Antisense nucleic acid

Abstract

An oligonucleotide, which expresses a functional human WRN protein for exon 26 or exon 28 skipping mutation in human WRN gene, or a pharmaceutically acceptable salt thereof, wherein: the individual nucleotides in the oligonucleotide are bonded via a phosphate group and / or a modified phosphate group; the oligonucleotide contains a modified nucleic acid having at least one modified sugar; the base length of the oligonucleotide is 10-30 mer; and the base sequence of the oligonucleotide is a base sequence having a 90-100% inclusive sequence identity, on the basis of a base sequence that is complementary to at least one target region having the same base length as the aforesaid oligonucleotide in the base sequence represented by SEQ ID NO:1, 2, 3, 4, 5 or 6, a base sequence that is complementary to a base sequence derived from the aforesaid target region by the deletion, substitution, insertion or addition of one to several bases, or a base sequence that is hybridizable, under stringent conditions, with an oligonucleotide having the aforesaid target region.

Description

antisense nucleic acids

[0001] The present invention relates to an antisense oligonucleotide (antisense nucleic acid) that enables skipping of the 27th exon of the WRN gene, which is the causative gene for Werner syndrome, and a pharmaceutical composition containing said oligonucleotide.

[0002] Werner syndrome is a rare disease that causes premature aging. It is known that patients exhibit typical elderly features in their early years, such as graying of hair, baldness, diabetes, heart disease, cancer, skin atrophy, scleroderma-like changes in the skin, premature cataracts, premature aging, and hypogonadism. More specifically, signs of aging, such as skin atrophy and hardening, changes in hair such as graying and baldness, and cataracts, appear in their 20s and 30s. Furthermore, patients frequently develop complications such as diabetes, arteriosclerosis, and malignant tumors, and many die in their 50s. Furthermore, intractable skin ulcers, which frequently occur on the elbows, knees, and heels, can worsen, leading to limb amputation and impaired quality of life in many cases (Non-Patent Document 1).

[0003] Werner syndrome is a rare, autosomal recessive genetic disorder. The causative gene is the WRN gene, which encodes a RecQ-type DNA / RNA helicase (WRN helicase) located on the short arm of chromosome 8 (8p12) (Non-Patent Document 2, Non-Patent Document 3). The WRN protein consists of 1,432 amino acids and possesses ATP-dependent DNA / RNA helicase activity and 3'→5' exonuclease activity. Its physiological functions include DNA replication, base excision repair, DNA double-strand break repair, transcription, and telomere maintenance, and it contributes to gene repair and chromosomal stability. Therefore, significant genomic instability is observed in Werner syndrome. Fibroblasts derived from Werner syndrome patients have a shorter replicative lifespan and significantly reduced proliferation rate compared to normal cells derived from healthy individuals (Non-Patent Document 4). Reduced fibroblast mitotic activity is thought to be one of the factors behind delayed wound healing of skin ulcers in Werner syndrome (Non-Patent Document 5).

[0004] The WRN protein contains a nuclear localization signal (NLS) near its C-terminus, which induces nuclear localization and thereby performs its normal physiological function (Figure 1). However, when an incomplete WRN protein lacks the NLS due to the appearance of a premature stop codon (termination codon) caused by splicing, nonsense, or frameshift mutations, the WRN helicase, which normally functions in the nucleus, is unable to translocate into the nucleus and is therefore unable to perform its function, resulting in Werner syndrome (Figures 1 and 2) (Non-Patent Document 3). Approximately 70% of Japanese patients with Werner syndrome have a skipping mutation c.3139-1G>C in exon 26 of the human WRN gene, which causes a shift in the amino acid reading frame (out-of-frame), resulting in the appearance of a stop codon in exon 27 (hereinafter sometimes referred to as "exon 27"). As a result, a WRN protein is produced that lacks the nuclear localization signal present at the C-terminus of the human WRN gene after exon 27, resulting in the loss of its original function (Figure 2) (Non-patent Document 3).

[0005] International Publication No. WO 2011 / 052436 International Publication No. WO 2014 / 046212 International Publication No. WO 2015 / 125783 International Publication No. WO 1991 / 009033 International Publication No. WO 2009 / 064471

[0006] Geriatr Gerontol Int (2013) 13:475-481 Nature (1992) 355:735-738 Am. J. Hum. Genet. (1997) 60:330-341 Human Genetics (1981) 58:310-316 Dermatology (2000) 40:1512-1513 Annu. Rev. Pharmacol. Toxicol. (2010) 50:259-293 J. Med. Chem. (2016) 59:9645-9667J. Am. Chem. Soc (2016) 138:15663-15672

[0007] Werner syndrome is an incurable disease that threatens the life and quality of life of patients, but there is currently no effective treatment for Werner syndrome, and there is a strong need to develop new therapeutic drugs.

[0008] In recent years, nucleic acid drugs have been commercialized as new therapeutic agents. Nucleic acid drugs are composed of oligonucleotides, consisting of nucleic acids or modified nucleic acids linked together by a dozen or so bases. They are chemically synthesized and include drugs that act directly on messenger RNA or non-translated RNA involved in protein translation, as well as drugs that act on target proteins, such as antibody drugs. Splicing-controlling antisense oligonucleotides, a type of nucleic acid drug, inhibit the binding of splicing factors to pre-mRNA (pre-mRNA), thereby switching the splicing of nearby exons (splicing out) and normalizing the reading frame of abnormal RNA that has undergone frameshifting. As a result, splicing out exons of a gene results in the expression of the protein, retaining the N- and C-termini that are important for functional expression. In this way, skipping targeted exons with splicing-controlling antisense oligonucleotides allows the expression of functional proteins that are translated up to the C-terminus, potentially improving the pathology of certain diseases. This is called exon skipping therapy, and although the protein expressed by this treatment is shorter than normal, it can restore the function lost due to the mutation (Non-Patent Document 6).

[0009] As described above, the most frequently reported mutation in Werner syndrome is the c.3139-1G>C skipping mutation in exon 26 of the human WRN gene. This c.3139-1G>C skipping mutation results in the appearance of a stop codon in exon 27, resulting in the production of WRN protein lacking the amino acid sequence following the stop codon, including the nuclear localization signal at the C-terminus, resulting in the loss of its inherent physiological function. To improve this condition, the present inventors focused on splicing-controlling antisense oligonucleotides and investigated the creation of antisense oligonucleotides that induce exon 27 skipping of the human WRN gene.

[0010] The present invention has been made in consideration of the above circumstances, and an object of the present invention is to provide an oligonucleotide that enables skipping of exon 27 of the human WRN gene, and a drug containing the oligonucleotide that efficiently skips exon 27 of the human WRN gene.

[0011] The present inventors have found that exon 27 skipping can be induced with high efficiency by targeting a sequence consisting of exon 27 and surrounding nucleotides in the pre-mRNA of the human WRN gene with an antisense oligonucleotide. Based on this finding, the present inventors have completed the present invention. Specifically, the present invention is as follows.

[0012] [1] The oligonucleotide of the present invention is an oligonucleotide or a pharmaceutically acceptable salt thereof that expresses a functional human WRN protein in response to a skipping mutation of exon 26 or 28 of the human WRN gene, wherein each nucleotide in the oligonucleotide is linked via a phosphate group and / or a modified phosphate group, the oligonucleotide comprises a modified nucleic acid having at least one modified sugar, the oligonucleotide has a base length of 10 to 30 mer, and the base sequence of the oligonucleotide is: a base sequence that has 90% to 100% sequence identity with respect to a base sequence complementary to at least one target region of the same base length as the oligonucleotide in the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6; a base sequence complementary to a base sequence in the target region in which one or several bases have been deleted, substituted, inserted, or added; or a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region.

[0013] The oligonucleotide having the above-described structure or a pharmaceutically acceptable salt thereof enables skipping of exon 27 of the human WRN gene.

[0014] [2] The base sequence of the oligonucleotide is preferably a base sequence having a sequence identity of 95% or more and 100% or less with respect to a base sequence complementary to at least one target region of the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, the base sequence having the same base length as the oligonucleotide.

[0015] [3] The base sequence of the oligonucleotide is preferably a base sequence complementary to at least one target region of the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, the target region having the same base length as the oligonucleotide.

[0016] [4] The base length of the oligonucleotide is preferably 15 to 25 mer.

[0017] [5] It is preferred that the sugar constituting the oligonucleotide is D-ribofuranose, and the sugar modification is a sugar modification of the 2'-hydroxyl group of D-ribofuranose.

[0018] [6] The sugar modification constituting the oligonucleotide is preferably a group represented by the following formula (A1): (In the formula, Bx is a nucleic acid base, and each Bx is independently a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine, or uracil; X is a phosphate bond, and each X is independently a phosphodiester bond, phosphorothioate bond, phosphorodithioate bond, phosphoamidate bond, boranophosphate bond, or alkylphosphonate bond; Y is independently a hydrogen atom, a hydroxyl group, a fluorine atom, an optionally substituted alkoxy group having 1 to 6 carbon atoms, or an optionally substituted amino group; and Z is independently a hydrogen atom, a carbon-oxygen double bond, or an alkyl group having 1 to 5 carbon atoms which may have a cyclic structure, or some carbon atoms of the alkyl group and Y combine to form a ring.)

[0019] [7] The sugar modification constituting the oligonucleotide is preferably a group represented by the following formula (A2) or (A3): (wherein Bx is a nucleic acid base, each independently a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine, or uracil; X is a phosphate bond, each independently a phosphodiester bond, a phosphorothioate bond, a phosphorodithioate bond, a phosphoramidate bond, a boranophosphate bond, or an alkylphosphonate bond; R ４ are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, Y' is an oxygen atom or an optionally substituted nitrogen atom, and R ５ and R ６ are each independently a hydrogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, or R ５ and R ６ are taken together to form a carbonyl group or a ring, and n is 0 or 1.

[0020] [8] The modification of the sugar constituting the oligonucleotide is a group represented by the formula (A2), and the R ４ is preferably each independently a methyl group, a methoxyethyl group, or an N-methylpropanamide group.

[0021] [9] The sugar modification constituting the oligonucleotide is a group represented by the formula (A3), the Y' is an oxygen atom, or a nitrogen atom which may be substituted with a hydrogen atom, a methyl group, a methoxyethyl group, or an N-methylpropanamide group, and the R ５ and R ６ are each independently a hydrogen atom, an alkyl group having 1 to 2 carbon atoms, or the R ５ and R ６ are taken together to form a carbonyl group or a ring having 3 to 6 carbon atoms, and n is preferably 0 or 1.

[0022]

[10] Preferably, at least one internucleotide bond of the oligonucleotide is a phosphorothioate bond.

[0023]

[11] Preferably, at least one internucleotide bond of the oligonucleotide is a phosphodiester bond.

[0024]

[12] Preferably, the internucleotide bond of the oligonucleotide is a phosphodiester bond or a phosphorothioate bond.

[0025]

[13] The base sequence of the oligonucleotide is preferably a morpholino oligonucleotide having a base sequence complementary to at least one target region of the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, which has the same base length as the oligonucleotide. (In the formula, Bx is a nucleic acid base, each independently a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine, or uracil, and X' is a phosphate bond, each independently a phosphorodiamidate bond, a phosphorodiamidioate bond, or a phosphorodiamidiodithioate bond.)

[0026]

[14] The base sequence of the oligonucleotide is preferably a base sequence having 90% to 100% sequence identity to a base sequence complementary to a target region consisting of a 15- to 25-mer contiguous bases from bases located at positions 1 to 40, 46, 51, 56, or 62 to 63, counting from the 5' end, in the base sequence of SEQ ID NO: 1, or a 15- to 25-mer contiguous bases from bases located at positions 108873, 108878 to 108917, 108923, 108928, 108933, or 108939 to 108940, counting from the 5' end, in the base sequence of SEQ ID NO: 2; a base sequence complementary to a base sequence in the target region in which one or several bases have been deleted, substituted, inserted, or added; or a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region.

[0027]

[15] The base sequence of the oligonucleotide is preferably a base sequence having 90% to 100% sequence identity based on a base sequence complementary to a target region consisting of a 15- to 25-mer contiguous bases located at positions 1 to 32, 34 to 40, 46, 51, or 62 to 63 in the base sequence of SEQ ID NO: 1, counting from the 5' end, or a target region consisting of a 15- to 25-mer contiguous bases located at positions 108878 to 108909, 108911 to 108917, 108923, 108928, or 08939 to 108940 in the base sequence of SEQ ID NO: 2, counting from the 5' end.

[0028]

[16] The base sequence of the oligonucleotide is a target region consisting of a continuous 15-25mer from the bases located at positions 1, 3, 5 to 12, 14 to 19, 21, 25, 29, 30, 31, 34, 46, or 51 from the 5' end in the base sequence of SEQ ID NO: 1, or 108878, 10888, or 10888 from the 5' end in the base sequence of SEQ ID NO: 2. It is preferable that the base sequence has a sequence identity of 90% or more and 100% or less based on a base sequence complementary to a target region composed of a consecutive 15-25mer from bases located at positions 0, 108882 to 108889, 108891 to 108896, 108898, 108902, 108906 to 108908, 108911, 108923, or 108928.

