A method for detecting mutant tRNA Leu caused by adenine to guanine mutation at position 3243 of mitochondrial DNA.

Abstract

To provide: a method of detecting mutant tRNA Leu caused by an adenine-to-guanine mutation at position 3243 of mitochondrial DNA; and a method of introducing mitochondrial tRNA Leu to a cell in which mutant tRNA Leu caused by an adenine-to-guanine mutation at position 3243 of mitochondrial DNA is detected.SOLUTION: Primers are found that may be advantageously used for detecting mutant tRNA Leu caused by an adenine-to-guanine mutation at position 3243 of mitochondrial DNA, and the primer set can be used to obtain the mutation rate, which is the rate of mutant tRNA Leu in all mitochondria. Also provided is a method of introducing wild-type tRNA Leu to a cell having mutant tRNA Leu.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting a mutant tRNALeu caused by a mutation from adenine to guanine at position 3243 of mitochondrial DNA. The present invention also relates to a method for introducing mitochondrial tRNALeu into a cell in which a mutant tRNALeu caused by a mutation from adenine to guanine at position 3243 of mitochondrial DNA has been detected. [Background technology]

[0002] Mitochondrial diseases are diseases caused by abnormalities in mitochondrial function. The main cause of mitochondrial diseases is mutations in mitochondrial DNA (mtDNA). One mitochondrion contains 2 to 10 copies of mtDNA, and the mitochondria of many mitochondrial disease patients contain a mixture of normal and mutant mtDNA (Non-Patent Documents 1 and 2). It is known that when the proportion of mutant mtDNA exceeds a certain threshold, it causes mitochondrial dysfunction (Non-Patent Document 3). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Bogenhagen D, Clayton DA., J Biol Chem 249, 7991-7995(1974) [Non-Patent Document 2] Holt IJ et al., J Neurol Neurosurg Psychiatry 51, 1075-1077(1988) [Non-Patent Document 3] Schapira AH., Lancet 379, 1825-1834(2006) Summary of the Invention

[0004] The present invention relates to a method for detecting a mutant tRNALeu caused by a mutation from adenine to guanine at position 3243 of mitochondrial DNA. The present invention also relates to a method for introducing mitochondrial tRNALeu into a cell in which a mutant tRNALeu caused by a mutation from adenine to guanine at position 3243 of mitochondrial DNA has been detected.

[0005] The present inventors have found primers that can be advantageously used to detect mutant tRNALeu caused by a mutation from adenine to guanine at position 3243 of mitochondrial DNA. The present inventors have found that the proportion of mutant tRNALeu in total mitochondria (mutation rate) can be determined by using this primer set. The present inventors have also found a method for introducing wild-type tRNALeu into cells having mutant tRNALeu.

[0006] According to the present invention, the following inventions can be provided. (1) An oligonucleotide having a base sequence set forth in SEQ ID NO: 12 or a part thereof, which includes the base sequence set forth in SEQ ID NO: 3 and consists of a base sequence of 15 or more consecutive bases. (2) An oligonucleotide having a base sequence set forth in SEQ ID NO: 13 or a part thereof, which includes the base sequence set forth in SEQ ID NO: 7 and consists of a base sequence of 15 or more consecutive bases. (3) A combination comprising the oligonucleotide according to claim 1 and the oligonucleotide according to claim 2. (4) A combination comprising the oligonucleotide according to claim 1 and an oligonucleotide having a base sequence capable of hybridizing to the N-terminal region of mitochondrial tRNA Leu. (5) A combination comprising the oligonucleotide according to claim 2 and an oligonucleotide having a base sequence capable of hybridizing to the N-terminal region of mitochondrial tRNA Leu. (6) A combination comprising the combination of claim 3 and an oligonucleotide having a base sequence capable of hybridizing to the N-terminal region of mitochondrial tRNA Leu. (7) A method for examining the base sequence of tRNA Leu produced by the mitochondria of a cell, comprising the steps of: A method comprising carrying out a quantitative polymerase chain reaction (quantitative PCR) using a primer set consisting of the combination according to claim 3 and a primer set consisting of the combination according to claim 4, and using RNA extracted from mitochondria of the cell as a template. (8) The method according to (7) above, further comprising determining a ratio of tRNA Leu having a wild-type base sequence and / or a tRNA having a mutant base sequence relative to tRNA Leu. (9) Lipid nanoparticles encapsulating a complex of mitochondrial tRNA Leu and a cationic polymer. (10) A pharmaceutical composition comprising the lipid nanoparticles described in (9) above. (11) The pharmaceutical composition according to (10) above for use in treating MELAS. [Brief description of the drawings]

[0007] [Figure 1] 1 shows the relationship between the theoretical value and the quantitative ratio of the template (actual value) estimated by quantitative PCR when wild-type mitochondrial tRNA Leu and mutant mitochondrial tRNA Leu due to the 3243A→G mutation were mixed at a constant ratio (theoretical value) and analyzed using the forward primer set and common reverse primer shown at the top of each graph. The primer names at the top of each graph correspond to the sequence names listed in Table 1. [Diagram 2] 2 shows a method for forming lipid nanoparticles for delivering wild-type mitochondrial tRNA Leu having the base sequence set forth in SEQ ID NO: 10 into cells. tRNA was mixed with protamine to obtain a complex (nanoparticle) of tRNA and protamine. The obtained complex of tRNA and protamine was mixed with an alcohol solution containing the described lipid, and after dilution and filtration, lipid nanoparticles (mt-tRNA Leu / MITO) encapsulating mitochondrial tRNA Leu were obtained. [Diagram 3]FIG. 3 shows the ratio of mutant mt-tRNA Leu to total mt-tRNA Leu (mutation rate) in MELAS cells 24 hours after contact with mt-tRNA Leu / MITO in a culture medium. [Figure 4] FIG. 4 shows the relationship between the amount of mt-tRNA Leu contacted with MELAS cells in a culture medium and the mutation rate of mt-tRNA Leu in the MELAS cells subjected to the treatment. [Diagram 5] FIG. 5 shows the maximum mitochondrial respiratory capacity of MELAS cells 24 hours after contact with mt-tRNA Leu / MITO in culture medium. [Figure 6] FIG. 6 shows the relationship between the amount of mt-tRNA Leu contacted with MELAS cells in a culture medium and the maximum mitochondrial respiratory capacity of the treated MELAS cells. Detailed Description of the Invention

[0008] As used herein, a "subject" is a eukaryotic organism, e.g., an animal, e.g., a mammal, e.g., a primate, e.g., a human, a dog, a cat, a mouse, a guinea pig, a hamster, a horse, a cow, a sheep, a pig, and a goat.

[0009] As used herein, "treatment" includes therapeutic treatment and prophylactic treatment. Therapeutic treatment can mean slowing or stopping the progression of, or alleviating or curing, symptoms of a disease. Prophylactic treatment can mean slowing or preventing the onset of symptoms of a disease.

