JP2023040852A - Single-stranded nucleic acid molecules and compositions for inducing a -1 frameshift - Google Patents

Abstract

To provide substances that are safe for living organisms and capable of expressing a protein having an amino acid sequence more similar to that of a normal protein in the state that the length is suppressed from being deleted even from a gene with an out-of-frame mutation, and to provide compositions containing the substance.SOLUTION: The foregoing problem is solved by a single-stranded nucleic acid molecule containing a first sequence complementary to a target sequence of a gene of interest and a second sequence forming a stem-loop structure, in the direction from the 5'end side to the 3'end side; and a composition or the like comprising the single-stranded nucleic acid molecule as an active ingredient.SELECTED DRAWING: Figure 7

Description

The present invention relates to single-stranded nucleic acid molecules for inducing a -1 frameshift and compositions comprising the single-stranded nucleic acid molecules.

A hereditary disease is a disease caused by abnormal expression of a gene, such as a decrease in the expression level of the gene or an increase in the expression level of the gene due to some kind of abnormality in the gene. Genetic diseases include, for example, muscular dystrophy, cancer, multiple sclerosis, spinal muscular atrophy, Rett syndrome, amyotrophic lateral sclerosis, fragile X syndrome, Prader-Willi syndrome, xeroderma pigmentosum, porphyrin disease, Werner's syndrome, fibrodysplasia ossificans progressive, infantile neuronal ceroid lipofuscinosis, Alzheimer's disease, Tay-Sachs disease, neurodegeneration, Parkinson's disease, rheumatoid arthritis, graft-versus-host disease, arthritis , hemophilia, von Willebrand disease, ataxia-telangiectasia, thalassemia, kidney stones, osteogenesis imperfecta, liver cirrhosis, neurofibromatosis, bullous disease, lysosomal storage disease, Hurler's disease, cerebellar ataxia, nodular sclerosis, familial polycythemia, immunodeficiency, renal disease, pulmonary disease, cystic fibrosis, familial hypercholesterolemia, retinopathy pigmentosa, amyloidosis, atherosclerosis, gigantism, dwarfism, Hypothyroidism, hyperthyroidism, aging, obesity, diabetes, Niemann-Pick disease, Marfan syndrome, etc. are known.

Among hereditary diseases, nonsense mutation-type hereditary diseases are diseases caused by inhibition of protein expression due to premature termination codons (PTC) formed by point mutations on genes. be.

When PTC, which is a stop codon that appears upstream (5′ end side) from the original stop codon, is formed on the mRNA obtained by gene transcription, the protein obtained by translation becomes shorter and functional. Protein may not be obtained. In addition, when PTC occurs due to incorrect translation of mRNA, NMD (Nonsense-mediated mRNA decay: nonsense mutation-dependent mRNA degradation mechanism), which is an intracellular mRNA quality control system, is activated, and mRNA having PTC in the cell may be actively degraded, resulting in decreased or no protein expression. Such abnormalities in gene expression cause hereditary diseases.

PTCs can also result from frameshift mutations. A PTC is formed by a deletion or insertion of a number of bases other than a multiple of 3 in a gene, resulting in a shift of the reading frame during translation (frame shift).

Therefore, when PTC is formed by out-of-frame mutations such as nonsense mutations and frameshift mutations, the normal protein cannot be obtained. In-frame mutations, on the other hand, are mutations that maintain the reading frame, and the protein is more likely to be expressed even if some parts differ from the normal protein. Therefore, in general, out-of-frame mutation-type diseases tend to be more serious than in-frame mutation-type diseases.

Duchenne muscular dystrophy (DMD), one of out-of-frame mutational genetic diseases, is caused by mutation of the dystrophin gene on the X chromosome. For example, when a part of the exon of the dystrophin gene on the X chromosome is deleted, PTC is formed on the mRNA, and translation is interrupted or terminated, resulting in normal expression of dystrophin and dystrophin-related proteins. It will not work properly. This leads to a lack of functional dystrophin protein in muscle tissue, leading to the development of DMD.

Chemotherapy by administering a substance that induces exon skipping and a substance that has read-through activity is expected as a therapeutic method for out-of-frame mutation-type genetic diseases such as DMD.

Exon skipping is a technique in which an exon containing an out-of-frame mutation is skipped, thereby converting the out-of-frame mutation into an in-frame mutation to obtain a protein encoded by mRNA lacking some exons. be. A protein obtained by exon skipping is a protein (truncated protein) with an amino acid length shorter than that of a normal protein. For example, compounds and antisense nucleic acids described in Patent Documents 1 and 2 below (the entire descriptions of these documents are incorporated herein by reference) are known as substances that induce exon skipping.

On the other hand, the read-through activity refers to an activity that acts on the ribosome and causes the ribosome to read over the PTC formed by out-of-frame mutation and translate. As a result of readthrough, normal protein can be synthesized. For example, as a substance having read-through activity, a compound described in Patent Document 3 below (the entire description of the document is incorporated herein by reference) is known.

Non-Patent Document 1 below (the entire description of which is incorporated herein by reference) describes a method for identifying frameshift genes in the genome and circular code information used in the method.

Japanese Patent No. 5950818 Japanese Patent No. 5363655 Japanese Patent No. 5705136

J Comput Sci Syst Biol, 2011, Vol.4(1), pp.7-15

Exon skipping using compounds and antisense nucleic acids described in Patent Documents 1 and 2 makes it possible to read genetic information to the end without generating PTC by skipping mutated exons. Become. However, since it is not possible to correct the disordered reading of genetic information by simply skipping only the mutated exon, exon skipping requires skipping the mutated exon and multiple exons around it. There's a problem. Furthermore, there is a problem that the protein is shortened by the skipped exons, and as a result, a functional protein cannot be obtained.

If a substance having read-through activity such as the compound described in Patent Document 3 is used, PTC can be skipped. However, even if a substance having read-through activity is used, the deviated reading frame is not restored, so there is a problem that the resulting protein has an amino acid composition different from that of the normal protein. In addition, whether or not read-through occurs largely depends on probability theory, and there is a problem that other amino acids are not incorporated into PTC, and protein synthesis does not continue in many cases. Furthermore, arbekacin, which is the compound described in Patent Document 3, is an antibiotic and has the problem of being highly toxic to living organisms.

In addition, a method for expressing a protein having an amino acid sequence more similar to that of a normal protein from a gene with an out-of-frame mutation while suppressing the length loss as much as possible. Few cases have been reported.

Therefore, the present invention provides a protein having an amino acid sequence more similar to that of a normal protein, even from a gene with an out-of-frame mutation, compared to a method using a substance having exon skipping and read-through activity. It is an object of the present invention to provide a substance that is safe for living organisms and a composition containing the substance, which can be expressed in a state in which deletion of the gene is suppressed.

In order to solve the above problems, the present inventors have made intensive studies on correcting disordered reading of genetic information without skipping exons. As a result, if the reading frame can be shifted in the 5' or 3' direction by 1 base or in the 3' direction by 2 bases in the mutated portion or its surroundings, the length of the expressed protein can be maintained as long as possible, and the normal expression can be obtained. This led to the idea that it might be possible to express a protein having an amino acid sequence more similar to that of the original protein.

Based on the above considerations, the present inventors have, as a result of repeated trial and error, surprisingly found a single-stranded nucleic acid containing a sequence complementary to the target sequence of the target gene and a sequence forming a specific stem-loop structure. By using a molecule to induce a −1 frame shift around the mutation site to shift the reading frame, we succeeded in correcting the disordered reading of genetic information without skipping exons. In addition, since the single-stranded nucleic acid molecule can be degraded by nucleolytic enzymes in living organisms, it is safer for living organisms than synthetic compounds.

Finally, the present inventor succeeded in creating the single-stranded nucleic acid molecule and a composition containing the single-stranded nucleic acid molecule as an active ingredient. The present invention is an invention that has been completed based on such successful examples and findings.

Accordingly, according to one aspect of the present invention, the following single-stranded nucleic acid molecules and compositions are provided.
[1] A single strand comprising a first sequence complementary to the target sequence of the gene of interest and a second sequence forming a stem-loop structure in the direction from the 5' end side to the 3' end side nucleic acid molecule.
[2] The single-stranded nucleic acid molecule according to [1], wherein the ends of the stem portion of the stem-loop structure are cytosine and guanine.
[3] The single-stranded nucleic acid molecule according to any one of [1] to [2], wherein the stem-loop structure is a stem-loop structure in which the loop portion is mainly composed of cytosine.
[4] The single-stranded nucleic acid molecule according to any one of [1] to [3], wherein the stem-loop structure has a stem portion of 4 to 20 base pairs.
[5] The single-stranded nucleic acid molecule according to any one of [1] to [4], wherein the second sequence is the sequence set forth in SEQ ID NO:17 or SEQ ID NO:18.
[6] The single-stranded nucleic acid molecule according to any one of [1] to [5], wherein the first sequence has a length of 8 to 16 bases.
[7] The single-stranded nucleic acid molecule according to any one of [1] to [6], wherein the gene has two or more consecutive identical bases in 9 to 15 bases upstream of the target sequence.
[8] Any one of [1] to [6], wherein the gene has two or more portions in which two or more identical bases are consecutive in 9 to 15 bases upstream of the target sequence. A single-stranded nucleic acid molecule as described.
[9] A composition for inducing a -1 frameshift, comprising the single-stranded nucleic acid molecule of any one of [1] to [8] as an active ingredient.
[10] The composition is a pharmaceutical composition for preventing and/or treating a genetic disease caused by abnormal expression of the gene by inducing -1 frameshift to normalize the expression of the gene. The composition according to [9], which is a product.
[11] The genetic disease includes muscular dystrophy, cancer, multiple sclerosis, spinal muscular atrophy, Rett syndrome, amyotrophic lateral sclerosis, fragile X syndrome, Prader-Willi syndrome, xeroderma pigmentosum, Porphyria, Werner's syndrome, fibrodysplasia ossificans progressis, infantile neuronal ceroid lipofuscinosis, Alzheimer's disease, Tay-Sachs disease, neurodegeneration, Parkinson's disease, rheumatoid arthritis, graft-versus-host disease, Arthritis, hemophilia, von Willebrand disease, ataxia-telangiectasia, thalassemia, kidney stones, osteogenesis imperfecta, liver cirrhosis, neurofibromatosis, bullous disease, lysosomal storage disease, Hurler's disease, cerebellar ataxia, nodules Sclerosis, familial polycythemia, immunodeficiency, kidney disease, lung disease, cystic fibrosis, familial hypercholesterolemia, retinopathy pigmentosa, amyloidosis, atherosclerosis, gigantism, dwarfism , hypothyroidism, hyperthyroidism, aging, obesity, diabetes, Niemann-Pick disease, and Marfan syndrome.
[12] having a genetic disease caused by abnormal expression of the single-stranded nucleic acid molecule of any one of [1] to [8] or the composition of [9], or a method of treating and/or preventing a genetic disease, including administration to an organism at risk thereof.
[13] An effective amount of the single-stranded nucleic acid molecule of any one of [1] to [8] or the composition of [9] is applied to a cell or tissue in which abnormal expression of the gene of interest is observed. , a method of improving abnormal gene expression, including administering to an organ or an individual organism.
[14] The genetic disease includes muscular dystrophy, cancer, multiple sclerosis, spinal muscular atrophy, Rett syndrome, amyotrophic lateral sclerosis, fragile X syndrome, Prader-Willi syndrome, xeroderma pigmentosum, Porphyria, Werner's syndrome, progressive fibrodysplasia ossificans, infantile neuronal ceroid lipofuscinosis, Alzheimer's disease, Tay-Sachs disease, nerve tissue degeneration, Parkinson's disease, rheumatoid arthritis, graft-versus-host disease, Arthritis, hemophilia, von Willebrand disease, ataxia-telangiectasia, thalassemia, kidney stones, osteogenesis imperfecta, liver cirrhosis, neurofibromatosis, bullous disease, lysosomal storage disease, Hurler's disease, cerebellar ataxia, nodules Sclerosis, familial polycythemia, immunodeficiency, kidney disease, lung disease, cystic fibrosis, familial hypercholesterolemia, retinopathy pigmentosa, amyloidosis, atherosclerosis, gigantism, dwarfism , hypothyroidism, hyperthyroidism, aging, obesity, diabetes, Niemann-Pick disease, and Marfan syndrome.

