WO2011010737A1

WO2011010737A1 - Guide nucleic acid for cleavage of micro-rna

Info

Publication number: WO2011010737A1
Application number: PCT/JP2010/062470
Authority: WO
Inventors: 正之梨本; 陽史山田; 哲郎吉田
Original assignee: 学校法人新潟科学技術学園新潟薬科大学; 協和発酵キリン株式会社
Priority date: 2009-07-24
Filing date: 2010-07-23
Publication date: 2011-01-27

Abstract

A guide nucleic acid for use in the cleavage of micro-RNA, wherein the guide nucleic acid comprises a single-stranded nucleic acid which has a complementary nucleotide sequence to a nucleotide sequence that is composed of contiguous nucleotides starting from any nucleotide selected from the 1^st to 4^th nucleotides numbered from the 5'-terminal of micro-RNA to be cleaved or a nucleotide sequence having a 90% or higher identity to the complementary nucleotide sequence, and which has a length of 11 to 15 nucleotides; a method for cleaving micro-RNA, which comprises bringing the guide nucleic acid into contact with micro-RNA to be cleaved in the presence of tRNaseZ; and a therapeutic agent for diseases associated with the overexpression of micro-RNA, which comprises the guide nucleic acid as an active ingredient.

Description

Guide nucleic acids for microRNA cleavage

The present invention relates to a guide nucleic acid for cleaving microRNA useful as a therapeutic agent for diseases, and a method for cleaving microRNA using the same.

MicroRNA (miRNA) is a small non-coding single-stranded RNA of about 22 nucleotides that is not translated into protein, and has been confirmed to exist in many organisms including humans (Non-Patent Documents 1 and 2).
MicroRNAs are generated from genes that are transcribed into single or clustered microRNA precursors. That is, the primary transcript, primary-microRNA (pri-miRNA), is first transcribed from the gene, and then a characteristic hairpin structure is formed from the pri-miRNA in stepwise processing from the pri-miRNA to the mature microRNA. Precursor-microRNA (pre-miRNA) having a length of about 70 bases is generated. Further, mature microRNA (also simply referred to as microRNA) is generated from pre-miRNA by Dicer-mediated processing (Non-patent Document 3).

MicroRNAs are thought to be involved in post-transcriptional control of gene expression by binding to target mRNAs in a complementary manner to suppress translation of mRNA, or by degrading mRNA. As of June 2009, the microRNA database miRBase (http://microrna.sanger.ac.uk/) has registered 885 microRNAs for humans and 10097 types for all biological species. Physiologically, microRNAs that have been revealed to be involved in diseases such as differentiation into specific functional cells or canceration, especially miR-21, suggesting an association with carcinogenesis There is also.
It is known that there is a relationship between microRNA overexpression and disease, and suppressing microRNA expression is useful for the treatment of diseases associated with microRNA overexpression. Antisense RNA having a sequence complementary to microRNA (Non-patent Document 4) and decoy expression vector having a microRNA recognition sequence (Non-patent Documents 5 and 6) have been developed as suppression of microRNA expression. In both cases, expression is suppressed by hybridization with microRNA, and the microRNA itself is not cleaved.

tRNaseZ is one of endonucleases involved in maturation of tRNA (transfer RNA). Usually, tRNA is first transcribed as a precursor tRNA, and then becomes a mature tRNA having a function through a processing process. When the enzyme involved in the processing is a eukaryote, RNaseP is involved in the 5′-side endonuclease, and tRNaseZ is involved in the 3′-side endonuclease (Non-patent Documents 7 and 8).
An example of cleaving a specific mRNA artificially using the activity of tRNaseZ has been reported. It is important how tRNA-like structure is recognized by using a suitable guide RNA for the target mRNA to be cleaved. In mammals, it is known that there are two types of tRNaseZ, tRNaseZS and tRNaseZL. As a result of in vitro studies mainly using tRNaseZ purified from swine in the 1990s on how to make a tRNA-like structure by combining the target sequence and the guide nucleic acid, it was necessary to recognize tRNaseZ. As a structure, it was found that only Acceptor-Stem and T-Stem-Loop were sufficient, and it was shown that a guide nucleic acid with a minimum length of 7 bases is sufficient (Non-patent Documents 9 and 10). From around 2003, research on the concept of cleaving mRNA using endogenous tRNaseZ by introducing a guide nucleic acid from the outside into cells has been proceeding, and synthetic 2'-O-Me with a minimum length of 7 bases Inhibition of the targeted Luciferase gene by introduction of -RNA (Non-patent Document 11), but it is sufficient that the target sequence also has a structure consisting of one stem loop (Micro-Pre-tRNA). It is known (Non-patent Document 12). Furthermore, it is known that a mere 12-18 bp stem structure without even one loop is weak but shows a cleavage activity (Non-patent Document 13). Regarding the suppression of the expression of genes inherent in cells, some suppression of mRNA expression has been reported by introducing a guide nucleic acid into GSK-3 or VEGF (Non-patent Documents 14 and 15). However, it is not known that tRNaseZ cleaves microRNA. In addition, a method of cleaving microRNA by introducing an appropriate guide nucleic acid and allowing RNaseP to recognize it is known (Patent Document 1), but this method requires a guide nucleic acid having a length of at least 30 bases or more. It is.

WO2009 / 026576

An object of the present invention is to provide a guide nucleic acid for cleaving microRNA, a method for cleaving microRNA using the same, and a therapeutic drug for diseases caused by overexpression of microRNA.