[0010] As used herein, "mt-tRNA Leu" is a type of transfer RNA (tRNA). As used herein, tRNA encoded by mitochondrial DNA is referred to as mt-tRNA. As used herein, mt-tRNA Leu is a mitochondrial tRNA, and is a tRNA having an anticodon encoding leucine. mt-tRNA Leu is a type of transfer RNA (tRNA). Leumt-tRNA Leu is expressed as pre-tRNA (pre-mt-tRNA Leu) and is processed to become mature. Human mitochondrial DNA includes, for example, the base sequence registered in NCBI Reference Sequence: NC_012920.1, in which pre-mt-tRNA Leu is encoded by the region of 3182-3331, and mature mt-tRNA Leu (SEQ ID NO: 10) is encoded by the region of 3230-3304. Here, in RNA, T (thymine) in DNA is transcribed as U (uracil). In this specification, "mt-tRNA Leu" is used to include mature mt-tRNA Leu and pre-mt-tRNA Leu. mt-tRNA Leu includes mt-tRNA Leu encoded by the above base sequence, but also includes functional mt-tRNA Leu having identity to the above base sequence. Examples of functional mt-tRNA Leu having identity to the above-mentioned base sequence include functional mt-tRNA Leu having sequence identity of 90% or more, 95% or more, or 97% or more. Functional means having a normal function as a tRNA or pre-tRNA.

[0011] In this specification, "a base sequence corresponding to the 3243rd base adenine" means a base (particularly adenine) in another mitochondrial DNA that corresponds to the 3243rd base adenine in the base sequence registered in NCBI Reference Sequence: NC_012920.1. The 3243rd base is present in the DNA encoding mt-tRNA Leu in the base sequence registered in NCBI Reference Sequence: NC_012920.1. Therefore, "a base sequence corresponding to the 3243rd base adenine" means a base of mt-tRNA Leu corresponding to the 3243rd base in another mitochondrial DNA. The corresponding base can be determined, for example, by aligning the base sequences of the nucleic acid encoding mt-tRNA Leu. By the alignment, a continuous region of matching bases in two base sequences is a corresponding region, and each base in the region can be a corresponding base.

[0012] In this specification, "mitochondria" refers to an intracellular organelle of a eukaryotic cell that produces energy through oxidative phosphorylation. Mitochondrial dysfunction causes various diseases called mitochondrial diseases. Multiple mitochondria exist within a cell, and the mitochondria themselves have multiple mitochondrial genomes (mitochondrial DNA). Thus, a cell has multiple mitochondrial genomes, and each mitochondrial genome is not necessarily identical. The onset of mitochondrial diseases and the severity of symptoms are thought to be related to the proportion of mitochondrial genomes and the proportion of abnormal RNA or protein expressed from the mitochondrial genome.

[0013] In this specification, "MELAS" refers to a type of mitochondrial disease, mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes. MELAS patients are known to suffer from repeated stroke-like episodes. In 80% of patients with MELAS, the base sequence corresponding to the 3243rd base adenine is mutated to guanine. The 3243rd base adenine is a base contained in mitochondrial mt-tRNA Leu, and the mutation of this adenine to guanine reduces the function of the mt-tRNA Leu, resulting in impaired respiration and ATP production by mitochondria. It is believed that the severity of MELAS is related to the proportion of mutant mt-tRNA Leu (SEQ ID NO: 11) having a mutation of the 3243rd adenine to guanine in the total mt-tRNA Leu.

[0014] As used herein, "isolating mitochondria" means removing mitochondria from the interior of a cell to the exterior and separating them from at least one other component.

[0015] In this specification, "polymerase chain reaction" (PCR) is a type of technique for amplifying DNA. In PCR, DNA between oligonucleotides called primers, which have short base sequences designed for +strand and -strand, can be amplified. When RNA is the subject of PCR, RNA amplification can be substituted by synthesizing a DNA (cDNA) complementary to the RNA and amplifying a DNA encoding the RNA using the cDNA as a template. As the primer, an oligonucleotide having a length of about 15 to 30 mers can be used. The primer can be, for example, 15mer, 16mer, 17mer, 18mer, 19mer, 20mer, 21mer, 22mer, 23mer, 24mer, 25mer, 26mer, 27mer, 28mer, 29mer, or 30mer, and preferably 20 to 25mer (for example, 20 to 22mer, for example, 21mer). In this specification, "quantitative PCR" means a method for quantifying the amount of template present using PCR. An example of quantitative PCR is a fluorescent probe method. In the fluorescent probe method, a probe that emits fluorescence when decomposed can be used. The probe is a single-stranded DNA having a fluorescent dye and a quencher that quenches the fluorescence, has a base sequence complementary to the target PCR amplification product, and can hybridize to the amplification product. When the temperature is raised and polymerase is activated, the probe is decomposed by the exonuclease activity of the polymerase, and the fluorescent dye dissociates from the quencher, which allows the fluorescent dye to emit fluorescence. As the amplification product increases by PCR, the amount of fluorescent dye that dissociates from the quencher increases. In the probe method, this mechanism allows the initial amount of DNA to be estimated from the degree of increase in the amount of fluorescence.

[0016] As used herein, "oligonucleotide" refers to a single-stranded oligonucleotide. An oligonucleotide is a polymer of nucleotides. The nucleotides are, for example, DNA.

[0017] In the present invention, a method is provided for examining mutations in mt-tRNA Leu encoded by mitochondrial DNA, rather than examining mutations in mitochondrial DNA itself. Specifically, in the present invention, the proportion of mutant mt-tRNA Leu in mt-tRNA Leu can be determined by performing quantitative PCR using the pre-RNA of mt-tRNA Leu as a template.

[0018] A method for extracting mt-tRNA Leu from a cell can include isolating mitochondria from the cell and extracting RNA containing mt-tRNA Leu from the isolated mitochondria.

[0019] Isolation of mitochondria from cells can be appropriately carried out using well-known techniques. Isolation of mitochondria from cells can be carried out, for example, by mechanically disrupting the cells with a water stream such as homogenization, and / or chemically disrupting the cells with a surfactant. After the cells are disrupted to obtain a cell lysate, the cell debris can be precipitated, for example, by centrifugation (for example, 700×g, 4° C., 10 minutes), and the supernatant containing mitochondria can be collected. The supernatant containing mitochondria may be further purified, for example, by density gradient centrifugation. Further purification by density gradient centrifugation may further include, for example, adding the supernatant containing mitochondria to a 60% Percoll solution, centrifuging (for example, 400×g, 4° C., 15 minutes), and collecting the interface part at the center of the solution.

