Artificial RNA molecules

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

[Technical Field]

[0001] This invention relates to novel artificial RNA molecules, and more particularly to artificial RNA molecules that regulate mRNA splicing. [Background technology]

[0002] 4.5SH RNA is a non-coding RNA specific to rodents (Myodonta Clade), and its nucleotide sequence has been identified (Non-Patent Literature 1). However, the function of 4.5SH RNA remains unclear.

[0003] On the other hand, gene therapy has been attracting attention in recent years. Examples of gene therapy include nucleic acid drugs and technologies that utilize genome editing with CRISPR / Cas9.

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

[0005] Technologies applying genome editing using CRISPR / Cas9 include, for example, the development of a technique to transplant patient-derived stem cells into patients in which the expression of disease-causing genes, specifically β-globin genes with disease-causing mutations, is reduced, while the expression of corresponding normal genes is increased (see, for example, Patent Document 2). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2014-54250 [Patent Document 2] Japanese Patent Publication No. 2021-166514 [Non-patent literature]

[0007] [Non-Patent Document 1] Harada, F and Kato, N. (1980) NAR., 8:1273-J285 [Summary of the Invention] [Problems to be Solved by the Invention]

[0008] However, since antisense nucleic acid pharmaceuticals generally have a length of about 14 to 25 bases, screening is necessary to search for effective sites for mRNA splicing regulation from within the target gene, and development takes time and cost. In addition, because of the short half-life, the drug effect cannot be maintained unless repeated administration is carried out every few weeks to several months, which imposes a significant burden on the medical economy.

[0009] As one method for solving this problem, a technique applying genome editing by CRISPR / Cas9 can be cited. Since it is a technique applying genome editing, it acts directly on the genome, so the effect can be maintained for a long time.

[0010] As the CRISPR / Cas9 system expression vector, an adeno-associated virus vector (AAV) is often used from the viewpoint of safety and the like, but the Cas9 gene has a large molecular weight and there is a problem that it is difficult to load it onto AAV.

[0011] Under the above circumstances, a new technology capable of controlling gene expression different from antisense nucleic acids and genome editing by CRISPR / Cas9 is required.

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

[0013] As a result of intensive studies, the inventors of the present invention have found that a non-coding RNA specific to rodents of the order Rodentia is involved in mRNA splicing. Through further studies, they have found that the characteristic secondary structure of a specific sequence of the non-coding RNA specific to rodents of the order Rodentia plays a role in recruiting splicing regulators and stabilizing RNA molecules. By using an artificial RNA molecule in which this secondary structure is combined with a sequence complementary to the target mRNA precursor, mRNA splicing can be regulated and the above problems can be solved, thus completing the present invention.

[0014] That is, the present invention provides: [1] An artificial RNA molecule comprising the following (a) and (b), wherein (a) and (b) are arranged in this order from the 5'-end to the 3'-end: (a) A polynucleotide potentially having a secondary structure represented by the following 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 the polynucleotide potentially having a secondary structure represented by the following formula (I) (b) An mRNA precursor targeting polynucleotide containing a sequence complementary to a target sequence that is a partial sequence of an mRNA precursor

Chemical Formula

[10] The artificial RNA molecule described in any of [1] to [9] above, wherein the target sequence is a sequence of part of the MAPT gene.

[11] An expression vector containing a DNA sequence encoding an artificial RNA molecule as described in any of [1] to

[10] above,

[12] A method for regulating mRNA splicing, (A) A step of preparing an artificial RNA molecule comprising the following (a) and the following (b), wherein (a) and (b) are arranged in this order from the 5' side to the 3' side. (a) Polynucleotides that potentially have the secondary structure represented by formula (I), or polynucleotides in which 1 to 3 bases are substituted, deleted, or added to the 7 bases on the 3' side of a polynucleotide that potentially has the secondary structure represented by formula (I). (b) mRNA precursor-targeting polynucleotides containing a complementary sequence to the target sequence, which is a part of the mRNA precursor sequence, and (B) A step in which the artificial RNA molecule prepared in step (A) is brought into contact with the mRNA precursor, a complementary strand is formed between the artificial RNA molecule and the mRNA precursor, thereby regulating mRNA splicing. Methods including

[13] The method described in

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

[14] The method described in

[12] or

[13] above, wherein the base sequence of (a) is the sequence shown in Sequence ID No. 2, or a sequence that has 80% or more sequence identity with the sequence shown in Sequence ID No. 2.

[15] The method according to any one of the above

[12] to

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

[16] The method according to any of the above

[12] to

[15] , 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 has a polynucleotide containing a transcription termination signal sequence recognized by RNApolIII enzyme positioned on the 3' side of (b),

[18] A method for producing mature mRNA, (A) A step of preparing an artificial RNA molecule comprising the following (a) and the following (b), wherein (a) and (b) are arranged in this order from the 5' side to the 3' side. (a) Polynucleotides that potentially have the secondary structure represented by formula (I), or polynucleotides in which 1 to 3 bases are substituted, deleted, or added to the 7 bases on the 3' side of a polynucleotide that potentially has the secondary structure represented by formula (I). (b) mRNA precursor-targeted polynucleotides containing a complementary sequence to the target sequence, which is a part of the mRNA precursor sequence. (B') A step of contacting the artificial RNA molecule prepared in step (A) with the mRNA precursor to form a complementary strand between the artificial RNA molecule and the mRNA precursor, and (C) Step (B') is a process in which mRNA splicing is regulated and mature mRNA is produced. Methods including

[19] The method described in

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

[20] The method described in

[18] or

[19] above, wherein the base sequence of (a) is the sequence shown in Sequence ID No. 2, or a sequence that has 80% or more sequence identity with the sequence shown in Sequence ID No. 2.

[21] The method according to any one of the above

[18] to

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

[22] The method according to any of the above

[18] to

[21] , 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 has a polynucleotide containing a transcription termination signal sequence recognized by RNApolIII enzyme positioned on the 3' side of (b), Regarding. [Effects of the Invention]

[0015] According to the present invention, a novel technique is provided for regulating mRNA splicing, which differs from conventional techniques, by combining an artificial RNA molecule with a characteristic secondary structure and a sequence complementary to the target mRNA precursor, and by using the artificial RNA molecule. [Brief explanation of the drawing]