According to the present invention, by inducing a −1 frameshift, a protein having an amino acid sequence more similar to that of a normal protein is expressed from a gene with an out-of-frame mutation while maintaining a longer full length. be able to. Thus, according to the present invention, expression of functional proteins from mutated genes is expected.

Therefore, according to the present invention, the expression of a gene of interest is normalized, and a hereditary disease caused by an abnormality in which the expression of the gene is decreased or increased, such as muscular dystrophy, cancer, multiple sclerosis, and spinal cord muscle. Atrophy, Rett syndrome, amyotrophic lateral sclerosis, fragile X syndrome, Prader-Willi syndrome, xeroderma pigmentosum, porphyria, Werner syndrome, fibrodysplasia ossificans progressis, infant neuronal ceroid lipofuscin Deposition disease, Alzheimer's disease, Tay-Sachs disease, neurodegeneration, Parkinson's disease, rheumatoid arthritis, graft-versus-host disease, arthritis, hemophilia, von Willebrand's disease, ataxia-telangiectasia, thalassemia, kidney stones , osteogenesis imperfecta, cirrhosis, neurofibromatosis, bullous disease, lysosomal storage disease, Hurler's disease, cerebellar ataxia, tuberous sclerosis, familial polycythemia, immunodeficiency, kidney disease, lung disease, cystic Fibrosis, familial hypercholesterolemia, retinopathy pigmentosa, amyloidosis, atherosclerosis, gigantism, dwarfism, hypothyroidism, hyperthyroidism, aging, obesity, diabetes, Niemann-Pick disease , is expected to treat and/or prevent genetic disorders such as Marfan syndrome.

FIG. 1 shows the coding region (CDS) of EGFP-Human α-Tubulin, as described in the Examples below. FIG. 2 is a diagram showing the photographing results of the fluorescence microscope of Example 1, as described in Examples below. FIG. 3 is a diagram showing the CDS of the DHFR gene, as described in Examples below. Figure 4A shows the nucleotide sequence of the reverse primer used in Example 2, as described in the Examples below. FIG. 4B shows the amino acid sequence of the DHFR protein and the amino acid sequence of the DHFR-His protein, as described in the examples below. FIG. 5A is an image of the entire gel after electrophoresis and staining in Example 2, as described in the Examples below. FIG. 5B is an enlarged view of FIG. 5A where the His-tagged DHFR proteins of TEST2 Elution1 and Elution2 appeared. Figure 5C is an enlarged view of Figure 5A where DHFR proteins and DHFR protein fragments of NC, PC and TEST1 appear. FIG. 6A is a diagram showing the target sequence of Position 1 set around the breakpoint between exons 44 and 51 of the mutant dystrophin gene, as described in Examples below. FIG. 6B is a diagram showing the target sequence of Position 2 set around the breakpoint between exons 44 and 51 of the mutant dystrophin gene, as described in Examples below. FIG. 6C is a diagram showing the target sequence of Position 3 set around the breakpoint between exons 44 and 51 of the mutant dystrophin gene, as described in Examples below. FIG. 7 is a graph showing the results of the relative amount of dystrophin mRNA expression level with respect to the GAPDH mRNA expression level described in Example 3, as described in Examples below.

The details of each aspect of the present invention will be described below, but the present invention is not limited only by the matters of this item, and can take various aspects as long as the object of the present invention is achieved.

Unless otherwise specified, each term in the present specification is used in the meaning commonly used by those skilled in the art in the technical fields such as the pharmaceutical field and the biological field, and is interpreted as having an unduly restrictive meaning. shouldn't. In addition, the speculations and theories made in this specification are based on the present inventor's knowledge and experience, so the present invention is not bound only by such speculations and theories. .

The term "composition" is not particularly limited in its commonly used meaning, but includes, for example, a combination of two or more components.
"Active ingredient" means an ingredient that characterizes the use of the composition.
The term "and/or" means any one, or any or all combinations of two or more of the associated listed items.
"Content" is synonymous with concentration and added amount, and means the ratio of the amount of an ingredient to the total amount of the composition. However, the total content of the components does not exceed 100%. "Effective amount" means an amount that produces the effects of an active ingredient or composition.
"-" in a numerical range is a range including the numerical values before and after it, for example, "0% to 100%" means a range of 0% or more and 100% or less. "greater than" and "less than" mean the lower and upper limits, respectively, excluding the preceding number; means.
"Contains" means that it can add elements other than those explicitly included (which is synonymous with "including at least"), but includes "consisting of" and "consisting essentially of" . That is, "comprising" can mean including the specified element and any one or more elements, consisting of, or consisting essentially of the specified element. . Factors include limitations such as components, steps, conditions, parameters, and the like.

The "stem-loop structure" is also referred to as a hairpin loop, and is not particularly limited in its commonly used meaning. A structure formed in a third sequence that is between the sequences of In this case, the first and second sequences form a base pair to form a double-stranded portion (stem portion), and the third sequence forms a loop portion without base pairing to form the entire as a stem-loop structure.
"Complementary base pair" or simply "base pair" means Watson-Crick base pairs and wobble base pairs, and combinations of adenine (A) with thymine (T) or uracil (U) and guanine (G) and cytosine (C), as well as guanine (G) and uracil (U). In the present specification, adenine, thymine, uracil, guanine, and cytosine, and A, T, U, G, and C representing these, respectively, are referred to as "bases" or "nucleotides." As used herein, the terms "base" and "nucleotide" are used interchangeably.
The term “end of stem portion” refers to the sequence of the end that is not linked to the sequence forming the loop portion, out of the two ends of the stem portion.

“−1 frameshift” means a frameshift that shifts the codon reading frame by one base to the 5′ side, among frameshifts that mean a codon reading frame shift. For example, exemplifying scheme (I) citing Figure 2 of Non-Patent Document 1, the mRNA shown as scheme (I) is formed by ribosomal RNA from the start codon (AUG) to CGU, GCU, and GUG in order of 3 bases. Read (Frame 0). However, when the reading frame shifts by one base to the 5′ side at the illustrated frameshift site (also referred to as a slippage site), GGC is read, then GAA, AUA are read (Frame 2). Such a frameshift caused by shifting one base to the 5' side at the frameshift site is referred to as -1 frameshift.

スキーム（Ｉ）

Scheme (I)

An “out-of-frame mutation” means a mutation that shifts the reading frame of an mRNA open reading frame (ORF). When an out-of-frame mutation occurs, a protein having an amino acid sequence different from that of a normal protein without mutation is synthesized, and a premature stop codon (PTC) occurs 5' (upstream) from the original stop codon, resulting in a mutation. Synthesis of proteins shorter than normal proteins without

A “nucleic acid molecule”, also simply referred to as a nucleic acid, means a polymer in which nucleotides composed of ribonucleotides, deoxyribonucleotides, and analogues thereof are linked together by phosphodiester bonds, as is usually meant. A nucleotide is sometimes called a base, and a nucleotide sequence and a base sequence are synonymous. Nucleotide and base sequences are sometimes simply referred to as sequences of nucleic acid molecules. Representative examples of nucleic acid molecules include ribonucleic acid (RNA) consisting of ribonucleotides and deoxyribonucleic acid (DNA) consisting of deoxyribonucleotides.
A "single-stranded nucleic acid molecule" means a single nucleic acid molecule that has not formed complementary base pairs with other nucleic acid molecules. A single-stranded nucleic acid molecule may have a stem portion in which sequences within the molecule form complementary base pairs.
"Expression" refers to the synthesis of mRNA from a gene (transcription), protein synthesis from mRNA (translation), or both.
"Upstream" with respect to a sequence means 5' for nucleotide sequences and N-terminal for amino acid sequences. "Downstream" with respect to a sequence means 3' for nucleotide sequences and C-terminal for amino acid sequences.

The number of digits in the integer value and the number of significant digits are the same. For example, 1 has 1 significant digit and 10 has 2 significant digits. Also, for decimal values, the number of digits after the decimal point and the number of significant digits are the same. For example, 0.1 has one significant digit and 0.10 has two significant digits.

[Single-stranded nucleic acid molecule]
The single-stranded nucleic acid molecule of one aspect of the present invention forms a stem-loop structure with the first sequence complementary to the target sequence of the gene of interest in the direction from the 5' end side to the 3' end side. and a second array.

A single-stranded nucleic acid molecule can be used to induce a -1 frameshift when the mRNA of the gene of interest is translated. That is, a single-stranded nucleic acid molecule has a -1 frameshift-inducing activity. The mechanism of the −1 frameshift-inducing action of the single-stranded nucleic acid molecule will be explained using the schematic diagram of the mRNA of the target gene shown as Scheme (II).

