JP2022105226A - Kit including primer dna set for detecting mitochondrial ribosomal rna mutation, nucleic acid for expressing mitochondrial ribosomal rna, lipid membrane structure obtained by encapsulating the nucleic acid, and uses of these - Google Patents

Abstract

To provide a method for detecting a point mutation of mt-12S rRNA, RNA or DNA for efficiently expressing mt-rRNA in mitochondria, and a method for introducing them into mitochondria.SOLUTION: The present invention relates to a kit for detecting a point mutation of mitochondrial DNA or a corresponding point mutation of mitochondrial ribosomal RNA including a primer DNA set for wild type detection and a primer DNA set for mutant type detection, a mitochondria-targeted lipid membrane structure obtained by encapsulating a nucleic acid including a base sequence of mitochondrial ribosomal RNA or a base sequence complementary thereto, a pharmaceutical composition containing the nucleic acid as an active ingredient, and a method for producing a cell formulation for treating and/or preventing mitochondrial disease that includes introducing the nucleic acid in vitro into cells derived from a mitochondrial disease patient or a person at risk of developing mitochondrial disease.SELECTED DRAWING: None

Description

The present invention is a kit containing a primer DNA set for detecting point mutations in 12S ribosomal RNA (hereinafter referred to as mt-12S rRNA), which is mitochondrial ribosomal RNA (hereinafter referred to as mt-rRNA), in mitochondria. With respect to nucleic acids for expressing mt-rRNA in, mitochondrial-directed lipid membrane structures encapsulated therein, and use in pharmaceutical compositions of said nucleic acids and said lipid membrane structures and in the manufacture of cell preparations.

Mitochondria, which is one of the organelles of cells, has mitochondrial genomic DNA (hereinafter referred to as mt-DNA) independent of chromosomes in the cell nucleus. Mitochondria are mitochondrial proteins (mitochondrial proteins derived from nuclear DNA) that are transcribed and translated from nuclear genomic DNA and transferred into mitochondria, and mitochondrial proteins encoded by mt-DNA and transcribed and translated in mitochondria (mitochondrial proteins derived from mitochondrial DNA). , Mitochondrial t-RNA (hereinafter referred to as mt-tRNA) and mt-rRNA. Many associations between mutations in genomic DNA encoding these proteins and RNA and various diseases (cerebral myopathy, neurodegenerative diseases, cancer, diabetes, hearing loss, etc.) have been reported, and these are collectively referred to as mitochondrial diseases. .. So far, symptomatic treatments that supplement vitamins and the like have been the mainstream treatments for mitochondrial diseases, and the development of radical treatments is desired. Hereinafter, unless otherwise specified, the present invention will be described with human mitochondria as a representative example.

One of the therapeutic methods for mitochondrial disease currently under research and development is gene therapy aimed at delivering a protein expected to have a therapeutic effect, for example, a wild-type protein corresponding to a mutated mitochondrial protein, into the mitochondria. Is. In order to realize gene therapy, a foreign gene expression system that can transfer the target protein transcribed in the cell nucleus and translated in the cytoplasm into the mitochondria, or a foreign gene that can directly express the target protein in the mitochondria. Gene expression systems and the like have been proposed.

A wide variety of expression vectors have been developed in connection with the transfer of target proteins expressed in the cytoplasm into mitochondria. Most of them utilize the mitochondrial targeting signal peptide (MTS) possessed by the mitochondrial protein derived from nuclear DNA. Specifically, an expression vector encoding a target protein (MTS-added protein) having MTS upstream is delivered to the cell nucleus, the MTS-added protein is expressed in the cytoplasm, and the MTS-added protein is delivered to mitochondria.

Although this method may be useful for certain proteins of interest, it has the problem of low applicability to mitochondrial DNA-derived mitochondrial proteins. This is because most mitochondrial DNA-derived mitochondrial proteins are insoluble in the cytoplasm, so that the mitochondrial DNA-derived mitochondrial proteins expressed in the cytoplasm aggregate and the transfer to mitochondria becomes insufficient.

Patent Document 1 discloses a method for suppressing aggregation of a target protein in the cytoplasm by using an expression vector encoding an MTS-added protein consisting of an MTS having increased water solubility and a mitochondrial protein derived from a specific mitochondrial DNA. However, since mitochondrial DNA-derived mitochondrial proteins often show cytotoxicity when they are present in intracellular organelles other than mitochondria, the range of target proteins for which the method described in Patent Document 1 can be used is limited.

In addition, the above method using MTS-added protein may cause fatal damage to cells by interfering with or competing with the intracellular transport of the original mitochondrial protein, and there is concern as a tool for gene therapy. Remain.

On the other hand, in the use of a foreign gene expression system capable of directly expressing a target protein in mitochondria, firstly, a necessary and sufficient amount of transcriptional expression of the target protein in mitochondria is required. To meet this purpose, several expression vectors have been designed in which a promoter derived from a gene present in mt-DNA, for example, HSP (heavy strand promoter), is selected and a DNA encoding the target protein is placed under the control thereof. ing. This expression vector has been devised to use triplet codons, which are frequently used in mt-DNA. However, so far, the expression levels of these expression vectors have not reached the necessary and sufficient range for disease treatment.

For example, in Non-Patent Document 1, a DNA that is transcriptionally induced in mitochondria under HSP control is encapsulated inside an artificial virus vector to which MTS is added, and the viral vector is directly introduced into the mitochondria to obtain a target protein. A method for expressing it has been proposed. While this method has the advantage of relatively high expression levels of the protein of interest from the introduced viral vector, it has the safety issues associated with being a viral vector and the protein of interest is as expected. It has not been completely verified whether it is expressed in mitochondria.

As one of the foreign gene expression systems capable of directly expressing the target protein in mitochondria, the present inventors have a promoter sequence showing transcriptional activity in the cell nucleus of an animal cell, and a codon corresponding to tryptophan. We have proposed a recombinant expression vector having a coding region encoding a target protein containing one or more TGAs under the control of the promoter sequence (Patent Document 2). By delivering this expression vector into mitochondria using a mitochondrial-directed lipid membrane structure, translation of an undesired target protein in the cytoplasm is avoided, and the expression level of the target protein in mitochondria is further increased. It becomes possible.

