Artificial RNA molecule

Abstract

Provided is a novel technique for regulating mRNA splicing. An artificial RNA molecule comprising the following (a) and (b), wherein (a) and (b) are arranged in this order from the 5'-side to the 3'- side. (a) A polynucleotide potentially having a secondary structure represented by formula (I), or a polynucleotide in which 1 to 3 bases are substituted, deleted, or added in the 7 bases on the 3'-side of a polynucleotide potentially having the secondary structure represented by formula (I). (b) An mRNA precursor-targeting polynucleotide containing a sequence complementary to a target sequence which is a partial sequence of an mRNA precursor.

Description

artificial RNA molecule

[0001] The present invention relates to novel artificial RNA molecules, particularly to artificial RNA molecules that regulate mRNA splicing.

[0002] 4.5SH RNA is a non-coding RNA specific to rodents (Myodonta Clade), and its base sequence has been identified (Non-Patent Document 1). However, the function of 4.5SH RNA has not been elucidated.

[0003] On the other hand, gene therapy has been attracting attention in recent years. Known gene therapy techniques include nucleic acid drugs and genome editing using CRISPR / Cas9.

[0004] As nucleic acid drugs, for example, antisense nucleic acid drugs that treat neurological and muscular diseases by regulating mRNA splicing of disease-causing genes are available on the market (for example, Patent Document 1).

[0005] As a technology that applies genome editing using CRISPR / Cas9, for example, progress is being made in the development of a technology in which the expression of disease-causing genes, specifically β-globin genes with disease-causing mutations, is reduced, and the expression of corresponding normal genes is increased, and patient-derived stem cells are then transplanted into the patient (e.g., Patent Document 2).

[0006] JP 2014-54250 A JP 2021-166514 A

[0007] Harada, F and Kato, N. (1980) NAR.,8:1273-J285

[0008] However, because antisense nucleic acid drugs are generally around 14 to 25 bases in length, they require screening to find sites within target genes that are effective in regulating mRNA splicing, which makes development time-consuming and expensive.In addition, because they have a short half-life, they require repeated administration every few weeks to several months to maintain their efficacy, which places a significant burden on the medical economy.

[0009] One method to solve this problem is to use genome editing technology using CRISPR / Cas9, which acts directly on the genome and can therefore have long-lasting effects.

[0010] Adeno-associated virus vectors (AAV) are often used as CRISPR / Cas9 system expression vectors from the standpoint of safety, etc. However, the Cas9 gene has a large molecular weight, making it difficult to incorporate into AAV.

[0011] Given the above situation, there is a need for new technologies that can control gene expression different from genome editing using antisense nucleic acids or CRISPR / Cas9.

[0012] Therefore, an object of the present invention is to provide a new technique for regulating mRNA splicing.

[0013] As a result of extensive research, the present inventors have found that rodent-specific non-coding RNA is involved in mRNA splicing. Further research led to the discovery that a characteristic secondary structure of a specific sequence of the rodent-specific non-coding RNA serves to attract splicing regulatory factors and stabilize the RNA molecule, and that by using an artificial RNA molecule in which this secondary structure is combined with a sequence complementary to a target mRNA precursor, mRNA splicing can be regulated, thereby solving the above-mentioned problems, and thus the present invention has been completed.

[0014] That is, the present invention provides: [1] an artificial RNA molecule comprising the following (a) and (b), wherein the following (a) and (b) are arranged in this order from the 5' side to the 3' side: (a) a polynucleotide potentially having a secondary structure represented by the following formula (I), or a polynucleotide in which 1 to 3 bases have been substituted, deleted or added among the 7 bases on the 3' side of a polynucleotide potentially having a secondary structure represented by the following formula (I); and (b) a pre-mRNA-targeting polynucleotide comprising a sequence complementary to a target sequence which is a partial sequence of a pre-mRNA. (In the formula, N1 to N 25each independently represents A, C, G, or U; N and N 25 That is, N2 and N 24 That is, N3 and N 23 That is, N4 and N 22 That is, N5 and N 21 That is, N6 and N 20 That is, N8 and N 19 That is, N9 and N 18 That is, N 10 and N 17and (b) form base pairs with each other.), [2] The artificial RNA molecule according to [1] above, which regulates splicing of a pre-mRNA targeted by (b); [3] The artificial RNA molecule according to [1] or [2] above, wherein the base sequence of (a) is the sequence shown in SEQ ID NO: 2 or a sequence having 80% or more sequence identity to the sequence shown in SEQ ID NO: 2; [4] The artificial RNA molecule according to any of [1] to [3] above, wherein the length of (b) is 15 to 300 bases, more preferably 20 to 60 bases; [5] The artificial RNA molecule according to any of [1] to [4] above, wherein the target sequence is an exon sequence; [6] The artificial RNA molecule according to any of [1] to [5] above, wherein the following (c) is located on the 3' side of (b): (c) a polynucleotide comprising a sequence that contributes to terminal stability; [7] The artificial RNA molecule according to [6] above, wherein (c) is a polynucleotide comprising a transcription termination signal sequence recognized by an RNA pol III enzyme; [8] The artificial RNA molecule according to any one of [1] to [7] above, wherein (a) is a polynucleotide that binds to a protein that controls mRNA splicing; [9] The artificial RNA molecule according to any one of [1] to [8] above, wherein the target sequence is a partial sequence of a gene selected from the group consisting of the FAS gene, the dystrophin gene, and the fukutin gene;

[10] The artificial RNA molecule according to any one of [1] to [9] above, wherein the target sequence is a partial sequence of the MAPT gene;

[11] An expression vector comprising a DNA sequence encoding the artificial RNA molecule according to any one of [1] to

[10] above;

[12] A method for regulating mRNA splicing, comprising: (A) preparing an artificial RNA molecule comprising the following (a) and (b), wherein the following (a) and (b) are arranged in this order from the 5' side to the 3' side: (a) a polynucleotide potentially having a secondary structure represented by formula (I), or a polynucleotide in which 1 to 3 bases have been substituted, deleted, or added among the 7 bases on the 3' side of a polynucleotide potentially having a secondary structure represented by formula (I); (b) a pre-mRNA targeting polynucleotide comprising a sequence complementary to a target sequence that is a partial sequence of a pre-mRNA; and(B) contacting the artificial RNA molecule prepared in step (A) with a pre-mRNA to form a complementary strand between the artificial RNA molecule and the pre-mRNA, thereby regulating mRNA splicing;

[13] The method according to

[12] above, wherein the regulation of mRNA splicing is by exon skipping;

[14] The method according to

[12] or

[13] above, wherein the base sequence of (a) is the sequence shown in SEQ ID NO: 2 or a sequence having 80% or more sequence identity with the sequence shown in SEQ ID NO: 2;

[15] The method according to any one of

[12] to

[14] above, wherein the length of (b) is 15 to 300 bases, more preferably 20 to 60 bases;

[16] The method according to any one of

[12] to

[15] above, wherein the target sequence is an exon sequence;

[17] The method according to any one of

[12] to

[16] above, wherein the artificial RNA molecule comprises a polynucleotide comprising a transcription termination signal sequence recognized by an RNA pol III enzyme arranged on the 3' side of (b);

[18] A method for producing mature mRNA, comprising: (A) a step of preparing an artificial RNA molecule comprising the following (a) and (b), wherein the following (a) and (b) are arranged in this order from the 5' side to the 3' side: (a) a polynucleotide potentially having a secondary structure represented by formula (I), or a polynucleotide in which 1 to 3 bases have been substituted, deleted, or added among the 7 bases on the 3' side of the polynucleotide potentially having a secondary structure represented by formula (I); (b) a pre-mRNA-targeting polynucleotide comprising a sequence complementary to a target sequence which is a partial sequence of a pre-mRNA; (B') a step of contacting the artificial RNA molecule prepared in step (A) with a pre-mRNA to form a complementary strand between the artificial RNA molecule and the pre-mRNA; (C) a step of regulating mRNA splicing by step (B') to produce mature mRNA;

[19] The method according to

[18] above, wherein the regulation of mRNA splicing is by exon skipping;

[20] The method according to

[18] or

[19] above, wherein the base sequence of (a) is the sequence shown in SEQ ID NO: 2 or a sequence having 80% or more sequence identity with the sequence shown in SEQ ID NO: 2;

[21] The method according to any one of

[18] to

[20] above, wherein the length of (b) is 15 to 300 bases, more preferably 20 to 60 bases;

[22] The method according to any one of

[18] to

[21] above, wherein the target sequence is an exon sequence; and

[23] The method according to any one of

[18] to

[22] above, wherein the artificial RNA molecule comprises a polynucleotide comprising a transcription termination signal sequence recognized by an RNA pol III enzyme located on the 3' side of (b).

[0015] According to the present invention, an artificial RNA molecule that combines a characteristic secondary structure with a sequence complementary to a target mRNA precursor, and a method using the artificial RNA molecule, are provided, which provide a new technology for regulating mRNA splicing that differs from conventional technologies.