スキーム（ＩＩ）

Scheme (II)

The first sequence in the single-stranded nucleic acid molecule forms complementary base pairs and binds to a target sequence in the mRNA of the gene of interest. When the mRNA of the target gene is translated in a state in which the single-stranded nucleic acid molecules are bound, a -1 frameshift occurs, in which the reading frame shifts forward by 1 base 14 bases upstream of the target sequence. To explain using scheme (II), the sense codon N ₃₆ N ₃₅ N ₃₄ starting from N ₃₆ , which is 14 bases upstream of the target sequence, is shifted forward by 1 base due to -1 frame shift, and the sense codon N ₃₇ N ₃₆ N ₃₅ shift to However, the -1 frameshift occurs after the addition of the amino acids corresponding to the first sense codon N ₃₆ N ₃₅ N ₃₄ followed by the second sense codon N ₃₃ N ₃₂ N ₃₁ , It is believed that the ribosome slips to the positions of sense codons N ₃₇ N ₃₆ N ₃₅ and sense codons N ₃₄ N ₃₃ N ₃₂ . As a result, the amino acid sequence of the synthesized protein consists of the amino acid encoded by the first sense codon N ₃₆ N ₃₅ N ₃₄ , the amino acid encoded by the second sense codon N ₃₃ N ₃₂ N ₃₁ , and the sense codon N ₃₁ N ₂₆ N ₂₅ . It is presumed that the amino acid sequence encoded by the sense codon N ₂₄ N ₂₃ N _{22 .} . . The base (nucleotide) upstream of a certain sequence means the base (nucleotide) on the 5′ side of the 5′ end of the sequence. A base (nucleotide) downstream of a certain sequence means a base (nucleotide) on the 3′ side of the 3′ end of the sequence.

The target sequence can be appropriately determined according to the site at which the -1 frameshift is desired. That is, the target sequence can be a site beginning 14 bases downstream of the site (N ₃₆ in scheme (II)) where a -1 frameshift is desired. However, in the 9 to 15 bases upstream of the target sequence (N ₃₇ N ₃₆ N ₃₅ N ₃₄ N ₃₃ N ₃₂ N ₃₁ in scheme (II)), two identical bases such as CC, AA, UU or GG It is preferable that at least one of them is continuous. In addition, it is preferable that there are two or more portions in which two or more of the same bases are consecutive in the 9 to 15 bases upstream of the target sequence. The above-mentioned "upstream 9 bases to upstream 15 bases of the target sequence" is the number of bases when the sequence N ₂₆ N ₂₅ N ₂₄ N ₂₃ N ₂₂ N ₂₁ (referred to as a spacer sequence) in scheme (II) is 6 bases. is. The spacer sequence may be 3 bases to 8 bases. ”.

The first sequence may be any sequence that includes a sequence complementary to the target sequence. The length of the first sequence is not particularly limited as long as it is long enough to bind to the target sequence. Considering that the portion that binds to the target sequence does not bind and interact with other portions of the single-stranded nucleic acid molecule of one embodiment of the present invention to disrupt the stem-loop structure, the length is 6 to 20 bases. preferably 8 to 16 bases in length, more preferably 9 to 14 bases in length, even more preferably 10 to 13 bases in length.

The first sequence may be either completely complementary or partially complementary to the target sequence, but it should be completely complementary considering the binding strength with the target sequence. is preferred.

The first sequence can be determined based on the nucleotide sequence, which is the alignment of nucleotides of the target sequence.

The second sequence forms a stem-loop structure. When the single-stranded nucleic acid molecule and the mRNA of the target gene bind by forming complementary base pairs between the target sequence and the first sequence, the end of the stem portion of the stem-loop structure is an intramolecular double strand It maintains its structure and does not form complementary base pairs with the mRNA of the gene of interest. This will be explained by exemplifying Scheme (III), which shows a non-limiting single-stranded nucleic acid molecule of one embodiment of the present invention.

スキーム（ＩＩＩ）

Scheme (III)

In scheme (III), "○" (open circle) represents one nucleotide. Solid lines indicate complementary base pair relationships (hydrogen bonds) and dotted lines indicate bonds between adjacent nucleotides (phosphodiester bonds). As shown in scheme (III), the two nucleotides at the end of the stem portion as indicated by the black arrows are complementary to the sequences (N ₁₂ , N ₁₁ ) of the mRNA of the opposite gene of interest as indicated by the white arrows. do not form organic base pairs. Thus, in the second sequence, the ends of the stem-loop structure maintain the intramolecular double-stranded structure as the stem portion.

In the stem-loop structure of the second sequence, the length of the stem portion is not particularly limited as long as it is long enough to form an intramolecular double-stranded structure. For example, 3 base pairs to 50 bases. It is a length that forms a pair, and considering that the intramolecular double-stranded structure is stably maintained, it is preferably a length that forms 4 base pairs to 20 base pairs, and 5 base pairs to 10 base pairs. More preferably, it has a length that forms a base pair, and more preferably a length that forms about 8 base pairs.

The stem portion may be either a complete intramolecular double-stranded structure or a double-stranded structure with a single-stranded structure in the middle. A complete intramolecular double-stranded structure is preferred in consideration of maintaining the In addition, even if the double-stranded structure has a portion that assumes a single-stranded structure in the middle, it is preferable that the two sequences at the ends of the stem portion form complementary base pairs.

The nucleotide sequence of the stem portion is not particularly limited as long as it is a sequence that forms an intramolecular double-stranded structure. However, the terminal two nucleotides of the stem portion are preferably a combination of cytosine and guanine, a combination of adenine and uracil, and more preferably a combination of cytosine and guanine. A specific example of the nucleotide sequence of one of the stem portions is the sequence shown in SEQ ID NO: 15 (5'-CCGCAUUA-3'), but is not limited thereto. The sequence (5'-UAGUGUGG-3') shown in SEQ ID NO: 16 forms a complementary base pair with the sequence shown in SEQ ID NO: 15 and forms a complete intramolecular double-stranded structure.

In the stem-loop structure of the second sequence, the length of the loop portion is not particularly limited as long as it is long enough to maintain the single-stranded structure. Considering stably maintaining the single-stranded structure, it is preferably 5 to 20 bases long, more preferably 8 to 15 bases long, and about 11 bases long. It is even more preferable to have

The loop portion may be either a complete single-stranded structure or a single-stranded structure with a double-stranded structure in the middle, but the single-stranded structure must be stably maintained. Considering the complete single-stranded structure is preferred.

The nucleotide sequence of the loop portion is not particularly limited as long as it is a sequence that maintains a single-stranded structure. Examples thereof include sequences that do not form an intramolecular double-stranded structure. A sequence consisting of guanine and adenine, a sequence consisting of cytosine, guanine, adenine or uridine, more preferably a sequence consisting mainly of cytosine in which 80% or more is cytosine, still more preferably completely consisting of cytosine is an array of A specific example of the nucleotide sequence of the loop portion is, but not limited to, a sequence consisting of 11 cytosines.

The length of the second sequence may be the sum of the length of the stem portion and the length of the loop portion. Considering that the length is 15 to 50 bases, it is more preferably 18 to 35 bases, and even more preferably about 27 bases.

In the second sequence, the length of the loop portion can be longer, shorter, or identical compared to the length of the stem portion, but given the efficiency of −1 frameshift induction, The length of the loop portion is preferably longer than or the same as the length of the stem portion, more preferably longer, more preferably 1.2 to 8 times longer, and 1.3 times longer. ~1.5 times longer is even more preferred.

The second sequence may be any sequence that forms a stem-loop structure consisting of the above-described stem portion and loop portion, and specific examples include the sequence shown in SEQ ID NO: 17, the sequence shown in SEQ ID NO: 18, and the like. In addition, sequences shown in SEQ ID NO: 19 to SEQ ID NO: 30 and the like can be mentioned as those having a stable structure like these sequences.

A single-stranded nucleic acid molecule may contain a first sequence and a second sequence in the direction from the 5' end side to the 3' end side, and the 5' end side of the first sequence, the second sequence 1, 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides between the 3′ end of and/or the first sequence and the second sequence Although it may be added, it preferably consists of the first sequence and the second sequence.

The length of the single-stranded nucleic acid molecule consisting of the first sequence and the second sequence may be the sum of the length of the first sequence and the length of the second sequence. It is 14 to 90 bases long, preferably 21 to 70 bases long, more preferably 26 to 51 bases long, and even more preferably 38 to 40 bases long. . In the single-stranded nucleic acid molecule, the length of the second sequence is preferably longer than the length of the first sequence, and the length of the first sequence is longer than the length of the stem portion in the second sequence. is preferred.

Each nucleotide constituting a single-stranded nucleic acid molecule may be either a natural nucleotide (natural nucleotide) or a modified nucleotide (modified nucleotide). A single-stranded nucleic acid molecule may be composed of natural nucleotides, modified nucleotides, or a combination of natural and modified nucleotides. .

Modified nucleotides are not particularly limited, but for example, sugar-modified nucleotides (2'-O-alkylated D-ribofuranose, 2'-O,4'-C-alkylenated nucleotides, etc.); nucleotides with modified phosphodiester bonds (thioated modified nucleotides); nucleotides with modified bases; and nucleotides with combinations of these modifications. A single-stranded nucleic acid molecule having modified nucleotides can have advantages such as high binding strength to RNA and high resistance to nucleases. -1 frame shift induction effect can be expected in some cases. A modified nucleotide may be a peptide nucleic acid (PNA).

Examples of nucleotide sugar modifications include 2′-O-alkylation of D-ribofuranose (e.g., 2′-O-methylation, 2′-O-aminoethylation, 2′-O-propylation, 2′-O-allylation, 2′-O-methoxyethylation, 2′-O-butylation, 2′-O-pentylation, 2′-O-propargylation, etc.), 2′ of D-ribofuranose —O,4′-C-alkylenated (e.g., 2′-O,4′-C-ethylenated, 2′-O,4′-C-methylenated, 2′-O,4′-C-propylene 2′-O,4′-C-tetramethylation, 2′-O,4′-C-pentamethylene, etc.), 3′-deoxy-3′-amino-2′-deoxy-D-ribo Furanose, 3'-deoxy-3'-amino-2'-deoxy-2'-fluoro-D-ribofuranose and the like can be mentioned.

Examples of nucleotide phosphodiester bond modifications include phosphorothioate bonds, methylphosphonate bonds, methylthiophosphonate bonds, phosphorodithioate bonds, phosphoramidate bonds, and the like.

