the mitochondrial A3243G mutation and a healthy donor (GJ) were analyzed using a single-cell ddPCR protocol. After isolation of mononuclear cells by density gradient centrifugation, the cells were further sorted to CD3+ or CD11b+ cells, which express surface markers of T cell and macrophage-monocyte lineages, respectively.

[0098] A conventional single nucleotide polymorphism (SNP) genotyping assay using TaqMan polymerase was applied to the whole cell population, which was divided into three samples. The heteroplasmy of the A3243G mutation in mononuclear cells from MELAS patients was 27%, while the level of A3243G in healthy controls was negligible (Figure 14).

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

[0100] Control samples showed negligible A3243G heteroplasmy and no sample-to-sample variation, consistent with conventional SNP genotyping assays (see, e.g., Figure 15). Blood samples from MELAS patients showed that, for the entire mononuclear population, approximately 18.4%, 72.4%, and 9.1% of cells had mutant A3243G homoplasmy, healthy homoplasmy, and intracellular A3243G heteroplasmy, respectively (see, e.g., Figure 16). T cells from MELAS patients showed a lower ratio of mutant A3243G homoplasmy (approximately 6.9%) and a higher ratio of healthy homoplasmy (approximately 88.2%) compared to the overall population (see, e.g., Figure 17). Cells with intracellular A3243G heteroplasmy were rare in T cells from MELAS patients (see, e.g., Figure 17). On the other hand, cells of the macrophage-monocyte lineage showed higher mutant A3243G homoplasmy (approximately 25%) and a lower ratio of healthy homoplasmy (approximately 62.4%) compared to the overall mononuclear population (see, e.g., Figure 18). Intracellular heteroplasmy was much higher in the macrophage-monocyte lineage compared to the overall T cell population (see, e.g., Figure 18).

[0101] These results indicated that there were differences in the expression of mutant A3243G between lineages with respect to intercellular A3243G heteroplasmy. Cells with mutant A3243G homoplasmy were predominant in each lineage compared with intracellular A3243G heteroplasmy, suggesting that intracellular A3243G heteroplasmy is unstable in contrast to the stability of homoplasmy (Figure 19).

[0102] The above-described embodiments are intended to be merely illustrative; those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific compounds, materials, and procedures. All such equivalents are considered to be within the scope of the invention and are encompassed by the appended claims. The present application provides the following aspects of the invention. (Aspect 1) 1. A method for detecting or monitoring the presence of mitochondrial DNA (mtDNA) heteroplasmy, 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) determining the ratio of wild-type and mutant forms of the intracellular mtDNA sequence in the one or more single cells; and (d) calculating the amount of intercellular and / or intracellular variation in the sequences of intracellular mtDNA between the one or more single cells and within the one or more single cells, thereby determining the heteroplasmy of mtDNA in the sample. (Aspect 2) 1. A method for use in diagnosing a mitochondrial-related disease or disorder in a subject based on said method, 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) determining the ratio of wild-type and mutant forms of the intracellular mtDNA sequence in the one or more single cells; (d) calculating the amount of cell-to-cell and / or intracellular variation in the sequence of the cellular mtDNA between the one or more single cells and within the one or more single cells, thereby determining the heteroplasmy of mtDNA in the sample; and (e) diagnosing the subject as having or suspected of having a mitochondrial-related disease or disorder if mtDNA heteroplasmy is present in the sample. (Aspect 3) 1. A method for monitoring the effectiveness of a treatment affecting mitochondrial DNA (mtDNA) in a subject having or suspected of having a mitochondrial-related disease or disorder, comprising: (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) determining the ratio of wild-type and mutant forms of the intracellular mtDNA sequence in the one or more single cells; (e) calculating the amount of cell-to-cell and / or intracellular variation in the sequence of the cellular mtDNA between the one or more single cells and within the one or more single cells, thereby determining the level of mtDNA heteroplasmy in the sample; and (f) comparing the level of mtDNA heteroplasmy in the sample with the level of mtDNA heteroplasmy obtained from a reference sample, wherein a change in the level of mtDNA heteroplasmy indicates the effectiveness of the treatment in the 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 method of embodiment 3, wherein said mtDNA-affecting treatment comprises administering mitochondrial replacement cells (MirCs). (Aspect 7) The method of any one of aspects 3 to 6, wherein said reference sample is obtained from the same subject prior to administering said treatment to said subject. (Aspect 8) 1. A method for identifying a threshold level of heteroplasmy for a pathogenic mitochondrial DNA (mtDNA) mutation for use in stratifying a patient population having or suspected of having a mitochondrial-related disease or disorder, 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) determining the ratio of wild-type and mutant forms of the intracellular mtDNA sequence in the one or more single cells; (d) calculating the amount of cell-to-cell and / or intracellular variation in the sequence of intracellular mtDNA between and within the one or more single cells, thereby determining the level of mtDNA heteroplasmy in the sample; and (e) identifying a minimum level of heteroplasmy that positively correlates with a mitochondrial disease or disorder, thereby determining a threshold level of heteroplasmy that is manifest in the mitochondrial-related disease or disorder. (Aspect 9) The method according to any one of aspects 1 to 8, comprising calculating the amount of cell-to-cell variation in the sequence of the intracellular mtDNA among the one or more single cells. (Aspect 10) The method according to any one of aspects 1 to 8, comprising calculating intracellular variation in the sequence of the intracellular mtDNA within the one or more single cells. (Aspect 11) The method according to any one of aspects 1 to 8, comprising calculating intercellular and intracellular variations in the sequences of the intracellular mtDNA between and within the one or more single cells. (Aspect 12) 12. The method of any one of embodiments 1 to 11, wherein determining the sequence of the intracellular mtDNA in the one or more single cells is performed in a single assay. (Aspect 13) 12. The method of any one of embodiments 1 to 11, wherein 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 sequence of the intracellular mtDNA in the one or more single cells are performed in a single assay. (Aspect 14) 14. The method of any one of embodiments 1 to 13, wherein said determining the sequence of intracellular mtDNA comprises a quantitative polymerase chain reaction (PCR) assay. (Aspect 15) 15. The method of embodiment 14, wherein said quantitative PCR assay is a digital droplet PCR (ddPCR) assay. (Aspect 16) 16. The method of embodiment 14 or 15, wherein the quantitative PCR assay comprises TaqMan polymerase. (Aspect 17) The method of any one of embodiments 1 to 16, wherein the one or more single cells have heterologous intercellular mtDNA. (Aspect 18) The method of any one of embodiments 1 to 16, wherein the one or more single cells have heterologous intracellular mtDNA.