[0020] RNA including tRNA can be collected from the obtained mitochondria. RNA can be collected by those skilled in the art as appropriate, for example, by using a commercially available RNA extraction kit. Complementary DNA (cDNA) for mt-tRNA Leu can be synthesized from RNA. cDNA synthesis can be performed by those skilled in the art as appropriate using primers.

[0021] The method of the present invention can be carried out by synthesizing cDNA using an RNA solution extracted from isolated mitochondria as a template, and then using the obtained cDNA as a template.

[0022] Primer DNA and its set for detecting point mutations in mt-tRNA Leu First Oligonucleotide The present invention provides an oligonucleotide (first oligonucleotide) having a base sequence which is a part of the base sequence set forth in SEQ ID NO: 12, includes the base sequence set forth in SEQ ID NO: 3, and consists of a base sequence of 15 or more consecutive bases.

[0023] In the base sequence of the first oligonucleotide, the base at the 3' end is A, and the second base from the 3' end is other than G (i.e., H (not G), specifically A, T, or C, for example, C). The second base being other than G changes the annealing conditions of the primer to the template, and this allows the primer to increase selectivity for wild type and mutant type. This oligonucleotide contains a sequence complementary to the 14-mer sequence at the 3' end of mt-tRNA Leu, and further contains a longer sequence (additional sequence). The additional sequence is considered to be advantageous for binding to pre-tRNA.

[0024] The first oligonucleotide may be a polynucleotide having a length of 15 bases or more, preferably 16 bases or more, preferably 17 bases or more, preferably 18 bases or more, preferably 19 bases or more, preferably 20 bases or more, or 21 bases or more (e.g., 21 bases, 22 bases, 23 bases, 24 bases, or 25 bases, preferably 21 bases). The first oligonucleotide may bind to the pre-tRNA of wild-type mt-tRNA Leu under annealing conditions for polymerase chain reaction (PCR).

[0025] The first oligonucleotide preferably has the base sequence set forth in SEQ ID NO:4.

[0026] The first oligonucleotide is combined with a common oligonucleotide, i.e., mt-tRNA Leu under PCR conditions. 3 The first oligonucleotide can be used as a PCR primer in combination with a polynucleotide (common oligonucleotide) having a length of 15 bases or more, preferably 16 bases or more, preferably 17 bases or more, preferably 18 bases or more, preferably 19 bases or more, preferably 20 bases or more, or 21 bases or more (for example, 21 bases, 22 bases, 23 bases, 24 bases, or 25 bases, preferably 21 bases) that hybridizes to or has a sequence complementary to the DNA encoding the 3280-3331st region of the mitochondrial DNA. The common oligonucleotide has, for example, the base sequence of SEQ ID NO: 1. That is, the first oligonucleotide can be used in combination with the common oligonucleotide to amplify mt-tRNA Leu encoded by a gene in which the 3243rd position of the mitochondrial DNA is A by PCR. For example, the first oligonucleotide can be used in combination with the common oligonucleotide to amplify wild-type mt-tRNA Leu by PCR.

[0027] Second Oligonucleotide The second oligonucleotide is an oligonucleotide having a base sequence which is a part of the base sequence set forth in SEQ ID NO: 13, includes the base sequence set forth in SEQ ID NO: 7, and is composed of a base sequence of 15 or more consecutive bases.

[0028] In the base sequence of the second oligonucleotide, the base at the 3' end is G, and the second base from the 3' end is other than G (i.e., H (not G), specifically, A, T, or C, for example, C). The second base being other than G changes the annealing conditions of the primer to the template, and this allows the primer to increase selectivity for wild type and mutant type. This oligonucleotide contains a sequence complementary to the 14-mer sequence at the 3' end of mt-tRNA Leu, and further contains a longer sequence (additional sequence). The additional sequence is considered to be advantageous for binding to pre-tRNA.

[0029] The second oligonucleotide may be a polynucleotide having a length of 15 bases or more, preferably 16 bases or more, preferably 17 bases or more, preferably 18 bases or more, preferably 19 bases or more, preferably 20 bases or more, or 21 bases or more (e.g., 21 bases, 22 bases, 23 bases, 24 bases, or 25 bases, preferably 21 bases). The second oligonucleotide may bind to the pre-tRNA of mutant mt-tRNA Leu having a mutation of adenine at position 3243 to guanine under annealing conditions for polymerase chain reaction (PCR).

[0030] The second oligonucleotide preferably has the base sequence of SEQ ID NO:8.

[0031] The second oligonucleotide can be used as a PCR primer in combination with the common oligonucleotide defined above to amplify mt-tRNALeu encoded by a gene in which the 3243rd position of the mitochondrial DNA is G. That is, the second oligonucleotide can be used in combination with the common oligonucleotide to amplify, by PCR, mt-tRNALeu encoded by a gene in which the 3243rd position of the mitochondrial DNA is G. For example, the second oligonucleotide can be used in combination with the common oligonucleotide to amplify, by PCR, mutant mt-tRNALeu having a mutation from adenine at position 3243 to guanine.

[0032] Primer set According to the present invention, a combination (first combination) of a first oligonucleotide and a common oligonucleotide is provided. The first combination can be used as a primer set to detect wild-type mitochondrial mt-tRNA Leu by PCR. In the primer set, one of the first oligonucleotide and the common oligonucleotide is a forward primer, and the other is a reverse primer.

[0033] According to the present invention, a combination (second combination) of a second oligonucleotide and a common oligonucleotide is provided. The second combination can be used as a primer set to detect mutant mitochondrial mt-tRNA Leu by PCR. Mutant mitochondrial mt-tRNA Leu can be the cause of MELAS. In the primer set, one of the second oligonucleotide and the common oligonucleotide is a forward primer, and the other is a reverse primer.

[0034] According to the present invention, a combination (third combination) of a first oligonucleotide, a second oligonucleotide, and a common oligonucleotide is provided. The third combination can be used as a primer set to detect either or both of wild-type mt-tRNA Leu and mutant mt-tRNA Leu by PCR.

[0035] The third combination can also be used in quantitative PCR, which allows the ratio of wild-type mt-tRNA Leu to mutant mt-tRNA Leu or the mutation rate (i.e., mutant mt-tRNA Leu amount / total mt-tRNA Leu amount) to be determined, as described below.

[0036] The primer set of the present invention (eg, the third combination) may further comprise a probe that binds to mt-tRNA Leu.

[0037] Disease Presumption According to the present invention, there is provided a method for measuring intracellular mitochondrial mt-tRNA Leu, comprising the steps of: synthesizing complementary DNA (cDNA) of mt-tRNA Leu from RNA isolated from the isolated mitochondria; and amplifying a DNA corresponding to mt-tRNA Leu by PCR using a template containing the synthesized cDNA and the first combination.