In the above-mentioned conventional technique, the nucleic acid delivered into mitochondria was exclusively DNA, but with the recent progress in RNA synthesis technology, a gene therapy strategy by delivering RNA directly into mitochondria instead of DNA. Has been proposed. All of these conventional techniques are techniques for delivering a relatively short-chain nucleic acid encoding the protein into the mitochondria, with the aim of exerting the function of the target protein in the mitochondria.

In addition, the establishment of a diagnostic method for mitochondrial disease remains an important issue, as there are cases in which mitochondrial disease is not properly diagnosed and becomes severe without effective treatment. For example, the point mutation A1555G present in the mt-DNA base sequence encoding the mt-12S rRNA gene is the cause of drug-induced hearing loss, which is a type of mitochondrial disease, but currently only DNA tests are available for this point mutation. It has been carried out, and no testing technique has been established for mutations in mt-12S rRNA that directly cause the disease.

Yu, H. et al., Proc.Natl.Acad.Sci.USA, 2012, 109: E1238-47

US2015 / 0225740 WO2017 / 090763

An object of the present invention is to provide a method for detecting a point mutation in mt-12S rRNA, RNA or DNA for efficiently expressing mt-rRNA in mitochondria, and a method for introducing these into mitochondria. ..

The present inventors can detect and quantify point mutations in mt-12S rRNA by using a primer DNA set consisting of a specific base sequence, and deliver nucleic acids having certain characteristics in the base sequence into mitochondria. We found that this enables efficient expression of mt-rRNA in mitochondria, and completed the following invention.

(1) A wild-type detection primer DNA set consisting of a combination of a forward primer DNA containing the base sequence shown in SEQ ID NO: 1 and a reverse primer DNA consisting of the base sequence shown in SEQ ID NO: 3 or 4.
Mitochondrial DNA comprising a mutant detection primer DNA set consisting of a combination of a forward primer DNA containing the base sequence shown in SEQ ID NO: 1 and a reverse primer DNA consisting of the base sequence shown in SEQ ID NO: 16, 17 or 20. Point mutation A kit for detecting point mutations in A1555G or the corresponding mitochondrial 12S ribosome DNA.
(2) The kit according to (1), wherein the forward primer DNA containing the base sequence shown in SEQ ID NO: 1 is the primer DNA consisting of the base sequence shown in SEQ ID NO: 1 or 2.
(3) The kit according to (1) or (2), wherein the reverse primer DNA of the primer DNA set for detecting a variant is a primer DNA consisting of the base sequence shown in SEQ ID NO: 17.
(4) A mitochondria-directed lipid membrane structure in which the nucleic acid shown in the following a) or b) is encapsulated.
a) RNA containing the base sequence of the first mitochondrial tRNA, the base sequence of the mitochondrial ribosome RNA, and the base sequence of the second mitochondrial tRNA in this order.
b) DNA containing the promoter base sequence and the base sequence complementary to the RNA of a)
(5) The lipid membrane structure according to (4), wherein the base sequence of mitochondrial ribosomal RNA and the base sequence of two mitochondrial tRNAs in a) are continuous.
(6) The lipid membrane structure according to (4) or (5), wherein the mitochondrial ribosomal RNA is a wild-type 12S mitochondrial ribosomal RNA.
(7) The lipid membrane structure according to any one of (4) to (6), which contains dioleylphosphatidylethanolamine and sphingomyelin as constituent lipids of the lipid membrane.
(8) The lipid membrane structure according to any one of (4) to (7), which has a membrane-permeable peptide on the surface of the lipid membrane.
(9) The lipid membrane structure according to (8), wherein the membrane-permeable peptide is a polyarginine peptide consisting of 4 to 20 consecutive arginine residues.
(10) The lipid membrane structure according to any one of (4) to (9), which has a peptide consisting of the amino acid sequence represented by SEQ ID NO: 23 on the surface of the lipid membrane.
(11) A pharmaceutical composition for treating and / or preventing mitochondrial disease, which comprises the nucleic acid shown in the following a) or b) as an active ingredient.
a) RNA containing the base sequence of the first mitochondrial tRNA, the base sequence of the mitochondrial ribosome RNA, and the base sequence of the second mitochondrial tRNA in this order.
b) DNA containing the promoter base sequence and the base sequence complementary to the RNA of a)
(12)
The pharmaceutical composition according to (11), wherein the base sequence of mitochondrial ribosomal RNA and the base sequence of two mitochondrial tRNAs in a) are continuous.
(13) The pharmaceutical composition according to (11) or (12), wherein the mitochondrial ribosomal RNA is a wild-type 12S mitochondrial ribosomal RNA.
(14) The pharmaceutical composition according to any one of (11) to (13), wherein the nucleic acid is encapsulated in a mitochondria-directed lipid membrane structure.
(15) Treatment and / or prevention of mitochondrial disease, which comprises introducing the nucleic acid shown in a) or b) below into cells derived from a patient with mitochondrial disease or a person who may develop mitochondrial disease. A method for producing a cell preparation for.
a) RNA containing the base sequence of the first mitochondrial tRNA, the base sequence of the mitochondrial ribosome RNA, and the base sequence of the second mitochondrial tRNA in this order.
b) DNA containing the promoter base sequence and the base sequence complementary to the RNA of a)
(16) The production method according to (15), wherein the base sequence of mitochondrial ribosomal RNA and the base sequence of two mitochondrial tRNAs in a) are continuous.
(17) The production method according to (15) or (16), wherein the mitochondrial ribosomal RNA is a wild-type 12S mitochondrial ribosomal RNA.
(18) The production method according to any one of (15) to (17), wherein the nucleic acid is encapsulated in a mitochondria-directed lipid membrane structure.

The primer DNA set of the present invention can easily detect and quantify a point mutation of A1555G, which is a point mutation of mt-DNA, or a point mutation of mt-12S rRNA corresponding thereto, by performing PCR using the primer DNA set. Further, since the lipid membrane structure and nucleic acid of the present invention can deliver or express mt-rRNA more efficiently and selectively in mitochondria, they are safer and more effective for treating mitochondrial diseases. It can be used as a medicine or in the manufacture of cell preparations.

