ANTISENSE NUCLEIC ACID ENABLING EXON SKIPPING

TECHNICAL FIELD

The present invention relates to an antisense oligomer capable of causing simultaneous skipping of a plurality of exons in a target gene, and a pharmaceutical composition comprising the oligomer.

BACKGROUND ART

In recent years, exon skipping therapy has received attention which involves causing exon skipping of a gene having a mutation that causes a disease so that a protein having partial functions arises, thereby treating the disease. Examples of the disease that may be treated by such exon skipping therapy include Duchenne muscular dystrophy (DMD).

DMD is the most frequent form of hereditary progressive muscular disease that affects one in about 3,500 newborn boys. Although DMD patients exhibit motor functions rarely different from healthy humans in their infancy and childhood, muscle weakness is observed in children from around 4 to 5 years old. Then, muscle weakness in DMD patients progresses with age to the loss of ambulation by about 12 years old and death due to cardiac or respiratory insufficiency in their twenties. Therefore, it has been strongly desired to develop an effective therapeutic agent.

DMD is known to be caused by a mutation in the dystrophin gene. The dystrophin gene is located on X chromosome and is a huge gene consisting of 2.2 million DNA base pairs. DNA is transcribed into pre-mRNA, and introns are removed by splicing to form mRNA of 13,993 bases in which 79 exons are joined together. This mRNA is translated into 3,685 amino acids to produce the dystrophin protein. The dystrophin protein is associated with the maintenance of membrane stability in muscle cells and necessary to make muscle cells less fragile. Patients with DMD have a mutation in the dystrophin gene and hence, the functional dystrophin protein is rarely expressed in muscle cells of the patients. Therefore, the structure of muscle cells cannot be maintained at the time of muscle contraction in the body of the patients with DMD, leading to a large influx of calcium ions into muscle cells. Consequently, muscle cell necrosis and fibrosis progress so that muscle cells become progressively more difficult to regenerate.

Becker muscular dystrophy (BMD) is also caused by a mutation in the dystrophin gene. The symptoms involve muscle weakness but are typically mild and slow in the progress of muscle weakness, when compared to DMD. In many cases, its onset is in adulthood. Differences in clinical symptoms between DMD and BMD are considered to reside in whether the reading frame for amino acids on the translation of dystrophin mRNA into the dystrophin protein is disrupted by the mutation or not (Non Patent Literature 1). More specifically, in DMD, the presence of mutation shifts the amino acid reading frame so that the expression of functional dystrophin protein is abolished, whereas in BMD the dystrophin protein that is capable of functioning, though imperfectly, is produced because the amino acid reading frame is preserved, while part of the exons are deleted by the mutation.

Exon skipping is expected to serve as a method for treating DMD. This method involves modifying splicing to restore the amino acid reading frame of dystrophin mRNA and induce expression of the dystrophin protein having the function partially restored (Non Patent Literature 2). The amino acid sequence part to be translated from an exon, which is a target for exon skipping, will be lost. For this reason, the dystrophin protein expressed by this treatment becomes shorter than normal one but since the amino acid reading frame is maintained, the function to stabilize muscle cells is partially retained. Consequently, it is expected that exon skipping will lead DMD to the similar symptoms to that of BMD which is milder. The exon skipping approach has passed the animal tests using mice or dogs and now is currently assessed in clinical trials on human DMD patients.

The skipping of an exon can be induced by binding of antisense nucleic acids targeting site(s) surrounding either 5′ or 3′ splice site or both sites, or exon-internal sites. An exon will only be included in the mRNA when both splice sites thereof are recognized by the spliceosome complex. Thus, exon skipping can be induced by targeting the sites surrounding the splice sites with antisense nucleic acids. Furthermore, the binding of an SR protein rich in serine and arginine to an exonic splicing enhancer (ESE) is considered necessary for an exon to be recognized by the splicing mechanism. Accordingly, exon skipping can also be induced by targeting ESE.

Since a mutation of the dystrophin gene may vary depending on DMD patients, antisense nucleic acids need to be designed based on the site or type of respective genetic mutation. There is a plurality of reports on an antisense nucleic acid that induces exon skipping targeting one sequence of consecutive bases for a single exon in the dystrophin gene (Patent Literatures 1 to 6 and Non Patent Literatures 1 and 2). It has also been reported that when two types of antisense nucleic acids that target the same exon in the dystrophin gene are mixed and allowed to act (dual targeting), skipping activity may be enhanced as compared to use of each antisense nucleic acid alone (Patent Literature 7).

A method called multi-exon skipping has received attention which involves causing skipping of a plurality of exons (exon group), not one exon as described above. This method enables a wide range of mutations in the dystrophin gene to be treated by exon skipping. For example, exons 45 to 55 in the dystrophin gene are known as hot spots of genetic mutation, and it has been reported that skipping of these 11 exons enables about 60% of DMD patients having a deletion mutation to be treated (Non Patent Literature 3). Most of patients congenitally lacking exons 45 to 55 are known to manifest no or mild symptoms, though developing BMD (Non Patent Literature 4). Thus, it is expected that drugs capable of inducing exon 45 to 55 skipping are promising as therapeutic agents for DMD.

For example, a method using antisense nucleic acids respectively targeting all exons in a region which is the target of exon skipping (Non Patent Literatures 5, 7, 8, and 10), and a method using antisense nucleic acids respectively targeting two different exons on the 3′ side and 5′ side of a region which is the target of exon skipping (Non Patent Literatures 6 and 9 and Patent Literature 8) have been reported as methods for inducing multi-exon skipping.

However, there is still no report stating that an antisense nucleic acid targeting a region including the vicinity of a donor of an intron or the vicinity of an acceptor induces multi-exon skipping.

CITATION LIST
Patent Literature

Patent Literature 1: International Publication WO2004/048570

Patent Literature 2: International Publication WO2009/139630

Patent Literature 3: International Publication WO2010/048586

Patent Literature 4: U.S. Patent Publication Nos. 2010/0168212

Patent Literature 5: International Publication WO2011/057350

Patent Literature 6: International Publication WO2006/000057

Patent Literature 7: International Publication WO2007/135105

Patent Literature 8: International Publication WO2004/083446

Non Patent Literature

Non Patent Literature 1: Annemieke Aartsma-Rus et al., (2002) Neuromuscular Disorders 12: S71-S77

Non Patent Literature 2: Wilton S. D., e t al., Molecular Therapy 2007: 15: p. 1288-96

Non Patent Literature 3: Christophe Beroud et al., Human Mutation, 28(2), 2007, 196-202

Non Patent Literature 4: Yusuke Echigoya et al., Molecular Therapy-Nucleic Acids, 4(2), 2015, e225

Non Patent Literature 5: Yoshitsugu Aoki et al., PNAS, 109(34), 2012, 13763-13768

Non Patent Literature 6: Laura van Vliet et al., BMC Medical Genetics, 9, 105, 2008

Non Patent Literature 7: Joshua Lee et al., PLoS ONE, 13(5), e0197084, 2018

Non Patent Literature 8: Joshua Lee et al., Methods in Molecular Biology, 1828, 141-150, 2018

Non Patent Literature 9: Annemieke Aartsma-Rus et al, Am. J. Hum. Genet. 74(1), 83-92, 2004

Non Patent Literature 10: Yusuke Echigoya et al., Molecular Therapy, 27(11), 1-13, 2019

SUMMARY OF INVENTION
Technical Problem

Under the foregoing circumstances, medicaments for treating patients having various mutations by causing simultaneous skipping of a plurality of exons (exon group) in pre-mRNA of interest have been desired.

Solution to Problem

As a result of detailed studies of the technical contents of the above documents and the structure of the dystrophin gene, the present inventors have found that an antisense nucleic acid targeting a specific region in human dystrophin pre-mRNA is capable of causing simultaneous skipping of a plurality of exons among exons 45 to 55. Based on this finding, the present inventors have accomplished the present invention.

Specifically, the present invention is as follows.

[1]

An antisense oligomer or a pharmaceutically acceptable salt thereof, or hydrate thereof which causes simultaneous skipping of any two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA,

the antisense oligomer comprising a base sequence complementary to a base sequence of at least one region selected from the group consisting of regions R1 to R24 represented by

region Rn (wherein n is an odd number of 1 to 23) which consists of a base sequence of NX bases in the upstream direction from the 3′ end of the NAth exon and a base sequence of NY bases in the downstream direction from the 5′ end of the NBth intron in the human dystrophin pre-mRNA, and

region Rn (wherein n is an even number of 2 to 24) which consists of a base sequence of NX bases in the upstream direction from the 3′ end of the NAth intron and a base sequence of NY bases in the downstream direction from the 5′ end of the NBth exon in the human dystrophin pre-mRNA,

or a partial base sequence thereof, wherein

when n is 1, NA=44, NB=44, NX=20, and NY=400,

when n is 2, NA=44, NB=45, NX=600, and NY=50,

when n is 3, NA=45, NB=45, NX=20, and NY=400,

when n is 4, NA=45, NB=46, NX=400, and NY=50,

when n is 5, NA=46, NB=46, NX=20, and NY=400,

when n is 6, NA=46, NB=47, NX=400, and NY=50,

when n is 7, NA=47, NB=47, NX=20, and NY=400,

when n is 8, NA=47, NB=48, NX=400, and NY=50,

when n is 9, NA=48, NB=48, NX=20, and NY=400,

when n is 10, NA=48, NB=49, NX=400, and NY=50,

when n is 11, NA=49, NB=49, NX=20, and NY=400,

when n is 12, NA=49, NB=50, NX=400, and NY=50,

when n is 13, NA=50, NB=50, NX=20, and NY=400,

when n is 14, NA=50, NB=51, NX=400, and NY=50,

when n is 15, NA=51, NB=51, NX=20, and NY=400,

when n is 16, NA=51, NB=52, NX=400, and NY=50,

when n is 17, NA=52, NB=52, NX=20, and NY=400,

when n is 18, NA=52, NB=53, NX=400, and NY=50,

when n is 19, NA=53, NB=53, NX=20, and NY=400,

when n is 20, NA=53, NB=54, NX=400, and NY=50,

when n is 21, NA=54, NB=54, NX=20, and NY=400,

when n is 22, NA=54, NB=55, NX=400, and NY=50,

when n is 23, NA=55, NB=55, NX=20, and NY=400, or

when n is 24, NA=55, NB=56, NX=400, and NY=50.

[2]

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to [1], wherein

the region R1 is a region that consists of a base sequence of 20 bases in the upstream direction from the 3′ end of the 44th exon and a base sequence of 400 bases in the downstream direction from the 5′ end of the 44th intron in the human dystrophin pre-mRNA,

the region R2 is a region that consists of a base sequence of 600 bases in the upstream direction from the 3′ end of the 44th intron and a base sequence of 50 bases in the downstream direction from the 5′ end of the 45th exon in the human dystrophin pre-mRNA,

the region R3 is a region that consists of a base sequence of 20 bases in the upstream direction from the 3′ end of the 45th exon and a base sequence of 400 bases in the downstream direction from the 5′ end of the 45th intron in the human dystrophin pre-mRNA,

the region R4 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 45th intron and a base sequence of 50 bases in the downstream direction from the 5′ end of the 46th exon in the human dystrophin pre-mRNA,

the region R5 is a region that consists of a base sequence of 20 bases in the upstream direction from the 3′ end of the 46th exon and a base sequence of 400 bases in the downstream direction from the 5′ end of the 46th intron in the human dystrophin pre-mRNA,

the region R6 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 46th intron and a base sequence of 50 bases in the downstream direction from the 5′ end of the 47th exon in the human dystrophin pre-mRNA,

the region R7 is a region that consists of a base sequence of 20 bases in the upstream direction from the 3′ end of the 47th exon and a base sequence of 400 bases in the downstream direction from the 5′ end of the 47th intron in the human dystrophin pre-mRNA,

the region R8 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 47th intron and a base sequence of 50 bases in the downstream direction from the 5′ end of the 48th exon in the human dystrophin pre-mRNA,

the region R9 is a region that consists of a base sequence of 20 bases in the upstream direction from the 3′ end of the 48th exon and a base sequence of 400 bases in the downstream direction from the 5′ end of the 48th intron in the human dystrophin pre-mRNA,

the region R10 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 48th intron and a base sequence of 50 bases in the downstream direction from the 5′ end of the 49th exon in the human dystrophin pre-mRNA,

the region R11 is a region that consists of a base sequence of 20 bases in the upstream direction from the 3′ end of the 49th exon and a base sequence of 400 bases in the downstream direction from the 5′ end of the 49th intron in the human dystrophin pre-mRNA,

the region R12 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 49th intron and a base sequence of 50 bases in the downstream direction from the 5′ end of the 50th exon in the human dystrophin pre-mRNA,

the region R13 is a region that consists of a base sequence of 20 bases in the upstream direction from the 3′ end of the 50th exon and a base sequence of 400 bases in the downstream direction from the 5′ end of the 50th intron in the human dystrophin pre-mRNA,

the region R14 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 50th intron and a base sequence of 50 bases in the downstream direction from the 5′ end of the 51st exon in the human dystrophin pre-mRNA,

the region R15 is a region that consists of a base sequence of 20 bases in the upstream direction from the 3′ end of the 51st exon and a base sequence of 400 bases in the downstream direction from the 5′ end of the 51st intron in the human dystrophin pre-mRNA,

the region R16 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 51st intron and a base sequence of 50 bases in the downstream direction from the 5′ end of the 52nd exon in the human dystrophin pre-mRNA,

the region R17 is a region that consists of a base sequence of 20 bases in the upstream direction from the 3′ end of the 52nd exon and a base sequence of 400 bases in the downstream direction from the 5′ end of the 52nd intron in the human dystrophin pre-mRNA,

the region R18 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 52nd intron and a base sequence of 50 bases in the downstream direction from the 5′ end of the 53rd exon in the human dystrophin pre-mRNA,

the region R19 is a region that consists of a base sequence of 20 bases in the upstream direction from the 3′ end of the 53rd exon and a base sequence of 400 bases in the downstream direction from the 5′ end of the 53rd intron in the human dystrophin pre-mRNA,

the region R20 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 53rd intron and a base sequence of 50 bases in the downstream direction from the 5′ end of the 54th exon in the human dystrophin pre-mRNA,

the region R21 is a region that consists of a base sequence of 20 bases in the upstream direction from the 3′ end of the 54th exon and a base sequence of 400 bases in the downstream direction from the 5′ end of the 54th intron in the human dystrophin pre-mRNA,

the region R22 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 54th intron and a base sequence of 50 bases in the downstream direction from the 5′ end of the 55th exon in the human dystrophin pre-mRNA,

the region R23 is a region that consists of a base sequence of 20 bases in the upstream direction from the 3′ end of the 55th exon and a base sequence of 400 bases in the downstream direction from the 5′ end of the 55th intron in the human dystrophin pre-mRNA, or

the region R24 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 55th intron and a base sequence of 50 bases in the downstream direction from the 5′ end of the 56th exon in the human dystrophin pre-mRNA.

[3]

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to [1] or [2], wherein

the antisense oligomer comprises a base sequence complementary to

(a) any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389,

(b) a base sequence that hybridizes under stringent conditions to a base sequence complementary to any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389,

(c) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±15% of the length of the any one base sequence selected, or

(d) a partial base sequence of any one base sequence selected from the group consisting of the base sequences (a), (b), and (c).

[4]

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [1] to [3], wherein

the antisense oligomer is an antisense oligomer comprising two or more unit oligomers linked to each other, wherein

each of the unit oligomers comprises a base sequence complementary to a base sequence of any one region selected from the group consisting of the regions R1 to R24, or a partial base sequence thereof, and the respective base sequences of the unit oligomers are neither consecutive nor overlapped with each other.

[5]

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [1] to [3], wherein

the antisense oligomer is an antisense oligomer comprising two or more unit oligomers linked to each other, wherein

each of the unit oligomers comprises a base sequence complementary to

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to [4] or [5], wherein each of the unit oligomers comprises a base sequence complementary to a consecutive base sequence of 5- to 20-base length in the region.

[7]

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [1] to [3], wherein the antisense oligomer consists of

(1) any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, or

(2) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, and has a length within ±15% of the length of the any one base sequence selected.

[8]

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [1] to [3] and [7], wherein the antisense oligomer consists of any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232.

[9]

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [1] to [3], [7] and [8], wherein the antisense oligomer consists of any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228.

[10]

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [1] to [9], wherein the antisense oligomer is an oligonucleotide.

[11]

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to [10], wherein the sugar moiety and/or the phosphate bond moiety of at least one nucleotide constituting the oligonucleotide is modified.

[12]

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to [10] or [11], wherein the sugar moiety of at least one nucleotide constituting the oligonucleotide is a ribose in which the 2′-OH group is replaced by any one selected from the group consisting of —OR, —R, —R′OR, —SH, —SR, —NH₂, —NHR, —NR₂, —N₃, —CN, —F, —Cl, —Br, and —I (wherein R is an alkyl or an aryl and R′ is an alkylene).

[13]

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [10] to [12], wherein the phosphate bond moiety of at least one nucleotide constituting the oligonucleotide is any one selected from the group consisting of a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoramidate bond and a boranophosphate bond.

[14]

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [1] to [9], wherein the antisense oligomer is a morpholino oligomer.

[15]

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to [14], wherein the antisense oligomer is a phosphorodiamidate morpholino oligomer.

[16]

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to [14] or [15], wherein the 5′ end is any one of chemical formulae (1) to (3) below:

embedded image

[17]

A suppressor antisense oligomer or a pharmaceutically acceptable salt thereof, or hydrate thereof which suppresses single skipping of any one exon selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA,

the suppressor antisense oligomer comprising a base sequence complementary to

(a) any one base sequence of a base sequence selected from the group consisting of SEQ ID NOs: 370 to 384,

(b) a base sequence that has at least 85% identity with any one base sequence of a base sequence selected from the group consisting of SEQ ID NOs: 370 to 384, and has a length within ±15% of the length of the any one base sequence selected, or

(c) a partial base sequence of the base sequence (a) or (b).

[18]

The suppressor antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to [17], wherein the suppressor antisense oligomer consists of

(1) any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, or (2) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, and has a length within ±15% of the length of the any one base sequence selected.

[19]

The suppressor antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to [17] or [18], wherein the suppressor antisense oligomer consists of any one base sequence selected from the group consisting of SEQ ID NOs: 260, 261, and 263.

[20]

The suppressor antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [17] to [19], wherein the suppressor antisense oligomer is an oligonucleotide.

[21]

The suppressor antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to [20], wherein the sugar moiety and/or the phosphate bond moiety of at least one nucleotide constituting the oligonucleotide is modified.

[22]

The suppressor antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to [20] or [21], wherein the sugar moiety of at least one nucleotide constituting the oligonucleotide is a ribose in which the 2′-OH group is replaced by any one selected from the group consisting of —OR, —R, —R′OR, —SH, —SR, —NH₂, —NHR, —NR₂, —N₃, —CN, —F, —Cl, —Br, and —I

(wherein R is an alkyl or an aryl and R′ is an alkylene).

[23]

The suppressor antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [20] to [22], wherein the phosphate bond moiety of at least one nucleotide constituting the oligonucleotide is any one selected from the group consisting of a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoramidate bond and a boranophosphate bond.

[24]

[25]

The suppressor antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to [24], wherein the suppressor antisense oligomer is a phosphorodiamidate morpholino oligomer.

[26]

The suppressor antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to [24] or [25], wherein the 5′ end is any one of chemical formulae (1) to (3) below:

embedded image

[27]

A pharmaceutical composition comprising the antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [1] to [16].

[28]

The pharmaceutical composition according to [27], further comprising the suppressor antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [17] to [26].

[29]

A pharmaceutical composition comprising the antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [1] to [16], and the suppressor antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [17] to [26].

[30]

The pharmaceutical composition according to [28] or [29], wherein

(1) the antisense oligomer is an oligomer consisting of SEQ ID NO: 75, and the suppressor antisense oligomer is an oligomer consisting of SEQ ID NO: 260,

(2) the antisense oligomer is an oligomer consisting of SEQ ID NO: 75, and the suppressor antisense oligomer is an oligomer consisting of SEQ ID NO: 261, or

(3) the antisense oligomer is an oligomer consisting of SEQ ID NO: 75, and the suppressor antisense oligomer is an oligomer consisting of SEQ ID NO: 263.

[31]

The pharmaceutical composition according to any one of [27] to [30], further comprising a pharmaceutically acceptable carrier.

[32]

The pharmaceutical composition according to any one of [27] to [31] for treatment of muscular dystrophy.

[33]

The pharmaceutical composition according to any one of [27] to [32] for being administered to a human patient.

[34]

A method for treatment of muscular dystrophy, which comprises administering to a patient with muscular dystrophy the antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [1] to [16], or the pharmaceutical composition according to any one of [27] to [33].

[35]

The method for treatment according to [34], wherein the patient with muscular dystrophy is a patient with a mutation that is amenable to exon 45 to 55 skipping in the dystrophin gene.

[36]

The method for treatment according to [34] or [35], wherein the patient is a human.

[37]

Use of the antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [1] to [16], or the pharmaceutical composition according to any one of [27] to [33] in manufacturing of a medicament for the treatment of muscular dystrophy.

[38]

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [1] to [16], or the pharmaceutical composition according to any one of [27] to [33] for use in the treatment of muscular dystrophy.

[39]

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof, or the pharmaceutical composition according to [38], wherein the treatment involves performing skipping of any two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA.

[40]

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof, or the pharmaceutical composition according to [38] or [39], wherein in the treatment, a patient with muscular dystrophy is a human.

[41]

A method for enhancing the efficiency of skipping of two or more numerically consecutive exons, which comprises

inhibiting a splicing silencer sequence, a splice site sequence, or a branch site sequence of pre-mRNA of interest when the two or more numerically consecutive exons are skipped from the pre-mRNA of interest.

[42]

The method according to [41], wherein the splicing silencer sequence is a recognition sequence of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1).

[43]

The method according to [41] or [42], wherein the pre-mRNA of interest is human dystrophin pre-mRNA.

[44]

The method according to any one of [41] to [43], wherein the two or more numerically consecutive exons are selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA.

[45]

The method according to [44], wherein the skipping of the two or more numerically consecutive exons of the pre-mRNA of interest is performed using the antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [1] to [16].

[46]

The method according to [44] or [45], wherein specific inhibition of the splicing silencer sequence, the splice site sequence, or the branch site sequence is performed using the suppressor antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to any one of [17] to [26].

The present invention may also include the following embodiments.

[47]

An antisense oligomer or a pharmaceutically acceptable salt thereof, or hydrate thereof which causes simultaneous skipping of two or more numerically consecutive exons from pre-mRNA of interest,

the antisense oligomer comprising a base sequence complementary to a base sequence of any one region selected from the group consisting of

(1) a region that is constituted by a base sequence of 20 bases in the upstream direction from the 3′ end of any exon and a base sequence of 400 bases in the downstream direction from the 5′ end of an intron which is adjacent to the 3′ end of the exon in the pre-mRNA of interest, or

(2) a region that is constituted by a base sequence of 400 bases or 600 bases in the upstream direction from the 3′ end of any intron and a base sequence of 50 bases in the downstream direction from the 5′ end of an exon which is adjacent to the 3′ end of the intron in the pre-mRNA of interest,

or a partial base sequence thereof.

[48]

The antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof according to [47], wherein the pre-mRNA of interest is human dystrophin pre-mRNA.

Advantageous Effects of Invention

The present invention provides an antisense oligomer that causes simultaneous skipping of a plurality of exons in a target. Another embodiment of the present invention provides a pharmaceutical composition for treating patients having various mutations by causing simultaneous skipping of a plurality of exons in pre-mRNA of interest. An alternative embodiment of the present invention provides a suppressor antisense oligomer that suppresses single skipping of an exon in pre-mRNA of interest or a pharmaceutical composition comprising the oligomer. An alternative embodiment of the present invention enables simultaneous skipping of exons 45 to 55 in human dystrophin pre-mRNA to be caused with high efficiency.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing results of studying exon 45 to 55 skipping in human dystrophin pre-mRNA in RD cells (human rhabdomyosarcoma cells) by RT-PCR.

FIG. 2 is a diagram showing results of studying exon 45 to 55 skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 3 is a diagram showing results of studying exon 45 to 55 skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 4 is a diagram showing results of studying exon 45 to 55 skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 5 is a diagram showing results of studying exon 45 to 55 skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 6 is a diagram showing results of studying exon 45 to 55 skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 7 is a diagram showing results of studying exon 45 to 55 skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 8 is a diagram showing results of studying exon 45 to 55 skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 9 is a diagram showing results of studying exon 45 to 55 skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 10 is a diagram showing results of studying exon 45 to 55 skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 11 is a diagram showing results of studying exon 45 to 55 skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 12 is a diagram showing results of studying exon 45 to 55 skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 13 is a diagram showing results of studying exon 45 to 55 skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 14 is a diagram showing results of studying exon 45 to 55 skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 15 is a diagram showing results of studying exon 45 to 55 skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 16 is a diagram showing results of studying exon 45 to 55 skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 17 is a diagram showing results of studying exon 45 to 55 multi-skipping and exon 45 single skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 18 is a diagram showing results of studying exon 45 to 55 multi-skipping and exon 45 single skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 19 is a diagram showing results of studying multi-skipping of exons selected from exons 45 to 49 and exon 45 single skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 20 is a diagram showing results of studying multi-skipping of exons selected from exons 45 to 49 in human dystrophin pre-mRNA in RD cells by RT-PCR, and calculating the total amounts of the respective skips of exons 45 and 46, exons 45 to 47, exons 45 to 48, and exons 45 to 49 (total multi-skipping products). Each of these skips is considered to have therapeutic effects.

FIG. 21 is a diagram showing results of studying multi-skipping of exons selected from exons 45 to 52 and exon 45 single skipping in human dystrophin pre-mRNA in RD cells by RT-PCR.

FIG. 22 is a diagram showing results of studying multi-skipping of exons selected from exons 45 to 52 in human dystrophin pre-mRNA in RD cells by RT-PCR, and calculating the total amounts of the respective skips of exons 45 to 47, exons 45 to 48, exons 45 to 49, exons 45 to 50, exons 45 to 51, and exons 45 to 52 (left) or the total amounts of the respective skips of exons 45 to 47, exons 45 to 48, exons 45 to 49, and exons 45 to 51 (right). Each of the skips of exons 45 to 47, exons 45 to 48, exons 45 to 49, and exons 45 to 51 has therapeutic effects.

DESCRIPTION OF EMBODIMENTS

Hereinafter, the present invention is described in detail. The embodiments described below are intended to be presented by way of example merely to describe the invention but not limited only to the following embodiments. The present invention may be implemented in various ways without departing from the gist of the invention.

1. Antisense Oligomer

The present invention provides an antisense oligomer or a pharmaceutically acceptable salt thereof, or hydrate thereof which causes simultaneous skipping of two or more numerically consecutive exons from pre-mRNA of interest,

the antisense oligomer comprising a base sequence complementary to a base sequence of any one region selected from the group consisting of

or a partial base sequence thereof.

As used herein, the term “cause simultaneous skipping” of two or more numerically consecutive exons includes not only removal of the respective exons from pre-mRNA at completely the same timings but also sequential removal of the respective exons within a period from pre-mRNA to mature mRNA. Specifically, the term “cause simultaneous skipping” of two or more numerically consecutive exons refers to removal of a plurality of (two or more) numerically consecutive exons from pre-mRNA.

As used herein, the term “two or more numerically consecutive exons” means a plurality of exons that increase one by one in exon number among exons (the total number of exons is referred to as Texon) included in pre-mRNA of interest. The exon number means a number assigned to exons in order from the 5′ end to the 3′ end with an exon at the most upstream position of pre-mRNA defined as the first exon, followed by the second, the third, . . . . In the case of skipping of two or more numerically consecutive exons in a certain gene, its exon numbers a₁, . . . , a_jcan be represented by the sequence {a_j}. The general term a_jin the sequence {a_j} is represented by the expression below:

a
_j
=m+(j−1) [Expression 1]

wherein m is a given natural number that satisfies 1≤m≤(Texon−1), and j is a natural number that satisfies 2≤(m+j)≤Texon+1.

When the pre-mRNA of interest is, for example, human dystrophin pre-mRNA, Texon is 79.

In a certain embodiment, j is a given natural number selected from 1 to 11. In another embodiment, j is 11, j is 10, j is 9, j is 8, j is 7, j is 6, j is 5, j is 4, j is 3, j is 2, or j is 1.

As used herein, the term “gene” is intended to mean a genomic gene and also include cDNA, pre-mRNA and mRNA. Preferably, the gene is pre-mRNA. As used herein, the term “pre-mRNA” is an RNA molecule comprising an exon and an intron transcribed from a target gene on the genome and is a mRNA precursor.

In a certain embodiment, the present invention provides an antisense oligomer or a pharmaceutically acceptable salt thereof, or hydrate thereof which causes simultaneous skipping of any two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA (hereinafter, the antisense oligomer and the pharmaceutically acceptable salt thereof and the hydrate of the antisense oligomer or the salt are also collectively referred to as the “antisense oligomer of the present invention”; the antisense oligomer of the present invention may refer to any of the antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof).

The human dystrophin pre-mRNA is an RNA molecule comprising an exon and an intron transcribed from the human dystrophin gene on the genome and is a mRNA precursor. Those skilled in the art can obtain information on the base sequence of the human dystrophin pre-mRNA by analogy from the genomic sequence of the human dystrophin gene (GenBank Accession Nos. NG_012232.1).

In the human genome, the human dystrophin gene locates at locus Xp21.2. The human dystrophin gene has a size of about 3.0 Mbp and is the largest gene among known human genes. However, the coding regions of the human dystrophin gene are only about 14 kb, distributed as 79 exons throughout the human dystrophin gene (Roberts, R G, et al., Genomics, 16: 536-538 (1993)). The pre-mRNA, which is the transcript of the human dystrophin gene, undergoes splicing to form mature mRNA of about 14 kb. The base sequence of mature mRNA of human wild-type dystrophin gene is known (GenBank Accession No. NM_004006).

The antisense oligomer of the present invention according to the foregoing embodiment targets at least any one region selected from the group consisting of regions R1 to R24 represented by

Hereinafter, the regions R1 to R24 are each or collectively referred to as a “target region of the antisense oligomer of the present invention” or interchangeably the “target region of the present invention”.