[0016] [Figure 1] This is a schematic diagram showing a portion of the secondary structure of 4.5SH RNA as predicted by the secondary structure prediction program RNAfold. [Figure 2]These are electrophoresis maps of three genes containing asSINEB1, performed using primers that specifically amplify each gene using RT-PCR in wild-type and 4.5SH knockout mice. From left to right, the results are for Smchd1, Slc25a40, and Smg6. In the schematic diagram on the left, the upper panel shows mRNA containing the novel exon in gray, and the lower panel shows mRNA without the novel exon. The exon in question is shown in gray, with its sides in black and white. [Figure 3] This is a schematic diagram of a reporter containing the U6 promoter and mouse 4.5SH sequences. The U6 promoter sequence and the 4.5SH sequence are arranged in order from the 5' side of the reporter. [Figure 4] Figure 3 is an electrophoresis diagram showing the results of confirming, by Northern blotting, that 4.5SH RNA is expressed in human cells after introducing and expressing the promoter shown in the diagram. [Figure 5] This is an electrophoresis map of human 4.5SH-expressing cells into which minigenes containing two genes with asSINEB1 were introduced, and RT-PCR was performed using primers that specifically amplify each gene. The results from left to right are for Slc25a40 and Smg6. In the schematic diagram on the left, the upper panel shows mRNA containing the novel exon in gray, and the lower panel shows mRNA without the novel exon. [Figure 6] Figure 5 is a bar graph showing the skipping efficiency obtained from the electrophoresis diagram. The results for Slc25a40 are shown on the left, and the results for Smg6 are shown on the right. [Figure 7] From top to bottom, these are excerpts of DNA sequences from plasmids expressing the wild-type (WT) Slc25a40, the wild-type (WT) 4.5SH, the mutant (MUT) Slc25a40, and the mutant (MUT) 4.5SH. The parts where the bases differ between WT and MUT are shown in gray. [Figure 8] This is an electrophoresis diagram of human cells into which mutant plasmids and wild-type plasmids were introduced, and then RT-PCR was performed using primers that specifically amplify each plasmid. [Figure 9]This bar graph shows the skipping efficiency obtained from the electrophoresis diagram in Figure 8. From left to right, the graphs show Slc25a40 wild-type only, wild-type only, Slc25a40 mutant and 4.5SH wild-type, Slc25a40 wild-type and 4.5SH mutant, and mutant only. [Figure 10] This figure shows the exon skipping efficiency of B1 modified 4.5SH (SL-B1-4.5SH). From top to bottom, the figures are: an electrophoresis graph obtained using BioAnalyzer, an electrophoresis graph showing the results of confirming RNA expression by Northern blotting, and a bar graph showing the exon skipping efficiency. In the schematic diagram on the left of the BioAnalyzer electrophoresis graph, the markers shown indicate mRNA containing the novel Slc25a40 exon (exon 8.5) in the upper panel and mRNA not containing Slc25a40 exon 8.5 in the lower panel. [Figure 11] This is an electrophoresis diagram showing the co-expression of a minigene for detecting FAS gene exon skipping with wild-type 4.5SH or modified FAS 4.5SH. [Figure 12] This is an electrophoresis diagram showing the co-expression of a minigene containing Slc25a40 exon 8.5 with wild-type 4.5SH or FAS-modified 4.5SH. [Figure 13] These are bar graphs showing the skipping efficiency obtained from the electrophoresis diagrams in Figures 11 and 12. [Figure 14] This is a schematic diagram of the target sequence of the DMD gene modification 4.5SH. [Figure 15] This is an electrophoresis diagram showing the co-expression of a minigene for detecting DMD gene exon skipping and the modified DMD gene 4.5SH. In the schematic diagram on the left, the markers in the upper panel represent mRNA containing DMD exon 53 (gray), and the markers in the lower panel represent mRNA without DMD exon 53. [Figure 16] This is an electrophoresis map obtained by BioAnalyzer when a minigene for detecting DMD gene exon skipping and the modified DMD gene 4.5SH are co-expressed. The schematic diagram above illustrates the targeting of DMD exon 53 by the modified DMD gene 4.5SH. In the schematic diagram on the left, the markers shown in the upper row represent mRNA containing DMD exon 53, and the markers shown in the lower row represent mRNA without DMD exon 53. [Figure 17] This is a schematic diagram of the target sequence of the DMD gene modification 4.5SH. [Figure 18] This figure shows the results of co-expressing a minigene for detecting DMD gene exon skipping with the DMD gene modification 4.5SH. From top to bottom, the images are: an electrophoresis graph obtained using BioAnalyzer, an electrophoresis graph showing the results of confirming RNA expression by Northern blotting, and a bar graph showing the exon skipping efficiency. In the schematic diagram on the left of the BioAnalyzer electrophoresis graph, the markers shown in the upper row represent mRNA containing DMD exon 53, and the markers in the lower row represent mRNA not containing DMD exon 53. [Figure 19] This figure shows the exon skipping efficiency of the MAPT gene-modified 4.5SH. From top to bottom, the figures are a schematic diagram illustrating the targeting of MAPT exon 10 by the MAPT gene-modified 4.5SH, an electrophoresis graph obtained using BioAnalyzer, an electrophoresis graph showing the results of confirming RNA expression by Northern blotting, and a bar graph showing the exon skipping efficiency. In the schematic diagram on the left of the BioAnalyzer electrophoresis graph, the markers shown indicate mRNA containing MAPT exon 10 in the upper row and mRNA not containing MAPT exon 10 in the lower row. [Modes for carrying out the invention]

[0017] While we do not intend to be constrained by theory, the mechanism by which the effects of this invention are exerted can be considered as follows, for example.

[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 1 to 3 bases are substituted, deleted, or added to the 7 bases on the 3' side of a polynucleotide potentially having a secondary structure represented by formula (I), forms a robust stem-loop structure to which a splicing regulatory factor binds, and (b) an mRNA precursor-targeted polynucleotide (polynucleotide(b)) containing a complementary sequence to the target sequence, which is a part of the mRNA precursor sequence, binds to the target sequence within the mRNA precursor. The splicing regulatory factor bound to polynucleotide(a) then acts in an inhibitory or promoting manner on the splicing reaction, so that the portion containing the target sequence, such as an exon, is skipped or included during mRNA splicing.

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

[0020] In this specification, "mRNA precursor" refers to RNA that has been transcribed in the cell nucleus using DNA as a template by the RNA polymerase enzyme, and which has not yet undergone mRNA splicing.

[0021] In this specification, "mRNA splicing" refers to the process by which introns are removed from mRNA precursors to produce mature mRNA. The same applies when the term "splicing" is used without further explanation.

[0022] In this specification, “exon” means a sequence contained in mature mRNA. A person skilled in the art can obtain annotated sequences of exons from publicly accessible databases such as Ensembl, the National Center for Biotechnology Information (NCBI), GenBank, or other NCBI databases using a computer with the necessary software. Specifically, for example, with respect to human genes, a person 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, a person skilled in the art can browse genomes in the Ensembl database (http: / / www.ensembl.org) and obtain information about exons.

[0023] In this specification, “intron” means a sequence that is not included in mature mRNA. As with “exons” above, those 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 with the necessary software.

[0024] In this specification, "alternative splicing" means splicing performed by removing specific exons.

[0025] In this specification, "base pairing" refers not only to the so-called Watson-Crick type base pairing, where adenine (A) and thymine (T) (or uracil (U)) or guanine (G) and cytosine (C) of nucleotides form a hydrogen bond, but also to fluctuating base pairings such as G and U, inosine (I) and U, and I and A.

[0026] In this specification, "exonization" refers to the inclusion of a sequence that is originally an intron into mature mRNA as a result of regulated mRNA splicing.

[0027] In this specification, "novel exon" refers to a sequence that was originally an intron and was created by exonization.

[0028] In this specification, "stem-loop structure" refers to a secondary structure formed within a single nucleic acid molecule, which is formed by nucleotide sequences held by the nucleic acid molecule itself. A stem-loop structure consists of a stem region created by the base pairing of a nucleotide sequence located at the 3' end and a nucleotide sequence located at the 5' end of the same molecule, and a loop region formed by a nucleotide sequence located between the nucleotide sequence at the 3' end and the nucleotide sequence at the 5' end that constitute the stem region. Those skilled in the art can confirm the formation of a stem-loop structure and its strength through thermodynamic simulations using secondary structure prediction programs such as RNA Fold (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi), and experimental methods such as RNA footprint assays, nuclear magnetic resonance (NMR) and crystal structure analysis of nucleic acid molecules.

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

[0030] In this specification, "exon skipping" refers to a form of alternative splicing in which mature mRNA is produced that does not contain certain exons that were originally present.

[0031] In this specification, "exon skipping efficiency" is defined as the amount of polynucleotides (X) in the band from which exons have been skipped, and the length of those nucleotides (L). X ) and the amount of polynucleotides (Y) in the band where the exons were not skipped, and the length of those nucleotides (L) Y ) can be calculated using the following formula. Skipping efficiency (%) = {X / L} X ÷(X / L X +Y / L Y )} × 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 the following (b), wherein (a) and (b) are arranged in this order from the 5' side to the 3' side. (a) Polynucleotides that potentially have the secondary structure represented by formula (I) above, or polynucleotides in which 1 to 3 bases are substituted, deleted, or added to the 7 bases on the 3' side of a polynucleotide that potentially has the secondary structure represented by formula (I) above. (b) mRNA precursor-targeted polynucleotides containing a complementary sequence to the target sequence, which is a part of the mRNA precursor sequence.

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

[0034] <Polynucleotide (a)> A polynucleotide (a) is a polynucleotide that potentially has the secondary structure represented by formula (I) below, or a polynucleotide in which 1 to 3 bases are substituted, deleted, or added to the 7 bases on the 3' side of a polynucleotide that potentially has the secondary structure represented by formula (I) below. [ka] (In the formula, N1~N 25 Each of these independently represents A, C, G, or U, and N1 and N 25 This refers to N2 and N 24 This refers to N3 and N 23 This refers to N4 and N 22 This refers to N5 and N 21 This refers to N6 and N 20 This refers to N8 and N 19 This refers to N9 and N 18 That is, N 10 and N 17 These two elements form base pairs.

[0035] Polynucleotide (a) is a secondary structure that exists stably under physiological conditions, as shown in formula (I), N1~N 25 It forms a robust stem-loop structure related to this.

[0036] N1~N 25 In order to form a strong stem-loop structure by base-pairing within a single RNA molecule, N1~N 10 and N 17 ~N 25 However, it is preferable that it be rich in guanine (G) and cytosine (C) or G and uracil (U). N1 and N 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 17The nine base pair bonds formed are preferably G and C or G and U. Of these nine base pair bonds, it is preferable that 5 to 9 are G and C or G and U base pair bonds, more preferably 6 to 9, even more preferably 7 to 9, even more preferably 8 to 9, even more preferably 5 to 8, even more preferably 6 to 8, particularly preferably 7 to 8, and most preferably 8. 11 ~N 16 It is preferable that the loop structure is RYRRYR, where R represents a purine base (A or G) and Y represents a pyrimidine base (C or U).