Examples of nucleotide base modifications include cytosine 5-methylation, 5-fluorination, 5-bromination, 5-iodination, N4-methylation, thymidine 5-demethylation (uracil), 5-fluoro 5-bromination, 5-iodination, N6-methylation of adenine, 8-bromination, N2-methylation of guanine, 8-bromination and the like.

A single-stranded nucleic acid molecule may be in salt form, ie, a pharmaceutically acceptable salt of a single-stranded nucleic acid molecule. Examples of salts include alkali metal salts such as sodium salts, potassium salts and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salts, iron salts, zinc salts, copper salts, nickel salts; metal salts such as cobalt salts; inorganic salts such as ammonium salts; t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts, guanidine salts , diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris(hydroxymethyl ) organic salts such as aminomethane salts; hydrohalogenates such as hydrofluoride, hydrochloride, hydrobromide, hydroiodide; nitrates, perchlorates, sulfates, inorganic acid salts such as phosphate; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate and ethanesulfonate; arylsulfonic acids such as benzenesulfonate and p-toluenesulfonate Salt; organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate; glycine, lysine, arginine, ornithine, glutamate , amino acid salts such as aspartate, and the like, but are not limited to these.

A single-stranded nucleic acid molecule may be in the form of a solvate such as a hydrate.

A single-stranded nucleic acid molecule may be in the form of a prodrug. Prodrugs can include amides, esters, carbamates, carbonates, ureides, phosphates, and the like.

Single-stranded nucleic acid molecules can be synthesized using previously known methods and equipment. For example, DNA according to the method described in Sinha et al. (ND Sinha et al., Nucleic Acids Research, 12, 4539 (1984); the entire description of this document is incorporated herein by reference). can be synthesized. Alternatively, the DNA synthesized in this manner can be incorporated into a vector, and RNA can be synthesized by transcription reaction using an RNA polymerase such as T7 RNA polymerase using this as a template. Single-stranded nucleic acid molecules may be synthesized in vivo or in vitro. In vitro synthesis is preferred.

Commercially available nucleotides may be used for the in vitro synthesis of single-stranded nucleic acid molecules, and those synthesized from commercially available nucleotides according to a known method may be used. good too. For example, various modified nucleotides are described in Blommers et al., Biochemistry (1998), 37, 17714-17725, Lesnik et al., Biochemistry (1993), 32, 7832-7838.), Patent Document (US6261840), Martin Document (Martin, P. Helv. Chim. Acta. (1995) 78, 486-504.), Patent Document (WO99/14226), Patent Document ( WO00/47599), Huynh et al. (Huynh Vu et al., Tetrahedron Letters, 32, 3005-3008 (1991)), Radhakrishnan et al. (Radhakrishnan P. Iyer et al., J. Am. Chem. Soc. 112, 1253 (1990)), patent documents (PCT/WO98/54198), reference documents (Oligonucleotide Synthesis, Edited by MJ Gait, Oxford University Press, 1984), patent documents (JP-A-7-87982), etc. It can be produced according to the method described. In addition, all the descriptions of the above-mentioned documents cited here are incorporated herein as disclosure.

A synthesized single-stranded nucleic acid molecule may be purified using previously known methods and devices such as extraction with solvents or resins, precipitation, electrophoresis, chromatography, and the like. When obtaining a single-stranded nucleic acid molecule, a contract manufacturing service using a contract manufacturing company such as Genedesign, Dharmacon, QIAGEN, and Sigma-Aldrich may be used.

The single-stranded nucleic acid molecule is preferably RNA because it binds to mRNA, which is the transcription product of the gene of interest.

When the single-stranded nucleic acid molecule is RNA, it may be designed to be synthesized within the subject, for example, a gene (DNA fragment) encoding the single-stranded nucleic acid molecule may be used as the subject. may be inserted into a vector suitable for When such a vector is introduced into a subject and the gene encoding the single-stranded nucleic acid molecule is transcribed, the single-stranded nucleic acid molecule will be synthesized within the subject. Vectors that can be used for such purposes include, but are not limited to, viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses (AAV), and the like.

Single-stranded nucleic acid molecules can be used in vivo or in vitro. The subject to which the single-stranded nucleic acid molecule is applied in vivo is not particularly limited, and may be any organism such as an animal, a plant, or a microorganism. Examples of animals include mammals, and examples of mammals include humans, dogs, cats, cows, horses, pigs, and sheep, among which humans are preferred. The subject of application may be a healthy individual organism, but may be an individual organism for which prevention or treatment of a hereditary disease is desired, such as an organism having a genetic disease described later or an organism at risk of such disease. preferable. Moreover, the application target may be the individual organism itself, or a part of the individual organism, such as a cell, tissue, or organ.

Single-stranded nucleic acid molecules can be used in vitro for any purpose, such as reagents, experiments, research, and medicine. For example, for medical purposes, stem cells collected from a person suffering from a genetic disease are modified to constantly synthesize single-stranded nucleic acid molecules, cultured, and then transplanted to a person suffering from a genetic disease. can do. Thus, the single-stranded nucleic acid molecule of one embodiment of the present invention can be used for medical purposes such as regenerative medicine and cell therapy.

A single-stranded nucleic acid molecule is preferably used together with a nucleic acid introduction aid so that it can be easily transferred into the target cell. Nucleic acid introduction aids include, but are not limited to, lipofectamine, oligofectamine, liposomes, polyamines, DEAE dextran, calcium phosphate, dendrimers, lipid nanoparticles, and the like.

The amount of the single-stranded nucleic acid molecule to be used is not particularly limited as long as it is an amount that induces -1 frameshift when the single-stranded nucleic acid molecule is applied. 500 nmol/kg/day to 500 mmol/kg in consideration of the cost of synthesizing a single-stranded nucleic acid molecule and obtaining an excellent -1 frameshift-inducing action. /day, more preferably 40 nmol/kg/day to 500 μmol/kg/day.

The method for evaluating the -1 frameshift-inducing action of single-stranded nucleic acid molecules is not particularly limited. and a method of confirming an increase or decrease in the amount of protein or both of them. It may also be confirmed by examining and/or testing that the disease of the person to whom the single-stranded nucleic acid molecule has been applied has been prevented or treated, or that the symptoms have been alleviated.

The degree of -1 frameshift-inducing action of single-stranded nucleic acid molecules is not particularly limited, for example, the amount of mRNA and / or protein related to the target gene compared to the case where the single-stranded nucleic acid molecule is not used (control) It is sufficient if the fluctuation (increase or decrease) is observed in the

[Composition]
The single-stranded nucleic acid molecule is used alone or mixed with other components in the form of a composition. Another aspect of the present invention is a composition used to induce −1 frameshift, comprising the single-stranded nucleic acid molecule of one aspect of the present invention as an active ingredient. Specific aspects of the composition include pharmaceutical compositions, food and drink compositions, quasi-drug compositions, cosmetic compositions, and animal feed compositions. The composition can be oral preparations such as tablets, capsules, granules, powders and syrups; parenteral preparations such as injections, suppositories, patches and external preparations.

The other components are not particularly limited as long as the -1 frameshift is induced when the single-stranded nucleic acid molecule is applied. include excipients (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol, sorbitol; starch derivatives such as maize starch, bailesh starch, alpha starch, dextrin; cellulose derivatives such as crystalline cellulose; gum; dextran; organic excipients such as pullulan; silicate derivatives such as light silicic anhydride, synthetic aluminum silicate, calcium silicate and magnesium aluminometasilicate; phosphates such as calcium hydrogen phosphate; inorganic excipients such as sulfates such as calcium sulfate), lubricants (such as stearic acid, calcium stearate, metal stearates such as magnesium stearate; talc; colloidal silica; beads Waxes such as Wax, Gay Wax; Boric Acid; Adipic Acid; Sulfates such as Sodium Sulfate; Glycol; Fumaric Acid; Sodium Benzoate; Silicic acid, silicic acid such as silicic acid hydrate; the above starch derivatives, etc.), binders (e.g., hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, compounds similar to the above excipients, etc.), disintegrants (e.g., low-substituted hydroxypropylcellulose, carboxymethylcellulose, calcium carboxymethylcellulose, cellulose derivatives such as sodium carboxymethylcellulose; chemically modified starch cellulose such as carboxymethylstarch, sodium carboxymethylstarch, crosslinked polyvinylpyrrolidone; etc.), emulsifiers (e.g. colloidal clays such as bentonite, Veegum; metal hydroxides such as magnesium hydroxide, aluminum hydroxide; anionic surfactants such as sodium lauryl sulfate, calcium stearate; Cationic surfactants such as ruconium; nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, sucrose fatty acid esters, etc.), stabilizers (paraoxy Benzoates alcohols such as chlorobutanol, benzyl alcohol, phenylethyl alcohol; benzalkonium chloride; phenols such as phenol, cresol; thimerosal; dehydroacetic acid; sweeteners, acidulants, flavoring agents, etc.), diluents, and the like.

The composition may be supplied in solution. In this case, considering the storage stability of the single-stranded nucleic acid molecule, the composition is preferably stored under refrigerated conditions, frozen conditions, or freeze-dried. The freeze-dried composition may be dissolved in a dissolving solution (distilled water for injection, etc.) at the time of use to return to a solution state before use.

The amount of the composition to be applied is not particularly limited as long as the -1 frameshift-inducing action is expressed. Yes, preferably 1 mg/day to 1,000 mg/day. The composition of one embodiment of the present invention can be applied once a day or in multiple doses. When the -1 frameshift-inducing action of the composition of one aspect of the present invention is desired to be expressed continuously, the composition of one aspect of the present invention is applied for several days to several weeks to several months to several years, Continuous or intermittent application is preferred. The specific usage and dosage of the composition of one aspect of the present invention are appropriately determined depending on the type of individual organism to be applied, the type and degree of disease or symptoms that the individual organism has or is at risk of, age, administration method, etc. can change.

