JP2012217421A - Lna modified heptamer-type small guide nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a heptamer-type small guide nucleic acid maintaining cleaving efficiency of a target RNA in an allowable range and excellent in biological stability.SOLUTION: The heptamer-type small guide nucleic acid comprises 7 bases including a base sequence complementary to at least continuous 5 bases in the target RNA, wherein 1-4 bases in the sequence are LNA (locked nucleic acid) modified, and the activity of a gene for transcribing the target RNA is reduced or extinguished by cleaving the target RNA with the action of tRNaseZin cells.

Description

  The present invention effectively suppresses or eliminates the expression of a gene that transcribes RNA that is cleaved as a target, and is excellent in in vivo stability, so that it is effective in treating or preventing a disease caused by the gene expression or the like. It relates to a small heptamer type small guide nucleic acid.

The inventors of the present invention, TRUE gene silencing ^{(tR Nase Z L - u tilizing} ef ficacious gene silencing) is developing a new gene silencing technology named (Patent Document 1, Non-Patent Document 1-4) . tRNase Z ^L is an enzyme having a large molecular weight of endoribonuclease (tRNase Z or 3'tRNase) that processes 3 'of tRNA, and excises the 3' end of precursor tRNA (Patent Document 1, Non-patent) Literature 5, 6). This TRUE gene silencing can cleave any portion of RNA by recognizing the pre-tRNA-like or micro-pre-tRNA-like complex formed by the target RNA and the synthetic small guide nucleic acid Based on the unique enzymatic properties of mammalian tRNase Z ^L (Non-patent Documents 7-13). The small guide nucleic acid (especially small guide RNA) includes the following 4 types: 5′-half tRNA (Non-patent Document 8), ˜12-16-nt linear RNA (Non-patent Document 13), heptamer RNA (non-patent) Reference 9) and hook-type RNA (Non-patent Document 12) (FIG. 1).

  The inventors have introduced a small guide nucleic acid (hereinafter sometimes referred to as sg nucleic acid) targeting various mRNAs into various mammals by introducing its expression plasmid or 2′-O-methyl RNAs. It demonstrates the effectiveness of TRUE gene silencing in cells (Non-Patent Documents 1-4). For example, HIV-1 expression in Jurkat cells was suppressed by 5′-half tRNA-type sgRNA for 18 days or longer (Non-patent Document 2), and luciferase expression in mouse liver was suppressed by heptamer-type sg nucleic acid (Non-patent Document 3). ). Furthermore, the effect of TRUE gene silencing is equivalent to RNA interference (RNA interference: RNAi) (Non-Patent Documents 3 and 14), and in some cases, it has been confirmed that it exceeds the effect of RNAi (Non-Patent Documents). Reference 4).

Recently, the inventors have found that tRNase Z ^L is present not only in the nucleus but also in the cytosol, and that the same 5′-half tRNA used as sg nucleic acid is present in the cytoplasm. (Non-patent Document 15). We also found that human cytosolic tRNase Z ^L regulates gene expression by cleaving mRNA under the direction of 5'-half tRNA, and PPM1F mRNA is the true target of tRNase Z ^L (Non-patent Document 15). Furthermore, the inventors have demonstrated that part of the microRNA containing miR-103 acts as a hook-type sg nucleic acid and can down-regulate gene expression via mRNA cleavage by cytosolic tRNase Z ^L (Non-patent document 16). From the above, it is clear that TRUE gene silencing functions on the basis of the newly elucidated physiological role of cytosolic tRNase Z ^L operated by small non-coding RNA in cells.

  One of the ultimate goals of TRUE gene silencing is the use of sg nucleic acid as a therapeutic or prophylactic agent for diseases caused by the expression of specific genes. For example, the inventors have already reported that intracellular mRNA levels encoding Bcl-2 and VEGF, which are regarded as promising molecular targets for cancer therapy, are 5′-half tRNA type sg nucleic acid, 14-nt linear type sg nucleic acid. Alternatively, it has been found that expression is suppressed by heptamer-type sg nucleic acid (Non-Patent Documents 1, 4, and 17).