As used herein, the term “targeting” means that an intended base sequence is a base sequence complementary to the base sequence of a target region or a partial base sequence of the target sequence.

The antisense oligomer of the present invention according to the foregoing embodiment comprises a base sequence complementary to a base sequence of at least any one region selected from the group consisting of the target regions R1 to R24 of the present invention, or a partial base sequence thereof.

Examples of the target region Rn of the present invention according to the foregoing embodiment include regions wherein

when n is 1, NA=44, NB=44, NX=20, and NY=400,

when n is 2, NA=44, NB=45, NX=600, and NY=50,

when n is 3, NA=45, NB=45, NX=20, and NY=400,

when n is 4, NA=45, NB=46, NX=400, and NY=50,

when n is 5, NA=46, NB=46, NX=20, and NY=400,

when n is 6, NA=46, NB=47, NX=400, and NY=50,

when n is 7, NA=47, NB=47, NX=20, and NY=400,

when n is 8, NA=47, NB=48, NX=400, and NY=50,

when n is 9, NA=48, NB=48, NX=20, and NY=400,

when n is 10, NA=48, NB=49, NX=400, and NY=50,

when n is 11, NA=49, NB=49, NX=20, and NY=400,

when n is 12, NA=49, NB=50, NX=400, and NY=50,

when n is 13, NA=50, NB=50, NX=20, and NY=400,

when n is 14, NA=50, NB=51, NX=400, and NY=50,

when n is 15, NA=51, NB=51, NX=20, and NY=400,

when n is 16, NA=51, NB=52, NX=400, and NY=50,

when n is 17, NA=52, NB=52, NX=20, and NY=400,

when n is 18, NA=52, NB=53, NX=400, and NY=50,

when n is 19, NA=53, NB=53, NX=20, and NY=400,

when n is 20, NA=53, NB=54, NX=400, and NY=50,

when n is 21, NA=54, NB=54, NX=20, and NY=400,

when n is 22, NA=54, NB=55, NX=400, and NY=50,

when n is 23, NA=55, NB=55, NX=20, and NY=400, or

when n is 24, NA=55, NB=56, NX=400, and NY=50.

In another embodiment, examples of the target region Rn of the present invention include, but not limited to, regions wherein

when n is 1, NA=44, NB=44, NX=20, and NY=400,

when n is 2, NA=44, NB=45, NX=600, and NY=19,

when n is 3, NA=45, NB=45, NX=20, and NY=400,

when n is 4, NA=45, NB=46, NX=400, and NY=50,

when n is 5, NA=46, NB=46, NX=20, and NY=400,

when n is 6, NA=46, NB=47, NX=400, and NY=37,

when n is 7, NA=47, NB=47, NX=20, and NY=400,

when n is 8, NA=47, NB=48, NX=400, and NY=19,

when n is 9, NA=48, NB=48, NX=20, and NY=400,

when n is 10, NA=48, NB=49, NX=400, and NY=42,

when n is 11, NA=49, NB=49, NX=20, and NY=400,

when n is 12, NA=49, NB=50, NX=400, and NY=44,

when n is 13, NA=50, NB=50, NX=20, and NY=400,

when n is 14, NA=50, NB=51, NX=400, and NY=25,

when n is 15, NA=51, NB=51, NX=20, and NY=400,

when n is 16, NA=51, NB=52, NX=400, and NY=24,

when n is 17, NA=52, NB=52, NX=20, and NY=400,

when n is 18, NA=52, NB=53, NX=400, and NY=34,

when n is 19, NA=53, NB=53, NX=20, and NY=400,

when n is 20, NA=53, NB=54, NX=400, and NY=43,

when n is 21, NA=54, NB=54, NX=20, and NY=400,

when n is 22, NA=54, NB=55, NX=400, and NY=25,

when n is 23, NA=55, NB=55, NX=20, and NY=400, or

when n is 24, NA=55, NB=56, NX=400, and NY=50.

In another embodiment, R1 to R24 which are examples of the target region Rn of the present invention are, but not limited to, as follows:

for example, in a certain embodiment,

In an alternative embodiment, R1 to R24 which are examples of the target region Rn of the present invention are, but not limited to, as follows:

for example, in a certain embodiment,

the region R2 is a region that consists of a base sequence of 600 bases in the upstream direction from the 3′ end of the 44th intron and a base sequence of 19 bases in the downstream direction from the 5′ end of the 45th exon in the human dystrophin pre-mRNA,

the region R6 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 46th intron and a base sequence of 37 bases in the downstream direction from the 5′ end of the 47th exon in the human dystrophin pre-mRNA,

the region R8 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 47th intron and a base sequence of 19 bases in the downstream direction from the 5′ end of the 48th exon in the human dystrophin pre-mRNA,

the region R10 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 48th intron and a base sequence of 42 bases in the downstream direction from the 5′ end of the 49th exon in the human dystrophin pre-mRNA,

the region R12 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 49th intron and a base sequence of 44 bases in the downstream direction from the 5′ end of the 50th exon in the human dystrophin pre-mRNA,

the region R14 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 50th intron and a base sequence of 25 bases in the downstream direction from the 5′ end of the 51st exon in the human dystrophin pre-mRNA,

the region R16 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 51st intron and a base sequence of 24 bases in the downstream direction from the 5′ end of the 52nd exon in the human dystrophin pre-mRNA,

the region R18 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 52nd intron and a base sequence of 34 bases in the downstream direction from the 5′ end of the 53rd exon in the human dystrophin pre-mRNA,

the region R20 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 53rd intron and a base sequence of 43 bases in the downstream direction from the 5′ end of the 54th exon in the human dystrophin pre-mRNA,

the region R22 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 54th intron and a base sequence of 25 bases in the downstream direction from the 5′ end of the 55th exon in the human dystrophin pre-mRNA,

In an alternative embodiment, R1 to R24 which are examples of the target region Rn of the present invention are, but not limited to, as follows:

(A1) Region R1

The region R1 is a region indicated by the range of −20 bases to +400 bases when the boundary between the 3′ end of exon 44 and the 5′ end of intron 44 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “+”, and a base sequence region on the 3′ side (downstream) therefrom is indicated by “−” (minus). In this respect, the region indicated by the range of −20 bases to −1 base belongs to exon 44, and the region indicated by the range of +1 base to +400 bases belongs to intron 44.

(A2) Region R2

The region R2 is a region indicated by the range of −600 bases to +50 bases when the boundary between the 3′ end of intron 44 and the 5′ end of exon 45 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −600 bases to −1 base belongs to intron 44, and the region indicated by the range of +1 base to +50 bases belongs to exon 45.

(A3) Region R3

The region R3 is a region indicated by the range of −20 bases to +400 bases when the boundary between the 3′ end of exon 45 and the 5′ end of intron 45 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −20 bases to −1 base belongs to exon 45, and the region indicated by the range of +1 base to +400 bases belongs to intron 45.

(A4) Region R4

The region R4 is a region indicated by the range of −400 bases to +50 bases when the boundary between the 3′ end of intron 45 and the 5′ end of exon 46 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 45, and the region indicated by the range of +1 base to +50 bases belongs to exon 46.

(A5) Region R5

The region R5 is a region indicated by the range of −20 bases to +400 bases when the boundary between the 3′ end of exon 46 and the 5′ end of intron 46 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −20 bases to −1 base belongs to exon 46, and the region indicated by the range of +1 base to +400 bases belongs to intron 46.

(A6) Region R6

The region R6 is a region indicated by the range of −400 bases to +50 bases when the boundary between the 3′ end of intron 46 and the 5′ end of exon 47 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 46, and the region indicated by the range of +1 base to +50 bases belongs to exon 47.

(A7) Region R7

The region R7 is a region indicated by the range of −20 bases to +400 bases when the boundary between the 3′ end of exon 47 and the 5′ end of intron 47 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −20 bases to −1 base belongs to exon 47, and the region indicated by the range of +1 base to +400 bases belongs to intron 47.

(A8) Region R8

The region R8 is a region indicated by the range of −400 bases to +50 bases when the boundary between the 3′ end of intron 47 and the 5′ end of exon 48 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 47, and the region indicated by the range of +1 base to +50 bases belongs to exon 48.

(A9) Region R9

The region R9 is a region indicated by the range of −20 bases to +400 bases when the boundary between the 3′ end of exon 48 and the 5′ end of intron 48 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −20 bases to −1 base belongs to exon 48, and the region indicated by the range of +1 base to +400 bases belongs to intron 48.

(A10) Region R10

The region R10 is a region indicated by the range of −400 bases to +50 bases when the boundary between the 3′ end of intron 48 and the 5′ end of exon 49 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 48, and the region indicated by the range of +1 base to +50 bases belongs to exon 49.

(A11) Region R11

The region R11 is a region indicated by the range of −20 bases to +400 bases when the boundary between the 3′ end of exon 49 and the 5′ end of intron 49 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −20 bases to −1 base belongs to exon 49, and the region indicated by the range of +1 base to +400 bases belongs to intron 49.

(A12) Region R12

The region R12 is a region indicated by the range of −400 bases to +50 bases when the boundary between the 3′ end of intron 49 and the 5′ end of exon 50 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 49, and the region indicated by the range of +1 base to +50 bases belongs to exon 50.

(A13) Region R13

The region R13 is a region indicated by the range of −20 bases to +400 bases when the boundary between the 3′ end of exon 50 and the 5′ end of intron 50 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −20 bases to −1 base belongs to exon 50, and the region indicated by the range of +1 base to +400 bases belongs to intron 50.

(A14) Region R14

The region R14 is a region indicated by the range of −400 bases to +50 bases when the boundary between the 3′ end of intron 50 and the 5′ end of exon 51 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 50, and the region indicated by the range of +1 base to +50 bases belongs to exon 51.

(A15) Region R15

The region R15 is a region indicated by the range of −20 bases to +400 bases when the boundary between the 3′ end of exon 51 and the 5′ end of intron 51 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −20 bases to −1 base belongs to exon 51, and the region indicated by the range of +1 base to +400 bases belongs to intron 51.

(A16) Region R16

The region R16 is a region indicated by the range of −400 bases to +50 bases when the boundary between the 3′ end of intron 51 and the 5′ end of exon 52 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 51, and the region indicated by the range of +1 base to +50 bases belongs to exon 52.

(A17) Region R17

The region R17 is a region indicated by the range of −20 bases to +400 bases when the boundary between the 3′ end of exon 52 and the 5′ end of intron 52 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −20 bases to −1 base belongs to exon 52, and the region indicated by the range of +1 base to +400 bases belongs to intron 52.

(A18) Region R18

The region R18 is a region indicated by the range of −400 bases to +50 bases when the boundary between the 3′ end of intron 52 and the 5′ end of exon 53 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 52, and the region indicated by the range of +1 base to +50 bases belongs to exon 53.

(A19) Region R19

The region R19 is a region indicated by the range of −20 bases to +400 bases when the boundary between the 3′ end of exon 53 and the 5′ end of intron 53 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −20 bases to −1 base belongs to exon 53, and the region indicated by the range of +1 base to +400 bases belongs to intron 53.

(A20) Region R20

The region R20 is a region indicated by the range of −400 bases to +50 bases when the boundary between the 3′ end of intron 53 and the 5′ end of exon 54 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 53, and the region indicated by the range of +1 base to +50 bases belongs to exon 54.

(A21) Region R21

The region R21 is a region indicated by the range of −20 bases to +400 bases when the boundary between the 3′ end of exon 54 and the 5′ end of intron 54 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −20 bases to −1 base belongs to exon 54, and the region indicated by the range of +1 base to +400 bases belongs to intron 54.

(A22) Region R22

The region R22 is a region indicated by the range of −400 bases to +50 bases when the boundary between the 3′ end of intron 54 and the 5′ end of exon 55 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 54, and the region indicated by the range of +1 base to +50 bases belongs to exon 55.

(A23) Region R23

The region R23 is a region indicated by the range of −20 bases to +400 bases when the boundary between the 3′ end of exon 55 and the 5′ end of intron 55 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −20 bases to −1 base belongs to exon 55, and the region indicated by the range of +1 base to +400 bases belongs to intron 55.

(A24) Region R24

The region R24 is a region indicated by the range of −400 bases to +50 bases when the boundary between the 3′ end of intron 55 and the 5′ end of exon 56 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 55, and the region indicated by the range of +1 base to +50 bases belongs to exon 56.

In an alternative aspect, R2, R6, R8, R10, R12, R14, R16, R18, R20, and R22 which are examples of the target region Rn of the present invention are, but not limited to, as follows:

(A2) Region R2

The region R2 is a region indicated by the range of −600 bases to +19 bases when the boundary between the 3′ end of intron 44 and the 5′ end of exon 45 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −600 bases to −1 base belongs to intron 44, and the region indicated by the range of +1 base to +19 bases belongs to exon 45.

(A6) Region R6

The region R6 is a region indicated by the range of −400 bases to +37 bases when the boundary between the 3′ end of intron 46 and the 5′ end of exon 47 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 46, and the region indicated by the range of +1 base to +37 bases belongs to exon 47.

(A8) Region R8

The region R8 is a region indicated by the range of −400 bases to +19 bases when the boundary between the 3′ end of intron 47 and the 5′ end of exon 48 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 47, and the region indicated by the range of +1 base to +19 bases belongs to exon 48.

(A10) Region R10

The region R10 is a region indicated by the range of −400 bases to +42 bases when the boundary between the 3′ end of intron 48 and the 5′ end of exon 49 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 48, and the region indicated by the range of +1 base to +42 bases belongs to exon 49.

(A12) Region R12

The region R12 is a region indicated by the range of −400 bases to +44 bases when the boundary between the 3′ end of intron 49 and the 5′ end of exon 50 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 49, and the region indicated by the range of +1 base to +44 bases belongs to exon 50.

(A14) Region R14

The region R14 is a region indicated by the range of −400 bases to +25 bases when the boundary between the 3′ end of intron 50 and the 5′ end of exon 51 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 50, and the region indicated by the range of +1 base to +25 bases belongs to exon 51.

(A16) Region R16

The region R16 is a region indicated by the range of −400 bases to +24 bases when the boundary between the 3′ end of intron 51 and the 5′ end of exon 52 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 51, and the region indicated by the range of +1 base to +24 bases belongs to exon 52.

(A18) Region R18

The region R18 is a region indicated by the range of −400 bases to +34 bases when the boundary between the 3′ end of intron 52 and the 5′ end of exon 53 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 52, and the region indicated by the range of +1 base to +34 bases belongs to exon 53.

(A20) Region R20

The region R20 is a region indicated by the range of −400 bases to +43 bases when the boundary between the 3′ end of intron 53 and the 5′ end of exon 54 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 53, and the region indicated by the range of +1 base to +43 bases belongs to exon 54.

(A22) Region R22

The region R22 is a region indicated by the range of −400 bases to +25 bases when the boundary between the 3′ end of intron 54 and the 5′ end of exon 55 is defined as basing point 0, a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus), and a base sequence region on the 3′ side (downstream) therefrom is indicated by “+”. In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 54, and the region indicated by the range of +1 base to +25 bases belongs to exon 55.

Specific examples of the base sequences of the regions R1 to R24 will be given below. As used herein, thymine “T” and uracil “U” are interchangeable with each other. It does not essentially affect the exon skipping activity of the antisense oligomer of the present invention whether it is “T” or “U”. Therefore, as used herein, identical base sequences, even when “T” are replaced with “U”, are also included and represented by the same SEQ ID NO. In the tables below, “U” may be described as “T” even in the base sequence of pre-mRNA. Those skilled in the art can understand an RNA sequence by appropriately replacing “T” with “U”.

TABLE 1

Table 1

Region
Base sequence of target region
SEQ ID NO

R1
Region in vicinity of donor of intron 44 (in range of −20 to
SEQ No. 233

+400 bases from 5′ end of intron 44 as basing point)

AATACAAATGGTATCTTAAGGTAAGTCTTTGATTTGTTTTTTCGAAATTGTATTTATCTTC

AGCACATCTGGACTCTTTAACTTCTTAAAGATCAGGTTCTGAAGGGTGATGGAAATTACTT

TTGACTGTTGTTGTCATCATTATATTACTAGAAAGAAAATTATCATAATGATAATATTAGA

GCACGGTGCTATGGACTTTTTGTGTCAGGATGAGAGAGTTTGCCTGGACGGAGCTGGTTTA

TCTGATAAACTGCAAAATATAATTGAATCTGTGACAGAGGGAAGCATCGTAACAGCAAGGT

GTTTTGTGGCTTTGGGGCAGTGTGTATTTCGGCTTTATGTTGGAACCTTTCCAGAAGGAGA

ACTTGTGGCATACTTAGCTAAAATGAAGTTGCTAGAAATATCCATCATGATAAA

R2
Region in vicinity of acceptor of intron 44 (in range of −600
SEQ No. 234

to +50 bases from 3′ end of intron 44 as basing point)

TCTTGATGGGATGCTCCTGAAAGCAATTAATTCTCAGTTTTTTGTGGCTTCTAATGCAAAA

TACATTGACGCAGACAGAATTTGAAATGAATTTTCTTCTAATATAGCAATTAATTTTATTT

AAATATCTCTAGAGTTTTTTTTTAATACTGTGACTAACCTATGTTTGTTCTTTTTCACCTC

TCGTATCCACGATCACTAAGAAACCCAAATACTTTGTTCATGTTTAAATTTTACAACATTT

CATAGACTATTAAACATGGAACATCCTTGTGGGGACAAGAAATCGAATTTGCTCTTGAAAA

GGTTTCCAACTAATTGATTTGTAGGACATTATAACATCCTCTAGCTGACAAGCTTACAAAA

ATAAAAACTGGAGCTAACCGAGAGGGTGCTTTTTTCCCTGACACATAAAAGGTGTCTTTCT

GTCTTGTATCCTTTGGATATGGGCATGTCAGTTTCATAGGGAAATTTTCACATGGAGCTTT

TGTATTTCTTTCTTTGCCAGTACAACTGCATGTGGTAGCACACTGTTTAATCTTTTCTCAA

ATAAAAAGACATGGGGCTTCATTTTTGTTTTGCCTTTTTGGTATCTTACAGGAACTCCAGG

ATGGCATTGGGCAGCGGCAAACTGTTGTCAGAACATTGAA

R3
Region in vicinity of donor of intron 45 (in range of −20 to
SEQ No. 235

+400 bases from 5′ end of intron 45 as basing point)

CTGTCAGACAGAAAAAAGAGGTAGGGCGACAGATCTAATAGGAATGAAAACATTTTAGCAG

ACTTTTTAAGCTTTCTTTAGAAGAATATTTCATGAGAGATTATAAGCAGGGTGAAAGGCAC

TAACATTAAAGAACCTATCAACCATTAATCAACAGCAGTAAAGAAATTTTTTATTTCTTTT

TTTCATATACTAAAATATATACTTGTGGCTAGTTAGTGGTTTTCTGCTATTTTAAACTTGA

AGTTTGCTTTAAAAATCACCCATGATTGCTTAAAGGTGAATATCTTCAATATATTTTAACT

TCAACAAGCTGAATCTCAGTTGTTTTTCAAGAAGATTTTAGAAAGCAATTATAAATGATTG

TTTTGTAGGAAAGACAGATCTTTGCTTAGTTTTAAAAATAGCTATGAATATGAC

R4
Region in vicinity of acceptor of intron 45 (in range of −400
SEQ No. 236

to +50 bases from 3′ end of intron 45 as basing point)

ATTACAGGCGCCTGCCACCAAACCTGGCAAATTTTTGTATTTTTAGTGTAGACGGGGTTTC

ACCATATTTGCCAGGCTGGTCGCAAACTCCTGACCTCAAGTGATCCGCCCACATCGGCCTC

CCTAAGCGCTAGGGTTACAGGCATGAGCCACTGCGCCTGGCCAGGAATTTTTGAATCAGAA

TTTTTCTTGTTCGATTTTAATCTCTTATCATTTAGAGATTCTTGAAATATTGAAATTACTT

TGTTCAAAGTGAATGAATTTTCTTAAATTATGTATGGTTAACATCTTTTAAATTGCTTATT

TTTAAATTGCCATGTTTGTGTCCCAGTTTGCATTAACAAATAGTTTGAGAACTATGTTGGA

AAAAAAAATAACAATTTTATTCTTCTTTCTCCAGGCTAGAAGAACAAAAGAATATCTTGTC

AGAATTTCAAAGAGATTTAAATG

R5
Region in vicinity of donor of intron 46 (in range of −20 to
SEQ No. 237

+400 bases from 5′ end of intron 46 as basing point)

AAAAGCTTGAGCAAGTCAAGGTAATTTTATTTTCTCAAATCCCCCAGGGCCTGCTTGCATA

AAGAAGTATATGAATCTATTTTTTAATTCAATCATTGGTTTTCTGCCCATTAGGTTATTCA

TAGTTCCTTGCTAAAGTGTTTTTCTCACAACTTTATTTCTTCTTAACCCTGCAGTTCTGAA

CCAGTGCACATAAGAACATATGTATATATGTGTGTGTGTGTATTTATATATACACACACAC

ATATTGCATCTATACATCTACACATATAGATGTATAGATTCAATATGTCTAAATGTATATA

ATTCACAGTTTTTATCTTTGATTTGAAATTAATTTTAGATTTTACTTGAGAACTTCACAAC

TTCATATAATTTTAAAAACTGAAGACCAGATTGTGGAATCATAAAATCTAAATC

R6
Region in vicinity of acceptor of intron 46 (in range of −400
SEQ No. 238

to +50 bases from 3′ end of intron 46 as basing point)

ATAATGCAATTTCTCAGCAGCAAGCTACGGTATGCTATGGCATGCTATGATACCCAAGAGG

CTGATGAATTTGTTCACATTGTTCTATTTCTGATAGAGAGATAGGTTTTCAGACACTAACT

TTATTTGGAGTGTTGCTTTACCATCTCACATTTTTCTCTTAAAAAATTTATGAGGGATAAT

ATAATCGTTTATTTTCTACAGAGATTTATCTACTGAGGGGGTGAGTGTTTCAGTCAATCAG

CTCTGTGCTCAGATAGAAAACTGTTGGTATTTGAGGTACCACTGGGCCCTCGGTCAAGTCG

CTTCATTTTGATAGACTAATCAATAGAAGCAAAGACAAGGTAGTTGGAATTGTGCTGTAAT

TCATTTTAAACGTTGTTGCATTTGTCTGTTTCAGTTACTGGTGGAAGAGTTGCCCCTGCGC

CAGGGAATTCTCAAACAATTAAA

R7
Region in vicinity of donor of intron 47 (in range of −20 to
SEQ No. 239

+400 bases from 5′ end of intron 47 as basing point)

CAAATCTCCAGTGGATAAAGGTTAGACATTAACCATCTCTTCCGTCACATGTGTTAAATGT

TGCAAGTATTTGTATGTATTTTGTTTCCTGGGTGCTTCATTGGTCGGGGAGGAGGCTGGTA

TGTGGATTGTTGTTTTGTTTTGTTTTTTTAACCTGACCGTTTGCTTTGGCTATATGTTTTG

TTGTGGCTAGAAAAAATGATGATGGTGAATGGCTTTACATTAATGACCAAATGCCAAAATT

TATACCACAATTTTTTGCATAAATTATTCTGAAGAATCAGACTGAAGAAATGGCGAAGTAT

TTAATTCAGTGGCCAGGCATGTACTGACAGTATTTAAGCTGAAAGGACGTGGTCTGGTTCT

AGTTAAACAAGTGTCATAAATCAAAATTAATTATTCACACCTGTGGTATGGACT

R8
Region in vicinity of acceptor of intron 47 (in range of −400

to +50 bases from 3′ end of intron 47 as basing point)
SEQ No. 240

TATTCATTTTTATAACTGCAAAGGAAGCGCGTATGGCATATAATACACAACACACCAGTAT

ATTTAGTAACTGAGTGAATAAATGAAAGATGTATTTCTTTACTTTATCAGTTGCAGTTGGC

TATGCCTTTGTGTAAGGTGTGTGTTTTGAAATTCCAAAAAGGTATTAGTTTCTTTAAAGCA

AAGAATTTTTGTAGCAGGTTAATGAATAATTTTGAATACATTGGTTAAATCCCAACATGTA

ATATATGTAAATAATCAATATTATGCTGCTAAAATAACACAAATCAGTAAGATTCTGTAAT

ATTTCATGATAAATAACTTTTGAAAATATATTTTTAAACATTTTGGCTTATGCCTTGAGAA

TTATTTACCTTTTTAAAATGTATTTTCCTTTCAGGTTTCCAGAGCTTTACCTGAGAAACAA

GGAGAAATTGAAGCTCAAATAAA

R9
Region in vicinity of donor of intron 48 (in range of −20 to
SEQ No. 241

+400 bases from 5′ end of intron 48 as basing point)

AAGGACCATTTGACGTTCAGGTAGGGAACTTTTTGCTTTAAATATTTTTGTCTTTTTTAAG

AAAAATGGCAATATCACTGAATTTTCTCATTTGGTATCATTATTAAAGACAAAATATTACT

TGTTAAAGTGTGGTAAGGAAGACTTTATTCAGGATAACCACAATAGGCACAGGGACCACTG

CAATGGAGTATTACAGGAGGTTGGATAGAGAGAGATTGGGCTCAACTCTAAATACAGCACA

GTGGAAGTAGGAATTTATAGCCAAGGAGCAGTGTAGGAGTCAGTAGATGGAAAATTATTAA

GAGGAAACATCAGGGGTAAGTGGGATTCTGGCTAAACCAACCTCACAGGATTCTTGCTGAA

GATAGGCCAGGGTTATCTTATCAGACAACCCTTGGGGAATGGTGGAGAATACTG

R10
Region in vicinity of acceptor of intron 48 (in range of −400
SEQ No. 242

to +50 bases from 3′ end of intron 48 as basing point)

TACTAAACACAGAATTTTGTAAAACAATAAGTGTATAAAGTAAAATGAACATTAGGATTAT

TGAGATTATTGTAGCTAAAACTAGTGTTTATTCATATAAATTATGTTAATAAATTGTATTG

TCATTATTGCATTTTACTTTTTTGAAAAGTAGTTAATGCCTGTGTTTCTATATGAGTATTA

TATAATTCAAGAAGATATTGGATGAATTTTTTTTAAGTTTAATGTGTTTCACATCTCTGTT

TCTTTTCTCTGCACCAAAAGCTACATTTTTGTGCCCTTATGTACCAGGCAGAAATTGATCT

GCAATACATGTGGAGTCTCCAAGGGTATATTTAAATTTAGTAATTTTATTGCTAACTGTGA

AGTTAATCTGCACTATATGGGTTCTTTTCCCCAGGAAACTGAAATAGCAGTTCAAGCTAAA

CAACCGGATGTGGAAGAGATTTT

R11
Region in vicinity of donor of intron 49 (in range of −20 to
SEQ No. 243

+400 bases from 5′ end of intron 49 as basing point)

CAGCCACTCAGCCAGTGAAGGTAATGAAGCAACCTCTAGCAATATCCATTACCTCATAATG

GGTTATGCTTCCCCTGTTGTACATTTGCCATTGACGTGGACTATTTATAATCAGTGAAATA

ACTTGTAAGGAAATACTGGCCATACTGTAATAGCAGAGGCAAAGCTGTCTTTTTGATCAGC

ATATCCTATTTATATATTGTGATCTTAAGGCTATTAACGAGTCATTGCTTTAAAGGACTCA

TTTGTGTCCTGGTGTGCTGCCATCAATACAAAAGTAGTCCCACCTTCAAGGTAGATTAAAT

TCTTTGGGGCTTTATTGCTTTGCTTGCCAGCCTTGATGCTTTTCATATTGTTTGGTTTAAT

TCAAATCAAGCTACTGCATCATAGTGTCTGTCTCCAACAGCTGTAAAGAATCAC

R12
Region in vicinity of acceptor of intron 49 (in range of −400
SEQ No. 244

to +50 bases from 3′ end of intron 49 as basing point)

ATATAATTGACTGGGGGTGAGCCAGTACATTAGGATTTTCCTAAAGTTATCTGGATAATTT

TAGTATGCAACCACAATAGATACTCTTCAAGAATTAAGCTAGTTGCTGAGAGGGAACTGTT

TTTTGTTGGTTTGTTTTCACTAATGTTTGCACTCTACTTCCTTTAAATAAAATTATGCCTG

GAGAAAGGGTTTTTGTATGGAGCAATTGATAAATATTTGTAGGGTGGTTGGCTAAAATAAT

TATAATTCCTTTAAAAGAAATTCTACCCACTAAAGTTAATTTAGAAGTAAAATATAATAGA

AATCCAATAATATATTCACCAAATGGATTAAGATGTTCATGAATTATCTTCAAAGTGTTAA

TCGAATAAGTAATGTGTATGCTTTTCTGTTAAAGAGGAAGTTAGAAGATCTGAGCTCTGAG

TGGAAGGCGGTAAACCGTTTACT

R13
Region in vicinity of donor of intron 50 (in range of −20 to
SEQ No. 245

+400 bases from 5′ end of intron 50 as basing point)

ACTGACCACTATTGGAGCCTGTAAGTATACTGGATCCCATTCTCTTTGGCTCTAGCTATTT

GTTCAAAAGTGCAACTATGAAGTGATGACTGGGTGAGAGAGAAAATTTGTTTCAATTCTAA

AGATAGAGATAAACCTTTGTGTTATTGACTGTGCAAAAAGTCTTAGAGTACATTCCTTGGA

AATTGACTCTGATTCAAAGTGTTGCATGACAACGGGATATGGGGAGTGTTCTCTGGAGATA

CACCCACAAGGAAGAGAAGAGCACAAGGGAGATTGTGGGAGAGTCTGAAATGTGATTTGTC

TGCAGCAGAGGCCTAAGCCAGTCTCGCAGGAGCCCTACATCTGGGCTGGCTGTGCAGAGCT

GTCCTGAATTGCAGGCAGTGGGCCTGGCCCTTGTATTCCTGATCCAGCCAGCCA

R14
Region in vicinity of acceptor of intron 50 (in range of −400
SEQ No. 246

to +50 bases from 3′ end of intron 50 as basing point)