[0038] According to the present invention, there is provided a method for measuring intracellular mitochondrial mt-tRNA Leu, comprising the steps of: synthesizing complementary DNA (cDNA) of mt-tRNA Leu from RNA isolated from the isolated mitochondria; and amplifying a DNA corresponding to mt-tRNA Leu by PCR using a template containing the synthesized cDNA and a second combination.

[0039] According to the present invention, there is provided a method for measuring intracellular mitochondrial mt-tRNA Leu, comprising the steps of: synthesizing complementary DNA (cDNA) of mt-tRNA Leu from RNA isolated from the isolated mitochondria; and amplifying a DNA corresponding to mt-tRNA Leu by PCR using a template containing the synthesized cDNA and a third combination.

[0040] The third method is preferably quantitative PCR, in particular the probe method.

[0041] The method of the present invention (e.g., the third method) can determine the mutation rate of mt-tRNA Leu. For example, the method of the present invention (e.g., the third method) may further include determining the mutation rate of mt-tRNA Leu.

[0042] According to the method of the present invention (e.g., the third method), it is possible to determine whether a cell or a subject from which mt-tRNA Leu is derived has a mitochondrial disease from the mutation rate of mt-tRNA Leu. For example, the method of the present invention (e.g., the third method) may further include determining whether a cell or a subject from which mt-tRNA Leu is derived has a mitochondrial disease from the mutation rate of mt-tRNA Leu.

[0043] The method of the present invention (e.g., the third method) can evaluate the risk of developing a mitochondrial disease in a cell or subject from which it is derived, based on the mutation rate of mt-tRNA Leu. For example, the method of the present invention (e.g., the third method) may further include evaluating the risk of developing a mitochondrial disease in a cell or subject from which it is derived, based on the mutation rate of mt-tRNA Leu.

[0044] The method of the present invention (e.g., the third method) may further include administering a functional mt-tRNA Leu to a subject determined to have a mitochondrial disease or a subject at risk of developing a mitochondrial disease as described above. The functional mt-tRNA Leu can be administered by encapsulating a complex of the functional mt-tRNA Leu and a cationic polymer in a lipid nanoparticle, as described below.

[0045] Detection of mutant mt-tRNA Leu abundance ratio There is a correlation between the onset of mitochondrial disease and an increase in the ratio of mutant mt-tRNA Leu in the total mitochondrial mt-tRNA Leu. Therefore, examining the abundance ratio (mutation rate) of mutant mt-tRNA Leu at the RNA level can be useful for evaluating the presence of mitochondrial disease or the risk of developing mitochondrial disease.

[0046] The mutation rate can be quantified using known methods that can be used to quantify gene mutations, such as allele-specific PCR, invader, allele-specific hybridization, and next-generation sequencing. One of these methods that is preferably used is quantitative ARMS-PCR (Amplification Refractory Mutation Systems PCR, e.g., Newton, CR et al, Nucleic Acids Res. 1989, 17, 2503-2516; V. Venegas et al, Methods Mol. Biol. 2012, 837, 313-326, the entirety of which is incorporated herein by reference). In this method, a primer DNA set for amplifying a wild-type sequence (primer DNA set for wild-type detection) and a primer DNA set for amplifying a sequence containing a point mutation (primer DNA set for mutant detection) are designed based on the base of the point mutation to be detected and the base sequence before and after it, and quantitative PCR is performed using each primer DNA set with cDNA obtained by reverse transcription reaction of the mitochondrial RNA to be measured as a template. C when using the wild-type detection primer DNA set determined by quantitative PCR T Value (C Twild ) and C when using the mutation detection primer DNA set T Value (C Tmut ) can be applied to the following formula to calculate the mutation rate.

number

[0047] C T The value is the cycle number of PCR at which a signal level is achieved that is significantly increased over baseline, as is well known to those skilled in the art. T The value can be appropriately set by a person skilled in the art.

[0048] For absolute quantification, a dilution series of a known amount of standard sample is prepared to determine the amount of pre-amplified DNA and C TA calibration curve showing the relationship between the C value and the T The amount of DNA before amplification can be calculated using a standard curve from the value. Absolute quantification can be performed for both the wild type and the mutant type.

[0049] The kit of this embodiment may contain a primer DNA set for detecting the wild-type and a primer DNA set for detecting the mutant-type, and may further contain a reverse transcriptase, a reagent for PCR reaction, a buffer, and the like.

[0050] The kit of this embodiment may further include a probe. The probe is modified with a fluorescent dye and a quencher. Here, the quencher is a substance that quenches the fluorescence of the fluorescent dye, and a person skilled in the art can appropriately determine the combination of the fluorescent dye and the quencher to create a probe. Before decomposition, the probe does not emit light because the fluorescence of the fluorescent dye is quenched by the quencher, but after decomposition by the polymerase, the fluorescent dye can be dissociated from the quencher and the fluorescent dye can emit fluorescence. The probe may be a partial sequence of mt-tRNA Leu (particularly the sequence portion excluding the primer binding portion), and a person skilled in the art can appropriately design it.

[0051] The kit of this embodiment can be used to determine the mutation rate of mt-tRNA Leu in a cell.

[0052] Lipid structure (lipid nanoparticle) encapsulating mt-tRNA Leu Another aspect of the present invention relates to a lipid membrane structure encapsulating nanoparticles containing mt-tRNA Leu and a cationic polymer (referred to as the "lipid membrane structure of the first embodiment"). The lipid membrane structure of the present invention encapsulating nanoparticles containing a nucleic acid and a cationic polymer can encapsulate large-sized nucleic acids. Here, "encapsulation" means that the lipid membrane structure contains a nanoparticle, and preferably that the lipid membrane structure completely encapsulates the nanoparticle. One effect of complete encapsulation is that the stability of the encapsulated substance is improved.

[0053] In the lipid membrane structure of the first embodiment, the cationic polymer is used to form a complex with the nucleic acid. Since the nucleic acid is a polyanion, a polycation is suitable for complex formation. The particles thus formed do not necessarily need to be electrically neutralized, but may be electrically neutralized. The cationic polymer includes a polymer containing a cationic amino acid, for example, a cationic protein. The cationic protein includes, for example, protamine. Protamine is a protein necessary for high-level aggregation of chromatin in sperm nuclei, and is known to strongly bind to negatively charged DNA, and can be preferably used in the present invention. When a cationic protein is used as the cationic polymer, the mixing ratio of the protein and the nucleic acid can be determined so that the N / P ratio is, for example, 0.5 to 1.5, 0.7 to 1.3, or 0.8 to 1.2 (for example, 0.9). Here, in the N / P ratio, N represents the number of nitrogen atoms in the side chain in the protein, and P represents the number of phosphorus atoms in the nucleic acid.