[0037] The N shown in equation (I) 25 The seven bases closer to the 3' end do not need to be bases that form a stem-loop structure; 1 to 3 bases may be substituted, deleted, or added. The substitution, deletion, or addition of bases may be made to any of the seven bases shown in formula (I), with 1 to 2 bases being preferred and 1 base being more preferred. In the case of substitution, it is preferable that purine bases are substituted with other purine bases, and pyrimidine bases are substituted with other pyrimidine bases. 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 shown in formula (I), however, it is preferable that it contains the sequence shown in SEQ ID NO: 2 or a sequence that has 80% or more sequence identity with the sequence shown in SEQ ID NO: 2. The sequence shown in SEQ ID NO: 2 is rich in G and C and can form a robust stem-loop structure as shown in formula (I), as described later (Figure 1).

[0039] The polynucleotide (a) is more preferably 85% or more in sequence identity with the sequence shown in Sequence 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 RNA molecule is stabilized when the strength of the stem-loop structure is within the above range.

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

[0042] Polynucleotide(a), as described above, is thought to contribute to the stabilization of artificial RNA molecules by containing a stem-loop structure and the subsequent 3' base. Furthermore, polynucleotide(a) is thought to play a role in attracting splicing regulators.

[0043] In this specification, "splicing regulator" refers to all factors present in living organisms that regulate mRNA splicing, and specifically includes proteins and nucleic acids that regulate mRNA splicing, as well as protein-RNA complexes.

[0044] Polynucleotide(a) is thought to have the function of attracting and binding to splicing regulators. The splicing regulators that bind to polynucleotide(a) determine how the artificial RNA molecule of the present invention regulates mRNA splicing, that is, what kind of alternative splicing occurs.

[0045] More specifically, the splicing regulators that bind to polynucleotide (a) include proteins that make up the nuclear small RNA U2 complex and their binding proteins. More specifically, these include SF3b1, U2AF2, Nono, Sfpq, and Hnrnpf. Furthermore, based on mass spectrometry results, it is inferred that Hnrnpm, Hnrnpa1, Hnrnpa2b1, Ddx5, Hnrnpa3, Fus, Hnrnpa0, Pabpc1, Snrpa, Dhx9, Myef2, Ddx17, Hnrnph1, Prpf8, Rps3a, and Rbm14 also bind to these proteins.

[0046] The functions of these splicing regulators are known to include suppressing sequences such as splice donor sequences or splice acceptor sequences necessary for enabling splicing reactions, suppressing exon inclusion signals, and being involved in the accurate recognition of 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 in the suborder Muridae. In 4.5SH, the sequence shown in SEQ ID NO: 2 forms a stem-loop structure as a secondary structure that exists stably under physiological conditions, recruits splicing regulators, and has been found to be an essential sequence in regulating mRNA splicing of mRNA precursor sequences targeted by 4.5SH. The sequences targeted by 4.5SH and the regulation of their mRNA splicing will be described later. Note that 4.5SH is transcribed from DNA by the RNA polymerase III enzyme (RNApol 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 recruit mRNA splicing repressors, thereby inhibiting accurate intron recognition. Furthermore, the sequence shown in Sequence ID No. 2 is thought to contribute to the overall stability of 4.5SH, as it forms a robust stem-loop structure as shown in Equation (I) and Figure 1.

[0049] Hereinafter, the polynucleotide (a) in the present invention will be described with reference to the drawings, using the sequence shown in Sequence ID No. 2, which is a specific example thereof, as an example.

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

[0051] In this specification, "the Xth base" means the Xth base counting from the 5' side. In Figure 1, the sequence shown as Sequence ID No. 2 in 4.5SH forms a stem region through the base pairing of the 1st G and 25th U, the 2nd C and 24th G, the 3rd C and 23rd G, the 4th G and 22nd C, the 5th G and 21st C, the 6th U and 20th G, the 8th G and 19th C, the 9th U and 18th A, and the 10th G and 17th C. The 11th through 16th bases are bases that do not have a base pairing partner and form a loop structure.

[0052] In Figure 1, the bases from the 26th A to the 32nd U are not bases that form a stem-loop structure. Furthermore, in 4.5SH, the bases from the 26th A to the 32nd U in the sequence shown in Sequence ID No. 2 are neither bases that form a stem-loop structure nor bases that form a complementary strand with the target sequence of 4.5SH, as described later.

[0053] <Polynucleotide (b)> Polynucleotide(b) is an mRNA precursor-targeting polynucleotide that contains a complementary sequence to the target sequence, which is a part of the mRNA precursor sequence. Polynucleotide(b) can be designed with any sequence depending on the target sequence.

[0054] A polynucleotide (b) contains a sequence complementary to the target sequence, which is a part of the mRNA precursor sequence. That is, polynucleotide (b) forms a complementary strand with the target sequence. For the formation of the complementary strand to occur, the two nucleic acid molecules do not need to be perfectly (100%) complementary.

[0055] A complementary sequence is a sequence that allows two nucleic acid molecules to complement each other through base pairing. For example, a sequence with 80-100% complementarity (e.g., a sequence where 4 or 5 out of 5 bases are complementary) or a sequence with 90-100% identity (e.g., a sequence where 9 or 10 out of 10 bases are complementary) is acceptable.

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

[0057] 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 the stability of the complementary strand with respect to the target sequence and the specificity of the complementary strand with respect to the target sequence. Furthermore, from the viewpoint of the ease of synthesis of artificial RNA molecules, 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 not only depending on the complementarity of the base sequences as described above, but also on the number of hydrogen bonds between the bases (for example, the G-C pair has more hydrogen bonds than the A-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 part of the base sequence of an mRNA precursor that is a target for regulating mRNA splicing, but it is preferably an exon sequence relating to an exon whose presence or absence in mature mRNA alters the function of the protein encoded by the mRNA. In this case, the exon sequence may be the entire base sequence of the exon or a part of the base sequence of the exon, depending on the length of the target exon. Furthermore, the target sequence of polynucleotide(b) may include a part of the intron sequence located within 300 bases upstream (5' side) or downstream (3' side) of the exon, or it may be only a part of the intron sequence that does not include the exon sequence.

[0060] The polynucleotide (b) may contain multiple sequences that are each complementary to multiple target sequences. These multiple target sequences are preferably, but not limited to, selected from a region encompassing the same exon and the 300 base pairs upstream or downstream of it.

[0061] If the target sequence is an exon sequence and the exon sequence is equal to or greater than the average length of an exon (approximately 150 bases), then it is preferable that polynucleotide(b) contains multiple sequences that complement each of the 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. Furthermore, it is preferably 400 bases or less, and more preferably 300 bases or less.

[0062] <Polynucleotide (c)> The artificial RNA molecule of the present invention preferably includes, in addition to the polynucleotide (a) and polynucleotide (b) described above, a polynucleotide (polynucleotide (c)) containing a sequence that contributes to terminal stability, and it is preferable that this polynucleotide (c) is positioned on the 3' side of polynucleotide (b).

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

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

[0065] <Artificial RNA molecules> The artificial RNA molecule of the present invention regulates mRNA splicing of mRNA precursors targeted by polynucleotide (b). Modification of mRNA splicing includes intron retention and modification of alternative splicing, with modification of alternative splicing being preferred. Modification of alternative splicing includes induction of exon inclusion or induction of exon skipping. Of these, induction of exon skipping is preferred because the function of 4,5SH is to suppress the creation of new exons by 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' side to the 3' side. If polynucleotide (c) is included, polynucleotide (c) is arranged on the 3' side of polynucleotide (a) and polynucleotide (b).

[0067] Polynucleotides (a), (b), and (c) may each be arranged in groups of two or more, or individually. Since polynucleotide (a) is an essential sequence 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 of the structures shown in formula (I) may be arranged via a multibranched loop. Furthermore, it is unlikely that arranging three or more polynucleotides (a) will further increase the effect, and from the viewpoint of the stability of the artificial RNA molecule, it is preferable to use three or fewer. 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] Spacers may or may not be present between polynucleotides (a), polynucleotide (b), and polynucleotide (c). If spacers are present, the base lengths of the spacers can be, for example, 200 bases or less, 150 bases or less, 100 bases or less, 50 bases or less, 40 bases or less, and 30 bases or less.