[0029]

[17] The base sequence of the oligonucleotide is preferably one selected from the group consisting of the base sequences of SEQ ID NOs: 9 to 14, 16 to 26, 28 to 39, 41 to 42, 44 to 50, 52, 55 to 56, 69, 73, 80 to 105, 107 to 108, 110, 112, 114, 116, 118 to 131, 133, and 135.

[0030]

[18] The oligonucleotide is preferably one selected from the group consisting of the sequence names 6-20-A, 8-20-A, 8-20-B, 8-20-C, 9-20-A, 10-20-A, 12-20-A, 14-20-A, 16-20-A, 17-20-A, 18-20-A, 19-20-A, 15-25-A, 10-17-A, 10-20-B, 12-20-B, 14-20-B, 15-20-B, 16-20-B, 19-20-B, 31-20-B, 34-20-B, 6-20-B, 7-20-B, 8-20-D, and 9-20-B shown in Tables 2-1 to 2-7.

[0031]

[19] The oligonucleotide is preferably a single-stranded antisense oligonucleotide.

[0032]

[20] The double-stranded antisense oligonucleotide according to the present invention is a double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, comprising the oligonucleotide according to

[19] above and a second strand oligonucleotide hybridized to the single-stranded antisense oligonucleotide, wherein the base sequence of the second strand oligonucleotide has a sequence identity of 90% or more and 100% or less, based on a base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide.

[0033]

[21] The oligonucleotide complex according to the present invention is an oligonucleotide complex or a pharmaceutically acceptable salt thereof, comprising the oligonucleotide according to any one of [1] to

[19] above or a pharmaceutically acceptable salt thereof, or the double-stranded antisense oligonucleotide according to

[20] above or a pharmaceutically acceptable salt thereof, and an additional substance bound to the oligonucleotide or the second strand oligonucleotide directly or via a linker bond, wherein the additional substance is selected from the group consisting of polyethylene glycol, a peptide, an alkyl chain, a ligand compound, an antibody, a protein, and a sugar chain, and the linker bonds are each independently a phosphodiester bond, a phosphorothioate bond, a phosphorodithioate bond, a phosphoramidate bond, a boranophosphate bond, an alkylphosphonate bond, a phosphorodiamidate bond, a phosphorodiamidothioate bond, or a phosphorodiamidodithioate bond.

[0034]

[22] A pharmaceutical product according to the present invention is a pharmaceutical product comprising, as an active ingredient, the oligonucleotide according to any one of [1] to

[19] above or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide according to

[20] above or a pharmaceutically acceptable salt thereof, or the oligonucleotide complex according to

[21] above or a pharmaceutically acceptable salt thereof.

[0035]

[23] The agent for skipping exon 27 of the human WRN gene according to the present invention is an agent for skipping exon 27 of the human WRN gene, comprising, as an active ingredient, the oligonucleotide described in any of [1] to

[19] above or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide described in

[20] above or a pharmaceutically acceptable salt thereof, or the oligonucleotide conjugate described in

[21] above or a pharmaceutically acceptable salt thereof.

[0036]

[24] The functional WRN restorer of the present invention is a functional WRN restorer that contains, as an active ingredient, the oligonucleotide described in any of [1] to

[19] above or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide described in

[20] above or a pharmaceutically acceptable salt thereof, or the oligonucleotide conjugate described in

[21] above or a pharmaceutically acceptable salt thereof, and that possesses a nuclear localization signal resulting from skipping of the 27th exon of the human WRN gene.

[0037]

[25] A therapeutic agent for Werner syndrome according to the present invention is a therapeutic agent for Werner syndrome, comprising, as an active ingredient, the oligonucleotide according to any one of [1] to

[19] above or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide according to

[20] above or a pharmaceutically acceptable salt thereof, or the oligonucleotide complex according to

[21] above or a pharmaceutically acceptable salt thereof.

[0038]

[26] A preventive agent for Werner syndrome according to the present invention is a preventive agent for Werner syndrome, comprising, as an active ingredient, the oligonucleotide according to any one of [1] to

[19] above or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide according to

[20] above or a pharmaceutically acceptable salt thereof, or the oligonucleotide complex according to

[21] above or a pharmaceutically acceptable salt thereof.

[0039]

[27] A method for treating or preventing Werner syndrome according to the present invention is a method for treating or preventing Werner syndrome, comprising administering to an individual the oligonucleotide described in any of [1] to

[19] above or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide described in

[20] above or a pharmaceutically acceptable salt thereof, or the oligonucleotide complex described in

[21] above or a pharmaceutically acceptable salt thereof.

[0040]

[28] The present invention provides the oligonucleotide according to any one of [1] to

[19] above or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide according to

[20] above or a pharmaceutically acceptable salt thereof, or the oligonucleotide complex according to

[21] above or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of Werner syndrome.

[0041]

[29] The present invention provides the oligonucleotide according to any one of [1] to

[19] above or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide according to

[20] above or a pharmaceutically acceptable salt thereof, or the oligonucleotide complex according to

[21] above or a pharmaceutically acceptable salt thereof, which are used for producing an agent for treating or preventing Werner syndrome.

[0042] According to the present invention, it is possible to provide an oligonucleotide that enables skipping of exon 27 of the human WRN gene, and a drug containing the oligonucleotide that enables highly efficient skipping of exon 27 of the human WRN gene.

[0043] FIG. 1 is a schematic diagram showing the structure of the human WRN gene product. FIG. 2 is a schematic diagram illustrating the mechanism of Werner syndrome, which has a skipping mutation c.3139-1G>C in exon 26. FIG. 3 is a schematic diagram illustrating the mechanism of exon 27 skipping using a single-stranded antisense oligonucleotide according to the present embodiment. FIG. 4 is a schematic diagram showing the relative position of an antisense oligonucleotide designed to induce WRN exon 27 skipping in exon 27. FIG. 5 is an electrophoretic photograph showing the efficiency of exon 27 skipping in the WRN gene in HEK293T. FIG. 6 is an electrophoretic photograph showing the skipping activity of exon 27 in the WRN gene in fibroblasts from a Werner syndrome patient with a skipping mutation c.3139-1G>C in exon 26. FIG. 7 shows fluorescence microscopic images showing the exon 27 skipping activity of the WRN gene in fibroblasts from a Werner syndrome patient carrying the exon 26 skipping mutation c.3139-1G>C.

[0044] <Oligonucleotide causing exon 27 skipping of human WRN gene> The oligonucleotide of this embodiment is an oligonucleotide or a pharmaceutically acceptable salt thereof that expresses a functional human WRN protein in response to a skip mutation of exon 26 or 28 of the human WRN gene, wherein each nucleotide in the oligonucleotide is linked via a phosphate group and / or a modified phosphate group, and the oligonucleotide comprises a modified nucleic acid having at least one modified sugar, and the base length of the oligonucleotide is 10 to 30 mer, and the base sequence of the oligonucleotide is: a base sequence that has 90% to 100% sequence identity with respect to a base sequence complementary to at least one target region of the same base length as the oligonucleotide in the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, or a base sequence complementary to a base sequence in the target region in which one or several bases have been deleted, substituted, inserted, or added, or The oligonucleotide or a pharmaceutically acceptable salt thereof has a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region. The oligonucleotide is preferably a single-stranded antisense oligonucleotide. In this embodiment, the term "modified nucleic acid having a modified sugar" includes the morpholino nucleic acid described below.

[0045] The oligonucleotide or pharmaceutically acceptable salt thereof having the above-described configuration enables skipping of exon 27 of the human WRN gene. In one aspect of this embodiment, the oligonucleotide or pharmaceutically acceptable salt thereof can also be understood as "an oligonucleotide or pharmaceutically acceptable salt thereof that skips exon 27 of the human WRN gene." The present invention can improve symptoms of Werner syndrome, including intractable skin ulcers, by inducing skipping of exon 27 in pre-mRNA with a shift in amino acid reading frame caused by a splicing abnormality caused by the skipping mutation c.3139-1G>C in exon 26 of the human WRN gene, thereby resolving the shift in reading frame. Furthermore, the present invention can also be applied to the treatment of patients with Werner syndrome who have, other than the 26th exon skipping mutation c.3139-1G>C, for example, the 26th exon skipping mutation c.3233+1G>T, the 26th exon skipping mutation c.3233+1G>C, and the 28th exon skipping mutation c.IVS28+2T>C. These will be explained in detail below. For convenience, the oligonucleotide according to this embodiment may be referred to as an "antisense oligonucleotide," "single-stranded antisense oligonucleotide," etc., but it is not intended to be limited to these terms.

[0046] <Definitions of Terms, etc.> First, definitions of terms used in this specification will be explained below.

[0047] (WRN Gene) In this embodiment, the "WRN gene" can be defined according to Nature (1992) 355:735-738 (Non-Patent Document 2) and Am. J. Hum. Genet. (1997) 60:330-341 (Non-Patent Document 3).

[0048] (Single-Stranded Antisense Oligonucleotide) In this embodiment, a "single-stranded antisense oligonucleotide" or "antisense oligonucleotide" (hereinafter sometimes referred to as "ASO") refers to an oligonucleotide or a pharmacologically acceptable salt thereof that is complementary to the mRNA, pre-mRNA, or ncRNA (non-coding RNA) of a target gene (hereinafter these three may be collectively referred to as "target RNA"). Antisense oligonucleotides are composed of DNA, RNA, and / or analogs thereof. Antisense oligonucleotides form a duplex with the target mRNA, pre-mRNA, or ncRNA, thereby suppressing the activity of the target mRNA, pre-mRNA, or ncRNA. Antisense oligonucleotides include those that have a base sequence that is completely complementary to the base sequence of the target mRNA, pre-mRNA, or ncRNA; those that have a base sequence in which one or more bases have been deleted, substituted, inserted, or added in the complementary base sequence; and those that contain bases that form wobble base pairs in their base sequence. Furthermore, the antisense oligonucleotide of the present invention may further contain modified nucleotides known in the art other than "modified nucleic acids in which the sugar moiety is a modified sugar" (sugar-modified modified nucleotides) described below. Examples of modified nucleotides known in the art include sugar-modified modified nucleotides, as well as phosphate-modified nucleotides and nucleobase-modified nucleotides described below. The structures of both termini of the antisense oligonucleotide in this embodiment are not particularly limited, and may be, for example, -OH or -OR (wherein R represents an alkyl chain, a phosphate ester, or an additional substance described below). The single-stranded antisense oligonucleotide in this embodiment may be in a single-stranded form, or may hybridize with a second-strand oligonucleotide described below to form a double-stranded form.A double-stranded oligonucleotide consisting of the single-stranded antisense oligonucleotide and a second-stranded oligonucleotide hybridized to the single-stranded antisense oligonucleotide may be referred to as a "double-stranded antisense oligonucleotide."

[0049] (Oligonucleotide) In this embodiment, "oligonucleotide" refers to a polymer of nucleotides in which 2 to 30 identical or different nucleotides are linked together via phosphodiester bonds or other bonds. The oligonucleotide can also be understood to be composed of a nucleobase moiety, a phosphate moiety, and a sugar moiety or a morpholino ring moiety, as shown in the following structural formula:

[0050]

[0051]

[0052] The oligonucleotides are broadly classified into natural oligonucleotides and non-natural oligonucleotides. "Natural oligonucleotides" refer to oligonucleotides composed of naturally occurring nucleotides. "Non-natural oligonucleotides" refer to oligonucleotides containing at least one modified nucleotide as a constituent unit, as described below. "Non-natural oligonucleotides" preferably include modified sugar derivatives in which the sugar moiety is modified; phosphorothioate derivatives in which one non-bridging oxygen atom in the phosphodiester bond is replaced with a sulfur atom; phosphorodithioate derivatives in which two non-bridging oxygen atoms in the phosphodiester bond are replaced with sulfur atoms; ester derivatives in which the phosphodiester bond is triesterized; phosphoamide derivatives in which the phosphodiester bond is amidated; boranophosphate derivatives in which the phosphodiester bond is boronated; alkylphosphonate (e.g., methylphosphonate, methoxypropylphosphonate, etc.) derivatives in which the non-bridging oxygen atom in the phosphodiester bond is replaced with an alkyl group; amide derivatives in which the phosphodiester bond is replaced with an amide bond; and modified base derivatives in which the nucleobase is modified. More preferably, the unnatural oligonucleotide includes a cross-linked modified sugar derivative in which the sugar moiety is modified; a phosphorothioate derivative in which one non-bridging oxygen atom in the phosphodiester bond is replaced with a sulfur atom; an ester derivative in which the phosphodiester bond is esterified; and an alkylphosphonate derivative in which the sugar moiety is modified with a modified sugar (e.g., a cross-linked sugar) described below and one non-bridging oxygen atom in the phosphodiester bond is replaced with a sulfur atom or the non-bridging oxygen atom in the phosphodiester bond is substituted with an alkyl group.