TCTTGAATAAAAAAAAAATAAGTAAAATTTATTTCCCTGGCAAGGTCTGAAAACTTTTGTT

TTCTTTACCACTTCCACAATGTATATGATTGTTACTGAGAAGGCTTATTTAACTTAAGTTA

CTTGTCCAGGCATGAGAATGAGCAAAATCGTTTTTTAAAAAATTGTTAAATGTATATTAAT

GAAAAGGTTGAATCTTTTCATTTTCTACCATGTATTGCTAAACAAAGTATCCACATTGTTA

GAAAAAGATATATAATGTCATGAATAAGAGTTTGGCTCAAATTGTTACTCTTCAATTAAAT

TTGACTTATTGTTATTGAAATTGGCTCTTTAGCTTGTGTTTCTAATTTTTCTTTTTCTTCT

TTTTTCCTTTTTGCAAAAACCCAAAATATTTTAGCTCCTACTCAGACTGTTACTCTGGTGA

CACAACCTGTGGTTACTAAGGAA

R15
Region in vicinity of donor of intron 51 (in range of −20 to
SEQ No. 247

+400 bases from 5′ end of intron 51 as basing point)

AGATGATCATCAAGCAGAAGGTATGAGAAAAAATGATAAAAGTTGGCAGAAGTTTTTCTTT

AAAATGAAGATTTTCCACCAATCACTTTACTCTCCTAGACCATTTCCCACCAGTTCTTAGG

CAACTGTTTCTCTCTCAGCAAACACATTACTCTCACTATTCAGCCTAAGTATAATCAAGGA

TATAAATTAATGCAAATAACAAAAGTAGCCATACATTAAAAAGGAAATATACAAAAAAAAA

AAAAAAAAAAAGCAGAAACCTTACAAGAATAGTTGTCTCAGTTAAATTTACTAAACAACCT

GGTATTTTAAAAATCTATTTTATACCAAATAAGTCACTCAACTGAGCTATTTACATTTAAA

CTGTTTGTTTTGGCACTACGCAGCCCAACATATTGCAGAATCAAATATAATAGT

R16
Region in vicinity of acceptor of intron 51 (in range of −400
SEQ No. 248

to +50 bases from 3′ end of intron 51 as basing point)

TTTAAAATAAATATACCTTAATTTTGACGTCACACAGAATGATATTATAAGTATAAATAGT

TATCTATCTTTTAAATACATTGTCGTAATTCAGAATAACATTTCTTACTCAAGGCATTCAG

ACAGTGGTTTAAGTAATCCGAGGTACTCCGGAATGTCTCCATTTGAGCCTTTAAATGAAGA

AAATCTATAGTCAAGATTTTCATTTGAAATATTTTTGATATCTAAGAATGAAACATATTTC

CTGTTAAATTGTTTTCTATAAACCCTTATACAGTAACATCTTTTTTATTTCTAAAAGTGTT

TTGGCTGGTCTCACAATTGTACTTTACTTTGTATTATGTAAAAGGAATACACAACGCTGAA

GAACCCTGATACTAAGGGATATTTGTTCTTACAGGCAACAATGCAGGATTTGGAACAGAGG

CGTCCCCAGTTGGAAGAACTCAT

R17
Region in vicinity of donor of intron 52 (in range of −20 to
SEQ No. 249

+400 bases from 5′ end of intron 52 as basing point)

AACAATCATTACGGATCGAAGTAAGTTTTTTAACAAGCATGGGACACACAAAGCAAGATGC

ATGACAAGTTTCAATAAAAACTTAAGTTCATATATCCCCCTCACATTTATAAAAATAATGT

GAAATAATTGTAAATGATAACAATTGTGCTGAGATTTTCAGTCCATAATGTTACCTTTTAA

TAAATGAATGTAATTCCATTGAATAGAAGAAATACATTTTTAAATCAATTCAGGGCTTATA

TAGTTGCAAAGCATGCATTGATGGGTGTGGTGACCACAGTGTGGCAGAACATTTGTGGCAG

AACATTTGTTCTTTAGTTGTCATCTGGGCTGGCATCCATGGAGATGCCAGTCTCTCCCTCA

TATCCTTGGCTGTTGGTCCAAGCAGGCAGTGGCTTCTTCCTGGGCCATCTTTCA

R18
Region in vicinity of acceptor of intron 52 (in range of −400
SEQ No. 250

to +50 bases from 3′ end of intron 52 as basing point)

CAAACTCCTGTGGCAACAGAAAGCCTTCAGGCAATGAAATGCTGGCACTGGGAAATCAGGC

TGATGGGTGCTGAAGTGGCAAGGATGAGGGGATATGGATATTCTGCTGTAGTGCTTTTCTA

ACAGATGATTCATATTTGGTTCTAGGGATCAAGAATTGAGTTAAAATTTTATATATATGTT

GATGTTCTATGTCACCTTCAGGAAAATAATTTAACAGAAACTAATATTTGCCATCAAAAAA

GCAAAGAATCCTGTTGTTCATCATCCTAGCCATAACACAATGAATAATTTTTTAAATAAGC

AACATAAATGTGAGATAACGTTTGGAAGTTACATTTAAAATGTCTCCTCCAGACTAGCATT

TACTACTATATATTTATTTTTCCTTTTATTCTAGTTGAAAGAATTCAGAATCAGTGGGATG

AAGTACAAGAACACCTTCAGAAC

R19
Region in vicinity of donor of intron 53 (in range of −20 to
SEQ No. 251

+400 bases from 5′ end of intron 53 as basing point)

AGAAAATCACAGAAACCAAGGTTAGTATCAAAGATACCTTTTTAAAATAAAATACTGGTTA

CATTTGATAAAATTATACCATAGATTGTAATTTAATGATGTTTAATGTAAAGTTATTAACA

GAAAATCACGTTAAAGCTGAAATGAACAGTAGACTTTGTATATTTATTTTCTTAGAGACAG

AGTCTCACTGTCACCCAGGCTAAAGTGCAGTGGCACAATCATAGCTCACTGAGCCTTGAAC

TCTGGGGCTCAAGCAGTCCTCCTGCCTCAGCCTCCCTAGTAGCTGGGACTACTAGCCAGGC

GTGTACCACCACGCCTGGCTAATTTTTTAAAAATTTTTGTTTTCTGTAGAGATGGGTTCTT

GAACTCTTGGCCTCAAGCAATTCTCCTTCCTTGGCCTCCCAAAGCACTAGGATT

R20
Region in vicinity of acceptor of intron 53 (in range of −400
SEQ No. 252

to +50 bases from 3′ end of intron 53 as basing point)

CAACAAGTTAATGAAGAGGGAAAGAAATGTATGAGGTTTTTTTCGTTCAAATGTTGTTATA

TGTCACATATTCAACAATTATATATGAGCTTATTTTTGTAGTTTTTTTCTCTTGTGATAAA

AACAATTAAGCCCACTTTATTGCCAATTAATTGCTACTAAGTTGAAATACTTGATACTGGT

TATTGCTCAAGATGCTGCATTTGAAAAGTTTGTCCTGAAAGGTGGGTTACCTTATACTGTC

ATGATTGACTAAATCATATGGTAGGTTAAAAGCAATCTAATATATGTATTCTGACCTGAGG

ATTCAGAAGCTGTTTACGAAGTATTTTAAGACACTCCAACTAGAGATTTCATAAAAAAAAC

TGACATTCATTCTCTTTCTCATAAAAATCTATAGCAGTTGGCCAAAGACCTCCGCCAGTGG

CAGACAAATGTAGATGTGGCAAA

R21
Region in vicinity of donor of intron 54 (in range of −20 to
SEQ No. 253

+400 bases from 5′ end of intron 54 as basing point)

TGGAGAAGCATTCATAAAAGGTATGAATTACATTATTTCTAAAACTACTGTTGGCTGTAAT

AATGGGGTGGTGAAACTGGATGGACCATGAGGATTTGTTTTTCCAATCCAGCTAAACTGGA

GCTTGGGAGGGTTCAAGACGATAAATACCAACTAAACTCACGGACTTGGCTCAGACTTCTA

TTTTAAAAACGAGGAACATAAGATCTCATTTGCCCGCTGTCACAAAAGTAGTGACATAACC

AAGAGATTAAACAAAAAGCAAAATACTGATTTATAGCTAGAAGAGCCATTTATCAGTCTAC

TTTGATAACTCTATCCAAAGGAATATCTTTCTATCTCATCATGGCGCACACTGCCTTACCT

GTTATCTGATAAATAAGTCACTTTGGGATTCATGATAGAGTTATAGCTGTACAT

R22
Region in vicinity of acceptor of intron 54 (in range of −400
SEQ No. 254

to +50 bases from 3′ end of intron 54 as basing point)

TCTCAAATTTGGCAGTATATTAAAAATAAGCTTTCAAAATTGACCAACAAAAACTACAAAA

TTGAAAAAAAGGTACTTTGAACTTTCACATGTTCAAATATATGTATATATATTTCACATAT

ATATATGAAACCTCCTCTGTGGAGAGGGGTTTATAGAAATCTGTAATTGTCATTCTTGCAT

GCCTTCCCCCATACAAACGCCTTTAAGTTAAATAAAAATGAAAGTAAATAGACTGCACAAT

ATTATAGTTGTTGCTTAAAGGAAGAGCTGTAGCAACAACTCACCCCATTGTTGGTATATTA

CAATTTAGTTCCTCCATCTTTCTCTTTTTATGGAGTTCACTAGGTGCACCATTCTGATATT

TAATAATTGCATCTGAACATTTGGTCCTTTGCAGGGTGAGTGAGCGAGAGGCTGCTTTGGA

AGAAACTCATAGATTACTGCAAC

R23
Region in vicinity of donor of intron 55 (in range of −20 to
SEQ No. 255

+400 bases from 5′ end of intron 55 as basing point)

AGCTGATGAAACAATGGCAAGTAAGTCAGGCATTTCCGCTTTAGCACTCTTGTGGATCCAA

TTGAACAATTCTCAGCATTTGTACTTGTAACTGACAAGCCAGGGACAAAACAAAATAGTTG

CTTTTATACAGCCTGATGTATTTCGGTATTTGGACAAGGAGGAGAGAGGCAGAGGGAGAAG

GAAACATCATTTATAATTCCACTTAACACCCTCGTCTTAGAAAAAGTACATGCTCTGACCA

GGAAAACATTTGCATATAAAACCAGAGCTTCGGTCAAGGAGAAACTTTGCTCAGAGAAATA

ACTTAGGGATTGGTTTATTAAATTTTAAAAGTTGACATTTTTGAGTGTTTATTTAATATTT

TACAGGGAAAGCATCTGTATGAATTGTCTGTTTTATTTAGCGTTGCTAACTGAA

R24
Region in vicinity of acceptor of intron 55 (in range of −400
SEQ No. 256

to +50 bases from 3′ end of intron 55 as basing point)

ACTTGATCCATATAGTAATGAAATTATTGGCACTGGGGTACACTTTATCATAGAATTTTAT

TGCCTATCACTTCCATAAAATAATACATTTTGTCCATAGACTAGAAGATATAACTTGTGAA

CTTTATAAAGTTATAAATACATTACTTTCCAACTCATAATGGCAAGGAATAAATCTATTAC

AACTAATAAGATGCCCATTTTAAATCTACATAATAACAGGAGAAGGCAATACGCCAAGAAA

AGGGATTTGAGATGTATCTTCTTGTTAGTTTAGCCTGATTGAAATGTCTTTTGAACTAATA

ATTATTTATATTTTGCAATTCTCCAAATTCACATTCATCGCTTGTTTCTTTTGTTTGGTAA

TTCTGCACATATTCTTCTTCCTGCTGTCCTGTAGGACCTCCAAGGTGAAATTGAAGCTCAC

ACAGATGTTTATCACAACCTGGA

In an alternative aspect, specific examples of the base sequences of the regions R2, R6, R8, R10, R12, R14, R16, R18, R20, and R22 are as follows.

TABLE 2

Table 2

Region
Base sequence of target region
SEQ ID NO

R2
Region in vicinity of acceptor of intron 44 (in range of −600
SEQ No. 341

to +19 bases from 3′ end of intron 44 as basing point)

TCTTGATGGGATGCTCCTGAAAGCAATTAATTCTCAGTTTTTTGTGGCTTCTAATGCAAAA

TACATTGACGCAGACAGAATTTGAAATGAATTTTCTTCTAATATAGCAATTAATTTTATTT

AAATATCTCTAGAGTTTTTTTTTAATACTGTGACTAACCTATGTTTGTTCTTTTTCACCTC

TCGTATCCACGATCACTAAGAAACCCAAATACTTTGTTCATGTTTAAATTTTACAACATTT

CATAGACTATTAAACATGGAACATCCTTGTGGGGACAAGAAATCGAATTTGCTCTTGAAAA

GGTTTCCAACTAATTGATTTGTAGGACATTATAACATCCTCTAGCTGACAAGCTTACAAAA

ATAAAAACTGGAGCTAACCGAGAGGGTGCTTTTTTCCCTGACACATAAAAGGTGTCTTTCT

GTCTTGTATCCTTTGGATATGGGCATGTCAGTTTCATAGGGAAATTTTCACATGGAGCTTT

TGTATTTCTTTCTTTGCCAGTACAACTGCATGTGGTAGCACACTGTTTAATCTTTTCTCAA

ATAAAAAGACATGGGGCTTCATTTTTGTTTTGCCTTTTTGGTATCTTACAGGAACTCCAGG

ATGGCATTG

R6
Region in vicinity of acceptor of intron 46 (in range of −400
SEQ No. 385

to +37 bases from 3′ end of intron 46 as basing point)

ATAATGCAATTTCTCAGCAGCAAGCTACGGTATGCTATGGCATGCTATGATACCCAAGAGG

CTGATGAATTTGTTCACATTGTTCTATTTCTGATAGAGAGATAGGTTTTCAGACACTAACT

TTATTTGGAGTGTTGCTTTACCATCTCACATTTTTCTCTTAAAAAATTTATGAGGGATAAT

ATAATCGTTTATTTTCTACAGAGATTTATCTACTGAGGGGGTGAGTGTTTCAGTCAATCAG

CTCTGTGCTCAGATAGAAAACTGTTGGTATTTGAGGTACCACTGGGCCCTCGGTCAAGTCG

CTTCATTTTGATAGACTAATCAATAGAAGCAAAGACAAGGTAGTTGGAATTGTGCTGTAAT

TCATTTTAAACGTTGTTGCATTTGTCTGTTTCAGTTACTGGTGGAAGAGTTGCCCCTGCGC

CAGGGAATTC

R8
Region in vicinity of acceptor of intron 47 (in range of −400
SEQ No. 386

to +19 bases from 3′ end of intron 47 as basing point)

TATTCATTTTTATAACTGCAAAGGAAGCGCGTATGGCATATAATACACAACACACCAGTAT

ATTTAGTAACTGAGTGAATAAATGAAAGATGTATTTCTTTACTTTATCAGTTGCAGTTGGC

TATGCCTTTGTGTAAGGTGTGTGTTTTGAAATTCCAAAAAGGTATTAGTTTCTTTAAAGCA

AAGAATTTTTGTAGCAGGTTAATGAATAATTTTGAATACATTGGTTAAATCCCAACATGTA

ATATATGTAAATAATCAATATTATGCTGCTAAAATAACACAAATCAGTAAGATTCTGTAAT

ATTTCATGATAAATAACTTTTGAAAATATATTTTTAAACATTTTGGCTTATGCCTTGAGAA

TTATTTACCTTTTTAAAATGTATTTTCCTTTCAGGTTTCCAGAGCTTTACCTG

R10
Region in vicinity of acceptor of intron 48 (in range of −400
SEQ No. 387

to +42 bases from 3′ end of intron 48 as basing point)

TACTAAACACAGAATTTTGTAAAACAATAAGTGTATAAAGTAAAATGAACATTAGGATTAT

TGAGATTATTGTAGCTAAAACTAGTGTTTATTCATATAAATTATGTTAATAAATTGTATTG

TCATTATTGCATTTTACTTTTTTGAAAAGTAGTTAATGCCTGTGTTTCTATATGAGTATTA

TATAATTCAAGAAGATATTGGATGAATTTTTTTTAAGTTTAATGTGTTTCACATCTCTGTT

TCTTTTCTCTGCACCAAAAGCTACATTTTTGTGCCCTTATGTACCAGGCAGAAATTGATCT

GCAATACATGTGGAGTCTCCAAGGGTATATTTAAATTTAGTAATTTTATTGCTAACTGTGA

AGTTAATCTGCACTATATGGGTTCTTTTCCCCAGGAAACTGAAATAGCAGTTCAAGCTAAA

CAACCGGATGTGGAA

R12
Region in vicinity of acceptor of intron 49 (in range of −400
SEQ No. 388

to +44 bases from 3′ end of intron 49 as basing point)

ATATAATTGACTGGGGGTGAGCCAGTACATTAGGATTTTCCTAAAGTTATCTGGATAATTT

TAGTATGCAACCACAATAGATACTCTTCAAGAATTAAGCTAGTTGCTGAGAGGGAACTGTT

TTTTGTTGGTTTGTTTTCACTAATGTTTGCACTCTACTTCCTTTAAATAAAATTATGCCTG

GAGAAAGGGTTTTTGTATGGAGCAATTGATAAATATTTGTAGGGTGGTTGGCTAAAATAAT

TATAATTCCTTTAAAAGAAATTCTACCCACTAAAGTTAATTTAGAAGTAAAATATAATAGA

AATCCAATAATATATTCACCAAATGGATTAAGATGTTCATGAATTATCTTCAAAGTGTTAA

TCGAATAAGTAATGTGTATGCTTTTCTGTTAAAGAGGAAGTTAGAAGATCTGAGCTCTGAG

TGGAAGGCGGTAAACCG

R14
Region in vicinity of acceptor of intron 50 (in range of −400
SEQ No. 342

to +25 bases from 3′ end of intron 50 as basing point)

TCTTGAATAAAAAAAAAATAAGTAAAATTTATTTCCCTGGCAAGGTCTGAAAACTTTTGTT

TTCTTTACCACTTCCACAATGTATATGATTGTTACTGAGAAGGCTTATTTAACTTAAGTTA

CTTGTCCAGGCATGAGAATGAGCAAAATCGTTTTTTAAAAAATTGTTAAATGTATATTAAT

GAAAAGGTTGAATCTTTTCATTTTCTACCATGTATTGCTAAACAAAGTATCCACATTGTTA

GAAAAAGATATATAATGTCATGAATAAGAGTTTGGCTCAAATTGTTACTCTTCAATTAAAT

TTGACTTATTGTTATTGAAATTGGCTCTTTAGCTTGTGTTTCTAATTTTTCTTTTTCTTCT

TTTTTCCTTTTTGCAAAAACCCAAAATATTTTAGCTCCTACTCAGACTGTTACTCTGGTGA

CAC

R16
Region in vicinity of acceptor of intron 51 (in range of −400
SEQ No. 343

to +24 bases from 3′ end of intron 51 as basing point)

TTTAAAATAAATATACCTTAATTTTGACGTCACACAGAATGATATTATAAGTATAAATAGT

TATCTATCTTTTAAATACATTGTCGTAATTCAGAATAACATTTCTTACTCAAGGCATTCAG

ACAGTGGTTTAAGTAATCCGAGGTACTCCGGAATGTCTCCATTTGAGCCTTTAAATGAAGA

AAATCTATAGTCAAGATTTTCATTTGAAATATTTTTGATATCTAAGAATGAAACATATTTC

CTGTTAAATTGTTTTCTATAAACCCTTATACAGTAACATCTTTTTTATTTCTAAAAGTGTT

TTGGCTGGTCTCACAATTGTACTTTACTTTGTATTATGTAAAAGGAATACACAACGCTGAA

GAACCCTGATACTAAGGGATATTTGTTCTTACAGGCAACAATGCAGGATTTGGAACAGAGG

CGTCCCCAGTTGGAAGAACTCA

R18
Region in vicinity of acceptor of intron 52 (in range of −400
SEQ No. 344

to +34 bases from 3′ end of intron 52 as basing point)

CAAACTCCTGTGGCAACAGAAAGCCTTCAGGCAATGAAATGCTGGCACTGGGAAATCAGGC

TGATGGGTGCTGAAGTGGCAAGGATGAGGGGATATGGATATTCTGCTGTAGTGCTTTTCTA

ACAGATGATTCATATTTGGTTCTAGGGATCAAGAATTGAGTTAAAATTTTATATATATGTT

GATGTTCTATGTCACCTTCAGGAAAATAATTTAACAGAAACTAATATTTGCCATCAAAAAA

GCAAAGAATCCTGTTGTTCATCATCCTAGCCATAACACAATGAATAATTTTTTAAATAAGC

AACATAAATGTGAGATAACGTTTGGAAGTTACATTTAAAATGTCTCCTCCAGACTAGCATT

TACTACTATATATTTATTTTTCCTTTTATTCTAGTTGAAAGAATTCAGAATCAGTGGGATG

AAGTACA

R20
Region in vicinity of acceptor of intron 53 (in range of −400
SEQ No. 389

to +43 bases from 3′ end of intron 53 as basing point)

CAACAAGTTAATGAAGAGGGAAAGAAATGTATGAGGTTTTTTTCGTTCAAATGTTGTTATA

TGTCACATATTCAACAATTATATATGAGCTTATTTTTGTAGTTTTTTTCTCTTGTGATAAA

AACAATTAAGCCCACTTTATTGCCAATTAATTGCTACTAAGTTGAAATACTTGATACTGGT

TATTGCTCAAGATGCTGCATTTGAAAAGTTTGTCCTGAAAGGTGGGTTACCTTATACTGTC

ATGATTGACTAAATCATATGGTAGGTTAAAAGCAATCTAATATATGTATTCTGACCTGAGG

ATTCAGAAGCTGTTTACGAAGTATTTTAAGACACTCCAACTAGAGATTTCATAAAAAAAAC

TGACATTCATTCTCTTTCTCATAAAAATCTATAGCAGTTGGCCAAAGACCTCCGCCAGTGG

CAGACAAATGTAGATG

R22
Region in vicinity of acceptor of intron 54 (in range of −400
SEQ No. 345

to +25 bases from 3′ end of intron 54 as basing point)

TCTCAAATTTGGCAGTATATTAAAAATAAGCTTTCAAAATTGACCAACAAAAACTACAAAA

TTGAAAAAAAGGTACTTTGAACTTTCACATGTTCAAATATATGTATATATATTTCACATAT

ATATATGAAACCTCCTCTGTGGAGAGGGGTTTATAGAAATCTGTAATTGTCATTCTTGCAT

GCCTTCCCCCATACAAACGCCTTTAAGTTAAATAAAAATGAAAGTAAATAGACTGCACAAT

ATTATAGTTGTTGCTTAAAGGAAGAGCTGTAGCAACAACTCACCCCATTGTTGGTATATTA

CAATTTAGTTCCTCCATCTTTCTCTTTTTATGGAGTTCACTAGGTGCACCATTCTGATATT

TAATAATTGCATCTGAACATTTGGTCCTTTGCAGGGTGAGTGAGCGAGAGGCTGCTTTG

In a further alternative aspect, the antisense oligomer of the present invention targets at least any one region selected from the group consisting of regions R1 to R24 represented by

region Rn (wherein n is an odd number of 1 to 23) which consists of a base sequence of NY bases in the downstream direction from the 5′ end of the NBth intron in the human dystrophin pre-mRNA, and

region Rn (wherein n is an even number of 2 to 24) which consists of a base sequence of NX bases in the upstream direction from the 3′ end of the NAth intron in the human dystrophin pre-mRNA.

In the foregoing embodiment, examples of the target region Rn of the present invention include, but not limited to, regions wherein

when n is 1, NB=44 and NY=400,

when n is 2, NA=44 and NX=600,

when n is 3, NB=45 and NY=400,

when n is 4, NA=45 and NX=400,

when n is 5, NB=46 and NY=400,

when n is 6, NA=46 and NX=400,

when n is 7, NB=47 and NY=400,

when n is 8, NA=47 and NX=400,

when n is 9, NB=48 and NY=400,

when n is 10, NA=48 and NX=400,

when n is 11, NB=49 and NY=400,

when n is 12, NA=49 and NX=400,

when n is 13, NB=50 and NY=400,

when n is 14, NA=50 and NX=400,

when n is 15, NB=51 and NY=400,

when n is 16, NA=51 and NX=400,

when n is 17, NB=52 and NY=400,

when n is 18, NA=52 and NX=400,

when n is 19, NB=53 and NY=400,

when n is 20, NA=53 and NX=400,

when n is 21, NB=54 and NY=400,

when n is 22, NA=54 and NX=400,

when n is 23, NB=55 and NY=400, or

when n is 24, NA=55 and NX=400.

R1 to R24 which are examples of the target region Rn of the present invention according to the foregoing embodiment are, but not limited to, as follows:

for example, in a certain embodiment,

the region R1 is a region that consists of a base sequence of 400 bases in the downstream direction from the 5′ end of the 44th intron in the human dystrophin pre-mRNA,

the region R2 is a region that consists of a base sequence of 600 bases in the upstream direction from the 3′ end of the 44th intron in the human dystrophin pre-mRNA,

the region R3 is a region that consists of a base sequence of 400 bases in the downstream direction from the 5′ end of the 45th intron in the human dystrophin pre-mRNA,

the region R4 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 45th intron in the human dystrophin pre-mRNA,

the region R5 is a region that consists of a base sequence of 400 bases in the downstream direction from the 5′ end of the 46th intron in the human dystrophin pre-mRNA,

the region R6 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 46th intron in the human dystrophin pre-mRNA,

the region R7 is a region that consists of a base sequence of 400 bases in the downstream direction from the 5′ end of the 47th intron in the human dystrophin pre-mRNA,

the region R8 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 47th intron in the human dystrophin pre-mRNA,

the region R9 is a region that consists of a base sequence of 400 bases in the downstream direction from the 5′ end of the 48th intron in the human dystrophin pre-mRNA,

the region R10 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 48th intron in the human dystrophin pre-mRNA,

the region R11 is a region that consists of a base sequence of 400 bases in the downstream direction from the 5′ end of the 49th intron in the human dystrophin pre-mRNA,

the region R12 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 49th intron in the human dystrophin pre-mRNA,

the region R13 is a region that consists of a base sequence of 400 bases in the downstream direction from the 5′ end of the 50th intron in the human dystrophin pre-mRNA,

the region R14 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 50th intron in the human dystrophin pre-mRNA,

the region R15 is a region that consists of a base sequence of 400 bases in the downstream direction from the 5′ end of the 51st intron in the human dystrophin pre-mRNA,

the region R16 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 51st intron in the human dystrophin pre-mRNA,

the region R17 is a region that consists of a base sequence of 400 bases in the downstream direction from the 5′ end of the 52nd intron in the human dystrophin pre-mRNA,

the region R18 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 52nd intron in the human dystrophin pre-mRNA,

the region R19 is a region that consists of a base sequence of 400 bases in the downstream direction from the 5′ end of the 53rd intron in the human dystrophin pre-mRNA,

the region R20 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 53rd intron in the human dystrophin pre-mRNA,

the region R21 is a region that consists of a base sequence of 400 bases in the downstream direction from the 5′ end of the 54th intron in the human dystrophin pre-mRNA,

the region R22 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 54th intron in the human dystrophin pre-mRNA,

the region R23 is a region that consists of a base sequence of 400 bases in the downstream direction from the 5′ end of the 55th intron in the human dystrophin pre-mRNA, or

the region R24 is a region that consists of a base sequence of 400 bases in the upstream direction from the 3′ end of the 55th intron in the human dystrophin pre-mRNA.

In another embodiment, R1 to R24 which are examples of the target region Rn of the present invention according to the foregoing embodiment are, but not limited to, as follows:

(A1) Region R1

The region R1 is a region indicated by the range of +1 base to +400 bases when the boundary between the 3′ end of exon 44 and the 5′ end of intron 44 is defined as basing point 0 and a base sequence region on the 3′ side (downstream) from the basing point is indicated by “+”. In this respect, the region indicated by the range of +1 base to +400 bases belongs to intron 44.

(A2) Region R2

The region R2 is a region indicated by the range of −600 bases to −1 base when the boundary between the 3′ end of intron 44 and the 5′ end of exon 45 is defined as basing point 0 and a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus). In this respect, the region indicated by the range of −600 bases to −1 base belongs to intron 44.

(A3) Region R3

The region R3 is a region indicated by the range of +1 base to +400 bases when the boundary between the 3′ end of exon 45 and the 5′ end of intron 45 is defined as basing point 0 and a base sequence region on the 3′ side (downstream) from the basing point is indicated by “+”. In this respect, the region indicated by the range of +1 base to +400 bases belongs to intron 45.

(A4) Region R4

The region R4 is a region indicated by the range of −400 bases to −1 base when the boundary between the 3′ end of intron 45 and the 5′ end of exon 46 is defined as basing point 0 and a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus). In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 45.

(A5) Region R5

The region R5 is a region indicated by the range of +1 base to +400 bases when the boundary between the 3′ end of exon 46 and the 5′ end of intron 46 is defined as basing point 0 and a base sequence region on the 3′ side (downstream) from the basing point is indicated by “+”. In this respect, the region indicated by the range of +1 base to +400 bases belongs to intron 46.

(A6) Region R6

The region R6 is a region indicated by the range of −400 bases to −1 base when the boundary between the 3′ end of intron 46 and the 5′ end of exon 47 is defined as basing point 0 and a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus). In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 46.

(A7) Region R7

The region R7 is a region indicated by the range of +1 base to +400 bases when the boundary between the 3′ end of exon 47 and the 5′ end of intron 47 is defined as basing point 0 and a base sequence region on the 3′ side (downstream) from the basing point is indicated by “+”. In this respect, the region indicated by the range of +1 base to +400 bases belongs to intron 47.

(A8) Region R8

The region R8 is a region indicated by the range of −400 bases to −1 base when the boundary between the 3′ end of intron 47 and the 5′ end of exon 48 is defined as basing point 0 and a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus). In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 47.

(A9) Region R9

The region R9 is a region indicated by the range of +1 base to +400 bases when the boundary between the 3′ end of exon 48 and the 5′ end of intron 48 is defined as basing point 0 and a base sequence region on the 3′ side (downstream) from the basing point is indicated by “+”. In this respect, the region indicated by the range of +1 base to +400 bases belongs to intron 48.

(A10) Region R10

The region R10 is a region indicated by the range of −400 bases to −1 base when the boundary between the 3′ end of intron 48 and the 5′ end of exon 49 is defined as basing point 0 and a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus). In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 48.