The composition of one aspect of the present invention prevents and/or treats various genetic diseases by inducing −1 frameshift and increasing or decreasing the expression of the target gene. can be done. The composition of one aspect of the present invention preferably induces -1 frameshift to normalize the expression of the gene containing the target sequence, thereby preventing and/or causing a genetic disease caused by abnormal expression of the gene. or a pharmaceutical composition for treatment. Genetic diseases are not particularly limited, but for example, muscular dystrophy, cancer, multiple sclerosis, spinal muscular atrophy, Rett syndrome, amyotrophic lateral sclerosis, fragile X syndrome, Prader-Willi syndrome, dry skin pigment disease, porphyria, Werner's syndrome, fibrodysplasia ossificans progressis, infantile neuronal ceroid lipofuscinosis, Alzheimer's disease, Tay-Sachs disease, neurodegeneration, Parkinson's disease, rheumatoid arthritis, graft versus host arthritis, hemophilia, von Willebrand disease, ataxia-telangiectasia, thalassemia, kidney stones, osteogenesis imperfecta, liver cirrhosis, neurofibromatosis, bullous disease, lysosomal storage disease, Hurler's disease, cerebellar ataxia , tuberous sclerosis, familial polycythemia, immunodeficiency, kidney disease, pulmonary disease, cystic fibrosis, familial hypercholesterolemia, retinopathy pigmentosa, amyloidosis, atherosclerosis, gigantism, small Human disease, hypothyroidism, hyperthyroidism, aging, obesity, diabetes, Niemann-Pick disease, Marfan syndrome and the like can be mentioned. As described above, an organism having or at risk of having a genetic disease caused by abnormal expression of a target gene by administering an effective amount of the single-stranded nucleic acid molecule and composition of one embodiment of the present invention. An inherited disease can be treated and/or prevented for an individual. Furthermore, by administering an effective amount of the single-stranded nucleic acid molecule and composition of one aspect of the present invention, expression of the gene in a cell, tissue, organ, or individual organism in which abnormal expression of the gene of interest is observed. Abnormality can be improved.

Among hereditary diseases, hereditary diseases caused by out-of-frame mutations in gene exons are those in which exons with out-of-frame mutations are not expressed partially or wholly due to a shift in the reading frame of the exons. , often resulting in NMD and decreased gene expression. In such cases, the composition of one embodiment of the present invention can be used to induce a −1 frameshift to correct the misalignment of the exon reading frame of the gene to generate an in-frame mutation, resulting in an out-of-frame mutation. Mutated exons can be partially or wholly expressed, resulting in decreased NMD effects and increased gene expression. Hereditary diseases caused by decreased expression of such genes include muscular dystrophy, cystic fibrosis, xeroderma pigmentosum, β-thalassemia, and colon cancer.

On the other hand, for some reason, a mutant gene that was not originally expressed or whose expression level was low may be expressed, resulting in a hereditary disease. In such a case, the composition of one embodiment of the present invention can be used to induce a −1 frameshift to shift the reading frame of the exon of the gene to generate an out-of-frame mutation to partially replace the exon. , or may not be expressed at all, resulting in the induction of NMD and reduced expression of the deleterious mutant gene. Examples of hereditary diseases caused by abnormal protein synthesis resulting from the expression of such mutant genes include cancer, fibrodysplasia ossificans progressive, familial Alzheimer's disease, and familial amyloid polypoliosis. These include common dominant genetic disorders such as neuropathy and Huntington's disease.

For example, muscular dystrophy develops due to non-expression or low expression of the dystrophin gene. The dystrophin gene is a gene consisting of 79 exons. Among them, the dystrophin gene of Duchenne muscular dystrophy (DMD) patients with out-of-frame mutations has either exons 45 to 55 (exons 45 to 55) or multiple exons deleted. often GM03429, which is a DMD patient-derived fibroblast used in the examples described later, has a mutant dystrophin gene in which 6 exons from exon 45 to exon 50 (871 bases = 290 × 3 + 1 bases) of the dystrophin gene are deleted. . The deleted 6 exons consist of 871 bases, which is a multiple of 3 plus 1 base, so that the sense strand is short of 2 bases for in-frame formation. That is, when one base is deleted, the frame is shifted backward (in the 3' direction) by +1, because two bases are short of three bases. To resolve this, the frame must either be shifted -1 (5' direction) or shifted +2 and backed up in the 3' direction to make it a multiple of 3. Scheme (IV) illustrates this.

スキーム（ＩＶ）

Scheme (IV)

In the normal dystrophin gene mRNA, U, the last base of exon 50, and CU at the tip of exon 51 form one sense codon UCU (corresponding to serine), and then CCU of exon 51 forms one sense codon. (corresponding to proline). However, in the mRNA of the mutated dystrophin gene in which exons 45 to 50 are deleted, U, the last base of exon 50, is deleted, and the reading frame is shifted, so that the CUC at the tip of exon 51 is replaced by one sense codon ( After that, CUA in exon 51 forms one sense codon (corresponding to leucine), which encodes an amino acid sequence different from that of the normal dystrophin protein.

On the other hand, in scheme (IV), by using the composition of one embodiment of the present invention with the underlined portion as the target sequence, it is believed that -1 frameshift occurs and ribosome slipping occurs. be done. That is, after the last sense codon (AAG) of exon 44, a -1 frameshift results in a sense codon GCU, thus forming a triplet codon. As a result, the second sense codon (CCU) of exon 51 encodes the same amino acids as those of normal dystrophin gene mRNA.

In this case, as a result, the mRNA of the mutant dystrophin gene with a -1 frameshift is different from the mRNA of the normal dystrophin gene, between the last base of exon 50 and the top two bases of exon 51. Instead of the serine corresponding to the formed sense codon (UCU), an alanine corresponding to the sense codon (GCU) generated by the -1 frameshift is added. These guesses are guesses obtained by experiments, and the number of nucleotides between the target sequence and the -1 frameshift occurrence site may be changed as appropriate. In addition, the theory of such a -1 frameshift is based on FARABAUGH's article (PHILIP J. FARABAUGH, MICROBIOLOGICAL REVIEWS, Mar. 1996, p. 103-134; the entire description of the article is incorporated herein by reference). of FIG. 4, which fit well with the model of Jacks et al. and Weiss et al. for the −1 frameshift.

Based on the above speculation, by using the composition of one aspect of the present invention, the amino acid sequence of the mutant dystrophin protein expressed by the mutant dystrophin gene is the amino acid encoded by exon 45 to exon 50 and the last of exon 50 Only the amino acid (serine→alanine) corresponding to the sense codon formed by the base and the two bases at the top of exon 51 is different, and the rest has the same amino acid sequence as that of the normal dystrophin protein. Therefore, by using the composition of one aspect of the present invention, it is possible to obtain a mutant dystrophin protein having an amino acid sequence that differs from the normal dystrophin protein by only one amino acid, except for the deleted exon.

As another example, if the amino acid sequence of the expression product of an oncogene such as the c-myc gene can be changed, or if the expression of the oncogene can be reduced, the progression of cancer can be expected to be stagnated or alleviated. Therefore, by targeting mRNA, which is a transcription product of an oncogene, and inducing a -1 frameshift using the composition of one embodiment of the present invention, the structure of the expression product of the oncogene is changed to reduce its activity. It is possible to prevent and/or treat cancer through, for example, reducing the expression level of expression products of oncogenes. The c-myc gene is also used in producing so-called "induced pluripotent stem cells" (iPS cells). Therefore, the composition of one embodiment of the present invention, which targets the c-myc gene used for producing iPS cells, is used simultaneously with introduction and expression of the c-myc gene, or at different times to produce iPS cells. It is expected to prevent iPS cells from becoming cancerous while being manufactured.

The method for producing the composition of one embodiment of the present invention is not particularly limited, and for example, a method of mixing the single-stranded nucleic acid molecule as an active ingredient with optionally other ingredients and molding it into a desired dosage form. is mentioned. The packaging form of the composition of one embodiment of the present invention is not particularly limited, and can be appropriately selected according to the applied form and dosage form. Examples include glass containers such as vials and ampoules; Examples include vials, ampoules, bottles, cans, pouches, blister packs, strips, single-layer or laminated film bags made of paper, plastics such as PET, and the like.

As a non-limiting specific example, when the composition of one embodiment of the present invention is applied to DMD patients, it can be carried out as follows. That is, a single-stranded nucleic acid molecule as an active ingredient is prepared by a conventionally known method, sterilized by a conventional method, and, for example, a 1 ml injection containing the single-stranded nucleic acid molecule is prepared. This injection is administered intravenously by infusion so that the dosage of the single-stranded nucleic acid molecule is 0.1 mg to 100 mg per 1 kg body weight of the patient. Administration is repeated several times, for example, at intervals of 1 week to 2 weeks, and thereafter, examination; imaging such as X-ray, CT, MRI; observation by endoscopy, laparoscope, etc.; Administration is repeated as appropriate while confirming the enhancing effect.

The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these Examples, and the present invention can take various aspects as long as the problems of the present invention can be solved. .

[Example 1. -1 frameshift evaluation of ORF of EGFP-Human α-Tubulin gene]
1. Overview of Evaluation Using an artificially produced nucleic acid molecule, the ORF (Open Reading Frame) of EGFP-Human α-Tubulin in the pEGFP-Tub vector introduced into 143Btk-osteosarcoma cells was artificially frame-shifted by -1. let me By shifting the ORF by -1 frame in the vicinity of the target sequence of the nucleic acid molecule, the amino acid sequence of the EGFP fluorescent protein is changed from the middle, and furthermore, at the boundary of the connecting portion between the EGFP gene sequence and the α-Tubulin gene sequence. A premature termination codon (PTC) was generated as a termination codon (TGA) for .

2. Preparation of Test Cell Line 143Btk-osteosarcoma cells cultured in 10% FCS-added DMEM were transfected with a pEGFP-Tub vector (CLONTECH Laboratories; 6.0 kb) using a transfection reagent "Lipofectamine2000" (Thermo Fisher Scientific). transfected. By drug selection using G418 sulfate (Cosmo Bio), EGFP fluorescent protein (Human α-Tubulin protein) fused to the C-terminus of human α-Tubulin protein is stably highly expressed in 143Btk-bone. A clone of sarcoma cells was selected as a test cell line.

3. Preparation of Nucleic Acid Molecules for Frameshifting -1 Regarding nucleic acid molecules that cause frameshifting, several kinds of nucleic acid molecules were molecularly designed based on the conditions described in this specification. The conformation in the reaction solution was simulated using the RNA secondary structure prediction program "CentroidFold" (available from https://github.com/satoken/centroid-rna-package), and highly stable ones were custom-synthesized. .

The EGFP-Human α-Tubulin coding region (CDS) is shown in FIG. As shown in FIG. 1, the EGFP gene (SEQ ID NO: 7) is linked to the Human α-Tubulin gene (SEQ ID NO: 9) via a linker (5'-TCCGGACTCAGATCTCGA-3'; SEQ ID NO: 8).