[0016] This is a schematic diagram showing a portion of the secondary structure of 4.5SH RNA predicted by the secondary structure prediction program RNAfold. This is an electrophoresis diagram of RT-PCR performed using primers that specifically amplify three genes containing asSINEB1 in wild-type and 4.5SH knockout mice. From left to right, the results are for Smchd1, Slc25a40, and Smg6. The markers shown in the schematic diagram on the left indicate mRNA containing the new exon in gray on the top row and mRNA not containing the new exon in the bottom row. The exon in question is shown in gray, with black and white on either side. This is a schematic diagram of a reporter equipped with the U6 promoter and mouse 4.5SH sequence. The U6 promoter sequence and 4.5SH sequence are arranged in order from the 5' end of the reporter. This is an electrophoresis diagram showing the results of Northern blotting, confirming that 4.5SH RNA is expressed in human cells by introducing and expressing the promoter shown in Figure 3. Minigenes incorporating two genes with asSINEB1 were introduced into human 4.5SH-expressing cells, and RT-PCR was performed using primers that specifically amplify each gene. From left to right, the results are for Slc25a40 and Smg6. The markers shown in the schematic diagram on the left indicate mRNAs containing the new exon (gray) at the top and mRNAs not containing the new exon (gray) at the bottom. A bar graph shows the skipping efficiency determined from the electrophoresis diagram in Figure 5. The results for Slc25a40 are shown on the left and Smg6 on the right. From top to bottom, the graphs show portions of the DNA base sequence of the plasmids expressing the wild-type 4.5SH (WT), the wild-type Slc25a40 (WT), the mutant 4.5SH (MUT), and the mutant Slc25a40 (MUT). The bases that differ between WT and MUT are shown in gray. This is an electrophoresis diagram of RT-PCR performed by introducing a mutant plasmid and a wild-type plasmid into human cells and using primers that specifically amplify each. 8 is a bar graph showing the skipping efficiency determined from the electrophoresis diagram of Figure 8. From left to right, the diagrams show Slc25a40 wild-type only, both wild-types, Slc25a40 mutant and 4.5SH wild-type, Slc25a40 wild-type and 4.5SH mutant, and both mutants. This diagram shows the exon skipping efficiency of B1 modified 4.5SH (SL-B1-4.5SH).From top to bottom, they are electrophoresis diagrams by BioAnalyzer, electrophoresis diagrams showing the results of confirming RNA expression by Northern blotting, and bar graphs showing exon skipping efficiency. The markers shown in the schematic diagram on the left of the electrophoresis diagram by BioAnalyzer indicate mRNA containing the Slc25a40 novel exon (exon 8.5) in the upper row, and mRNA not containing Slc25a40 exon 8.5 in the lower row. This is an electrophoresis diagram when a minigene for detecting FAS gene exon skipping and wild-type 4.5SH or FAS modified 4.5SH are co-expressed. This is an electrophoresis diagram when a minigene containing Slc25a40 exon 8.5 and wild-type 4.5SH or FAS modified 4.5SH are co-expressed. This is a bar graph showing the skipping efficiency calculated from the electrophoresis diagrams of Figures 11 and 12. This is a schematic diagram of the target sequence of DMD gene modified 4.5SH. This is an electrophoresis diagram when a minigene for detecting DMD gene exon skipping and DMD gene modification 4.5SH are co-expressed. The markers shown in the schematic diagram on the left indicate mRNA containing DMD exon 53 in gray at the top, and mRNA not containing DMD exon 53 at the bottom. This is an electrophoresis diagram using a BioAnalyzer when a minigene for detecting DMD gene exon skipping and DMD gene modification 4.5SH are co-expressed. The upper schematic diagram is a schematic diagram illustrating targeting of DMD gene modification 4.5SH to DMD exon 53. The markers shown in the schematic diagram on the left indicate mRNA containing DMD exon 53 at the top, and mRNA not containing DMD exon 53 at the bottom. This is a schematic diagram of the target sequence of DMD gene modification 4.5SH. This is a diagram showing the results when a minigene for detecting DMD gene exon skipping and DMD gene modification 4.5SH are co-expressed. From the top, it is a BioAnalyzer electrophoresis diagram, an electrophoresis diagram showing the results of confirming RNA expression by Northern blotting, and a bar graph showing exon skipping efficiency. The markers shown in the schematic diagram on the left of the BioAnalyzer electrophoresis diagram indicate mRNA containing DMD exon 53 in the upper row and mRNA not containing DMD exon 53 in the lower row. This is a diagram showing the exon skipping efficiency of MAPT gene modification 4.5SH.From top to bottom, the diagram shows a schematic diagram illustrating targeting of MAPT gene modification 4.5SH to MAPT exon 10, a BioAnalyzer electrophoresis diagram, an electrophoresis diagram showing the results of confirming RNA expression by Northern blotting, and a bar graph showing exon skipping efficiency. The markers shown in the schematic diagram on the left of the BioAnalyzer electrophoresis diagram indicate mRNA containing MAPT exon 10 at the top and mRNA not containing MAPT exon 10 at the bottom.

[0017] Although it is not intended to be bound by theory, the mechanism by which the effects of the present invention are exhibited is thought to be, for example, as follows.

[0018] In the artificial RNA molecule of the present invention, (a) a polynucleotide potentially having a secondary structure represented by formula (I), or a polynucleotide (polynucleotide (a)) in which one to three bases have been substituted, deleted, or added among the seven bases on the 3' side of a polynucleotide potentially having a secondary structure represented by formula (I), forms a strong stem-loop structure, to which a splicing regulator binds, and (b) a pre-mRNA-targeting polynucleotide (polynucleotide (b)) containing a sequence complementary to the target sequence, which is a portion of the pre-mRNA, binds to a target sequence within the pre-mRNA. The splicing regulator bound to polynucleotide (a) then acts to suppress or promote the splicing reaction, resulting in the skipping or inclusion of a portion containing the target sequence, such as an exon, during mRNA splicing.

[0019] <Definition> In this specification, the term "artificial RNA molecule" refers to an RNA molecule that does not exist in nature. Even if an artificially produced RNA molecule corresponds to an artificial RNA molecule containing polynucleotide (a) and polynucleotide (b) of the present invention, if the sequence of the entire molecule exists in nature as is, it is not included in the artificial RNA molecule of the present invention.

[0020] As used herein, the term "pre-mRNA" refers to RNA that is transcribed in the cell nucleus by an RNA polymerase enzyme using DNA as a template and has not yet been subjected to mRNA splicing.

[0021] As used herein, "mRNA splicing" refers to the process of removing introns from a pre-mRNA to produce a mature mRNA from the pre-mRNA. The same applies when simply referring to "splicing."

[0022] As used herein, "exon" refers to a sequence contained in mature mRNA. Those skilled in the art can obtain annotated sequences of exons from publicly accessible databases, such as Ensembl, National Center for Biotechnology Information (NCBI), GenBank, or other NCBI databases, using a computer equipped with software. Specifically, for example, with respect to human genes, those skilled in the art can select genes from the following NCBI search query: http: / / www.ncbi.nlm.nih.gov / gene / ?term=Homo+sapiens[Orgn]. Alternatively, those skilled in the art can view genomes at the Ensembl database (http: / / www.ensembl.org) and obtain information about exons.

[0023] As used herein, the term "intron" refers to a sequence that is not contained in mature mRNA. As with the above-mentioned "exon," a person skilled in the art can obtain annotated sequences of introns from publicly accessible databases such as NCBI, GenBank, or other NCBI databases using a computer equipped with software.

[0024] As used herein, the term "alternative splicing" refers to splicing that removes specific exons.

[0025] In this specification, the term "base pairing" refers not only to so-called Watson-Crick base pairing in which the nucleotides adenine (A) and thymine (T) (or uracil (U)), and guanine (G) and cytosine (C) form hydrogen bonds, but also to wobble base pairing such as between G and U, inosine (I) and U, and I and A.

[0026] As used herein, "exonization" refers to the inclusion of a sequence that is originally an intron in a mature mRNA as a result of regulated mRNA splicing.

[0027] As used herein, the term "novel exon" refers to a sequence that was originally an intron and that was generated by exonization.

[0028] As used herein, the term "stem-loop structure" refers to a secondary structure formed within a single nucleic acid molecule, which is formed by a nucleotide sequence folded by the nucleic acid molecule itself. The stem-loop structure comprises a stem region created by base pairing between a nucleotide sequence located at the 3'-end and a nucleotide sequence located at the 5'-end of the same molecule, and a loop structure region formed by a nucleotide sequence located between the nucleotide sequence located at the 3'-end and the nucleotide sequence located at the 5'-end that constitute the stem region. Those skilled in the art can confirm the formation and strength of a stem-loop structure by thermodynamic simulation using a secondary structure prediction program such as RNA Fold (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi), or experimentally by RNA footprinting assays, nuclear magnetic resonance (NMR) spectroscopy, and crystal structure analysis of nucleic acid molecules.

[0029] The strength of the stem-loop structure can be evaluated by the minimum free energy (MFE), which is an index of secondary structure stability. A smaller MFE value indicates a more stable secondary structure and a stronger stem-loop structure. MFE can be calculated using a secondary structure prediction program such as RNA Fold (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi).

[0030] As used herein, "exon skipping" refers to a form of alternative splicing that results in a mature mRNA that does not contain a particular exon that was originally present.

[0031] As used herein, "exon skipping efficiency" refers to the ratio of the polynucleotide amount (X) of an exon-skipped band to its nucleotide length (L X ), and the polynucleotide amount (Y) and its nucleotide length (L Y ) can be calculated by the following formula: Skipping efficiency (%) = {X / L X ÷(X / L X +Y / L Y ) x 100

[0032] In a first embodiment, the present invention provides an artificial RNA molecule (also referred to as the artificial RNA molecule of the present invention) comprising the following (a) and (b), wherein the following (a) and (b) are arranged in this order from the 5' side to the 3' side: (a) a polynucleotide potentially having a secondary structure represented by the above formula (I), or a polynucleotide in which 1 to 3 bases have been substituted, deleted or added among the 7 bases on the 3' side of a polynucleotide potentially having a secondary structure represented by the above formula (I); and (b) a pre-mRNA-targeting polynucleotide comprising a sequence complementary to a target sequence which is a partial sequence of a pre-mRNA.

[0033] Hereinafter, in this specification, (a) a polynucleotide potentially having a secondary structure represented by formula (I), or a polynucleotide in which 1 to 3 bases have been substituted, deleted, or added among the 7 bases on the 3' side of a polynucleotide potentially having a secondary structure represented by formula (I) below, may be simply referred to as a "polynucleotide (a)," (b) a pre-mRNA-targeting polynucleotide containing a sequence complementary to a target sequence that is a portion of a pre-mRNA sequence may be simply referred to as a "polynucleotide (b)," and (c) a polynucleotide containing a sequence that contributes to terminal stability, as described below, may be simply referred to as a "polynucleotide (c)."

[0034] <Polynucleotide (a)> Polynucleotide (a) is a polynucleotide potentially having a secondary structure represented by the following formula (I), or a polynucleotide in which 1 to 3 bases have been substituted, deleted, or added among the 7 bases on the 3' side of a polynucleotide potentially having a secondary structure represented by the following formula (I): (In the formula, N1 to N 25 each independently represents A, C, G, or U; N and N 25 That is, N2 and N 24 That is, N3 and N 23 That is, N4 and N 22 That is, N5 and N 21 That is, N6 and N 20 That is, N8 and N 19 That is, N9 and N 18 That is, N 10 and N 17 and form base pairs, respectively.)

[0035] The polynucleotide (a) has a secondary structure that is stable under physiological conditions, and has N1 to N2 as shown in formula (I). 25 It forms a strong stem-loop structure related to

[0036] N1 to N 25 N1 to N2 form a strong stem-loop structure by base pairing within a single RNA molecule. 10 and N 17 ~N 25 However, it is preferable that N1 and N2 are rich in guanine (G) and cytosine (C) or G and uracil (U). 25, N2 and N 24 , N3 and N 23 , N4 and N 22 , N5 and N 21 , N6 and N 20 , N8 and N 19 , N9 and N 18 , and N 10 and N 17 The nine base pairs formed in are preferably those formed by G and C or G and U. Of these nine base pairs, the number of base pairs formed by G and C or G and U is preferably 5 to 9, more preferably 6 to 9, even more preferably 7 to 9, still more preferably 8 to 9, even more preferably 5 to 8, still more preferably 6 to 8, particularly preferably 7 to 8, and most preferably 8. 11 ~N 16 is preferably RYRRYR to form a loop structure, where R represents a purine base (A or G) and Y represents a pyrimidine base (C or U).