  When sg nucleic acid is used as a drug component, heptamer type sg nucleic acid is most preferable from a pharmacological viewpoint. This is because heptamers (7-base nucleic acids) can be synthesized much more easily and cheaply than long sg nucleic acids. In addition, heptamer-type sg nucleic acid can be easily taken into cells without using stimulants (such as transfection reagents) (Non-patent Document 18).

Various chemical modifications of heptamer-type sg nucleic acids have been studied for the purpose of improving the ability to induce cleavage of target RNA by tRNase Z ^L and in vivo stability. The inventors previously investigated 2′-O-methyl RNA, phosphorothioate RNA, DNA, and phosphorothioate DNA. As a result, the 2′-O-methyl-modified sg nucleic acid was found to be TRUE due to its ability to induce cleavage and stability to the enzyme. It has been concluded that it is most preferable for gene silencing (Non-patent Document 1).

On the other hand, the appearance of Locked Nucleic Acid (LNA) in which the 2'-position oxygen atom and the 4'-position carbon atom are cross-linked via methylene has a wide influence on nucleic acid research (non-patent literature). 19, 20). In other words, LNA is used as a chemical modification of antisense RNA and siRNA to impart both biological stability and temperature stability to nucleic acids.
Japanese Patent No. 3660718 Tamura, M., Nashimoto, C., Miyake, N., Daikuhara, Y., Ochi, K. And Nashimoto, M. (2003) Intracellular mRNA cleavage by 3 ′ tRNase under the direction of 2′-O-methylRNA heptamers Nucleic Acids Res., 31, 4354-4360. Habu, Y., Miyano-Kurosaki, N., Kitano, M., Endo, Y., Yukita, M., Ohira, S., Takaku, H., Nashimoto, M. and Takaku, H. (2005) Inhibition of HIV-1 gene expression by retroviral vector-mediated small-guide RNAs that direct specific RNA cleavage by tRNase ZL. Nucleic Acids Res., 33, 235-243. Nakashima, A., Takaku, H., Shibata, HS, Negishi, Y., Takagi, M., Tamura, M. and Nashimoto, M. (2007) Gene-silencing by the tRNA maturase tRNase ZL under the direction of small guide RNA. Gene Therapy, 14, 78-85. Elbarbary, RA, Takaku, H., Tamura, M. And Nashimoto, M. (2009) Inhibition of vascular endothelial growth factor expression by TRUE gene silencing.Biochem.and Biophys.Res. Commun., 379, 924-927. Nashimoto, M. (1997) Distribution of both lengths and 5 ′ terminal nucleotides of mammalian pre-tRNA 3 ′ trailers representing properties of 3 ′ processing endoribonuclease. Nucleic Acids Res., 25, 1148-1155. Takaku, H., Minagawa, A., Masamichi, T. and Nashimoto, M. (2003) A candidate prostate cancer susceptibility gene encodes tRNA 3 ′ processing endoriobonuclease. Nucleic Acids Res., 31, 2272-2278. Nashimoto, M. (1995) Conversion of mammalian tRNA 3 ′ processing endoribonuclease to four-base-recognizing RNA cutters.Nucleic Acids Res., 23, 3642-3647. Nashimoto, M. (1996) Specific cleavage of target RNAs from HIV-1 with 5 ′ half tRNA by mammalian tRNA 3 ′ processing endoribonuclease. RNA, 2, 2523-2524. Nashimoto, M., Geary, S., Tamura, M. and Kasper, R. (1998) RNA heptamers that directs RNA cleavage by mammalian tRNA 3 ′ processing endoribonuclease. Nucleic Acids Res., 26, 2565-2571. Nashimoto, M. (2000) Anomalous RNA substrates for mammalian tRNA 3′processing endoribonuclease. FEBS Letters, 472, 179-186. Takaku, H., Minagawa, A., Takagi, M. And Nashimoto, M. (2004) The N-terminal half-domain of the long form of tRNase Z is required for the RNase 65 activity.Nucleic Acids Res., 32 , 4429-4438. Takaku, H., Minagawa, A., Takagi, M. And Nashimoto, M. (2004) A novel four-base-recognizing RNA cutter that can remove the single 3 ′ terminal nucleotides from RNA molecules.Nucleic Acids Res., 32 , e91. Shibata, HS, Takaku, H., Takagi, M. and Nashimoto, M. (2005) The T loop structure is dispensable for substrate recognition by tRNase ZL. J. Biol. Chem., 280, 22326-22334. Appasani, K. (2005) RNA interference technology: from basic science to drug development.Cambridge University Press, Cambridge. Elbarbary, RA, Takaku, H., Uchiumi, N., Tamiya, H., Abe, M., Takahashi, M., Nishida, H. And Nashimoto, M. (2009) Modulation of gene expression by human cytosolic tRNase ZL through 5′-half-tRNA. PLoS ONE, 4, e5908. Elbarbary, RA, Takaku, H., Uchiumi, N., Tamiya, H., Abe, M., Nishida, H. And Nashimoto, M. (2009) Human cytosolic tRNase ZL can downregulate gene expression through miRNA.FEBS Lett. , 583, 3241-3246. Hu, W. and Kavanagh, JJ (2003) Anticancer therapy targeting the apoptotic pathway. Lancet Oncol., 4, 721-729. Loke, SL, Stein, CA, Zhang XH, Mori, K., Nakanishi, M., Subasinghe, C., Cohen, JS and Neckers, LM (1989) Characterization of oligonucleotide transport into living cells.Proc. Natl. Acad. Sci. USA, 86, 3474-3478. Koch, T. and Orum, H. (2008) Locked nucleic acid. In Crooke, ST (ed.), Antisense Drug Technology. CRC Press, Boca Raton, pp. 519-564. Straarup, EM, Fisker, N., Hedtjarn, M., Lindholm, MW, Rosenbohm, C., Aarup, V., Hansen, HF, Orum, H., Hansen, JB and Koch, T. (2010) Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates.Nucleic Acids Res., 38, 7100-7111.