(A11) Region R11

The region R11 is a region indicated by the range of +1 base to +400 bases when the boundary between the 3′ end of exon 49 and the 5′ end of intron 49 is defined as basing point 0 and a base sequence region on the 3′ side (downstream) from the basing point is indicated by “+”. In this respect, the region indicated by the range of +1 base to +400 bases belongs to intron 49.

(A12) Region R12

The region R12 is a region indicated by the range of −400 bases to −1 base when the boundary between the 3′ end of intron 49 and the 5′ end of exon 50 is defined as basing point 0 and a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus). In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 49.

(A13) Region R13

The region R13 is a region indicated by the range of +1 base to +400 bases when the boundary between the 3′ end of exon 50 and the 5′ end of intron 50 is defined as basing point 0 and a base sequence region on the 3′ side (downstream) from the basing point is indicated by “+”. In this respect, the region indicated by the range of +1 base to +400 bases belongs to intron 50.

(A14) Region R14

The region R14 is a region indicated by the range of −400 bases to −1 base when the boundary between the 3′ end of intron 50 and the 5′ end of exon 51 is defined as basing point 0 and a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus). In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 50.

(A15) Region R15

The region R15 is a region indicated by the range of +1 base to +400 bases when the boundary between the 3′ end of exon 51 and the 5′ end of intron 51 is defined as basing point 0 and a base sequence region on the 3′ side (downstream) from the basing point is indicated by “+”. In this respect, the region indicated by the range of +1 base to +400 bases belongs to intron 51.

(A16) Region R16

The region R16 is a region indicated by the range of −400 bases to −1 base when the boundary between the 3′ end of intron 51 and the 5′ end of exon 52 is defined as basing point 0 and a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus). In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 51.

(A17) Region R17

The region R17 is a region indicated by the range of +1 base to +400 bases when the boundary between the 3′ end of exon 52 and the 5′ end of intron 52 is defined as basing point 0 and a base sequence region on the 3′ side (downstream) from the basing point is indicated by “+”. In this respect, the region indicated by the range of +1 base to +400 bases belongs to intron 52.

(A18) Region R18

The region R18 is a region indicated by the range of −400 bases to −1 base when the boundary between the 3′ end of intron 52 and the 5′ end of exon 53 is defined as basing point 0 and a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus). In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 52.

(A19) Region R19

The region R19 is a region indicated by the range of +1 base to +400 bases when the boundary between the 3′ end of exon 53 and the 5′ end of intron 53 is defined as basing point 0 and a base sequence region on the 3′ side (downstream) from the basing point is indicated by “+”. In this respect, the region indicated by the range of +1 base to +400 bases belongs to intron 53.

(A20) Region R20

The region R20 is a region indicated by the range of −400 bases to −1 base when the boundary between the 3′ end of intron 53 and the 5′ end of exon 54 is defined as basing point 0 and a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus). In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 53.

(A21) Region R21

The region R21 is a region indicated by the range of +1 base to +400 bases when the boundary between the 3′ end of exon 54 and the 5′ end of intron 54 is defined as basing point 0 and a base sequence region on the 3′ side (downstream) from the basing point is indicated by “+”. In this respect, the region indicated by the range of +1 base to +400 bases belongs to intron 54.

(A22) Region R22

The region R22 is a region indicated by the range of −400 bases to −1 base when the boundary between the 3′ end of intron 54 and the 5′ end of exon 55 is defined as basing point 0 and a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus). In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 54.

(A23) Region R23

The region R23 is a region indicated by the range of +1 base to +400 bases when the boundary between the 3′ end of exon 55 and the 5′ end of intron 55 is defined as basing point 0 and a base sequence region on the 3′ side (downstream) from the basing point is indicated by “+”. In this respect, the region indicated by the range of +1 base to +400 bases belongs to intron 55.

(A24) Region R24

The region R24 is a region indicated by the range of −400 bases to −1 base when the boundary between the 3′ end of intron 55 and the 5′ end of exon 56 is defined as basing point 0 and a base sequence region on the 5′ side (upstream) from the basing point is indicated by “−” (minus). In this respect, the region indicated by the range of −400 bases to −1 base belongs to intron 55.

In the foregoing embodiment, specific examples of the base sequences of the regions R1 to R24 are as follows.

TABLE 3

SEQ

Region
Base sequence of target region
ID NO

R1
Region in vicinity of donor of intron 44 (in range of +1
SEQ

to +400 bases from 5′ end of intron 44 as basing point)
No.

GTAAGTCTTTGATTTGTTTTTTCGAAATTGTATTTATCTTCAGCACATCTGGACTCTTTAA
346

CTTCTTAAAGATCAGGTTCTGAAGGGTGATGGAAATTACTTTTGACTGTTGTTGTCATCAT

TATATTACTAGAAAGAAAATTATCATAATGATAATATTAGAGCACGGTGCTATGGACTTTT

TGTGTCAGGATGAGAGAGTTTGCCTGGACGGAGCTGGTTTATCTGATAAACTGCAAAATAT

AATTGAATCTGTGACAGAGGGAAGCATCGTAACAGCAAGGTGTTTTGTGGCTTTGGGGCAG

TGTGTATTTCGGCTTTATGTTGGAACCTTTCCAGAAGGAGAACTTGTGGCATACTTAGCTA

AAATGAAGTTGCTAGAAATATCCATCATGATAAA

R2
Region in vicinity of acceptor of intron 44 (in range of
SEQ

−600 to −1 bases from 3′ end of intron 44 as basing point)
No.

TCTTGATGGGATGCTCCTGAAAGCAATTAATTCTCAGTTTTTTGTGGCTTCTAATGCAAAA
347

TACATTGACGCAGACAGAATTTGAAATGAATTTTCTTCTAATATAGCAATTAATTTTATTT

AAATATCTCTAGAGTTTTTTTTTAATACTGTGACTAACCTATGTTTGTTCTTTTTCACCTC

TCGTATCCACGATCACTAAGAAACCCAAATACTTTGTTCATGTTTAAATTTTACAACATTT

CATAGACTATTAAACATGGAACATCCTTGTGGGGACAAGAAATCGAATTTGCTCTTGAAAA

GGTTTCCAACTAATTGATTTGTAGGACATTATAACATCCTCTAGCTGACAAGCTTACAAAA

ATAAAAACTGGAGCTAACCGAGAGGGTGCTTTTTTCCCTGACACATAAAAGGTGTCTTTCT

GTCTTGTATCCT7TGGATATGGGCATGTCAGTTTCATAGGGAAATTTTCACATGGAGCTTT

TGTATTTCTTTCTTTGCCAGTACAACTGCATGTGGTAGCACACTGTTTAATCTTTTCTCAA

ATAAAAAGACATGGGGCTTCATTTTTGTTTTGCCTTTTTGGTATCTTACAG

R3
Region in vicinity of donor of intron 45 (in range of +1 to
SEQ

+400 bases from 5′ end of intron 45 as basing point)
No.

GTAGGGCGACAGATCTAATAGGAATGAAAACATTTTAGCAGACTTTTTAAGCTTTCTTTAG
348

AAGAATATTTCATGAGAGATTATAAGCAGGGTGAAAGGCACTAACATTAAAGAACCTATCA

ACCATTAATCAACAGCAGTAAAGAAATTTTTTATTTCTTTTTTTCATATACTAAAATATAT

ACTTGTGGCTAGTTAGTGGTTTTCTGCTATTTTAAACTTGAAGTTTGCTTTAAAAATCACC

CATGATTGCTTAAAGGTGAATATCTTCAATATATTTTAACTTCAACAAGCTGAATCTCAGT

TGTTTTTCAAGAAGATTTTAGAAAGCAATTATAAATGATTGTTTTGTAGGAAAGACAGATC

TTTGCTTAGTTTTAAAAATAGCTATGAATATGAC

R4
Region in vicinity of acceptor of intron 45 (in range of
SEQ

−400 to −1 bases from 3′ end of intron 45 as basing point)
No.

ATTACAGGCGCCTGCCACCAAACCTGGCAAATTTTTGTATTTTTAGTGTAGACGGGGTTTC
349

ACCATATTTGCCAGGCTGGTCGCAAACTCCTGACCTCAAGTGATCCGCCCACATCGGCCTC

CCTAAGCGCTAGGGTTACAGGCATGAGCCACTGCGCCTGGCCAGGAATTTTTGAATCAGAA

TTTTTCTTGTTCGATTTTAATCTCTTATCATTTAGAGATTCTTGAAATATTGAAATTACTT

TGTTCAAAGTGAATGAATTTTCTTAAATTATGTATGGTTAACATCTTTTAAATTGCTTATT

TTTAAATTGCCATGTTTGrGTCCCAGTTTGCATTAACAAATAGTTTGAGAACTATGTTGGA

AAAAAAAATAACAATTTTATTCTTCTTTCTCCAG

R5
Region in vicinity of donor of intron 46 (in range of +1 to
SEQ

+400 bases from 5′ end of intron 46 as basing point)
No.

GTAATTTTATTTTCTCAAATCCCCCAGGGCCTGCTTGCATAAAGAAGTATATGAATCTATT
350

TTTTAATTCAATCATTGGTTTTCTGCCCATTAGGTTATTCATAGTTCCTTGCTAAAGTGTT

TTTCTCACAACTTTATTTCTTCTTAACCCTGCAGTTCTGAACCAGTGCACATAAGAACATA

TGTATATATGTGTGTGTGTGTATTTATATATACACACACACATATTGCATCTATACATCTA

CACATATAGATGTATAGATTCAATATGTCTAAATGTATATAATTCACAGTTTTTATCTTTG

ATTTGAAATTAATTTTAGATTTTACTTGAGAACTTCACAACTTCATATAATTTTAAAAACT

GAAGACCAGATTGTGGAATCATAAAATCTAAATC

R6
Region in vicinity of acceptor of intron 46 (in range of
SEQ

−400 to −1 n bases from 3′ end of intron 46 as basing point)
No.

ATAATGCAATTTCTCAGCAGCAAGCTACGGTATGCTATGGCATGCTATGATACCCAAGAGG
351

CTGATGAATTTGTTCACATTGTTCTATTTCTGATAGAGAGATAGGTTTTCAGACACTAACT

TTATTTGGAGTGTTGCTTTACCATCTCACATTTTTCTCTTAAAAAATTTATGAGGGATAAT

ATAATCGTTTATTTTCTACAGAGATTTATCTACTGAGGGGGTGAGTGTTTCAGTCAATCAG

CTCTGTGCTCAGATAGAAAACTGTTGGTATTTGAGGTACCACTGGGCCCTCGGTCAAGTCG

CTTCATTTTGATAGACTAATCAATAGAAGCAAAGACAAGGTAGTTGGAATTGTGCTGTAAT

TCATTTTAAACGTTGTTGCATTTGTCTGTTTCAG

R7
Region in vicinity of donor of intron 47 (in range of +1 to
SEQ

+400 bases from 5′ end of intron 47 as basing point)
No.

GTTAGACATTAACCATCTCTTCCGTCACATGTGTTAAATGTTGCAAGTATTTGTATGTATT
352

TTGTTTCCTGGGTGCTTCATTGGTCGGGGAGGAGGCTGGTATGTGGATTGTTGTTTTGTTT

TGTTTTTTTAACCTGACCGTTTGCTTTGGCTATATGTTTTGTTGTGGCTAGAAAAAATGAT

GATGGTGAATGGCTTTACATTAATGACCAAATGCCAAAATTTATACCACAATTTTTTGCAT

AAATTATTCTGAAGAATCAGACTGAAGAAATGGCGAAGTATTTAATTCAGTGGCCAGGCAT

GTACTGACAGTATTTAAGCTGAAAGGACGTGGTCTGGTTCTAGTTAAACAAGTGTCATAAA

TCAAAATTAATTATTCACACCTG7GGTATGGACT

R8
Region in vicinity of acceptor of intron 47 (in range of
SEQ

−400 to −1 bases from 3′ end of intron 47 as basing point)
No.

TATTCATTTTTATAACTGCAAAGGAAGCGCGTATGGCATATAATACACAACACACCAGTAT
353

ATTTAGTAACTGAGTGAATAAATGAAAGATGTATTTCTTTACTTTATCAGTTGCAGTTGGC

TATGCCTTTGTGTAAGGTGTGTGTTTTGAAATTCCAAAAAGGTATTAGTTTCTTTAAAGCA

AAGAATTTTTGTAGCAGGTTAATGAATAATTTTGAATACATTGGTTAAATCCCAACATGTA

ATATATGTAAATAATCAATATTATGCTGCTAAAATAACACAAATCAGTAAGATTCTGTAAT

ATTTCA7GATAAATAACTTTTGAAAATATATTTTTAAACATTTTGGCTTATGCCTTGAGAA

TTATTTACCTTTTTAAAATGTATTTTCCTTTCAG

R9
Region in vicinity of donor of intron 48 (in range of +1 to

+400 bases from 5′ end of intron 48 as basing point)

GTAGGGAACTTTTTGCTTTAAATATTTTTGTCTTTTTTAAGAAAAATGGCAATATCACTGA
SEQ

ATTTTCTCATTTGGTATCATTATTAAAGACAAAATATTACTTGTTAAAGTGTGGTAAGGAA
No.

GACTTTATTCAGGATAACCACAATAGGCACAGGGACCACTGCAATGGAGTATTACAGGAGG
354

TTGGATAGAGAGAGATTGGGCTCAACTCTAAATACAGCACAGTGGAAGTAGGAATTTATAG

CCAAGGAGCAGTGTAGGAGTCAGTAGATGGAAAATTATTAAGAGGAAACATCAGGGGTAAG

TGGGATTCTGGCTAAACCAACCTCACAGGATTCTTGCTGAAGATAGGCCAGGGTTATCTTA

TCAGACAACCCTTGGGGAATGGTGGAGAATACTG

R10
Region in vicinity of acceptor of intron 48 (in range of
SEQ

−400 to −1 bases from 3′ end of intron 48 as basing point)
No.

TACTAAACACAGAATTTTGTAAAACAATAAGTGTATAAAGTAAAATGAACATTAGGATTAT
355

TGAGATTATTGTAGCTAAAACTAGTGTTTATTCATATAAATTATGTTAATAAATTGTATTG

TCATTATTGCATTTTACTTTTTTGAAAAGTAGTTAATGCCTGTGTTTCTATATGAGTATTA

TATAATTCAAGAAGATATTGGATGAATTTTTTTTAAGTTTAATGTGTTTCACATCTCTGTT

TCTTTTCTCTGCACCAAAAGCTACATTTTTGTGCCC7TATGTACCAGGCAGAAATTGATCT

GCAATACATGTGGAGTCTCCAAGGGTATATTTAAATTTAGTAATTTTATTGCTAACTGTGA

AGTTAATCTGCACTATATGGGTTCTTTTCCCCAG

R11
Region in vicinity of donor of intron 49 (in range of +1 to
SEQ

+400 bases from 5′ end of intron 49 as basing point)
No.

GTAATGAAGCAACCTCTAGCAATATCCATTACCTCATAATGGGTTATGCTTCCCCTGTTGT
356

ACATTTGCCATTGACGTGGACTATTTATAATCAGTGAAATAACTTGTAAGGAAATACTGGC

CATACTGTAATAGCAGAGGCAAAGCTGTCTTTTTGATCAGCATATCCTATTTATATATTGT

GATCT7AAGGCTATTAACGAGTCATTGCTTTAAAGGACTCATTTCTGTCCTGGTGTGCTGC

CATCAATACAAAAGTAGTCCCACCTTCAAGGTAGATTAAATTCTTTGGGGCTTTATTGCTT

TGCTTGCCAGCCTTGATGCTTTTCATATTGTTTGGTTTAATTCAAATCAAGCTACTGCATC

ATAGTGTCTGTCTCCAACAGCTGTAAAGAATCAC

R12
Region in vicinity of acceptor of intron 49 (in range of
SEQ

−400 to −1 bases from 3′ end of intron 49 as basing point)
No.

ATATAATTGACTGGGGGTGAGCCAGTACATTAGGATTTTCCTAAAGTTATCTGGATAATTT
357

TACTATGCAACCACAATAGATACTCTTCAAGAATTAAGCTAGTTGCTGAGAGGGAACTGTT

TTTTGTTGGTTTGTTTTCACTAATGTTTGCACTCTACTTCCTTTAAATAAAATTATGCCTG

GAGAAAGGGTTTTTGTATGGAGCAATTGATAAATATTTGTAGGGTGGTTGGCTAAAATAAT

TATAATTCCTTTAAAAGAAATTCTACCCACTAAAGTTAATTTAGAAGTAAAATATAATAGA

AATCCAATAATATATTCACCAAATGGATTAAGATGTTCATGAATTATCTTCAAAGTGTTAA

TCGAATAAGTAATGTGTATGCTTTTCTGTTAAAG

R13
Region in vicinity of donor of intron 50 (in range of +1 to
SEQ

+400 bases from 5′ end of intron 50 as basing point)
No.

GTAAGTATACTGGATCCCATTCTCTTTGGCTCTAGCTATTTGTTCAAAAGTGCAACTATGA
358

AGTGATGACTGGGTGAGAGAGAAAATTTGTTTCAATTCTAAAGATAGAGATAAACCTTTGT

GTTATTGACTGTGCAAAAAGTCTTAGAGTACATTCCTTGGAAATTGACTCTGATTCAAAGT

GTTGCATGACAACGGGATATGGGGAGTGTTCTCTGGAGATACACCCACAAGGAAGAGAAGA

GCACAAGGGAGATTGTGGGAGAGTCTGAAATGTGATTTGTCTGCAGCAGAGGCCTAAGCCA

GTCTCGCAGGAGCCCTACATCTGGGCTGGCTGTGCAGAGCTGTCCTGAATTGCAGGCAGTG

GGCCTGGCCCTTGTATTCCTGATCCAGCCAGCCA

R14
Region in vicinity of acceptor of intron 50 (in range of
SEQ

−400 to −1 bases from 3′ end of intron 50 as basing point)
No.

TCTTGAATAAAAAAAAAATAAGTAAAATTTATTTCCCTGGCAAGGTCTGAAAACTTTTGTT
359

TTCTTTACCACTTCCACAATGTATATGATTGTTACTGAGAAGGCTTATTTAACTTAAGTTA

CTTGTCCAGGCATGAGAATGAGCAAAATCGTTTTTTAAAAAATTGTTAAATGTATATTAAT

GAAAAGGTTGAATCTTTTCATTTTCTACCATGTATTGCTAAACAAAGTATCCACATTGTTA

GAAAAAGATATATAATGTCATGAATAAGAGTTTGGCTCAAATTGTTACTCTTCAATTAAAT

TTGACTTATTGTTATTGAAATTGGCTCTTTAGCTTGTGTTTCTAATTTTTCTTTTTCTTCT

TTTTTCCTTTTTGCAAAAACCCAAAATATTTTAG

R15
Region in vicinity of donor of intron 51 (in range of +1 to
SEQ

+400 bases from 5′ end of intron 51 as basing point)
No.

GTATGAGAAAAAATGATAAAAGTTGGCAGAAGTTTTTCTTTAAAATGAAGATTTTCCACCA
360

ATCACTTTACTCTCCTAGACCATTTCCCACCAGTTCTTAGGCAACTGTTTCTCTCTCAGCA

AACACATTACTCTCACTATTCAGCCTAAGTATAATCAAGGATATAAATTAATGCAAATAAC

AAAAGTAGCCATACATTAAAAAGGAAATATACAAAAAAAAAAAAAAAAAAAAGCAGAAACC

TTACAAGAATAGTTGTCTCAGTTAAATTTACTAAACAACCTGGTATTTTAAAAATCTATTT

TATACCAAATAAGTCACTCAACTGAGCTATTTACATTTAAACTGTTTGTTTTGGCACTACG

CAGCCCAACATATTGCAGAATCAAATATAATAGT

R16
Region in vicinity of acceptor of intron 51 (in range of
SEQ

−400 to −1 bases from 3′ end of intron 51 as basing point)
No.

TTTAAAATAAATATACCTTAATTTTGACGTCACACAGAATGATATTATAAGTATAAATAGT
361

TATCTATCTTTTAAATACATTGTCGTAATTCAGAATAACATTTCTTACTCAAGGCATTCAG

ACAGTGGTTTAAGTAATCCGAGGTACTCCGGAATGTCTCCATTTGAGCCTTTAAATGAAGA

AAATCTATAGTCAAGATTTTCATTTGAAATATTTTTGATATCTAAGAATGAAACATATTTC

CTGTTAAATTGTTTTCTATAAACCCTTATACAGTAACATCTTTTTTATTTCTAAAAGTGTT

TTGGCTGGTCTCACAATTGTACTTTACTTTGTATTATGTAAAAGGAATACACAACGCTGAA

GAACCCTGATACTAAGGGATATTTGTTCTTACAG

R17
Region in vicinity of donor of intron 52 (in range of +1 to

+400 bases from 5′ end of intron 52 as basing point)

GTAAGTTTTTTAACAAGCATGGGACACACAAAGCAAGATGCATGACAAGTTTCAATAAAAA
SEQ

CTTAAGTTCATATATCCCCCTCACATTTATAAAAATAATGTGAAATAATTGTAAATGATAA
No.

CAATTGTGCTGAGATTTTCAGTCCATAATGTTACCTTTTAATAAATGAATGTAATTCCATT
362

GAATAGAAGAAATACATTTTTAAATCAATTCAGGGCTTATATAGTTGCAAAGCATGCATTG

ATGGGTGTGGTGACCACAGTGTGGCAGAACATTTGTGGCAGAACATTTGTTCTTTAGTTGT

CATCTGGGCTGGCATCCATGGAGATGCCAGTCTCTCCCTCATATCCTTGGCTGTTGGTCCA

AGCAGGCAGTGGCTTCTTCCTGGGCCATCTTTCA

R18
Region in vicinity of acceptor of intron 52 (in range of
SEQ

−400 to −1 bases from 3′ end of intron 52 as basing point)
No.

CAAACTCCTGTGGCAACAGAAAGCCTTCAGGCAATGAAATGCTGGCACTGGGAAATCAGGC
363

TGATGGGTGCTGAAGTGGCAAGGATGAGGGGATATGGATATTCTGCTGTAGTGCTTTTCTA

ACAGATGATTCATATTTGGTTCTAGGGATCAAGAATTGAGTTAAAATTTTATATATATGTT

GATGTTCTATGTCACCTTCAGGAAAATAATTTAACAGAAACTAATATTTGCCATCAAAAAA

GCAAAGAATCCTGTTGTTCATCATCCTAGCCATAACACAATGAATAATTTTTTAAATAAGC

AACATAAATGTGAGATAACGTTTGGAAGTTACATTTAAAATGTCTCCTCCAGACTAGCATT

TACTACTATATATTTATTTTTCCTTTTATTCTAG

R19
Region in vicinity of donor of intron 53 (in range of +1 to
SEQ

+400 bases from 5′ end of intron 53 as basing point)
No.

GTTAGTATCAAAGATACCTTTTTAAAATAAAATACTGGTTACATTTGATAAAATTATACCA
364

TAGATTGTAATTTAATGATGTTTAATGTAAAGTTATTAACAGAAAATCACGTTAAAGCTGA

AATGAACAGTAGACTTTGTATATTTATTTTCTTAGAGACAGAGTCTCACTGTCACCCAGGC

TAAAGTGCAGTGGCACAATCATAGCTCACTGAGCCTTGAACTCTGGGGCTCAAGCAGTCCT

CCTGCCTCAGCCTCCCTAGTAGCTGGGACTACTAGCCAGGCGTGTACCACCACGCCTGGCT

AATTTTTTAAAAATTTTTGTTTTCTGTAGAGATGGGTTCTTGAACTCTTGGCCTCAAGCAA

TTCTCCTTCCTTGGCCTCCCAAAGCACTAGGATT

R20
Region in vicinity of acceptor of intron 53 (in range of
SEQ

−400 to −1 bases from 3′ end of intron 53 as basing point)
No.

CAACAAGTTAATGAAGAGGGAAAGAAATGTATGAGGTTTTTTTCGTTCAAATGTTGTTATA
365

TGTCACATATTCAACAATTATATATGAGCTTATTTTTGTAGTTTTTTTCTCTTG7GATAAA

AACAATTAAGCCCACTTTATTGCCAATTAATTGCTACTAAGTTGAAATACTTGATACTGGT

TATTGCTCAAGATGCTGCATTTGAAAAGTTTGTCCTGAAAGGTGGGTTACCTTATACTGTC

ATGATTGACTAAATCATATGGTAGGTTAAAAGCAATCTAATATATGTATTCTGACCTGAGG

ATTCAGAAGCTGTTTACGAAGTATTTTAAGACACTCCAACTAGAGATTTCATAAAAAAAAC

TGACATTCATTCTCTTTCTCATAAAAATCTATAG

R21
Region in vicinity of donor of intron 54 (in range of +1 to
SEQ

+400 bases from 5′ end of intron 54 as basing point)
No.

GTATGAATTACATTATTTCTAAAACTACTGTTGGCTGTAATAATGGGGTGGTGAAACTGGA
366

TGGACCATGAGGATTTGTTTTTCCAATCCAGCTAAACTGGAGCTTGGGAGGGTTCAAGACG

ATAAATACCAACTAAACTCACGGACTTGGCTCAGACTTCTATTTTAAAAACGAGGAACATA

AGATCTCATTTGCCCGCTGTCACAAAAGTAGTGACATAACCAAGAGATTAAACAAAAAGCA

AAATACTGATTTATAGCTAGAAGAGCCATTTATCAGTCTACTTTGATAACTCTATCCAAAG

GAATATCTTTCTATCTCATCATGGCGCACACTGCCTTACCTGTTATCTGATAAATAAGTCA

CTTTGGGATTCATGATAGAGTTATAGCTGTACAT

R22
Region in vicinity of acceptor of intron 54 (in range of
SEQ

−400 to −1 bases from 3′ end of intron 54 as basing point)
No.

TCTGAAATTTGGCAGTATATTAAAAATAAGCTTTCAAAATTGACCAACAAAAACTACAAAA
367

TTGAAAAAAAGGTACTTTGAACTTTCACATGTTCAAATATATGTATATATATTTCACATAT

ATATATGAAACCTCCTCTGTGGAGAGGGGTTTATAGAAATCTGTAATTGTCATTCTTGCAT

GCCTTCCCCCATACAAACGCCTTTAAGTTAAATAAAAATGAAAGTAAATAGACTGCACAAT

ATTATAGTTGTTGCTTAAAGGAAGAGCTGTAGCAACAACTCACCCCATTGTTGGTATATTA

CAATTTAGTTCCTCCATCTTTCTCTTTTTATGGAGTTCACTAGGTGCACCATTCTGATATT

TAATAATTGCATCTGAACATTTGGTCCTTTGCAG

R23
Region in vicinity of donor of intron 55 (in range of +1 to
SEQ

+400 nucleotides from 5′ end of intron 55 as basing point)
No.

GTACTTGTAACTGACAAGCCAGGGACAAAACAAAATAGTTGCTTTTATACAGCCTGATGTA
368

TTTCGGTATTTGGACAAGGAGGAGAGAGGCAGAGGGAGAAGGAAACATCATTTATAATTCC

ACTTAACACCCTCGTCTTAGAAAAAGTACATGCTCTGACCAGGAAAACATTTGCATATAAA

ACCAGAGCTTCGGTCAAGGAGAAACTTTGCTCAGAGAAATAACTTAGGGATTGGTTTATTA

AATTTTAAAAGTTGACATTTTTGAGTGTTTATTTAATATTTTACAGGGAAAGCATCTGTAT

GAATTGTCTGTTTTATTTAGCGTTGCTAACTGAA

R24
Region in vicinity of acceptor of intron 55 (in range of
SEQ

−400 to −1 bases from 3′ end of intron 55 as basing point)
No.

ACTTGATCCATATAGTAATGAAATTATTGGCACTGGGGTACACTTTATCATAGAATTTTAT
369

TGCCTATCACTTCCATAAAATAATACATTTTGTCCATAGACTAGAAGATATAACTTGTGAA

CTTTATAAAGTTATAAATACATTACTTTCCAACTCATAATGGCAAGGAATAAATCTATTAC

AACTAATAAGATGCCCATTTTAAATCTACATAATAACAGGAGAAGGCAATACGCCAAGAAA

AGGGATTTGAGATGTATCTTCTTGTTAGTTTAGCCTGATTGAAATGTCTTTTGAACTAATA

ATTATTTATATTTTGCAATTCTCCAAATTCACATTCATCGCTTGTTTCTTTTGTTTGGTAA

TTCTGCACATATTCTTCTTCCTGCTGTCCTGTAG

The target regions R1 to R24 of the antisense oligomer of the present invention include both wild (e.g., the regions represented by SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389) and mutant types in relation to the human dystrophin pre-mRNA. Such a mutant type specifically has any one base sequence selected from the group consisting of base sequences (B0) and (B1) to (B16) below:

(B0) a base sequence that hybridizes under stringent conditions to a base sequence complementary to any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389;

(B1) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±15% of the length of the any one base sequence selected;

(B2) a base sequence that has at least 86% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±14% of the length of the any one base sequence selected;

(B3) a base sequence that has at least 87% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±13% of the length of the any one base sequence selected;

(B4) a base sequence that has at least 88% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±12% of the length of the any one base sequence selected;

(B5) a base sequence that has at least 89% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±11% of the length of the any one base sequence selected;

(B6) a base sequence that has at least 90% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±10% of the length of the any one base sequence selected;

(B7) a base sequence that has at least 91% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±9% of the length of the any one base sequence selected;

(B8) a base sequence that has at least 92% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±8% of the length of the any one base sequence selected;

(B9) a base sequence that has at least 93% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±7% of the length of the any one base sequence selected;

(B10) a base sequence that has at least 94% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±6% of the length of the any one base sequence selected;

(B11) a base sequence that has at least 95% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±5% of the length of the any one base sequence selected;

(B12) a base sequence that has at least 96% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±4% of the length of the any one base sequence selected;

(B13) a base sequence that has at least 97% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±3% of the length of the any one base sequence selected;

(B14) a base sequence that has at least 98% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±2% of the length of the any one base sequence selected;

(B15) a base sequence that has at least 99% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±1% of the length of the any one base sequence selected;

and

(B16) a base sequence that has at least 99.5% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±0.5% of the length of the any one base sequence selected.

As used herein, the term “base sequence that hybridizes under stringent conditions” refers to, for example, a base sequence obtained by colony hybridization, plaque hybridization, Southern hybridization or the like, using as a probe all or part of a base sequence complementary to, e.g., any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389. The hybridization method which may be used includes methods described in, for example, “Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor, Laboratory Press, 2001,” “Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons, 1987-1997,” etc.