The present invention relates to the following (1) to (10).
(1) A base sequence complementary to a base sequence continuous from any one of the first to fourth bases from the 5 ′ end of the microRNA to be cleaved, or a base having 90% or more identity to the complementary base sequence A guide nucleic acid for cleaving microRNA, comprising a single-stranded nucleic acid having a sequence and a length of 11 to 15 bases.
(2) The guide nucleic acid according to (1) above, wherein the microRNA to be cleaved is a microRNA that is overexpressed in cancer, allergic disease, neurodegenerative disease, cardiovascular disease or hepatitis.
(3) The microRNA to be cleaved is hsa-miR-16, 17, 18a, 19a, 19b, 20a, 21, 92a, 103, 122, 125b, 142-3p, 155, 181a, 208a, 221, 222, The guide nucleic acid of (1) or (2) above, which is 372 or 373.
(4) The guide nucleic acid according to (1) or (2) above, wherein the complementary base sequence is the base sequence represented by any one of SEQ ID NOs: 1 to 19.
(5) A method of cleaving microRNA using the guide nucleic acid of any one of (1) to (4) above.
(6) A method for determining the function of a microRNA using the guide nucleic acid according to any one of (1) to (4) above.
(7) A therapeutic agent for a disease caused by overexpression of microRNA, comprising the guide nucleic acid according to any one of (1) to (4) as an active ingredient.
(8) The therapeutic agent according to (7) above, wherein the disease caused by overexpression of microRNA is cancer, allergic disease, neurodegenerative disease, cardiovascular disease or hepatitis.
(9) A method for treating a disease caused by overexpression of microRNA in a subject, comprising administering an effective amount of the guide nucleic acid according to any one of (1) to (4) to the subject.
(10) The method according to (9) above, wherein the disease caused by overexpression of microRNA is cancer, allergic disease, neurodegenerative disease, cardiovascular disease or hepatitis.

The present invention can provide a guide nucleic acid for cleaving microRNA, a method for cleaving microRNA using the same, and a therapeutic drug for diseases caused by overexpression of microRNA.

FIG. 1 (1) is an electrophoresis diagram of miR-103 in which various guide nucleic acids are allowed to act. 5'product indicates a cleavage product. FIG. 1 (2) is a diagram of the expression level of miR-103 in which various guide nucleic acids are allowed to act. FIG. 2 (1) is a diagram of miR-16 electrophoresis using various guide nucleic acids. 5'product indicates a cleavage product. FIG. 2 (2) is a diagram of miR-16 electrophoresis with and without various guide nucleic acids and tRNasetZL siRNA. FIG. 2 (3) is a graph showing the relative expression level of miR-16 on which various guide nucleic acids were allowed to act. FIG. 3 (1) shows the relationship between the sequence of miR-16 and the base sequences of various guide nucleic acids. The arrow is the position where it is expected to be cut. FIG. 3 (2) is a diagram of miR-16 electrophoresis using various guide nucleic acids. FIG. 4 (1) is a graph showing the relative expression level of miR-16 in 293 cells to which various guide nucleic acids were allowed to act. FIG. 4 (2) is a graph showing the relative expression level of miR-122 in Huh-7 cells to which various guide nucleic acids were allowed to act. FIG. 4 (3) is a graph showing the relative expression level of miR-19a in RPMI-8226 cells with various guide nucleic acids. FIG. 4 (4) is a graph showing the relative expression level of miR-142-3p in human lymphoma cell line Daudi with various guide nucleic acids. FIG. 4 (5) is a graph showing the relative expression level of miR-142-3p in HL-60 cells on which various guide nucleic acids were allowed to act. FIG. 5 is a graph showing the relative number of cells when various guide nucleic acids are allowed to act on melanoma cells.

The guide nucleic acid for cleaving microRNA of the present invention (hereinafter also simply referred to as guide nucleic acid) is any one of the first to fourth bases from the 5 ′ end of the microRNA to be cleaved, preferably the 5 ′ end base. Having a base sequence complementary to a continuous base sequence or a base sequence having 90% or more, preferably 95% or more identity with the complementary base sequence, and a length of 11 to 15 bases, preferably 12 to Examples thereof include nucleic acids consisting of single-stranded RNA having a length of 14 bases, more preferably 14 bases.

In the present invention, microRNA refers to a single-stranded RNA having a length of 17 to 28 bases present in a cell. RNA transcribed from the surrounding genome including DNA encoding microRNA has a sequence capable of forming a hairpin structure, and microRNA can be excised from any one strand of the hairpin. MicroRNAs complementarily bind to their target mRNA and suppress mRNA translation, or promote post-transcriptional regulation of mRNA expression by promoting mRNA degradation.

In the present invention, a complementary base sequence refers to a base sequence composed of nucleotides capable of forming base pairs by hydrogen bonding with respect to nucleotides constituting RNA. Specific examples of nucleotides constituting RNA include adenine (A), cytosine (C), guanine (G), and uracil (U). Adenine is uracil, cytosine is guanine, and guanine is cytosine or Uracil and uracil can base pair with adenine.

In the present invention, a guide nucleic acid comprising a base sequence having 90% or more identity is BLAST [J. Mol. Biol., 215, 403 (1990)] or FASTA [Methods Enzymology, 183, 63 (1990). ], Which is a nucleic acid having a base sequence of 90% or more when calculated using an analysis software such as Examples of the base sequence having 90% or more identity with the complementary base sequence include base sequences in which the complementary base sequence is substituted with 2 bases or less, preferably 1 base.

The base sequence of microRNA is registered in, for example, a database called miRBase (http://microrna.sanger.ac.uk/), and this base sequence can be used as the base sequence of the microRNA to be cleaved.
The base sequence of the guide nucleic acid can be determined as a sequence complementary to a base sequence continuous from any one of the first to fourth bases from the 5 ′ end of the microRNA. For example, hsa-miR-16 (SEQ ID NO: 31), 17 (SEQ ID NO: 32), 18a (SEQ ID NO: 33), 19a (SEQ ID NO: 34), 19b (SEQ ID NO: 35), 20a (SEQ ID NO: 36), 21 ( SEQ ID NO: 37), 92a (SEQ ID NO: 38), 103 (SEQ ID NO: 39), 122 (SEQ ID NO: 40), 125b (SEQ ID NO: 41), 142-3p (SEQ ID NO: 42), 155 (SEQ ID NO: 43), 181a The nucleotide sequences of the guide nucleic acids of (SEQ ID NO: 44), 208a (SEQ ID NO: 45), 221 (SEQ ID NO: 46), 222 (SEQ ID NO: 47), 372 (SEQ ID NO: 48) and 373 (SEQ ID NO: 49) are respectively SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 can be exemplified.