One challenge when using heptamer-type sg nucleic acid as a drug component is that the main effect (induction of target RNA cleavage by tRNase Z ^L ) is sustained when administered in vivo. And improve temperature stability. For such problems, LNA modification, which has been proven as a modification to antisense RNA and siRNA, is considered promising.

However, it is not known at all how the main effect of heptamer-type sg nucleic acid (induction of cleavage of target RNA by tRNase Z ^L ) is altered by LNA modification. If the main effect of the heptamer-type sg nucleic acid is greatly impaired by LNA modification, the stability improvement by LNA modification is not preferable for sg nucleic acid as a drug component.

  The present invention provides a heptamer type sg nucleic acid from the viewpoint of the main effect and stability of the heptamer type sg nucleic acid by providing LNA modification conditions that allow the heptamer type sg nucleic acid to exert its main effect to the maximum in vivo. It is an object to further improve the value of sg nucleic acid as a drug component.

The present invention provides the following inventions for solving the above-mentioned problems.
(1) It consists of 7 bases including a base sequence complementary to at least 5 consecutive bases of the target RNA, and 1 to 4 bases in that sequence are LNA (locked nucleic acid) modified, and the action of tTNaseZ ^{L in} the cell A heptamer-type small guide nucleic acid that reduces or eliminates the activity of a gene that transcribes the target RNA by cleaving the target RNA.
(2) The heptamer type small guide nucleic acid according to (1), wherein a base other than the LNA-modified base is 2′-O methylated.
(3) The heptamer type small guide nucleic acid according to (1) or (2), wherein the target RNA is RNA transcribed from a gene associated with a disease.
(4) The heptamer type small guide nucleic acid according to (3) above, wherein the gene associated with the disease is a gene associated with the onset of cancer.
(5) A drug containing an effective amount of the heptamer type small guide nucleic acid of (3) or (4).

  According to the present invention (1), a heptamer type small guide nucleic acid that maintains the target RNA cleavage efficiency within an allowable range and is extremely excellent in vivo stability is provided. When this heptamer type small guide nucleic acid is administered in vivo as a drug component, it is possible to maximize the target RNA cleavage effect due to its excellent stability.