The molecular design was designed based on the position of the target sequence (Target sequence). Specifically, a target sequence of ten or more bases was designed after two bases corresponding to the bottom of the Axis of the stem loop with a 6-base spacer base interposed from the position of the last original codon that causes slipping. The target sequence must have sufficient binding to the frameshifting nucleic acid molecule to inhibit the progress of the ribosome that has migrated and cause a single nucleotide slip at the forward sequence, and the -1 orientation. After inducing base slipping, translation was restarted and the bond was also loose enough to strip the frameshift molecule from the mRNA. For this purpose, the target sequence is preferably tens of bases long, and the portion that binds to the target sequence does not bind and interact with other portions of the frameshifting nucleic acid molecule to break the stem-loop structure. set. Experimentally, the target sequence was set to a length of about 10 to 13 bases, and the chain length was adjusted while confirming that the basic structure of the stem loop was not disrupted using CentroidFold, an RNA secondary structure prediction program. and designed as the most structurally stable nucleic acid molecule for frameshifting. As a result, it was speculated that the frameshifting nucleic acid molecule for the target sequence would have a more stable molecular structure.

Theoretically, the designed nucleic acid molecule for frameshifting induces a -1 frameshift in the vicinity of the target sequence, thereby generating a PTC near the boundary between the EGFP gene sequence and the human α-Tubulin gene sequence, resulting in 143Btk-bone Only EGFP fluorescent protein is expressed without α-Tubulin protein in sarcoma cells. However, since the structure of the pre-mRNA of the fluorescent protein expressed from the pEGFP-Tub vector is a single exon, NMD (Nonsense-mediated mRNA decay), an intracellular mRNA quality control system, is used as described in Example 3 below. mutation-dependent mRNA degradation mechanism). That is, there is still mRNA expression of the EGFP gene.

“Stem38egfp(CG)” (SEQ ID NO: 1) was designed as a nucleic acid molecule for frameshifting against the target sequence. In addition, “acGFP2” (SEQ ID NO: 6), which is similar in sequence and structure to Stem38egfp (CG) and does not target the EGFP gene, was designed. The structures of Stem38egfp (CG) and acGFP2 are shown in Schemes (V) and (VI), respectively.

スキーム（Ｖ）

Scheme (V)

スキーム（ＶＩ）

Scheme (VI)

Each nucleic acid molecule was adjusted to 100 μM using TE buffer to prepare a nucleic acid molecule solution.

4. Induction of frameshift A test cell line was seeded in each well of a 6-well plate and cultured for 2 days at 37°C under 5% _CO2 conditions. After reaching 80% confluency, the medium in each well was replaced with Opti-MEM® (Thermo Fisher Scientific) to which each nucleic acid molecule and transfection reagent (“Lipofectamine2000”) were mixed and added. After the cells were transfected in this Opti-MEM for 6 hours, the 6-well plates were subjected to drug treatment by incubating for 48 hours at 37° C., 5% CO ₂ conditions, and then assayed.

The test group using only Opti-MEM without adding each nucleic acid molecule and transfection reagent is defined as an untreated (Untreated: Unt) group; -The test group using MEM was called the Mock group; the test group using the molecule "Stem39egfp (GC)" targeting the target sequence was called the P group; Five test groups were prepared, with the test group used as the NC (Negative Control) group. In the P and NC groups, the final concentration of each nucleic acid molecule in Opti-MEM was adjusted to 40 nM.

After transfection, the medium in each well was replaced with 15% FCS-added DMEM, and the 6-well plate was incubated at 37° C., 5% CO ₂ for 48 hours. After incubation, the cells in each well were observed using a fluorescence microscope “BZ-X9000” (Keyence) to confirm stable expression of a green fluorescent protein transcribed and translated from the pEGFP-Tub vector.

5. Evaluation of Frameshift Induction FIG. 2 shows the results of observing the cells of each test group with a fluorescence microscope. As shown in FIG. 2, in the Unt group, the Mock group and the NC group, fluorescence due to orientation of the human α-Tubulin protein-fused EGFP protein along the cytoskeleton was confirmed in the cytoplasm except the nucleus. . On the other hand, in the P group, weak fluorescence that seemed to diffuse and blur was confirmed in the cells.

From the above observation results, the nucleic acid molecule Stem38egfp (CG) used in group P induced a -1 frameshift; Human α-Tubulin-deficient and misoriented EGFP protein diffused randomly in the cell; the weak fluorescence indicated that the expressed EGFP protein had a shift of the ORF from the normal amino acid sequence. It was found that the amino acid sequence was changed from the first portion, and the amino acid sequence as a whole was such that the fluorescence was weak.

[Example 2. -1 frameshift evaluation of the ORF of the DHFR gene]
1. Overview of Evaluation Using a DNA fragment containing two types of dihydrofolate reductase (DHFR) genes, the -1 frameshift of the ORF was verified by an in vitro cell-free translation system.

FIG. 3 shows the CDS of the DHFR gene (SEQ ID NO: 10) derived from E. coli. The first DNA fragment, DHFR, contains the T7 promoter and ribosome binding site (SD sequence) positioned upstream of the initiation codon (ATG) of the DHFR gene.

Transcription and translation using DHFR yields the normal DHFR protein. On the other hand, when the −1 frameshift occurs, the reading frame changes, the −1 stop codon (PTC) shown in FIG. be done.

The second DNA fragment, DHFR-His, was prepared by using the first DNA fragment as a template and forward primer "Primer DHFR-His_Fw" (sequence No. 11) and a reverse primer "Primer DHFR-His_Rv" (SEQ ID NO: 12) complementary to the "3' Reverse PCR primer binding site" in the middle of the DHFR gene (Fig. 3). is. The nucleotide sequence of the reverse primer is shown in Figure 4A.

As shown in FIG. 4A, Primer DHFR-His_Rv has a sequence configuration such that a -1 frameshift generates a His tag. Normally, when transcription and translation are performed using DHFR-His, translation proceeds up to the stop codon (TAA, TAG) on the 3' side of the reverse primer, resulting in a protein without histidine (His) tag. On the other hand, when the -1 frameshift occurs, the reading frame changes, translation ends at the -1 stop codon (TAA), and a protein having a His tag at the C-terminus is obtained.

As described above, by using DHFR and DHFR-His, different proteins can be obtained depending on the presence or absence of the -1 frameshift. FIG. 4B shows the amino acid sequence of the DHFR protein (SEQ ID NO: 13) and the amino acid sequence of the DHFR-His protein (SEQ ID NO: 14). Using such a DNA fragment, an artificially designed nucleic acid molecule binds to a target sequence in the DHFR gene by a reconstituted cell-free translation system, and shifts the original ORF by -1 to produce a synthesized protein. The amino acid sequence is changed and a -1 frameshift becomes detectable.

2. Preparation of Nucleic Acid Molecules for Frameshifting Target sequences were selected in the vicinity of sites predicted to easily cause −1 slippage. FIG. 3 shows the position of the target sequence (Target sequence) on the DHFR gene.

As described above, when DHFR-His is transcribed and translated, if -1 frameshift occurs, immediately before the position of the first stop codon (-1 Stop codon (PTC)), a His tag at the C-terminus It is estimated that a protein of about 9.6 kDa fused with is synthesized. A frameshifting nucleic acid molecule having a stable molecular structure was designated as "Stem39dhfr(GC)" (SEQ ID NO: 2). The structure of Stem39dhfr(GC) is shown in scheme (VII).

スキーム（ＶＩＩ）

Scheme (VII)

3. Induction of frameshift “PUREfrex (registered trademark) 2.0” (Cosmo Bio) was used as an in vitro cell-free translation kit. For DHFR, "DHFR DNA" attached to the kit was used. DHFR-His was obtained by performing PCR according to a conventional method using DHFR as a template and DHFR-His_Fw and DHFR-His_Rv, and purifying the resulting PCR product.

Table 1 shows the composition of the reaction solution of the kit used. Here, the sample solutions shown in Table 2 were used.

Distilled water (ddH ₂ O) was used as a sample solution for negative control (NC) (test group 1). For positive control (PC), a solution containing DHFR having the full-length DHFR gene was used as a sample solution (test group 2). In frameshift reaction system 1, a solution containing both Stem39dhfr (GC) and DHFR, which are nucleic acid molecules for frameshifting, was used as a sample solution (test group 3). In the frameshift reaction system 2, a solution containing both Stem39dhfr (GC) as a sample solution and a part of the DHFR gene and DHFR-His designed to have a His tag by -1 frameshift was used ( test group 4). For Positive Negative Control (PN), a solution containing only DHFR-His was used as a sample solution (test group No. 5). In the sample solution, Stem39dhfr (GC) was adjusted to a concentration of 500 nM in the reaction solution (20 μl). Similarly, in the sample solution, DHFR and DHFR-His were prepared in amounts of 20 μg and 60 μg, respectively, in the reaction solution (20 μl).

Each reaction solution was placed in a tube for a thermal cycler and subjected to a protein synthesis reaction using a thermal cycler at 37° C. for 4 hours.

20 μL of ddH ₂ O was added to 20 μL of the reacted solutions of Test Groups 1 to 3 to dilute them 2-fold. To the obtained diluted solution, 13.3 μL of 4×SDS sample buffer containing 10 vol % β-mercaptoethanol was added and heated at 95° C. for 5 minutes to thermally denature the protein.

To 20 μL of the reacted solutions of test groups 4 and 5, 275 μL of xTractor buffer attached to the His-tagged protein purification kit “Capturem ^™ His-tagged Purification Miniprep Kit” (Clontech) was added to prepare about 300 μL of solution A. bottom. The prepared solution A was added to the equilibrated His-tagged fusion protein adsorption column. Column equilibration was performed by adding xTractor buffer and centrifuging at 9,800 g for 1 minute and 45 seconds.

The column to which solution A was added was centrifuged at 9,800 g for 1 minute and 45 seconds, and the obtained supernatant was collected as "Lysate". Next, 300 μL of wash buffer was added to the column, centrifuged at 9,800 g for 1 minute and 45 seconds, and the obtained supernatant was collected as “Wash”. Next, in order to elute the His-tagged protein from the column, 300 μL of elution buffer was added to the column, centrifuged at 9,800 g for 1 minute and 45 seconds, and the resulting supernatant was collected as “Elution1”. Then, "Elution2" was collected by performing the same operation.

45 μL of 4×SDS sample buffer was added to 150 μL of each collected solution, mixed, and then dispensed every 50 μL. The dispensed solution was placed in a tube, and the protein was thermally denatured at 95° C. for 5 minutes in a thermal cycler. Heat-denatured protein samples were stored at -20°C.