Once the base sequence of the guide nucleic acid is determined, the guide nucleic acid can be synthesized based on it. The method for synthesizing the guide nucleic acid of the present invention is not particularly limited, and the guide nucleic acid can be produced by a method using a known chemical synthesis or an enzymatic transcription method. Examples of known chemical synthesis methods include phosphoramidite method, phosphorothioate method, phosphotriester method, and the like. For example, ABI3900 high-throughput nucleic acid synthesizer (Applied Biosystems) or NTS H- 6Nucleic acid synthesizer (manufactured by Nippon Techno Service) or Oligopilot 10 nucleic acid synthesizer (GE Healthcare) Examples of the enzymatic transcription method include transcription using a typical phage RNA polymerase, for example, T7, T3, or SP6 RNA polymerase, using a plasmid or DNA having the target base sequence as a template.

As the nucleic acid used for the guide nucleic acid of the present invention, any nucleic acid may be used as long as it is a polymer obtained by polymerizing nucleotides or molecules having functions equivalent to the nucleotides. As the nucleotide polymer, for example, RNA, which is a polymer of ribonucleotides, DNA which is a polymer of deoxyribonucleotides, a polymer in which a polymer in which ribonucleotides and deoxyribonucleotides are mixed is a molecule in which molecules having functions equivalent to those nucleotides are polymerized Examples thereof include nucleotide polymers containing nucleotide analogues. As the nucleic acid, RNA is preferably used.

Nucleotide analogs include, for example, to improve or stabilize the nuclease resistance of nucleotide polymers, to increase affinity with complementary strand nucleic acids, to increase cell permeability, or to be visualized compared to RNA or DNA. In addition, any molecule may be used as long as it is a ribonucleotide, deoxyribonucleotide, RNA or DNA modified. Examples thereof include sugar moiety-modified nucleotide analogs and phosphodiester bond-modified nucleotide analogs.

The sugar moiety-modified nucleotide analog may be any one obtained by adding or substituting any chemical structural substance to a part or all of the chemical structure of the sugar of the nucleotide. For example, 2'-O-methyl Nucleotide analogues substituted with ribose, nucleotide analogues substituted with 2'-O-propylribose, nucleotide analogues substituted with 2'-methoxyethoxyribose, substituted with 2'-O-methoxyethylribose Nucleotide analogues, nucleotide analogues substituted with 2'-O- [2- (guanidinium) ethyl] ribose, nucleotide analogues substituted with 2'-O-fluororibose, introducing a bridging structure into the sugar moiety Bridged synthetic nucleic acid (BNA) having two circular structures, more specifically, an oxygen atom at the 2 ′ position And 4'-position carbon atoms cross-linked via methylene (Locked Nucleic Acid) (LNA), and ethylene-bridged artificial nucleic acid (Ethylene bridged nucleic acid) (ENA) [Nucleic Acid Research, 32, e175 (2004)], peptide nucleic acids (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxypeptide nucleic acids (OPNA) [J. Am. Chem. Soc., 123, 4653 ( 2001)], and peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)].

The phosphodiester bond-modified nucleotide analog may be any one obtained by adding or substituting an arbitrary chemical substance to a part or all of the chemical structure of a phosphodiester bond of a nucleotide. Examples include nucleotide analogues substituted with thioate linkages, nucleotide analogues substituted with N3'-P5 'phosphoramidate linkages [Cell engineering, 16, 1463-1473 (1997)] [RNAi method And Antisense, Kodansha (2005)].

Other nucleotide analogues include atoms (for example, hydrogen atoms, oxygen atoms) or functional groups (for example, hydroxyl groups, amino groups) of nucleic acid base moieties, ribose moieties, phosphodiester bond moieties, etc. For example, a hydrogen atom, a sulfur atom), a functional group (for example, an amino group), a group substituted with an alkyl group having 1 to 6 carbon atoms, or a group protected with a protecting group (for example, a methyl group or an acyl group), a nucleic acid In addition, for example, a molecule to which another chemical substance is added such as lipid, phospholipid, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and a dye may be used.
Examples of molecules obtained by adding another chemical substance to nucleic acid include 5′-polyamine addition derivatives, cholesterol addition derivatives, steroid addition derivatives, bile acid addition derivatives, vitamin addition derivatives, Cy5 addition derivatives, Cy3 addition derivatives, and 6-FAM. Examples include addition derivatives, biotin addition derivatives, and the like.

By using the guide nucleic acid of the present invention, the target microRNA can be cleaved. The cleavage can be performed by bringing the guide nucleic acid of the present invention into contact with the microRNA to be cleaved in the presence of tRNaseZ.
tRNaseZ is one of endonucleases involved in tRNA maturation, and has an activity of cleaving the 3 ′ side when a precursor tRNA is matured into a mature tRNA. As tRNaseZ, either tRNaseZL or tRNaseZS may be used, but tRNaseZL is preferably used.
When cleaving a microRNA expressed in a cell or an animal individual using the guide nucleic acid of the present invention, tRNaseZ endogenous to the cell or animal individual can be used. Specifically, the nucleic acid described below can be used. It can be carried out in the same manner as in the case of determining the function of microRNA using or the use of the nucleic acid as a medicine.