  According to the present invention (2), since a base other than the LNA-modified base is further 2'-O methylated, a heptamer-type small guide nucleic acid with further excellent target RNA cleavage efficiency is provided.

  According to the present invention (3) to (5), a means for treating or preventing a disease such as cancer is provided.

Examples of four types of small guide nucleic acids (2, 4, 5, 6th from the left). Of each base sequence, the 5′NNNN3 ′ region is a small guide nucleic acid. A is the secondary structure of the heptamer sgRNA / T3H RNA complex used in Example 1. Of each base sequence, the 5'NNNN3 'region is a tammer-type sgRNA. Uppercase letters are 2'-O methylated modified bases, lowercase letters are LNA modified bases. The arrow is the target RNA cleavage site. B is an electrophoretic image of the cleavage product of the target RNA (T3H) on a denaturing 10% polyacrylamide gel. The cutting efficiency and relative percentage are shown below. It is a result of Example 2. 2ME: All 2'-O-methylated 3'-FITC-labeled hepL0, LNA: 3'- nucleotides with LNA modification of 2nd, 4th and 6th nucleotides (2'-O-methylated in 4 other positions) FITC labeled hepL3b. *: Heptamer-type sgRNA extracted without being degraded from cells. The band above it appears to be a complex of heptamer sgRNA and other RNA in the cell.

  The heptamer type small guide nucleic acid of the present invention (1) consists of 7 bases including a base sequence complementary to at least 5 consecutive bases of the target RNA, and 1 to 4 bases in the sequence are modified with LNA (locked nucleic acid). ing.

  LNA is an artificial nucleic acid in which 2'-position oxygen atom and 4'-position carbon atom are bridged via methylene, for example, nucleic acid containing 2'-O-4'C-methylene-β-D-ribofuranose. is there.

  The target RNA is a transcript RNA of a gene (target gene) to be suppressed or eliminated, particularly mRNA. The expression suppression of the target gene means that the amount of protein transcribed and synthesized from the gene is reduced to 25% or less, preferably 50% or less, more preferably 75% or less, particularly preferably 95% or less. A target gene is a gene whose suppression or loss of expression has industrial utility value, and in particular, a gene whose enhanced expression causes a specific disease (hereinafter sometimes referred to as a pathogenic gene) ). Examples of such pathogenic genes include, for example, ras, erbb2, myc, apc; brca1, rb, Bcl-2, BGEF oncogene, genes involved in hypertension such as renin, diabetes-related genes such as insulin, LDL receptor Examples include hyperlipidemia-related genes such as leptin, obesity-related genes such as leptin, arteriosclerotic disease-related genes such as angiotensin, apolipoproteins and preserinin known to cause dementia, genes associated with aging, and the like. These genes and their mRNA sequences are known in known databases (such as the NCBI nucleotide database).

The heptamer-type sg nucleic acid forms a tRNA acceptor stem-like duplex with the target RNA through base pairing, thereby enabling specific cleavage of the target RNA by tRNase Z ^L. The target site is directly under the stable hairpin structure region similar to tRNA T-arm, and the heptamer-type sg nucleic acid is not only 7 bases, but also RNA by the specificity of about 12 bases forming a stable hairpin structure. Disconnect. Therefore, the base sequence of heptamer type sg nucleic acid can be determined from the mRNA sequence information of these target genes.

  The heptamer-type sg nucleic acid whose sequence has been determined can be produced by a method using known chemical synthesis, an enzymatic transcription method or the like. Known methods using chemical synthesis 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), Oligolot 10 nucleic acid synthesizer (GE Healthcare) Examples of the enzymatic transcription method include transcription using RNA polymerase such as T7, T3, and SP6 RNA polymerase using a plasmid or DNA having a target base sequence as a template. The heptamer sg nucleic acid produced by the synthesis method or transcription method is then purified by HPLC or the like. For example, during HPLC purification, heptamer-type sg nucleic acid is eluted from the column using a mixed solution of triethylammonium acetate (TEAA) or hexalamonium acetate (HAA) and acetonitrile. Then, the elution solution is dialyzed for 10 hours with distilled water 1000 times the elution volume, and the dialysis solution is freeze-dried and stored frozen until use. When used, dissolve in distilled water to a final concentration of about 100 μM.