On the day of the test, the frozen and heat-denatured protein samples were collected in 1.5 mL tubes for each of "Lysate", "Wash", "Elution1" and "Elution2". The entire volume of each sample solution was then transferred to a filter column "Amicon® Ultra-0.5 (3k)" (Max Volume 500 μL; Merck Millipore) and centrifuged at 12,000 g for about 45 minutes. The filter column was then inverted and centrifuged at 12,000 g for 2 minutes to recover the concentrated protein sample from the filter column. The total amount (45 μL) of the recovered concentrated protein sample was applied to each well of a polyacrylamide gel for protein electrophoresis “10-20% Criterion ^™ TGX ^™ Precast Gel” (Bio-Rad). Similarly, the entire amount of heat-denatured protein samples of test groups 1-3 was applied to each well. The gel to which each protein sample was applied was subjected to electrophoresis at 200 V for 50 minutes. The arrangement of protein samples applied to each well is as shown in Table 3. "Precision Plus Protein Dual Extra Standard"(Bio-Rad; product number 1610377) was used as the molecular weight marker (M).

After electrophoresis, the gel was stained with Coomassie chromoprotein gel staining reagent "Simply Blue Safe Stein" (Thermo Fisher Scientific) to visualize the bands.

4. Induction Assessment of Frameshifting Results after gel staining are shown in FIGS. 5A-5C. FIG. 5A is an image of the entire gel after staining. FIG. 5B is an enlarged view of FIG. 5A where the His-tagged DHFR proteins of TEST2 Elution1 and Elution2 appeared. Figure 5C is an enlarged view of Figure 5A where DHFR proteins and DHFR protein fragments of NC, PC and TEST1 appear.

His-tagged fusion protein bands were observed in Lane 6 (TEST2 Elution 1) and Lane 7 (TEST2 Elution 2) of the reaction solution (TEST2) to which DHFR-His and the frameshifting nucleic acid molecule Stem39dhfr (GC) were added. In particular, a clear band was observed at Lane 6. On the other hand, in Lane 11 (PN Elution 1) and Lane 12 (PN Elution 2) of the reaction solution (PN) in which only DHFR-His was added and the frameshifting nucleic acid molecule Stem39dhfr (GC) was not added, a His-tagged fusion protein was synthesized. No band was observed where it would have appeared had it been done.

The above results demonstrate that in the frameshifting nucleic acid molecule Stem39dhfr (GC), when DHFR-His is translated, the ORF undergoes a −1 frameshift, resulting in the synthesis of a His-tagged fusion protein. rice field.

On the other hand, in Lane 3 (TEST 1) of the reaction solution (TEST 1) to which both the frameshifting nucleic acid molecule Stem39dhfr (GC) molecule was added, similar to Lane 2 by the reaction solution (PC) to which only DHFR was added, the full length of the DHFR protein band was recognized. However, in Lane 3, a band due to a partial fragment of the DHFR protein (truncated DHFR protein) was strongly observed.

From the above results, the Stem39dhfr (GC) molecule for frameshifting caused a -1 frameshift of the ORF, and a termination codon that did not exist in the original DHFR gene sequence, that is, PTC appeared, resulting in translation being interrupted. Proved to stop.

These results prove that an artificially designed frameshifting nucleic acid molecule can induce an artificial −1 frameshift.

[Example 3. -1 frameshift evaluation of the ORF of the dystrophin gene]
1. Overview of evaluation ORF is shifted due to deletion of exons, etc., and PTC (premature stop codon) occurs in the middle of ORF of mRNA that has become out-of-frame. mediated mRNA decay (nonsense mutation-dependent mRNA degradation mechanism) is induced, and PTC-bearing mRNA is actively degraded.

In Duchenne muscular dystrophy (DMD), the dystrophin gene (HGNC ID HGNC: 2928, chromosomal location: Xp21.2-p21.1) with 79 exons is partially deleted and has PTC. is produced and degraded, the onset occurs without the dystrophin protein being synthesized. Therefore, if a -1 frameshift of ORF can be artificially induced using a nucleic acid molecule for frameshifting in living cells derived from patients with genetic diseases such as DMD, comparisons that avoid NMD and are not degraded It can synthesize a large dystrophin protein.

In this example, a nucleic acid molecule for frameshifting, which is a therapeutic molecule molecularly designed to target dystrophin mRNA in fibroblasts derived from patients with Duchenne muscular dystrophy in which exons 45 to 50 of the dystrophin gene are deleted, to induce a −1 frameshift of the ORF. By correcting the out-of-frame during ribosomal translation of dystrophin mRNA to in-frame due to the occurrence of -1 frameshift and confirming the increase in the expression level of dystrophin gene mRNA, -1 frameshift in living cells and demonstrated its potential for treating genetic diseases.

2. Test Cell Line GM03429, a DMD patient-derived fibroblast, has a mutated dystrophin gene that lacks 6 exons from exon 45 to exon 50 (871 bases=290×3+1 bases) of the dystrophin gene. GM05118, a fibroblast derived from a healthy subject, has a normal dystrophin gene. GM03429 and GM05118 were obtained from the Coriell Institute for Medical Research.

3. Preparation of Nucleic Acid Molecule for Frameshifting The mutant dystrophin gene lacks exons 45 to 50, so that the 3′ end of exon 44 (AAG) and the 5′ end of exon 51 (CTC) are directly linked. It is a concatenated array. A breakpoint was defined between AAG and CTC at both ends.

In the vicinity of the breakpoint between exon 44 and exon 51 of the mutated dystrophin gene, Position 1, Position 2 and Position 3 were set as three types of target sequences, and a frameshifting nucleic acid molecule "P1Stem38dmd45-50 (GC )” (SEQ ID NO:3), “P2Stem39dmd45-50(CG)” (SEQ ID NO:4) and “P3Stem39dmd45-50(CG)” (SEQ ID NO:5) were designed and made. Stability of the designed nucleic acid molecule for frameshifting was confirmed by calculation using "Centroid Fold". The target sequences for Position 1, Position 2 and Position 3 are shown in Figures 6A-6C. The structures of P1Stem38dmd45-50 (GC), P2Stem39dmd45-50 (CG) and P3Stem39dmd45-50 (CG) are shown in schemes (VIII) to (X), respectively.

スキーム（ＶＩＩＩ）

Scheme (VIII)

スキーム（ＩＸ）

Scheme (IX)

スキーム（Ｘ）

Scheme (X)

4. Cell Culture Test cell lines (GM03429 and GM05118) were subcultured in 6-well plates in DMEM supplemented with 15% FCS and Antibiotic Antimycotic at 37° C., 5% CO ₂ . After culturing, each cell was collected.

5. Induction of Frameshift and Synthesis of cDNA The recovered test cell lines were inoculated into each well of a 96-well plate and cultured for 2 days at 37° C. under 5% CO ₂ conditions. After confirming 90% confluency, the medium in each well was replaced with Opti-MEM (registered trademark) (Thermo Fisher Scientific) added with a mixture of each nucleic acid molecule and transfection reagent (“Lipofectamine2000”). Six hours later, the plate was replaced with normal DMEM, and after transfection treatment, the 96-well plate was subjected to drug treatment for 48 hours at 37°C under 5% _CO2 conditions.

The test cell line is GM03429, and the test group using only Opti-MEM without adding each nucleic acid molecule and transfection reagent is called an untreated (Unt) group (n = 1); A test group using Opti-MEM to which only the transfection reagent was added without addition was designated as a Mock group (n = 6); The group was designated as P1 group (n=3); the test group using the molecule "P2Stem39dmd45-50 (CG)" targeting Position2 was designated as P2 group (n=3); and the molecule targeting Position3 as "P3Stem39dmd45- Five test groups were prepared, with the test group using "50 (CG)" as the P3 group (n=3). In addition, the test cell line was GM05118, and the test group using only Opti-MEM without adding each nucleic acid molecule and transfection reagent was defined as a wild type (WT) group (n=2). In the P1, P2 and P3 groups, the final concentration of each nucleic acid molecule in Opti-MEM was adjusted to 40 nM.

After completion of transfection, the collected cells in each well were lysed using the cDNA synthesis kit "SuperScript ^™ III Cells Direct cDNA Synthesis System" (Thermo Fisher Scientific), and the total RNA in the resulting lysate was reverse transcribed. A reaction was performed to generate cDNA.

6. Quantification of dystrophin mRNA by qPCR Dystrophin mRNA was quantified using real-time PCR "TaqMan (registered trademark) Gene Expression Master Mix" and "TaqMan (registered trademark) Gene Expression Assay" (both from Thermo Fisher Scientific) and real-time PCR device "TaKaRa PCR". qPCR was performed by the ΔΔCT method using the Thermal Cycler Dice Real Time System II. As an endogenous control, the GAPDH gene was used, and the expression level of dystrophin mRNA was calculated as a relative amount with respect to the expression level.

4. Evaluation of frameshift induction FIG. 7 shows a graph of the relative amount of dystrophin mRNA expression level with respect to the GAPDH mRNA expression level for each test group.

The expression level of dystrophin gene mRNA in the untreated group (Unt) was lower than that in the WT group using normal fibroblasts. Similarly, the Mock group, in which only the transfection reagent was added to the medium, showed a very low expression level. In these groups of studies, the dystrophin mRNA, which had a PTC due to exon deletion, was captured by the mRNA quality control machinery and degraded by NMD, suggesting.

On the other hand, in the P1, P2 and P3 groups using frameshifting nucleic acid molecules P1Stem38dmd45-50 (GC), P2Stem39dmd45-50 (CG) and P3Stem39dmd45-50 (CG) for Position1, Position2 and Position3, any An increase in dystrophin mRNA expression was also observed in DMD patient-derived fibroblasts transfected with the nucleic acid molecule. These results suggest that the frameshifting nucleic acid molecule binds to and acts on mRNA to bring the ORF out-of-frame to in-frame when the ORF is translated by the ribosome, thereby avoiding NMD. When the frameshifting nucleic acid molecule P2Stem39dmd45-50 (CG) for Position 2 is used, a UAA premature termination codon (PTC) occurs before the axis. When using the frameshifting nucleic acid molecule P3Stem39dmd45-50 (CG) for Position 3, UAA PTC occurs before the axis and after the target sequence. This may have reduced the amount of mRNA in the P2 and P3 groups compared to the P1 group. However, since the P2 and P3 groups had a higher amount of mRNA than the Mock group, it is considered that the frameshifting nucleic acid molecules used in these groups were able to avoid NMD. The finding that NMD is not induced only by the development of PTC is a surprising finding first discovered by the present inventors.

The above results show that the use of the nucleic acid molecule for frameshifting enabled the expression to be maintained without degrading the dystrophin mRNA by, for example, avoiding NMD. Thus, it is very surprising that the very low expression level of dystrophin gene mRNA in DMD patient-derived fibroblasts was brought into expression by the frameshifting nucleic acid molecule. This event is directly linked to the usefulness of the frameshifting nucleic acid molecule for the treatment of DMD.