[0037] N shown in formula (I) 25 The seven bases closest to the 3'-side do not need to be bases that form a stem-loop structure, and one to three bases may be substituted, deleted, or added. The base substitution, deletion, or addition may be made to any of the seven bases represented by formula (I), with one to two being preferred, and one being more preferred. In the case of substitution, it is preferable that a purine base is substituted with another purine base, and a pyrimidine base is substituted with another pyrimidine base. In the case of addition, it is particularly preferable that a base is added to the U at the 3'-end.

[0038] The sequence (primary structure) of polynucleotide (a) is not particularly limited as long as it is a base sequence that potentially has the secondary structure represented by formula (I), but it preferably contains the sequence represented by SEQ ID NO: 2 or a sequence having 80% or more sequence identity to the sequence represented by SEQ ID NO: 2. The sequence represented by SEQ ID NO: 2 is rich in G and C and can form a strong stem-loop structure represented by formula (I) as described below ( FIG. 1 ).

[0039] It is more preferable that polynucleotide (a) has a sequence identity of 85% or more with the sequence shown in SEQ ID NO: 2, even more preferably 90% or more, particularly preferably 92% or more, and most preferably 95% or more.

[0040] The MFE of the stem-loop structure of polynucleotide (a) is preferably -10.00 kcal / mol or less, more preferably -11.00 kcal / mol or less, even more preferably -12.00 kcal / mol or less, particularly preferably -12.80 kcal / mol or less, and most preferably -12.90 kcal / mol or less. It is believed that the strength of the stem-loop structure within the above range stabilizes the RNA molecule.

[0041] The MFE of a stem-loop structure can vary depending on the number of base pairs in the stem region (the length of the complementary strand), the number of mismatched or bulged bases, the number of bases forming the loop structure, and the like.

[0042] Polynucleotide (a) is thought to contribute to the stabilization of the artificial RNA molecule by including the stem-loop structure and the subsequent 3'-terminal base as described above. Polynucleotide (a) is also thought to play a role in recruiting splicing regulatory factors.

[0043] As used herein, the term "splicing control factor" refers to all factors present in the body that control mRNA splicing, and specifically includes proteins and nucleic acids that control mRNA splicing, as well as complexes of proteins and RNA.

[0044] Polynucleotide (a) is thought to have the function of attracting and binding to splicing regulatory factors. The splicing regulatory factors that bind to polynucleotide (a) determine how the artificial RNA molecule of the present invention regulates mRNA splicing, i.e., what type of alternative splicing will occur.

[0045] More specifically, splicing regulatory factors that bind to polynucleotide (a) include proteins that constitute the small nuclear RNA U2 complex and their binding proteins. More specifically, SF3b1, U2AF2, Nono, Sfpq, Hnrnpf, etc. are mentioned. In addition, from the results of mass spectrometry, it is presumed that Hnrnpm, Hnrnpa1, Hnrnpa2b1, Ddx5, Hnrnpa3, Fus, Hnrnpa0, Pabpc1, Snrpa, Dhx9, Myef2, Ddx17, Hnrnph1, Prpf8, Rps3a, Rbm14, etc. bind.

[0046] The functions of these splicing regulatory factors include suppressing sequences such as splice donor or splice acceptor sequences necessary for splicing reactions, suppressing exon inclusion signals, and accurately recognizing intron sequences.

[0047] A specific example of polynucleotide (a) is the sequence (SEQ ID NO: 2) discovered in 4.5SH RNA (hereinafter also referred to as 4.5SH), a non-coding RNA specific to rodents. It was discovered that the sequence shown in SEQ ID NO: 2 in 4.5SH forms a stem-loop structure as a secondary structure that is stable under physiological conditions, attracts splicing regulatory factors, and is an essential sequence for regulating mRNA splicing of the pre-mRNA sequence targeted by 4.5SH. The sequence targeted by 4.5SH and the regulation of its mRNA splicing will be described later. 4.5SH is transcribed from DNA by RNA polymerase III enzyme (RNA pol III enzyme).

[0048] The sequence corresponding to polynucleotide (a) in 4.5SH does not form a complementary strand with the target sequence, and is therefore thought to attract mRNA splicing inhibitors and inhibit accurate intron recognition. Furthermore, the sequence shown in SEQ ID NO: 2 is thought to contribute to the stability of 4.5SH as a whole by forming a strong stem-loop structure, as shown in formula (I) and Figure 1.

[0049] The polynucleotide (a) of the present invention will be described below with reference to the drawings, taking as an example the sequence shown in SEQ ID NO: 2 as a specific example.

[0050] Figure 1 shows the secondary structure of a portion of the sequence shown in SEQ ID NO: 2 in 4.5SH predicted by RNA Fold (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi). In Figure 1, G, which is the terminal of the stem-loop structure, corresponds to the 5'-terminal base in formula (I) above.

[0051] As used herein, "Xth base" refers to the Xth base counting from the 5' side. In FIG. 1 , the sequence shown by SEQ ID NO: 2 in 4.5SH is such that the first G and the 25th U are base-paired, the second C and the 24th G, the third C and the 23rd G, the fourth G and the 22nd C, the fifth G and the 21st C, the sixth U and the 20th G, the eighth G and the 19th C, the ninth U and the 18th A, and the tenth G and the 17th C form a stem region. The 11th to 16th bases are unpaired bases, forming a loop structure.

[0052] 1, the bases from A at position 26 to U at position 32 are not bases that form a stem-loop structure. In addition, in 4.5SH, the bases from A at position 26 to U at position 32 in the sequence shown in SEQ ID NO: 2 are not bases that form a stem-loop structure, nor are they bases that form a complementary strand with the target sequence of 4.5SH described below.

[0053] <Polynucleotide (b)> Polynucleotide (b) is a pre-mRNA-targeting polynucleotide that contains a sequence complementary to a target sequence, which is a partial sequence of a pre-mRNA. Any sequence can be designed for polynucleotide (b) depending on the target sequence.

[0054] Polynucleotide (b) contains a sequence complementary to a target sequence, which is a partial sequence of a pre-mRNA. That is, polynucleotide (b) forms a complementary strand with the target sequence. The formation of a complementary strand does not require that the two nucleic acid molecules be completely (100%) complementary.

[0055] A complementary sequence refers to a sequence that allows two nucleic acid molecules to complement each other through base pairing. For example, the sequence may be 80 to 100% complementary (e.g., a sequence in which 4 or 5 of 5 bases are complementary) or 90 to 100% identical (e.g., a sequence in which 9 or 10 of 10 bases are complementary).

[0056] The complementarity of the base sequence between polynucleotide (b) and the target sequence is preferably 80% or more, more preferably 85% or more, and even more preferably 90% or more.

[0057] From the viewpoints of the stability of the complementary strand to the target sequence and the specificity of the complementary strand to the target sequence, the base length of polynucleotide (b) is preferably 15 bases or more, more preferably 20 bases or more, even more preferably 40 bases or more, and particularly preferably 50 bases or more. From the viewpoint of ease of synthesis of the artificial RNA molecule, the base length of polynucleotide (b) is preferably 300 bases or less, more preferably 200 bases or less, even more preferably 100 bases or less, particularly preferably 80 bases or less, and most preferably 60 bases or less.

[0058] The stability of the complementary strand between polynucleotide (b) and the target sequence can vary depending not only on the complementarity of the base sequences, but also on the number of hydrogen bonds between the bases (for example, the number of hydrogen bonds between a G and C pair is greater than that between an A and U pair) and the position of the non-complementary sequence (whether it is in the middle or at the end of the complementary strand, etc.).

[0059] The target sequence of polynucleotide (b) is not particularly limited as long as it is a partial nucleotide sequence of a precursor mRNA that serves as a target for regulating mRNA splicing, but is preferably an exon sequence of an exon whose presence or absence in the mature mRNA changes the function of the protein encoded by the mRNA. In this case, the exon sequence may be the entire nucleotide sequence of the exon or a partial nucleotide sequence of the exon, depending on the length of the targeted exon. Furthermore, the target sequence of polynucleotide (b) may include, in addition to the exon sequence, a portion of the intron sequence located within 300 bases upstream (5' side) or downstream (3' side) of the exon, or may be only a portion of the intron sequence excluding the exon sequence.

[0060] The polynucleotide (b) may contain multiple sequences complementary to multiple target sequences, respectively. Such multiple target sequences are preferably selected from a region including the same exon and 300 bases upstream or downstream thereof, but are not limited thereto.

[0061] When the target sequence is an exon sequence and the exon sequence is equal to or longer than the average exon length (approximately 150 bases), polynucleotide (b) preferably contains multiple sequences complementary to multiple target sequences. In such cases, the base length of polynucleotide (b) is preferably 60 bases or more, more preferably 90 bases or more, and even more preferably 100 bases or more. It is also preferably 400 bases or less, more preferably 300 bases or less.

[0062] <Polynucleotide (c)> In addition to the above-mentioned polynucleotide (a) and polynucleotide (b), the artificial RNA molecule of the present invention preferably contains a polynucleotide (polynucleotide (c)) containing a sequence that contributes to the stability of the terminal end, and this polynucleotide (c) is preferably located on the 3' side of polynucleotide (b).

[0063] In order for the artificial RNA molecule of the present invention to function as a non-coding RNA, the polynucleotide (c) is preferably a polynucleotide containing a transcription termination signal sequence recognized by an RNA pol III enzyme. The transcription termination signal sequence recognized by an RNA pol III enzyme is a sequence consisting of seven or more uracils (U) linked together.

[0064] The transcription termination signal sequence recognized by RNA pol III enzymes not only functions as a signal to terminate transcription when non-coding RNA is transcribed from DNA, but may also contribute to the stabilization of the entire non-coding RNA. In addition to the transcription termination signal sequence recognized by RNA pol III enzymes, the structure of the 3' end of a non-coding RNA may also contribute to the stabilization of the entire non-coding RNA (see, for example, Trends in Genetics, 2007 Dec; vol23: 614-622, Biochim Biophys Acta. 2013 Mar; 1829(3-4): 318-330). The 3' end structure of non-coding RNA may include a miRNA sequence or a self-cleaving ribozyme encoded upstream of a transcription termination signal sequence recognized by RNA pol III enzyme (see, for example, Trends in Genetics, 2007 Dec; vol23: 614-622; Biochim Biophys Acta. 2013 Mar; 1829(3-4): 318-330).