As used herein, the term “complementary base sequence” is not limited to a base sequence that forms Watson-Crick pairs with an intended base sequence, and also includes a base sequence that forms wobble base pairs therewith. Herein, the Watson-Crick pair means a base pair that forms a hydrogen bond between adenine and thymine, between adenine and uracil, or between guanine and cytosine, and the wobble base pair means a base pair that forms a hydrogen bond between guanine and uracil, between inosine and uracil, between inosine and adenine, or between inosine and cytosine. The term “complementary base sequence” does not have to have 100% complementarity with the intended base sequence and may contain, for example, 1, 2, 3, 4, or 5 noncomplementary bases to the intended base sequence or may be a base sequence shorter by 1 base, 2 bases, 3 bases, 4 bases, or 5 bases than the intended base sequence.

As used herein, the term “stringent conditions” may be any of low stringent conditions, moderate stringent conditions or high stringent conditions. The term “low stringent condition” is, for example, 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide at 32° C. The term “moderate stringent condition” is, for example, 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide at 42° C., or 5×SSC, 1% SDS, 50 mM Tris-HCl (pH 7.5), 50% formamide at 42° C. The term “high stringent condition” is, for example, 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide at 50° C., or 0.2×SSC, 0.1% SDS at 65° C. Under these conditions, base sequences with higher identity are expected to be obtained efficiently at higher temperatures, although multiple factors are involved in hybridization stringency including temperature, probe concentration, probe length, ionic strength, time, salt concentration and others, and those skilled in the art may appropriately select these factors to achieve similar stringency.

When commercially available kits are used for hybridization, for example, an Alkphos Direct Labelling and Detection System (GE Healthcare) may be used. In this case, according to the attached protocol, after incubation with a labeled probe overnight, the membrane can be washed with a primary wash buffer containing 0.1% (w/v) SDS at 55° C., thereby detecting hybridization. Alternatively, when the probe is labeled with digoxigenin (DIG) using a commercially available reagent (e.g., a PCR Labelling Mix (Roche Diagnostics), etc.) in producing a probe based on all or part of the complementary sequence to any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, hybridization can be detected with a DIG Nucleic Acid Detection Kit (Roche Diagnostics) or the like.

The identity between base sequences may be determined using algorithm BLAST (Basic Local Alignment Search Tool) by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc. Natl. Acad. Sci. USA 90: 5873, 1993). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul S F, et al: J. Mol. Biol. 215: 403, 1990). When a base sequence is analyzed using BLASTN, the parameters are, for example, score=100 and wordlength=12. When BLAST and Gapped BLAST programs are used, the default parameters for each program are employed.

The antisense oligomer of the present invention comprises a base sequence complementary to a base sequence of at least any one region selected from the group consisting of the target regions R1 to R24 of the present invention, or a partial base sequence thereof. The term “partial” means a region, except for the full length, of at least any one region selected from the group consisting of the target regions R1 to R24, i.e., a partial region of at least any one region selected from the group consisting of the target regions R1 to R24. The partial region may be 10 to 60 bases long, 10 to 55 bases long, 10 to 50 bases long, 10 to 45 bases long, 10 to 40 bases long, 10 to 35 bases long, 10 to 30 bases long, 10 to 25 bases long, 15 to 60 bases long, 15 to 55 bases long, 15 to 50 bases long, 15 to 45 bases long, 15 to 40 bases long, 15 to 35 bases long, 15 to 30 bases long, 15 to 25 bases long, 16 to 60 bases long, 16 to 55 bases long, 16 to 50 bases long, 16 to 45 bases long, 16 to 40 bases long, 16 to 35 bases long, 16 to 30 bases long, 16 to 25 bases long, 17 to 60 bases long, 17 to 55 bases long, 17 to 50 bases long, 17 to 45 bases long, 17 to 40 bases long, 17 to 35 bases long, 17 to 30 bases long, 17 to 25 bases long, 18 to 60 bases long, 18 to 55 bases long, 18 to 50 bases long, 18 to 45 bases long, 18 to 40 bases long, 18 to 35 bases long, 18 to 30 bases long, 18 to 25 bases long, 19 to 60 bases long, 19 to 55 bases long, 19 to 50 bases long, 19 to 45 bases long, 19 to 40 bases long, 19 to 35 bases long, 19 to 30 bases long, 19 to 25 bases long, 20 to 60 bases long, 20 to 55 bases long, 20 to 50 bases long, 20 to 45 bases long, 20 to 40 bases long, 20 to 35 bases long, 20 to 30 bases long, 20 to 25 bases long, 15 to 30 bases long, 15 to 29 bases long, 15 to 28 bases long, 15 to 27 bases long, 15 to 26 bases long, 15 to 25 bases long, 15 to 24 bases long, 15 to 23 bases long, 15 to 22 bases long, 15 to 21 bases long, 15 to 20 bases long, 15 to 19 bases long, 15 to 18 bases long, 16 to 30 bases long, 16 to 29 bases long, 16 to 28 bases long, 16 to 27 bases long, 16 to 26 bases long, 16 to 25 bases long, 16 to 24 bases long, 16 to 23 bases long, 16 to 22 bases long, 16 to 21 bases long, 16 to 20 bases long, 16 to 19 bases long, 16 to 18 bases long, 17 to 30 bases long, 17 to 29 bases long, 17 to 28 bases long, 17 to 27 bases long, 17 to 26 bases long, 17 to 25 bases long, 17 to 24 bases long, 17 to 23 bases long, 17 to 22 bases long, 17 to 21 bases long, 17 to 20 bases long, 17 to 19 bases long, 17 to 18 bases long, 18 to 30 bases long, 18 to 29 bases long, 18 to 28 bases long, 18 to 27 bases long, 18 to 26 bases long, 18 to 25 bases long, 18 to 24 bases long, 18 to 23 bases long, 18 to 22 bases long, 18 to 21 bases long, 18 to 20 bases long, 18 to 19 bases long, 19 to 30 bases long, 19 to 29 bases long, 19 to 28 bases long, 19 to 27 bases long, 19 to 26 bases long, 19 to 25 bases long, 19 to 24 bases long, 19 to 23 bases long, 19 to 22 bases long, 19 to 21 bases long, 19 to 20 bases long, 20 to 30 bases long, 20 to 29 bases long, 20 to 28 bases long, 20 to 27 bases long, 20 to 26 bases long, 20 to 25 bases long, 20 to 24 bases long, 20 to 23 bases long, 20 to 22 bases long, 20 to 21 bases long, 5 to 25 bases long, 5 to 24 bases long, 5 to 23 bases long, 5 to 22 bases long, 5 to 21 bases long, 5 to 20 bases long, 5 to 19 bases long, 5 to 18 bases long, 5 to 17 bases long, 5 to 16 bases long, 5 to 15 bases long, 5 to 14 bases long, 5 to 13 bases long, 5 to 12 bases long, 7 to 25 bases long, 7 to 24 bases long, 7 to 23 bases long, 7 to 22 bases long, 7 to 21 bases long, 7 to 20 bases long, 7 to 19 bases long, 7 to 18 bases long, 7 to 17 bases long, 7 to 16 bases long, 7 to 15 bases long, 7 to 14 bases long, 7 to 13 bases long, 7 to 12 bases long, 9 to 25 bases long, 9 to 24 bases long, 9 to 23 bases long, 9 to 22 bases long, 9 to 21 bases long, 9 to 20 bases long, 9 to 19 bases long, 9 to 18 bases long, 9 to 17 bases long, 9 to 16 bases long, 9 to 15 bases long, 9 to 14 bases long, 9 to 13 bases long, 9 to 12 bases long, 10 to 25 bases long, 10 to 24 bases long, 10 to 23 bases long, 10 to 22 bases long, 10 to 21 bases long, 10 to 20 bases long, 10 to 19 bases long, 10 to 18 bases long, 10 to 17 bases long, 10 to 16 bases long, 10 to 15 bases long, 10 to 14 bases long, 10 to 13 bases long, 10 to 12 bases long, 60 bases long, 59 bases long, 58 bases long, 57 bases long, 56 bases long, 55 bases long, 54 bases long, 53 bases long, 52 bases long, 51 bases long, 50 bases long, 49 bases long, 48 bases long, 47 bases long, 46 bases long, 45 bases long, 44 bases long, 43 bases long, 42 bases long, 41 bases long, 40 bases long, 39 bases long, 38 bases long, 37 bases long, 36 bases long, 35 bases long, 34 bases long, 33 bases long, 32 bases long, 31 bases long, 30 bases long, 29 bases long, 28 bases long, 27 bases long, 26 bases long, 25 bases long, 24 bases long, 23 bases long, 22 bases long, 21 bases long, 20 bases long, 19 bases long, 18 bases long, 17 bases long, 16 bases long, 15 bases long, 14 bases long, 13 bases long, 12 bases long, 11 bases long, 10 bases long, 9 bases long, 8 bases long, 7 bases long, 6 bases long, or 5 bases long, but not limited thereto. These lengths may be increased or decreased by 1, 2, or 3 bases.

The antisense oligomer of the present invention has an activity to cause simultaneous skipping of any two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA. As used herein, such skipping of two or more numerically consecutive exons from pre-mRNA of interest is referred to as “multi-exon skipping” or “multi-skipping”, and this activity is referred to as “multi-exon skipping activity” or “multi-skipping activity”.

Herein, the any two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon mean a plurality of exons that increase one by one in exon number among 11 exons from the 45th exon to the 55th exon included in pre-mRNA. The exon number means a number assigned to exons in order from the 5′ end to the 3′ end with an exon at the most upstream position of pre-mRNA defined as the first exon, followed by the second, the third, . . . , and the 79th exons among 79 exons included in human dystrophin pre-mRNA. An intron is numbered as the same number as that of an exon positioned on the 5′ side thereof. Specifically, the 45th intron is flanked by the 45th exon positioned on the 5′ side thereof and the 46th exon positioned on the 3′ side thereof. As used herein, the “nth” exon or intron means the nth exon or intron counted from the 5′ end toward the 3′ end in pre-mRNA.

Table 4 shows combinations of exons included in the any two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon.

TABLE 4

Combination
Exons included

Combination 1
45, 46

Combination 2
45-47

Combination 3
45-48

Combination 4
45-49

Combination 5
45-50

Combination 6
45-51

Combination 7
45-52

Combination 8
45-53

Combination 9
45-54

Combination 10
45-55

Combination 11
46, 47

Combination 12
46-48

Combination 13
46-49

Combination 14
46-50

Combination 15
46-51

Combination 16
46-52

Combination 17
46-53

Combination 18
46-54

Combination 19
46-55

Combination 20
47, 48

Combination 21
47-49

Combination 22
47-50

Combination 23
47-51

Combination 24
47-52

Combination 25
47-53

Combination 26
47-54

Combination 27
47-55

Combination 28
48, 49

Combination 29
48-50

Combination 30
48-51

Combination 31
48-52

Combination 32
48-53

Combination 33
48-54

Combination 34
48-55

Combination 35
49, 50

Combination 36
49-51

Combination 37
49-52

Combination 38
49-53

Combination 39
49-54

Combination 40
49-55

Combination 41
50, 51

Combination 42
50-52

Combination 43
50-53

Combination 44
50-54

Combination 45
50-55

Combination 46
51, 52

Combination 47
51-53

Combination 48
51-54

Combination 49
51-55

Combination 50
52, 53

Combination 51
52-54

Combination 52
52-55

Combination 53
53, 54

Combination 54
53-55

Combination 55
54, 55

Among the combinations of exons described in Table 4, for example, the combination 1, 2, 3, 4, 6, 8, 10, 18, 20, 21, 23, 25, 27, 28, 30, 32, 34, 36, 38, 40, 41, 43, 45, 46, 50, 52, or 55 is a skipping pattern expected to exert higher therapeutic effects on DMD. Multi-exon skipping in such a combination is expected to exert therapeutic effects on more patients with DMD.

The any two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon may include a plurality of groups of consecutive exons and may be, for example, but not limited to, (example 1) exons 45 and 46 (first exon group) and exons 48 to 53 (second exon group), or (example 2) exons 46 and 47 (first exon group), exons 49 and 50 (second exon group), and exons 52 to 54 (third exon group).

In the present invention, the term “activity to cause skipping” (i.e., multi-skipping activity) means, when human dystrophin pre-mRNA is taken as an example, an activity to produce human dystrophin mRNA having deletion of any two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in the human dystrophin pre-mRNA.

In other words, this activity means that by binding of the antisense oligomer of the present invention to a target site in human dystrophin pre-mRNA, the 5′-terminal nucleotide of an exon immediately downstream of the exons to be deleted is linked to the 3′-terminal nucleotide of an exon immediately upstream of the exons to be deleted when the pre-mRNA undergoes splicing, thus resulting in formation of mature mRNA which is free of codon frame shift (i.e., mature mRNA having deletion of the exons without frame shift).

The antisense oligomer of the present invention exhibits a multi-skipping activity under physiological conditions. The term “under physiological conditions” refers to conditions set to mimic the in vivo environment in terms of pH, salt composition and temperature. The conditions are, for example, 25 to 40° C., preferably 37° C., pH 5 to 8, preferably pH 7.4 and 150 mM of sodium chloride concentration. When multi-skipping is induced, one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 antisense oligomers, can be used in combination as the antisense oligomer of the present invention.

Whether multi-skipping is caused or not can be confirmed by introducing the antisense oligomer of the present invention into a dystrophin expression cell (e.g., human rhabdomyosarcoma cells), amplifying the region surrounding exons 45 to 55 of mRNA of the human dystrophin gene from the total RNA of the dystrophin expression cell by RT-PCR and performing nested PCR or sequence analysis on the PCR amplified product. The multi-skipping efficiency can be determined as follows. The mRNA for the human dystrophin gene is collected from test cells; in the mRNA, the polynucleotide level “A” of the band where any two or more numerically consecutive exons among exons 45 to 55 are skipped, the polynucleotide level “B” of the band where any one exon among exons 45 to 55 is skipped, and the polynucleotide level “C” of the band where no skipping is caused are measured. Using these measurement values of “A”, “B”, and “C”, the efficiency is calculated by the following equation.

Skipping efficiency (%)=A/(A+B+C)×100

For example, the multi-skipping efficiency of exons 45 to 55 can be determined by using a forward primer for exon 44 and a reverse primer for exon 56 to measure the polynucleotide level “A” of the band where exons 45 to 55 are multi-skipped, using the forward primer for exon 44 and a reverse primer for exon 46 to measure the polynucleotide level “B” of the band where exon 45 is single-skipped, and using the forward primer for exon 44 and the reverse primer for exon 46 to measure the polynucleotide level “C” of the band where no skipping is caused, followed by calculation by the equation using these measurement values of “A”, “B”, and “C”.

The number of exons to be deleted in human dystrophin mRNA by the antisense oligomer of the present invention is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. This is referred to as a deletion pattern, and various deletion patterns may exist in admixture in results obtained in one skipping experiment or skipping treatment. For example, mRNA admixture having deletion of 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 exons is obtained by introducing the antisense oligomer of the present invention to cells expressing human dystrophin pre-mRNA, and collecting its mRNA.

In a certain embodiment, the term “activity to cause skipping” can be defined as (C1) to (C10) below.

(C1) Any two numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.

Herein, the two numerically consecutive exons may be the 45th and the 46th exons, the 46th and the 47th exons, the 47th and the 48th exons, the 48th and the 49th exons, the 49th and the 50th exons, the 50th and the 51st exons, the 51st and the 52nd exons, the 52nd and the 53rd exons, the 53rd and the 54th exons, or the 54th and the 55th exons.

(C2) Any three numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.

Herein, the three numerically consecutive exons may be the 45th to the 47th exons, the 46th to the 48th exons, the 47th to the 49th exons, the 48th to the 50th exons, the 49th to the 51st exons, the 50th to the 52nd exons, the 51st to the 53rd exons, the 52nd to the 54th exons, or the 53rd to the 55th exons.

(C3) Any four numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.

Herein, the four numerically consecutive exons may be the 45th to the 48th exons, the 46th to the 49th exons, the 47th to the 50th exons, the 48th to the 51st exons, the 49th to the 52nd exons, the 50th to the 53rd exons, the 51st to the 54th exons, or the 52nd to the 55th exons.

(C4) Any five numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.

Herein, the five numerically consecutive exons may be the 45th to the 49th exons, the 46th to the 50th exons, the 47th to the 51st exons, the 48th to the 52nd exons, the 49th to the 53rd exons, the 50th to the 54th exons, or the 51st to the 55th exons.

(C5) Any six numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.

Herein, the six numerically consecutive exons may be the 45th to the 50th exons, the 46th to the 51st exons, the 47th to the 52nd exons, the 48th to the 53rd exons, the 49th to the 54th exons, or the 50th to the 55th exons.

(C6) Any seven numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.

Herein, the seven numerically consecutive exons may be the 45th to the 51st exons, the 46th to the 52nd exons, the 47th to the 53rd exons, the 48th to the 54th exons, or the 49th to the 55th exons.

(C7) Any eight numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.

Herein, the eight numerically consecutive exons may be the 45th to the 52nd exons, the 46th to the 53rd exons, the 47th to the 54th exons, or the 48th to the 55th exons.

(C8) Any nine numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.

Herein, the nine numerically consecutive exons may be the 45th to the 53rd exons, the 46th to the 54th exons, or the 47th to the 55th exons.

(C9) Any ten numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.

Herein, the ten numerically consecutive exons may be the 45th to the 54th exons, or the 46th to the 55th exons.

(C10) Eleven numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA are skipped with the efficiency of 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher.

Herein, the eleven numerically consecutive exons may be the 45th to the 55th exons.

The antisense oligomer of the present invention may be 10 to 60 bases long, 10 to 55 bases long, 10 to 50 bases long, 10 to 45 bases long, 10 to 40 bases long, 10 to 35 bases long, 10 to 30 bases long, 10 to 25 bases long, 15 to 60 bases long, 15 to 55 bases long, 15 to 50 bases long, 15 to 45 bases long, 15 to 40 bases long, 15 to 35 bases long, 15 to 30 bases long, 15 to 25 bases long, 16 to 60 bases long, 16 to 55 bases long, 16 to 50 bases long, 16 to 45 bases long, 16 to 40 bases long, 16 to 35 bases long, 16 to 30 bases long, 16 to 25 bases long, 17 to 60 bases long, 17 to 55 bases long, 17 to 50 bases long, 17 to 45 bases long, 17 to 40 bases long, 17 to 35 bases long, 17 to 30 bases long, 17 to 25 bases long, 18 to 60 bases long, 18 to 55 bases long, 18 to 50 bases long, 18 to 45 bases long, 18 to 40 bases long, 18 to 35 bases long, 18 to 30 bases long, 18 to 25 bases long, 19 to 60 bases long, 19 to 55 bases long, 19 to 50 bases long, 19 to 45 bases long, 19 to 40 bases long, 19 to 35 bases long, 19 to 30 bases long, 19 to 25 bases long, 20 to 60 bases long, 20 to 55 bases long, 20 to 50 bases long, 20 to 45 bases long, 20 to 40 bases long, 20 to 35 bases long, 20 to 30 bases long, 20 to 25 bases long, 15 to 30 bases long, 15 to 29 bases long, 15 to 28 bases long, 15 to 27 bases long, 15 to 26 bases long, 15 to 25 bases long, 15 to 24 bases long, 15 to 23 bases long, 15 to 22 bases long, 15 to 21 bases long, 15 to 20 bases long, 15 to 19 bases long, 15 to 18 bases long, 16 to 30 bases long, 16 to 29 bases long, 16 to 28 bases long, 16 to 27 bases long, 16 to 26 bases long, 16 to 25 bases long, 16 to 24 bases long, 16 to 23 bases long, 16 to 22 bases long, 16 to 21 bases long, 16 to 20 bases long, 16 to 19 bases long, 16 to 18 bases long, 17 to 30 bases long, 17 to 29 bases long, 17 to 28 bases long, 17 to 27 bases long, 17 to 26 bases long, 17 to 25 bases long, 17 to 24 bases long, 17 to 23 bases long, 17 to 22 bases long, 17 to 21 bases long, 17 to 20 bases long, 17 to 19 bases long, 17 to 18 bases long, 18 to 30 bases long, 18 to 29 bases long, 18 to 28 bases long, 18 to 27 bases long, 18 to 26 bases long, 18 to 25 bases long, 18 to 24 bases long, 18 to 23 bases long, 18 to 22 bases long, 18 to 21 bases long, 18 to 20 bases long, 18 to 19 bases long, 19 to 30 bases long, 19 to 29 bases long, 19 to 28 bases long, 19 to 27 bases long, 19 to 26 bases long, 19 to 25 bases long, 19 to 24 bases long, 19 to 23 bases long, 19 to 22 bases long, 19 to 21 bases long, 19 to 20 bases long, 20 to 30 bases long, 20 to 29 bases long, 20 to 28 bases long, 20 to 27 bases long, 20 to 26 bases long, 20 to 25 bases long, 20 to 24 bases long, 20 to 23 bases long, 20 to 22 bases long, 20 to 21 bases long, 60 bases long, 59 bases long, 58 bases long, 57 bases long, 56 bases long, 55 bases long, 54 bases long, 53 bases long, 52 bases long, 51 bases long, 50 bases long, 49 bases long, 48 bases long, 47 bases long, 46 bases long, 45 bases long, 44 bases long, 43 bases long, 42 bases long, 41 bases long, 40 bases long, 39 bases long, 38 bases long, 37 bases long, 36 bases long, 35 bases long, 34 bases long, 33 bases long, 32 bases long, 31 bases long, 30 bases long, 29 bases long, 28 bases long, 27 bases long, 26 bases long, 25 bases long, 24 bases long, 23 bases long, 22 bases long, 21 bases long, 20 bases long, 19 bases long, 18 bases long, 17 bases long, 16 bases long, 15 bases long, 14 bases long, 13 bases long, 12 bases long, 11 bases long, or 10 bases long, but not limited thereto. These lengths may be increased or decreased by 1, 2, or 3 bases.

One embodiment of the antisense oligomer of the present invention is a linked-type antisense oligomer configured to comprise a plurality of unit oligomers linked to each other, a pharmaceutically acceptable salt thereof, or hydrate thereof (hereinafter, referred to as the “linked-type antisense oligomer of the present invention”). The unit oligomers mean respective oligomers constituting the linked-type antisense oligomer of the present invention. Specifically, the unit oligomers mean moieties (units) comprising base sequences that hybridize with target base sequences having consecutive base sequences when the linked-type antisense oligomer of the present invention binds to the target base sequences in human dystrophin pre-mRNA.

Specifically, the linked-type antisense oligomer of the present invention is an antisense oligomer comprising two or more unit oligomers linked to each other, or a pharmaceutically acceptable salt thereof, or hydrate thereof, wherein

each of the unit oligomers comprises a base sequence complementary to a base sequence of any one region selected from the group consisting of the regions R1 to R24 (which may include the wild-type regions represented by SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389 and their mutant types), or a partial base sequence thereof, and the respective base sequences of the unit oligomers are neither consecutive nor overlapped with each other.

The unit oligomers may be linked via a linker that does not contribute to hybridization, or may be linked directly without the mediation of a linker. When the unit oligomers are linked directly to each other, the 3′ end of the unit positioned on the 5′ side and the 5′ end of the unit positioned on the 3′ side form a phosphate bond or any one of the following groups.

embedded image

wherein X represents —OH, —CH₂R¹, —O—CH₂R¹, —S—CH₂R¹, —NR²R³or F;

R¹represents H or an alkyl;

R²and R³, which may be the same or different, each represents H, an alkyl, a cycloalkyl or an aryl;

Y₁represents O, S, CH₂, or NR¹;

Y₂represents O, S, or NR¹;

Z represents O or S.

In the linked-type antisense oligomer of the present invention, the unit oligomers may respectively target base sequences included in separate regions among the regions R1 to R24, or may respectively target base sequences included in the same region. In the linked-type antisense oligomer of the present invention, the respective base sequences of the unit oligomers are neither consecutive nor overlapped with each other. The respective base sequences of the unit oligomers that are not consecutive mean that the respective target base sequences of the unit oligomers constituting the linked-type antisense oligomer of the present invention are not consecutive as base sequences in human dystrophin pre-mRNA. Thus, when the respective target base sequences of the unit oligomers are compared to the base sequence of human dystrophin pre-mRNA, at least one base intervenes between the target base sequences as the base sequence of human dystrophin pre-mRNA. On the other hand, the respective base sequences of the unit oligomers that do not overlapped with each other mean that the respective target base sequences of the unit oligomers do not overlapped with each other as base sequences in human dystrophin pre-mRNA. The accidental presence of a match of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases between the respective base sequences of the unit oligomers is accepted.

The linked-type antisense oligomer of the present invention may be configured such that each of the unit oligomers comprises a base sequence complementary to

(D1) any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389,

(D2) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±15% of the length of the any one base sequence selected,

(D3) a base sequence that hybridizes under stringent conditions to a base sequence complementary to any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, or

(D4) a partial base sequence of any one base sequence selected from the group consisting of the base sequences (D1), (D2), and (D3), and the respective base sequences of the unit oligomers are neither consecutive nor overlapped with each other.

(D2-1) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±15% of the length of the any one base sequence selected,

(D2-2) a base sequence that has at least 86% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±14% of the length of the any one base sequence selected,

(D2-3) a base sequence that has at least 87% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±13% of the length of the any one base sequence selected,

(D2-4) a base sequence that has at least 88% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±12% of the length of the any one base sequence selected,

(D2-5) a base sequence that has at least 89% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±11% of the length of the any one base sequence selected,

(D2-6) a base sequence that has at least 90% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±10% of the length of the any one base sequence selected,

(D2-7) a base sequence that has at least 91% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±9% of the length of the any one base sequence selected,

(D2-8) a base sequence that has at least 92% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±8% of the length of the any one base sequence selected,

(D2-9) a base sequence that has at least 93% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±7% of the length of the any one base sequence selected,

(D2-10) a base sequence that has at least 94% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±6% of the length of the any one base sequence selected,

(D2-11) a base sequence that has at least 95% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±5% of the length of the any one base sequence selected,

(D2-12) a base sequence that has at least 96% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±4% of the length of the any one base sequence selected,

(D2-13) a base sequence that has at least 97% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±3% of the length of the any one base sequence selected,

(D2-14) a base sequence that has at least 98% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±2% of the length of the any one base sequence selected,

(D2-15) a base sequence that has at least 99% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±1% of the length of the any one base sequence selected, and

(D2-16) a base sequence that has at least 99.5% identity consisting of SEQ ID NOs: 233 to 256, 341 to 369, and 385 to 389, and has a length within ±0.5% of the length of the any one base sequence selected.

Thus, each of the unit oligomers constituting the linked-type antisense oligomer of the present invention may comprise a base sequence complementary to any one base sequence selected from the group consisting of the base sequences (D1), (D2-1) to (D2-16), (D3), and (D4).

The linked-type antisense oligomer of the present invention may comprise, as described above, a base sequence complementary to any one base sequence selected from the group consisting of (D1), (D2), (D3), and (D4). The base sequence (D4) relates to a partial base sequence of any one base sequence selected from the group consisting of the base sequences (D1), (D2), and (D3). In relation to (D4), the term “partial” means a partial region of consecutive bases, except for the full length, of (D1), (D2), or (D3). The partial region may be 5 to 25 bases long, 5 to 24 bases long, 5 to 23 bases long, 5 to 22 bases long, 5 to 21 bases long, 5 to 20 bases long, 5 to 19 bases long, 5 to 18 bases long, 5 to 17 bases long, 5 to 16 bases long, 5 to 15 bases long, 5 to 14 bases long, 5 to 13 bases long, 5 to 12 bases long, 7 to 25 bases long, 7 to 24 bases long, 7 to 23 bases long, 7 to 22 bases long, 7 to 21 bases long, 7 to 20 bases long, 7 to 19 bases long, 7 to 18 bases long, 7 to 17 bases long, 7 to 16 bases long, 7 to 15 bases long, 7 to 14 bases long, 7 to 13 bases long, 7 to 12 bases long, 9 to 25 bases long, 9 to 24 bases long, 9 to 23 bases long, 9 to 22 bases long, 9 to 21 bases long, 9 to 20 bases long, 9 to 19 bases long, 9 to 18 bases long, 9 to 17 bases long, 9 to 16 bases long, 9 to 15 bases long, 9 to 14 bases long, 9 to 13 bases long, 9 to 12 bases long, 10 to 25 bases long, 10 to 24 bases long, 10 to 23 bases long, 10 to 22 bases long, 10 to 21 bases long, 10 to 20 bases long, 10 to 19 bases long, 10 to 18 bases long, 10 to 17 bases long, 10 to 16 bases long, 10 to 15 bases long, 10 to 14 bases long, 10 to 13 bases long, 10 to 12 bases long, 25 bases long, 24 bases long, 23 bases long, 22 bases long, 21 bases long, 20 bases long, 19 bases long, 18 bases long, 17 bases long, 16 bases long, 15 bases long, 14 bases long, 13 bases long, 12 bases long, 11 bases long, 10 bases long, 9 bases long, 8 bases long, 7 bases long, 6 bases long, or 5 bases long, but not limited thereto. These lengths may be increased or decreased by 1, 2, or 3 bases.