Claims

1. 1. A method for detecting or monitoring the presence of mitochondrial DNA (mtDNA) heteroplasmy, comprising: (a) determining the sequence of intracellular mtDNA in a plurality of single cells contained in a biological sample, wherein determining the sequence of intracellular mtDNA in the plurality of single cells comprises performing 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; (b) determining the ratio of wild-type and mutant forms of the intracellular mtDNA sequence in the plurality of single cells; and (c) calculating the amount of intracellular variation in the sequences of intracellular mtDNA within the plurality of single cells, and calculating the amount of cell-to-cell variation in the sequences of intracellular mtDNA between the plurality of single cells, thereby determining the heteroplasmy of mtDNA in the sample.

2. A method for obtaining data for use in diagnosing a mitochondrial-related disease or disorder in a subject, comprising: (a) determining the sequence of intracellular mitochondrial DNA (mtDNA) in a plurality of single cells contained in a biological sample obtained from the subject, wherein determining the sequence of intracellular mtDNA in the plurality of single cells comprises performing 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; (b) determining the ratio of wild-type and mutant forms of the intracellular mtDNA sequence in the plurality of single cells; and (c) calculating intracellular variation in the sequences of intracellular mtDNA within the plurality of single cells and calculating intercellular variation in the sequences of intracellular mtDNA among the plurality of single cells, thereby determining mtDNA heteroplasmy in the sample; The method, wherein the presence of mtDNA heteroplasmy in the sample indicates that the subject has or is suspected of having a mitochondrial-related disease or disorder.

3. 1. A method of obtaining data for monitoring the effectiveness of a treatment affecting mitochondrial DNA (mtDNA) in a subject having or suspected of having a mitochondrial-related disease or disorder, comprising: (a) determining the sequence of intracellular mtDNA in a plurality of single cells contained in a biological sample obtained from the subject, wherein the subject has been treated with a treatment that affects mtDNA, and determining the sequence of intracellular mtDNA in the plurality of single cells comprises performing 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; (b) determining the ratio of wild-type and mutant forms of the intracellular mtDNA sequence in the plurality of single cells; and (c) calculating intracellular variation in the sequences of intracellular mtDNA within the plurality of single cells and calculating intercellular variation in the sequences of intracellular mtDNA among the plurality of single cells, thereby determining the level of mtDNA heteroplasmy in the sample; The method, wherein when the level of mtDNA heteroplasmy in the sample is compared with the level of mtDNA heteroplasmy obtained from a reference sample, a change in the level of mtDNA heteroplasmy indicates the effectiveness of the treatment in the subject.

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 method of claim 3, wherein the treatment affecting mtDNA comprises administering mitochondrial replacement cells (MirCs).

7. The method of any one of claims 3 to 6, wherein said reference sample is obtained from the same subject before administering said treatment to said subject.

8. 1. A method for identifying a threshold level of heteroplasmy for a pathogenic mitochondrial DNA (mtDNA) mutation for use in stratifying a patient population having or suspected of having a mitochondrial-related disease or disorder, comprising: (a) determining the sequence of intracellular mtDNA in a plurality of single cells contained in a biological sample obtained from the subject, wherein determining the sequence of intracellular mtDNA in the plurality of single cells comprises performing 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; (b) determining the ratio of wild-type and mutant forms of the intracellular mtDNA sequence in the plurality of single cells; (c) calculating the intracellular variation in the sequences of intracellular mtDNA within the plurality of single cells and calculating the intercellular variation in the sequences of intracellular mtDNA among the plurality of single cells, thereby determining the level of mtDNA heteroplasmy in the sample; and (d) identifying a minimum level of mtDNA heteroplasmy that positively correlates with a mitochondrial disease or disorder, thereby determining a threshold level of mtDNA heteroplasmy that is manifest in the mitochondrial-related disease or disorder.

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

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

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

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