[0065] <Artificial RNA molecule> The artificial RNA molecule of the present invention regulates mRNA splicing of a pre-mRNA targeted by polynucleotide (b). Regulation of mRNA splicing includes intron retention and correction of alternative splicing, of which correction of alternative splicing is preferred. Correction of alternative splicing includes induction of exon inclusion or induction of exon skipping (exon skipping). Of these, induction of exon skipping is preferred, since the function of 4.5SH is to suppress new exon formation due to exon skipping.

[0066] In the present invention, at least one polynucleotide (a) and at least one polynucleotide (b) are arranged in this order from the 5' to the 3' side. Furthermore, when polynucleotide (c) is included, polynucleotide (c) is arranged on the 3' side of polynucleotide (a) and polynucleotide (b).

[0067] Two or more of each of polynucleotide (a), polynucleotide (b), and polynucleotide (c) may be arranged, or one of each may be arranged. Since polynucleotide (a) is a sequence essential for regulating mRNA splicing, arranging two or more polynucleotides (a) may further enhance the effects of the present invention. When two or more polynucleotides (a) are arranged, at least two structures represented by formula (I) may be arranged via a multi-branched loop. Furthermore, even if three or more polynucleotides (a) are arranged, further enhancement of the effect is unlikely, and from the viewpoint of the stability of the artificial RNA molecule, it is preferable to arrange three or less. When two or more polynucleotides (a) are arranged, it is preferable that the second polynucleotide (a) is arranged on the 3' side of polynucleotide (b).

[0068] A spacer may or may not be present between each of the polynucleotides (a), (b), and (c). When a spacer is present, the base length of the spacer may be, for example, 200 bases or less, 150 bases or less, 100 bases or less, 50 bases or less, 40 bases or less, or 30 bases or less.

[0069] The nucleic acid in the artificial RNA molecule of the present invention may be a natural or modified nucleic acid. Modified nucleic acids may include, for example, nucleic acids in which the OH group at the 2'-position of ribose is substituted with an F group or an OMe group, phosphorothioate nucleic acids, morpholino nucleic acids, bridged nucleic acids (LNA), etc., from the viewpoint of increasing resistance to nucleases and hydrolysis. Furthermore, from the viewpoint of reducing cytotoxicity, pseudouridine (Ψ), N1-methylpseudouridine (N1mΨ), etc., may be contained instead of uridine (U). The positions of the modified nucleic acids may be all or part of the nucleic acid, and if part of the nucleic acid, they may be randomly positioned in any proportion.

[0070] <5'-end> To stabilize the artificial RNA molecule of the present invention, a structure commonly used in genetic engineering can be added to the 5'-end. For example, a triphosphate or a cap structure may be added.

[0071] <3'-Terminal> A structure commonly used in genetic engineering can be added to the 3'-terminus of the artificial RNA molecule of the present invention to stabilize the molecule. For example, a phosphate group may be added.

[0072] <Other Sequences> The RNA molecule of the present invention may contain polynucleotides other than the above-mentioned polynucleotides (a), (b), and (c). Examples of other sequences include polynucleotides for gene expression and drug delivery. More specifically, examples of such other sequences include polynucleotides containing a promoter sequence of an expression vector, polynucleotides containing a transcription termination sequence of an expression vector, polynucleotides containing a sequence for avoiding degradation by RNA nuclease enzymes, and polynucleotides containing a sequence with affinity for a splicing regulatory factor.

[0073] <Target Gene> The artificial RNA molecule of the present invention targets a pre-mRNA using polynucleotide (b), thereby regulating the splicing of the pre-mRNA, thereby enabling the control of the gene (target gene) that is translated and expressed. Examples of such target genes include the FAS gene, dystrophin gene (DMD), tau (MAPT) gene, fukutin gene (FKTN), survival motor neuron (SMN) 2 gene, myotonin protein kinase (DMPK) gene, IKBKAP gene, GFAP gene, titin gene (TTN), C1-INH gene (SERPING1 gene), ishat homolog (EYS) gene, and huntingtin gene (HTT). The target gene may be a gene known to form multiple isoforms from a single gene by alternative splicing, or may be a gene for which isoforms have not generally been identified in vivo. Here, examples of genes known to form multiple isoforms from a single gene by alternative splicing include the FAS gene and the tau (MAPT) gene. Examples of genes for which isoforms have not generally been identified in vivo include the dystrophin gene (DMD) and the fukutin gene (FKTN).

[0074] <Expression Vector> The present invention also provides an expression vector containing a DNA sequence encoding the above-mentioned artificial RNA molecule of the present invention. By using such an expression vector, the artificial RNA of the present invention can be expressed in vivo and used to regulate mRNA splicing of a target gene.

[0075] Expression vectors include plasmids, bacteriophages, viral vectors, and cosmids.

[0076] Examples of the plasmid include PCDNA3 and PCDNA5FRT / TO.

[0077] Viral vectors are vectors based on one or more nucleic acid sequences comprising a viral genome, including adenoviral vectors, adeno-associated viral vectors (AAV), retroviral vectors, and lentiviral vectors.

[0078] In the present invention, when a viral vector is used as an expression vector, a parvovirus vector such as an AAV vector is preferred. Parvoviruses are small viruses with a single-stranded DNA genome. AAV belongs to the parvovirus family.

[0079] The expression promoter possessed by the expression vector may be any promoter for the RNA pol III enzyme, since the artificial RNA molecule of the present invention is transcribed by the RNA pol III enzyme. Specific examples include the U6 promoter and the H1 promoter, with the U6 promoter being particularly preferred.

[0080] An expression vector containing a DNA sequence encoding an artificial RNA molecule of the present invention can be obtained by chemically synthesizing the artificial RNA molecule of the present invention and all expression vectors such as expression promoters and plasmids, or by chemically or biochemically synthesizing the artificial RNA molecule of the present invention and expression promoters and recombining them into an expression vector such as a virus or plasmid.

[0081] A method for recombining an expression vector so that it contains a DNA sequence encoding the artificial RNA of the present invention can be a method commonly used in molecular biology, such as a method using restriction enzymes and ligase or a method using Gibson assembly.

[0082] In a second embodiment, the present invention provides a method for regulating mRNA splicing, comprising: (A) preparing an artificial RNA molecule comprising the following (a) and (b), wherein the following (a) and (b) are arranged in this order from the 5' side to the 3' side; (a) a polynucleotide potentially having a secondary structure represented by formula (I), or a polynucleotide in which 1 to 3 bases have been substituted, deleted, or added among the 7 bases on the 3' side of a polynucleotide potentially having a secondary structure represented by formula (I); (b) a pre-mRNA-targeting polynucleotide comprising a sequence complementary to a target sequence, which is a partial sequence of a pre-mRNA; and (B) contacting the artificial RNA molecule prepared in step (A) with a pre-mRNA to form a complementary strand between the artificial RNA molecule and the pre-mRNA, thereby regulating mRNA splicing (also referred to as the regulating method of the present invention).

[0083] <Step (A)> Step (A) is a step of preparing an artificial RNA molecule. The step of preparing the artificial RNA molecule of the present invention is not particularly limited and may include a method of chemically synthesizing an RNA molecule or a method of synthesizing by a biochemical technique. Examples of chemical synthesis methods include the phosphoramitide method and its modifications, and the H-phosphonate method and its modifications. Examples of biochemical synthesis methods include a method of synthesizing an RNA molecule using an in vitro transcription system (enzymatic synthesis method). Furthermore, artificial RNA manufactured on a contract basis can also be applied to the present invention.

[0084] <Step (B)> Step (B) is a step in which the artificial RNA molecule prepared in step (A) is contacted with a pre-mRNA to form a complementary strand between the artificial RNA molecule and the pre-mRNA, thereby regulating mRNA splicing. The method of contacting the artificial RNA molecule with the target pre-mRNA can be any method commonly used in the art, such as introducing the artificial RNA molecule into a cell ex vivo or administering the artificial RNA molecule to a living body.

[0085] Methods for introducing artificial RNA molecules into cells ex vivo can be known, such as lipofection, liposome, electroporation, calcium phosphate co-precipitation, microinjection, gene gun, and reverse transfection.

[0086] Here, mRNA splicing typically proceeds via a two-step mechanism. In the first step, the 5' splice site is cleaved, generating a free 5' exon and a lariat intermediate. In the second step, this 5' exon is joined to the 5' exon, resulting in the release of the intron as a lariat product. These reactions are catalyzed by the spliceosome (see, e.g., Moore, MJ, PA Sharp (1993) Nature, 365:364-368).

[0087] How mRNA splicing is regulated depends on the type of splicing regulator recruited by polynucleotide (a), the target sequence targeted by polynucleotide (b), and the like.

[0088] Specific examples of methods for regulating mRNA splicing include methods for inducing intron retention and methods for correcting alternative splicing, with methods for correcting alternative splicing being preferred. Methods for correcting alternative splicing include methods for inducing exon inclusion or exon skipping (exon skipping), with methods for inducing exon skipping being preferred.

[0089] The regulation of mRNA splicing can be confirmed by RT-PCR, etc. Specifically, total RNA is extracted from cells or tissues into which the artificial RNA molecule of the present invention has been introduced, and cDNA is obtained by reverse transcription using reverse transcriptase to purify the mRNA, and PCR is performed using PCR primers that specifically amplify the target sequence, thereby confirming that mature mRNA in which splicing of the target sequence has been regulated has been obtained.

[0090] In a third embodiment, the present invention provides a method for producing mature mRNA, comprising: (A) a step of preparing an artificial RNA molecule comprising the following (a) and (b), wherein the following (a) and (b) are arranged in this order from the 5' side to the 3' side: (a) a polynucleotide potentially having a secondary structure represented by formula (I), or a polynucleotide in which 1 to 3 bases have been substituted, deleted, or added among the 7 bases on the 3' side of a polynucleotide potentially having a secondary structure represented by formula (I); (b) a pre-mRNA-targeting polynucleotide comprising a sequence complementary to a target sequence, which is a partial sequence of a pre-mRNA; (B') a step of contacting the artificial RNA molecule prepared in step (A) with a pre-mRNA to form a complementary strand between the artificial RNA molecule and the pre-mRNA; and (C) a step of regulating mRNA splicing by step (B') to produce mature mRNA (also referred to as the production method of the present invention).