As a method for confirming the cleavage of microRNA using the guide nucleic acid of the present invention, any method can be used as long as it can detect a cleavage product, and examples thereof include a labeled RNA cleavage assay. Alternatively, the cleavage product may be detected by collecting the sample RNA and further collecting the low molecular weight RNA fraction and determining the base sequence. Alternatively, the cleavage may be detected indirectly by measuring the expression level of microRNA using Northern hybridization or quantitative PCR.

In the labeled RNA cleavage assay, the microRNA to be cleaved is labeled with a radioisotope or a fluorescent reagent, reacted with a guide nucleic acid and tRNaseZ, and then electrophoresed on an acrylamide denaturing gel, and then the gel A method of analyzing the band length by exposing the film to an imaging plate or the like is used. tRNaseZ can be produced and purified as a recombinant protein, for example, according to the method described in PLoS ONE 4, e5908 等 (2009).

In Northern blotting, sample-derived RNA is separated by gel electrophoresis, then transferred to a support such as a nylon filter, and a probe labeled appropriately based on the base sequence of the microRNA is prepared for hybridization and washing. By performing this, a method for detecting a band specifically bound to microRNA (or its cleavage product) is used. Specifically, for example, it can be performed according to the method described in Science, 294, 853-858 (2001). The labeled probe is complementary to the base sequence of the microRNA by, for example, radioisotope, biotin, digoxigenin, fluorescent group, chemiluminescent group, etc. by methods such as nick translation, random priming, or phosphorylation at the 5 ′ end. It can be prepared by incorporating it into DNA or RNA having an appropriate sequence or LNA. Since the binding amount of the labeled probe reflects the amount of microRNA (or its cleavage product), the amount of microRNA (or its cleavage product) can be quantified by quantifying the amount of bound labeled probe. Electrophoresis, transfer to a membrane, probe preparation, hybridization, and detection of nucleic acid can be performed, for example, by the method described in Molecular Cloning 3rd Edition (Cold Spring Harbor Laboratory Press).

The quantitative PCR method is a method for quantifying cDNA synthesized from a sample-derived RNA by PCR using a reverse transcription primer and a reverse transcriptase (hereinafter referred to as a sample-derived cDNA). As a reverse transcription primer to be used for cDNA synthesis, a specific primer such as a random primer or a primer having a sequence complementary to a base sequence corresponding to microRNA can be used.
For example, after synthesizing a sample-derived cDNA, using this as a template, PCR is performed using a template-specific primer designed from a base sequence corresponding to microRNA or a base sequence corresponding to a primer for reverse transcription, and microRNA is The contained cDNA fragment is amplified, and the amount of microRNA contained in the sample-derived RNA is detected from the number of cycles until a certain amount is reached. As a template-specific primer, an appropriate region corresponding to the microRNA can be selected, and a DNA or LNA pair consisting of a sequence complementary to that region can be used. Specifically, it can be performed according to the method described in Nucleic Acids Research, 32, e43 (2004).
A specific RT primer having a stem-loop structure can also be used as a reverse transcription primer for cDNA synthesis. Specifically, it can be performed using the method described in Nucleic Acids Research, 33, e179 (2005) or TaqMan MicroRNA Assays (Applied Biosystems).

As another method, the sample-derived cDNA is hybridized to a filter or slide glass, silicon, or other substrate on which DNA corresponding to a base sequence containing at least one microRNA or a DNA sequence or LNA is immobilized, and washed. By doing so, fluctuations in the amount of microRNA can be detected. Examples of methods based on such hybridization include methods using differential hybridization [Trends Genet., 7, 314 (1991)] and microarrays [Genome Res., 6, 639 (1996)]. In both methods, the amount of microRNA between the control sample (without the guide nucleic acid) and the target sample (with the guide nucleic acid) is fixed by immobilizing an internal control such as a nucleotide sequence corresponding to U6 RNA on a filter or substrate. It is possible to accurately detect the difference. In addition, labeled cDNA synthesis using differently labeled dNTPs (mixtures of dATP, dGTP, dCTP, and dTTP) based on RNA derived from the control sample and the target sample, and two filters on one filter or one substrate. MicroRNA can be quantified by simultaneously hybridizing labeled cDNA. For example, microRNA can be detected using a microarray described in Proc. Natl. Acad. Sci. USA, 101, 9740-9744 (2004), Nucleic Acids Research, 32, e188 (2004). Specifically, it can be detected or quantified in the same manner as mirVana miRNA Bioarray (Ambion).

A guide nucleic acid for cleaving microRNA used in the present invention is introduced into a cell or animal individual to reduce microRNA expression, and the function of microRNA is determined by observing and measuring changes occurring in the cell or individual. Can do. There are lipofection reagents such as Lipofectamine 2000 (Invitrogen) and electroporation to introduce a guide nucleic acid for cleaving microRNA into cells, but it can also be introduced by adding the guide nucleic acid directly into the cell culture medium. be able to. When introducing a guide nucleic acid into an animal individual, it can be carried out according to the administration method as a pharmaceutical described later.

The guide nucleic acid for cleaving microRNA used in the present invention can be used as a therapeutic agent for diseases caused by overexpression of microRNA by suppressing the expression of microRNA. Examples of the disease caused by overexpression of microRNA include cancer, allergic disease, cardiovascular disease, neurodegenerative disease, hepatitis and the like.
Examples of microRNAs related to canceration by overexpression include miR-16, miR-17, miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-92a, miR- 103, miR-155, miR-181a, miR-372, miR-373 and the like. Examples of microRNAs that increase the number of mast cells that contribute to the development of allergic diseases include miR-221 and miR-222. As microRNAs that promote degranulation activity of mast cells, miR-142- 3p can be given. MiR-208a is a microRNA that leads to cardiac hypertrophy due to overexpression, miR-125b is a microRNA highly expressed in the brain of Alzheimer's disease, and miR-122 is a microRNA that propagates hepatitis C virus. , You can give each.