The size of each unit oligomer may be 5 to 30 bases long, 5 to 29 bases long, 5 to 28 bases long, 5 to 27 bases long, 5 to 26 bases long, 5 to 25 bases long, 5 to 24 bases long, 5 to 23 bases long, 5 to 22 bases long, 5 to 21 bases long, 5 to 20 bases long, 5 to 19 bases long, 5 to 18 bases long, 5 to 17 bases long, 5 to 16 bases long, 5 to 15 bases long, 5 to 14 bases long, 5 to 13 bases long, 5 to 12 bases long, 7 to 30 bases long, 7 to 29 bases long, 7 to 28 bases long, 7 to 27 bases long, 7 to 26 bases long, 7 to 25 bases long, 7 to 24 bases long, 7 to 23 bases long, 7 to 22 bases long, 7 to 21 bases long, 7 to 20 bases long, 7 to 19 bases long, 7 to 18 bases long, 7 to 17 bases long, 7 to 16 bases long, 7 to 15 bases long, 7 to 14 bases long, 7 to 13 bases long, 7 to 12 bases long, 9 to 30 bases long, 9 to 29 bases long, 9 to 28 bases long, 9 to 27 bases long, 9 to 26 bases long, 9 to 25 bases long, 9 to 24 bases long, 9 to 23 bases long, 9 to 22 bases long, 9 to 21 bases long, 9 to 20 bases long, 9 to 19 bases long, 9 to 18 bases long, 9 to 17 bases long, 9 to 16 bases long, 9 to 15 bases long, 9 to 14 bases long, 9 to 13 bases long, 9 to 12 bases long, 10 to 30 bases long, 10 to 29 bases long, 10 to 28 bases long, 10 to 27 bases long, 10 to 26 bases long, 10 to 25 bases long, 10 to 24 bases long, 10 to 23 bases long, 10 to 22 bases long, 10 to 21 bases long, 10 to 20 bases long, 10 to 19 bases long, 10 to 18 bases long, 10 to 17 bases long, 10 to 16 bases long, 10 to 15 bases long, 10 to 14 bases long, 10 to 13 bases long, 10 to 12 bases long, 30 bases long, 29 bases long, 28 bases long, 27 bases long, 26 bases long, 25 bases long, 24 bases long, 23 bases long, 22 bases long, 21 bases long, 20 bases long, 19 bases long, 18 bases long, 17 bases long, 16 bases long, 15 bases long, 14 bases long, 13 bases long, 12 bases long, 11 bases long, 10 bases long, 9 bases long, 8 bases long, 7 bases long, 6 bases long, 5 bases long, but not limited thereto. These lengths may be increased or decreased by 1, 2, or 3 bases. The unit oligomers may have the same size or different sizes.

In a certain embodiment, the linked-type antisense oligomer of the present invention may be an antisense oligomer consisting of

(E1) any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232, or

(E2) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232, and has a length within ±15% of the length of the any one base sequence selected, or a pharmaceutically acceptable salt thereof, or hydrate thereof.

Herein, the base sequence (E2) is a mutant type of the base sequence (E1), and examples of such a mutant type also include

(E2-1) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232, and has a length within ±15% of the length of the any one base sequence selected,

(E2-2) a base sequence that has at least 86% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232, and has a length within ±14% of the length of the any one base sequence selected,

(E2-3) a base sequence that has at least 87% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232, and has a length within ±13% of the length of the any one base sequence selected,

(E2-4) a base sequence that has at least 88% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232, and has a length within ±12% of the length of the any one base sequence selected,

(E2-5) a base sequence that has at least 89% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232, and has a length within ±11% of the length of the any one base sequence selected,

(E2-6) a base sequence that has at least 90% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232, and has a length within ±10% of the length of the any one base sequence selected,

(E2-7) a base sequence that has at least 91% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232, and has a length within ±9% of the length of the any one base sequence selected,

(E2-8) a base sequence that has at least 92% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232, and has a length within ±8% of the length of the any one base sequence selected,

(E2-9) a base sequence that has at least 93% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232, and has a length within ±7% of the length of the any one base sequence selected,

(E2-10) a base sequence that has at least 94% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232, and has a length within ±6% of the length of the any one base sequence selected,

(E2-11) a base sequence that has at least 95% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232, and has a length within ±5% of the length of the any one base sequence selected,

(E2-12) a base sequence that has at least 96% identity consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232, and has a length within ±4% of the length of the any one base sequence selected,

(E2-13) a base sequence that has at least 97% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232, and has a length within ±3% of the length of the any one base sequence selected,

(E2-14) a base sequence that has at least 98% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232, and has a length within ±2% of the length of the any one base sequence selected,

(E2-15) a base sequence that has at least 99% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232, and has a length within ±1% of the length of the any one base sequence selected, and

(E2-16) a base sequence that has at least 99.5% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232, and has a length within ±0.5% of the length of the any one, base sequence selected.

Thus, the linked-type antisense oligomer of the present invention may consist of any one base sequence selected from the group consisting of the base sequences (E1) and (E2-1) to (E2-16).

Preferably, the linked-type antisense oligomer of the present invention comprises any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232. In a certain embodiment, the linked-type antisense oligomer of the present invention consists of any one base sequence selected from the group consisting of SEQ ID NOs: 18 to 21, 80 to 111, and 225 to 232.

Further, the antisense oligomer of the present invention may be an antisense oligomer consisting of (E′1) any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, or

(E′2) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, and has a length within ±15% of the length of the any one base sequence selected, or

a pharmaceutically acceptable salt thereof, or hydrate thereof.

Herein, the base sequence (E′2) is a mutant type of the base sequence (E′1), and examples of such a mutant type also include

(E′2-1) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, and has a length within ±15% of the length of the any one base sequence selected,

(E′2-2) a base sequence that has at least 86% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, and has a length within ±14% of the length of the any one base sequence selected,

(E′2-3) a base sequence that has at least 87% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, and has a length within ±13% of the length of the any one base sequence selected,

(E′2-4) a base sequence that has at least 88% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, and has a length within ±12% of the length of the any one base sequence selected,

(E′2-5) a base sequence that has at least 89% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, and has a length within ±11% of the length of the any one base sequence selected,

(E′2-6) a base sequence that has at least 90% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, and has a length within ±10% of the length of the any one base sequence selected,

(E′2-7) a base sequence that has at least 91% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, and has a length within ±9% of the length of the any one base sequence selected,

(E′2-8) a base sequence that has at least 92% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, and has a length within ±8% of the length of the any one base sequence selected,

(E′2-9) a base sequence that has at least 93% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, and has a length within ±7% of the length of the any one base sequence selected,

(E′2-10) a base sequence that has at least 94% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, and has a length within ±6% of the length of the any one base sequence selected,

(E′2-11) a base sequence that has at least 95% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, and has a length within ±5% of the length of the any one base sequence selected,

(E′2-12) a base sequence that has at least 96% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, and has a length within ±4% of the length of the any one base sequence selected,

(E′2-13) a base sequence that has at least 97% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, and has a length within ±3% of the length of the any one base sequence selected,

(E′2-14) a base sequence that has at least 98% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, and has a length within ±2% of the length of the any one base sequence selected,

(E′2-15) a base sequence that has at least 99% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, and has a length within ±1% of the length of the any one base sequence selected, and

(E′2-16) a base sequence that has at least 99.5% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, and has a length within ±0.5% of the length of the any one base sequence selected.

Thus, the antisense oligomer of the present invention may consist of any one base sequence selected from the group consisting of the base sequences (E′1) and (E′2-1) to (E′2-16).

Preferably, the antisense oligomer of the present invention comprises any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232. In a certain embodiment, the antisense oligomer of the present invention consists of any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232.

In a certain embodiment, the antisense oligomer of the present invention may be an antisense oligomer consisting of

(E″1) any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, or

(E″2) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, and has a length within ±15% of the length of the any one base sequence selected,

or a pharmaceutically acceptable salt thereof, or hydrate thereof.

Herein, the base sequence (E″2) is a mutant type of the base sequence (E″1), and examples of such a mutant type also include

(E″2-1) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, and has a length within ±15% of the length of the any one base sequence selected, (E″2-2) a base sequence that has at least 86% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, and has a length within ±14% of the length of the any one base sequence selected,

(E″2-3) a base sequence that has at least 87% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, and has a length within ±13% of the length of the any one base sequence selected,

(E″2-4) a base sequence that has at least 88% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, and has a length within ±12% of the length of the any one base sequence selected,

(E″2-5) a base sequence that has at least 89% identity consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, and has a length within ±11% of the length of the any one base sequence selected,

(E″2-6) a base sequence that has at least 90% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, and has a length within ±10% of the length of the any one base sequence selected,

(E″2-7) a base sequence that has at least 91% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, and has a length within ±9% of the length of the any one base sequence selected,

(E″2-8) a base sequence that has at least 92% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, and has a length within ±8% of the length of the any one base sequence selected,

(E″2-9) a base sequence that has at least 93% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, and has a length within ±7% of the length of the any one base sequence selected,

(E″2-10) a base sequence that has at least 94% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, and has a length within ±6% of the length of the any one base sequence selected,

(E″2-11) a base sequence that has at least 95% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, and has a length within ±5% of the length of the any one base sequence selected,

(E″2-12) a base sequence that has at least 96% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, and has a length within ±4% of the length of the any one base sequence selected,

(E″2-13) a base sequence that has at least 97% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, and has a length within ±3% of the length of the any one base sequence selected,

(E″2-14) a base sequence that has at least 98% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, and has a length within ±2% of the length of the any one base sequence selected,

(E″2-15) a base sequence that has at least 99% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, and has a length within ±1% of the length of the any one base sequence selected, and

(E″2-16) a base sequence that has at least 99.5% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, and has a length within ±0.5% of the length of the any one base sequence selected.

Thus, the antisense oligomer of the present invention may consist of any one base sequence selected from the group consisting of the base sequences (E″1) and (E″2-1) to (E″2-16).

In a certain embodiment, the antisense oligomer of the present invention may be an antisense oligomer consisting of

(E′″1) any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, or

(E′″2) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, and has a length within ±15% of the length of the any one base sequence selected,

or a pharmaceutically acceptable salt thereof, or hydrate thereof.

Herein, the base sequence (E2) is a mutant type of the base sequence (E1), and examples of such a mutant type also include

(E′″2-1) a base sequence that has at least 85% identity consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, and has a length within ±15% of the length of the any one base sequence selected,

(E′″2-2) a base sequence that has at least 86% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, and has a length within ±14% of the length of the any one base sequence selected,

(E′″2-3) a base sequence that has at least 87% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, and has a length within ±13% of the length of the any one base sequence selected,

(E′″2-4) a base sequence that has at least 88% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, and has a length within ±12% of the length of the any one base sequence selected,

(E′″2-5) a base sequence that has at least 89% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, and has a length within ±11% of the length of the any one base sequence selected,

(E′″2-6) a base sequence that has at least 90% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, and has a length within ±10% of the length of the any one base sequence selected,

(E′″2-7) a base sequence that has at least 91% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, and has a length within ±9% of the length of the any one base sequence selected,

(E′″2-8) a base sequence that has at least 92% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, and has a length within ±8% of the length of the any one base sequence selected,

(E′″2-9) a base sequence that has at least 93% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, and has a length within ±7% of the length of the any one base sequence selected,

(E′″2-10) a base sequence that has at least 94% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, and has a length within ±6% of the length of the any one base sequence selected,

(E′″2-11) a base sequence that has at least 95% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, and has a length within ±5% of the length of the any one base sequence selected,

(E′″2-12) a base sequence that has at least 96% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, and has a length within ±4% of the length of the any one base sequence selected,

(E′″2-13) a base sequence that has at least 97% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, and has a length within ±3% of the length of the any one base sequence selected,

(E′″2-14) a base sequence that has at least 98% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, and has a length within ±2% of the length of the any one base sequence selected,

(E′″2-15) a base sequence that has at least 99% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, and has a length within ±1% of the length of the any one base sequence selected, and

(E′″2-16) a base sequence that has at least 99.5% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, and has a length within ±0.5% of the length of the any one base sequence selected.

Thus, the antisense oligomer of the present invention may consist of any one base sequence selected from the group consisting of the base sequences (E′″1) and (E′″2′2-1) to (E′″2-16).

Preferably, the antisense oligomer of the present invention is an antisense oligomer comprising any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, or a pharmaceutically acceptable salt thereof, or hydrate thereof. In a certain embodiment, the antisense oligomer of the present invention is an antisense oligomer consisting of any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, or a pharmaceutically acceptable salt thereof, or hydrate thereof.

More preferably, the antisense oligomer of the present invention is an antisense oligomer comprising any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, or a pharmaceutically acceptable salt thereof, or hydrate thereof. In a certain embodiment, the antisense oligomer of the present invention is an antisense oligomer consisting of any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228, or a pharmaceutically acceptable salt thereof, or hydrate thereof.

In a certain embodiment, the linked-type antisense oligomer of the present invention is an antisense oligomer comprising any one base sequence selected from the group consisting of SEQ ID NOs: 80, 82, 86, 92, 97, 98, 100, 102, 225, 228, 231, and 232, or a pharmaceutically acceptable salt thereof, or hydrate thereof. In a certain embodiment, the linked-type antisense oligomer of the present invention consists of any one base sequence selected from the group consisting of SEQ ID NOs: 80, 82, 86, 92, 97, 98, 100, 102, 225, 228, 231, and 232. Further preferably, the linked-type antisense oligomer of the present invention comprises any one base sequence selected from the group consisting of SEQ ID NOs: 102, 225, and 228. In a certain embodiment, the linked-type antisense oligomer of the present invention is an antisense oligomer consisting of any one base sequence selected from the group consisting of SEQ ID NOs: 102, 225, and 228, or a pharmaceutically acceptable salt thereof, or hydrate thereof.

The antisense oligomer of the present invention comprises a base sequence complementary to a base sequence of at least any one region selected from the group consisting of the regions R1 to R24, or a partial base sequence thereof. Any base sequence selected from the group consisting of (E1) and (E2-1) to (E2-16), the group consisting of (E′1) and (E′2-1) to (E′2-16), the group consisting of (E″1) and (E″2-1) to (E″2-16), or the group consisting of (E′″1) and (E′″2-1) to (E′″2-16) may be selected as the base sequence complementary to the base sequence of each region included in the regions R1 to R24, or a partial base sequence thereof. For example, as the base sequences complementary to the base sequences of the regions R1 to R24, or partial base sequences thereof, a base sequence complementary to the base sequence of each region, or a partial base sequence thereof may be used as to the regions R4 to R24, and a base sequence that is selected from the group consisting of (E1) and (E2-1) to (E2-16) and is included in each of the regions R1 to R3 may be used as to the regions R1 to R3.

The antisense oligomer of the present invention (including the linked-type antisense oligomer of the present invention) may be an oligonucleotide, morpholino oligomer or peptide nucleic acid (PNA) oligomer (hereinafter, also referred to as the “antisense oligonucleotide of the present invention”, the “antisense morpholino oligomer of the present invention”, or the “antisense peptide nucleic acid oligomer of the present invention”).

The antisense oligonucleotide of the present invention is an antisense oligomer composed of nucleotides as constituent units. Such nucleotides may be any of ribonucleotides, deoxyribonucleotides and modified nucleotides.

The modified nucleotide refers to one having fully or partly modified nucleobases, sugar moieties and/or phosphate bond moieties, which constitute the ribonucleotide or deoxyribonucleotide.

The nucleobase includes, for example, adenine, guanine, hypoxanthine, cytosine, thymine, uracil, and modified bases thereof. Examples of such modified bases include, but not limited to, pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosines (e.g., 5-methylcytosine), 5-alkyluracils (e.g., 5-ethyluracil), 5-halouracils (e.g., 5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidines (e.g., 6-methyluracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5′-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine, indole, imidazole, xanthine, etc.

Modification of the sugar moiety may include, for example, modifications at the 2′-position of ribose and modifications of the other positions of the sugar. The modification at the 2′-position of ribose includes a modification of replacing the 2′-OH of ribose with —OR, —R, —R′OR, —SH, —SR, —NH₂, —NHR, —NR₂, —N₃, —CN, —F, —Cl, —Br or —I, wherein R represents an alkyl or an aryl and R′ represents an alkylene.

The modification for the other positions of the sugar includes, for example, replacement of O at the 4′ position of ribose or deoxyribose with S, bridging between 2′ and 4′ positions of the sugar, e.g., LNA (locked nucleic acid) or ENA (2′-0,4′-C-ethylene-bridged nucleic acids), but is not limited thereto.

A modification of the phosphate bond moiety includes, for example, a modification of replacing phosphodiester bond with phosphorothioate bond, phosphorodithioate bond, alkyl phosphonate bond, phosphoramidate bond or boranophosphate bond (cf., e.g., Enya et al: Bioorganic & Medicinal Chemistry, 2008, 18, 9154-9160) (cf., e.g., Japan Domestic Re-Publications of PCT Application Nos. 2006/129594 and 2006/038608).

As used herein, the alkyl is preferably a straight or branched alkyl having 1 to 6 carbon atoms. Specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl and isohexyl. The alkyl may optionally be substituted. Examples of such substituents are a halogen, an alkoxy, cyano and nitro. The alkyl may be substituted with 1 to 3 substituents.

As used herein, the cycloalkyl is preferably a cycloalkyl having 3 to 12 carbon atoms. Specific examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl and cyclododecyl.

As used herein, the halogen includes fluorine, chlorine, bromine and iodine.

As used herein, the alkoxy is a straight or branched alkoxy having 1 to 6 carbon atoms such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy, isohexyloxy, etc. Among others, an alkoxy having 1 to 3 carbon atoms is preferred.

As used herein, the aryl is preferably an aryl having 6 to 10 carbon atoms. Specific examples include phenyl, α-naphthyl and β-naphthyl. Among others, phenyl is preferred. The aryl may optionally be substituted. Examples of such substituents are an alkyl, a halogen, an alkoxy, cyano and nitro. The aryl may be substituted with one to three of such substituents.

As used herein, the alkylene is preferably a straight or branched alkylene having 1 to 6 carbon atoms. Specific examples include methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, 2-(ethyl) trimethylene and 1-(methyl) tetramethylene.

As used herein, the acyl includes a straight or branched alkanoyl or aroyl. Examples of the alkanoyl include formyl, acetyl, 2-methylacetyl, 2,2-dimethylacetyl, propionyl, butyryl, isobutyryl, pentanoyl, 2,2-dimethylpropionyl, hexanoyl, etc. Examples of the aroyl include benzoyl, toluoyl and naphthoyl. The aroyl may optionally be substituted at substitutable positions and may be substituted with an alkyl(s).

Preferably, the antisense oligonucleotide of the present invention is the antisense oligomer of the present invention having a group represented by general formula below as a constituent unit wherein the —OH group at position 2′ of ribose is substituted with methoxy and the phosphate bond moiety is a phosphorothioate bond:

embedded image

wherein Base represents a nucleobase.

The antisense oligonucleotide of the present invention may be easily synthesized using various automated synthesizer (e.g., AKTA oligopilot plus 10/100 (GE Healthcare)). Alternatively, the synthesis may also be entrusted to a third-party organization (e.g., Promega Corp. or Takara Co.), etc.

The antisense morpholino oligomer of the present invention is an antisense oligomer comprising the constituent unit represented by general formula below:

embedded image

wherein Base has the same significance as defined above, and,

W represents a group shown by any one of the following groups:

embedded image

wherein X represents —CH₂R1, —O—CH₂R1, —S—CH₂R1, —NR²R3, or F;

R¹represents H or an alkyl;

R²and R³, which may be the same or different, each represents H, an alkyl, a cycloalkyl, or an aryl;

Y₁represents O, S, CH₂, or NR¹;

Y₂represents O, S, or NR¹;

Z represents O or S.

Examples of morpholino monomer compounds that are used in synthesis of the antisense morpholino oligomer of the present invention include, but not limited to, the following morpholino monomer compound (A), morpholino monomer compound (C), morpholino monomer compound (T), and morpholino monomer compound (G) shown in Table 5.

TABLE 5

Morpholino monomer
Morpholino monomer

compound
compound

(A)
(C)

embedded image

Morpholino monomer
Morpholino monomer

compound
compound

(T)
(G)

embedded image

In the present invention, preferably, the morpholino oligomer is an oligomer having a group represented by general formula below as a constituent unit (phosphorodiamidate morpholino oligomer (hereinafter referred to as “PMO”)).

embedded image

wherein Base, R²and R³have the same significance as defined above.

The morpholino oligomer may be produced by the procedure described in, e.g., WO 1991/009033 or WO 2009/064471. In particular, PMO can be produced by the procedure described in WO 2009/064471 or WO2013/100190.

The antisense peptide nucleic acid oligomer of the present invention is an antisense oligomer having a group represented by general formula below as a constituent unit:

embedded image

wherein Base has the same significance as defined above.

The peptide nucleic acid oligomer can be produced in accordance with, e.g., the following literatures:

1) P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, Science, 254, 1497 (1991)
2) M. Egholm, O. Buchardt, P. E. Nielsen, R. H. Berg, JACS, 114, 1895 (1992)
3) K. L. Dueholm, M. Egholm, C. Behrens, L. Christensen, H. F. Hansen, T. Vulpius, K. H. Petersen, R. H. Berg, P. E. Nielsen, O. Buchardt, J. Org. Chem., 59, 5767 (1994)
4) L. Christensen, R. Fitzpatrick, B. Gildea, K. H. Petersen, H. F. Hansen, T. Koch, M. Egholm, O. Buchardt, P. E. Nielsen, J. Coull, R. H. Berg, J. Pept. Sci., 1, 175 (1995)
5) T. Koch, H. F. Hansen, P. Andersen, T. Larsen, H. G. Batz, K. Otteson, H. Orum, J. Pept. Res., 49, 80 (1997)

The antisense oligomer of the present invention (including the linked-type antisense oligomer of the present invention) may be in the form of a pharmaceutically acceptable salt thereof, in the form of a hydrate thereof, or in the form of a hydrate of the pharmaceutically acceptable salt.

Examples of the pharmaceutically acceptable salt of the antisense oligomer of the present invention are alkali metal salts such as salts of sodium, potassium and lithium; alkaline earth metal salts such as salts of calcium and magnesium; metal salts such as salts of aluminum, iron, zinc, copper, nickel, cobalt, etc.; ammonium salts; organic amine salts such as salts of t-octylamine, dibenzylamine, morpholine, glucosamine, phenylglycine alkyl ester, ethylenediamine, N-methylglucamine, guanidine, diethylamine, triethylamine, dicyclohexylamine, N,N′-dibenzylethylenediamine, chloroprocaine, procaine, diethanolamine, N-benzyl-phenethylamine, piperazine, tetramethylammonium, tris(hydroxymethyl)aminomethane; hydrohalide salts such as salts of hydrofluorides, hydrochlorides, hydrobromides and hydroiodides; inorganic acid salts such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkane sulfonates such as methanesulfonates, trifluoromethanesulfonates and ethanesulfonates; arylsulfonates such as benzenesulfonates and p-toluenesulfonates; organic acid salts such as acetates, malates, fumarates, succinates, citrates, tartarates, oxalates, maleates, etc.; and, amino acid salts such as salts of glycine, lysine, arginine, ornithine, glutamic acid and aspartic acid. These salts may be produced by known methods. Alternatively, the antisense oligomer of the present invention may be in the form of a hydrate thereof.

2. Suppressor Antisense Oligomer

Another embodiment of the present invention provides a suppressor antisense oligomer which suppresses single exon skipping (hereinafter, referred to as “single skipping”). The suppressor antisense oligomer can suppress single skipping and thereby enhance an effect of multi-exon skipping by an antisense oligomer. The single skipping relates to skipping of only one exon, not simultaneous skipping of a plurality of exons.

Specifically, the present invention provides a suppressor antisense oligomer or a pharmaceutically acceptable salt thereof, or hydrate thereof which suppresses single skipping of any one exon selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA. Hereinafter, the suppressor antisense oligomer and the pharmaceutically acceptable salt thereof and the hydrate of the antisense oligomer or the salt are also collectively referred to as the “suppressor antisense oligomer of the present invention”. The suppressor antisense oligomer of the present invention may refer to each of the suppressor antisense oligomer or the pharmaceutically acceptable salt thereof, or hydrate thereof.

The suppressor antisense oligomer of the present invention suppresses single skipping by targeting the site of a splicing silencer sequence, a branch site sequence, or a splice site sequence in human dystrophin pre-mRNA and inhibiting splicing. The suppressor antisense oligomer of the present invention reduces the efficiency of single skipping of an intended exon as compared to a control.

For example, the suppressor antisense oligomer that suppresses single skipping of any one exon selected from the group consisting of the 45th exon to the 55th exon targets a splicing silencer sequence of any one of the 44th to 56th exons, or a splicing silencer sequence, a branch site sequence, or a splice site sequence of any one of the 44th to 55th introns.

As used herein, the splicing silencer sequence is a base sequence element that functions to suppress recognition of an exon in pre-mRNA. Examples of the splicing silencer sequence include recognition sequences of proteins or protein complexes, such as heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), hnRNP A2/B1, DAZAP1, hnRNP I, Fox-1, Fox-2, hnRNP H1, hnRNP H2, hnRNP H3, hnRNP L, Sam68, and SRp40.

As used herein, the splice site is the boundary between an exon and an intron. The splice site can be predicted from the 5′ end of the intron starting at GU, AU, or the like, and the 3′ end of the intron ending at AG, AC, or the like.

As used herein, the branch site is a sequence within an intron that initiates an attack on a 5′ splice site during splicing reaction and forms a covalent bond. The branch site sequence refers to a base sequence in an intron containing the branch site.

Examples of the splicing silencer sequence, the branch site sequence, or the splice site sequence which is the target (hereinafter, referred to as a “suppressor sequence”) of the suppressor antisense oligomer of the present invention include base sequences given below (SEQ ID NOs: 370 to 384 and 390). The positions of the suppressor sequence, the branch site sequence, and the splice site sequence can be detected using software such as SpliceAid (Francesco Piva et al., Bioinformatics, 25 (9), 1211-1213, 2009), SpliceAid-2 (Francesco Piva et al., Human Mutation, 33 (1), 81-85, 2012), SpliceAid-F (Matteo Giulietti et al., Nucleic Acids Res., 41, D125-D131, 2012), ATtRACT-a (Girolamo Giudice et al., Database (Oxford), baw035, 2016), SROOGLE (Schraga Schwartz et al., Nucleic Acids Res., 37, W189-W192, 2009), Reg RNA (Hsi-Yuan Huang et al., Nucleic Acids Res., 34, W429-W434, 2006), Reg RNA 2.0 (Tzu-Hao Chang et al., BMC bioinformatics, 14 (Suppl 2), S4, 2013), Human Splicing Finder (Francois-Olivier Desmet et al., Nucleic Acids Res., 37, 9, e67, 2009), or SVM-BPfinder (Andre Corvelo et al., PLoS Comput Biol., 6, 11, e1001016, 2010).

TABLE 6

SEQ ID
Name of protein or protein complex
Base

NO
or sequence site to be bound
sequence

370
hnRNP A1
UAGGA

371

UAGGAU

372

UAGGAA

373

UAGGCA

374

UAGGCU

375

UAGGGA

376

UAGGGC

377

UAGGGU

378

GGUAGGGC

379

AGAAC

380
Fox-1
AGCAUG

381

UGACUG

382

UGCAUG

383
Branch site
yUnAy

384
Splice site
GU

390

AG

y: A or U,

n: A, G, C, U, or T

Herein, specific examples of the splicing silencer sequence include SEQ ID NOs: 370 to 382. Examples of mutants of SEQ ID NOs: 370 to 382 also include

(F1) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 382, and has a length within ±15% of the length of the any one base sequence selected,

(F2) a base sequence that has at least 86% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 382, and has a length within ±14% of the length of the any one base sequence selected,

(F3) a base sequence that has at least 87% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 382, and has a length within ±13% of the length of the any one base sequence selected,

(F4) a base sequence that has at least 88% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 382, and has a length within ±12% of the length of the any one base sequence selected,

(F5) a base sequence that has at least 89% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 382, and has a length within ±11% of the length of the any one base sequence selected,

(F6) a base sequence that has at least 90% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 382, and has a length within ±10% of the length of the any one base sequence selected,

(F7) a base sequence that has at least 91% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 382, and has a length within ±9% of the length of the any one base sequence selected,

(F8) a base sequence that has at least 92% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 382, and has a length within ±8% of the length of the any one base sequence selected,

(F9) a base sequence that has at least 93% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 382, and has a length within ±7% of the length of the any one base sequence selected,

(F10) a base sequence that has at least 94% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 382, and has a length within ±6% of the length of the any one base sequence selected,

(F11) a base sequence that has at least 95% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 382, and has a length within ±5% of the length of the any one base sequence selected,

(F12) a base sequence that has at least 96% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 382, and has a length within ±4% of the length of the any one base sequence selected,

(F13) a base sequence that has at least 97% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 382, and has a length within ±3% of the length of the any one base sequence selected,

(F14) a base sequence that has at least 98% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 382, and has a length within ±2% of the length of the any one base sequence selected,

(F15) a base sequence that has at least 99% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 382, and has a length within ±1% of the length of the any one base sequence selected, and

(F16) a base sequence that has at least 99.5% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 382, and has a length within ±0.5% of the length of the any one base sequence selected.

In a certain embodiment of the present invention, the splicing silencer sequence may be a recognition sequence of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). Examples of the recognition sequence of human hnRNP A1 are shown in SEQ ID NOs: 370 to 379.

Herein, specific examples of the splice site sequence include SEQ ID NOs: 384 and 390. Examples of mutants of SEQ ID NOs: 384 and 390 also include

(G1) a base sequence that has at least 80% identity with the base sequence of SEQ ID NO: 384 or 390, and has a length within ±20% of the length of the any one base sequence selected.

Herein, specific examples of the branch site sequence include SEQ ID NO: 383. Examples of a mutant of SEQ ID NO: 383 also include

(H1) a base sequence that has at least 80% identity with the base sequence of SEQ ID NO: 383, and has a length within ±20% of the length of the any one base sequence selected.

As for a mutant type of the suppressor sequence, the following target regions are also included in the target base sequence of the suppressor antisense oligomer of the present invention:

(I1) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 384 and 390, and has a length within ±15% of the length of the any one base sequence selected,

(I2) a base sequence that has at least 86% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 384 and 390, and has a length within ±14% of the length of the any one base sequence selected,

(I3) a base sequence that has at least 87% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 384 and 390, and has a length within ±13% of the length of the any one base sequence selected,

(I4) a base sequence that has at least 88% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 384 and 390, and has a length within ±12% of the length of the any one base sequence selected,

(I5) a base sequence that has at least 89% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 384 and 390, and has a length within ±11% of the length of the any one base sequence selected,

(I6) a base sequence that has at least 90% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 384 and 390, and has a length within ±10% of the length of the any one base sequence selected,

(I7) a base sequence that has at least 91% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 384 and 390, and has a length within ±9% of the length of the any one base sequence selected,

(I8) a base sequence that has at least 92% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 384 and 390, and has a length within ±8% of the length of the any one base sequence selected,

(I9) a base sequence that has at least 93% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 384 and 390, and has a length within ±7% of the length of the any one base sequence selected,

(I10) a base sequence that has at least 94% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 384 and 390, and has a length within ±6% of the length of the any one base sequence selected,

(I11) a base sequence that has at least 95% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 384 and 390, and has a length within ±5% of the length of the any one base sequence selected,

(I12) a base sequence that has at least 96% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 384 and 390, and has a length within ±4% of the length of the any one base sequence selected,

(I13) a base sequence that has at least 97% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 384 and 390, and has a length within ±3% of the length of the any one base sequence selected,

(I14) a base sequence that has at least 98% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 384 and 390, and has a length within ±2% of the length of the any one base sequence selected,

(I15) a base sequence that has at least 99% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 384 and 390, and has a length within ±1% of the length of the any one base sequence selected, and

(I16) a base sequence that has at least 99.5% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 370 to 384 and 390, and has a length within ±0.5% of the length of the any one base sequence selected.