[0054] In the lipid membrane structure of the first embodiment, the complex of the nucleic acid and the cationic polymer can be formed in a solution by mixing the nucleic acid and the cationic polymer in the solution. The solution used for the above mixing is not particularly limited as long as the nucleic acid and the cationic polymer form a complex, but can be, for example, a neutral aqueous solution, for example, an aqueous solution of pH 7 to 8, for example, an aqueous solution of pH 7.4. The pH can be adjusted using a buffer such as HEPES.

[0055] In the lipid membrane structure of the first embodiment, an aqueous solution containing a complex of nucleic acid and cationic polymer is mixed with an alcohol solution containing phospholipids to obtain a lipid membrane structure encapsulating a complex of nucleic acid and cationic polymer.

[0056] In the lipid membrane structure of the first embodiment, the phospholipid is not particularly limited, and examples thereof include phosphatidylcholine (e.g., dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, etc.), phosphatidylglycerol (e.g., dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, etc.), phosphatidylethanolamine (e.g., dioleoylphosphatidylcholine, dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, etc.), and phosphatidylethanolamine (e.g., dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, etc.). phosphatidylethanolamine, dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidiethanolamine, etc.), phosphatidylserine, phosphatidylinositol, phosphatidic acid, cardiolipin, sphingomyelin, ceramide phosphorylethanolamine, ceramide phosphorylglycerol, ceramide phosphorylglycerol phosphate, 1,2-dimyristoyl-1,2-deoxyphosphatidylcholine, plasmalogen, egg yolk lecithin, soybean lecithin, hydrogenated products thereof, and the like. The phospholipid is preferably phosphatidylethanolamine (e.g., dioleylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidiethanolamine, etc.) or sphingomyelin, more preferably dioleylphosphatidylethanolamine or sphingomyelin. In a preferred embodiment, from the viewpoint of increasing the transfer rate of the lipid membrane structure to mitochondria, the phospholipid may contain one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin, and dioleylphosphatidylethanolamine.

[0057] In the lipid membrane structure of the first embodiment, the alcohol used in the alcohol solution is not particularly limited, and examples thereof include ethanol, t-butanol, 1-propanol, 2-propanol, and 2-butoxyethanol, and ethanol is preferably used. The ethanol may be, for example, 100% ethanol.

[0058] In a preferred embodiment of the present invention, in order to impart membrane permeability to the lipid membrane structure, the lipid membrane structure may display a cationic polymer such as a cell membrane-permeable peptide (e.g., a cationic amino acid polymer, e.g., polyarginine or polylysine). In this way, it is possible to promote the introduction of the lipid membrane structure into cells. When the lipid membrane structure introduced into a cell displays a mitochondria-targeting peptide, it can make the nucleic acid function in the mitochondria. Thus, the present invention is advantageous in that even a type of RNA that forms a specific three-dimensional structure to exhibit activity, such as tRNA, can be made to function after being introduced into a cell. In order to display a cationic polymer on the lipid membrane structure, a conjugate of a cationic polymer and a lipid (e.g., myristoyl group, but not limited thereto) is prepared, and the lipid portion of the conjugate is anchored to the lipid membrane structure, whereby the cationic polymer can be stably displayed on the lipid membrane structure. Examples of membrane-permeable peptides include tat peptide (having a peptide sequence corresponding to amino acid sequences 48 to 60 of the amino acid sequence of the human immunodeficiency virus tat protein (GenBank registration number: AAF35362.1), peptides rich in basic amino acids such as oligoarginine (R9) and oligolysine (K10), amphipathic peptides having a basic portion and a hydrophobic portion such as penetratin, transportin, and TP10. Examples of membrane-permeable peptides having mitochondrial tropism include lipid-soluble cationic substances such as octaarginine (R8), lipophilic triphenylphosphonium cation (TPP) or rhodamine 123, and polypeptides such as Mitochondrial Targeting Sequence (MTS) peptide (Kong, BW. et al., Biochimica et Biophysica Acta 2003, 1625, pp. 98-108) or S2 peptide (Szeto, HH et al., Pharm. Res. 2011, 28, pp. 2669-2679).Here, the S2 peptide was Dmt-D-Arg-FK-Dmt-D-Arg-FK-NH. 2 {wherein Dmt is 2,6-dimethyltyrosine, D-Arg is D-arginine, F is L-phenylalanine, and K is L-lysine}. In one embodiment, the zeta potential of the lipid membrane structure (or lipid nanoparticle) can be 5 mV or more, 10 mV or more, 15 mV or more, 16 mV or more, 17 mV or more, 18 mV or more, 19 mV or more, or 20 mV or more, for example, about 20 mV.

[0059] In the lipid membrane structure of the first embodiment, the lipid membrane structure encapsulating the complex of nucleic acid and cationic polymer may have an average particle size (number average particle size) measured by dynamic light scattering (DLS) of 200 nm or less, 190 nm or less, or 180 nm or less. A small average particle size is preferable from the viewpoint of increasing the efficiency of uptake into cells. In the lipid membrane structure of the first embodiment, the lipid membrane structure encapsulating the complex of nucleic acid and cationic polymer may have an average particle size measured by dynamic light scattering (DLS) of, for example, 20 nm or more, 30 nm or more, 40 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, 110 nm or more, 120 nm or more, 130 nm or more, 140 nm or more, or 150 nm or more. In the lipid membrane structure of the first embodiment, the lipid membrane structure encapsulating a complex of a nucleic acid and a cationic polymer may have an average particle size measured by dynamic light scattering (DLS) of, for example, 20 nm or more and 200 nm or less, 50 nm or more and 150 nm or less, 150 nm or more and 200 nm or less, or 100 nm or more and 150 nm or less.

[0060] In the lipid membrane structure of the first embodiment, the lipid membrane structure encapsulating the complex of nucleic acid and cationic polymer may have a polydispersity index (PDI) measured by dynamic light scattering (DLS) of, for example, 0.4 or less, 0.35 or less, or 0.3 or less. The polydispersity index may be, for example, 0.1 or more, 0.15 or more, or 0.2 or more.

[0061] In the lipid membrane structure of the first embodiment, the lipid membrane structure encapsulating the complex of nucleic acid and cationic polymer may have an average particle size measured by dynamic light scattering (DLS) of, for example, 20 nm to 200 nm and a PDI of 0.4 or less, an average particle size of 150 nm to 200 nm and a PDI of 0.4 or less, or an average particle size of 100 nm to 150 nm and a PDI of 0.4 or less. In the lipid membrane structure of the first embodiment, the lipid membrane structure encapsulating the complex of nucleic acid and cationic polymer may have an average particle size measured by dynamic light scattering (DLS) of, for example, 20 nm to 200 nm and a PDI of 0.3 or less, an average particle size of 150 nm to 200 nm and a PDI of 0.3 or less, or an average particle size of 100 nm to 150 nm and a PDI of 0.3 or less.