[0053] (Nucleoside) In this embodiment, "nucleoside" refers to a compound in which a purine base or a pyrimidine base is bound to a sugar. A naturally occurring nucleoside may be referred to as a "natural nucleoside." A modified nucleoside that does not exist in nature may be referred to as a "modified nucleoside." A modified nucleoside in which the sugar moiety is particularly modified may be referred to as a "modified sugar nucleoside."

[0054] (Nucleotide) In this embodiment, "nucleotide" refers to a compound in which a phosphate group is bound to the sugar of the above-mentioned nucleoside. A naturally occurring nucleotide may be referred to as a "natural nucleotide." A non-naturally occurring modified nucleotide may be referred to as a "modified nucleotide" or "modified nucleic acid." Examples of "modified nucleotide" or "modified nucleic acid" include a compound in which a phosphate group is bound to the sugar moiety of the above-mentioned modified nucleoside, and a compound in which a modified phosphate group described below is bound to the sugar moiety of a natural nucleoside.

[0055] (Sugar modification, modified sugar) In this embodiment, "sugar modification" means that the sugar moiety of the nucleotide is modified. The modified sugar moiety may be specifically referred to as a "modified sugar". Modified nucleotides with sugar modifications can be used as modified nucleic acids, and examples thereof include 2'-O-alkylated nucleic acids, 2'-fluorinated nucleic acids, 5'-methylated nucleic acids, 2',4'-BNA (bridged nucleic acid, hereinafter sometimes referred to as "LNA"), AmNA (amide-bridged artificial nucleic acid, amido-bridged nucleic acid), GuNA (guanidine-bridged artificial nucleic acid, guanidino-bridged nucleic acid), scpBNA (2'-O,4'-C-spirocyclopropylene-bridged nucleic acid), ENA (2'-O,4'-C-ethylene-bridged nucleic acid), and the like. Acid), S-cEt (2',4'-constrained Ethyl Nucleic Acid), etc. Examples of 2'-O-alkylated nucleic acids include those containing structures represented by the symbols "A(M)", "A(m)", "C(M)", "5(m)", "G(M)", "G(m)", "U(M)", and "T(m)" as described below. Examples of LNAs include those containing structures represented by the symbols "A(L)", "5(L)", "G(L)", and "T(L)" as described below. Examples of AmNAs include those containing structures represented by the symbols "A(Y)", "5(Y)", "G(Y)", and "T(Y)" as described below. Examples of GuNAs include those containing structures represented by the symbols "A(Gx)", "5(Gx)", "G(Gx)", and "T(Gx)" as described below. Examples of scpBNA include those containing structures represented by the symbols "A(S)", "5(S)", "G(S)", and "T(S)" described below.

[0056] In one aspect of this embodiment, the modification of the sugar constituting the oligonucleotide is preferably a group represented by the following formula (A1): In another aspect of this embodiment, the modified sugar constituting the oligonucleotide can also be understood to be represented by the following formula (A1):

[0057]

[0058] In the above formula, Bx is a nucleic acid base, each independently a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine, or uracil; X is a phosphate bond, each independently a phosphodiester bond, phosphorothioate bond, phosphorodithioate bond, phosphoamidate bond, boranophosphate bond, or alkylphosphonate bond; Y is each independently a hydrogen atom, a hydroxyl group, a fluorine atom, an optionally substituted alkoxy group having 1 to 6 carbon atoms, or an optionally substituted amino group; and Z is each independently a hydrogen atom, a carbon-oxygen double bond, or an alkyl group having 1 to 5 carbon atoms which may have a cyclic structure, or a ring formed by combining some carbon atoms of the alkyl group with Y. Here, the "alkyl group having 1 to 5 carbon atoms" can also be understood as a monovalent group formed by combining some carbon atoms of an alkylene group having 1 to 5 carbon atoms with a hydrogen atom.

[0059] Examples of the "optionally substituted alkoxy group having 1 to 6 carbon atoms" include an unsubstituted alkoxy group having 1 to 6 carbon atoms, as well as a hydroxyl group, a fluorine atom, a linear or branched alkoxy group having 1 to 6 carbon atoms, a cyclic alkoxy group having 3 to 8 carbon atoms, a carbamoyl group, and an alkylamido group having 1 to 8 carbon atoms (-(CO)-NR ７ R ８ and an alkoxy group having 1 to 6 carbon atoms substituted with a substituent selected from the group consisting of groups represented by the following formula: ７ and R ８ are each independently a hydrogen atom or a linear or branched alkyl group having 1 to 8 carbon atoms.

[0060] Examples of the "optionally substituted amino group" include an unsubstituted amino group and a linear or branched alkyl group having 1 to 6 carbon atoms (the alkyl group is a linear or branched alkoxy group having 1 to 6 carbon atoms, or -(CO)-NR ９ R １０and an amino group substituted with a substituent selected from the group consisting of a cyclic alkyl group having 3 to 8 carbon atoms, an acyl group having 1 to 6 carbon atoms, and an amidine group substituted at 1 to 3 positions with a linear or branched alkyl group having 1 to 6 carbon atoms. ９ and R １０ are each independently a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms.

[0061] Examples of the "alkyl group having 1 to 5 carbon atoms, which may have a carbon-oxygen double bond or a cyclic structure" include an unsubstituted alkyl group having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms, which has a carbon-oxygen double bond, and a cycloalkyl group having 3 to 5 carbon atoms (such as a cyclopropyl group). Here, the number of carbon atoms in the "alkyl group having 1 to 5 carbon atoms, which has a carbon-oxygen double bond" may include the carbon that constitutes the carbon-oxygen double bond. Therefore, in this embodiment, the "alkyl group having 1 to 5 carbon atoms, which has a carbon-oxygen double bond" may include an aldehyde group. When Z is an aldehyde group and Y is an amino group which may be substituted, the ring formed together by Y and Z may have a structure containing an amide bond.

[0062] In one aspect of this embodiment, the modification of the sugar constituting the oligonucleotide is preferably a group represented by the following formula (A2) or formula (A3): In another aspect of this embodiment, the modified sugar constituting the oligonucleotide can also be understood to be represented by the following formula (A2) or formula (A3):

[0063]

[0064] In the formula, Bx is a nucleic acid base, each independently a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine, or uracil; X is a phosphate bond, each independently a phosphodiester bond, a phosphorothioate bond, a phosphorodithioate bond, a phosphoramidate bond, a boranophosphate bond, or an alkylphosphonate bond; R ４are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, Y′ is an oxygen atom or an optionally substituted nitrogen atom, and R ５ and R ６ are each independently a hydrogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, or R ５ and R ６ are taken together to form a carbonyl group or a ring, and n is 0 or 1.

[0065] In another aspect of this embodiment, the modification of the sugar constituting the oligonucleotide is a group represented by formula (A2) above, and the R ４ are each independently a methyl group, a methoxyethyl group, or an N-methylpropanamide group (—CH ２ CH ２ -CONH-CH ３ , methyliminocarbonylethyl group).

[0066] In another aspect of this embodiment, the sugar modification constituting the oligonucleotide is a group represented by formula (A3), Y′ is an oxygen atom, or a nitrogen atom optionally substituted with a hydrogen atom, a methyl group, a methoxyethyl group, or an N-methylpropanamide group, and R ５ and R ６ are each independently a hydrogen atom, an alkyl group having 1 to 2 carbon atoms, or the R ５ and R ６ are taken together to form a carbonyl group or a ring having 3 to 6 carbon atoms, and n is preferably 0 or 1.

[0067] (Nucleotide Modifications Known in the Art Other Than Sugar Modifications) Nucleotide modifications known in the art other than the sugar modifications described above can be used as modified nucleic acids for producing the single-stranded antisense oligonucleotide of the present invention. Known nucleotide modifications include phosphate group modifications and nucleobase modifications, which will be described later. Examples of such nucleotide modifications include the nucleotide modifications described in J. Med. Chem. (2016) 59:9645-9667 (Non-Patent Document 5) and the like. These nucleotide modifications can be carried out based on methods known in the art described in the literature cited in the above literature.

[0068] (Phosphate Group) In this embodiment, the term "phosphate group" refers to a nucleotide in which the bond at the phosphate moiety is a naturally occurring phosphodiester bond (a bond indicated by the symbol "-" described below).

[0069] (Phosphate group modification, modified phosphate group) In this embodiment, "phosphate group modification" means that the phosphate moiety of the nucleotide is modified. The modified phosphate moiety may be particularly referred to as a "modified phosphate group." Examples of bond types containing the modified phosphate group include a phosphorothioate bond (a bond indicated by the symbol "^" described below), a phosphorodithioate bond, a phosphoamidate bond, or a boranophosphate bond, an alkylphosphonate bond, a phosphorodiamidate bond (a bond indicated by the symbol "*" described below), a phosphorodiamidethioate bond, and a phosphorodiamidedithioate bond.

[0070] (Nucleobase modification, modified nucleobase) In this embodiment, "nucleobase modification" means that the nucleobase portion of the nucleotide is modified. The modified nucleobase portion may be particularly referred to as a "modified nucleobase". Examples of modified nucleobases include 5-methylcytosine, 5-hydroxymethylcytosine, and 5-propynylcytosine.

[0071] (DNA or RNA Analogs) The term "DNA or RNA analogs" refers to molecules with a structure similar to that of DNA or RNA. Examples include peptide nucleic acids (pNA) and morpholino nucleic acids. The nucleic acids used in the present invention are not limited to those modified in the sugar moiety of the nucleic acid, and morpholino nucleic acids or peptide nucleic acids may also be used. That is, in another embodiment of the single-stranded antisense oligonucleotide of the present invention, the oligonucleotide may be composed of a DNA or RNA analog in the sequence described in the "Single-Stranded Antisense Oligonucleotide Base Sequence" section below. This DNA or RNA analog includes at least a peptide nucleic acid or a morpholino nucleic acid. The antisense oligonucleotide of the present invention, including a peptide nucleic acid or a morpholino nucleic acid, is synthesized according to a standard method. Morpholino oligonucleotides are described, for example, in WO 1991 / 009033 (Patent Document 4), WO 2009 / 064471 (Patent Document 5), J. Am. Chem. Soc (2016) 138: 15663-15672 (Non-Patent Document 8). Examples of morpholino nucleic acids include those containing structures represented by the symbols "A(N)", "C(N)", "G(N)", and "T(N)" described below.

[0072] In one aspect of this embodiment, the oligonucleotide is preferably a morpholino oligonucleic acid composed of a morpholino nucleic acid represented by the following formula (A4):

[0073]

[0074] In the formula, Bx is a nucleic acid base, each independently a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine, or uracil, and X' is a phosphate bond, each independently a phosphorodiamidate bond, a phosphorodiamidioate bond, or a phosphorodiamidiodithioate bond.

[0075] (ncRNA) In this embodiment, "ncRNA" refers to a general term for RNA that is not involved in protein translation. Examples of the ncRNA include ribosomal RNA, transfer RNA, miRNA, and Natural Antisense Transcript (NAT).

[0076] (Nucleobase Moiety of Oligonucleotide) Examples of the nucleobase moiety of the oligonucleotide include thyminyl, cytosinyl, adeninyl, guaninyl, 5-methylcytosinyl, uracilyl, 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl, 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl, and 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl groups. Preferred examples of the nucleobase moiety include thyminyl, cytosinyl, adeninyl, guaninyl, 5-methylcytosinyl, and uracilyl groups. Among these nucleobases, uracil (U) and thymine (T) are interchangeable. Both uracil (U) and thymine (T) can form base pairs with adenine (A) in a complementary strand, as well as in the nucleobase portion of antisense oligonucleotides.

[0077] (Target RNA) In this embodiment, the term "target RNA" refers to the RNA to which the single-stranded antisense oligonucleotide binds. In other words, in this embodiment, the target RNA refers to a pre-mRNA of the WRN gene. Examples of the target RNA include a pre-mRNA of the human WRN gene having the nucleotide sequence set forth in SEQ ID NO: 2, a pre-mRNA of the human WRN gene having a nucleotide sequence set forth in any of SEQ ID NOs: 3 to 5 and having an exon 26 skipping mutation, and a pre-mRNA of the human WRN gene having the nucleotide sequence set forth in SEQ ID NO: 6 and having an exon 28 skipping mutation.

[0078] (Binding to target RNA) In this embodiment, "binding to target RNA" means that the nucleic acid bases of the single-stranded antisense oligonucleotide form a double-stranded nucleic acid together with the nucleic acid bases of the target RNA due to complementarity with the target RNA. The double-stranded nucleic acid may be formed in at least a portion of the target RNA. The strength of binding to the target RNA can be measured, for example, by an index of thermal stability. An example of the index of thermal stability is the melting temperature (Tm value) of the double-stranded nucleic acid. The Tm value is preferably 40 to 90°C, more preferably 50 to 70°C.

[0079] (Target region) The target region refers to a region in the WRN gene pre-mRNA to which the single-stranded antisense oligonucleotide binds, including the target region consisting of the indicated base sequence and a region in the WRN pre-mRNA.

[0080] (Pre-mRNA) The pre-mRNA refers to the primary transcript of RNA transcribed from DNA. That is, the pre-mRNA is RNA containing exon regions, intron regions, and untranslated regions (UTRs). The pre-mRNA can also be understood as RNA before splicing after transcription. When the pre-mRNA is spliced, it becomes mRNA.