In one embodiment, the suppressor antisense oligomer of the present invention comprises a base sequence complementary to any one base sequence of a base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 370 to 384 and 390 and the mutant type base sequences shown in (I1) to (I16) above, or a partial base sequence thereof. In another embodiment, the suppressor antisense oligomer of the present invention consists of a base sequence complementary to any one base sequence of a base sequence selected from the group consisting of the base sequences represented by SEQ ID NOs: 370 to 384 and 390, or a partial base sequence thereof.

Herein, the term “partial” is as defined in the section about the antisense oligomer of the present invention.

As a further embodiment, the suppressor antisense oligomer of the present invention is a suppressor antisense oligomer which consists of

(J1) any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, or

(J2) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, and has a length within ±15% of the length of the any one base sequence selected, or a pharmaceutically acceptable salt thereof, or hydrate thereof.

Herein, the base sequence (J2) is a mutant type of the base sequence (J1). Such a mutant type also includes

(J2-1) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, and has a length within ±15% of the length of the any one base sequence selected,

(J2-2) a base sequence that has at least 86% identity consisting of SEQ ID NOs: 257 to 275, and has a length within ±14% of the length of the any one base sequence selected,

(J2-3) a base sequence that has at least 87% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, and has a length within ±13% of the length of the any one base sequence selected,

(J2-4) a base sequence that has at least 88% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, and has a length within ±12% of the length of the any one base sequence selected,

(J2-5) a base sequence that has at least 89% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, and has a length within ±11% of the length of the any one base sequence selected,

(J2-6) a base sequence that has at least 90% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, and has a length within ±10% of the length of the any one base sequence selected,

(J2-7) a base sequence that has at least 91% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, and has a length within ±9% of the length of the any one base sequence selected,

(J2-8) a base sequence that has at least 92% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, and has a length within ±8% of the length of the any one base sequence selected,

(J2-9) a base sequence that has at least 93% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, and has a length within ±7% of the length of the any one base sequence selected,

(J2-10) a base sequence that has at least 94% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, and has a length within ±6% of the length of the any one base sequence selected,

(J2-11) a base sequence that has at least 95% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, and has a length within ±5% of the length of the any one base sequence selected,

(J2-12) a base sequence that has at least 96% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, and has a length within ±4% of the length of the any one base sequence selected,

(J2-13) a base sequence that has at least 97% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, and has a length within ±3% of the length of the any one base sequence selected,

(J2-14) a base sequence that has at least 98% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, and has a length within ±2% of the length of the any one base sequence selected,

(J2-15) a base sequence that has at least 99% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, and has a length within ±1% of the length of the any one base sequence selected, and

(J2-16) a base sequence that has at least 99.5% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, and has a length within ±0.5% of the length of the any one base sequence selected.

(J2-1) to (J2-16) or a pharmaceutically acceptable salt thereof, or hydrate thereof.

A further embodiment of the suppressor antisense oligomer of the present invention is a suppressor antisense oligomer consisting of

(J′1) any one base sequence selected from the group consisting of SEQ ID NO: 260, 261, and 263, or

(J′2) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 260, 261, and 263, and has a length within ±15% of the length of the any one base sequence selected, or

a pharmaceutically acceptable salt thereof, or hydrate thereof.

Herein, the base sequence (J′2) is a mutant type of the base sequence (J′1). Such a mutant type also includes

(J′2-1) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 260, 261, and 263, and has a length within ±15% of the length of the any one base sequence selected,

(J′2-2) a base sequence that has at least 86% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 260, 261, and 263, and has a length within ±14% of the length of the any one base sequence selected,

(J′2-3) a base sequence that has at least 87% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 260, 261, and 263, and has a length within ±13% of the length of the any one base sequence selected,

(J′2-4) a base sequence that has at least 88% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 260, 261, and 263, and has a length within ±12% of the length of the any one base sequence selected,

(J′2-5) a base sequence that has at least 89% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 260, 261, and 263, and has a length within ±11% of the length of the any one base sequence selected,

(J′2-6) a base sequence that has at least 90% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 260, 261, and 263, and has a length within ±10% of the length of the any one base sequence selected,

(J′2-7) a base sequence that has at least 91% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 260, 261, and 263, and has a length within ±9% of the length of the any one base sequence selected,

(J′2-8) a base sequence that has at least 92% identity consisting of SEQ ID NOs: 260, 261, and 263, and has a length within ±8% of the length of the any one base sequence selected,

(J′2-9) a base sequence that has at least 93% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 260, 261, and 263, and has a length within ±7% of the length of the any one base sequence selected,

(J′2-10) a base sequence that has at least 94% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 260, 261, and 263, and has a length within ±6% of the length of the any one base sequence selected,

(J′2-11) a base sequence that has at least 95% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 260, 261, and 263, and has a length within ±5% of the length of the any one base sequence selected,

(J′2-12) a base sequence that has at least 96% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 260, 261, and 263, and has a length within ±4% of the length of the any one base sequence selected,

(J′2-13) a base sequence that has at least 97% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 260, 261, and 263, and has a length within ±3% of the length of the any one base sequence selected,

(J′2-14) a base sequence that has at least 98% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 260, 261, and 263, and has a length within ±2% of the length of the any one base sequence selected,

(J′2-15) a base sequence that has at least 99% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 260, 261, and 263, and has a length within ±1% of the length of the any one base sequence selected, and

(J′2-16) a base sequence that has at least 99.5% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 260, 261, and 263, and has a length within ±0.5% of the length of the any one base sequence selected.

Thus, a further embodiment of the suppressor antisense oligomer of the present invention is a suppressor antisense oligomer consisting of any one base sequence selected from the group consisting of (J′1) and (J′2-1) to (J′2-16) or a pharmaceutically acceptable salt thereof, or hydrate thereof.

The suppressor antisense oligomer of the present invention enhances a multi-skipping effect under physiological conditions. The term “under physiological conditions” refers to conditions set to mimic the in vivo environment in terms of pH, salt composition and temperature. The conditions are, for example, 25 to 40° C., preferably 37° C., pH 5 to 8, preferably pH 7.4 and 150 mM of sodium chloride concentration.

Whether the suppressor antisense oligomer of the present invention enhances a multi-exon skipping effect or not can be confirmed by providing (i) an experimental system for multi-exon skipping using only the antisense oligomer of the present invention alone and (ii) an experimental system for multi-exon skipping using the antisense oligomer of the present invention and the suppressor antisense oligomer of the present invention such that the other conditions are the same therebetween, and observing the difference between a multi-exon skipping effect obtained in the experimental system (ii) and a multi-exon skipping effect obtained in the experimental system (i).

The multi-skipping effect is measured as mentioned in the section “1. Antisense oligomer” about the antisense oligomer of the present invention.

The suppressor antisense oligomer of the present invention is 10 to 60 bases long and may be 10 to 55 bases long, 10 to 50 bases long, 10 to 45 bases long, 10 to 40 bases long, 10 to 35 bases long, 10 to 30 bases long, 10 to 25 bases long, 15 to 60 bases long, 15 to 55 bases long, 15 to 50 bases long, 15 to 45 bases long, 15 to 40 bases long, 15 to 35 bases long, 15 to 30 bases long, 15 to 25 bases long, 16 to 60 bases long, 16 to 55 bases long, 16 to 50 bases long, 16 to 45 bases long, 16 to 40 bases long, 16 to 35 bases long, 16 to 30 bases long, 16 to 25 bases long, 17 to 60 bases long, 17 to 55 bases long, 17 to 50 bases long, 17 to 45 bases long, 17 to 40 bases long, 17 to 35 bases long, 17 to 30 bases long, 17 to 25 bases long, 18 to 60 bases long, 18 to 55 bases long, 18 to 50 bases long, 18 to 45 bases long, 18 to 40 bases long, 18 to 35 bases long, 18 to 30 bases long, 18 to 25 bases long, 19 to 60 bases long, 19 to 55 bases long, 19 to 50 bases long, 19 to 45 bases long, 19 to 40 bases long, 19 to 35 bases long, 19 to 30 bases long, 19 to 25 bases long, 20 to 60 bases long, 20 to 55 bases long, 20 to 50 bases long, 20 to 45 bases long, 20 to 40 bases long, 20 to 35 bases long, 20 to 30 bases long, 20 to 25 bases long, 15 to 30 bases long, 15 to 29 bases long, 15 to 28 bases long, 15 to 27 bases long, 15 to 26 bases long, 15 to 25 bases long, 15 to 24 bases long, 15 to 23 bases long, 15 to 22 bases long, 15 to 21 bases long, 15 to 20 bases long, 15 to 19 bases long, 15 to 18 bases long, 16 to 30 bases long, 16 to 29 bases long, 16 to 28 bases long, 16 to 27 bases long, 16 to 26 bases long, 16 to 25 bases long, 16 to 24 bases long, 16 to 23 bases long, 16 to 22 bases long, 16 to 21 bases long, 16 to 20 bases long, 16 to 19 bases long, 16 to 18 bases long, 17 to 30 bases long, 17 to 29 bases long, 17 to 28 bases long, 17 to 27 bases long, 17 to 26 bases long, 17 to 25 bases long, 17 to 24 bases long, 17 to 23 bases long, 17 to 22 bases long, 17 to 21 bases long, 17 to 20 bases long, 17 to 19 bases long, 17 to 18 bases long, 18 to 30 bases long, 18 to 29 bases long, 18 to 28 bases long, 18 to 27 bases long, 18 to 26 bases long, 18 to 25 bases long, 18 to 24 bases long, 18 to 23 bases long, 18 to 22 bases long, 18 to 21 bases long, 18 to 20 bases long, 18 to 19 bases long, 19 to 30 bases long, 19 to 29 bases long, 19 to 28 bases long, 19 to 27 bases long, 19 to 26 bases long, 19 to 25 bases long, 19 to 24 bases long, 19 to 23 bases long, 19 to 22 bases long, 19 to 21 bases long, 19 to 20 bases long, 20 to 30 bases long, 20 to 29 bases long, 20 to 28 bases long, 20 to 27 bases long, 20 to 26 bases long, 20 to 25 bases long, 20 to 24 bases long, 20 to 23 bases long, 20 to 22 bases long, 20 to 21 bases long, 60 bases long, 59 bases long, 58 bases long, 57 bases long, 56 bases long, 55 bases long, 54 bases long, 53 bases long, 52 bases long, 51 bases long, 50 bases long, 49 bases long, 48 bases long, 47 bases long, 46 bases long, 45 bases long, 44 bases long, 43 bases long, 42 bases long, 41 bases long, 40 bases long, 39 bases long, 38 bases long, 37 bases long, 36 bases long, 35 bases long, 34 bases long, 33 bases long, 32 bases long, 31 bases long, 30 bases long, 29 bases long, 28 bases long, 27 bases long, 26 bases long, 25 bases long, 24 bases long, 23 bases long, 22 bases long, 21 bases long, 20 bases long, 19 bases long, 18 bases long, 17 bases long, 16 bases long, 15 bases long, 14 bases long, 13 bases long, 12 bases long, 11 bases long, or 10 bases long, but not limited thereto. These lengths may be increased or decreased by 1, 2, or 3 bases.

The suppressor antisense oligomer of the present invention may be a linked-type antisense oligomer configured to comprise a plurality of unit oligomers linked to each other, or a pharmaceutically acceptable salt thereof, or hydrate thereof. The unit oligomers may be linked via a linker that does not contribute to hybridization, or may be linked directly without the mediation of a linker, as mentioned above. When the suppressor antisense oligomer of the present invention is a linked type, the respective base sequences of the unit oligomers are neither consecutive nor overlapped with each other.

The suppressor antisense oligomer of the present invention may be an oligonucleotide, morpholino oligomer, or peptide nucleic acid (PNA) oligomer.

When the suppressor antisense oligomer of the present invention is an oligonucleotide (hereinafter, referred to as the “suppressor antisense oligonucleotide of the present invention”), the suppressor antisense oligonucleotide of the present invention is a suppressor antisense oligomer composed of nucleotides as constituent units. Such nucleotides may be any of ribonucleotides, deoxyribonucleotides and modified nucleotides.

The modified nucleotide is as mentioned above.

Preferably, the suppressor antisense oligonucleotide of the present invention is the suppressor antisense oligomer of the present invention having a group represented by general formula below as a constituent unit wherein the —OH group at position 2′ of ribose is substituted with methoxy and the phosphate bond moiety is a phosphorothioate bond:

embedded image

wherein Base has the same significance as defined above.

The suppressor antisense oligonucleotide of the present invention may be easily synthesized using various automated synthesizer (e.g., AKTA oligopilot plus 10/100 (GE Healthcare)). Alternatively, the synthesis may also be entrusted to a third-party organization (e.g., Promega Corp. or Takara Co.), etc.

When the suppressor antisense oligomer of the present invention is a morpholino oligomer (hereinafter, referred to as the “suppressor antisense morpholino oligomer of the present invention”), the suppressor antisense morpholino oligomer of the present invention is a suppressor antisense oligomer having a group represented by general formula below as a constituent unit:

embedded image

wherein Base and W have the same significance as defined above.

Examples of morpholino monomer compounds that are used in synthesis of the suppressor antisense morpholino oligomer of the present invention include, but not limited to, the following morpholino monomer compound (A), morpholino monomer compound (C), morpholino monomer compound (T), and morpholino monomer compound (G) shown in Table 7.

TABLE 7

Morpholino monomer
Morpholino monomer

compound
compound

(A)
(C)

embedded image

Morpholino monomer
Morpholino monomer

compound
compound

(T)
(G)

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In the suppressor antisense morpholino oligomer of the present invention, preferably the morpholino oligomer is PMO.

When the suppressor antisense oligomer of the present invention is a peptide nucleic acid oligomer (hereinafter, referred to as the “suppressor antisense peptide nucleic acid oligomer of the present invention”), the suppressor antisense peptide nucleic acid oligomer of the present invention is a suppressor antisense oligomer having a group represented by general formula (I) below as a constituent unit:

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wherein Base has the same significance as defined above.

The peptide nucleic acid oligomer can be produced as mentioned above.

The suppressor antisense oligomer of the present invention may be in the form of a pharmaceutically acceptable salt thereof, in the form of a hydrate thereof, or in the form of a hydrate of the pharmaceutically acceptable salt.

Examples of the pharmaceutically acceptable salt of the suppressor antisense oligomer of the present invention are alkali metal salts such as salts of sodium, potassium and lithium; alkaline earth metal salts such as salts of calcium and magnesium; metal salts such as salts of aluminum, iron, zinc, copper, nickel, cobalt, etc.; ammonium salts; organic amine salts such as salts of t-octylamine, dibenzylamine, morpholine, glucosamine, phenylglycine alkyl ester, ethylenediamine, N-methylglucamine, guanidine, diethylamine, triethylamine, dicyclohexylamine, N,N′-dibenzylethylenediamine, chloroprocaine, procaine, diethanolamine, N-benzyl-phenethylamine, piperazine, tetramethylammonium, tris(hydroxymethyl)aminomethane; hydrohalide salts such as salts of hydrofluorides, hydrochlorides, hydrobromides and hydroiodides; inorganic acid salts such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkane sulfonates such as methanesulfonates, trifluoromethanesulfonates and ethanesulfonates; arylsulfonates such as benzenesulfonates and p-toluenesulfonates; organic acid salts such as acetates, malates, fumarates, succinates, citrates, tartarates, oxalates, maleates, etc.; and, amino acid salts such as salts of glycine, lysine, arginine, ornithine, glutamic acid and aspartic acid. These salts may be produced by known methods. Alternatively, the suppressor antisense oligomer of the present invention may be in the form of a hydrate thereof.

[Method for Producing PMO]

Each of the antisense oligomer of the present invention and the suppressor antisense oligomer of the present invention may be PMO. An embodiment of PMO is, for example, the compound represented by general formula (I) below (hereinafter, referred to as PMO (I)).

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wherein Base, R²and R³have the same significance as defined above; and,

n is a given integer of 1 to 99, preferably a given integer of 18 to 28.

PMO (I) can be produced in accordance with a known method (cf., e.g., WO2009/064471 or WO2013/100190).

In the antisense oligomer of the present invention or the suppressor antisense oligomer of the present invention, the 5′ end may be a group represented by any of chemical structures (1) to (3) below, and preferably is (3) —OH.

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Hereinafter, the groups shown by (1), (2) and (3) above are referred to as “Group (1),” “Group (2)” and “Group (3),” respectively.

3. Medical Application

The present invention provides a pharmaceutical composition comprising the antisense oligomer of the present invention (also including the pharmaceutically acceptable salt thereof, or hydrate thereof) (hereinafter, referred to as the “pharmaceutical composition of the present invention”). The pharmaceutical composition of the present invention may further comprise the suppressor antisense oligomer of the present invention (also including the pharmaceutically acceptable salt thereof, or hydrate thereof) and/or a pharmaceutically acceptable carrier.

The present invention also provides a pharmaceutical composition comprising the antisense oligomer of the present invention (also including the pharmaceutically acceptable salt thereof, or hydrate thereof) and the suppressor antisense oligomer of the present invention (also including the pharmaceutically acceptable salt thereof, or hydrate thereof) (hereinafter, referred to as the “pharmaceutical combination of the present invention”).

When the pharmaceutical composition of the present invention comprises the antisense oligomer of the present invention and the suppressor antisense oligomer of the present invention, any combination of these oligomers is included. In the pharmaceutical combination of the present invention, any combination of the antisense oligomer of the present invention and the suppressor antisense oligomer of the present invention is included. The antisense oligomer contained in the pharmaceutical composition of the present invention or the pharmaceutical combination of the present invention can be any antisense oligomer of the present invention and is, but not particularly limited to, preferably an antisense oligomer consisting of any one base sequence selected from the group consisting of SEQ ID NOs: 1 to 111, 113 to 177, 179, 180, 182, 183, 185 to 193, 195 to 198, 200 to 223, and 225 to 232, more preferably an antisense oligomer consisting of any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 7, 8, 10, 11, 14, 26, 27, 28, 29, 35, 38, 39, 40, 41, 43, 45, 46, 47, 50, 51, 52, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 68, 74, 75, 76, 77, 78, 80, 82, 86, 92, 97, 98, 100, 102, 113, 119, 121, 122, 124, 125, 126, 128, 130, 131, 132, 139, 142, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 225, 228, 231, and 232, and further preferably an antisense oligomer consisting of any one base sequence selected from the group consisting of SEQ ID NOs: 1, 5, 11, 27, 28, 35, 40, 41, 43, 45, 46, 47, 50, 51, 52, 54, 55, 59, 63, 64, 65, 66, 68, 74, 75, 76, 77, 78, 102, 113, 119, 128, 131, 132, 139, 142, 161, 162, 163, 225, and 228.

The suppressor antisense oligomer contained in the pharmaceutical composition of the present invention or the pharmaceutical combination of the present invention can be any suppressor antisense oligomer of the present invention and is, but not particularly limited to, preferably a suppressor antisense oligomer consisting of any one base sequence selected from the group consisting of SEQ ID NOs: 257 to 275, more preferably SEQ ID NO: 1, 5, 6, 7, 8, 10, 11, 14, 26, or 27, and further preferably a suppressor antisense oligomer consisting of any one base sequence selected from the group consisting of SEQ ID NOs: 260, 261, and 263.

When the pharmaceutical composition of the present invention comprises the antisense oligomer of the present invention and the suppressor antisense oligomer of the present invention, preferably, the pharmaceutical composition comprises a combination of the antisense oligomer of the present invention and the suppressor antisense oligomer of the present invention described above. Preferably, the pharmaceutical combination of the present invention comprises a combination of the antisense oligomer of the present invention and the suppressor antisense oligomer of the present invention described above.

In the pharmaceutical combination of the present invention, examples of the combination of the antisense oligomer of the present invention and the suppressor antisense oligomer of the present invention include

(K1) a combination in which the antisense oligomer is an oligomer consisting of SEQ ID NO: 75 and the suppressor antisense oligomer is an oligomer consisting of SEQ ID NO: 260,

(K2) a combination in which the antisense oligomer is an oligomer consisting of SEQ ID NO: 75 and the suppressor antisense oligomer is an oligomer consisting of SEQ ID NO: 261,

(K3) a combination in which the antisense oligomer is an oligomer consisting of SEQ ID NO: 75 and the suppressor antisense oligomer is an oligomer consisting of SEQ ID NO: 263,

(K4) a combination in which the antisense oligomer is an oligomer consisting of SEQ ID NO: 65 and the suppressor antisense oligomer is an oligomer consisting of SEQ ID NO: 260,

(K5) a combination in which the antisense oligomer is an oligomer consisting of SEQ ID NO: 65 and the suppressor antisense oligomer is an oligomer consisting of SEQ ID NO: 261,

(K6) a combination in which the antisense oligomer is an oligomer consisting of SEQ ID NO: 65 and the suppressor antisense oligomer is an oligomer consisting of SEQ ID NO: 263,

(K7) a combination in which the antisense oligomer is a combination of oligomers consisting of SEQ ID NOs: 55 and 59 and the suppressor antisense oligomer is an oligomer consisting of SEQ ID NO: 260,

(K8) a combination in which the antisense oligomer is a combination of oligomers consisting of SEQ ID NOs: 55 and 59 and the suppressor antisense oligomer is an oligomer consisting of SEQ ID NO: 261, and

(K9) a combination in which the antisense oligomer is a combination of oligomers consisting of SEQ ID NOs: 55 and 59 and the suppressor antisense oligomer is an oligomer consisting of SEQ ID NO: 263.

The pharmaceutical composition of the present invention may comprise the antisense oligomer of the present invention and the suppressor antisense oligomer of the present invention in these combinations.

The pharmaceutical composition of the present invention and the pharmaceutical combination of the present invention can each be used for the treatment of, for example, Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy (LGMD), congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, cerebral autosomal dominant arteriopathy with subcortical infarct and leukoencephalopathy (CADASIL), and Alport's syndrome. The pharmaceutical combination of the present invention and the pharmaceutical composition of the present invention can each be administered to a human patient and in particular, a human patient with muscular dystrophy. The patient to receive the pharmaceutical combination of the present invention or the pharmaceutical composition of the present invention may be a human patient having a mutation that is amenable to skipping of two or more exons selected from the group consisting of exons 45 to 55 in the dystrophin gene.

One embodiment of the present invention provides a method for treatment of muscular dystrophy, which comprises administering to a patient with muscular dystrophy the antisense oligomer of the present invention or a combination of the antisense oligomer of the present invention and the suppressor antisense oligomer of the present invention.

Another embodiment of the present invention provides a method for treatment of muscular dystrophy, which comprises administering to a patient with muscular dystrophy the pharmaceutical composition of the present invention or the pharmaceutical combination of the present invention.

The method for treatment may involve performing skipping of any two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA. In the method for treatment, the patient with muscular dystrophy may be a patient having a mutation that is amenable to exon 45 to 55 skipping in the dystrophin gene. The patient may be a human and may be a human patient having a mutation that is amenable to exon 45 to 55 skipping in the dystrophin gene.

The present invention further provides use of the antisense oligomer of the present invention or a combination of the antisense oligomer of the present invention and the suppressor antisense oligomer of the present invention, or the pharmaceutical composition of the present invention or the pharmaceutical combination of the present invention in manufacturing of a medicament for the treatment of muscular dystrophy.

The present invention further provides the antisense oligomer of the present invention or a combination of the antisense oligomer of the present invention and the suppressor antisense oligomer of the present invention, or the pharmaceutical composition of the present invention or the pharmaceutical combination of the present invention for use in the treatment of muscular dystrophy. The treatment may involve performing skipping of any two or more numerically consecutive exons selected from the group consisting of the 45th exon to the 55th exon in human dystrophin pre-mRNA. In the treatment, the patient with muscular dystrophy may be a patient having a mutation that is amenable to exon 45 to 55 skipping in the dystrophin gene. The patient may be a human and may be a human patient having a mutation that is amenable to exon 45 to 55 skipping in the dystrophin gene.

Administration route for the antisense oligomer of the present invention or the combination of the antisense oligomer of the present invention and the suppressor antisense oligomer of the present invention, or the pharmaceutical composition of the present invention or the pharmaceutical combination of the present invention is not particularly limited so long as it is pharmaceutically acceptable route for administration, and can be chosen depending upon method of treatment. In view of easiness in delivery to muscle tissues, preferred are intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, oral administration, tissue administration, transdermal administration, etc. Also, dosage forms which are available for the composition of the present invention are not particularly limited, and include, for example, various injections, oral agents, drips, inhalations, ointments, lotions, etc.

In administration of the antisense oligomer of the present invention and/or the suppressor antisense oligomer of the present invention to patients with muscular dystrophy, preferably, the composition of the present invention contains a carrier to promote delivery of the oligomer to muscle tissues. Such a carrier is not particularly limited as far as it is pharmaceutically acceptable, and examples include cationic carriers such as cationic liposomes, cationic polymers, etc., or carriers using viral envelope. The cationic liposomes are, for example, liposomes composed of 2-O-(2-diethylaminoethyl)carbamoyl-1,3-O-dioleoylglycerol and phospholipids as the essential constituents (hereinafter referred to as “liposome A”), Oligofectamine (registered trademark) (manufactured by Invitrogen Corp.), Lipofectin (registered trademark) (manufactured by Invitrogen Corp.), Lipofectamine (registered trademark) (manufactured by Invitrogen Corp.), Lipofectamine 2000 (registered trademark) (manufactured by Invitrogen Corp.), DMRIE-C(registered trademark) (manufactured by Invitrogen Corp.), GeneSilencer (registered trademark) (manufactured by Gene Therapy Systems), TransMessenger (registered trademark) (manufactured by QIAGEN, Inc.), TransIT TKO (registered trademark) (manufactured by Mirus) and Nucleofector II (Lonza). Among others, liposome A is preferred. Examples of cationic polymers are JetSI (registered trademark) (manufactured by Qbiogene, Inc.) and Jet-PEI (registered trademark) (polyethylenimine, manufactured by Qbiogene, Inc.). An example of carriers using viral envelop is GenomeOne (registered trademark) (HVJ-E liposome, manufactured by Ishihara Sangyo). Alternatively, the medical devices described in Japanese Patent Nos. 2924179 and the cationic carriers described in Japanese Domestic Re-Publication PCT Nos. 2006/129594 and 2008/096690 may be used as well.

A concentration of the antisense oligomer of the present invention contained in the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention may vary depending on kind of the carrier, etc., and is appropriately in a range of 0.1 nM to 100 μM, preferably in a range of 1 nM to 10 μM, and more preferably in a range of 10 nM to 1 μM. A weight ratio of the antisense oligomer of the present invention contained in the composition of the present invention and the carrier (carrier/antisense oligomer of the present invention) may vary depending on property of the oligomer, type of the carrier, etc., and is appropriately in a range of 0.1 to 100, preferably in a range of 1 to 50, and more preferably in a range of 10 to 20.

A concentration of the suppressor antisense oligomer of the present invention contained in the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention may vary depending on kind of the carrier, etc., and is appropriately in a range of 0.1 nM to 100 μM, preferably in a range of 1 nM to 10 μM, and more preferably in a range of 10 nM to 1 μM. A weight ratio of the suppressor antisense oligomer of the present invention contained in the composition of the present invention and the carrier (carrier/suppressor antisense oligomer of the present invention) may vary depending on property of the oligomer, type of the carrier, etc., and is appropriately in a range of 0.1 to 100, preferably in a range of 1 to 50, and more preferably in a range of 10 to 20.

The pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention may be in the form of an aqueous solution. In this case, the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention may comprise the antisense oligomer of the present invention in a concentration of 2.5 to 500 mg/mL, 5 to 450 mg/mL, 10 to 400 mg/mL, 15 to 350 mg/mL, 20 to 300 mg/mL, 20 to 250 mg/mL, 20 to 200 mg/mL, 20 to 150 mg/mL, 20 to 100 mg/mL, 20 to 50 mg/mL, 20 to 40 mg/mL, 20 to 30 mg/mL, 23 to 27 mg/mL, 24 to 26 mg/mL, or 25 mg/mL. Alternatively, the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention may comprise the antisense oligomer of the present invention in a concentration of 10 to 100 mg/mL, 15 to 95 mg/mL, 20 to 80 mg/mL, 25 to 75 mg/mL, 30 to 70 mg/mL, 35 to 65 mg/mL, 40 to 60 mg/mL, 45 to 55 mg/mL, 47 to 53 mg/mL, 48 to 52 mg/mL, 49 to 51 mg/mL, or 50 mg/mL.

The pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention in the form of an aqueous solution may comprise the suppressor oligomer of the present invention in a concentration of 2.5 to 500 mg/mL, 5 to 450 mg/mL, 10 to 400 mg/mL, 15 to 350 mg/mL, 20 to 300 mg/mL, 20 to 250 mg/mL, 20 to 200 mg/mL, 20 to 150 mg/mL, 20 to 100 mg/mL, 20 to 50 mg/mL, 20 to 40 mg/mL, 20 to 30 mg/mL, 23 to 27 mg/mL, 24 to 26 mg/mL, or 25 mg/mL. Alternatively, the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention may comprise the suppressor oligomer of the present invention in a concentration of 10 to 100 mg/mL, 15 to 95 mg/mL, 20 to 80 mg/mL, 25 to 75 mg/mL, 30 to 70 mg/mL, 35 to 65 mg/mL, 40 to 60 mg/mL, 45 to 55 mg/mL, 47 to 53 mg/mL, 48 to 52 mg/mL, 49 to 51 mg/mL, or 50 mg/mL.

The pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention may be in a dry form. In this case, in order to prepare the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention in an aqueous solution form, for example, 125 mg or 250 mg of the antisense oligomer of the present invention in a dry form may be mixed with 0.5 mL to 100 mL of water (which corresponds to the antisense oligomer of the present invention in a concentration of 1.25 mg/mL to 250 mg/mL or 2.5 mg/mL to 500 mg/mL), preferably with 1 mL to 50 mL of water (which corresponds to the antisense oligomer of the present invention in a concentration of 2.5 mg/mL to 125 mg/mL or 5 mg/mL to 250 mg/mL), more preferably with 5 mL to 10 mL of water (which correspond to the antisense oligomer of the present invention in a concentration of 12.5 mg/mL to 25 mg/mL or 25 mg/mL to 50 mg/mL) and used.