It is a figure which shows the structure of tR ^F -mt-12S rRNA-tR ^V which is one aspect of this invention, and the base sequence of the corresponding DNA. For 1 to 16 primer DNA sets designed to detect point mutations (A1555G) in the mitochondrial ribosome RNA gene, the horizontal axis is the theoretical mutation rate, and the vertical axis is the measured value measured by quantitative PCR. It is a calibration line which plotted the mutation rate. For 17-32 primer DNA sets designed to detect point mutations (A1555G) in the mitochondrial ribosome RNA gene, the horizontal axis is the theoretical mutation rate and the vertical axis is the measured value measured by quantitative PCR. It is a calibration line which plotted the mutation rate. For 33-48 of the primer DNA sets designed to detect point mutations (A1555G) in the mitochondrial ribosome RNA gene, the horizontal axis is the theoretical mutation rate, and the vertical axis is the measured value measured by quantitative PCR. It is a calibration line which plotted the mutation rate. For 49-64 primer DNA sets designed to detect point mutations (A1555G) in the mitochondrial ribosome RNA gene, the horizontal axis is the theoretical mutation rate and the vertical axis is the measured value measured by quantitative PCR. It is a calibration line which plotted the mutation rate. For 65-80 of the primer DNA set designed to detect point mutations (A1555G) in the mitochondrial ribosome RNA gene, the horizontal axis is the theoretical mutation rate, and the vertical axis is the measured value measured by quantitative PCR. It is a calibration line which plotted the mutation rate. For 81-96 of the primer DNA sets designed to detect point mutations (A1555G) in the mitochondrial ribosome RNA gene, the horizontal axis is the theoretical mutation rate, and the vertical axis is the measured value measured by quantitative PCR. It is a calibration line which plotted the mutation rate. For 97-108 of the primer DNA sets designed to detect point mutations (A1555G) in the mitochondrial ribosome RNA gene, the horizontal axis is the theoretical mutation rate, and the vertical axis is the measured value measured by quantitative PCR. It is a calibration line which plotted the mutation rate. For primer DNA sets 14, 24 and 6, the horizontal axis is the theoretical value of the mutation rate, and the vertical axis is the calibration curve plotting the average value of the mutation rate calculated from the measured values measured by 3 to 4 quantitative PCRs. be. When tR ^F -rRNA-tR ^V / MITO is introduced into the mitochondria of skin fibroblasts derived from mitochondrial disease patients, and when tR ^F -mt-12S rRNA-tR ^V is introduced using a commercially available RNA introduction reagent. It is a graph which shows the change of the A1555G mutation rate in the cDNA complementary to each mt-rRNA.

Primer DNA set for point mutation detection The first aspect of the present invention is
A wild-type detection primer DNA set consisting of a combination of a forward primer DNA containing the base sequence shown in SEQ ID NO: 1 and a reverse primer DNA consisting of the base sequence shown in SEQ ID NO: 3 or 4.
Mt-DNA containing a mutant detection primer DNA set consisting of a combination of a forward primer DNA containing the base sequence shown in SEQ ID NO: 1 and a reverse primer DNA consisting of the base sequence shown in SEQ ID NO: 16, 17 or 20. A kit for detecting a point mutation in A1555G or the corresponding point mutation in mt-12S rRNA. In the present specification, the base sequence of mt-DNA and the position of the base in the sequence are represented by the base sequence registered in NCBI Reference Sequence: NC_012920.1 and its base number.

The point mutation A1555G is a gene mutation in which adenine at position 1555 of the mt-DNA base sequence is replaced with guanine, which is a cause of drug-induced hearing loss, which is a type of mitochondrial disease. It is known that the risk of hearing loss when administering drugs is significantly high (Sven N. Hobbie et al., Pro. Nat. Acad. Sci. USA, 2008, 105, 52, 20888-20893 .; N. Raimundo et al., Cell, 148, 4, 17, 2012, 716-726). Therefore, the detection of this gene mutation or the corresponding mt-12S rRNA mutation can be used to determine the risk of developing deafness due to the administration of aminoglycoside drugs, and can contribute to the induction or suppression of the aggravation of deafness. Be expected. In particular, the detection of mt-12S rRNA mutations is expected to lead to more accurate determination of the risk of developing drug-induced deafness because it is the detection of mutations in the final product that directly causes the disease.

The wild-type detection primer DNA set consisting of a combination of the primer DNA containing the base sequence shown in SEQ ID NO: 1 and the primer DNA consisting of the base sequence shown in SEQ ID NO: 3 or 4 is mt-DNA to be detected or mt. To specifically detect mt-DNA whose base at position 1555 is adenine or mt-12S rRNA corresponding thereto by performing PCR using cDNA, which is a reverse transcription reaction product from -12S rRNA, as a template. Can be done.

Further, the variant detection primer DNA set consisting of a combination of a forward primer DNA containing the base sequence shown in SEQ ID NO: 1 and a reverse primer DNA consisting of the base sequence shown in SEQ ID NO: 16, 17 or 20 is a detection target. By performing PCR using mt-DNA or cDNA, which is a reverse transcription reaction product from mt-12S rRNA, as a template, mt-DNA whose 1555th base is guanine or mt-12S rRNA corresponding thereto can be obtained. It can be detected specifically.

The primer DNA containing the base sequence shown in SEQ ID NO: 1 is, for example, the primer DNA consisting of the base sequence shown in SEQ ID NO: 1, or the 5'end side and / or the 3'end side of the base sequence shown in SEQ ID NO: 1. A primer DNA consisting of a base sequence to which one or more bases, for example 1 to 10, preferably 1 to 5, more preferably 1 to 3, and particularly 1 or 2 bases are added. Can be done. The type of base to be added is not limited as long as the cDNA from the mt-DNA or mt-12S rRNA to be detected can be specifically detected, but the correspondence in the mt-DNA or the cDNA from the mt-12S rRNA is not limited. It is preferable that the base is complementary to the base at the position where it is formed. The primer DNA containing the base sequence shown in SEQ ID NO: 1 is preferably a primer DNA consisting of the base sequence shown in SEQ ID NO: 1 or 2.

The reverse primer DNA included in the variant detection primer DNA set is a primer DNA consisting of the base sequence shown in SEQ ID NO: 16, 17 or 20, preferably a primer DNA consisting of the base sequence shown in SEQ ID NO: 17. ..