  The nucleic acid other than the LNA-modified nucleic acid used for the heptamer-type sg nucleic acid of the present invention may be any molecule as long as it is a polymer of nucleotides or molecules having functions equivalent to those of the nucleotides. Examples of nucleotides include RNA, which is a polymer of ribonucleotides, DNA, which is a polymer of deoxyribonucleotides, a polymer in which RNA and DNA are mixed, and a nucleotide polymer containing nucleotide analogs. RNA is particularly preferable.

  Nucleotide analogs include, for example, ribonucleotides to improve or stabilize nuclease resistance, to increase affinity with complementary strand nucleic acids, to increase cell permeability, or to be visualized compared to RNA or DNA. , Deoxyribonucleotides, RNA or DNA modified molecules. 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 analogs, nucleotide analogs substituted with 2'-O- [2- (guanidinium) ethyl] ribose, nucleotide analogs substituted with 2'-O-fluororibose, Ethylene bridged structure artificial nucleic acid (Ethylene bridged nucleic acid: ENA) [Nucleic Acid Research, 32, e175 (2004)], and peptide nucleic acids (PNA) [Acc. Chem. Res., 32, 624 ( 1999)], oxypeptide nucleic acid (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)], and peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000 )] Etc. In particular, nucleotide analogs substituted with 2'-O-methylribose (nucleic acids modified with 2'-O methylation) are preferred.

  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.

  The present invention (5) is a drug containing an effective amount of the above heptamer type guide nucleic acid. This drug can be formulated with the heptamer-type guide nucleic acid of the present invention alone, but it is usually mixed with one or more pharmacologically acceptable carriers and used in the technical field of pharmaceutics. It is desirable to administer it as a pharmaceutical formulation produced by any well-known method.

  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.

  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 mg / kg per day for an adult.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated further in detail and concretely, this invention is not limited to the following examples.

Cleavage activity and stability test of LNA-modified heptamer sgRNA in vitro (1) Materials and methods (1-1) Preparation of recombinant human tRNase Z ^L Histidine-labeled human Δ30 tRNase Z ^L It was overexpressed from the expression plasmid pQE-80L (Quigen) in Rosetta (DE3) pLysS (Novagen) and purified using nickel-agarose beads (Non-patent Document 15).
(1-2) RNA synthesis Target RNA, T3H:
5'-GAUCAGAAGAUUCCAGGUUCGACUCCUGGCUGGCUCGGUGUAAGCAGGGUCGUUUU-3: SEQ ID NO: 1
Was synthesized from the corresponding synthetic DNA using T7 RNA polymerase (Promega) (Non-patent Document 8). The transcription reaction was performed under the conditions recommended by the manufacturer (Promega), and RNA was purified by denaturing gel electrophoresis. The T3H RNA transcript was then labeled with a fluorescent dye according to the manufacturer's protocol (Amersham Pharmacia Biotech) (Non-patent Document 13). That is, after removing 5′-phosphate of RNA transcripts with bacterial alkaline phosphatase (Takara Shuzo), RNA was phosphorylated using T4 polynucleotide kinase (Takara Shuzo) and ATPγS. Subsequently, a single fluorescent dye moiety was added to the 5 ′ phosphorylation site, and this fluorescent dye-labeled RNA was used after gel purification.