[0091] The mature mRNA obtained by the production method of the present invention can be used in gene therapy using in vivo and ex vivo approaches. An example of an in vivo approach is an embodiment in which an artificial mRNA molecule of the present invention is administered into a living body, where it is incorporated into the nucleus of a cell, and the mature mRNA produced in the body remains there, resulting in the production of a normal protein. An example of an ex vivo approach is an embodiment in which cells or tissues within a living body are removed and an artificial mRNA molecule of the present invention is administered thereto, where it is incorporated into the nucleus of a cell, where the mature mRNA produced in the cell remains there, resulting in the production of a normal protein, and the cells or tissues are then returned to the patient.

[0092] <Step (B')> Step (B') is a step of contacting the artificial RNA molecule prepared in step (A) with a pre-mRNA to form a complementary strand between the artificial RNA molecule and the pre-mRNA. As with step (B) in the regulation method of the present invention, step (B') can be performed using a method commonly used in the technical field, such as a method of introducing an artificial RNA molecule into a cell ex vivo or a method of administering an artificial RNA molecule to a living body.

[0093] <Step (C)> Step (C) is a step in which mRNA splicing is regulated by step (B') to produce mature mRNA. The regulation of mRNA splicing by step (B') to produce mature mRNA can be confirmed by RT-PCR or the like, as in the regulation method of the present invention.

[0094] The above description of the artificial RNA molecule of the present invention is similarly applied to the description of the artificial RNA molecule in the regulation method of the present invention and the production method of the present invention. Furthermore, the applicable content of the description of the regulation method of the present invention and the production method of the present invention (particularly the description of the above step (A)) is similarly applied to each embodiment.

[0095] [Pharmaceutical Composition] The present invention also provides a pharmaceutical composition containing the artificial RNA molecule of the present invention or the mature mRNA obtained by the production method of the present invention. This pharmaceutical composition can be used for the treatment or prevention of diseases that can be treated by regulating mRNA splicing.

[0096] As a drug delivery system (DDS) for such a pharmaceutical composition, any method commonly used in this technical field can be used without particular limitation, and examples thereof include a method using an expression vector, a method using lipid nanoparticles (LNPs), and a method using chemical modification.

[0097] Specific examples of methods using expression vectors include viral vectors and non-viral vectors such as plasmids and bacteria. Among these, viral vectors are widely used in gene therapy because they can introduce and deliver drugs with relatively high efficiency. Furthermore, among viral vectors, AAV vectors are preferably used from the viewpoint of safety. When the artificial RNA molecule of the present invention is loaded into AAV, multiple copies can be loaded. Therefore, the expression level of RNA can be increased, which is expected to enhance the effects of the present invention.

[0098] Lipid nanoparticle (LNP) delivery is a drug delivery method in which RNA molecules are encapsulated in LNPs. The RNA molecules are packaged in LNPs, which protect them from RNA degrading enzymes, enter cells, and are released into the cytoplasm. LNPs enable efficient delivery of chemically modified or unmodified RNA molecules. The production of LNPs requires PEGylated lipids, cholesterol, neutral phospholipids, etc.

[0099] Methods using chemical modification involve chemically modifying the nucleotides that make up an artificial RNA molecule or mature mRNA, and specific examples include methods of adding a peptide to a nucleotide, adding a 2'O-ME modification, a phosphorothioate modification, adding a pseudouridine, etc. Such chemical modifications can protect the RNA molecule from RNases, enable specific delivery to target tissues or cells, and enable stronger binding to the target sequence.

[0100] Diseases that can be treated by regulating mRNA splicing include systemic lupus erythematosus (SLE), autoimmune lymphoproliferative syndrome (ALPS), Dusenne muscular dystrophy (DMD), frontotemporal lobe dementia (FTDP-17), Fukuyama muscular dystrophy, myotonic dystrophy, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), familial dysautonomia (FD), Alexander disease, Angelman syndrome (AS), idiopathic dilated cardiomyopathy (DCM), Parkinson's disease, hereditary angioedema (HAE), retinitis pigmentosa, Huntington's disease, chronic kidney disease, non-alcoholic steatohepatitis (NASH), thalassemia, and various cancers.

[0101] The present invention will be explained in more detail below based on specific experimental examples and examples, but the present invention is not limited to these experimental examples and examples.

[0102] Experimental Example 1: Generation of Knockout Mice First, 4.5SH knockout mice were generated. Knockout mice were generated according to a standard method for large-scale genome deletion / insertion using the CRISPR / Cas9 system (see, for example, Luqing Zhang et al. (2015) PLoS One, Mar 24;10(3)). Specifically, a gene cassette containing a drug resistance gene with sequences complementary to both ends of the mouse 4.5SH gene cluster was designed and isolated, and the isolated gene was introduced into mouse ES cells by electroporation. To increase the efficiency of homologous recombination, CRISPR / Cas9, which cleaves the terminal portion of the complementary sequence, was simultaneously introduced.

[0103] Chimeric mice were produced from the established ES cells and mated with wild-type mice to obtain heterozygous recombinant mice. The heterozygous mice were mated with each other to obtain blastocysts of 4.5SH knockout mice. 4.5SH knockout ES cells were obtained from the blastocysts. RNA was extracted from the ES cells of the 4.5SH knockout mice, and RT-PCR was performed using primers that specifically amplify 4.5SH, with cDNA synthesized from the RNA as a template. The results showed that 4.5SH was significantly reduced in the 4.5SH knockout mice.

[0104] Experimental Example 2: RNA-Seq Analysis Total RNA was prepared from the established ES cells of 4.5SH knockout mice and wild-type mice using Trizol. RNA-Seq analysis was performed on the prepared total RNA. Sequencing was performed using an Illumina HiSeq4000. Analysis software was used: rMATS 4.1.1, and changes in alternative splicing were compared between the ES cells of 4.5SH knockout mice and the cells of wild-type mice, for each of the three groups.

[0105] The results of a comparison of five types of statistically significant alterations in alternative splicing between ES cells from 4.5SH knockout mice and wild-type mouse cells at a false discovery rate (FDR) of less than 0.05 are shown in Table 1. The five types of alternative splicing refer to cassette exon type (SE), alternative 5' splice site type (A5SS), alternative 3' splice site type (A3SS), mutually exclusive exon type (MXE), and intron retention type (IR).

[0106] RNA-Seq analysis revealed that there were 293 exons that were specifically expressed in ES cells of 4.5SH knockout mice.

[0107] Next, we analyzed the characteristics of exons specifically expressed in ES cells of 4.5SH knockout mice based on the results of RNA-Seq analysis. We found that many of the exons specifically expressed in ES cells of 4.5SH knockout mice matched the sequence of asSINEB1. Here, asSINEB1 is the complementary strand (as) of SINEB1. Therefore, we hypothesized that in rodents, 4.5SH suppresses the exonization of asSINEB1 present within introns, and we demonstrated this through the following experimental example.

[0108] Experimental Example 3: Confirmation of exon skipping in mouse cells RNA was extracted from ES cells of male and female 4.5SH knockout mice and ES cells of wild-type mice. Using cDNA synthesized from the RNA as a template, RT-PCR was performed using primers (SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, and SEQ ID NOs: 7 and 8) that specifically amplify three genes carrying asSINEB1 (Smchd1 exons 21-22, Slc25a40 exons 8-9, Smg6 exons 8-9). As a result, asSINEB1 is present within the introns between exon 24 and exon 25 of Smchd1 (NM_028887.3), between exon 8 and exon 9 of Slc25a40 (NM_178766.5), and between exon 8 and exon 9 of Smg6 (NM_001002764.1). Therefore, if these asSINEB1s are converted into exons and become new exons by knocking out 4.5SH, this would be one way to prove the above hypothesis.

[0109] As a result, exon skipping of novel exons was detected for Smcdh1, Slc25a40, and Smg6 in wild-type mouse cells, whereas no exon skipping of novel exons was detected in ES cells of 4.5SH knockout mice (Figure 2). Thus, novel exons were skipped in the presence of 4.5SH, but not in the presence of 4.5SH knockout, confirming the appearance of novel exons. Hereinafter, in the present specification and drawings, these novel exons are also referred to as Smcdh1 Ex24.5, Slc25a40 Ex8.5, and Smg6 Ex8.5, respectively, or as Smcdh1 exon 24.5, Slc25a40 exon 8.5, and Smg6 exon 8.5, respectively.

[0110] Experimental Example 4: Confirmation of exon skipping in a human cell overexpression system To further substantiate the above hypothesis, exon skipping was confirmed in a human cell overexpression system by confirming the activity of 4.5SH using human cells that do not naturally possess 4.5SH.

[0111] A plasmid carrying the U6 promoter and the mouse 4.5SH sequence (Figure 3) was constructed, and the plasmid (pUC57) was introduced into human cells (HEK293 cells) by liposome transfection. Expression of 4.5SH RNA was confirmed by Northern blotting, and human 4.5SH-expressing cells were isolated (Figure 4).

[0112] A DNA sequence (SEQ ID NO: 9) containing wild-type Slc25a40Ex8.5 and 100 bases upstream and downstream thereof, and a DNA sequence (SEQ ID NO: 10) containing wild-type Smg6Ex8.5 and 100 bases upstream and downstream thereof, were inserted into the SacII sequence in an intron of a plasmid derived from an adenovirus late gene (Ad2). The resulting minigenes were then inserted into a plasmid (pcDNA3 BamHI / XbaI). The resulting vector (Ad2 / pcDNA3 BamHI / XbaI, vector consensus sequence: SEQ ID NO: 28) was introduced into human 4.5SH-expressing cells and cultured at 37°C for 24 hours. The cells were then harvested and RNA was extracted. RT-PCR was performed using cDNA synthesized from the RNA as a template and primers (SEQ ID NO: 26 and SEQ ID NO: 27) that specifically amplify both minigenes. As a result, exon skipping was detected for the Slc25a40 exon 8.5 and Smg6 exon 8.5 minigenes (Figure 5). The left columns of Figures 5 and 6 show the results for a control in which the minigenes were transfected into human cells (HEK293 cells) before 4.5SH transfection, instead of into human 4.5SH-expressing cells.

[0113] 10 μL of the PCR reaction product was electrophoresed in a polyacrylamide gel, and the exon skipping efficiency was calculated from each band on the resulting electrophoresis diagram. The polynucleotide amount (X) and nucleotide length (L) of the exon-skipped band were calculated. X ), and the polynucleotide amount (Y) and nucleotide length (L) of the band in which the exon was not skipped. Y ) and the skipping efficiency was calculated using the following formula (Figure 6): Skipping efficiency (%) = {X / L X ÷(X / L X +Y / L Y ) x 100

[0114] From the above, it was found that even in human cells that do not naturally have 4.5SH, in the absence of 4.5SH, a new exon appears within an intron, and in the presence of 4.5SH, the new exon is skipped.