In a preferred embodiment, the kit comprises a wild-type detection primer DNA set consisting of a combination of a forward primer DNA consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer DNA consisting of the base sequence shown in SEQ ID NO: 3 or 4. , A variant detection primer DNA set comprising a combination of a forward primer DNA consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer DNA consisting of the base sequence shown in SEQ ID NO: 16, 17 or 20.

In a more preferred embodiment, the kit comprises a wild-type detection primer DNA set consisting of a combination of a forward primer DNA consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer DNA consisting of the base sequence shown in SEQ ID NO: 3. It includes a primer DNA set for detecting a variant consisting of a combination of a forward primer DNA consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer DNA consisting of the base sequence shown in SEQ ID NO: 17.

Using the wild type detection primer DNA set and the mutant type detection primer DNA set, PCR using mt-DNA or cDNA, which is a reverse transcription reaction product from mt-12S rRNA, as a template is performed to obtain the mt. -The proportion of molecules having the point mutation A1555G contained in DNA or cDNA, that is, the mutation rate can be quantified. Those skilled in the art can appropriately set PCR conditions capable of specifically amplifying and detecting a molecule having a point mutation.

In the present invention, the mutation rate means the ratio of the mutant molecule in all the mt-12S rRNA molecules to the mt-12S rRNA transcribed from the mutant mt-DNA that causes mitochondrial disease.

The mutation rate can be quantified by using a known method that can be used for quantification of gene mutation, for example, an allele-specific PCR method, an invader method, an allele-specific hybridization method, a next-generation sequencing method, or the like. One of these methods preferably used is the Quantitative ARMS-PCR Method (Amplification Refractory Mutation Systems PCR, eg Newton, CR et al, Nucleic Acids Res. 1989, 17, 2503-2516; V. Venegas et. al, Methods Mol. Biol. 2012, 837, 313-326, these documents are incorporated herein by reference in their entirety). In this method, a primer DNA set for amplifying a wild-type sequence (primer DNA set for wild-type detection) and a sequence containing a point mutation are amplified based on the base of the point mutation to be detected and the base sequences before and after it. Quantitative PCR using the DNA obtained by the reverse transcription reaction of the mt-rRNA to be measured using each primer DNA set by designing a primer DNA set (primer DNA set for mutation detection). I do. The C _T value (C Twild) when using the wild-type detection primer DNA set and the C _T value (C _Tmut ) when using the mutant detection primer DNA set determined by quantitative PCR are _expressed by the following formulas. By applying to this, the mutation rate can be calculated.

The kit of this embodiment may include a primer DNA set for wild-type detection and a primer DNA set for mutant detection, but may further include a reverse transcriptase, a reagent for a PCR reaction, a buffer, and the like.

Nucleic Acids and Lipid Membrane Structures Encapsulating The Nucleic Acids Another aspect of the present invention relates to the nucleic acids shown in a) or b) below, and the mitochondria-directed lipid membrane structures encapsulating the nucleic acids;
a) RNA containing the base sequence of the first mitochondrial tRNA, the base sequence of mt-rRNA and the base sequence of the second mitochondrial tRNA in this order.
b) DNA containing the promoter base sequence and the base sequence complementary to the RNA of a)

The RNA of a) contains the base sequence of the first mitochondrial tRNA (hereinafter referred to as mt-tRNA), the base sequence of mt-rRNA, and the base sequence of the second mt-tRNA in this order.

In the RNA of a), the base sequence of mt-tRNA can be arbitrarily selected and used from the base sequences of a total of 22 types of mt-tRNA genes in humans contained in the mitochondrial genome. Further, the base sequences of the first and second mt-tRNAs may be different from each other or may be the same.

In the RNA of a), the mt-rRNA may be any mt-rRNA expressed in mitochondria, in other words, mt-rRNA whose function is desired to be exerted, and is expected to be effective for the treatment of mitochondrial disease. In addition to rRNA, mt-rRNA and the like, which have an academic interest in expression in mitochondria, can be mentioned. In the present invention, the mt-rRNA is preferably a wild-type 12S ribosomal RNA (mt-12S rRNA). mt-12SrRNA is essential for mitochondrial translation, and dysfunction of mt-12SrRNA is thought to reduce cellular function and cause deafness.

In the RNA of a), the base sequence of the first mt-tRNA, the base sequence of mt-rRNA and the base sequence of the second mt-tRNA are intervening sequences, for example, the 5'end side and 3'respective of in the mitochondrial genome. It may be linked via a flanking sequence on the terminal side, but it is directly linked without the presence of an intervening sequence, that is, the base sequence of mt-rRNA and the base sequence of two mt-tRNAs. Is preferable to be continuous. When an intervening sequence is present, its length is about 1 to 10 bases, for example 1 to 6 bases, preferably 1 to 3 bases. In addition, the RNA of a) has 1 to 10 bases of the above flanking sequence or the like on the 5'end side of the base sequence of the first mt-tRNA and the 3'end side of the base sequence of the second mt-tRNA. It may have a degree, for example 1 to 6 bases, preferably 1 to 3 bases, and further.

In this embodiment, the RNA of a) contains a plurality of mt-rRNA base sequences, and each of the mt-rRNA base sequences has a mitochondrial tRNA base sequence on the 5'end side and the 3'end side thereof. It may be RNA having. When such RNA contains, for example, two base sequences of mt-rRNA, "the base sequence of the first mitochondrial tRNA, the base sequence of the first mt-rRNA, the base sequence of the second mitochondrial tRNA, the second mt -The base sequence of rRNA and the base sequence of the third mitochondrial tRNA can be expressed as RNA containing in this order. The details of such RNA are as described above for RNA in a), and a plurality of RNAs contained therein. The base sequence of mt-rRNA and the base sequences of a plurality of mitochondrial tRNAs may be different from each other or may be the same.

The DNA of b) is a DNA containing a promoter base sequence and an RNA of a), that is, a base sequence complementary to the RNA of a) above. Specifically, the DNA of b) has a promoter base sequence, a base sequence complementary to the first mt-tRNA base sequence, a base sequence complementary to the mt-rRNA base sequence, and a second mt-. It is a DNA containing a base sequence complementary to the base sequence of tRNA in this order. Here, the characteristics of the first and second mt-tRNA base sequences and the mt-rRNA base sequences are as described for a) RNA.