[0069] The nucleic acid in the artificial RNA molecule of the present invention may be of the natural type or a modified nucleic acid. Modified nucleic acids may include, for example, nucleic acids in which the OH group at the 2' position of ribose is replaced with an F group or an OMe group, as well as phosphorothioate nucleic acids, morpholino nucleic acids, and cross-linked nucleic acids (LNA), from the viewpoint of increasing resistance to nucleases and hydrolysis. Furthermore, from the viewpoint of reducing cytotoxicity, pseudouridine (Ψ), N1-methylpseudridine (N1mΨ), etc., may be included instead of uridine (U). The modified nucleic acid may be all or part of the molecule, and if only part, it may be at random positions in any proportion.

[0070] <5´ end> The 5' end of the artificial RNA molecule of the present invention may be modified with a genetically engineered structure to stabilize the molecule. For example, a triphosphate group may be added, or a cap structure may be added.

[0071] <3´ end> The 3' end of the artificial RNA molecule of the present invention may be modified with a structure commonly used in genetic engineering to stabilize the molecule. For example, a phosphate group may be added.

[0072] <Other arrangements> The RNA molecule of the present invention may contain polynucleotides other than the polynucleotides (a), (b), and (c) described above. Examples of other sequences include polynucleotides for gene expression and drug delivery. More specifically, examples include polynucleotides containing promoter sequences of expression vectors, polynucleotides containing transcription termination sequences of expression vectors, polynucleotides containing sequences to avoid degradation by RNA nuclease enzymes, and polynucleotides containing sequences that have affinity for splicing regulators.

[0073] <Target gene> The artificial RNA molecule of the present invention targets mRNA precursors with polynucleotides (b), thereby regulating the splicing of mRNA precursors and consequently enabling the control of translated and expressed genes (target genes). 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 Huntington gene (HTT). Target genes may be genes from which multiple isoforms are known to be formed from a single gene by alternative splicing, or they may be genes from which isoforms have not generally been identified in vivo. Examples of genes from which multiple isoforms are known to be formed from a single gene by alternative splicing include the FAS gene and the tau (MAPT) gene. Generally, genes for which isoforms have not been identified in vivo include, for example, 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 artificial RNA molecule of the present invention described above. 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 plasmids include PCDNA3 and PCDNA5FRT / TO.

[0077] A viral vector is a vector based on one or more nucleic acid sequences containing a viral genome. Examples of viral vectors include adenovirus vectors, adeno-associated virus vectors (AAVs), 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 that have a single-stranded DNA genome. AAV belongs to the parvovirus family.

[0079] As for the expression promoter of the expression vector, since the artificial RNA molecule of the present invention is transcribed by the RNApolIII enzyme, any promoter of the RNApolIII enzyme is acceptable. Specifically, examples include the U6 promoter and the H1 promoter, with the U6 promoter being preferred.

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

[0081] Methods for recombining an expression vector to include the DNA sequence encoding the artificial RNA of the present invention include molecularly biologically common methods. For example, methods using restriction enzymes and ligases, or methods using Gibson assemblies.

[0082] In a second embodiment, the present invention relates to a method for regulating mRNA splicing, (A) A step of preparing an artificial RNA molecule comprising the following (a) and the following (b), wherein (a) and (b) are arranged in this order from the 5' side to the 3' side. (a) Polynucleotides that potentially have the secondary structure represented by formula (I), or polynucleotides in which 1 to 3 bases are substituted, deleted, or added to the 7 bases on the 3' side of a polynucleotide that potentially has the secondary structure represented by formula (I). (b) mRNA precursor-targeting polynucleotides containing a complementary sequence to the target sequence, which is a part of the mRNA precursor sequence, and A method (also referred to as the regulatory method of the present invention) is provided, which includes the step of contacting the artificial RNA molecule prepared in step (A) with an mRNA precursor to form a complementary strand between the artificial RNA molecule and the mRNA precursor, thereby regulating mRNA splicing.

[0083] <Process (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 can be a chemical synthesis method or a biochemical synthesis method. Chemical synthesis methods include the phosphoramitide method and its modifications, the H-phosphonate method and its modifications, etc. Biochemical synthesis methods include the synthesis of RNA molecules using an in vitro transcription system (enzyme synthesis method), etc. Artificial RNA manufactured under contract can also be used in the present invention.

[0084] <Process (B)> Step (B) involves contacting the artificial RNA molecule prepared in step (A) with the mRNA precursor to form a complementary strand between the artificial RNA molecule and the mRNA precursor, thereby regulating mRNA splicing. Methods for contacting the artificial RNA molecule with the target mRNA precursor include conventional methods in this technology, such as introducing the artificial RNA molecule into cells in vitro or administering the artificial RNA molecule into a living organism.

[0085] Known methods can be used to introduce artificial RNA molecules into cells outside the body, such as lipofection, liposomes, electroporation, calcium phosphate coprecipitation, microinjection, gene guns, and reverse transfection.

[0086] Here, mRNA splicing typically proceeds via a two-step mechanism. In the first step, the 5' splicing site is divided, producing a free 5' exon and a lasso-type intermediate. In the second step, this 5' exon binds to another 5' exon, accompanied by the release of an intron as a lasso-type product. These reactions are catalyzed by spliceosomes (see Moore, MJ, PASharp (1993) Nature, 365:364~368, etc.).

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

[0088] Methods for regulating mRNA splicing include, specifically, methods for inducing intron retention and methods for modifying alternative splicing, but methods for modifying alternative splicing are preferred. Methods for modifying alternative splicing include methods for inducing exon inclusion or methods for inducing exon skipping, and methods for inducing exon skipping are preferred.

[0089] The regulation of mRNA splicing can be confirmed by RT-PCR or the like. Specifically, total RNA is extracted from cells or tissues into which the artificial RNA molecule of the present invention has been introduced, and mRNA is purified by obtaining cDNA through a reverse transcription reaction using reverse transcriptase. By performing PCR using PCR primers that specifically amplify the target sequence, it can be confirmed that mature mRNA with regulated target sequence splicing has been obtained.

[0090] In a third embodiment, the present invention relates to a method for producing mature mRNA, (A) A step of preparing an artificial RNA molecule comprising the following (a) and the following (b), wherein (a) and (b) are arranged in this order from the 5' side to the 3' side. (a) Polynucleotides that potentially have the secondary structure represented by formula (I), or polynucleotides in which 1 to 3 bases are substituted, deleted, or added to the 7 bases on the 3' side of a polynucleotide that potentially has the secondary structure represented by formula (I). (b) mRNA precursor-targeted polynucleotides containing a complementary sequence to the target sequence, which is a part of the mRNA precursor sequence. (B') A step of contacting the artificial RNA molecule prepared in step (A) with the mRNA precursor to form a complementary strand between the artificial RNA molecule and the mRNA precursor, and The present invention provides a method (also referred to as the production method of the present invention) that includes a step (C) in which mRNA splicing is regulated by step (B') to produce mature mRNA.

[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 in vivo approach could involve administering the artificial mRNA molecule of the present invention into a living organism, where the artificial mRNA molecule is taken up into the cell nucleus, and mature mRNA produced within the organism remains there, leading to the production of normal proteins. An ex vivo approach could involve extracting cells or tissues from a living organism, administering the artificial mRNA molecule of the present invention, where the artificial mRNA molecule is taken up into the cell nucleus, and mature mRNA produced within the cell remains there, leading to the production of normal proteins. These cells or tissues could then be returned to the patient.

[0092] <Process (B')> Step (B') is a step in which the artificial RNA molecule prepared in step (A) is brought into contact with the mRNA precursor to form a complementary strand between the artificial RNA molecule and the mRNA precursor. Step (B') can be performed using conventional methods in the art, such as introducing the artificial RNA molecule into cells in vitro or administering the artificial RNA molecule into a living organism, similar to step (B) in the regulatory method of the present invention.

[0093] <Process (C)> Step (C) is a step in which mRNA splicing is regulated by step (B') and mature mRNA is produced. Confirmation that mRNA splicing has been regulated by step (B') and that mature mRNA has been produced can be performed by RT-PCR or the like, similar to the regulatory method of the present invention.

[0094] The description of the artificial RNA molecule in the regulatory method and production method of the present invention is similar to the description of the artificial RNA molecule described above. Furthermore, the descriptions of the regulatory method and production method of the present invention, in their applicable parts (particularly the description relating to step (A) described above), are similarly applicable to each embodiment.

[0095] [Pharmaceutical composition] The present invention can also provide a pharmaceutical composition comprising the artificial RNA molecule of the present invention or mature mRNA obtained by the production method of the present invention. This pharmaceutical composition can be used to treat or prevent treatable diseases by regulating mRNA splicing.