[0081] (Binding to target region) The binding to the target region means that the single-stranded antisense oligonucleotide of the present invention forms a double strand with the target region.However, the single-stranded antisense oligonucleotide of the present invention does not necessarily need to form a double strand with the entire target region, but can form a double strand with a part of the target region.That is, the single-stranded antisense oligonucleotide of the present invention is preferably one that has complete complementarity with the target region, but as long as it binds to the target region of WRN, it can be complementary to at least a part of the target region.

[0082] (Part of the target region) The part of the target region means a region of the target region having a length of 10 to 15 nucleotide bases.

[0083] (Complementary to at least a portion of the target region) "Complementary to at least a portion of the target region" means complementary to the bases of at least a portion of the target region on the target RNA, including complementary to the bases of a region on a pre-mRNA corresponding to at least a portion of the region.

[0084] The present inventors focused on splicing-controlling antisense oligonucleotides and investigated the creation of antisense oligonucleotides that induce skipping of exon 27 of the human WRN gene.

[0085] <Design of Single-Stranded Antisense Oligonucleotides> Single-stranded antisense oligonucleotides are compounds that can restore the abnormal reading frame of abnormal RNA caused by frameshifts, etc., and cause exon skipping, which is the target region. Therefore, the structure of the antisense oligonucleotide is designed so that it can bind to the target region of the target RNA, and in addition, sugar-modified nucleic acids are arranged so that it cannot be recognized by nucleases such as RNase H.

[0086] <Base Sequence of Single-Stranded Antisense Oligonucleotide> The base sequence of the single-stranded antisense oligonucleotide according to this embodiment is: (A) a base sequence having 90% to 100% sequence identity based on a base sequence complementary to a target region consisting of a 10-30 mer (preferably a 15-25 mer) contiguous bases from bases located at positions 1 to 40, 46, 51, 56, or 62-63, counting from the 5' end, in the base sequence set forth in SEQ ID NO: 1, or a 15-25 mer contiguous base from bases located at positions 108873, 108878 to 108917, 108923, 108928, 108933, or 108939 to 108940, counting from the 5' end, in the base sequence set forth in SEQ ID NO: 2; (B) a base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted, or added in the target region, or (C) a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region. In this embodiment, each base sequence shown in the sequence listing is used to indicate only the sequence information of the nucleic acid base portion. The structural information of the oligonucleotide, including the sugar portion and phosphate portion in addition to the nucleic acid base portion, is shown in the format shown in Tables 2-1 to 2-7 below.

[0087] In this embodiment, "sequence identity" refers to the percentage (%) of identical bases in the total overlapping base sequence in the optimal alignment when two base sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm can take into account the introduction of gaps into one or both of the sequences for optimal alignment). The "sequence identity" of a base sequence can be easily confirmed by those skilled in the art. For example, NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) can be used.

[0088] The base sequence of the single-stranded antisense oligonucleotide of this embodiment preferably has a sequence identity of 95% to 100% with the base sequence complementary to the above-mentioned predetermined target region in the base sequence set forth in SEQ ID NO: 1, more preferably 98% to 100%, and even more preferably 100% sequence identity.

[0089] In this embodiment, examples of "a base sequence in which one or several bases have been deleted, substituted, inserted or added" include a base sequence that has, due to the deletion, substitution, insertion or addition, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the base sequence before the deletion, substitution, insertion or addition. The specific number of "one or several bases" may be one, two, three, four, or five of the above-mentioned deletions, substitutions, insertions or additions, each independently, or a combination of multiple deletions, substitutions, insertions or additions.

[0090] In this embodiment, "stringent conditions" refers to conditions in which the sample is incubated for 12 hours at room temperature in a solution containing 6xSSC (1xSSC has the composition: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5x Denhardt's solution, 100 μg / mL denatured salmon sperm DNA, and 50% (v / v) formamide, followed by washing with 0.5xSSC at a temperature of 50°C or higher. More stringent conditions are also encompassed, such as incubation for 12 hours at 45°C or 60°C, washing with 0.2xSSC or 0.1xSSC, or washing at a temperature of 60°C or 65°C or higher.

[0091] In one aspect of this embodiment, the base sequence of the single-stranded antisense oligonucleotide is preferably a base sequence complementary to a target region consisting of a 15- to 25-mer contiguous base from bases located at positions 1 to 40, 46, 51, 56, or 62 to 63, counting from the 5' end, in the base sequence set forth in SEQ ID NO: 1, or a target region consisting of a 15- to 25-mer contiguous base from bases located at positions 108873, 108878 to 108917, 108923, 108928, 108933, or 108939 to 108940, counting from the 5' end, in the base sequence set forth in SEQ ID NO: 2.

[0092] In one aspect of this embodiment, the base sequence of the single-stranded antisense oligonucleotide is more preferably a base sequence complementary to a target region consisting of a 15- to 25-mer contiguous base from bases located at positions 1 to 32, 34 to 40, 46, 51, or 62 to 63, counting from the 5' end, in the base sequence set forth in SEQ ID NO: 1, or a target region consisting of a 15- to 25-mer contiguous base from bases located at positions 108878 to 108909, 108911 to 108917, 108923, 108928, or 108939 to 108940, counting from the 5' end, in the base sequence set forth in SEQ ID NO: 2.

[0093] In one aspect of this embodiment, the base sequence of the single-stranded antisense oligonucleotide is more preferably a base sequence complementary to a target region consisting of a contiguous 15-25mer from bases located at positions 1, 3, 5 to 12, 14 to 19, 21, 25, 29, 30, 31, 34, 46, or 51 in the base sequence set forth in SEQ ID NO: 1, counting from the 5' end; or a target region consisting of a contiguous 15-25mer from bases located at positions 108878, 108880, 108882 to 108889, 108891 to 108896, 108898, 108902, 108906 to 108908, 108911, 108923, or 108928 in the base sequence set forth in SEQ ID NO: 2, counting from the 5' end.

[0094] The single-stranded antisense oligonucleotide can bind to a target region of the WRN gene. As used herein, the term "binding to a target region of the WRN gene" of the single-stranded antisense oligonucleotide of the present invention includes direct binding of the single-stranded antisense oligonucleotide of the present invention to the WRN pre-mRNA.

[0095] One embodiment of the single-stranded antisense oligonucleotide of the present invention is a single-stranded antisense oligonucleotide capable of skipping exon 27 of the WRN gene, which has any of the nucleotide sequences listed in Tables 1-1 to 1-4, and which is complementary to a target region in the pre-mRNA of the human WRN gene. Furthermore, as long as the single-stranded antisense oligonucleotide contains a nucleotide sequence listed in Tables 1-1 to 1-4, it may extend by 1 to 5 nucleotides on the 3' and / or 5' ends. The target region can be considered to be a region in the human WRN pre-mRNA that is particularly involved in the regulation of expression of the human WRN gene (e.g., a region having a secondary structure of mRNA that is easily bound by antisense nucleotides). For example, in Table 1-1, if the 5'-terminal position of SEQ ID NO: 1 is "5" and the 3'-terminal position is "24," the contiguous 20-mer nucleotide sequence from the 5th to the 24th nucleotide counting from the 5'-terminal in the nucleotide sequence of SEQ ID NO: 1 (corresponding to exon 27 of SEQ ID NO: 2) is the target region in exon 27 of human WRN targeted by the corresponding single-stranded antisense oligonucleotide (sequence name "5-20"). Also, in Table 1-1, if the 5'-terminal position of SEQ ID NO: 1 is "(-)5" and the 3'-terminal position is "15," the 20-mer nucleotide sequence from the 5th nucleotide counting from the position binding to exon 27 to the 15th nucleotide of exon 27 relative to the intron region binding to the 5'-terminal in the nucleotide sequence of SEQ ID NO: 1 (corresponding to exon 27 of SEQ ID NO: 2) is the target region in exon 27 of human WRN targeted by the corresponding single-stranded antisense oligonucleotide (sequence name "(-)5-20").

[0096]

[0097]

[0098]

[0099]

[0100] In Tables 1-1 to 1-4 above, the symbols "A'", "C'", "G'", and "T'" are each selected from natural nucleosides (a, c, g, and t, described below) or modified nucleosides (including modified sugar nucleosides). Among the modified sugar nucleosides, as 2'-O-alkylated nucleic acids, the symbol "A'" is selected from A(M) or A(m), described below; the symbol "C'" is selected from C(M) or S(m), described below; the symbol "G'" is selected from G(M) or G(m), described below; and the symbol "T'" is selected from U(M) or T(m), described below. As for bridged modified nucleosides, the symbol "A'" is selected from A(L), A(Y), A(Gx), or A(S), as described below; the symbol "C'" is selected from 5(x), 5(L), 5(Y), 5(Gx), or 5(S), as described below; the symbol "G'" is selected from G(L), G(Y), G(Gx), or G(S), as described below; and the symbol "T'" is selected from T(L), T(Y), T(Gx), or T(S), as described below. As for morpholino nucleic acids, the symbol "A'" is A(N), as described below; the symbol "C'" is C(N), as described below; the symbol "G'" is G(N), as described below; and the symbol "T'" is T(N), as described below.

[0101] <Pharmacologically acceptable salt> The single-stranded antisense oligonucleotide according to this embodiment may be in the form of a pharmacologically acceptable salt. Here, "pharmacologically acceptable salt" refers to a salt of the single-stranded antisense oligonucleotide of the present invention, which is a physiologically acceptable salt of the single-stranded antisense oligonucleotide of the present invention, that is, a salt that retains the desired biological activity of the single-stranded antisense oligonucleotide and does not retain undesired toxicological effects. The same applies to the double-stranded antisense oligonucleotide and antisense oligonucleotide complex described below.

[0102] Pharmaceutically Acceptable Salts In one aspect of this embodiment, the single-stranded antisense oligonucleotide may be in the form of a pharmaceutically acceptable salt. Here, "pharmaceutically acceptable salts" refers to the pharmacologically acceptable salts described above that are acid addition salts or base addition salts. Examples of acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate, as well as organic acid salts such as citrate, oxalate, phthalate, fumarate, maleate, succinate, malate, acetate, formate, propionate, benzoate, trifluoroacetate, methanesulfonate, benzenesulfonate, para-toluenesulfonate, and camphorsulfonate. Examples of base addition salts include inorganic base salts such as sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, and aluminum salts, as well as organic base salts such as trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, dicyclohexylamine, and N,N-dibenzylethylamine. Further examples include salts with basic or acidic amino acids such as arginine, lysine, ornithine, aspartic acid, and glutamic acid (amino acid salts). The same applies to the double-stranded antisense oligonucleotides and antisense oligonucleotide complexes described below.

[0103] <Structure of Single-Stranded Antisense Oligonucleotide> The single-stranded antisense oligonucleotide according to this embodiment is composed of a natural oligonucleotide and / or a non-natural oligonucleotide. The single-stranded antisense oligonucleotide is preferably in a single-stranded form. In one aspect of this embodiment, the single-stranded antisense oligonucleotide may hybridize with a second-strand oligonucleotide described below to form a double-stranded form (double-stranded antisense oligonucleotide). The base sequence of the second-strand oligonucleotide preferably has a sequence identity of 90% to 100% based on the base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide.

[0104] <Mechanism of Exon Skipping by Single-Stranded Antisense Oligonucleotides> The single-stranded antisense oligonucleotides described above restore the reading frame of an aberrantly spliced ​​target RNA to its original reading frame, thereby inducing the expression of a functional WRN protein, through the following mechanism. An exon is included in mRNA only when both splice sites are recognized by the spliceosome complex. Targeting a splice site with an antisense oligonucleotide inhibits splicing and induces exon skipping. Exon skipping can be induced by binding to an exon with a single-stranded antisense oligonucleotide that targets either or both of the 5' splice site and 3' splice site of an exon, or the interior of an exon. That is, the single-stranded antisense oligonucleotide binds to the target region of the target RNA and inhibits splicing of the exon, thereby allowing the targeted exon to be skipped, thereby inducing the translation of a functional protein based on the expressed mRNA.

[0105] Furthermore, in this embodiment, the single-stranded antisense oligonucleotide can be suitably used to efficiently skip the 27th exon of the human WRN gene using the mechanism described above (including through regulation of maturation of WRN pre-mRNA). More specifically, the single-stranded antisense oligonucleotide binds to exon 27, the target region of the target RNA (WRN pre-mRNA) (top panel of Figure 3), and inhibits splicing of exon 27. As a result, the stop codon in exon 27, which is generated by the exon 26 skipping mutation seen in Werner syndrome patients, is skipped, resulting in the generation of WRN mRNA (in-frame, corrected to the reading frame of the WRN gene in healthy individuals) in which exons 25 and 28 are linked (middle panel of Figure 3). This mRNA is then used to translate functional WRN protein containing the nuclear localization signal contained in exon 34. Furthermore, according to this embodiment, the single-stranded antisense oligonucleotide can exert its effect of regulating functional WRN gene expression even when administered via transdermal, intravenous, or intradermal routes commonly used in clinical applications. Here, "highly efficient skipping of exon 27 of the human WRN gene" at least means highly efficient skipping of exon 27 of the human WRN gene, thereby restoring functional WRN protein expression.