The promoter sequence has a base sequence complementary to the RNA of a) under its control. Having under the control of the promoter sequence means that the base sequence complementary to the RNA of a) is transcribeably linked to the promoter sequence, that is, within the range in which transcription is initiated by the transcriptional activity of the promoter sequence a). It means that there is a base sequence complementary to the RNA of. An example is the case where the first mt-tRNA is within the range of approximately 1 to 200 bases from the 3'end of the promoter sequence, but such a range varies depending on the type of promoter sequence. It can be adjusted as appropriate by a person skilled in the art.

The promoter is not particularly limited as long as it exhibits transcriptional activity, that is, transcription-inducing ability of mt-rRNA, but in order to express more efficient and selective mt-rRNA when delivered to mitochondria, patent literature. Promoters that exhibit transcriptional activity in the cell nucleus of mammals are preferred, as described in WO2017 / 090763 and US 2018/0362999, which are incorporated herein by reference in their entirety. Examples of promoters that exhibit transcriptional activity in mammalian cell nuclei are cytomegalovirus (CMV) promoter, Simian virus (SV) 40 promoter, Raus sarcomavirus (RSV) promoter, EF1α promoter, β-actin promoter and T7 promoter. Etc., and the use of CMV promoter or RSV promoter is preferable. Further, these may be those to which a substitution or other mutation is added to the base sequence as long as the transcriptional activity is not impaired.

The DNA of b) may be in a single-stranded form, or may be in a double-stranded form with a DNA having a base sequence complementary thereto.

When the DNA of b) is delivered to the mitochondria, the promoter functions and the RNA of a) is transcribed and synthesized in the mitochondria to synthesize mt-rRNA.

The mitochondrial-directed lipid membrane structure in this embodiment contains sphingomyelin (SM), preferably dioleylphosphatidylethanolamine (DOPE) and SM as constituent lipids of the lipid membrane.

The mitochondrial directional lipid membrane structure is preferably modified with a membrane permeable peptide. Membrane-permeable peptides include polyarginine, Trans-Activator of Transcription Protein (TAT; eg H. Nagahara et al., Nat. Med. 4 (1998) 1449-1452 and V. P. Torchilin et al., Proc. Natl. Acad. . Sci. U.S.A. 98 (2001) 8786-8791. These references are incorporated herein by reference in their entirety). The polyarginine is preferably a polyarginine peptide consisting of 4 to 20 consecutive arginine residues, particularly preferably octaarginine.

In addition, the mitochondrial directional lipid membrane structure is preferably modified with a mitochondrial directional molecule. Examples of mitochondrial directional molecules include MTS peptide (Kong, BW. Et al., Biochimica et Biophysica Acta 2003, 1625, p.98-108) or S2 peptide (Szeto, H.H. et al., Pharm.Res. 2011, 28, p.2669-2679).

Further, it is also preferable that the mitochondrial directional lipid membrane structure has a peptide having the amino acid sequence represented by SEQ ID NO: 23 (hereinafter referred to as KALA peptide) on the surface of the lipid membrane. The KALA peptide is described in Shaheen et al. (Biomaterials, 2011, 32: 6342-6350) as a lipid bilayer fusion peptide having a function of promoting membrane fusion between lipid membranes. The KALA peptide has the ability to selectively deliver lipid membrane structures to the intracellular organelle mitochondria by being placed on the surface of the lipid membrane structures.

A preferred embodiment of a mitochondrial directional lipid membrane structure comprises DOPE and SM as constituent lipids of the lipid membrane, and the surface of the lipid membrane is modified with at least one of octaarginine peptide, MTS peptide and S2 peptide. Lipid membrane structure MITO-Porter (Patent No. 5067733; WO2006 / 095837; Yamada Y. et al., Biochim Biophys Acta. 2008, 1778, p.423-432) and multiplex lipid membrane structure DF-MITO-Porter (Yamada, Y. et al., Mol.Ther., 2011, 19, p.1449-1456; Kawamura, E. et al., Mitochondrion 2013, 13, p.610-614), and WO 2017/090763 and US 2018 In the lipid membrane structure described in detail together with the production method in / 0362999, a lipid membrane structure containing DOPE and SM as constituent lipids of the lipid membrane and the surface of the lipid membrane being modified with KALA peptide is mentioned. Can be done. These documents are incorporated herein by reference in their entirety.

The most preferable lipid membrane structure in this embodiment is a lipid membrane structure in which the nucleic acid of the above a) or b) is encapsulated, contains DOPE and SM as constituent lipids of the lipid membrane, and has a KALA peptide on the surface of the lipid membrane. Is. This lipid membrane structure is prepared by converting DNA into the nucleic acid of a) or b) above according to the method for producing a lipid membrane structure modified with KALA peptide and encapsulated with DNA described in WO2017 / 090763 and US2018 / 0362999. It can be manufactured by replacing each of them.

Pharmaceutical Composition The present invention contains a nucleic acid shown in a) or b) having the characteristics as described above as an active ingredient, and mitochondrial disease caused by a mutation in mt-rRNA (hereinafter, unless otherwise specified). , Simply referred to as mitochondrial disease), particularly pharmaceutical compositions for the treatment and / or prevention of drug-induced deafness caused by mutations in mt-12S rRNA are provided as a further embodiment. Such a pharmaceutical composition may contain the nucleic acid shown in a) or b) having the characteristics as described above as it is, but the nucleic acid in the form of the mitochondrial directional lipid membrane structure is effective. It is particularly preferable that the pharmaceutical composition is contained as an ingredient.

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

The pharmaceutical composition of this embodiment may contain components such as pharmaceutically acceptable buffers, stabilizers, preservatives and other additives. The pharmaceutically acceptable ingredients are well known to those skilled in the art, and within the range of normal practicability, for example, from the ingredients described in the 17th revised Japanese Pharmacopoeia and other standards, depending on the form of the formulation. Can be appropriately selected and used.

The administration method of the pharmaceutical composition of this embodiment is not particularly limited, but in the case of a parenteral preparation, for example, inner ear administration such as ear drop, intratympanic administration, intravascular administration (preferably intravenous administration), intraperitoneal administration, etc. Intravenous administration, subcutaneous administration and the like can be mentioned. In one of the preferred embodiments, the therapeutic agent of the present invention is administered to a living body by ear instillation or intratympanic administration.