The therapeutic agent containing the nucleic acid used in the present invention as an active ingredient can be administered alone, but it is usually mixed with one or more pharmacologically acceptable carriers to prepare pharmaceutical technology. It is desirable to administer as a pharmaceutical formulation produced by any method well known in the art.
It is desirable to use the most effective route for treatment, and oral administration or parenteral administration such as buccal, respiratory tract, rectal, subcutaneous, intramuscular and intravenous is desirable. Can be given intravenously.
Examples of the dosage form include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.

Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p-hydroxybenzoic acid Preservatives such as esters, and flavors such as strawberry flavor and peppermint can be used as additives.
For capsules, tablets, powders, granules, etc., excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxy A binder such as propylcellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin can be used as additives.

Formulations suitable for parenteral administration include injections, suppositories, sprays and the like.
The injection is prepared using a carrier made of a salt solution, a glucose solution, or a mixture of both. Suppositories are prepared using a carrier such as cocoa butter, hydrogenated fat or carboxylic acid. The spray is prepared using a carrier that does not irritate the recipient's oral cavity and airway mucosa, and that facilitates absorption by dispersing the active ingredient as fine particles.
Specific examples of the carrier include lactose and glycerin. Depending on the nature of the nucleic acid used in the present invention and the carrier used, preparations such as aerosols and dry powders are possible. In these parenteral preparations, the components exemplified as additives for oral preparations can also be added.

The dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 μg / kg to 20 μg / kg per day for an adult.

The guide nucleic acid for microRNA cleavage used in the present invention can also be transferred by a non-viral nucleic acid transfer method. For example, calcium phosphate coprecipitation method (Virology, 52, 456-467 (1973); Science, 209, 1414-1422 (1980)), microinjection method (Proc.cNatl. Acad. Sci. USA, 77, 5399-5403 ( 1980); Proc. Natl. Acad. Sci. USA, 77, 7380-7384 (1980); Cell, 27, 223-231 (1981); Nature, 294, 92-94 (1981)), liposome-mediated membrane Fusion-mediated transfer (Proc. Natl.tlAcad. Sci. USA, 84, 7413-7417 (1987); Biochemistry, 28, 9508-9514 (1989); J. Biol. Chem., 264, 12126-12129 (1989) ); Hum. Gene Ther., 3, 267-275, (1992); Science, 249, 1285-1288 (1990); Circulation, 83, 2007-2011 (1992)) or direct DNA uptake and receptor-mediated DNA Transfer method (Science, 247, 1465-1468 (1990); J. Biol. Chem., 266, 14338-14342 (1991); Proc. Natl. Acad. Sci. USA, 87, 3655-3659 (1991); J Biol. Chem., 264, 16985-16987 (1989); BioTechniques, 11, 474-485 (1991); Proc. Natl. Acad. Sci. USA, 87, 3410-3414 (1990); Proc. Natl. Acad. Sci. USA, 88, 4255-4259 (1991); Proc. Natl. Acad. Sci. USA, 87, 4033-4037 (1990); Proc. Natl. Acad. Sci. USA, 88, 8850-8854 (1991); Hum. Gene Ther., 3, 147-154 (1991)).

The membrane fusion-mediated transfer method via liposomes allows the nucleic acid used in the present invention to be taken up locally and expressed in the tissue by directly administering the liposome preparation to the target tissue [Hum. Gene Ther., 3, 399 (1992)]. In order to target the nucleic acid directly to the lesion, it is preferable to use a technique for directly incorporating the nucleic acid.
For example, receptor-mediated nucleic acid transfer can be performed by binding a nucleic acid to a protein ligand via polylysine. The ligand is selected based on the presence of the corresponding ligand receptor on the cell surface of the target cell or tissue. The ligand-nucleic acid conjugate can be injected directly into the blood vessel, if desired, and can be directed to a target tissue where receptor binding and internalization of the nucleic acid-protein complex occurs. To prevent intracellular destruction of nucleic acids, adenovirus can be co-infected to disrupt endosomal function.

Hereinafter, the invention of this application will be specifically described with reference to examples, but the invention of this application is not limited by the following examples.

Guide nucleic acid that cleaves miR-103 A guide nucleic acid that induces cleavage of miR-103 was designed and examined to cleave miR-103.
(1) Verification in cell-free system It was examined whether miR-103 was cleaved by reacting labeled miR-103, various guide nucleic acids for miR-103, and tRNaseZL in vitro.
First, miR-103 was prepared by transcription using T7 RNA polymerase (manufactured by Promega) using a synthetic DNA corresponding to the base sequence of miR-103 as a template, and extraction by denaturing gel electrophoresis. Transcription with T7 RNA polymerase followed the method described in the instructions attached to the product. Then, miR-103 was treated with alkaline phosphatase (Takara Bio) to remove the 5'-terminal phosphate group, and then reacted with T4 polynucleotide kinase (Takara Bio) and ATPγS. After the reaction, fluorescein And fluorescently labeled. The labeling procedure was in accordance with the method described in the GE Healthcare manual.
SgR103-10 represented by SEQ ID NO: 20, sgR103-14 represented by SEQ ID NO: 9, sgR103-16 represented by SEQ ID NO: 21, and sgR103 represented by SEQ ID NO: 22 synthesized by Nippon Bioservice Co., Ltd. -23 was used as the guide nucleic acid for miR-103. sgR103-10, sgR103-14, sgR103-16, and sgR103-23 consist of base sequences that are complementary to base sequences that are 10, 14, 16, and 23 consecutive bases from the 5 ′ end of miR-103, respectively. It is a single-stranded RNA, and all bases are 2'-O-methylated.
tRNaseZL can be produced and purified by a conventional method as a recombinant protein using E. coli as a host. Specifically, it can be performed according to the method described in PLoS ONE 4, e5908 (2009), for example.
The cleavage assay was performed as follows. That is, 2 pmol labeled miR-103, 20 pmol guide nucleic acid, 50 ng tRNaseZL in 6 μL reaction solution (10 mM Tris-HCl (pH 7.5), 1.5 mM dithiothreitol, 3.3 mM MgCl ₂ ) The reaction was carried out at 37 ° C., and the presence or absence of degradation products was analyzed with Typhoon 9210 (GE Healthcare) after electrophoresis of 15% polyacrylamide-8M urea gel.
As a result, as shown in FIG. 1 (1), when tRNaseZL and sgR103-14 were added, a product having a length of about 14-15 bases was detected in addition to miR-103 consisting of 23 bases.