[0106] Examples of natural nucleotides in which the sugar moiety is deoxyribose include deoxyadenosine monophosphate, deoxyguanosine monophosphate, thymidine monophosphate, deoxycytidine monophosphate, deoxy-5-methylcytidine monophosphate, etc. In other words, natural nucleotides constituting the single-stranded antisense oligonucleotide include those containing structural formulas corresponding to the symbols a, g, t, c, and 5(x) described below.

[0107] Examples of non-natural nucleotides in which the sugar moiety is deoxyribose include 2-thio-thymidine phosphate, 2-aminoadenosine phosphate, and 7-deazaguanosine phosphate.

[0108] The base length of the single-stranded antisense oligonucleotide of the present invention is 10 to 30 mer, preferably 15 to 25 mer, more preferably 18 to 22 mer, even more preferably 16 to 20 mer, and particularly preferably 18 to 20 mer. When the base length of the single-stranded antisense oligonucleotide of the present invention is 15 to 25 mer, 18 to 22 mer, 16 to 20 mer, or 18 to 20 mer, it exhibits particularly strong binding to WRN pre-mRNA and can more effectively regulate highly efficient skipping of exon 27 of the human WRN gene.

[0109] In this embodiment, the single-stranded antisense oligonucleotide has each nucleotide linked via a phosphate group and / or a modified phosphate group, and is preferably linked via a phosphodiester bond or a phosphorothioate bond.

[0110] <Double-stranded antisense oligonucleotide> The double-stranded antisense oligonucleotide of this embodiment is a double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, comprising the single-stranded antisense oligonucleotide and a second-strand oligonucleotide hybridized to the single-stranded antisense oligonucleotide. The base sequence of the second-strand oligonucleotide preferably has a sequence identity of 90% to 100% with respect to a base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide.

[0111] The double-stranded antisense oligonucleotide can be dissociated in solution and separated into the single-stranded antisense oligonucleotide and the second-stranded oligonucleotide.The separated single-stranded antisense oligonucleotide can bind to the target RNA.The single-stranded antisense oligonucleotide can also be understood as a "first-stranded oligonucleotide" in relation to the second-stranded oligonucleotide.In addition, among the oligonucleotides constituting the double-stranded antisense oligonucleotide, the first-stranded oligonucleotide has an antisense strand to the target RNA, but for convenience, the double-stranded oligonucleotide consisting of the first-stranded oligonucleotide and the second-stranded oligonucleotide will be referred to as a "double-stranded antisense oligonucleotide".

[0112] <<Method for producing single-stranded antisense oligonucleotides>> The single-stranded antisense oligonucleotides of the present invention can be produced by solid-phase synthesis using the phosphoramidite method. For example, a single-stranded oligonucleotide having a predetermined base sequence is first synthesized on a solid support using a commercially available automated nucleic acid synthesizer. Next, the synthesized single-stranded oligonucleotide is cleaved from the solid support using a basic substance or the like, and deprotected to obtain a crude single-stranded oligonucleotide. The crude single-stranded oligonucleotide obtained is then purified using HPLC or the like. Not limited to the above-mentioned production method, the single-stranded antisense oligonucleotides of the present invention can be produced by appropriately modifying the base sequence, modification site, etc. of the nucleic acid according to methods known to those skilled in the art. In addition, AmNA, GuNA, and scpBNA can be produced by the methods described in WO 2011 / 052436 (Patent Document 1), WO 2014 / 046212 (Patent Document 2), and WO 2015 / 125783 (Patent Document 3), respectively. Furthermore, morpholino oligonucleotides (PMOs) can be produced according to the methods described in International Publication No. 1991 / 009033 (Patent Document 4), International Publication No. 2009 / 064471 (Patent Document 5), or Non-Patent Document 8.

[0113] <<Method for Producing Double-Stranded Antisense Oligonucleotide>> The double-stranded antisense oligonucleotide of the present invention can be produced by first producing an oligonucleotide (second strand oligonucleotide) having a predetermined sequence identity based on a base sequence complementary to the single-stranded antisense oligonucleotide using a production method similar to that of the single-stranded antisense oligonucleotide, and then hybridizing the single-stranded antisense oligonucleotide and the second strand oligonucleotide.

[0114] <<Antisense Oligonucleotide Conjugate>> The antisense oligonucleotide conjugate of this embodiment comprises the above-mentioned oligonucleotide (preferably a single-stranded antisense oligonucleotide) or a pharmaceutically acceptable salt thereof, or the above-mentioned double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, and an additional substance bound to the above-mentioned oligonucleotide or the above-mentioned second strand oligonucleotide. The additional substance is selected from the group consisting of polyethylene glycol, peptides, alkyl chains (e.g., saturated aliphatic hydrocarbons, etc.), ligand compounds, antibodies, proteins, and sugar chains (e.g., carbohydrates, polysaccharides, etc.).

[0115] In one aspect of this embodiment, the oligonucleotide conjugate is an oligonucleotide conjugate or a pharmaceutically acceptable salt thereof, comprising the oligonucleotide or a pharmaceutically acceptable salt thereof, or the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, and an additional substance bound to the oligonucleotide or the second strand oligonucleotide directly or via a linker bond, wherein the additional substance is selected from the group consisting of polyethylene glycol, a peptide, an alkyl chain, a ligand compound, an antibody, a protein, and a sugar chain, and the linker bond is each independently a phosphodiester bond, a phosphorothioate bond, a phosphorodithioate bond, a phosphoamidate bond, a boranophosphate bond, an alkylphosphonate bond, a phosphorodiamidate bond, a phosphorodiamidioate bond, or a phosphorodiamidiodithioate bond.

[0116] In one aspect of this embodiment, the antisense oligonucleotide conjugate comprises the oligonucleotide (preferably a single-stranded antisense oligonucleotide) or a pharmaceutically acceptable salt thereof, and an additional substance bound to the oligonucleotide, wherein the additional substance is selected from the group consisting of polyethylene glycol, a peptide, an alkyl chain, a ligand compound, an antibody, a protein, and a sugar chain.

[0117] In another aspect of this embodiment, the antisense oligonucleotide complex comprises the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, and an additional substance bound to the single-stranded antisense oligonucleotide or the second strand oligonucleotide, wherein the additional substance is selected from the group consisting of polyethylene glycol, peptides, alkyl chains (e.g., saturated aliphatic hydrocarbons, etc.), ligand compounds, antibodies, proteins, and sugar chains (e.g., carbohydrates, polysaccharides, etc.).

[0118] In this embodiment, the term "additional substance" refers to a substance bound to the single-stranded antisense oligonucleotide or the second-strand oligonucleotide and used to impart a predetermined effect. The additional substance may be bound to the 5'-end, the 3'-end, or both the 5'-end and the 3'-end of the single-stranded antisense oligonucleotide. The additional substance may be bound to the 5'-end, the 3'-end, or both the 5'-end and the 3'-end of the second-strand oligonucleotide. In one aspect of this embodiment, the additional substance is preferably bound to either the 5'-end or the 3'-end of the single-stranded antisense oligonucleotide or the second-strand oligonucleotide. The additional substance may be directly bound to the single-stranded antisense oligonucleotide or the second-strand oligonucleotide via a covalent bond. The additional substance may be bound to the single-stranded antisense oligonucleotide or the second-strand oligonucleotide via a linker substance. Examples of the linker substance include linkers composed of alkyl, polyethylene glycol, peptide, disulfide, phosphate bond, etc., and / or combinations thereof. The linker substance can also be understood as a linker bond.

[0119] Examples of peptides that can be used as the additional substance include, but are not limited to, CPPs (Cell Penetrating Peptides), nuclear transport peptides, TAT (Trans-Activator of Transcription Protein), polyarginine, glucagon-like peptide-1 analogous peptides, synthetic cyclic RGD peptides, and skin-penetrating peptides.

[0120] Examples of ligand compounds used as the additional substance include, but are not limited to, N-acetylgalactosamine (GalNAc), sugars (glucose, mannose, etc.), lipids (cholesterol, etc.), vitamins (folic acid, vitamin A, vitamin E, etc.), and amino acids.

[0121] Examples of antibodies that can be used as the additional substance include, but are not limited to, the following: anti-insulin receptor antibody, anti-transferrin receptor antibody, anti-LDL receptor-related protein antibody, anti-CD22 antibody, anti-CD30 antibody, and anti-HER2 antibody.

[0122] Examples of proteins that can be used as the additional substance include, but are not limited to, the following: Albumin.

[0123] In the morpholino oligonucleic acid, examples of the combination of the additional substance and the linker substance include, but are not limited to, those represented by the following structural formulas:

[0124] <<Agent for skipping exon 27 of human WRN gene>> The agent for skipping exon 27 of the human WRN gene according to this embodiment comprises, as an active ingredient, the single-stranded antisense oligonucleotide of the present invention, the double-stranded antisense oligonucleotide, or the antisense oligonucleotide conjugate. The single-stranded antisense oligonucleotide of the present invention can skip exon 27 of the human WRN gene by binding to WRN pre-mRNA. Any administration method and formulation known in the art can be used for the skipping agent.

[0125] <Functional WRN Restoration Agent> The functional WRN restoration agent of this embodiment is a functional WRN restoration agent that contains, as an active ingredient, the above-mentioned oligonucleotide or a pharmaceutically acceptable salt thereof, the above-mentioned double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the above-mentioned oligonucleotide conjugate or a pharmaceutically acceptable salt thereof, and that possesses a nuclear localization signal resulting from skipping of exon 27 of the human WRN gene. The single-stranded antisense oligonucleotide of the present invention can skip exon 27 of the human WRN gene by binding to WRN pre-mRNA. In particular, when the human WRN gene has an exon 26 skipping mutation found in Werner syndrome patients, the stop codon in exon 27 that appears in the human WRN gene is skipped, generating WRN mRNA in which exons 25 and 28 are linked, and this mRNA is used to translate a functional WRN protein that has a nuclear localization signal contained in exon 34.

[0126] <<Pharmaceutical Compositions Comprising Single-Stranded Antisense Oligonucleotides, etc., as Active Ingredients>> The pharmaceutical compositions of this embodiment comprise, as active ingredients, the single-stranded antisense oligonucleotides of the present invention or pharmaceutically acceptable salts thereof, the double-stranded antisense oligonucleotides or pharmaceutically acceptable salts thereof, or the antisense oligonucleotide complexes or pharmaceutically acceptable salts thereof. Any administration method and formulation known in the art can be used for the pharmaceutical compositions of this embodiment. Hereinafter, the pharmaceutical compositions may be referred to as "pharmaceutical compositions of antisense oligonucleotides, etc."

[0127] The pharmaceutical composition is used for the treatment or prevention of Werner syndrome. In other words, the pharmaceutical composition can be used for the treatment or prevention of Werner syndrome, which is expected to improve symptoms by highly efficient skipping of the 27th exon of the human WRN gene.

[0128] <Therapeutic and Preventive Agents for Werner Syndrome> A therapeutic agent for Werner syndrome according to this embodiment comprises, as an active ingredient, the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof. A preventive agent for Werner syndrome according to this embodiment comprises, as an active ingredient, the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof.

[0129] <Individual> In the present invention, an individual refers to a mammal. Preferably, it is a human, monkey, marmoset, dog, pig, rabbit, guinea pig, rat, or mouse. More preferably, it is a human.

[0130] When administering the single-stranded antisense oligonucleotide of the present invention or a pharmaceutical composition thereof (including a therapeutic or preventive agent for Werner's syndrome), the administration method and dosage form are not particularly limited. That is, any administration method and formulation known in the art can be used as the administration method and formulation of the antisense oligonucleotide of the present invention. Examples of administration methods include oral administration and parenteral administration. Examples of parenteral administration include ophthalmic administration, intravaginal administration, rectal administration, intranasal administration, transdermal administration, intravenous injection, infusion, subcutaneous administration, intraperitoneal administration or intramuscular injection, pulmonary administration by aspiration or inhalation, intrathecal administration, and intraventricular administration.

[0131] Various pharmaceutical additives such as excipients, binders, wetting agents, disintegrants, lubricants, diluents, flavoring agents, fragrances, solubilizers, suspending agents, emulsifiers, stabilizers, preservatives, and tonicity agents may be mixed into formulations of the antisense oligonucleotides and the like of the present invention as needed.

[0132] When pharmaceutical compositions such as the antisense oligonucleotides of the present invention are administered locally, formulations such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders can be used.

[0133] When pharmaceutical compositions of the present invention, such as antisense oligonucleotides, are orally administered, formulations such as powders, granules, suspensions or solutions dissolved in water or non-aqueous media, capsules, powders, tablets, etc. can be used.

[0134] When pharmaceutical compositions of the present invention, such as antisense oligonucleotides, are administered parenterally, intravenously, subcutaneously, or intradermally, formulations such as sterile aqueous solutions can be used.