The pharmaceutical composition of this embodiment exhibits a therapeutic and / or preventive effect on mitochondrial disease by supplying wild-type mt-rRNA corresponding to a genetic abnormality of mt-rRNA that causes mitochondrial disease into mitochondria. Is expected.

As used herein, treatment and / or prevention includes all types of medically acceptable therapeutic and / or prophylactic interventions aimed at cure, temporary remission or prevention of the disease. That is, treatment and / or prevention of mitochondrial disease includes medically acceptable interventions for various purposes, including delaying or stopping the progression of mitochondrial disease, retraction or disappearance of lesions, prevention of onset or prevention of recurrence, and the like. do.

The pharmaceutical composition of this embodiment can be used for the treatment and / or prevention of mitochondrial disease. Accordingly, the use of the pharmaceutical composition of this embodiment can also be expressed as a method of treating and / or preventing mitochondrial disease using the composition, and the present invention also provides an invention of such a method.

Furthermore, it is expected that mitochondrial disease can be treated and / or prevented by administering the pharmaceutical composition of this embodiment to a mitochondrial disease patient or a person who may develop mitochondrial disease. As described above, the present invention also provides a method for treating and / or preventing mitochondrial disease by administering an effective amount of the pharmaceutical composition of the present embodiment to a mitochondrial disease patient or a person who may develop mitochondrial disease. .. As used herein, the term "effective amount" means an amount effective for treating and / or preventing mitochondrial disease, and the amount is appropriately adjusted depending on the degree of symptoms of mitochondrial disease and other medical factors.

Method for Producing Cellular Preparation The present invention introduces the nucleic acid shown in a) or b) into cells derived from a mitochondrial disease patient or a person who may develop mitochondrial disease in vitro. Provided, as yet another embodiment, a method for producing a cell preparation for the treatment and / or prevention of mitochondrial disease, which comprises the above.

The cell derived from a mitochondrial disease patient who intends to introduce a nucleic acid or a person who may develop mitochondrial disease is preferably a somatic cell collected from a mitochondrial disease patient or a person who may develop mitochondrial disease. Such somatic cells include, for example, cells isolated from tissues damaged or at risk of damage or risk of heart, brain, skeletal muscle, skin or other mitochondrial diseases of these persons, or cells constituting such tissues. Examples include cells capable of differentiating into.

The nucleic acid introduced into cells derived from mitochondrial disease patients or those who may develop mitochondrial disease may be in the same form as it is, but from the viewpoint of introduction efficiency, the form of the above-mentioned mitochondrial-directed lipid membrane structure It is preferable to be in.

By introducing the nucleic acid shown in a) or b) having the characteristics as described above into cells derived from mitochondrial disease patients or those who may develop mitochondrial disease in vitro, mt- It is possible to produce somatic cells having a reduced mutation rate in rRNA. The cells can be used as a cell preparation for treating and / or preventing mitochondrial disease, or for producing such a cell preparation. The mutation rate and the method for measuring the mutation rate are as described in the first aspect.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1. Establishment of point mutation quantification protocol for mitochondrial RNA
1) Preparation of DNA encoding mt-12S rRNA Insert the inserts with EcoRI and EcoRV sites added to both ends of the following two types of base sequences into the plasmid pUC57-Amp opened with the restriction enzymes EcoRI and EcoRV, respectively. We prepared pT7-12S rRNA (WT) and pT7-12S rRNA (MT), which are plasmids to which mt-rRNA is transcribed by the T7 promoter.
pT7-12S rRNA (WT):
A base sequence in which 20 bp of the T7 promoter derived from pBluescript II SK (+) (Stratagene) and 1094 bp of the 577-1670th region of human mt-DNA (corresponding to mitochondrial tRNA ^Phe , normal 12S rRNA and mitochondrial tRNA ^Val ) are linked. (SEQ ID NO: 24).
pT7-12S rRNA (MT):
Corresponds to 20 bp of T7 promoter derived from pBluescript II SK (+) (Stratagene) and 1094 bp of human mt-DNA in which alanine at position 1555 is replaced with glycine (mitochondrial tRNA ^Phe , mutant 12S rRNA and mitochondrial tRNA ^Val ). It has a base sequence (SEQ ID NO: 25) linked to it.

2) Primer design Primer DNA consisting of the nucleotide sequences shown in Table 1 was chemically synthesized. Common forward primers are in the 1450th or 1451th to 1470th regions of mt-DNA, and wild-type reverse primers and mutant reverse primers are in the 1555th to 1574th, 1575th, 1576th, 1577th or 1578th regions of mt-DNA. Designed to combine with.

3) Quantitative PCR
Using a DNA mixture solution in which pT7-12S rRNA (WT) and pT7-12S rRNA (MT) are mixed at a specified ratio as a template, primer DNA consisting of the combinations shown in Table 2 (in the table, the numbers are shown in FIGS. 2 to 8). The graph numbers inside are shown, where (★) indicates that Fl1 was used as the common forward primer, and those without (★) indicate that F was used as the common forward primer.) And SYBR Green Realtime PCR Master Mix. Quantitative PCR was performed using (TOYOBO). The PCR conditions were 95 ° C / 1 min → {95 ° C / 15 sec → 60 ° C / 1 min} for 40 cycles.

C _T value (C _Twild ) obtained by PCR using a primer DNA set containing a reverse primer for wild type detection and C _T value (C T wild) obtained by PCR using a primer DNA set containing a reverse primer for mutant detection. C _Tmut ) was applied to the following formula to calculate the mutation rate in each DNA mixed solution.

The theoretical value of the mutation rate (mixing ratio of normal DNA and mutant DNA) is plotted on the horizontal axis, and the mutation rate calculated from the _CT value is plotted on the vertical axis (FIGS. 2 to 8). As a result, it was confirmed that there is a high correlation between the theoretical value and the measured value for some primer DNA sets.

Among the highly correlated primer DNA sets, the following three primer DNA sets were repeatedly tested 3 or 4 times, and all of them obtained highly reproducible results (FIG. 9). In particular, the primer DNA set 14 had a high correlation between the theoretical value and the measured value at all mutation rates, and the reproducibility was also good. It was confirmed that these primer DNA sets can be used to quantify the A1555G mutation rate without using a calibration curve.