(2) Verification in 293 cells Various guide nucleic acids for miR-103 were introduced into 293 cells, and the expression level of miR-103 was examined.
293 cells were cultured in a DMEM medium (manufactured by Sigma) containing 10% fetal bovine serum (FBS, manufactured by MP Biomedicals) in an incubator at 37 ° C. and 5% CO ₂ concentration. Four types of guide nucleic acids, sgR103-10, sgR103-14, sgR103-16, and sgR103-23 described in Example 1 (1) were introduced into cells.
A guide nucleic acid was introduced into a plate seeded with 293 cells using a lipofection method, specifically, Lipofectamine 2000 (manufactured by Invitrogen) to a final concentration of 0.1 μM. Lipofection followed the method described in the instructions attached to the product. 18 hours after introducing the guide nucleic acid by the lipofection method, total RNA was extracted from 293 cells using ISOGEN (Nippon Gene). Of this, 20 μg was electrophoresed on 15% polyacryamide-8M urea gel, transferred to Hybond N ⁺ membrane (GE Healthcare), and subjected to UV cross-linking treatment. A DNA oligo corresponding to the miR-103 sequence was labeled with ³² P as a probe, and the membrane was subjected to a hybridization reaction in QuickHyb buffer (Stratagene). After the membrane was washed, the presence or absence of a band corresponding to miR-103 was measured with Typhoon 9210. As a result, as shown in FIG. 1 (2), the amount of miR-103 decreased in the cells into which sgR103-14 was introduced.

Guide nucleic acid that cleaves miR-16 (1)
Similar to the guide nucleic acid for miR-103, a guide nucleic acid that induces cleavage of miR-16 was also designed and examined to cleave miR-16.
(1) Verification in cell-free system It was examined whether miR-16 is cleaved by reacting labeled miR-16, various guide nucleic acids for miR-16, and tRNaseZL in vitro.
MiR-16 synthesized by Nippon Bioservice Co., Ltd. was reacted with T4 polynucleotide kinase (manufactured by Takara Bio Inc.) and ATPγS, and then mixed with fluorescein to prepare fluorescently labeled miR-16. The labeling procedure was in accordance with the method described in the GE Healthcare manual.
The sgR16-14 represented by SEQ ID NO: 1 and the sgR16-14d represented by SEQ ID NO: 23, which were synthesized by Nippon Bioservice, were used as guide nucleic acids for miR-16. The former is a single-stranded RNA consisting of a base sequence complementary to the base sequence of 14 bases from the 5 'end of miR-16, and the latter is a base sequence of 14 bases from the 3' end of miR-16. It is a single-stranded RNA consisting of a complementary base sequence, both of which have all bases 2'-O-methylated.
tRNaseZL can be produced and purified by a conventional method as a recombinant protein using E. coli as a host. Specifically, it can be performed according to the method described in PLoS ONE 4, e5908 (2009), for example.
The cleavage assay was performed as follows. That is, 2 pmol labeled miR-16, 20 pmol guide nucleic acid, 50 ng tRNaseZL in 6 μL reaction solution (10 mM Tris-HCl (pH 7.5), 1.5 mM dithiothreitol, 3.3 mM MgCl ₂ ) The reaction was carried out at 37 ° C. for 30 or 60 minutes, and the presence or absence of degradation products was analyzed by Typhoon 9210 (GE Healthcare) after electrophoresis of 15% polyacrylamide-8M urea gel.
As a result, as shown in FIG. 2 (1), when tRNaseZL and sgR16-14 were added, a fragment shorter than miR-16 was confirmed apart from miR-16 consisting of 22 bases. This indicates that miR-16 has been cleaved. On the other hand, when tRNaseZL and sgR16-14d were added, short fragments other than miR-16 could not be confirmed.

(2) Verification in 293 cells Various guide nucleic acids for miR-16 were introduced into 293 cells, and the expression level of miR-16 was examined. At the same time, siRNA for tRNaseZL was also introduced to investigate the involvement of tRNaseZL.
293 cells were cultured in DMEM medium (manufactured by Sigma) containing 10% fetal bovine serum (FBS, manufactured by MP Biomedicals) in an incubator at 37 ° C. and 5% CO ₂ concentration. Three types of guide nucleic acids described in Example 2 (1), sgR16-14 and sgR16-14d, and sgR16-22 represented by SEQ ID NO: 24 were introduced into cells. sgR16-22 is a single-stranded RNA consisting of a base sequence complementary to a base sequence that is 22 bases continuous from the 5 ′ end of miR-16, and in any guide nucleic acid, all bases are 2′-O. -Methylated. As siRNA for human tRNaseZL, siRNA consisting of SEQ ID NO: 25 and SEQ ID NO: 26 was purchased from QIAGEN and used.
A guide nucleic acid was introduced into a plate seeded with 293 cells using a lipofection method, specifically, Lipofectamine 2000 (manufactured by Invitrogen) to a final concentration of 0.1 μM. In some cases, siRNA for tRNaseZL was simultaneously introduced by a lipofection method to a final concentration of 0.1 μM. Lipofection followed the method described in the instructions attached to the product. 18 hours after introduction of the guide nucleic acid by the lipofection method (42 hours after introduction of the tRNaseZL siRNA), total RNA was extracted from 293 cells using ISOGEN (Nippon Gene). Of this, 5 μg was electrophoresed with 15% polyacryamide-8M urea gel, transferred to Hybond N ⁺ membrane (GE Healthcare), and subjected to UV cross-linking treatment. A DNA oligo corresponding to the miR-16 sequence is labeled with ³² P as a probe, the membrane is hybridized in QuickHyb buffer (Stratagene), the membrane is washed, and miR-16 is supported with Typhoon 9210 The presence or absence of a band to be measured was measured.
As a result, as shown in Fig. 2 (2), the miR-16 band disappeared by the introduction of sgR16-14 and sgR16-22 in the group not introduced with tRNaseZL siRNA, whereas in the group introduced with tRNaseZL siRNA. The miR-16 band disappeared only by introducing sgR16-22. This indicates that miR-16 expression suppression by sgR16-22 does not depend on tRNaseZL, but miR-16 expression suppression by sgR16-14 depends on tRNaseZL.
Moreover, cDNA synthesis and real-time PCR were performed on the obtained total RNA using TaqMan MicroRNA Assays (Applied Biosystems), and the expression level of miR-16 was measured. The measurement followed the method described in the instructions attached to the product. At the same time, the expression level of sno234 RNA was measured and normalized to calculate the relative expression level.
As a result, as shown in FIG. 2 (3), as in the case of the northern analysis, miR-16 expression level was reduced by the introduction of sg16-14 and sgR16-22 in the group not introduced with tRNaseZL siRNA. In the introduced group, a decrease in the expression level was observed only with sgR16-22.