[0135] The effective dose of the single-stranded antisense oligonucleotide of the present invention can be determined arbitrarily depending on the sex, age, weight, symptoms, etc. of the individual to be administered. Furthermore, it can also be determined arbitrarily depending on the method, route, frequency, etc. of administration. For example, the dose may be 0.01 to 100 mg / kg, preferably 0.1 to 50 mg / kg, and more preferably 0.1 to 10 mg / kg.

[0136] <<Method for Highly Efficient Skipping of the 27th Exon of the Human WRN Gene>> The method for skipping the 27th exon of the human WRN gene in this embodiment comprises the step of administering, as an active ingredient, the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof to a cell, tissue, or individual expressing the WRN gene.

[0137] In this embodiment, the single-stranded antisense oligonucleotide or the like may be administered to a cell, tissue, or individual in vitro or in vivo. When administered in vivo, the administration route is the same as that described above.

[0138] In this embodiment, examples of "cells expressing the WRN gene" include fibroblasts, adipocytes, mesenchymal stem cells, keratinocytes, muscle cells, vascular endothelial cells, and smooth muscle cells.

[0139] In this embodiment, the method for treating or preventing Werner syndrome includes a step of administering, as an active ingredient, the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof to an individual suffering from Werner syndrome.

[0140] The antisense oligonucleotide according to this embodiment has been described above. The single-stranded antisense oligonucleotide having the above-described configuration enables highly efficient skipping of the 27th exon of the human WRN gene.

[0141] <<Method for Evaluating Activity of Exon 27 Skipping in the Human WRN Gene>> <Introduction of Antisense Oligonucleotides into Cells> Observation can be performed by treating "cells expressing the WRN gene" with antisense oligonucleotides for 6 hours to 1 month using methods such as lipofection, electroporation, or direct addition. The treatment time is preferably 48 hours to 7 days. Any cells expressing the WRN gene can be used, such as HEK293T cells or fibroblasts derived from Werner syndrome patients (hereinafter referred to as "Werner syndrome patient fibroblasts"). Cells treated with antisense oligonucleotides can be harvested immediately after treatment, or the antisense oligonucleotides can be removed and the cells can be continuously cultured.

[0142] <Evaluation of exon 27 skipping in the human WRN gene> Total RNA extracted from the recovered cells is subjected to a reverse transcription reaction, and the region surrounding exon 27 of the WRN gene is amplified by PCR using the resulting cDNA. The PCR amplification product can be subjected to quantification of polynucleotide amount or sequence analysis to confirm exon 27 skipping in the WRN gene. The skipping efficiency (%) can be calculated by quantifying the polynucleotide amount "A" of the PCR product in which exon 27 was skipped and the polynucleotide amount "B" of the PCR product in which exon 27 was not skipped, and then using the measured values ​​of "A" and "B" according to the following formula: Skipping efficiency (%) = 100 x A / (A + B)

[0143] The present invention is not limited to the above-described embodiments. For example, the single-stranded antisense oligonucleotide includes the following embodiments.

[0144] One embodiment of the single-stranded antisense oligonucleotide of the present invention is an oligonucleotide or a pharmaceutically acceptable salt thereof that expresses a functional human WRN protein in response to a skipping mutation of exon 26 or 28 of the human WRN gene, wherein each nucleotide in the oligonucleotide is linked via a phosphate group and / or a modified phosphate group, the oligonucleotide comprises a modified nucleic acid having at least one modified sugar, the base length of the oligonucleotide is 10 to 30 mer, and the base sequence of the oligonucleotide is: a base sequence that has 90% to 100% sequence identity with respect to a base sequence complementary to at least one target region of the same base length as the oligonucleotide in the base sequence set forth in SEQ ID NO: 1; a base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted, or added in the target region; or a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region.

[0145] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the base sequence of the single-stranded antisense oligonucleotide is a base sequence that has 95% to 100% sequence identity with respect to a base sequence complementary to at least one target region of the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, the base sequence having the same base length as the single-stranded antisense oligonucleotide.

[0146] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the base sequence of the single-stranded antisense oligonucleotide is a base sequence complementary to at least one target region in the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, the target region having the same base length as the single-stranded antisense oligonucleotide.

[0147] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the sugar constituting the oligonucleotide is D-ribofuranose, and the sugar modification is a sugar modification of the hydroxyl group at the 2'-position of D-ribofuranose.

[0148] The base length of the oligonucleotide is 15 to 25 mer.

[0149] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the sugar modification constituting the oligonucleotide is a group represented by the following formula (A1): (In the formula, Bx is a nucleic acid base, and each Bx is independently a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine, or uracil; X is a phosphate bond, and each X is independently a phosphodiester bond, phosphorothioate bond, phosphorodithioate bond, phosphoamidate bond, boranophosphate bond, or alkylphosphonate bond; Y is independently a hydrogen atom, a hydroxyl group, a fluorine atom, an optionally substituted alkoxy group having 1 to 6 carbon atoms, or an optionally substituted amino group; and Z is independently a hydrogen atom, a carbon-oxygen double bond, or an alkyl group having 1 to 5 carbon atoms which may have a cyclic structure, or a ring formed by combining with Y some carbon atoms of the alkyl group.) An example of the "carbon-oxygen double bond" possessed by the alkyl group having 1 to 5 carbon atoms is a carbonyl group. An example of the "cyclic structure" possessed by the alkyl group having 1 to 5 carbon atoms is a cyclopropane structure.

[0150] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, at least one internucleotide bond of the oligonucleotide is a phosphorothioate bond.

[0151] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, at least one internucleotide bond of the oligonucleotide is a phosphodiester bond.

[0152] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the base sequence of the oligonucleotide is a base sequence that has 90% to 100% sequence identity with respect to a base sequence complementary to a target region consisting of a consecutive 15-25mer starting from bases located at positions 1 to 12, 14 to 21, 24, 25, 29 to 31, 34, 36, 41, 46, 51, or 62, counting from the 5' end, in the base sequence set forth in SEQ ID NO: 1; a base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted, or added in the target region; or a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region.

[0153] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the base sequence of the oligonucleotide is a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to a target region consisting of a 15-25mer contiguous bases from bases located at positions 1 to 40, 46, 51, 56, or 62 to 63, counting from the 5' end, in the base sequence set forth in SEQ ID NO: 1, or a 15-25mer contiguous bases from bases located at positions 108873, 108878 to 108917, 108923, 108928, 108933, or 108939 to 108940, counting from the 5' end, in the base sequence set forth in SEQ ID NO: 2; a base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted, or added in the target region; or It is a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region.

[0154] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the base sequence of the oligonucleotide has a sequence identity of 90% to 100% based on the base sequence complementary to a target region consisting of a consecutive 15-25mer starting from the base at positions 1, 3 to 12, 14 to 19, 21, 25, 29, 30, 31, 34, 46, or 51 in the base sequence set forth in SEQ ID NO: 1, counting from the 5' end.

[0155] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the base sequence of the oligonucleotide has a sequence identity of 90% to 100% based on a base sequence complementary to a target region consisting of a 15- to 25-mer contiguous bases located at positions 1 to 32, 34 to 40, 46, 51, or 62 to 63, counting from the 5' end, in the base sequence set forth in SEQ ID NO: 1, or a 15- to 25-mer contiguous bases located at positions 108878 to 108909, 108911 to 108917, 108923, 108928, or 108939 to 108940, counting from the 5' end, in the base sequence set forth in SEQ ID NO: 2.

[0156] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the base sequence of the oligonucleotide has a sequence identity of 90% to 100% based on a base sequence complementary to a target region consisting of a consecutive 15-25mer starting from the base at positions 1, 3, 6 to 12, 14 to 19, 21, 25, 29, 30, 31, 34, 46, or 51 in the base sequence set forth in SEQ ID NO: 1, counting from the 5' end.

[0157] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the base sequence of the oligonucleotide is a target region consisting of a continuous 15-25mer from the bases located at positions 1, 3, 5 to 12, 14 to 19, 21, 25, 29, 30, 31, 34, 46, or 51 from the 5' end in the base sequence set forth in SEQ ID NO: 1, or from positions 108878, 108879, 108880, or 108881 from the 5' end in the base sequence set forth in SEQ ID NO: 2. The base sequence has 90% to 100% sequence identity with respect to a base sequence complementary to a target region consisting of a continuous 15-25mer starting from bases located at positions 80, 108882 to 108889, 108891 to 108896, 108898, 108902, 108906 to 108908, 108911, 108923, or 108928.

[0158] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the sugar modification constituting the oligonucleotide is a group represented by the following formula (A2) or (A3). (wherein Bx is a nucleic acid base, each independently a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine, or uracil; X is a phosphate bond, each independently a phosphodiester bond, a phosphorothioate bond, a phosphorodithioate bond, a phosphoramidate bond, a boranophosphate bond, or an alkylphosphonate bond; R ４ are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, Y' is an oxygen atom or an optionally substituted nitrogen atom, and R ５ and R ６ are each independently a hydrogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, or R ５ and R ６ are taken together to form a carbonyl group or a ring, and n is 0 to 1.

[0159] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the base sequence of the oligonucleotide is one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 9, 11, 14 to 22, 24 to 29, 31, 35, 39 to 41, 44, 52, and 53.

[0160] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the base sequence of the oligonucleotide is one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 9 to 14, 16 to 26, 28 to 39, 41 to 42, 44 to 50, 52, 55 to 56, 69, 73, 80 to 105, 107 to 108, 110, 112, 114, 116, 118 to 131, 133, and 135.

[0161] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the base sequence of the oligonucleotide is a single-stranded antisense oligonucleotide.

[0162] Examples of the present invention will be described below, but the present invention is not limited to these examples.

[0163] <<Preparation of Single-Stranded Antisense Oligonucleotide Against WRN Gene>> A single-stranded antisense oligonucleotide against the WRN gene was prepared by the following procedure.

[0164] Single-stranded antisense oligonucleotides containing 2'-OMe (O-methyl), 2'-MOE (O-methoxyethyl), AmNA, scpBNA, and GuNA were synthesized on a 0.2 μmol scale using an automated nucleic acid synthesizer (nS-8 model, manufactured by Gene Design Co., Ltd.). Chain elongation was carried out using a standard phosphoramidite protocol. CPG resin was used as the solid support. DDTT (((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazaoline-3-thione) or the like was used for sulfuration to form the phosphorothioated (PS) backbone. Antisense oligonucleotides containing AmNA and scpBNA were obtained such that the terminal 5'-position hydroxyl group was not protected with a DMTr (4,4'-dimethoxytrityl) group and the 3'-position was supported on a solid phase. Subsequently, the single-stranded antisense oligonucleotide was cleaved from the solid phase support by alkali treatment and recovered in solution. The solvent was then distilled off from the recovered solution to obtain a crude product. The resulting crude product was purified by reverse-phase HPLC to obtain purified single-stranded antisense oligonucleotides. The purity and structure of each resulting single-stranded antisense oligonucleotide were confirmed by LC-MS (Waters). Morpholino oligonucleotides containing morpholino nucleic acids were synthesized according to International Publication No. 2009 / 064471 (Patent Document 5) or J. Am. Chem. Soc (2016) 138:15663-15672 (Non-Patent Document 8).

[0165] The single-stranded antisense oligonucleotides prepared by the above-mentioned method are listed in Tables 2-1 to 2-7 below.

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173] In this specification, the following symbols or notations may be used to represent corresponding structures:

[0174]

[0175]

[0176]

[0177]

[0178] In the structural formula shown above, R １ , R ２ , and R ３ are each independently a hydrogen atom, a linear or branched alkyl group having 1 to 6 carbon atoms, or a cycloalkyl group having 3 to 7 carbon atoms. １ and R ３ are both hydrogen atoms, and R ２ When R is a methyl group, it is represented as "Gm" and R １ is a hydrogen atom, and R ２ and R ３ When both are methyl groups, it is represented as "Gdm", and R １ and R ３ is a hydrogen atom, and R ２ When is a tert-butyl group, it is represented as "GtB".

[0179] <<Evaluation of Exon Skipping of the 27th Exon of the Human WRN Gene>> Evaluation of exon skipping of the 27th exon of the human WRN gene was performed by confirming the induction of exon 27 skipping in HEK293T cells and Werner syndrome patient fibroblasts lacking exon 26. Gene expression evaluation in this example refers to assessing the sequence structure of the 27th exon of the WRN gene by measuring the amount of amplification, using PCR, of the region spanning exons 26 (HEK293T cells with a normal WRN gene) or 25 (Werner syndrome patient fibroblasts lacking exon 26) of the WRN gene from complementary DNA (cDNA) obtained by reverse transcription. Specific procedures for each expression evaluation are described below.

[0180] (Introduction of antisense oligonucleotides into cells) "Cells expressing the human WRN gene" were treated with antisense oligonucleotides for two days using methods such as lipofection or direct addition. The cells used were cells expressing the WRN gene (e.g., HEK293T cells or exon 26-deleted Werner syndrome patient fibroblasts). The antisense oligonucleotide-treated cells were either harvested immediately after treatment or harvested after continued culture following removal of the antisense oligonucleotides.