[0096] As a drug delivery system (DDS) for such pharmaceutical compositions, methods commonly used in the art can be used without particular limitation, but examples include methods using expression vectors, methods using lipid nanoparticles (LNPs), and methods using chemical modifications.

[0097] Methods using expression vectors specifically 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 preferred from a safety standpoint. When the artificial RNA molecule of the present invention is loaded onto an AAV, multiple copies can be loaded. Therefore, it is possible to increase the expression level of RNA, and it is expected that the effects of the present invention will be enhanced.

[0098] The lipid nanoparticle (LNP) method is a drug delivery method in which RNA molecules are encapsulated within LNPs. The RNA molecules are packaged in LNPs, which protect them from RNA-degrading enzymes, allowing them to enter cells and be released into the cytoplasm. Using LNPs makes it possible to efficiently deliver chemically modified or unmodified RNA molecules. LNPs require PEGylated lipids, cholesterol, neutral phospholipids, etc.

[0099] Chemical modification methods involve chemically modifying nucleotides that make up artificial RNA molecules or mature mRNA. Specifically, these methods include adding peptides to nucleotides, 2'O-ME modification, phosphorothioate modification, and pseudouridine. Such chemical modifications make it possible to protect RNA molecules from RNA-degrading enzymes, deliver them specifically to target tissues or cells, and achieve stronger binding to target sequences.

[0100] Diseases treatable by regulating mRNA splicing include systemic lupus erythematosus (SLE), autoimmune lymphoproliferative syndrome (ALPS), Dusenne muscular dystrophy (DMD), frontotemporal dementia (FTDP-17), Fukuyama muscular dystrophy, myotonic dystrophy, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), spinal and bulbar muscular atrophy (SBMA), familial autonomic dysfunction (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. [Examples]

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

[0102] Experimental Example 1: Creation of knockout mice First, 4.5SH knockout mice were created. The creation of knockout mice followed the general method of large-scale genome deletion / insertion using the CRISPR / Cas9 system (see Luqing Zhang et al, (2015) PLoS One, Mar 24;10(3), etc.). Specifically, gene cassettes containing drug resistance genes with sequences complementary to both ends of the mouse 4.5SH gene cluster were designed and isolated, and the isolated genes were introduced into mouse ES cells by electroporation. To enhance the efficiency of homologous recombination, CRISPR / Cas9, which cleaves the terminal portions of the complementary sequences, was introduced simultaneously.

[0103] Chimeric mice were created from the established ES cells and crossed with wild-type mice to obtain recombinant heterozygous mice. Heterozygous mice were crossed 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 4.5SH knockout mice, and RT-PCR was performed using cDNA synthesized from the RNA as a template and primers that specifically amplify 4.5SH. As a result, 4.5SH was significantly reduced in the 4.5SH knockout mice.

[0104] Experimental Example 2: RNA-Seq Analysis Total RNA was prepared using Trizol from ES cells of established 4.5SH knockout mice and wild-type mice. RNA-Seq analysis was performed on the prepared total RNA. An Illumina HiSeq4000 was used for sequencing. The analysis software rMATS4.1.1 was used to compare the changes in alternative splicing among the three groups of cells: ES cells from 4.5SH knockout mice and cells from wild-type mice.

[0105] Table 1 shows the results of a statistically significant comparison of five types of alternative splicing between ES cells from 4.5SH knockout mice and cells from wild-type mice, with a False Discovery Rate (FDR) of less than 0.05. Here, 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-retaining type (IR). [Table 1]

[0106] RNA-Seq analysis revealed that there are 293 exons that specifically appear in ES cells from 4.5SH knockout mice.

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

[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 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 amplified three genes containing asSINEB1 (Smchd1 exons 21-22, Slc25a40 exons 8-9, and Smg6 exons 8-9). Therefore, asSINEB1 exists within the introns between exons 24 and 25 of Smchd1 (NM_028887.3), between exons 8 and 9 of Slc25a40 (NM_178766.5), and between exons 8 and 9 of Smg6 (NM_001002764.1). If these asSINEB1 are exonized and become new exons through 4.5SH knockout, it would be one way to verify the above hypothesis.

[0109] As a result, exon skipping of novel exons was detected in wild-type mouse cells for Smcdh1, Slc25a40, and Smg6, while exon skipping of novel exons was not detected in ES cells of 4.5SH knockout mice (Figure 2). Therefore, in the presence of 4.5SH, novel exons were skipped, but not in the absence of 4.5SH, confirming the emergence of novel exons. Hereinafter, in this specification and in the drawings, these novel exons will also be 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 validate the above hypothesis by confirming the activity of 4.5SH using human cells that do not naturally possess 4.5SH, we confirmed exon skipping in a human cell overexpression system.

[0111] Plasmids containing the U6 promoter and mouse 4.5SH sequence (Figure 3) were constructed, and these plasmids (pUC57) were introduced into human cells (HEK293 cells) by liposome transfection. Northern blotting was used to confirm the expression of 4.5SH RNA, and human 4.5SH-expressing cells were obtained (Figure 4).

[0112] Minigenes were created by inserting the DNA sequence containing wild-type Slc25a40Ex8.5 and its upstream and downstream 100 bases (SEQ ID NO: 9), and the DNA sequence containing wild-type Smg6Ex8.5 and its upstream and downstream 100 bases (SEQ ID NO: 10) into the SacII sequence within the intron of a plasmid derived from the late adenovirus gene (Ad2). These minigenes were then inserted into plasmids (pcDNA3 BamHI / XbaI), and the resulting vector (Ad2 / pcDNA3 BamHI / XbaI, with SEQ ID NO: 28 as the common vector sequence) was introduced into human 4.5SH-expressing cells. After culturing at 37°C for 24 hours, the cells were harvested and RNA was extracted. Using cDNA synthesized from the RNA as a template, RT-PCR was performed using primers (SEQ ID NO: 26 and SEQ ID NO: 27) that specifically amplified both minigenes. As a result, exon skipping was detected for the minigenes of Slc25a40 exon 8.5 and Smg6 exon 8.5 (Figure 5). Note that the left-hand columns in Figures 5 and 6 show the results in a control group where the minigene was introduced into human cells (HEK293 cells) before 4.5SH expression, instead of into human 4.5SH-expressing cells.

[0113] 10 μL of the reaction product from the above PCR was subjected to electrophoresis in a polyacrylamide gel, and the exon skipping efficiency was calculated from each band in the resulting electrophoresis graph. The amount of polynucleotides (X) and the length of nucleotides (L) in the bands where exons were skipped were calculated. X ) and the amount of polynucleotides (Y) and nucleotide length (L) of the bands in which exons were not skipped. Y The skipping efficiency was calculated using the following formula (Figure 6). Skipping efficiency (%) = {X / L} X ÷(X / LX +Y / L Y )} × 100

[0114] Based on the above, it was found that even in human cells that do not normally possess 4.5SH, the absence of 4.5SH causes the emergence of new exons within introns, while the presence of 4.5SH causes the new exons to be skipped.

[0115] From Experimental Examples 3 and 4, it was found that 4.5SH suppresses the exonization of asSINEB1 present within the intron, and that this suppression of exonization is due to exon skipping of novel exons.

[0116] Experimental Example 5: Examination of base pair binding by co-expression using mutants To investigate the base pairing between 4.5SH and asSINEB1, mutant forms of 4.5SH and asSINEB1 were obtained, co-expressed in cells, and the presence or absence of exon skipping was detected.

[0117] A plasmid expressing the natural (wild-type) 4.5SH, obtained in the same manner as in Experimental Example 4, was modified to produce a plasmid expressing the mutant. Specifically, a plasmid was created in which the mouse 4.5SH sequence was modified by changing a portion of the asSINEB1 recognition region of the wild-type 4.5SH sequence to C, so that it would not base-pair with a novel exon (exon 8.5) between exons 8 and 9 of Slc25a40 (Figure 7). The obtained plasmid was transformed into E. coli. The transformed E. coli was plated in LB (ampicillin-containing) plates, colonies were formed at 37°C, and screening was performed. The DNA sequence of the plasmid held by ampicillin-resistant E. coli was determined, and a mutant 4.5SH expression plasmid was obtained (Figure 7).

[0118] A plasmid expressing a minigene containing the natural (wild-type) Slc25a40 exon 8.5, obtained in the same manner as in Experimental Example 4, was modified to obtain a plasmid expressing the mutant. Specifically, a DNA sequence fragment (SEQ ID NO: 11) was synthesized in which a portion of the wild-type Slc25a40 exon 8.5 sequence was modified from G to C, and designed not to base-pair with wild-type 4.5SH. The mutation was then inserted into the plasmid by Gibson assembly. The obtained plasmid was transformed into E. coli. The transformed E. coli were plated in LB (ampicillin-containing) plates, colonies were formed at 37°C, and screening was performed. The DNA sequence of the plasmid held by ampicillin-resistant E. coli was determined, and a plasmid expressing a minigene containing the mutant Slc25a40 exon 8.5 was obtained (Figure 7).