[0115] Experimental Examples 3 and 4 demonstrated that 4.5SH suppresses the exonization of asSINEB1 present in an intron, and that the suppression of exonization is due to exon skipping of a novel exon.

[0116] Experimental Example 5: Examination of base pairing by co-expression using mutant forms In order to examine the base pairing between 4.5SH and asSINEB1, mutant forms of 4.5SH and asSINEB1 were obtained and co-expressed in cells to detect the presence or absence of exon skipping.

[0117] Mutations were introduced into a plasmid expressing native (wild-type) 4.5SH obtained in the same manner as in Experimental Example 4, to obtain a plasmid expressing the mutant form. Specifically, a partial G in the asSINEB1 recognition region of the wild-type 4.5SH sequence was changed to C, and the sequence of mouse 4.5SH was modified so that it would not base pair with a novel exon (exon 8.5) between exons 8 and 9 of Slc25a40 (Figure 7). The resulting plasmid was transformed into Escherichia coli. The transformed E. coli were plated on LB (containing ampicillin) plates, and colonies were allowed to form at 37°C for screening. The DNA sequence of the plasmid carried by ampicillin-resistant E. coli was determined, and a mutant 4.5SH expression plasmid was obtained (Figure 7).

[0118] Mutations were introduced into a plasmid expressing a minigene containing native (wild-type) Slc25a40 exon 8.5, obtained as in Experimental Example 4, to obtain a plasmid expressing the mutant Slc25a40 exon 8.5. Specifically, a DNA fragment (SEQ ID NO: 11) was synthesized in which a partial G in the wild-type Slc25a40 exon 8.5 sequence was changed to C, preventing base pairing with wild-type 4.5SH. The mutation was then inserted into the plasmid by Gibson assembly. The resulting plasmid was transformed into Escherichia coli. The transformed E. coli was plated on LB (ampicillin-containing) plates, and colonies were allowed to form at 37°C for screening. The DNA sequence of the plasmid harbored by ampicillin-resistant E. coli was determined, and a plasmid expressing a minigene containing mutant Slc25a40 exon 8.5 was obtained (Figure 7).

[0119] The mutant and wild-type plasmids were co-transfected into human cells (HEK293 cells) by lipofection. After 24 hours of incubation at 37°C, the cells were harvested and RNA was extracted. RT-PCR was performed using cDNA synthesized from the RNA as a template and primers (SEQ ID NO: 26 and SEQ ID NO: 27) that specifically amplify both minigenes, as used in Experimental Example 4. 10 μL of the PCR reaction product was electrophoresed on a polyacrylamide gel, and the exon skipping efficiency was calculated using the formula described above. The results are shown in Figures 8 and 9. In Figures 8 and 9, the leftmost lane and bar graph represent a sample co-transfected with the plasmid before 4.5SH introduction (so-called empty vector) and a minigene expression plasmid containing wild-type Slc25a40 exon 8.5.

[0120] Exon skipping was detected in cells coexpressing wild-type 4.5SH and a minigene containing wild-type Slc25a40 exon 8.5 (Fig. 8, second lane from the left). In cells coexpressing mutant 4.5SH and a minigene containing wild-type Slc25a40 exon 8.5, or in cells coexpressing wild-type 4.5SH and a minigene containing mutant Slc25a40 exon 8.5, exon skipping was either not detected or the skipping efficiency was lower than that in the coexpression of wild-type variants (Fig. 8, third and fourth lanes from the left). In cells coexpressing mutant 4.5SH and a minigene containing mutant Slc25a40 exon 8.5, exon skipping was detected and the skipping efficiency was equivalent to that in the coexpression of wild-type variants (Fig. 8, fifth lane from the left; skipping efficiency is shown in Fig. 9, fifth lane from the left). This indicates that base pairing between 4.5SH and asSINEB1 is required for exon skipping by 4.5SH.

[0121] Experimental Example 6: Analysis of polynucleotide (a) In order to confirm the importance of polynucleotide (a) in 4.5SH, B1 modified 4.5SH was prepared by replacing the sequence of the polynucleotide (a) portion of 4.5SH (SEQ ID NO: 2) with the sequence of the corresponding portion of SINEB1, which is a homologous sequence of 4.5SH (RNA sequence corresponding to SEQ ID NO: 28), and the expression level in cells and the efficiency of exon skipping were investigated.

[0122] Obtaining B1 modified 4.5SH A mutation was introduced into a plasmid expressing wild-type 4.5SH obtained in the same manner as in Experimental Example 4, to obtain a plasmid expressing B1 modified 4.5SH. Specifically, the sequence of the polynucleotide (a) portion of wild-type 4.5SH (the DNA sequence corresponding to SEQ ID NO: 2) was modified to the DNA sequence of the corresponding portion of SINEB1 (SEQ ID NO: 29), and a plasmid (pUC57) carrying a U6 promoter on the 5' side of this DNA sequence was prepared. The resulting plasmid was transformed into Escherichia coli. The transformed E. coli was plated on an LB (containing ampicillin) plate, and colonies were allowed to form at 37°C and screened. The DNA sequence of the plasmid carried by ampicillin-resistant E. coli was determined, and the B1 modified 4.5SH expression plasmid shown in SEQ ID NO: 30 was obtained.

[0123] The B1-modified 4.5SH expression plasmid obtained above was introduced into human cells (HEK293 cells) by liposome transfection to obtain cells expressing B1-modified 4.5SH. Expression of B1-modified 4.5SH RNA was confirmed by Northern blotting (Figure 10, middle, rightmost lane: SL1-B1-4.5SH).

[0124] Comparison of skipping efficiency between B1 modified 4.5SH and wild-type 4.5SH A minigene containing wild-type Slc25a40 exon 8.5 and 100 bases upstream and downstream thereof, obtained in the same manner as in Experimental Example 4, was introduced into wild-type 4.5SH-expressing cells (obtained in the same manner as the human 4.5SH-expressing cells in Experimental Example 4) and the B1 modified 4.5SH-expressing cells obtained above. After culturing for 24 hours at 37°C, the cells were recovered and RNA was extracted. RT-PCR was performed using cDNA synthesized from the RNA as a template and primers (SEQ ID NO: 26 and SEQ ID NO: 27) that specifically amplify the minigene containing wild-type Slc25a40Ex8.5, which were also used in Experimental Example 4. As a result, the efficiency of exon skipping for Slc25a40 exon 8.5 was significantly reduced in the B1-modified 4.5SH-expressing cells (the rightmost lane in Fig. 10) compared to the wild-type 4.5SH-expressing cells (the center lane in Fig. 10) (lower panel in Fig. 10). Note that the leftmost column in Fig. 10 shows the results for a control in which, instead of using human 4.5SH-expressing cells or B1-modified 4.5SH-expressing cells, the plasmid (so-called empty vector) used in Experiment 4 before introducing mouse 4.5SH was introduced into human cells (HEK293 cells).

[0125] Example 1: FAS gene modified 4.5SH To confirm that exon skipping of asSINEB1 by 4.5SH can be applied to any exon skipping, human FAS gene modified 4.5SH was prepared and exon skipping was investigated.

[0126] Obtaining FAS Gene-Modified 4.5SH: A mutation was introduced into a plasmid expressing wild-type 4.5SH to obtain a plasmid expressing the mutant form. Specifically, a DNA fragment was synthesized in which the sequence of the asSINEB1 recognition region of the wild-type 4.5SH sequence was modified to a DNA sequence complementary to exon 6 of the FAS gene shown in SEQ ID NO: 12, and the DNA fragment was inserted into a plasmid (pUC57) by Gibson assembly. The resulting plasmid was transformed into Escherichia coli. The transformed E. coli was plated on an LB (containing ampicillin) plate, and colonies were formed at 37°C for screening. The DNA sequence of the plasmid carried by the ampicillin-resistant E. coli was determined, and a human FAS gene-modified 4.5SH expression plasmid shown in SEQ ID NO: 13 was obtained.

[0127] <Preparation of a minigene for detecting exon skipping> A DNA fragment containing exon 5, intron 5, exon 6, intron 6, and exon 7 of the FAS gene shown in SEQ ID NO: 14 was amplified by PCR, the PCR product was purified, and a mutation was inserted into a plasmid (pcDNA3 bamHI / xhoI) by Gibson assembly. The resulting plasmid was transformed into Escherichia coli. The transformed E. coli was plated on an LB (containing ampicillin) plate and allowed to form colonies at 37°C for screening. The DNA sequence of the plasmid carried by ampicillin-resistant E. coli was determined, and a minigene for detecting exon skipping of the FAS gene was obtained.

[0128] <Detection of FAS gene exon skipping> The FAS gene modified 4.5SH expression plasmid and the minigene for detecting FAS gene exon skipping were co-transfected into human cells (HEK293 cells) by lipofection. After 24 hours of incubation at 37°C, the cells were harvested and RNA was extracted. RT-PCR was performed using cDNA synthesized from the RNA as a template and reverse and forward primers that bind to pcDNA3 shown in SEQ ID NO: 15 and SEQ ID NO: 16. By using this primer set, the FAS gene endogenous to the cells is not amplified. The RT-PCR conditions are shown in Table 2.

[0129] 10 μL of the PCR reaction product was electrophoresed on a polyacrylamide gel to analyze skipping. The electrophoresis pattern is shown in the right lane of Figure 11. The exon skipping efficiency is shown in Figure 13.

[0130] In cells coexpressing the FAS gene modified 4.5SH expression plasmid and the minigene for detecting FAS gene exon skipping, exon skipping of the FAS gene exon 6 was confirmed (Fig. 11, right lane).Furthermore, the efficiency of exon skipping of the FAS gene exon 6 by the FAS gene modified 4.5SH was 70% (Fig. 13, right bar).

[0131] The left lane of the electrophoresis diagram in Figure 11 shows the results of RT-PCR of RNA extracted from human cells (HEK293 cells) co-expressing the wild-type 4.5SH expression plasmid and the minigene for detecting FAS gene exon skipping. The exon skipping efficiency was significantly lower than that of the FAS gene-modified 4.5SH expression plasmid.

[0132] <Confirmation of exon skipping by FAS gene modified 4.5SH> The FAS gene modified 4.5SH expression plasmid obtained above and a plasmid expressing a minigene containing wild-type Slc25a40 exon 8.5 obtained in the same manner as in Experimental Example 4 were co-transfected into human cells (HEK293 cells) by lipofection. After 24 hours of incubation at 37°C, the cells were harvested and RNA was extracted. RT-PCR was performed using cDNA synthesized from the RNA as a template and primers (SEQ ID NO: 26 and SEQ ID NO: 27) that specifically amplify the minigene also used in Experimental Example 4. 10 μL of the PCR reaction product was electrophoresed on a polyacrylamide gel to analyze skipping. An image of the electrophoresis is shown in the right lane of Figure 12.