[0181] (Analysis of WRN cDNA) Total RNA extracted from the recovered cells was reverse-transcribed, and the region of the WRN gene spanning exon 26 to exon 29 (when HEK293T cells with a normal WRN gene were used) or the region of the WRN gene spanning exon 25 to exon 29 (when fibroblasts from a Werner syndrome patient with exon 26 deletion were used) was amplified using PCR. The annealing temperature for these amplifications was set at 60°C.

[0182] (Confirmation of exon 27 skipping) When the region from exons 26 to 29 of the WRN cDNA was amplified in HEK293T cells carrying a normal WRN gene, a band of 290 base pairs was obtained without the addition of antisense oligonucleotide. The base sequence of this amplified product was determined by standard methods, and it was shown to consist of exons 26, 27, 28, and 29.

[0183] Similarly, when the region from exon 25 to exon 29 of WRN cDNA was amplified in fibroblasts from a Werner syndrome patient lacking exon 26, a band of 290 base pairs was obtained without the addition of antisense oligonucleotide. The base sequence of this amplified product was determined by standard methods and was shown to consist of exons 25, 27, 28, and 29. This result was in good agreement with the results of genetic analysis of the patient.

[0184] On the other hand, cDNA from HEK293T cells transfected with antisense oligonucleotides not only yielded an amplification product of the same size as that obtained from cells not transfected with antisense oligonucleotides, but also yielded a smaller amplification product after 48 hours of culture. The nucleotide sequence of this amplification product was determined by standard methods and showed that it consisted of exons 26, 28, and 29. Under these conditions, only a smaller transcript, i.e., one with exon 27 skipping, was obtained. cDNA from the above-mentioned Werner syndrome fibroblasts transfected with antisense oligonucleotides yielded an amplification product of a smaller size than that obtained from cells not transfected with antisense oligonucleotides. Sequencing of this smaller amplification product revealed that the sequence of exon 25 was directly connected to the sequence of exon 28, revealing deletion of exons 26 and 27. This indicates that exon 27 skipping was caused by antisense oligonucleotide treatment.

[0185] The present inventors have confirmed that the designed antisense oligonucleotides effectively induce exon 27 skipping in the splicing reaction of pre-mRNA transcribed from a gene with a normal human WRN gene structure. Furthermore, the present inventors have confirmed that the designed antisense oligonucleotides effectively induce exon 27 skipping in Werner syndrome patient fibroblasts carrying the exon 26 skipping mutation c.3139-1G>C (Werner syndrome patient fibroblasts lacking exon 26). The present invention will be described in more detail below based on various tests.

[0186] <<Evaluation of Induction of Exon 27 Skipping of the WRN Gene>> Evaluation of induction of exon 27 skipping of the WRN gene was carried out using human embryonic kidney HEK293T cells and Werner syndrome patient fibroblasts carrying the skipping mutation c.3139-1G>C in exon 26, depending on the single-stranded antisense oligonucleotide produced. Specific procedures are described below.

[0187] <Evaluation of Induction of Exon 27 Skipping Using Human Embryonic Kidney Cells, HEK293T Cells> Human embryonic kidney cells, HEK293T cells (ATCC (registered trademark) CRL-3216 (trademark)) were cultured in a culture medium at 37°C, 5% CO ２ The HEK293T cells were cultured under the following conditions. The culture medium used had the following composition:

[0188] Composition of culture medium for HEK293T cells Dulbecco's modified Eagle's medium (DMEM): Thermo Fisher Scientific, Cat. #11995 10% fetal bovine serum (FBS): Thermo Fisher Scientific, Cat. #10437028

[0189] First, on the day before transfection, HEK293T cells (12,000 cells / well) were seeded in a 96-well plate and incubated at 37°C, 5% CO ２The cells were cultured overnight under the conditions of 0.1% ethanol. Then, each single-stranded antisense oligonucleotide (50 nM) diluted with phosphate-buffered saline (PBS) was transfected into the above cells by lipofection. As a negative control, cells transfected with PBS containing no single-stranded antisense oligonucleotide were used. The transfected cells were incubated in culture medium at 37°C and 5% CO. ２ The cells were cultured for 48 hours under the conditions of

[0100] . The culture medium was then removed, and the extracted total RNA was reverse-transcribed using the Taqman Fast Cells-to-CT Kit (Thermo Fisher Scientific, Cat# 4399003). The complementary DNA (cDNA) obtained from this reverse transcription reaction was used to perform PCR spanning exon 25 to exon 29 of the WRN gene using predesigned specific primers (see below).

[0190] The PCR program used was as follows: 94°C, 1 minute: heat denaturation [98°C, 10 seconds; 60°C, 5 seconds; 68°C, 40 seconds] x 35 cycles: PCR amplification

[0191] The gene-specific primers used were WRN_Exon26_Fw and WRN_Exon29_Rv. WRN_Exon26_Fw: 5'-CTCAGAGCCTCATCCTTCAAGCTAATG-3' WRN_Exon29_Rv: 5'-CAATCTGAGTCTCCTGCTCTTGTGC-3'

[0192] The reaction products of the PCR reaction were separated by 3% agarose gel electrophoresis, and gel photographs were taken using a photography system combining an LED Transilluminator Gel Mieru (Wako) and an iPhone 8 (Apple).

[0193] The results of the experiment for measuring the exon 27 skipping activity of each single-stranded antisense oligonucleotide against human WRN pre-mRNA (FIG. 4), determined by the above-mentioned method, are shown in FIG.

[0194] When the region from exon 26 to exon 29 of WRN cDNA was amplified in HEK293T cells carrying a normal WRN gene, a 291-base pair band was obtained in the sample without antisense oligonucleotide transfection (labeled "No addition" in Figure 5). The base sequence of this amplification product was determined by DNA sequencing, and it was shown to consist of exons 26, 27, 28, and 29.

[0195] On the other hand, when cDNA from HEK293T cells was treated with each antisense oligonucleotide, not only was an amplified product of the same size as that obtained from cells not transfected with antisense oligonucleotides obtained, but after 48 hours of culture, a smaller amplified product (215 base pairs) was also obtained. Sequencing of the nucleotide sequence of this amplified product indicated that it consisted of exons 26, 28, and 29. Furthermore, under these conditions, only a small amplified product was observed, i.e., an antisense oligonucleotide with highly efficient exon 27 skipping activity was also obtained.

[0196] Using ImageJ (NIH, USA), the PCR product "215 bp, A" in which exon 27 was skipped and the PCR product "291 bp, B" in which exon 27 was not skipped were quantified. The skipping efficiency (%) was calculated from the measured values ​​of "A" and "B" using the following formula. The results are shown in Tables 3-1 to 3-3. Skipping efficiency (%) = 100 x A / (A + B)

[0197]

[0198]

[0199]

[0200] <Confirmation of WRN Gene Mutation in Werner Syndrome Patient Fibroblasts> Werner syndrome patient fibroblasts (Coriell Cell Repositories, Cat#AG12795) were cultured in a culture medium at 37°C and 5% CO ２The culture medium for Werner's syndrome patient fibroblasts had the following composition:

[0201] Culture medium composition for Werner syndrome patient fibroblasts Eagle's minimum essential medium (MEM): Sigma, Cat# M5650 15% FBS: Thermo Fisher Scientific, Cat# 10437028

[0202] Genetic mutation analysis was performed on the mutant type of the WRN gene in the Werner syndrome patient fibroblasts. Werner syndrome patient fibroblasts (10,000 cells / well) were seeded on a 96-well plate and incubated at 37°C, 5% CO ２ The cells were cultured overnight under the conditions of 1. Then, genomic DNA was extracted from the cells, and PCR spanning exon 25 to exon 29 of the WRN gene was performed using pre-designed specific primers (see below).

[0203] The PCR program used was as follows: 94°C, 1 minute: heat denaturation [98°C, 10 seconds; 60°C, 5 seconds; 68°C, 40 seconds] x 35 cycles: PCR amplification

[0204] The gene-specific primers used were WRN_Exon25_Fw and WRN_Exon29_Rv. WRN_Exon25_Fw: 5'-CTGGCAAGGATCAAACAGAGAGTTG-3' WRN_Exon29_Rv: 5'-CAATCTGAGTCTCCTGCTCTTGTGC-3'

[0205] The PCR reaction products were separated by 3% agarose gel electrophoresis, and the amplified PCR fragment was detected using an LED transilluminator Gel Mier (Wako Co.), and the gel containing the PCR fragment was excised. The amplified PCR fragment was purified and analyzed by sequencing, confirming that the Werner syndrome patient fibroblasts were homozygous for c.3139-1G>C in the WRN gene.

[0206] <Evaluation of the induction of exon 27 skipping using fibroblasts from Werner syndrome patients> Next, the induction of exon 27 skipping of the WRN gene was evaluated using fibroblasts from Werner syndrome patients who are homozygous for c.3139-1G>C in the WRN gene. On the day before transfection, Werner syndrome patient fibroblasts (10,000 cells / well) were seeded into a 96-well plate and incubated at 37°C and 5% CO. ２ The cells were cultured overnight under the conditions of 0.1% ethanol. Then, antisense oligonucleotides were treated for 48 hours to 10 days using the lipofection method. Each single-stranded antisense oligonucleotide diluted in PBS (final concentration 1-50 nM) was transfected into the above cells using the lipofection method. As a negative control, cells transfected with PBS containing no dissolved single-stranded antisense oligonucleotide were used. The cells treated with antisense oligonucleotides were incubated in culture medium at 37°C and 5% CO ２ The cells were cultured for 48 or 240 hours under these conditions. The culture medium was then removed, and the extracted total RNA was reverse-transcribed using the Taqman Fast Cells-to-CT Kit (Thermo Fisher Scientific, Cat# 4399003). The complementary DNA (cDNA) obtained from this reverse transcription reaction was used to perform PCR spanning exon 25 to exon 29 of the WRN gene using pre-designed specific primers (see below). The PCR program used was as follows:

[0207] 94°C, 1 minute: heat denaturation [98°C, 10 seconds; 60°C, 5 seconds; 68°C, 40 seconds] x 35 cycles: PCR amplification

[0208] The gene-specific primers used were WRN_Exon25_Fw and WRN_Exon29_Rv. WRN_Exon25_Fw: 5'-CTGGCAAGGATCAAACAGAGAGTTG-3' WRN_Exon29_Rv: 5'-CAATCTGAGTCTCCTGCTCTTGTGC-3'

[0209] The reaction products of the PCR reaction were separated by 3% agarose gel electrophoresis, and gel photographs were taken using a photography system combining an LED Transilluminator Gel Mieru (Wako) and an iPhone 8 (Apple).

[0210] The experimental results of measuring the exon 27 skipping activity of each single-stranded antisense oligonucleotide against human WRN pre-mRNA, as determined by the above-described method, are shown in Figure 6. When the region from exon 25 to exon 29 of WRN cDNA was amplified in fibroblasts from a Werner syndrome patient, a 352-base pair band was obtained in the sample without antisense oligonucleotide transfection (labeled "No addition" in Figure 6). The base sequence of this amplification product was determined by sequencing, and it was shown to consist of exons 25, 27, 28, and 29. This result was in good agreement with the results of genetic analysis of the patient.

[0211] On the other hand, the cDNA of Werner syndrome fibroblasts treated with antisense oligonucleotides yielded a smaller product (276 base pairs) than that obtained from cells not transfected with antisense oligonucleotides. Sequencing of this small amplified product revealed that the sequence of exon 25 was directly connected to the sequence of exon 28, revealing a deletion of exons 26 and 27. This indicates that exon 27 was skipped by antisense oligonucleotide treatment.

[0212] Using ImageJ (NIH, USA), the PCR product "276 bp, A" in which exon 27 was skipped and the PCR product "352 bp, B" in which exon 27 was not skipped were quantified. The skipping efficiency (%) was calculated from the measured values ​​of "A" and "B" using the following formula. The results are shown in Tables 4-1A to 4-1F, 4-2A to 4-2D, 4-3A to 4-3D, 4-4A to 4-4D, and 4-5. Skipping efficiency (%) = 100 x A / (A + B)

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232] <Induction of functional WRN protein expression by antisense oligonucleotides in Werner syndrome patient fibroblasts> We evaluated the induction of functional WRN protein expression by exon 27 skipping of the WRN gene using Werner syndrome patient fibroblasts homozygous for c.3139-1G>C in the WRN gene. On the day before the evaluation, Werner syndrome patient fibroblasts (10,000 cells / well) were seeded into a 96-well plate and incubated at 37°C, 5% CO. ２The cells were cultured overnight under the conditions of 0.1% ethanol. Thereafter, a single-stranded antisense oligonucleotide consisting of the base sequence of SEQ ID NO: 28 diluted with PBS (final concentration 50 nM) was transfected into the above cells by lipofection. As a negative control, cells transfected with PBS containing no dissolved single-stranded antisense oligonucleotide were used. The transfected cells were incubated in culture medium at 37°C and 5% CO ２ The cells were cultured for 48 hours under these conditions. The cells were then fixed with 4% paraformaldehyde (Fujifilm Wako Pure Chemical Industries, Ltd., Cat. #163-20145) for 15 minutes and incubated with 0.5% Triton-X (Nacalai Tesque, Cat. #12967-45) for 10 minutes. The incubated cells were then blocked with PBS containing 5% goat serum for 1 hour and incubated overnight in an anti-WRN antibody (Cell Signaling, Cat. #4666). The cells were then further incubated for 1 hour in an anti-mouse IgG antibody (Thermo Fisher Scientific, A32766) solution. Finally, the sections were incubated in Hoechst (registered trademark) 33342 nucleic acid staining solution (Thermo Fisher Scientific, Cat# H3570) for 15 minutes and observed under a fluorescence microscope.