[0119] Each of the above mutant plasmids and each wild-type plasmid were co-introduced into human cells (HEK293 cells) by lipofection. After culturing at 37°C for 24 hours, the cells were harvested and RNA was extracted. Using cDNA synthesized from the RNA as a template, RT-PCR was performed using primers (SEQ ID NO: 26 and SEQ ID NO: 27) that specifically amplify both minigenes, as also used in Experimental Example 4. 10 μL of the PCR reaction product was subjected to electrophoresis in a polyacrylamide gel, and the exon skipping efficiency was determined 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 samples in which the plasmid before 4.5SH introduction (so-called empty vector) and the minigene expression plasmid containing wild-type Slc25a40 exon 8.5 were co-introduced.

[0120] Exon skipping was detected in cells co-expressing wild-type 4.5SH and a minigene containing wild-type Slc25a40 exon 8.5 (the second lane from the left in Fig. 8). Exon skipping was not detected or the skipping efficiency was lower compared to the case of co-expressing wild-types in cells co-expressing mutant 4.5SH and a minigene containing wild-type Slc25a40 exon 8.5, and in cells co-expressing wild-type 4.5SH and a minigene containing mutant Slc25a40 exon 8.5 (the third lane from the left and the fourth lane from the left in Fig. 8). Exon skipping was detected in cells co-expressing mutant 4.5SH and a minigene containing mutant Slc25a40 exon 8.5, and the skipping efficiency was equivalent to that in the case of co-expressing wild-types (the fifth lane from the left in Fig. 8, skipping efficiency is the fifth from the left in Fig. 9). From this, it can be seen that base pair binding between 4.5SH and asSINEB1 is necessary for exon skipping by 4.5SH.

[0121] Experimental Example 6: Analysis of Polynucleotide (a) For the purpose of confirming that polynucleotide (a) is important in 4.5SH, B1-modified 4.5SH was prepared by replacing the sequence of the polynucleotide (a) part of 4.5SH (SEQ ID NO: 2) with the corresponding sequence of the homologous sequence of 4.5SH, the sequence of SINEB1 (RNA sequence corresponding to SEQ ID NO: 28), and the expression level in cells and the efficiency of exon skipping were examined.

[0122] <Obtaining B1-Modified 4.5SH> A mutation was introduced into the 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) with a U6 promoter mounted on the 5'-side of the DNA sequence was prepared. The obtained plasmid was transformed into Escherichia coli. The transformed Escherichia coli was spread on an LB (with ampicillin) plate and colonies were formed at 37°C for screening. The DNA sequence of the plasmid retained by the Escherichia coli with ampicillin resistance was determined to obtain the B1-modified 4.5SH expression plasmid shown in SEQ ID NO: 30.

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

[0124] <Comparison of Skipping Efficiencies between B1-Modified 4.5SH and Wild-Type 4.5SH> A mini - gene containing wild - type Slc25a40 exon 8.5 and 100 bases of its upstream and downstream regions, 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 the cells under the conditions of 37 °C for 24 hours, the cells were collected and RNA was extracted. Using the cDNA synthesized from the RNA as a template, RT - PCR was performed using primers (SEQ ID NO: 26 and SEQ ID NO: 27) that specifically amplify the mini - gene containing wild - type Slc25a40 Ex8.5, which was also used in Experimental Example 4. As a result, for Slc25a40 exon 8.5, the efficiency of exon skipping was significantly reduced in B1 - modified 4.5SH - expressing cells (the right - most lane in Figure 10) compared to wild - type 4.5SH - expressing cells (the middle lane in Figure 10) (the lower part of Figure 10). The left - most column in Figure 10 shows the results of the control in which a plasmid (so - called empty vector) before introducing mouse 4.5SH in Experimental Example 4 was introduced into human cells (HEK293 cells) instead of using human 4.5SH - expressing cells or B1 - modified 4.5SH - expressing cells.

[0125] Example 1: FAS gene - modified 4.5SH For the purpose of confirming that exon skipping by 4.5SH can be applied to any exon skipping, human FAS gene - modified 4.5SH was prepared and exon skipping was examined.

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

[0127] <Preparation of a Minigene for Exon Skipping Detection> 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 mutations were inserted into the plasmid (pcDNA3 bamHI / xhoI) by Gibson assembly. The resulting plasmid was transformed into Escherichia coli. The transformed Escherichia coli was spread on an LB (containing ampicillin) plate and colonies were formed at 37°C for screening. The DNA sequence of the plasmid retained by the ampicillin-resistant Escherichia coli was determined to obtain a minigene for detecting exon skipping of the FAS gene.

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

Table 2

[0129] 10 μL of the reaction product of PCR was electrophoresed on a polyacrylamide gel and skipping was analyzed. The electrophoresis diagram is shown in the right lane of FIG. 11. The exon skipping efficiency is shown in FIG. 13.

[0130] In the cells co-expressing the FAS gene-modified 4.5SH expression plasmid and the mini-gene for detecting FAS gene exon skipping, exon skipping of exon 6 of the FAS gene was confirmed (right lane of FIG. 11). Also, the exon skipping efficiency of exon 6 of the FAS gene by the FAS gene-modified 4.5SH was 70% (bar at the right end of FIG. 13).

[0131] The left lane of the electrophoresis diagram in FIG. 11 is the electrophoresis diagram of RT-PCR of the extracted RNA after co-expressing the wild-type 4.5SH expression plasmid and the mini-gene for detecting FAS gene exon skipping in human cells (HEK293 cells). 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-introduced into human cells (HEK293 cells) by lipofection. After culturing at 37°C for 24 hours, the cells were harvested and RNA was extracted. Using cDNA synthesized from the RNA as a template, RT-PCR was performed using primers (SEQ ID NOs. 26 and 27) that specifically amplify the minigene, which was also used in Experimental Example 4. 10 μL of the PCR reaction product was subjected to electrophoresis on a polyacrylamide gel, and skipping was analyzed. An image of the electrophoresis is shown in the right lane of Figure 12.

[0133] The left lane of the electrophoresis in Figure 12 shows the electrophoresis of human cells (HEK293 cells) co-expressing a plasmid expressing wild-type 4.5SH and a plasmid expressing a minigene containing wild-type Slc25a40 exon 8.5, and then RT-PCR of the extracted RNA. By comparing the results of co-expression of the wild-type 4.5SH plasmid and wild-type Slc25a40 exon 8.5 (left lane of Figure 12 and the leftmost bar of Figure 13) with the results of co-expression of the FAS gene-modified 4.5SH and the minigene for detecting FAS gene exon skipping (right lane of Figure 11 and the rightmost bar of Figure 13), it can be seen that the FAS gene-modified 4.5SH has FAS gene exon 6 skipping activity equivalent to that of wild-type 4.5SH for asSINEB1 (Figures 11, 12, and 13). Furthermore, since the FAS gene is known to have isoforms that contain exon 6 and isoforms that do not contain exon 6 in vivo, 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 third bar from the right in Figure 13) are thought to represent the isoform ratio in vivo.

[0134] Example 2: Human DMD gene modification 4.5SH To confirm that exon skipping of asSINEB1 by 4.5SH can be applied to arbitrary exon skipping, we created human dystrophin (DMD) gene-modified 4.5SH genes and investigated exon skipping. Since DMD exon 53 has a base length of 212 bases, which is longer than FAS gene exon 6 (63 bases), we first divided the target sequence into four locations (#1, #2, #3, #4) (see Figure 14) and created four DMD gene-modified 4.5SH genes (DMD gene-modified 4.5SH #1 to #4) with different polynucleotide (b) base sequences.

[0135] <Acquisition of four DMD gene-modified 4.5SH cells> We introduced mutations into plasmids expressing the wild-type 4.5SH and obtained four plasmids expressing the mutant. Specifically, we designed DNA sequences in which the asSINEB1 recognition region of the wild-type 4.5SH sequence was modified to a DNA sequence complementary to DMD gene exon 53, as shown in SEQ ID NOs. 17, 18, 19, and 20.

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

[0137] <Preparation of minigenes for exon skipping detection> The DNA sequence containing DMD gene exon 53 and the 100 base pairs upstream and downstream, as shown in Sequence ID No. 25, was inserted into the SacII sequence within the intron of a plasmid derived from the late adenovirus gene (Ad2) to obtain a minigene for detecting DMD gene exon skipping.