[0133] The left lane of the electrophoresis diagram in Figure 12 is an electrophoresis diagram of RT-PCR of RNA extracted from human cells (HEK293 cells) co-expressing a wild-type 4.5SH expression plasmid and a plasmid expressing a minigene containing wild-type Slc25a40 exon 8.5. Comparing the results of co-expressing the wild-type 4.5SH expression plasmid and wild-type Slc25a40 exon 8.5 (left lane in Figure 12 and the leftmost bar in Figure 13) with the results of co-expressing the FAS gene modified 4.5SH and a minigene for detecting FAS gene exon skipping (right lane in Figure 11 and the rightmost bar in Figure 13), it can be seen that the FAS gene modified 4.5SH has FAS gene exon 6 skipping activity equivalent to the skipping activity of wild-type 4.5SH against asSINEB1 (Figures 11, 12, and 13). It is known that the FAS gene has two isoforms in vivo: one containing exon 6 and one not containing exon 6. Therefore, the results of co-expressing the wild-type 4.5SH expression plasmid and the minigene for detecting FAS gene exon skipping (right lane in Figure 11 and the third bar from the right in Figure 13) are thought to indicate the ratio of isoform amounts in vivo.

[0134] Example 2: Human DMD Gene Modification 4.5SH 4.5SH-mediated asSINEB1 exon skipping To confirm that it can be applied to any exon skipping, human dystrophin (DMD) gene modification 4.5SH was created and exon skipping was examined. Because DMD exon 53 is 212 bases long, longer than the FAS gene exon 6 (63 bases), first, the target sequence was divided into four positions (#1, #2, #3, #4) (see Figure 14), and four DMD gene modifications 4.5SH (DMD gene modifications 4.5SH #1 to #4) with different base sequences of polynucleotide (b) were created.

[0135] Obtaining four DMD gene-modified 4.5SH constructs Mutations were introduced into a plasmid expressing wild-type 4.5SH to obtain four types of plasmids expressing mutant forms. Specifically, DNA sequences were designed in which the asSINEB1 recognition region of the wild-type 4.5SH sequence was modified to DNA sequences complementary to exon 53 of the DMD gene as shown in SEQ ID NOs: 17, 18, 19, and 20.

[0136] DNA fragments modified with the DMD sequence complementary to exon 53 of the DMD gene were synthesized, and mutations were inserted into a plasmid (pcDNA3 bamHI / xhoI) by Gibson assembly. The resulting plasmids were transformed into Escherichia coli. The transformed E. coli were plated on LB (containing ampicillin) plates and allowed to form colonies at 37°C for screening. The DNA sequences of the plasmids carried by ampicillin-resistant E. coli were determined, and four types of DMD gene-modified 4.5SH expression plasmids were obtained, as shown in SEQ ID NOs: 21, 22, 23, and 24.

[0137] <Preparation of minigene for detecting exon skipping> A DNA sequence including exon 53 of the DMD gene as shown in SEQ ID NO: 25 and 100 bases upstream and downstream thereof was inserted into the SacII sequence in an intron of a plasmid derived from an adenovirus late gene (Ad2) to obtain a minigene for detecting exon skipping of the DMD gene.

[0138] <Detection of DMD Gene Exon Skipping> The DMD gene modified 4.5SH expression plasmid and a vector (Ad2 / pcDNA3 BamHI / XbaI, vector consensus sequence SEQ ID NO: 28) in which the DMD gene exon skipping detection minigene was inserted into the plasmid were co-transfected into human cells (HEK293 cells) by lipofection. One type of DMD gene modified 4.5SH expression plasmid (plasmid expressing DMD gene modified 4.5SH#1 to #4) and a mixture of all four types were co-transfected with the DMD gene exon skipping detection minigene insertion plasmid. After culturing for 24 hours at 37°C, the cells were harvested and RNA was extracted. RT-PCR was performed using cDNA synthesized from the RNA as a template and primers (SEQ ID NO: 26 and SEQ ID NO: 27) that specifically amplify each minigene, as used in Experimental Example 4. The RT-PCR conditions are shown in Table 3.

[0139] 10 μL of the PCR reaction product was electrophoresed on a polyacrylamide gel to analyze skipping. An image of the electrophoresis pattern is shown in FIG.

[0140] Although the skipping efficiency was not high, skipping was detected in DMD gene exon 53. In particular, in the sample in which DMD gene modification 4.5SH#2 (simply represented as "2" in FIG. 15) and all four DMD gene modifications 4.5SH#1 to #4 were mixed ("Mix" in FIG. 15), bands after exon skipping were confirmed (FIG. 15, 3rd lane from the left and 6th lane from the left).

[0141] Example 2-2: Optimization of human DMD genetic modification 4.5SH <Design of polynucleotide (b) sequence> In order to create a DMD genetic modification 4.5SH with higher skipping efficiency, the target sequence of DMD exon 53 was optimized. The sequence of #4 (SEQ ID NO: 20), which is part of DMD exon 53, contains two transcription termination signal sequences (specifically, TTTT) recognized by the RNA pol III enzyme, and it was found that transcription was terminated. Therefore, the corresponding site was converted to TATT and designated #5 (SEQ ID NO: 31, FIG. 17). Using this DNA sequence, DMD genetic modification 4.5SH #5 was produced in the same manner as in Example 2. Figure 17 shows a schematic diagram of the complementary portion of each sequence to the exon of DMD exon 53.

[0142] <Obtaining DMD genetically modified 4.5SH> DNA fragments were synthesized in which the sequence of the asSINEB1 recognition region of the wild-type 4.5SH sequence was modified to the DNA sequences shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 31 (each a DNA sequence complementary to a part of DMD gene exon 53), and four types of DMD genetically modified 4.5SH #1, #2, #3, or #5 expression plasmids shown in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 32 were obtained.

[0143] The DMD gene-modified 4.5SH expression plasmid and the minigene for detecting DMD gene exon skipping prepared in the same manner as in Example 2 were co-transfected into human cells (HEK293 cells) by lipofection. All possible combinations of the DMD gene-modified 4.5SH expression plasmid were co-transfected with the minigene for detecting DMD gene exon skipping. After culturing for 24 hours at 37°C, the cells were harvested and RNA was extracted. RT-PCR was performed using cDNA synthesized from the RNA as a template and primers (SEQ ID NO: 26 and SEQ ID NO: 27) that specifically amplify the minigene used in Experimental Example 4. The RT-PCR conditions are shown in Table 4.

[0144] 1 μL of the PCR reaction product was analyzed using a BioAnalyzer (Agilent Technologies, Inc.) to calculate the exon skipping efficiency. As a result, in all combinations, skipping was detected, although some skipping efficiencies were not high. The combination with the highest exon skipping efficiency was the DMD gene modified 4.5SH # 2 and DMD gene modified 4.5SH # 5 (lane at the far right of Figure 16). In Figure 16, the leftmost lane shows the results of a sample co-introduced with a plasmid (so-called empty vector) and a minigene for detecting DMD gene exon skipping before introducing mouse 4.5SH in Experimental Example 4. The second lane from the left shows the results of a sample co-introduced with a vector expressing wild-type 4.5SH RNA (the same as the plasmid (pUC57) prepared when obtaining human 4.5SH-expressing cells in Experimental Example 4) and a minigene for detecting DMD gene exon skipping.

[0145] <Optimization of the design of polynucleotide (b) sequence> Here, the DNA sequence (SEQ ID NO: 18) corresponding to the polynucleotide (b) of the present invention among the DMD genetically modified 4.5SH # 2 was near the sequence of the existing antisense nucleic acid drug viltolarsen (see Figure 17). Therefore, the sequence of the polynucleotide (b) of DMD genetically modified 4.5SH # 2 was redesigned to include all of the viltolarsen sequence, and the DMD genetically modified 4.5SH # 6 shown in SEQ ID NO: 33 was designed, and a DNA fragment expressing the sequence of DMD genetically modified 4.5SH # 6 was synthesized, and the DMD genetically modified 4.5SH expression plasmid expressing the DMD genetically modified 4.5SH # 6 shown in SEQ ID NO: 34 was obtained. Furthermore, DMD genetically modified 4.5SH#7 was designed, containing a sequence (sequence number 35) that linked the sequences of polynucleotide (b) of DMD genetically modified 4.5SH#6 and polynucleotide (b) of DMD genetically modified 4.5SH#5, and a DNA fragment that expresses DMD genetically modified 4.5SH#7 was synthesized to obtain a DMD genetically modified 4.5SH expression plasmid that expresses #7 shown in sequence number 36.

[0146] <B1 type human DMD genetically modified 4.5SH (comparative example)> In addition, a comparative example was designed, which is a B1 modified sequence in which the polynucleotide (a) of 4.5SH of DMD genetically modified 4.5SH#7 was replaced with the sequence corresponding to the polynucleotide (a) of SINEB1, which is a homologous sequence of 4.5SH. A DNA fragment expressing the comparative example sequence was synthesized, and a DMD genetically modified B1 modified 4.5SH expression plasmid expressing the comparative example shown in sequence number 37 was obtained.

[0147] The DMD gene modified B1 modified 4.5SH expression plasmid obtained above and the minigene for detecting DMD gene exon skipping obtained in Example 2 were co-transfected into human cells (HEK293 cells) by lipofection. After culturing for 24 hours at 37°C, the cells were recovered and RNA was extracted. RT-PCR was performed using cDNA synthesized from the RNA as a template and primers (SEQ ID NO: 26 and SEQ ID NO: 27) that specifically amplify the minigene for detecting DMD gene exon skipping. The RT-PCR conditions are shown in Table 5.

[0148] As a result, in the combination of DMD gene modification 4.5SH#6 and DMD gene modification 4.5SH#5, the skipping efficiency of DMD exon 53 was 16.49% (lane 4 from the left in Figure 18). And, the skipping efficiency of DMD exon 53 in DMD gene modification 4.5SH#7 was 46.92% (lane 5 from the left in Figure 18).

[0149] The skipping efficiency of DMD exon 53 in the comparative sequence was 5.06%, a significantly reduced skipping efficiency (lane 6 from the left in Figure 18). In Figure 18, the leftmost lane shows the results for a sample co-transfected with a plasmid (so-called empty vector) before 4.5SH introduction and a minigene for detecting exon skipping of the DMD gene, while the second lane from the left shows the results for a sample co-transfected with a vector expressing wild-type 4.5SH RNA (the same as the plasmid (pUC57) prepared to obtain human 4.5SH-expressing cells in Experimental Example 4) and a minigene for detecting exon skipping of the DMD gene. Because exon skipping does not occur in vivo in the DMD gene, the skipping efficiency was 0% in the control transfected with an empty vector.