[0233] The results of induction of functional WRN protein expression by exon 27 skipping activity of the WRN gene by a single-stranded antisense oligonucleotide against human WRN pre-mRNA, as determined by the above-mentioned method, are shown in Figure 7. Figure 7 demonstrates that the functional WRN protein was translocated into the nucleus in cells transfected with a single-stranded antisense oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 28 ("ASO: No. 28" in Figure 7).

[0234] <Promotion of cell proliferation by antisense oligonucleotides in Werner syndrome patient fibroblasts> We evaluated the cell proliferation potential of exon 27 skipping of the WRN gene using Werner syndrome patient fibroblasts homozygous for c.3139-1G>C in the human WRN gene. On the day before the evaluation, Werner syndrome patient fibroblasts (12,000 cells / well) were seeded into a 96-well plate and incubated at 37°C, 5% CO ２ The cells were cultured overnight under the conditions of 0.1% ssDNA and 0.1% ssDNA. Then, each single-stranded antisense oligonucleotide diluted with PBS (final concentration 10 nM) was transfected into the above cells by lipofection. As a negative control, cells transfected with PBS containing no single-stranded antisense oligonucleotide were used. The transfected cells were incubated in culture medium at 37°C and 5% CO. ２ The cells were cultured for 288 hours under the conditions of . Thereafter, Celltiter-Glo 2.0 Assay (Promega, Cat# G9242) was added to the growth medium and the cell number was measured. The cell proliferation rate (%) was calculated using the following formula from the measured cell number "A" of the cells transfected with the single-stranded antisense oligonucleotide and the cell number "B" of the negative control group. The results are shown in Table 5. Cell proliferation rate (%) = 100 x (A) / B

[0235]

[0236] <Evaluation of Cytotoxicity of Single-Stranded Antisense Oligonucleotides> HepG2 cells, a human hepatoma-derived cell line, were cultured in growth medium at 37°C and 5% CO. ２ The growth medium used was one with the following composition:

[0237] Composition of growth medium used for cytotoxicity evaluation 10% FBS: GIBCO, CAT# 10270-106 Minimum Essential Medium (containing L-glutamine and phenol red) (GIBCO, CAT# 11095-080)

[0238] The day before the experiment, the above cells (1.5 × 10 ４The seeded cells were incubated at 37°C, 5% CO ２ After overnight culture under the conditions of

[0045] , each single-stranded antisense oligonucleotide diluted with PBS (final concentration: 3 to 30 nM) was transfected into the above cells by lipofection. After addition, the cells were incubated at 37°C, 5% CO ２ The cells were cultured for 24 hours under the above conditions. As a negative control, cells transfected with PBS containing no single-stranded antisense oligonucleotide were used. Caspase-Glo 3 / 7 Assay System (Promega, Cat# G8093) was then added to the growth medium to evaluate caspase activity. The caspase activity of each single-stranded antisense oligonucleotide determined by the above method is shown in Table 6.

[0239]

[0240] Although the embodiments and examples of the present invention have been described above, it is also planned from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined.

[0241] The embodiments and examples disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the present invention is defined by the claims, not by the embodiments and examples described above, and is intended to include any modifications within the scope of the claims and meanings equivalent to the claims.

Claims

1. An oligonucleotide or a pharmaceutically acceptable salt thereof that expresses a functional human WRN protein against a skip mutation in exon 26 or exon 28 of the human WRN gene, wherein each nucleotide of the oligonucleotide is linked by a phosphate group and / or a modified phosphate group, the oligonucleotide includes a modified nucleic acid having at least one modified sugar, the base length of the oligonucleotide is 10 to 30 mer, the base sequence of the oligonucleotide is a base sequence having a sequence identity of 90% or more and 100% or less, based on a base sequence complementary to at least one target region having the same base length as the oligonucleotide in the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, a base sequence complementary to a base sequence in which one or several bases are deleted, substituted, inserted, or added in the target region, or a base sequence that hybridizes under stringent conditions to the oligonucleotide having the target region, is the oligonucleotide or a pharmaceutically acceptable salt thereof.

2. The base sequence of the oligonucleotide is a base sequence having a sequence identity of 95% or more and 100% or less, based on a base sequence complementary to at least one target region having the same base length as the oligonucleotide in the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, is the oligonucleotide or a pharmaceutically acceptable salt thereof according to Claim 1.

3. The base sequence of the oligonucleotide is a base sequence complementary to at least one target region having the same base length as the oligonucleotide in the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, is the oligonucleotide or a pharmaceutically acceptable salt thereof according to Claim 1.

4. The base length of the oligonucleotide is 15 to 25 mer, is the oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of Claims 1 to 3.

5. The sugar constituting the oligonucleotide is D-ribofuranose, and the modification of the sugar is a sugar modification of the hydroxyl group at the 2'-position of the D-ribofuranose. The oligonucleotide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

6. The modification of the sugar constituting the oligonucleotide is a group represented by the following formula (A1). The oligonucleotide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof. 【Chemical 1】 (In the formula, Bx is a nucleobase, each independently a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine or uracil, X is a phosphate bond, each independently a phosphodiester bond, phosphorothioate bond, phosphorodithioate bond, phosphoroamidate bond, boranophosphate bond or alkylphosphonate bond, Y is each independently a hydrogen atom, a hydroxyl group, a fluorine atom, an optionally substituted alkoxy group having 1 to 6 carbon atoms, or an optionally substituted amino group, and Z is each independently a hydrogen atom, a carbon-oxygen double bond or an optionally cyclic alkyl group having 1 to 5 carbon atoms, or a part of the carbon atoms of the alkyl group and Y together form a ring.)

7. The modification of the sugar constituting the oligonucleotide is a group represented by the following formula (A2) or formula (A3). The oligonucleotide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof. [Chemical Formula 2] (In the formula, Bx is a nucleobase, each independently being a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine or uracil, X is a phosphate bond, each independently being a phosphodiester bond, phosphorothioate bond, phosphorodithioate bond, phosphoroamidate bond, boranophosphate bond or alkylphosphonate bond, R ４ is each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, Y' is an oxygen atom or an optionally substituted nitrogen atom, R ５ and R ６ are each independently a hydrogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, or R ５ and R ６ together form a carbonyl group or a ring, and n is 0 or 1.)

8. The modification of the sugar constituting the oligonucleotide is a group represented by the formula (A2), and the R ４ is, independently of each other, a methyl group, a methoxyethyl group, or an N-methylpropanamide group, the oligonucleotide according to claim 7, or a pharmaceutically acceptable salt thereof.

9. The modification of the sugar constituting the oligonucleotide is a group represented by the formula (A3), Y' is an oxygen atom, or a nitrogen atom optionally substituted with a hydrogen atom, a methyl group, a methoxyethyl group, or an N-methylpropanamide group, and R ５ and R ６ are each independently a hydrogen atom, an alkyl group having 1 to 2 carbon atoms, or the R ５ and R ６ together form a carbonyl group or a ring having 3 to 6 carbon atoms, and n is 0 or 1. The oligonucleotide according to claim 7 or a pharmaceutically acceptable salt thereof.

10. At least one nucleotide bond of the oligonucleotide is a phosphorothioate bond. The oligonucleotide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

11. At least one nucleotide bond of the oligonucleotide is a phosphodiester bond. The oligonucleotide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

12. The nucleotide bond of the oligonucleotide is a phosphodiester bond or a phosphorothioate bond. The oligonucleotide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

13. The base sequence of the oligonucleotide is The oligonucleotide is composed of morpholino nucleic acids represented by the following formula (A4), and has a base sequence complementary to at least one target region having the same base length as the oligonucleotide in the base sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO:

6. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3. 【Chemical Formula 3】 (In the formula, Bx is a nucleobase, and each is independently a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine or uracil. X' is a phosphate bond, and each is independently a phosphorodiamidate bond, a phosphorodiamidothioate bond, or a phosphorodiamidodithioate bond.)

14. The base sequence of the oligonucleotide is Based on a base sequence having a sequence identity of 90% or more and 100% or less, with respect to a target region composed of 15 to 25 mers consecutive from the bases located at positions 1 to 40, 46, 51, 56, or 62 to 63 counted from the 5'-end in the base sequence set forth in SEQ ID NO: 1, or a target region composed of 15 to 25 mers consecutive from the bases located at positions 108873, 108878 to 108917, 108923, 108928, 108933, or 108939 to 108940 counted from the 5'-end in the base sequence set forth in SEQ ID NO:

2. A base sequence complementary to a base sequence in which one or several bases are deleted, substituted, inserted or added in the target region, or A base sequence that hybridizes under stringent conditions to the oligonucleotide having the target region. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3.

15. The base sequence of the oligonucleotide is a base sequence having a sequence identity of 90% or more and 100% or less, based on a base sequence complementary to a target region composed of 15 to 25 mers consecutive from bases located at positions 1 to 32, 34 to 40, 46, 51, or 62 to 63, counting from the 5'-end in the base sequence set forth in SEQ ID NO: 1, or a target region composed of 15 to 25 mers consecutive from bases located at positions 108878 to 108909, 108911 to 108917, 108923, 108928, or 108939 to 108940, counting from the 5'-end in the base sequence set forth in SEQ ID NO:

2. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3.

16. The base sequence of the oligonucleotide is a base sequence having a sequence identity of 90% or more and 100% or less, based on a base sequence complementary to a target region composed of 15 to 25 mers consecutive from bases located at positions 1, 3, 5 to 12, 14 to 19, 21, 25, 29, 30, 31, 34, 46, or 51, counting from the 5'-end in the base sequence set forth in SEQ ID NO: 1, or a target region composed of 15 to 25 mers consecutive from bases located at positions 108878, 108880, 108882 to 108889, 108891 to 108896, 108898, 108902, 108906 to 108908, 108911, 108923, or 108928, counting from the 5'-end in the base sequence set forth in SEQ ID NO:

2. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3.

17. The base sequence of the oligonucleotide is one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 9 to 14, 16 to 26, 28 to 39, 41 to 42, 44 to 50, 52, 55 to 56, 69, 73, 80 to 105, 107 to 108, 110, 112, 114, 116, 118 to 131, 133, and 135. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3.

18. The oligonucleotide is one oligonucleotide selected from the group consisting of SEQ ID NOs: 6-20-A, 8-20-A, 8-20-B, 8-20-C, 9-20-A, 10-20-A, 12-20-A, 14-20-A, 16-20-A, 17-20-A, 18-20-A, 19-20-A, 15-25-A, 10-17-A, 10-20-B, 12-20-B, 14-20-B, 15-20-B, 16-20-B, 19-20-B, 31-20-B, 34-20-B, 6-20-B, 7-20-B, 8-20-D, and 9-20-B shown in Table 2-1 to Table 2-7, the oligonucleotide according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof.

19. The oligonucleotide is a single-stranded antisense oligonucleotide, the oligonucleotide according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof.

20. The oligonucleotide according to claim 19, a second-strand oligonucleotide hybridizing to the single-stranded antisense oligonucleotide, a double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the base sequence of the second-strand oligonucleotide has a sequence identity of 90% or more and 100% or less based on a base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide, a double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof.

21. The oligonucleotide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, an additional substance directly or via a linker bond bound to the oligonucleotide, an oligonucleotide complex or a pharmaceutically acceptable salt thereof, wherein the additional substance is selected from the group consisting of polyethylene glycol, peptide, alkyl chain, ligand compound, antibody, protein, and sugar chain, wherein the linker bond is independently a phosphodiester bond, phosphorothioate bond, phosphorodithioate bond, phosphoramidate bond, boranophosphate bond, alkylphosphonate bond, phosphorodiamidate bond, phosphorodiamidothioate bond, or phosphorodiamidodithioate bond, an oligonucleotide complex or a pharmaceutically acceptable salt thereof.

22. A double-stranded antisense oligonucleotide according to Claim 20 or a pharmaceutically acceptable salt thereof, and an additional substance directly or via a linker bond bonded to the oligonucleotide or the second-strand oligonucleotide, which is an oligonucleotide complex or a pharmaceutically acceptable salt thereof, wherein the additional substance is selected from the group consisting of polyethylene glycol, peptide, alkyl chain, ligand compound, antibody, protein, and sugar chain, wherein the linker bonds are each independently a phosphodiester bond, phosphorothioate bond, phosphorodithioate bond, phosphoroamidate bond, boranophosphate bond, alkylphosphonate bond, phosphorodiamidate bond, phosphorodiamidothioate bond, or phosphorodiamidodithioate bond, which is an oligonucleotide complex or a pharmaceutically acceptable salt thereof.

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