Primer DNA set 14
Wild-type detection set: forward primer F (SEQ ID NO: 1)
Reverse primer WT1 (SEQ ID NO: 3)
Mutant detection set: Forward primer F (SEQ ID NO: 1)
Reverse primer MT1-s2 (SEQ ID NO: 17)
Primer DNA set 24
Wild-type detection set: forward primer F (SEQ ID NO: 1)
Reverse primer WT1-l1 (SEQ ID NO: 4)
Mutant detection set: Forward primer F (SEQ ID NO: 1)
Reverse primer MT1-s1 (SEQ ID NO: 16)
Primer DNA set 6
Wild-type detection set: forward primer F (SEQ ID NO: 1)
Reverse primer WT1 (SEQ ID NO: 3)
Mutant detection set: Forward primer F (SEQ ID NO: 1)
Reverse primer MT2-l2 (SEQ ID NO: 20)

Example 2. Quantification of A1555G mutation rate of cells derived from mitochondrial disease patients
1) Preparation of cells derived from mitochondrial disease patients Healthy subjects (Control 1), mitochondrial disease (Leigh encephalopathy) patients with ND3 gene point mutation (T10158C) (Control 2), and mt-12S rRNA gene point mutation (A1555G) Fibroblasts were isolated from each skin biopsy of patients with mitochondrial disease who developed hearing loss, subcultured and then cryopreserved to prepare cell stock.

1 mL of cell stock was dissolved in a water bath at 37 ° C, 9 mL of DMEM (FBS +) was added, and the mixture was centrifuged (160 g, 4 ° C, 3 min). The supernatant was removed, 10 mL of DMEM (FBS +) was added to suspend the cells, the whole volume was transferred to a 10 cm dish, and the cells were incubated (37 ° C., 5% CO ₂ ). The passage was done when it became 80-90% confluent. After washing Dish with 5 mL of PBS (-), add 0.05% trypsin solution, incubate for 10 min (37 ° C, 5% CO ₂ ), add 9 mL of DMEM (FBS +), and centrifuge (160 g, 4 ° C, 3). min). After cell counting, the seeds were sown in a 10 cm dish at an appropriate concentration.

2) Preparation of mitochondrial fraction and quantification DNA Sustain skin fibroblasts (about 2 × 10 ⁶ cells) in 200 μL of Cell Scrub buffer, shake at 4 ° C for 15 min, and then centrifuge (700 g, 4 ° C,). 3 min). After removing the supernatant, it was suspended in 500 μL of MIB (EDTA +). The cells were disrupted with a syringe needle (27 G) and then centrifuged (700 g, 4 ° C, 10 min) to collect the supernatant, 0.45 μL of 0.1 mg / mL RNase solution was added, and the cells were incubated at 25 ° C for 10 min. .. Centrifuge (700 g, 4 ° C, 10 min) to collect the supernatant, add on top of 60% percoll solution and centrifuge (20,400 g, 4 ° C, 10 min) to recover 200 μL of solution at the interface. did. Centrifugation (20,400 g, 4 ° C, 15 min) was performed to remove the supernatant, and then 500 μL of MIB (-) was added. Centrifugation (20,400 g, 4 ° C, 15 min) was performed again to remove the supernatant, and a mitochondrial fraction was obtained.

RNA is extracted from the mitochondrial fraction using the RNase mini kit (QIAGEN N.V) and RNase-Free DNase Set (QIAGEN N.V), and then the reverse transcription reaction of RNA is performed using the High Capacity RNA-to-cDNA Kit (ABI). By doing so, quantification cDNA was prepared.

3) Quantification of mutation rate Using the primer DNA set 14 of Example 1, the mutation rate of A1555G in cDNA complementary to 12S rRNA of each skin fibroblast was quantified. The results are shown in Table 3.

In controls 1 and 2 without A1555G, 0.07% and 1.16% were substantially 0%, confirming that the A1555G quantitative system using the primer DNA set 14 was functioning normally.

Example 3. Introduction of normal mt-12S rRNA into cells derived from mitochondrial disease patients
1) Preparation of mt-12S rRNA
PT7-12S rRNA (WT) was treated with the limiting enzyme Eco RV to prepare a linearized plasmid DNA, and the T7 promoter was subjected to in vitro transcription reaction using RiboMAX ™ Large Scale RNA Production Systems-T7 (PROMEGA). RNA transcribed by the Nucleospin RNA clean-up and concentration of RNA XS kit (TAKARA BIO) was used for purification.

2) Preparation of MITO-Porter encapsulating mt-12S rRNA
An equal amount of 0.3 mg / mL purified mt-12S rRNA / HEPES Buffer solution was added to a 0.12 mg / mL protocol / HEPES Buffer solution while vortexing to prepare a nucleic acid nanoparticle solution (N / P ratio 0.6). A suspension is prepared by adding 65 μL of nucleic acid nanoparticle solution to a lipid solution (DOPE: SM: STR-R8 = 9: 2: 1, DOPE 18 μL, SM 8 μL, STR-R8 20 μL, EtOH 62 μL) while vortexing. did. The suspension was added to 630 μL of HEPES Buffer in a 5 mL tube while vortexing, then added to 4 mL of HEPES Buffer in Amicon Ultra-4 and centrifuged (1000 g, 25 ° C., 8 min). Further, 4 mL of HEPES Buffer was added and centrifugation was performed again (1000 g, 25 ° C., 12 min) to recover liposomes (mt-12S rRNA / MITO) containing mt-12S rRNA. The particle size and zeta potential (surface potential) of the liposome are shown in Table 4.

3) Introduction of mt-12S rRNA
RNA content in skin fibroblasts derived from mitochondrial disease patients or human normal skin fibroblasts NB1RGB cells (1 × 10 ⁵ cells / well) precultured in 6 well plates with 2 mL / well DMEM (FBS +) for about 24 hours. After adding mt-12S rRNA / MITO to 1.2, 12, 120 and 240 ng / dish in terms of conversion and incubating for 3 hours, the medium was changed and the cells were incubated for another 48 hours. After completion of the incubation, the same procedure as in Example 2 was carried out to prepare a quantitative cDNA from the mitochondrial fraction of skin fibroblasts, and quantitative PCR using the primer DNA set 14 was performed to complement the mt-12S rRNA. Changes in the A1555G mutation rate in the same cDNA were measured. In addition, the same amount of mt-12S rRNA in terms of RNA amount was introduced into skin fibroblasts using a commercially available nucleic acid-introducing reagent, lipofectamine iMAX (LFN iMAX, ThermoFisher Scientific), and the mutation rate was the same as above. The change in was measured.