The following 5 'phosphorylated, 2'-O methylated and / or LNA modified sgRNA was chemically synthesized.
hepL0: 5'-pGGGCCAG-3 '(SEQ ID NO: 2)
hepL2: 5'-pGgGCCaG-3 '(SEQ ID NO: 3)
hepL3a: 5′-pgGgCcAG-3 ′ (SEQ ID NO: 4)
hepL3b: 5'-pGgGcCaG-3 '(SEQ ID NO: 5)
hepL4: 5′-pGgGcCag-3 ′ (SEQ ID NO: 6)
hepL7: 5′-pgggccag-3 ′ (SEQ ID NO: 7)
In each of the above sgRNAs, a capital letter indicates a 2′-O methylation modified base, and a small letter indicates an LNA modified base.
(1-3) In vitro RNA cleavage assay
In vitro cleavage assay of target RNA labeled with fluorescent dye by mixing unlabeled RNA and recombinant human Δ30 tRNase Z ^L in a solution (6 μl) containing 10 mM Tris-HCl (pH 7.5) and 15 mM MgCl2. (Non-Patent Documents 15 and 16). After digesting the reaction products on a 5% or 10% polyacrylamide-8 M urea gel, the gel was analyzed with Typhoon 9210 (Amersham Pharmacia Biotech).
(1-4) Gel shift assay for Kd quantification To determine the dissociation constant of the heptamer / target RNA complex, fluorescently labeled T3H (0.1 pmol) was added to each unlabeled sgRNA (hepL0, hepL2, hepL3a, hepL3b, hepL4, hepL7: 4-100 pmol each) and incubated at 37 ° C. for 10 minutes. The reaction solution (6 μl) contains 10 mM Tris-HCl (pH 7.5) and 10 mM MgCl2. After incubation, the sample was mixed with an equal volume of loading buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol FF, 50% glycerol), then TBE buffer (90 Electrophoresis was performed on 8% non-denaturing polyacrylamide gel using mM Tris base, 90 mM boric acid, 1.5 mM EDTA, pH 8.3). After electrophoresis, the labeled RNA was quantified using Typhoon 9210. Similarly, the detachment constant of the substrate / tRNase Z ^L complex was measured by gel shift assay (Non-patent Document 13). RNA complexes of fluorescently labeled T3H (0.1 pmol) and each sgRNA (10 pmol) were treated with various amounts (0.5-2 μg) of recombinant human Δ30 tRNase Z ^{L in} the buffer at 4 ° C. for 10 minutes. Incubated and analyzed in the same manner as described above.
(2) Results
To examine whether heptamer-type sgRNA containing LNA modification induces cleavage of target RNA by human tRNase Z ^L, 5 heptamer-type sgRNAs having 2 to 7 LNA-modified nucleic acids were prepared, and each cleavage induction The ability was examined (FIG. 2). For this purpose, a heptamer / T3H RNA complex similar to micro-pre-tRNA was used as a test substrate (Non-patent Documents 8 and 13). The heptamer-type sgRNAs hepL2, hepL3a, hepL3b and hepL4 are LNA-modified at the 2-, 3-, 3- and 4-nucleic acid positions, respectively, and the 2 'ribose carbon of other nucleic acids is O-merylated. (FIG. 2A). In hepL7, all nucleic acids are LNA-modified, and in the control (hepL0), all nucleic acids are modified by 2′-O methylation.

RNA cleavage assays were performed with recombinant human tRNase Z ^L under single turnover conditions. Although cleavage of the target RNA T3H by hepL2 was observed, the cleavage rate was 56% of hepL0 (FIG. 2B). Overall, the cleavage rate decreased with increasing LNA modified nucleic acid, and the cleavage rate of hepL7 was only 8% of hepL0 (FIG. 2B).

Next, in order to clarify how each step in the entire reaction affects the cleavage efficiency, the dissociation constant of the heptamer sgRNA / target RNA complex and the dissociation constant of the substrate / tRNase Z ^L complex were determined. Then, the cutting efficiency constant was examined. The dissociation constants of heptamer-type sgRNA / target RNA complexes were determined by gel shift assay, and the effective concentrations of these complexes were calculated using their estimated dissociation constants. Observed cleavage efficiency constants were obtained under single turnover conditions based on the estimated effective concentration of the substrate. The dissociation constant of the substrate / tRNase Z ^L complex was also determined by gel shift assay using the estimated dissociation constant of the heptamer sgRNA / target RNA complex.