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

Table 3

[0139] 10 μL of the reaction product of PCR was electrophoresed on a polyacrylamide gel and skipping was analyzed. An image of the electrophoresis diagram is shown in Fig. 15.

[0140] In DMD gene exon 53, although the skipping efficiency was not high, skipping was detected. In particular, in the sample of DMD gene-modified 4.5SH #2 (simply represented as "2" in Fig. 15) and the mixture of all four of DMD gene-modified 4.5SH #1 to #4 ("Mix" in Fig. 15), the bands after exon skipping were confirmed (the third lane from the left and the sixth lane from the left in Fig. 15).

[0141] Example 2-2: Optimization of Human DMD Gene-Modified 4.5SH <Design of Polynucleotide (b) Sequence> To create a DMD gene-modified 4.5SH with even higher skipping efficiency, the target sequence of DMD exon 53 was optimized. In the sequence of #4 (SEQ ID NO: 20), which is part of DMD exon 53, two transcription termination signal sequences (specifically TTTT) recognized by the RNApolIII enzyme were found, indicating that transcription had terminated. Therefore, the corresponding site was converted to TATT to obtain #5 (SEQ ID NO: 31, Figure 17). Using this DNA sequence, DMD gene-modified 4.5SH#5 was created in the same manner as in Example 2. In Figure 17, the sites complementary to the exons of DMD exon 53 in each sequence are shown as schematic diagrams.

[0142] <Obtaining DMD Gene-Modified 4.5SH> DNA fragments were synthesized by modifying the asSINEB1 recognition region sequence of the wild-type 4.5SH sequence into the DNA sequences shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 31 (DNA sequences complementary to parts of DMD gene exon 53), and four types of DMD gene-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 above DMD gene-modified 4.5SH expression plasmid and the DMD gene exon skipping detection minigene prepared in the same manner as in Example 2 were co-introduced into human cells (HEK293 cells) by the lipofection method. Here, all possible combinations of two types of DMD gene-modified 4.5SH expression plasmids were co-introduced with the DMD gene exon skipping detection minigene. After culturing for 24 hours at 37°C, the cells were collected and RNA was extracted. Using the cDNA synthesized from the RNA as a template, RT-PCR was performed using the primers (SEQ ID NO: 26 and SEQ ID NO: 27) that specifically amplify the minigene used in Experimental Example 4. The conditions of RT-PCR are shown in Table 4.

Table 4

[0144] The reaction product of PCR was analyzed using BioAnalyzer (Agilent Technologies, Inc.) and the exon skipping efficiency was calculated. While some combinations did not exhibit high skipping efficiency, skipping was detected in all combinations. The combination with the highest exon skipping efficiency was DMD gene-modified 4.5SH#2 and DMD gene-modified 4.5SH#5 (rightmost lane in Figure 16). In Figure 16, the leftmost lane shows the results of a sample in which the plasmid used before introducing mouse 4.5SH (the so-called empty vector) and the DMD gene exon skipping detection minigene were co-introduced. The second lane from the left shows the results of a sample in which a vector expressing wild-type 4.5SH RNA (the same plasmid (pUC57) used to obtain human 4.5SH-expressing cells in Experimental Example 4) and the DMD gene exon skipping detection minigene were co-introduced.

[0145] <Optimization of polynucleotide (b) sequence design> Here, the DNA sequence corresponding to the polynucleotide (b) of the present invention in the modified DMD gene 4.5SH#2 (SEQ ID NO: 18) was in the vicinity of the sequence of the existing antisense nucleic acid drug viltolarsen (see Figure 17). Therefore, the sequence of polynucleotide (b) of the modified DMD gene 4.5SH#2 was redesigned to include the entire sequence of viltolarsen, and the modified DMD gene 4.5SH#6 shown in SEQ ID NO: 33 was designed. A DNA fragment expressing the sequence of the modified DMD gene 4.5SH#6 was synthesized, and a modified DMD gene 4.5SH expression plasmid expressing the modified DMD gene 4.5SH#6 shown in SEQ ID NO: 34 was obtained. Furthermore, DMD gene-modified 4.5SH#7 was designed, containing a sequence (SEQ ID NO: 35) obtained by linking the polynucleotide (b) of DMD gene-modified 4.5SH#6 and the polynucleotide (b) of DMD gene-modified 4.5SH#5. A DNA fragment expressing DMD gene-modified 4.5SH#7 was synthesized, and a DMD gene-modified 4.5SH expression plasmid expressing #7, as shown in SEQ ID NO: 36, was obtained.

[0146] <B1-type human DMD gene modification 4.5SH (comparative example)> In addition, a comparative example was designed, which was a B1-modified sequence obtained by replacing the polynucleotide (a) of 4.5SH in DMD gene modification 4.5SH#7 with the sequence of the corresponding part of the polynucleotide (a) of SINEB1, which is the homologous sequence of 4.5SH. A DNA fragment expressing the sequence of the comparative example was synthesized, and a DMD gene-modified B1-modified 4.5SH expression plasmid expressing the comparative example shown in SEQ ID NO: 37 was obtained.

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

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% (the fourth lane from the left in Fig. 18). And the skipping efficiency of DMD exon 53 in DMD gene modification 4.5SH#7 was 46.92% (the fifth lane from the left in Fig. 18).

[0149] In addition, the skipping efficiency of DMD exon 53 in the array of the comparative example was 5.06%, and the skipping efficiency was significantly reduced (the sixth lane from the left in FIG. 18). In FIG. 18, the leftmost lane shows the results of a sample in which a plasmid before the introduction of 4.5SH (so-called empty vector) and a mini-gene for detecting DMD gene exon skipping were co-introduced. The second lane from the left shows the results of a sample in which a vector expressing wild-type 4.5SH RNA (the same plasmid (pUC57) prepared when obtaining human 4.5SH-expressing cells in Experimental Example 4) and a mini-gene for detecting DMD gene exon skipping were co-introduced. Since exon skipping does not occur in the DMD gene in vivo, the skipping efficiency is 0% in the control into which the empty vector was introduced.

[0150] Example 3: MAPT gene-modified 4.5SH For the purpose of confirming that exon skipping of asSINEB1 by 4.5SH can be applied to arbitrary exon skipping, tau (MAPT) gene-modified 4.5SH was prepared and exon skipping was examined.

[0151] <Obtaining MAPT gene-modified 4.5SH> The DNA fragment with the sequence of the asSINEB1 recognition region of the wild-type 4.5SH sequence modified to the DNA sequence complementary to MAPT gene exon 10 shown in SEQ ID NO: 38 was synthesized, and the mutation was inserted into the plasmid (pcDNA3 bamHI / xhoI) by Gibson assembly. The obtained plasmid was transformed into Escherichia coli. The transformed Escherichia coli was spread on an LB (containing ampicillin) plate, and colonies were formed at 37°C for screening. The DNA sequence of the plasmid retained by the ampicillin-resistant Escherichia coli was determined to obtain the human MAPT gene-modified 4.5SH expression plasmid shown in SEQ ID NO: 39. A reporter carrying the U6 promoter and the sequence of the MAPT gene-modified 4.5SH was prepared, and the expression vector (pUC57) carrying the reporter was introduced into human cells (HEK293 cells) by the liposome transfection method. By Northern blotting, it was confirmed that the MAPT gene-modified 4.5SH RNA was expressed (the middle lane, the rightmost lane in Figure 20), and MAPT gene-modified 4.5SH-expressing cells were obtained.

[0152] <Production of Mini-Gene for Exon Skipping Detection> A DNA fragment containing MAPT gene exons 9, 10, and 11, etc. (for details, refer to Qingming Yu et al, (2004) J Neurochem, Jul;90(1):164-72) shown in SEQ ID NO: 40 was amplified by PCR, the PCR product was purified, and the mutation was inserted into the plasmid (pcDNA3 bamHI / xhoI) by Gibson assembly. The obtained plasmid was transformed into Escherichia coli. The transformed Escherichia coli was spread on an LB (containing ampicillin) plate, and colonies were formed at 37°C for screening. The DNA sequence of the plasmid retained by the ampicillin-resistant Escherichia coli was determined to obtain a mini-gene for detecting exon skipping of the MAPT gene.

[0153] <Detection of MAPT Gene Exon Skipping> The above-mentioned MAPT gene-modified 4.5SH expression plasmid and a minigene for detecting MAPT gene exon skipping were co-introduced into human cells (HEK293 cells) by lipofection. After culturing at 37°C for 24 hours, 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 (as shown in SEQ ID NOs. 41 and 42) and forward primers that bind to MAPT gene exon 11. By using this primer set, the MAPT gene inherent in the cells is not amplified. The RT-PCR conditions are shown in Table 6. [Table 6]

[0154] Figure 19 shows the results of analyzing 1 μL of the PCR reaction product using BioAnalyzer (Agilent Technologies, Inc.) and calculating the exon skipping efficiency.