Guide nucleic acid that cleaves miR-16 (2)
Three types of guide nucleic acids other than the guide nucleic acid shown in Example 2 were designed to examine whether the labeled miR-16 is cleaved.
SgR16 (1-14) represented by SEQ ID NO: 1, sgR16 (1-12) represented by SEQ ID NO: 27, and sgR16 (4-17) represented by SEQ ID NO: 28 synthesized by Nippon Bioservice Co., Ltd. Was used as a guide nucleic acid for miR-16. sgR16 (1-14), sgR16 (1-12) and sgR16 (4-17) are single-stranded RNAs each consisting of a sequence complementary to a base sequence that is 14 bases continuous from the 5 'end of miR-16. , A single-stranded RNA consisting of a sequence complementary to the base sequence of 12 bases from the 5 'end of miR-16, and a base sequence of 14 bases from the 4th base from the 5' end of miR-16 It is a single-stranded RNA consisting of a complementary sequence. In any guide nucleic acid, all bases are 2'-O-methylated. The relationship between miR-16 and each guide nucleic acid is shown in FIG.
Using the same method as in Example 2, labeled miR-16 and tRNaseZL cleavage assays were performed. As a result, as shown in FIG. 3 (2), in any case of sgR16 (1-14), sgR16 (1-12), sgR16 (4-17), apart from miR-16 consisting of 22 bases, A fragment shorter than miR-16 was confirmed.

Effect of guide nucleic acid in the absence of lipofection reagent The miRNA expression suppression when a guide nucleic acid was allowed to act on cultured cells without using a lipofection reagent was examined.
(1) Guide nucleic acid for miR-16 As for the guide nucleic acid, sgR16-14, sgR16-14d and sgR16-22 used in Example 2 (2) were each plated at a final concentration of 1 μM on a plate seeded with 293 cells. Added to. After 18 hours, total RNA was extracted from 293 cells using ISOGEN (Nippon Gene), and cDNA synthesis and real-time PCR were performed on total RNA using TaqMan MicroRNA Assays (Applied Biosystems). The expression level of miR-16 was measured. The measurement followed the method described in the instructions attached to the product. At the same time, the expression level of sno234 RNA, whose expression is considered not to change, was measured and corrected with that value to calculate the relative expression level. As a result, as shown in FIG. 4 (1), a decrease in the expression level of miR-16 was observed only when sgR16-14 was added.
(2) Guide nucleic acid for miR-122 The sgR122-14 represented by SEQ ID NO: 10 and sgR122-14d represented by SEQ ID NO: 29, synthesized by Nippon Bioservice, were used as the guide nucleic acid for miR-122. . The former is a single-stranded RNA consisting of a base sequence complementary to the base sequence of 14 bases from the 5 'end of miR-122, and the latter is a base sequence of 14 bases from the 3' end of miR-122. It is a single-stranded RNA consisting of a complementary base sequence, and all bases are 2′-O-methylated in any guide nucleic acid.
Huh-7 cells were cultured in a DMEM medium (Sigma) containing 10% fetal bovine serum (FBS, manufactured by MP Biomedicals) in an incubator at 37 ° C in a 5% CO ₂ concentration, and Huh-7 cells were seeded. It added to the plate so that it might become final concentration of 2 micromol. After 18 hours, total RNA was extracted from Huh-7 cells using ISOGEN (Nippon Gene), and cDNA synthesis and real-time analysis were performed on this total RNA using TaqMan MicroRNA Assays (Applied Biosystems). PCR was performed to measure the expression level of miR-122. The measurement followed the method described in the instructions attached to the product. At the same time, the expression level of sno234 RNA was measured and normalized to calculate the relative expression level. As a result, as shown in FIG. 4 (2), a decrease in the expression level of miR-122 was observed only when sgR122-14 was added.
(3) Guide nucleic acid for miR-19a As a guide nucleic acid for miR-19a, sgR19a-14 represented by SEQ ID NO: 4 was prepared. This is a single-stranded RNA consisting of a base sequence complementary to the base sequence that is 14 bases continuous from the 5 'end of miR-19a. All but 2a-O-methylated except for a at the 3' end, The 3 'terminal a was LNA and 3' phosphorylated, and was synthesized by Nippon Bioservice.
A guide nucleic acid was added to a plate seeded with RPMI-8226 cells to a final concentration of 1 μM. 18 hours later, total RNA was extracted from RPMI-8226 cells using ISOGEN (Nippon Gene), and cDNA synthesis and real-time analysis were performed on this total RNA using TaqMan MicroRNA Assays (Applied Biosystems). PCR was performed to measure the expression level of miR-19a. The measurement followed the method described in the instructions attached to the product. At the same time, the expression level of sno234 RNA was measured and normalized to calculate the relative expression level. As a result, as shown in FIG. 4 (3), it was confirmed that when sgR19a-14 was added, the expression level of miR-19a decreased.
(4) Guide nucleic acid for miR-142-3p The guide nucleic acid for miR-142-3p was prepared as sgR142-3p-14 represented by SEQ ID NO: 11 and sgR142-3p-14d represented by SEQ ID NO: 30. The former is a single-stranded RNA consisting of a base sequence complementary to a base sequence that is 14 bases continuous from the 5 'end of miR-142-3p. All bases are 2'-O-methylated, and 3' The terminal is phosphorylated. The latter is a single-stranded RNA consisting of a base sequence complementary to the base sequence that is 14 bases continuous from the 3 'end of miR-142-3p, and all bases are 2'-O-methylated, The terminal is phosphorylated. Both were synthesized by Nippon Bioservice.
Guide nucleic acid was added to a plate seeded with human lymphoma cell line Daudi to a final concentration of 1 μM. After 24 hours, total RNA was extracted from the cells using ISOGEN (Nippon Gene), and cDNA synthesis and real-time PCR were performed on the total RNA using TaqMan MicroRNA Assays (Applied Biosystems). The expression level of miR-142-3p was measured. The measurement followed the method described in the instructions attached to the product. At the same time, the expression level of sno234 RNA was measured and normalized to calculate the relative expression level. As a result, as shown in FIG. 4 (4), it was confirmed that when sgR142-3p-14 was added, the expression level of miR-142-3p decreased.
In addition, a guide nucleic acid was added to a plate seeded with HL-60 cells to a final concentration of 1 μM. 48 hours later, total RNA was extracted from the cells using ISOGEN (Nippon Gene), and cDNA synthesis and real-time PCR were performed on the total RNA using TaqMan MicroRNA Assays (Applied Biosystems). The expression level of miR-142-3p was measured. The measurement followed the method described in the instructions attached to the product. At the same time, the expression level of sno234 RNA was measured and normalized to calculate the relative expression level. As a result, as shown in FIG. 4 (5), it was confirmed that when sgR142-3p-14 was added, the expression level of miR-142-3p decreased.