Compared with the mutation rate when mt-rRNA is introduced using a commercially available nucleic acid introduction reagent, the mutation rate is significantly increased to about 15% by introducing mt-rRNA using the lipid membrane structure of the present invention. It was confirmed that the decrease was observed (Table 5, FIG. 10).

SEQ ID NO: 1: Forward primer F
SEQ ID NO: 2: Forward primer Fl1
SEQ ID NO: 3: Wild-type reverse primer WT1
SEQ ID NO: 4: Wild-type reverse primer WT1-l1
SEQ ID NO: 5: Wild-type reverse primer WT1-l2
SEQ ID NO: 6: Wild-type reverse primer WT1-s1
SEQ ID NO: 7: Wild-type reverse primer WT1-s2
SEQ ID NO: 8: Wild-type reverse primer WT2
SEQ ID NO: 9: Wild-type reverse primer WT2-l1
SEQ ID NO: 10: Wild-type reverse primer WT2-l2
SEQ ID NO: 11: Wild-type reverse primer WT2-s1
SEQ ID NO: 12: Wild-type reverse primer WT2-s2
SEQ ID NO: 13: Mutant reverse primer MT1
SEQ ID NO: 14: Mutant reverse primer MT1-l1
SEQ ID NO: 15: Mutant reverse primer MT1-l2
SEQ ID NO: 16: Mutant reverse primer MT1-s1
SEQ ID NO: 17: Mutant reverse primer MT1-s2
SEQ ID NO: 18: Mutant reverse primer MT2
SEQ ID NO: 19: Mutant reverse primer MT2-l1
SEQ ID NO: 20: Mutant reverse primer MT2-l2
SEQ ID NO: 21: Mutant reverse primer MT2-s1
SEQ ID NO: 22: Mutant reverse primer MT2-s2
SEQ ID NO: 23: KALA peptide SEQ ID NO: 24: tR ^F -rRNA-tR ^V sequence SEQ ID NO: 25: tR ^F -rRNA-tR ^V sequence with A1555G mutation

Claims (18)

A wild-type detection primer DNA set consisting of a combination of a forward primer DNA containing the base sequence shown in SEQ ID NO: 1 and a reverse primer DNA consisting of the base sequence shown in SEQ ID NO: 3 or 4.
Mitochondrial DNA comprising a mutant detection primer DNA set consisting of a combination of a forward primer DNA containing the base sequence shown in SEQ ID NO: 1 and a reverse primer DNA consisting of the base sequence shown in SEQ ID NO: 16, 17 or 20. Point mutation A kit for detecting point mutations in A1555G or the corresponding mitochondrial 12S ribosome DNA. The kit according to claim 1, wherein the forward primer DNA containing the base sequence shown in SEQ ID NO: 1 is a primer DNA consisting of the base sequence shown in SEQ ID NO: 1 or 2. The kit according to claim 1 or 2, wherein the reverse primer DNA of the primer DNA set for detecting a variant is a primer DNA consisting of the base sequence shown in SEQ ID NO: 17. A mitochondria-directed lipid membrane structure encapsulating the nucleic acid shown in a) or b) below.
a) RNA containing the base sequence of the first mitochondrial tRNA, the base sequence of the mitochondrial ribosome RNA, and the base sequence of the second mitochondrial tRNA in this order.
b) DNA containing the promoter base sequence and the base sequence complementary to the RNA of a) The lipid membrane structure according to claim 4, wherein the base sequence of mitochondrial ribosomal RNA and the base sequence of two mitochondrial tRNAs in a) are continuous. The lipid membrane structure according to claim 4 or 5, wherein the mitochondrial ribosomal RNA is a wild-type 12S mitochondrial ribosomal RNA. The lipid membrane structure according to any one of claims 4 to 6, which contains dioleylphosphatidylethanolamine and sphingomyelin as constituent lipids of the lipid membrane. The lipid membrane structure according to any one of claims 4 to 7, which has a membrane-permeable peptide on the surface of the lipid membrane. The lipid membrane structure according to claim 8, wherein the membrane-permeable peptide is a polyarginine peptide consisting of 4 to 20 consecutive arginine residues. The lipid membrane structure according to any one of claims 4 to 9, which has a peptide consisting of the amino acid sequence represented by SEQ ID NO: 23 on the surface of the lipid membrane. A pharmaceutical composition for treating and / or preventing mitochondrial disease, which comprises the nucleic acid shown in a) or b) below as an active ingredient.
a) RNA containing the base sequence of the first mitochondrial tRNA, the base sequence of the mitochondrial ribosome RNA, and the base sequence of the second mitochondrial tRNA in this order.
b) DNA containing the promoter base sequence and the base sequence complementary to the RNA of a) The pharmaceutical composition according to claim 11, wherein the base sequence of mitochondrial ribosomal RNA and the base sequence of two mitochondrial tRNAs in a) are continuous. The pharmaceutical composition according to claim 11 or 12, wherein the mitochondrial ribosomal RNA is a wild-type 12S mitochondrial ribosomal RNA. The pharmaceutical composition according to any one of claims 11 to 13, wherein the nucleic acid is encapsulated in a mitochondria-directed lipid membrane structure. For the treatment and / or prevention of mitochondrial disease, which comprises introducing the nucleic acid shown in a) or b) below into cells derived from mitochondrial disease patients or those who are at risk of developing mitochondrial disease. Method for manufacturing cell preparation.
a) RNA containing the base sequence of the first mitochondrial tRNA, the base sequence of the mitochondrial ribosome RNA, and the base sequence of the second mitochondrial tRNA in this order.
b) DNA containing the promoter base sequence and the base sequence complementary to the RNA of a) The production method according to claim 15, wherein the base sequence of mitochondrial ribosomal RNA and the base sequence of two mitochondrial tRNAs in a) are continuous. The production method according to claim 15 or 16, wherein the mitochondrial ribosomal RNA is a wild-type 12S mitochondrial ribosomal RNA. The production method according to any one of claims 15 to 17, wherein the nucleic acid is encapsulated in a mitochondria-directed lipid membrane structure.

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