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

[0156] The center lane (second from the left) of the electrophoresis in Figure 19 shows the electrophoresis of human cells co-expressing the FAS-modified 4.5SH expression plasmid obtained in Example 1 with a minigene for detecting MAPT gene exon skipping, and the extracted RNA obtained by RT-PCR. The exon skipping efficiency was 64.99%, which was significantly lower than the skipping efficiency when the above-mentioned MAPT gene-modified 4.5SH expression plasmid was expressed (Figure 19 4.5SH-asFAS). In Figure 19, the control on the far left shows the results of a sample in which the plasmid before mutation introduction (so-called empty vector) and the minigene for detecting MAPT gene exon skipping were co-introduced. Since it is known that the MAPT gene has isoforms that contain exon 10 and isoforms that do not contain exon 10 in vivo, the skipping efficiency of the control into which the empty vector was introduced is thought to represent the ratio of isoform amounts in vivo. [Industrial applicability]

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

Claims

1. An mRNA splicing regulator comprising an artificial RNA molecule having (a) and (b) below, wherein (a) and (b) are arranged in this order from the 5' side to the 3' side. (a) A polynucleotide that potentially has a secondary structure represented by the following formula (I), and whose base sequence is the sequence shown in Sequence ID No.

2. (b) mRNA precursor-targeted polynucleotides containing a complementary sequence to the target sequence, which is a part of the mRNA precursor sequence. 【Chemistry 1】 (where N 1 ~N 25 each independently represents A, C, G, or U, and N 1 and N 25 means that N 2 and N 24 means that N 3 and N 23 means that N 4 and N 22 means that N 5 and N 21 means that N 6 and N 20 means that N 8 and N 19 means that N 9 and N 18 means that N 10 and N 17 each form a base pair.)

2. A pharmaceutical composition comprising an artificial RNA molecule comprising (a) and (b) below, wherein (a) and (b) are arranged in this order from the 5' side to the 3' side. (a) A polynucleotide that potentially has a secondary structure represented by the following formula (I), and whose base sequence is the sequence shown in Sequence ID No.

2. (b) mRNA precursor-targeted polynucleotides containing a complementary sequence to the target sequence, which is a part of the mRNA precursor sequence. 【Chemistry 2】 (In the formula, N 1 ~N 25 Each of these independently represents A, C, G, or U, and N 1 and N 25 That is, N 2 and N 24 That is, N 3 and N 23 That is, N 4 and N 22 That is, N 5 and N 21 That is, N 6 and N 20 That is, N 8 and N 19 That is, N 9 and N 18 That is, N 10 and N 17 (These two bases each form a base pair.)

3. The pharmaceutical composition according to claim 2, wherein the splicing of the mRNA precursor targeted by (b) above is regulated.

4. The mRNA splicing regulator according to claim 1, wherein the length of (b) is 15 to 300 bases.

5. The mRNA splicing regulator according to claim 1, wherein the target sequence is an exon sequence.

6. The mRNA splicing regulator according to claim 1, wherein (c) is positioned on the 3' side of (b). (c) Polynucleotides containing sequences that contribute to terminal stability

7. The mRNA splicing regulator according to claim 6, wherein (c) is a polynucleotide containing a transcription termination signal sequence recognized by RNApol III enzyme.

8. The mRNA splicing regulator according to claim 1, wherein (a) is a polynucleotide that binds to a protein that controls mRNA splicing.

9. An artificial RNA molecule comprising (a) and (b) below, wherein (a) and (b) are arranged in this order from the 5' side to the 3' side. (a) A polynucleotide that potentially has a secondary structure represented by the following formula (I), and whose base sequence is the sequence shown in Sequence ID No.

2. (b) mRNA precursor-targeted polynucleotides containing a complementary sequence to the target sequence, which is a part of the mRNA precursor sequence. 【Transformation 3】 (In the formula, N 1 ~N 25 Each of these independently represents A, C, G, or U, and N 1 and N 25 That is, N 2 and N 24 That is, N 3 and N 23 That is, N 4 and N 22 That is, N 5 and N 21 That is, N 6 and N 20 That is, N 8 and N 19 That is, N 9 and N 18 That is, N 10 and N 17 (These two bases each form a base pair.) An artificial RNA molecule in which the target sequence is a sequence of a gene selected from the FAS gene, the dystrophin gene, and the fukutin gene.

10. An artificial RNA molecule comprising (a) and (b) below, wherein (a) and (b) are arranged in this order from the 5' side to the 3' side. (a) A polynucleotide that potentially has a secondary structure represented by the following formula (I), and whose base sequence is the sequence shown in Sequence ID No.

2. (b) mRNA precursor-targeted polynucleotides containing a complementary sequence to the target sequence, which is a part of the mRNA precursor sequence. 【Chemistry 4】 (In the formula, N 1 ~N 25 Each of these independently represents A, C, G, or U, and N 1 and N 25 That is, N 2 and N 24 That is, N 3 and N 23 That is, N 4 and N 22 That is, N 5 and N 21 That is, N 6 and N 20 That is, N 8 and N 19 That is, N 9 and N 18 That is, N 10 and N 17 (These two bases each form a base pair.) An artificial RNA molecule in which the target sequence is a part of the MAPT gene sequence.

11. An expression vector comprising a DNA sequence encoding an artificial RNA molecule according to claim 9 or 10.

12. A method for regulating mRNA splicing, (A) A step of preparing an artificial RNA molecule comprising (a) and (b) below, wherein (a) and (b) below are arranged in this order from the 5' side to the 3' side. (a) A polynucleotide that potentially has a secondary structure represented by the following formula (I), and whose base sequence is the sequence shown in Sequence ID No.

2. (b) mRNA precursor-targeted polynucleotides containing a complementary sequence to the target sequence, which is a part of the mRNA precursor sequence, and (B) A step in which the artificial RNA molecule prepared in step (A) is brought into contact with the mRNA precursor, a complementary strand is formed between the artificial RNA molecule and the mRNA precursor, thereby regulating mRNA splicing. Methods including (excluding medical procedures performed on humans). 【Transformation 5】 (where N 1 ~N 25 each independently represents A, C, G, or U, and N 1 and N 25 are such that N 2 and N 24 are such that N 3 and N 23 are such that N 4 and N 22 are such that N 5 and N 21 are such that N 6 and N 20 are such that N 8 and N 19 are such that N 9 and N 18 are such that N 10 and N 17 each form a base pair.)

13. The method according to claim 12, wherein the regulation of mRNA splicing is performed by exon skipping.

14. The method according to claim 12 or 13, wherein the target sequence is an exon sequence.

15. The method according to claim 12 or 13, wherein the artificial RNA molecule has a polynucleotide containing a transcription termination signal sequence recognized by RNApol III enzyme positioned on the 3' side of (b).

16. A method for producing mature mRNA, (A) A step of preparing an artificial RNA molecule comprising (a) and (b) below, wherein (a) and (b) below are arranged in this order from the 5' side to the 3' side. (a) A polynucleotide that potentially has a secondary structure represented by the following formula (I), and whose base sequence is the sequence shown in Sequence ID No.

2. (b) mRNA precursor-targeted polynucleotides containing a complementary sequence to the target sequence, which is a part of the mRNA precursor sequence. (B') A step of contacting the artificial RNA molecule prepared in step (A) with the mRNA precursor to form a complementary strand between the artificial RNA molecule and the mRNA precursor, and (C) Step (B') is a step in which mRNA splicing is regulated and mature mRNA is produced. Methods including (excluding medical procedures performed on humans). 【Transformation 6】 (In the formula, N 1 ~N 25 Each of these independently represents A, C, G, or U, and N 1 and N 25 That is, N 2 and N 24 That is, N 3 and N 23 That is, N 4 and N 22 That is, N 5 and N 21 That is, N 6 and N 20 That is, N 8 and N 19 That is, N 9 and N 18 That is, N 10 and N 17 (These two bases each form a base pair.)

17. The method according to claim 16, wherein the regulation of mRNA splicing is performed by exon skipping.

18. The method according to claim 16 or 17, wherein the target sequence is an exon sequence.

19. The method according to claim 16 or 17, wherein the artificial RNA molecule has a polynucleotide containing a transcription termination signal sequence recognized by RNApol III enzyme positioned on the 3' side of (b).

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