[0062] Pharmaceutical compositions, methods of treatment, etc. The present invention provides a pharmaceutical composition comprising the lipid membrane structure of the first embodiment.

[0063] As described above, the present invention provides, as a further aspect, a pharmaceutical composition for treating and / or preventing mitochondrial diseases caused by mutations in mt-tRNA Leu (hereinafter, unless otherwise specified, simply referred to as mitochondrial diseases), particularly drug-induced hearing loss caused by mutations in mt-tRNA Leu, which contains mitochondrial mt-tRNA Leu as an active ingredient. mt-tRNA Leu may be of the mature type or the pre-type. This is because pre-mt-tRNA Leu is converted to the mature type in cells.

[0064] The pharmaceutical composition of this embodiment can be used in the form of a parenteral preparation such as an injection, drip infusion, etc. Examples of carriers that can be used in such parenteral preparations include aqueous carriers such as physiological saline and isotonic solutions containing glucose, D-sorbitol, etc.

[0065] The pharmaceutical composition of this embodiment may contain components such as pharma- ceutically acceptable buffers, stabilizers, preservatives, and other additives. Pharmaceutically acceptable components are well known to those skilled in the art, and can be appropriately selected and used by those skilled in the art within the scope of their ordinary practice, for example, from components described in the 17th Revised Japanese Pharmacopoeia and other specifications, depending on the form of the preparation.

[0066] The method of administration of the pharmaceutical composition of this embodiment is not particularly limited, but in the case of a parenteral preparation, examples include ear drops, inner ear administration such as intratympanic administration, intravascular administration (preferably intravenous administration), intraperitoneal administration, intraintestinal administration, subcutaneous administration, etc. In one preferred embodiment, the therapeutic agent of the present invention is administered to a living body by ear drops or intratympanic administration.

[0067] The pharmaceutical composition of this embodiment is expected to be effective in treating and / or preventing mitochondrial disease by supplying functional mt-tRNA Leu (e.g., wild-type mt-tRNA Leu) into mitochondria that corresponds to the genetic abnormality of mt-tRNA Leu that causes the mitochondrial disease.

[0068] The pharmaceutical composition of the present invention can be administered to a subject having a genetic abnormality of mt-tRNA Leu. In this case, the pharmaceutical composition can include a lipid membrane structure containing functional mt-tRNA Leu (e.g., wild-type mt-tRNA Leu). The subject can be a subject who has developed a mitochondrial disease or a subject who is at risk of developing a mitochondrial disease. The subject can be a subject who has developed a mitochondrial disease or a subject who has been determined to be at risk of developing a mitochondrial disease by the method of the present invention. The onset of mitochondrial disease can be caused by an increase in the ratio of abnormal RNA to its normal counterpart. Therefore, the pharmaceutical composition of the present invention can be administered to a subject to the extent that the ratio of abnormal RNA to its normal counterpart is sufficiently low.

[0069] As used herein, treatment and / or prevention includes all types of medically acceptable therapeutic and / or preventive interventions aimed at curing, temporarily remitting or preventing a disease, etc. That is, treatment and / or prevention of a mitochondrial disease includes medically acceptable interventions for various purposes, including delaying or halting the progression of a mitochondrial disease, regressing or eliminating a lesion, preventing the onset or preventing recurrence, etc.

[0070] The pharmaceutical composition of this embodiment can be used for treating and / or preventing mitochondrial diseases. Therefore, the use of the pharmaceutical composition of this embodiment can also be expressed as a method for treating and / or preventing mitochondrial diseases using the composition, and the present invention also provides an invention of such a method.

[0071] Furthermore, it is expected that mitochondrial disease can be treated and / or prevented by administering the pharmaceutical composition of this embodiment to a patient with mitochondrial disease or a person at risk of developing mitochondrial disease. Thus, the present invention also provides a method for treating and / or preventing mitochondrial disease by administering an effective amount of the pharmaceutical composition of this embodiment to a patient with mitochondrial disease or a person at risk of developing mitochondrial disease. Here, "effective amount" means an amount effective for treating and / or preventing mitochondrial disease, and such an amount is appropriately adjusted depending on the degree of symptoms of mitochondrial disease and other medical factors.

[0072] According to the present invention, a method is provided, comprising administering the lipid membrane structure or pharmaceutical composition of the present invention to a subject having a mutation of adenine at position 3243 of mitochondrial DNA to guanine. The subject having a mutation of adenine at position 3243 of mitochondrial DNA to guanine may be a subject who has developed mitochondrial disease (particularly MELAS). The subject having a mutation of adenine at position 3243 of mitochondrial DNA to guanine may be a subject who is at risk of developing mitochondrial disease (particularly MELAS).

[0073] According to the present invention, there is provided a method for treating a mitochondrial disease (particularly MELAS) in a subject having a mutation of adenine to guanine at position 3243 of mitochondrial DNA, the method comprising administering to the subject a lipid membrane structure or pharmaceutical composition of the present invention.

[0074] According to the present invention, there is provided use of the lipid membrane structure or pharmaceutical composition of the present invention in the manufacture of a medicament for treating a mitochondrial disease (particularly MELAS).

[0075] According to the present invention, there is provided the lipid membrane structure or pharmaceutical composition of the present invention for use in treating mitochondrial diseases (particularly MELAS). EXAMPLES

[0076] Example 1. Establishment of a protocol for quantifying point mutations in mitochondrial RNA 1) Preparation of DNA encoding wild-type (WT) and mutant (MT) mt-tRNA Leu The plasmid pUC57-Amp was opened with the restriction enzymes EcoRI and EcoRV, and the following two types of inserts with EcoRI and EcoRV sites added to both ends were incorporated into the plasmid pUC57-Amp to prepare pT7-mt-tRNA Leu (WT) and pT7-mt-tRNA Leu mutant (MT), which are plasmids in which mt-tRNA Leu mutants are transcribed by the T7 promoter. In the following, the numbers in the base sequence of human mitochondrial DNA (human mt-DNA) are represented by the base sequence registered in NCBI Reference Sequence: NC_012920.1 and its base number. pT7-mt-tRNA Leu(WT): pBluescript II SK (+) (Stratagene)-derived T7 promoter 20 bp and the region from 3182 to 3331 of human mt-DNA (mitochondrial pre-mt-tRNA Leu The nucleic acid has the base sequence (SEQ ID NO: 10), which corresponds to pT7-mt-tRNA Leu(MT): pBluescript II SK (+) (Stratagene)-derived T7 promoter 20 bp and the region from bases 3182 to 3331 of human mt-DNA in which adenine at base 3243 was replaced with guanine (mitochondrial pre-mt-tRNA Leu (corresponding to

[0077] 2) Primer design The primer DNAs were chemically synthesized, each consisting of the base sequence shown in Table 1. The reverse primer had a sequence common to WT and MT, and was constructed to be completely complementary to mt-tRNA Leu.