The Kd value (0.39 μM) for the hepL2 / T3H complex was comparable to that for the hepL0 / T3H complex (0.44 μM). Overall, the Kd value of the heptamer sgRNA / target RNA complex increased as the number of LNA modifications increased and was greater than 3.43 μM for the hepL7 / T3H complex (Table 1). ^{Similarly, hepL2-T3H / tRNase Z L} Kd value of the complex (0.40) was De' those comparable with that of hepL0-T3H / tRNase Z ^L complex (0.32 [mu] M). The Kd value of the substrate / enzyme complex increased as the number of LNA modifications increased, and was 2.89 μM or more for hepL7-T3H / tRNase Z ^L (Table 1). The hepsL0 / T3H complex has a kobs value of 0.40 min ^-1 and the hepL2 / T3H complex has a value of 0.20 min ^-1 , whereas the kobs value of other complexes has decreased below 0.05 min ^-1 ( Table 1).

Based on the above detailed analysis, we can understand how each step affects cutting efficiency (Figure 2B). The decrease in the cleavage efficiency of T3H by hepL2 to 56% of the cleavage efficiency by hepL0 seems to depend on the decrease in kobs value to 50% (Table 1). The decrease in T3H cleavage efficiency by hepL3a, hepL3b and hepL4 to 18-22% means that the Kd value of the heptamer / T3H complex has increased to 32-70%, and the Kd value of the substrate / tRNase Z ^L complex has tripled. This is thought to be due to the increase above and the kobs value decreasing to 10% or less (Table 1). The dramatic reduction in cleavage efficiency by hepL7 by 8% increased the Kd value of the hepL7 / T3H complex by 8 times, the Kd value of the substrate / tRNase Z ^L complex by 9 times, and Kobs It is thought that this is because the value decreased to 13% (Table 1).

As described above, it was shown that LNA-modified heptamer RNA functions as sgRNA for tRNase Z ^L. However, their effectiveness in terms of cleavage efficiency generally decreases with increasing number of LNA modifications, Kd value of heptamer sgRNA / target RNA complex, Kd value of substrate / tRNase Z ^L complex, and Kobs value. (Fig. 2, Table 1).

  However, considering the high stability of LNA-modified heptamer RNA in serum and living cells, the cleavage efficiency of the tested 2-4 LNA-modified heptamer sgRNA is reduced to 18-56%, which When used as a component, the tolerance is sufficient.

LNA modified heptamer type SgRNA intracellular stability test (1) Methods Human 293 cells, KMM-1 cells or HeLa cells (0.8-1 X 10 ⁵ cells / well), Heptamer type in 24-well plates SgRNA, ( 1 μM) was added to the medium. All heptamer-type sgRNAs are FITC-labeled at the 3 ′ end,
hepL0: 5'-pGGGCCAG-3 '(SEQ ID NO: 2)
hepL3b: 5'-pGgGcCaG-3 '(SEQ ID NO: 5)
Was used.

After 24 hours of culture, the cells were collected and washed 4 times with PBS. Thereafter, total RNA was extracted from the cells using ISOGEN and separated on a 20% polyacrylamide-8 M urea gel. The gel was analyzed with Typhoon9210.
(2) Results As shown in FIG. 3, it was confirmed that LNA-modified heptamer-type sgRNA (LNA) can exist more stably in cells than those of only 2′-O-methylation. .

  According to the present invention, for example, a heptamer-type sg nucleic acid having excellent stability as an active ingredient of an effective therapeutic agent for a disease caused by overexpression of a specific gene is provided.

Claims (5)

It consists of 7 bases including a base sequence complementary to at least 5 consecutive bases of the target RNA, and 1 to 4 bases in the sequence are modified with LNA (locked nucleic acid), and the target RNA is activated by tRNaseZ ^{L in} the cell. A heptamer-type small guide nucleic acid that reduces or eliminates the activity of the gene that transcribes the target RNA by cleaving the target RNA. The heptamer-type small guide nucleic acid according to claim 1, wherein a base other than the LNA-modified base is 2'-O methylated. The heptamer-type small guide nucleic acid according to claim 1 or 2, wherein the target RNA is RNA transcribed from a gene associated with a disease. The heptamer type small guide nucleic acid according to claim 3, wherein the gene associated with the disease is a gene associated with the onset of cancer. A drug containing an effective amount of the heptamer-type small guide nucleic acid according to claim 3 or 4.