As described above, DMD, ie, Duchenne It can be an effective treatment for type muscular dystrophy.

In addition, other genetic diseases caused by a +1 ORF shift, such as Duchenne muscular dystrophy caused by the deletion of exons 45 to 50 of the dystrophin gene, also induce a -1 frameshift. Molecules can be used to treat.

On the other hand, by -1 frameshifting the ORF of the mRNA of the target gene functioning in the cell to cause incorrect translation, and by intentionally generating PTC on the mRNA, NMD is activated. reducing mRNA expression by promoting mRNA degradation, converting a functional gene product into a truncated protein with loss of function, and consequently targeting It is possible to disable the function of genes.

Such a frameshifting nucleic acid molecule that can be designed for various genes can be used, for example, to treat cancer by nullifying the functions of genes that are specifically expressed in cancer cells, and to produce proteins that are harmful to the body. It can also be used to treat dominant genetic diseases caused by expression.

The sequences listed in the sequence listing are as follows:
[SEQ ID NO: 1] Stem38egfp (CG)
CGCUGCCGUCCCCGCAUUACCCCCCCCCCCUAGUGUGG
[SEQ ID NO: 2] Stem39dhfr (GC)
CCGAUUGAUUCCGGCGCAUUACCCCCCCCCCCUAGUGUGC
[SEQ ID NO: 3] P1Stem38dmd45-50 (GC)
GUAACAGUCUGGCGCAUUACCCCCCCCCCCUAGUGUGC
[SEQ ID NO: 4] P2Stem39dmd45-50 (CG)
CUGAGUAGGAGCCCGCAUUACCCCCCCCCCCUAGUGUGG
[SEQ ID NO: 5] P3Stem39dmd45-50 (CG)
UACCAUUUGUAUGCGCAUUACCCCCCCCCCCUAGUGUGC
[SEQ ID NO: 6] acGFP2
CGAUGCCGGUGCCGCAUUACCCCCCCCCCCUAGUGUGG
[SEQ ID NO: 7] EGFP gene

[SEQ ID NO: 8] linker
TCCGGACTCAGATCTCGA
[SEQ ID NO: 9] α-Tubulin gene (Homo sapiens)

[SEQ ID NO: 10] DHFR gene (Escherichia coli)
ATGATCAGTCTGATTGCGGCGTTAGCGGTAGATCGCGTTATCGGCATGGAAAACGCCATGCCGTGGAACCTGCCTGCCGATCTCGCCTGGTTTAAACGCAACACCTTAAATAAACCCGTGATTATGGGCCGCCATACCTGGGAATCAATCGGTCGTCCGTTGCCAGGACGCAAAAATATTATCCTCAGCAGTCAACCGGGTACGGACGATCGCGTAACGTGGGTGAAGTCGGTGGATGAAGCCATCGCGGCGTGTGGTGACGTACCAGAAATCATGGTGATTGGCGGCGGTCGCGTTTATGAACAGTTCTTGCCAAAAGCGCAAAAACTGTATCTGACGCATATCGACGCAGAAGTGGAAGGCGACACCCATTTCCCGGATTACGAGCCGGATGACTGGGAATCGGTATTCAGCGAATTCCACGATGCTGATGCGCAGAACTCTCACAGCTATTGCTTTGAGATTCTGGAGCGGCGGTAA
[SEQ ID NO: 11] Primer DHFR-His_Fw
GAAATTAATACGACTC
[SEQ ID NO: 12] Primer DHFR-His_Rv
ATCCATTTAATTAGTGGTGATGGTGATGATGTCCACCGACTTCACCCACG
[SEQ ID NO: 13] DHFR protein
MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSYCFEILERR
[SEQ ID NO: 14] DHFR-His protein
MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRPYLGINRSSVARTQKYYPQQSTGYGRSRNVGEVGGHHHHHH
[SEQ ID NO: 15] First stem portion
CCGCAUUA
[SEQ ID NO: 16] second stem portion
UAGUGUGG
[SEQ ID NO: 17] Stem-loop structure 1
CCGCAUUACCCCCCCCCCCUAGUGUGG
[SEQ ID NO: 18] Stem-loop structure 2
GCGCAUUACCCCCCCCCCCUAGUGUGC
[SEQ ID NO: 19] Stem-loop structure 3
GCGCAUUAACUGCUAGAGGUAGUGUGC
[SEQ ID NO: 20] Stem-loop structure 4
GUACCUUUCGAAACACAUCCUAGGGUAU
[SEQ ID NO: 21] Stem-loop structure 5
GCCGGCGCGCGCCCAUCGCCGACGGC
[SEQ ID NO: 22] Stem-loop structure 6
GGCCGGCGGCCGCCCAUCGCCGACGGCC
[SEQ ID NO: 23] Stem-loop structure 7
CCGUACUCGCGGCUUCGUGAGUACGG
[SEQ ID NO: 24] Stem-loop structure 8
CGUACUCGCGGCUUCGUGAGUACG
[SEQ ID NO: 25] Stem-loop structure 9
CGCCCGAACGCGGCG
[SEQ ID NO: 26] Stem-loop structure 10
CGCCCGCGCCCGAACGCGGGCG
[SEQ ID NO: 27] Stem-loop structure 11
GCGCCCGCGCCCGAACGCGGGGCG
[SEQ ID NO: 28] stem-loop structure 12
CGCCCGCGCCCGCGCCCGAACGCGGGCG
[SEQ ID NO: 29] Stem-loop structure 13
GAACUUUCUGAUCAGUCC
[SEQ ID NO: 30] Stem-loop structure 14
CCCCGAACAUCGGGG

The single-stranded nucleic acid molecule and composition of one aspect of the present invention normalize gene expression through induction of -1 frameshift, and prevent and/or treat genetic diseases caused by genetic abnormalities. Available.

Claims (14)

A single-stranded nucleic acid molecule comprising a first sequence complementary to a target sequence of a gene of interest and a second sequence forming a stem-loop structure in the direction from the 5' end to the 3' end. 2. The single-stranded nucleic acid molecule according to claim 1, wherein the ends of the stem portion of said stem-loop structure are cytosine and guanine. The single-stranded nucleic acid molecule according to any one of claims 1 and 2, wherein the stem-loop structure is a stem-loop structure in which the loop portion is mainly composed of cytosine. The single-stranded nucleic acid molecule according to any one of claims 1 to 3, wherein the stem-loop structure has a stem portion of 4 to 20 base pairs. The single-stranded nucleic acid molecule according to any one of claims 1 to 4, wherein the second sequence is the sequence set forth in SEQ ID NO:17 or SEQ ID NO:18. The single-stranded nucleic acid molecule according to any one of claims 1 to 5, wherein the first sequence has a length of 8 to 16 bases. 7. The single-stranded nucleic acid molecule according to any one of claims 1 to 6, wherein the gene has two or more consecutive identical bases in 9 to 15 bases upstream of the target sequence. The single strand according to any one of claims 1 to 6, wherein the gene has two or more portions in which two or more of the same bases are consecutive in 9 bases to 15 bases upstream of the target sequence. nucleic acid molecule. A composition for inducing a -1 frameshift, comprising the single-stranded nucleic acid molecule according to any one of claims 1 to 8 as an active ingredient. The composition is a pharmaceutical composition for preventing and/or treating genetic diseases caused by abnormal expression of the gene by normalizing expression of the gene by inducing -1 frameshift. 10. The composition of claim 9. The genetic diseases include muscular dystrophy, cancer, multiple sclerosis, spinal muscular atrophy, Rett syndrome, amyotrophic lateral sclerosis, fragile X syndrome, Prader-Willi syndrome, xeroderma pigmentosum, porphyria, Werner's syndrome, progressive fibrodysplasia ossificans, infantile neuronal ceroid lipofuscinosis, Alzheimer's disease, Tay-Sachs disease, neurodegeneration, Parkinson's disease, rheumatoid arthritis, graft-versus-host disease, arthritis, blood Friendship, von Willebrand's disease, ataxia-telangiectasia, thalassemia, renal calculi, osteogenesis imperfecta, liver cirrhosis, neurofibromatosis, bullous disease, lysosomal storage disease, Hurler's disease, cerebellar ataxia, tuberous sclerosis , familial polycythemia, immunodeficiency, kidney disease, pulmonary disease, cystic fibrosis, familial hypercholesterolemia, retinopathy pigmentosa, amyloidosis, atherosclerosis, gigantism, dwarfism, thyroid function 11. The composition of claim 10, wherein the genetic disease is selected from the group consisting of hypothyroidism, hyperthyroidism, aging, obesity, diabetes, Niemann-Pick disease, and Marfan syndrome. An effective amount of the single-stranded nucleic acid molecule according to any one of claims 1 to 8 or the composition according to claim 9 has a genetic disease caused by abnormal expression of the target gene, or has a genetic disease. A method of treating and/or preventing a genetic disease comprising administering to an at-risk organism. An effective amount of the single-stranded nucleic acid molecule according to any one of claims 1 to 8 or the composition according to claim 9 is added to a cell, tissue, organ, or individual organism in which abnormal expression of the target gene is observed. A method for ameliorating abnormal gene expression, comprising administering to The genetic diseases include muscular dystrophy, cancer, multiple sclerosis, spinal muscular atrophy, Rett syndrome, amyotrophic lateral sclerosis, fragile X syndrome, Prader-Willi syndrome, xeroderma pigmentosum, porphyria, Werner's syndrome, progressive fibrodysplasia ossificans, infantile neuronal ceroid lipofuscinosis, Alzheimer's disease, Tay-Sachs disease, neurodegeneration, Parkinson's disease, rheumatoid arthritis, graft-versus-host disease, arthritis, blood Friendship, von Willebrand's disease, ataxia-telangiectasia, thalassemia, renal calculi, osteogenesis imperfecta, liver cirrhosis, neurofibromatosis, bullous disease, lysosomal storage disease, Hurler's disease, cerebellar ataxia, tuberous sclerosis , familial polycythemia, immunodeficiency, kidney disease, pulmonary disease, cystic fibrosis, familial hypercholesterolemia, retinopathy pigmentosa, amyloidosis, atherosclerosis, gigantism, dwarfism, thyroid function 14. The method of claim 12 or 13, wherein the genetic disease is selected from the group consisting of hypothyroidism, hyperthyroidism, aging, obesity, diabetes, Niemann-Pick disease, and Marfan syndrome.

Images