[0078] [Table 1] *In the table, lowercase letters indicate sequences that do not exist in mature tRNA Leu.

[0079] As described above, the wild-type forward primer was designed as the WT0 primer, which is completely complementary to the complementary strand of DNA encoding WT mt-tRNA Leu. The WT1 primer was mismatched at the second base from the 3' end of the WT0 primer. The WT1 Long primer was extended by 7 mer at the 5' end of the WT1 primer. The WT2 Long primer was mismatched at the third base from the 3' end of the WT1 Long primer. In addition, the mutant forward primer was designed as MT0 primer, which is completely complementary to the complementary strand of DNA encoding mt-tRNA Leu of MT. For the MT1 primer, the second base from the 3' end was mismatched with the MT0 primer. For the MT1 Long primer, the 5' end of the MT1 primer was extended by 7 mer. For the MT2 Long primer, the third base from the 3' end of the MT1 Long primer was mismatched.

[0080] 3) Quantitative PCR test Quantitative PCR was performed using the above primers and SYBR Green Realtime PCR Master Mix (TOYOBO) with DNA mixtures containing pT7-mt-tRNA Leu (WT) and pT7-mt-tRNA Leu (MT) at various ratios as samples. The PCR conditions were 95℃ / 1 min → {95℃ / 15 sec → 60℃ / 1 min} reaction for 40 cycles.

[0081] C obtained by PCR using a primer DNA set including a reverse primer for wild-type detection T Value (C Twild ) and the C obtained by PCR using a primer DNA set including a reverse primer for detecting the mutant type T Value (C Tmut ) was substituted into the following formula to calculate the mutation rate in each DNA mixture solution.

[0082]

number

[0083] The horizontal axis shows the theoretical mutation rate (mixture ratio of normal DNA and mutant DNA), and the vertical axis shows C T The mutation rates calculated from the values ​​were plotted (Figure 1). In Figure 1, the names of the wild-type detection primer and the mutant-type detection primer used are written at the top of each graph. For example, the description "WT0-MT0" at the top of the graph in the leftmost column of Figure 1 means that WT0 was used as the wild-type detection primer and MT0 was used as the mutant-type detection primer. The horizontal axis indicates the mixture ratio of pT7-mt-tRNA Leu (WT) and pT7-mt-tRNA Leu (MT), and the larger the value, the more MT DNA was mixed.

[0084] The results are shown in Figure 1. As shown in Figure 1, in WT0-MT1, an increase or decrease in the mixed ratio of mutant types in DNA could not be detected by PCR. However, in WT1 long-MT1 long, the theoretical values ​​correlated with the measured values, and the reproducibility was also good.

[0085] Example 2. Measurement of the mutation rate of mt-tRNA Leu in MELAS cells and normal cells Patients with mitochondrial DNA in which the 3243rd base is replaced by adenine to guanine develop symptoms of MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). It has been revealed that approximately 80% of MELAS patients have mitochondrial DNA in which the 3243rd base is replaced by adenine to guanine.

[0086] Dermal fibroblasts derived from a healthy individual were used as normal cells, and dermal fibroblasts derived from a MELAS patient with a mutation of the 3243rd base, adenine to guanine, were used as MELAS cells.

[0087] To collect mitochondria from normal and mutant cells, skin fibroblasts (2 × 10 6The cells (approximately 1000 cells) were suspended in 200μL of Cell Scrub buffer, shaken at 4℃ for 15 min, and centrifuged (700g, 4℃, 3 min). After removing the supernatant, the cells were suspended in 500μL of MIB(EDTA+). The cells were disrupted using a syringe needle (27G) and centrifuged (700g, 4℃, 10 min) to collect the supernatant, and 0.45μL of 0.1mg / mL RNase solution was added and incubated at 25℃ for 10 min. The supernatant was collected after centrifugation (700g, 4℃, 10 min), and added on top of 60% percoll solution, centrifuged (20,400g, 4℃, 10 min), and 200μL of the solution at the interface was collected. After centrifugation (20,400g, 4℃, 15 min) to remove the supernatant, 500μL of MIB(-) was added. The mixture was centrifuged again (20,400 g, 4° C., 15 min) and the supernatant was removed to obtain a mitochondrial fraction.

[0088] RNA was extracted from the mitochondrial fraction using RNase mini kit (QIAGEN NV) and RNase-Free DNase Set (QIAGEN NV), and then reverse transcription of the RNA was performed using the High Capacity RNA-to-cDNA Kit (ABI) to prepare cDNA for quantification.

[0089] The rRNA mutation rate in normal and MELAS cells was quantified using the WT1 long-MT1 long primer combination. The primers were thought to amplify pre-mt-tRNA Leu. The results are shown in Table 2.

[0090] [Table 2]

[0091] As shown in Table 2, 100% of mitochondrial DNA in MELAS cells encodes mutant forms of mt-tRNA Leu, and thus the theoretical mutation rate of mt-tRNA Leu is 100%, whereas the actual value measured using the above primer combination was 98.4%. Also, 0% of mitochondrial DNA in normal cells encodes mutant forms of mt-tRNA Leu, and thus the theoretical mutation rate of mt-tRNA Leu is 0%, whereas the actual value measured using the above primer combination was 0.02%.

[0092] Thus, it was demonstrated that the mutation rate of mt-tRNA Leu can be accurately estimated from the measured values ​​obtained using the WT1 long-MT1 long primer combination.

[0093] Example 3: Introduction of wild-type mt-tRNA Leu into MELAS cells and measurement of the resulting change in mutation rate A test was carried out to introduce wild-type mt-tRNA Leu into the above-mentioned MELAS cells. To introduce mt-tRNA Leu into cells, lipid nanoparticles loaded with mt-tRNA Leu were prepared according to the scheme shown in Figure 2. After that, the ratio of wild-type mt-tRNA Leu to mutant mt-tRNA Leu in cells after introduction of lipid nanoparticles was measured as in Example 2.

[0094] A vector was designed having a DNA encoding mt-tRNA Leu operably linked downstream of the T7 promoter. In this vector, a gene encoding pre-mt-tRNA Leu (3182-3331) was operably linked downstream of the T7 promoter. That is, in this example, pre-mt-tRNA Leu was used as mt-tRNA Leu. The vector was digested with a restriction enzyme to cut at the EcoRI site located downstream of pre-mt-tRNA Leu, linearized, and RNA transcribed by the T7 promoter was synthesized by an in vitro transcription reaction using RiboMAX (trademark) Large Scale RNA Production Systems-T7 (PROMEGA), and pre-mt-tRNA Leu was purified using Nucleospin RNA clean-up and concentration of RNA XS kit (TAKARA BIO).