[0150] Example 3: MAPT Gene Modified 4.5SH To confirm that exon skipping of asSINEB1 by 4.5SH can be applied to any exon skipping, tau (MAPT) gene modified 4.5SH was generated and exon skipping was investigated.

[0151] <Obtaining MAPT Gene-Modified 4.5SH> A DNA fragment was synthesized in which the sequence of the asSINEB1 recognition region of the wild-type 4.5SH sequence was modified to a DNA sequence complementary to exon 10 of the MAPT gene shown in SEQ ID NO: 38. The mutation was then inserted into a plasmid (pcDNA3 bamHI / xhoI) by Gibson assembly. The resulting plasmid was transformed into Escherichia coli. The transformed E. coli was plated on an LB (containing ampicillin) plate, allowed to form colonies at 37°C, and screened. The DNA sequence of the plasmid harbored by ampicillin-resistant E. coli was determined, and a human MAPT gene-modified 4.5SH expression plasmid shown in SEQ ID NO: 39 was obtained. A reporter carrying the U6 promoter and the sequence of the MAPT gene-modified 4.5SH was prepared, and an expression vector (pUC57) carrying the reporter was introduced into human cells (HEK293 cells) by liposome transfection. Expression of MAPT gene modified 4.5SH RNA was confirmed by Northern blotting (middle row, rightmost lane in Figure 20), and MAPT gene modified 4.5SH-expressing cells were obtained.

[0152] <Preparation of a minigene for detecting exon skipping> A DNA fragment containing exons 9, 10, and 11 of the MAPT gene shown in SEQ ID NO: 40 (for details, see Qingming Yu et al., (2004) J Neurochem, Jul;90(1):164-72) was amplified by PCR, the PCR product was purified, and a mutation was inserted into a plasmid (pcDNA3 bamHI / xhoI) by Gibson assembly. The resulting plasmid was transformed into Escherichia coli. The transformed E. coli was plated on an LB (containing ampicillin) plate and allowed to form colonies at 37°C for screening. The DNA sequence of the plasmid carried by ampicillin-resistant E. coli was determined, and a minigene for detecting exon skipping of the MAPT gene was obtained.

[0153] <Detection of MAPT gene exon skipping> The MAPT gene modified 4.5SH expression plasmid and the minigene for detecting MAPT gene exon skipping were co-transfected into human cells (HEK293 cells) by lipofection. After 24 hours of incubation at 37°C, the cells were harvested and RNA was extracted. Using cDNA synthesized from the RNA as a template, RT-PCR was performed using reverse primers that bind to pcDNA3 shown in SEQ ID NO: 41 and SEQ ID NO: 42 and a forward primer that binds to exon 11 of the MAPT gene. By using such a primer set, the MAPT gene endogenous to the cells is not amplified. The RT-PCR conditions are shown in Table 6.

[0154] 1 μL of the PCR reaction product was analyzed using BioAnalyzer (Agilent Technologies), and the exon skipping efficiency was calculated. The results are shown in FIG. 19 .

[0155] In cells co-expressing the MAPT gene modified 4.5SH expression plasmid and the minigene for detecting MAPT gene exon skipping, exon skipping of the MAPT gene exon 10 was confirmed (the lane at the right end of the upper electrophoresis diagram in Figure 19). Furthermore, the exon skipping efficiency of the MAPT gene exon 10 by the MAPT gene modified 4.5SH was 88.78% (4.5SH-asMAPT, the lane at the right end of the lower diagram in Figure 19).

[0156] The lane in the center of the electrophoresis diagram (second from the left) in Figure 19 represents an electrophoresis diagram of RNA extracted from human cells co-expressed with the FAS-modified 4.5SH expression plasmid obtained in Example 1 and a minigene for detecting MAPT gene exon skipping, followed by RT-PCR. The exon skipping efficiency was 64.99%, significantly lower than the skipping efficiency observed when the MAPT gene-modified 4.5SH expression plasmid was expressed (Figure 19, 4.5SH-asFAS). Note that the control on the left side of Figure 19 shows the results of a sample co-transfected with the pre-mutation plasmid (the so-called empty vector) and the minigene for detecting MAPT gene exon skipping. Since the MAPT gene is known to have isoforms containing exon 10 and those not containing exon 10 in vivo, the skipping efficiency of the control transfected with the empty vector is thought to represent the ratio of isoform abundance in vivo.

[0157] According to the present invention, a technology is provided in which artificial RNA molecules containing the sequence of a rodent-specific non-coding RNA molecule control gene expression through the regulation of mRNA splicing, and thus can be used to treat diseases that can be treated by regulating mRNA splicing.

Claims

1. An artificial RNA molecule comprising the following (a) and the following (b), wherein the following (a) and (b) are arranged in this order from the 5' side to the 3' side: (a) A polynucleotide potentially having a secondary structure represented by the following formula (I), or a polynucleotide in which 1 to 3 bases have been substituted, deleted or added among the 7 bases on the 3' side of a polynucleotide potentially having a secondary structure represented by the following formula (I): (b) a pre-mRNA targeting polynucleotide comprising a sequence complementary to a target sequence which is a portion of a pre-mRNA; 【Chemistry 1】 (In the formula, N 1 ~N 25 each independently represents A, C, G, or U; N 1 and N 25 By N 2 and N 24 By N 3 and N 23 By N 4 and N 22 By N 5 and N 21 By N 6 and N 20 By N 8 and N 19 By N 9 and N 18 By N 10 and N 17 and form base pairs.)

2. The artificial RNA molecule according to claim 1, which regulates splicing of a pre-mRNA targeted by (b).

3. 3. The artificial RNA molecule according to claim 1 or 2, wherein the base sequence of (a) is a sequence shown in SEQ ID NO: 2 or a sequence having a sequence identity of 80% or more with the sequence shown in SEQ ID NO:

2.

4. The artificial RNA molecule according to claim 1 or 2, wherein the length of (b) is 15 to 300 bases.

5. The artificial RNA molecule according to claim 1 or 2, wherein the target sequence is an exon sequence.

6. 2. The artificial RNA molecule according to claim 1, further comprising the following (c) located on the 3' side of (b): (c) a polynucleotide containing a sequence that contributes to terminal stability

7. The artificial RNA molecule according to claim 6 , wherein the (c) is a polynucleotide comprising a transcription termination signal sequence recognized by an RNA pol III enzyme.

8. The artificial RNA molecule according to claim 1 or 2, wherein the (a) is a polynucleotide that binds to a protein that controls mRNA splicing.

9. 3. The artificial RNA molecule according to claim 1 or 2, wherein the target sequence is a partial sequence of a gene selected from the group consisting of the FAS gene, the dystrophin gene, and the fukutin gene.

10. The artificial RNA molecule according to claim 1 or 2, wherein the target sequence is a partial sequence of the MAPT gene.

11. An expression vector comprising a DNA sequence encoding the artificial RNA molecule of claim 1 or 6.

12. 1. A method for modulating mRNA splicing, comprising: (A) preparing an artificial RNA molecule comprising the following (a) and the following (b), wherein the following (a) and (b) are arranged in this order from the 5' side to the 3' side: (a) A polynucleotide potentially having a secondary structure represented by formula (I), or a polynucleotide in which 1 to 3 bases have been substituted, deleted or added among the 7 bases on the 3' side of a polynucleotide potentially having a secondary structure represented by formula (I). (b) a pre-mRNA targeting polynucleotide comprising a sequence complementary to a target sequence that is a portion of a pre-mRNA; and (B) contacting the artificial RNA molecule prepared in step (A) with a pre-mRNA to form a complementary strand between the artificial RNA molecule and the pre-mRNA, thereby regulating mRNA splicing. A method comprising: 【Chemistry 2】 (In the formula, N 1 to N 25 each independently represent A, C, G, or U, and N 1 and N 25 , N 2 and N 24 , N 3 and N 23 , N 4 and N 22 , N 5 and N 21 , N 6 and N 20 , N 8 and N 19 , N 9 and N 18 , and N 10 and N 17 each form a base pair.)

13. The method of claim 12, wherein the modulation of mRNA splicing is by exon skipping.

14. The method according to claim 12 or 13, wherein the base sequence of (a) is the sequence shown in SEQ ID NO: 2 or a sequence having a sequence identity of 80% or more to the sequence shown in SEQ ID NO:

2.

15. The method of claim 12 or 13, wherein the target sequence is an exon sequence.

16. The method according to claim 12 or 13, wherein the artificial RNA molecule comprises a polynucleotide comprising a transcription termination signal sequence recognized by an RNA pol III enzyme located on the 3' side of (b).

17. 1. A method for producing mature mRNA, comprising: (A) preparing an artificial RNA molecule comprising the following (a) and the following (b), wherein the following (a) and (b) are arranged in this order from the 5' side to the 3' side: (a) A polynucleotide potentially having a secondary structure represented by formula (I), or a polynucleotide in which 1 to 3 bases have been substituted, deleted or added among the 7 bases on the 3' side of a polynucleotide potentially having a secondary structure represented by formula (I). (b) a pre-mRNA targeting polynucleotide comprising a sequence complementary to a target sequence which is a portion of a pre-mRNA; (B') contacting the artificial RNA molecule prepared in step (A) with a pre-mRNA to form a complementary strand between the artificial RNA molecule and the pre-mRNA; and (C) A step of regulating mRNA splicing by step (B') to produce mature mRNA. A method comprising: 【Chemistry 3】 (In the formula, N 1 to N 25 each independently represent A, C, G, or U, and N 1 and N 25 , N 2 and N 24 , N 3 and N 23 , N 4 and N 22 , N 5 and N 21 , N 6 and N 20 , N 8 and N 19 , N 9 and N 18 , and N 10 and N 17 each form a base pair.)

18. The method of claim 17, wherein the modulation of mRNA splicing is by exon skipping.

19. The method according to claim 17 or 18, wherein the base sequence of (a) is the sequence shown in SEQ ID NO: 2 or a sequence having a sequence identity of 80% or more to the sequence shown in SEQ ID NO:

2.

20. 19. The method of claim 17 or 18, wherein the target sequence is an exon sequence.

21. The method according to claim 17 or 18, wherein the artificial RNA molecule comprises a polynucleotide comprising a transcription termination signal sequence recognized by an RNA pol III enzyme located on the 3' side of (b).

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