Proliferation Assay of Guided Nucleic Acid-Introduced Cells It is known that miR-19a and miR-181a suppress their proliferation when their expression is suppressed by introduction of antisense in cancer cells [Proc. Natl. Acad. Sci. USA, 105, 12885-12890 (2008)]. Therefore, we designed guide nucleic acids for miR-19a and miR-181a and examined their effects on proliferation by introduction into cells.
SgR19a-14 represented by SEQ ID NO: 4 and sgR181-14 represented by SEQ ID NO: 14 synthesized by Nippon Bioservice Co., Ltd. were used as guide nucleic acids for miR-19a and miR-181a, respectively. The former is a single-stranded RNA consisting of a base sequence complementary to the base sequence that is 14 bases continuous from the 5 'end of miR-19a, and the latter is a base sequence that is 14 bases continuous from the 5' end of miR-181a. It is a single-stranded RNA consisting of a complementary base sequence. In both cases, all bases are 2'-O-methylated and the 3 'end is phosphorylated.
Guide nucleic acid was added to a plate seeded with KMM-1 cells and Oda cells to a final concentration of 0.5 μM, and 3 days later, the number of cells was measured using TetraColor ONE (Seikagaku Corporation). The measurement followed the method described in the instructions attached to the product.
As a result, as shown in FIG. 5, the number of cells decreased in cells into which sgR19a-14 or sgR181-14 was introduced, compared to control cells.

The present invention provides a guide nucleic acid that causes microRNA cleavage. By using the guide nucleic acid of the present invention, it is possible to treat a disease caused by overexpression of microRNA.

Claims

It has a base sequence complementary to the base sequence continuous from any of the 1st to 4th bases from the 5 ′ end of the microRNA to be cleaved or a base sequence having 90% or more identity to the complementary base sequence And a guide nucleic acid for cleaving microRNA, comprising a single-stranded nucleic acid having a length of 11 to 15 bases.
The guide nucleic acid according to claim 1, wherein the microRNA to be cleaved is a microRNA that is overexpressed in cancer, allergic disease, neurodegenerative disease, cardiovascular disease or hepatitis.
The microRNA to be cleaved is hsa-miR-16, 17, 18a, 19a, 19b, 20a, 21, 92a, 103, 122, 125b, 142-3p, 155, 181a, 208a, 221, 222, 372 or 373. The guide nucleic acid according to claim 1 or 2, wherein
The guide nucleic acid according to claim 1 or 2, wherein the complementary base sequence is a base sequence represented by any one of SEQ ID NOs: 1 to 19.
A method for cleaving microRNA using the guide nucleic acid according to any one of claims 1 to 4.
A method for determining the function of a microRNA using the guide nucleic acid according to any one of claims 1 to 4.
A therapeutic agent for a disease caused by overexpression of microRNA, comprising the guide nucleic acid according to any one of claims 1 to 4 as an active ingredient.
The therapeutic agent according to claim 7, wherein the disease caused by overexpression of microRNA is cancer, allergic disease, neurodegenerative disease, cardiovascular disease or hepatitis.
A method for treating a disease caused by overexpression of microRNA in a subject, comprising administering an effective amount of the guide nucleic acid according to any one of claims 1 to 4 to the subject.
The treatment method according to claim 9, wherein the disease caused by overexpression of microRNA is cancer, allergic disease, neurodegenerative disease, cardiovascular disease or hepatitis.