[0095] An equal amount of 0.3 mg / mL purified mt-tRNA Leu / HEPES Buffer solution was added to 0.12 mg / mL protamine / HEPES Buffer (pH 7.4) solution while vortexing to prepare a nucleic acid nanoparticle solution (N / P ratio 0.9) {where N represents the number of nitrogen atoms in the side chain in the protein, and P represents the number of phosphorus atoms in the nucleic acid}. The particle size and zeta potential (surface potential) of the liposomes were measured by dynamic light scattering. The results were that the average particle size was 163 nm ± 20 nm, the PdI was 0.06 ± 0.01, and the zeta potential was -33 mV ± 3 mV.

[0096] Next, 65 μL of the nucleic acid nanoparticle solution was added to the lipid solution (DOPE:SM:STR-R8=9:2:1, DOPE 18 μL, SM 8 μL, STR-R8 20 μL, EtOH 62 μL) while vortexing to prepare a suspension. The suspension was added to 630 μL of HEPES Buffer in a 5 mL tube while vortexing, and then added to 4 mL of HEPES Buffer in an Amicon Ultra-4 and centrifuged (1000 g, 25° C., 8 min). Further, 4 mL of HEPES Buffer was added and centrifuged again (1000 g, 25° C., 12 min) to recover liposomes (mt-tRNA Leu / MITO) in which pre-mt-tRNA Leu was encapsulated. The particle size and zeta potential (surface potential) of the liposomes were measured by dynamic light scattering. The average particle size was 179 nm ± 8 nm, the PdI was 0.30 ± 0.01, and the zeta potential was 41 mV ± 4 mV, and the percentage of encapsulated nanoparticles was 23% ± 4%.

[0097] MELAS cells (1×10 5 MELAS cells (cells) were seeded in 6-well plates, and mt-tRNA Leu / MITO was added to the medium after 24 hours. The medium (DMEM FBS+) was replaced after 3 hours, and the cells were harvested after 24 hours to measure the ratio of wild-type and mutant mt-tRNA Leu. As negative controls, untreated MELAS cells, MELAS cells treated with empty lipid nanoparticles, and MELAS cells transfected with mt-tRNA Leu using a commercially available Lipofectamine 2000 reagent were tested.

[0098] The results are shown in Figure 3. As shown in Figure 3, in MELAS cells into which mt-tRNA Leu / MITO was introduced, the proportion of mutant mt-tRNA Leu was statistically significantly decreased. In the figure, ** indicates a significant difference (p<0.05) in one-way analysis of variance (Bonferroni test).

[0099] In addition, the weight / well of mt-tRNA Leu introduced was changed, and as shown in Figure 4, the proportion of mutant mt-tRNA Leu in the transfected cells decreased with increasing weight of mt-tRNA Leu introduced.

[0100] Next, the respiratory activity of the above cells into which mt-tRNA Leu had been introduced was examined. 24±3 hours before the measurement, MELAS cells (1×10 5 Cells) were seeded (approximately 10,000 cells / well) onto Agilent Seahorse XFp Cell Culture Miniplates (Agilent Technologies, Santa Clara, CA, USA). A running medium containing 24.75 mM glucose and 4 mM glutamine was prepared by adding 1.0 M glucose solution and 200 mM glutamine solution (Agilent) to XF DMEM Medium (Agilent). The cells were cultured (CO 2 The oxygen consumption and mitochondrial function of the cells were measured using an Agilent Seahorse XFp extracellular flux analyzer (Agilent Technologies, Santa Clara, CA, USA). The XFp Cell Mito Stress Test Kit (Agilent) was used to measure the oxygen consumption and mitochondrial function of the cells. The XFp Cell Mito Stress Test Kit (Agilent) was used to measure the oxygen consumption and mitochondrial function of the cells using 1 μM oligomycin (an ATP synthase inhibitor of the electron transport chain complex V of the respiratory chain). Oxygen consumption was also measured when 1μM FCCP (uncoupler), 1μM rotenone + 1μM antimycin A (electron transport chain respiratory chain complex I and III inhibitor) were injected. TM The results were normalized by the cellular protein concentration quantified using a BCA Protein Assay Kit (Thermo Fisher Inc.). As negative controls, untreated MELAS cells, MELAS cells treated with empty lipid nanoparticles, and MELAS cells transfected with mt-tRNA Leu using a commercially available lipofectamine were tested. The results are shown in Figure 5.

[0101] As shown in Figure 5, mt-tRNA Leu / MITO significantly improved the respiratory capacity of MELAS cells. In MELAS cells, mitochondrial respiratory capacity is reduced due to the dysfunction of mt-tRNA Leu. It was revealed that mt-tRNA Leu / MITO has the effect of improving this reduced respiratory capacity.

[0102] As a negative control, MELAS cells administered with the complex of Lipofectamine 2000 and mt-tRNA Leu showed marginal improvement in respiratory capacity. Figure 3 suggests that while the mt-tRNA Leu molecule itself appears to have been introduced into the cells, this introduction into the cells does not lead to improvement in mitochondrial respiratory capacity.

[0103] The relationship between the amount of mt-tRNA Leu added to cells (ng / well) and the respiratory capacity of the cells is shown in Figure 6. As shown in Figure 6, the amount added was good when it was 200 to 800 ng / well, and was particularly good when it was 400 ng / well.

Claims

1. A method for testing for a mutation in tRNA Leu produced by mitochondria of a cell, comprising: The method includes combining a first oligonucleotide and a common oligonucleotide to amplify DNA corresponding to a wild-type mt-tRNALeu encoded by a gene in which the 3243rd position of the mitochondrial DNA is A, using a complementary DNA (cDNA) synthesized from RNA extracted from the mitochondria of the cell as a template, and combining a second oligonucleotide and a common oligonucleotide to amplify DNA corresponding to a mutant mt-tRNALeu encoded by a gene in which the 3243rd position of the mitochondrial DNA is G, the first oligonucleotide is a part of the base sequence shown in SEQ ID NO:12, includes the base sequence shown in SEQ ID NO:3, and is an oligonucleotide having a length of 19 to 23 consecutive bases; the second oligonucleotide is a part of the base sequence shown in SEQ ID NO:13, includes the base sequence shown in SEQ ID NO:7, and is an oligonucleotide having a length of 19 to 23 consecutive bases; A method in which the common oligonucleotide is an oligonucleotide consisting of the base sequence set forth in SEQ ID NO:

1.

2. The method described in claim 1, wherein the first oligonucleotide is an oligonucleotide consisting of the base sequence set forth in SEQ ID NO: 4, and the second oligonucleotide is an oligonucleotide consisting of the base sequence set forth in SEQ ID NO:

8.

3. The method according to claim 1 or 2, further comprising determining a ratio of tRNALeu having a wild-type base sequence and / or a tRNA having a mutant base sequence relative to tRNALeu.

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