When the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention is in a dry form, in order to prepare the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention in an aqueous solution form, for example, 125 mg or 250 mg of the suppressor oligomer of the present invention in a dry form may be mixed with 0.5 mL to 100 mL of water (which corresponds to the suppressor oligomer of the present invention in a concentration of 1.25 mg/mL to 250 mg/mL or 2.5 mg/mL to 500 mg/mL), preferably with 1 mL to 50 mL of water (which corresponds to the suppressor oligomer of the present invention in a concentration of 2.5 mg/mL to 125 mg/mL or 5 mg/mL to 250 mg/mL), more preferably with 5 mL to 10 mL of water (which correspond to the suppressor oligomer of the present invention in a concentration of 12.5 mg/mL to 25 mg/mL or 25 mg/mL to 50 mg/mL) and used.

The concentrations of the antisense oligomer of the present invention and the suppressor oligomer of the present invention contained in the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention may be the respective concentrations of the antisense oligomer of the present invention and the suppressor oligomer of the present invention or may be the total concentration of the antisense oligomer of the present invention and the suppressor oligomer of the present invention.

In addition to the antisense oligomer of the present invention and/or the suppressor antisense oligomer of the present invention and the carrier described above, pharmaceutically acceptable additives may also be optionally formulated in the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention. Examples of such additives are emulsification aids (e.g., fatty acids having 6 to 22 carbon atoms and their pharmaceutically acceptable salts, albumin and dextran), stabilizers (e.g., cholesterol, phosphatidic acid, mannitol, and sorbitol), isotonizing agents (e.g., sodium chloride, glucose, maltose, lactose, sucrose, and trehalose), and pH controlling agents (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide and triethanolamine). One or more of these additives can be used. The content of the additive in the composition of the present invention is appropriately 90 wt % or less, preferably 70 wt % or less and more preferably, 50 wt % or less.

The pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention can be prepared by adding the antisense oligomer of the present invention and/or the suppressor antisense oligomer of the present invention to a carrier dispersion and adequately stirring the mixture. Additives may be added at an appropriate step either before or after addition of the antisense oligomer of the present invention and/or the suppressor antisense oligomer of the present invention. An aqueous solvent that can be used in adding the antisense oligomer of the present invention and/or the suppressor antisense oligomer of the present invention is not particularly limited as far as it is pharmaceutically acceptable, and examples are injectable water or injectable distilled water, electrolyte fluid such as physiological saline, etc., and sugar fluid such as glucose fluid, maltose fluid, etc. A person skilled in the art can appropriately choose conditions for pH and temperature for such matter.

The pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention may be prepared into, e.g., a liquid form and its lyophilized preparation. The lyophilized preparation can be prepared by lyophilizing the composition of the present invention in a liquid form in a conventional manner. The lyophilization can be performed, for example, by appropriately sterilizing the composition of the present invention in a liquid form, dispensing an aliquot into a vial container, performing preliminary freezing for 2 hours at conditions in a range of about −40° C. to −20° C., performing a primary drying in a range of about 0° C. to 10° C. under reduced pressure, and then performing a secondary drying in a range of about 15° C. to 25° C. under reduced pressure. In general, the lyophilized preparation of the composition of the present invention can be obtained by replacing the content of the vial with nitrogen gas and capping.

The lyophilized preparation of the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention can be used in general upon reconstitution by adding an optional suitable solution (reconstitution liquid) and redissolving the preparation. Such a reconstitution liquid includes injectable water, physiological saline and other infusion fluids. A volume of the reconstitution liquid may vary depending on the intended use, etc., is not particularly limited, and is suitably 0.5-fold to 2-fold greater than the volume prior to lyophilization or no more than 500 mL.

It is desired to control a dose of the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention to be administered, by taking the following factors into account: the type and dosage form of the antisense oligomer of the present invention and/or the suppressor antisense oligomer of the present invention contained; patients' conditions including age, body weight, etc.; administration route; and the characteristics and extent of the disease. A single dose calculated as the amount of the antisense oligomer of the present invention and/or the suppressor antisense oligomer of the present invention can be 0.1 mg to 1 g per kg body weight, preferably 1 mg to 100 mg per kg body weight, more preferably 1 mg to 90 mg per kg body weight, and further preferably 1 mg to 80 mg per kg body weight. The frequency of administration may be once per 1 to 3 days, once per week, or once per 2 to 3 weeks. This numerical range may vary occasionally depending on type of the target disease, administration route and target molecule. Therefore, a dose or frequency of administration lower than the range may be sufficient in some occasion and conversely, a dose or frequency of administration higher than the range may be required occasionally.

In still another embodiment of the pharmaceutical composition of the present invention and/or the pharmaceutical combination of the present invention, there is provided a pharmaceutical composition comprising a vector capable of expressing the antisense oligomer of the present invention and/or the suppressor antisense oligomer of the present invention and the carrier described above. Such an expression vector may be a vector capable of expressing a plurality of the antisense oligomers of the present invention and/or the suppressor antisense oligomers of the present invention. The composition may be formulated with pharmaceutically acceptable additives as in the case with the composition of the present invention containing the antisense oligomer of the present invention and/or the suppressor antisense oligomer of the present invention. A concentration of the expression vector contained in the composition may vary depending upon type of the career, etc., and is appropriately in a range of 0.1 nM to 100 μM, preferably in a range of 1 nM to 10 μM, and more preferably in a range of 10 nM to 1 μM. A weight ratio of the expression vector contained in the composition and the carrier (carrier/expression vector) may vary depending on property of the expression vector, type of the carrier, etc., and is appropriately in a range of 0.1 to 100, preferably in a range of 1 to 50, and more preferably in a range of 10 to 20. The content of the carrier contained in the composition is the same as in the case with the composition of the present invention containing the antisense oligomer of the present invention and/or the suppressor antisense oligomer of the present invention, and a method for producing the same is also the same as in the case with the composition of the present invention.

4. Pre-mRNA of Interest Other than Human Dystrophin Pre-mRNA

As used herein, the case where the pre-mRNA of interest is human dystrophin pre-mRNA is specifically described, but is not limited thereto. As for pre-mRNA of interest other than human dystrophin pre-mRNA, methods for designing, producing, and using an antisense oligomer and a suppressor antisense oligomer can be carried out in accordance with techniques and methods disclosed herein or known in the art. Those skilled in the art can also design, produce and use an antisense oligomer or a suppressor antisense oligomer for pre-mRNA of interest other than human dystrophin pre-mRNA on the basis of techniques and methods disclosed herein or known in the art. Likewise, those skilled in the art can perform multi-exon skipping of the pre-mRNA of interest using the antisense oligomer and enhance the efficiency of multi-exon skipping using the suppressor antisense oligomer on the basis of techniques and methods disclosed herein or known in the art.

As used herein, examples of the pre-mRNA of interest include human γ-sarcoglycan (SGCG) pre-mRNA, human Notch3 pre-mRNA, and human dystrophin pre-mRNA. In a certain embodiment of the present invention, the pre-mRNA of interest may be human dystrophin pre-mRNA. In another embodiment of the present invention, the pre-mRNA of interest may be SGCG or human Notch3 pre-mRNA.

The base sequences of target regions of the antisense oligomer for use in multi-exon skipping in the pre-mRNA of interest of human SGCG or human Notch3 are as given below.

In the present invention, the target regions in human SGCG pre-mRNA are shown as SEQ ID NOs: 283 and 284 below.

TABLE 8

SEQ

Target region
Base sequence of target region
ID NO

Region in vicinity of
ATTACAAAAATATGGAAATAGAGAGTAGCAGGAAAAAGTATATGGCATT
283

acceptor of intron 3
TCTATTACCTAGAACCCCACTGTTGTAGGIGTCTTTCCTTGACATAGCT

(region indicated by
GTGACCA7ATAGCTCTGTTAAATTGCATATTTGTCTAAATAATTCATAT

range of −400 bases
TTTTTTCTATCCCTATCTTGCGTTTGGAGCTCATTTTATGATTGTTATT

to +50 bases with 3′
CTTTTATCTTTCAATAAATACTCAAAATGTTAAAAAAATATGATTCAGG

end of intron 3 defined
AATTTTTAATTGTCTTAGCCACAAATTTAIAGGATTTCCAGGATCTGTA

as basing point 0)
ACAATGGATAAATAATTTTATAAAAATCCTAAATTTACACAGAATTATA

AAGATATAATCATTTTAAACAGCACCTATTTTGCAAATTTTATAAATCT

CTTTCTAGGACTCATCTCTGCTTCTACAATCAACCCAGAATGTGACTGT

AAATGCGCG

Region in vicinity of
CACAGGCAGGTTAAAAGTCGGTGAGTCCAGCTTCATCATGGTGCTTTGC
284

donor of intron 4
ATGCATGTTGTCCATGAATAGTGCTAAATGAATGCATTGTTTTTTCTTC

(region indicated by
TAAAGAAATCAAAGCTACTTATGAACAAAATATGAATTTTCTAAATATC

range of −20 bases to
ATGCTGTGTTGACCACAGACTAGCACCACAGAGTGGGGTGGGGGGTGAG

+400 bases with 5′
GGGACAGCCTGAAGTGCTTTGATTAGAGTTATTTTTGTTCCAAGAAATA

end of intron 4 defined
GAGGACAATTAAAGCTAGTACAAGAAACAAAATGTTTA7TGTGGGTTAC

as basing point 0)
CAAAGACTCTTGCAGCAACCAAAGGGAAGGAACCCAAGIAGCTGGCCTC

AAAGAGCACAGAGACCAGTGGCTGAGAGCCACGAATACATTCAGTTCCC

TCCCCGTTCTTTCTTCTCTTTTCTCTCC

In the present invention, the target regions in human Notch3 pre-mRNA are shown below.

TABLE 9

SEQ

Target region
Base sequence of target region
ID NO

Region in vicinity of
AAGTGATCTGCCCGCCGCAGCCTCCCAAAGTTCTGGGATTACAGGCGTG
285

acceptor of intron 3
AGCCACTGCTCCCAGCAAATGTGTGTTTGCTGCTCTGTTTCCCTGCGTG

(region indicated by
TTTCTTGCCTGTCTTGTGTGTATCTTTGTGTCTGGGGCCATCCTGCCCT

range of −400 bases
GTGCTGCCCAACCAAGCCATCTCTGCCCACAGGTGCCCGCCTGGCTGGG

to +50 bases with 3′
TGGGTGAGCGGTGTCAGCTGGAGGACCCCTGTCACTCAGGCCCCTGTGC

end of intron 3 defined
TGGCCGTGGTGTCTGCCAGAGTTCAGTGGTGGCTGGCACCGCCCGATTC

as basing point 0)
TCATGCCGGTGCCCCCGTGGCTTCCGAGGTGAGAGGGGAAGAGTCTGGA

GGGGAGGTAGTCGGGGGTGTGGTCAGTCCTAAACTCACCCTGTCCTGGT

CCCTCCAGGCCCTGACTGCTCCCTGCCAGATCCCTGCCTCAGCAGCCCT

TGTGCCCAC

Region in vicinity of
CGACTGTGCCTGTCTTCCTGGTGAGTGAGCCCTACTCAGGAGAGTCAGA
286

donor of intron 4
GGGGTGGGCGTGGGGACAGCAGGCCAGCCCGGCGGTGACCATCCTTGCC

(region indicated by
CCCTTCCCTGCTAGGGTTTGAGGGTCAGAATTGTGAAGTGAACGTGGAC

range of −20 bases to
GACTGTCCAGGACACCGATGTCTCAATGGGGGGACATGCGTGGATGGCG

+400 bases with 5′ end
TCAACACCTATAACTGCCAGTGCCCTCCTGAGTGGACAGGTGGGCACTG

of intron 4 defined as
CGGCCAGAGGGAGCGGGGAGGCAGGCCTCGGGTGGACATGCGCCAGGTG

basing point 0)
GCTGGACTGCTGCATCTGTGTGCCACAGGCCAGTTCTGCACGGAGGACG

TGGATGAGTGTCAGCTGCAGCCCAACGCCTGCCACAATGGGGGTACCTG

CTTCAACACGCTGGGTGGCCACAGCTGC

The antisense oligomer for human SGCG that induces multi-exon skipping from human SGCG pre-mRNA targets, for example, the range of −400 to +50 bases with the 3′ end of intron 3 defined as a basing point, and as a specific example, is an antisense oligomer for human SGCG consisting of

(L1) any one base sequence selected from the group consisting of SEQ ID NOs: 287 to 308, or

(L2) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 287 to 308, and has a length within ±15% of the length of the any one base sequence selected, or a pharmaceutically acceptable salt thereof, or hydrate thereof.

Herein, the base sequence (L2) is a mutant type of the base sequence (L1). Such a mutant type also includes (L2-1) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 287 to 308, and has a length within ±15% of the length of the any one base sequence selected,

(L2-2) a base sequence that has at least 86% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 287 to 308, and has a length within ±14a of the length of the any one base sequence selected,

(L2-3) a base sequence that has at least 87% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 287 to 308, and has a length within ±13% of the length of the any one base sequence selected,

(L2-4) a base sequence that has at least 88% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 287 to 308, and has a length within ±12% of the length of the any one base sequence selected,

(L2-5) a base sequence that has at least 89% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 287 to 308, and has a length within ±11% of the length of the any one base sequence selected,

(L2-6) a base sequence that has at least 90% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 287 to 308, and has a length within ±10% of the length of the any one base sequence selected,

(L2-7) a base sequence that has at least 91% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 287 to 308, and has a length within ±9% of the length of the any one base sequence selected,

(L2-8) a base sequence that has at least 92% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 287 to 308, and has a length within ±8% of the length of the any one base sequence selected,

(L2-9) a base sequence that has at least 93% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 287 to 308, and has a length within ±7% of the length of the any one base sequence selected,

(L2-10) a base sequence that has at least 94% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 287 to 308, and has a length within ±6% of the length of the any one base sequence selected,

(L2-11) a base sequence that has at least 95% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 287 to 308, and has a length within ±5% of the length of the any one base sequence selected,

(L2-12) a base sequence that has at least 96% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 287 to 308, and has a length within ±4% of the length of the any one base sequence selected,

(L2-13) a base sequence that has at least 97% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 287 to 308, and has a length within ±3% of the length of the any one base sequence selected,

(L2-14) a base sequence that has at least 98% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 287 to 308, and has a length within ±2% of the length of the any one base sequence selected,

(L2-15) a base sequence that has at least 99% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 287 to 308, and has a length within ±1% of the length of the any one base sequence selected, and

(L2-16) a base sequence that has at least 99.5% identity consisting of SEQ ID NOs: 287 to 308, and has a length within ±0.5% of the length of the any one base sequence selected.

Thus, a further embodiment of the antisense oligomer for human SGCG is an antisense oligomer for human SGCG consisting of any one base sequence selected from the group consisting of (L1) and (L2-1) to (L2-16), or a pharmaceutically acceptable salt thereof, or hydrate thereof. The antisense oligomer for human SGCG is included in the present invention.

The suppressor antisense oligomer that suppresses single skipping from human SGCG pre-mRNA (suppressor antisense oligomer for human SGCG) targets, for example, a splicing silencer sequence of intron 4 or a splice site of single skipping, and as a specific example, is a suppressor antisense oligomer for human SGCG consisting of

(M1) any one base sequence selected from the group consisting of SEQ ID NOs: 331 to 335, or

(M2) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 331 to 335, and has a length within ±15% of the length of the any one base sequence selected, or a pharmaceutically acceptable salt thereof, or hydrate thereof.

Herein, the base sequence (M2) is a mutant type of the base sequence (M1). Such a mutant type also includes

(M2-1) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 331 to 335, and has a length within ±15% of the length of the any one base sequence selected,

(M2-2) a base sequence that has at least 86% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 331 to 335, and has a length within ±14% of the length of the any one base sequence selected,

(M2-3) a base sequence that has at least 87% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 331 to 335, and has a length within ±13% of the length of the any one base sequence selected,

(M2-4) a base sequence that has at least 88% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 331 to 335, and has a length within ±12% of the length of the any one base sequence selected,

(M2-5) a base sequence that has at least 89% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 331 to 335, and has a length within ±11% of the length of the any one base sequence selected,

(M2-6) a base sequence that has at least 90% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 331 to 335, and has a length within ±10% of the length of the any one base sequence selected,

(M2-7) a base sequence that has at least 91% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 331 to 335, and has a length within ±9% of the length of the any one base sequence selected,

(M2-8) a base sequence that has at least 92% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 331 to 335, and has a length within ±8% of the length of the any one base sequence selected,

(M2-9) a base sequence that has at least 93% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 331 to 335, and has a length within ±7% of the length of the any one base sequence selected,

(M2-10) a base sequence that has at least 94% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 331 to 335, and has a length within ±6% of the length of the any one base sequence selected,

(M2-11) a base sequence that has at least 95% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 331 to 335, and has a length within ±5% of the length of the any one base sequence selected,

(M2-12) a base sequence that has at least 96% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 331 to 335, and has a length within ±4% of the length of the any one base sequence selected,

(M2-13) a base sequence that has at least 97% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 331 to 335, and has a length within ±3% of the length of the any one base sequence selected,

(M2-14) a base sequence that has at least 98% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 331 to 335, and has a length within ±2% of the length of the any one base sequence selected,

(M2-15) a base sequence that has at least 99% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 331 to 335, and has a length within ±1% of the length of the any one base sequence selected, and

(M2-16) a base sequence that has at least 99.5% identity consisting of SEQ ID NOs: 331 to 335, and has a length within ±0.5% of the length of the any one base sequence selected.

Thus, a further embodiment of the suppressor antisense oligomer for human SGCG is a suppressor antisense oligomer for human SGCG consisting of any one base sequence selected from the group consisting of (M1) and (M2-1) to (M2-16), or a pharmaceutically acceptable salt thereof, or hydrate thereof. The suppressor antisense oligomer for human SGCG is included in the present invention. The suppressor antisense oligomer for human SGCG is included in the present invention. The suppressor antisense oligomer for human SGCG is capable of enhancing the efficiency of multi-skipping from SGCG pre-mRNA.

The antisense oligomer for human Notch3 that induces multi-exon skipping from human Notch3 pre-mRNA targets, for example, a region in the vicinity of an acceptor of intron 3, i.e., the range of −400 to +50 bases with the 3′ end of intron 3 defined as a basing point, and is, for example, an antisense oligomer for human Notch3 consisting of

(N1) any one base sequence selected from the group consisting of SEQ ID NOs: 309 to 330, or

(N2) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 309 to 330, and has a length within ±15% of the length of the any one base sequence selected, or a pharmaceutically acceptable salt thereof, or hydrate thereof.

Herein, the base sequence (N2) is a mutant type of the base sequence (N1). Such a mutant type also includes

(N2-1) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 309 to 330, and has a length within ±15% of the length of the any one base sequence selected,

(N2-2) a base sequence that has at least 86% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 309 to 330, and has a length within ±14% of the length of the any one base sequence selected,

(N2-3) a base sequence that has at least 87% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 309 to 330, and has a length within ±13% of the length of the any one base sequence selected,

(N2-4) a base sequence that has at least 88% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 309 to 330, and has a length within ±12% of the length of the any one base sequence selected,

(N2-5) a base sequence that has at least 89% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 309 to 330, and has a length within ±11% of the length of the any one base sequence selected,

(N2-6) a base sequence that has at least 90% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 309 to 330, and has a length within ±10% of the length of the any one base sequence selected,

(N2-7) a base sequence that has at least 91% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 309 to 330, and has a length within ±9% of the length of the any one base sequence selected,

(N2-8) a base sequence that has at least 92% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 309 to 330, and has a length within ±8% of the length of the any one base sequence selected,

(N2-9) a base sequence that has at least 93% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 309 to 330, and has a length within ±7% of the length of the any one base sequence selected,

(N2-10) a base sequence that has at least 94% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 309 to 330, and has a length within ±6% of the length of the any one base sequence selected,

(N2-11) a base sequence that has at least 95% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 309 to 330, and has a length within ±5% of the length of the any one base sequence selected,

(N2-12) a base sequence that has at least 96% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 309 to 330, and has a length within ±4% of the length of the any one base sequence selected,

(N2-13) a base sequence that has at least 97% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 309 to 330, and has a length within ±3% of the length of the any one base sequence selected,

(N2-14) a base sequence that has at least 98% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 309 to 330, and has a length within ±2% of the length of the any one base sequence selected,

(N2-15) a base sequence that has at least 99% identity consisting of SEQ ID NOs: 309 to 330, and has a length within ±1% of the length of the any one base sequence selected, and

(N2-16) a base sequence that has at least 99.5% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 309 to 330, and has a length within ±0.5% of the length of the any one base sequence selected.

Thus, a further embodiment of the antisense oligomer for human Notch3 is an antisense oligomer for human Notch3 consisting of any one base sequence selected from the group consisting of (N1) and (N2-1) to (N2-16), or a pharmaceutically acceptable salt thereof, or hydrate thereof. The antisense oligomer for human Notch3 is included in the present invention.

The suppressor antisense oligomer that suppresses single skipping from human Notch3 pre-mRNA (suppressor antisense oligomer for human Notch3) targets, for example, a splicing silencer sequence of intron 4 or a splice site of single skipping, and as a specific example, is a suppressor antisense oligomer for human Notch3 consisting of

(O1) any one base sequence selected from the group consisting of SEQ ID NOs: 336 to 340, or

(O2) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 336 to 340, and has a length within ±15% of the length of the any one base sequence selected, or a pharmaceutically acceptable salt thereof, or hydrate thereof.

Herein, the base sequence (02) is a mutant type of the base sequence (01). Such a mutant type also includes

(O2-1) a base sequence that has at least 85% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 336 to 340, and has a length within ±15% of the length of the any one base sequence selected,

(O2-2) a base sequence that has at least 86% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 336 to 340, and has a length within ±14% of the length of the any one base sequence selected,

(O2-3) a base sequence that has at least 87% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 336 to 340, and has a length within ±13% of the length of the any one base sequence selected,

(O2-4) a base sequence that has at least 88% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 336 to 340, and has a length within ±12% of the length of the any one base sequence selected,

(O2-5) a base sequence that has at least 89% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 336 to 340, and has a length within ±11% of the length of the any one base sequence selected,

(O2-6) a base sequence that has at least 90% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 336 to 340, and has a length within ±10% of the length of the any one base sequence selected,

(O2-7) a base sequence that has at least 91% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 336 to 340, and has a length within ±9% of the length of the any one base sequence selected,

(O2-8) a base sequence that has at least 92% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 336 to 340, and has a length within ±8% of the length of the any one base sequence selected,

(O2-9) a base sequence that has at least 93% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 336 to 340, and has a length within ±7% of the length of the any one base sequence selected,

(O2-10) a base sequence that has at least 94% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 336 to 340, and has a length within ±6% of the length of the any one base sequence selected,

(O2-11) a base sequence that has at least 95% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 336 to 340, and has a length within ±5% of the length of the any one base sequence selected,

(O2-12) a base sequence that has at least 96% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 336 to 340, and has a length within ±4% of the length of the any one base sequence selected,

(O2-13) a base sequence that has at least 97% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 336 to 340, and has a length within ±3% of the length of the any one base sequence selected,

(O2-14) a base sequence that has at least 98% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 336 to 340, and has a length within ±2% of the length of the any one base sequence selected,

(O2-15) a base sequence that has at least 99% identity consisting of SEQ ID NOs: 336 to 340, and has a length within ±1% of the length of the any one base sequence selected, and

(O2-16) a base sequence that has at least 99.5% identity with any one base sequence selected from the group consisting of SEQ ID NOs: 336 to 340, and has a length within ±0.5% of the length of the any one base sequence selected.

Thus, a further embodiment of the suppressor antisense oligomer for human Notch3 is a suppressor antisense oligomer for human Notch3 consisting of any one base sequence selected from the group consisting of (01) and (02-1) to (02-16), or a pharmaceutically acceptable salt thereof, or hydrate thereof. The suppressor antisense oligomer for human Notch3 is included in the present invention. The suppressor antisense oligomer for human Notch3 is capable of enhancing the efficiency of multi-skipping from Notch3 pre-mRNA.

The base sequences of SEQ ID NOs: 287 to 340 will be shown below.

SEQ

ID NO
Target sequence
Base sequence

287
SCGCex4_(−400)-(−371)
TGCTACTCTCTATTTCCATATTTTTGTAAT

288
SCGCex4_(−380)-(−351)
AAATGCCATATACTTTTTCCTGCTACTCTC

289
SCGCex4_(−360)-(−331)
AGTGGGGTTCTAGGTAATAGAAATGCCATA

290
SCGCex4_(−340)-(−311)
CAAGGAAAGACACCTACAACAGTGGGGTTC

291
SCGCex4_(−320)-(−291)
CTATATGGTCACAGCTATGTCAAGGAAAGA

292
SCGCex4_(−300)-(−271)
AAATATGCAATTTAACAGAGCTATATGGTC

293
SCGCex4_(−280)-(−251)
AAAATATGAATTATTTAGACAAATATGCAA

294
SCGCex4_(−260)-(−231)
ACGCAAGATAGGGATAGAAAAAAATATGAA

295
SCGCex4_(−240)-(−211)
AATCATAAAATGAGCTCCAAACGCAAGATA

296
SCGCex4_(−220)-(−191)
TTGAAAGATAAAAGAATAACAATCATAAAA

297
SCGCex4_(−200)-(−171)
TTTAACATTTTGAGTATTTATTGAAAGATA

298
SCGCex4_(−180)-(−151)
AAATTCCTGAATCATATTTTTTTAACATTT

299
SCGCex4_(−160)-(−131)
TTTGTGGCTAAGACAATTAAAAATTCCTGA

300
SCGCex4_(−140)-(−111)
GATCCTGGAAATCCTATAAATTTGTGGCTA

301
SCGCex4_(−120)-(−91)
ATTATTTATCCATTGTTACAGATCCTGGAA

302
SCGCex4_(−100)-(−71)
AAATTTAGGATTTTTATAAAATTATTTATC

303
SCGCex4_(−80)-(−51)
ATATCTTTATAATTCTGTGTAAATTTAGGA

304
SCGCex4_(−60)-(−31)
AGGTGCTGTTTAAAATGATTATATCTTTAT

305
SCGCex4_(−40)-(−11)
ATTTATAAAATTTGCAAAATAGGTGCTGTT

306
SCGCex4_(−20)-10
GAGATGAGTCCTAGAAAGAGATTTATAAAA

307
SCGCex4_l-30
CTGGGTTGATTGTAGAAGCAGAGATGAGTC

308
SCGCex4_21-50
CGCGCATTTACAGTCACATTCTGGGTTGAT

309
Notch3ex4_(−400)-(−371)
CTTTGGGAGGCTGCGGCGGGCAGATCACTT

310
Notch3ex4_(−380)-(−351)
TCACGCCTGTAATCCCAGAACTTTGGGAGG

311
Notch3ex4_(−360)-(−331)
CATTTGCTGGGAGCAGTGGCTCACGCCTGT

312
Notch3ex4_(−340)-(−311)
GAAACAGAGCAGCAAACACACATTTGCTGG

313
Notch3ex4_(−320)-(−291)
ACAGGCAAGAAACACGCAGGGAAACAGAGC

314
Notch3ex4_(−300)-(−271)
GACACAAAGATACACACAAGACAGGCAAGA

315
Notch3ex4_(−280)-(−251)
CACAGGGCAGGATGGCCCCAGACACAAAGA

316
Notch3ex4_(−260)-(−231)
GAGATGGCTTGGTTGGGCAGCACAGGGCAG

317
Notch3ex4_(−240)-(−211)
CAGGCGGGCACCTGTGGGCAGAGATGGCTT

318
NoLch3ex4_(−220)-(−191)
ACACCGCTCACCCACCCAGCCAGGCGGGCA

319
Notch3ex4_(−200)-(−171)
TGACAGGGGTCCTCCAGCTGACACCGCTCA

320
Notch3ex4_(−180)-(−151)
GGCCAGCACAGGGGCCTGAGTGACAGGGGT

321
Notch3ex4_(−160)-(−131)
TGAACTCTGGCAGACACCACGGCCAGCACA

322
Notch3ex4_(−140)-(−111)
CGGGCGGTGCCAGCCACCACTGAACTCTGG

323
Notch3ex4_(−120)-(−91)
GGGGGCACCGGCATGAGAATCGGGCGGTGC

324
Notch3ex4_(−100)-(−71)
CCTCTCACCTCGGAAGCCACGGGGGCACCG

325
Notch3ex4_(−80)-(−51)
CCTCCCCTCCAGACTCTTCCCCTCTCACCT

326
Notch3ex4_(−60)-(−31)
ACTGACCACACCCCCGACTACCTCCCCTCC

327
Notch3ex4_(−40)-(−11)
CAGGACAGGGTGAGTTTAGGACTGACCACA

328
Notch3ex4_(−20)-10
CAGTCAGGGCCTGGAGGGACCAGGACAGGG

329
Notch3ex4_1-30
GGCAGGGATCTGGCAGGGAGCAGTCAGGGC

330
Notch3ex4_21-50
GTGGGCACAAGGGCTGCTGAGGCAGGGATC

331
SGCGex4_216-246
TGTGGTGCTAGTCTGTGGTCAACACAGCAT

332
SGCGex4_226-256
CACCCCACTCTGTGGTGCTAGTCTGTGGTC

333
SGCGex4_236-266
CCTCACCCCCCACCCCACTCTGTGGTGCTA

334
SGCGex4_246-276
CACGCTGTCCCCTCACCCCCCACCCCACTC

335
SGCGex4_256-286
CAAAGCACTTCAGGCTGTCCCCTCACCCCC

336
Notch3ex4_397-426
AGGGAAGGGGGCAAGGATGGTCACCGCCGG

337
Notch3ex4_407-436
CAAACCCTAGCAGGGAAGGGGGCAAGGATG

338
Notch3ex4_417-446
TCTGACCCTCAAACCCTAGCAGGGAAGGGG

339
Notch3ex4_427-456
ACTTCACAATTCTGACCCTCAAACCCTAGC

340
Notch3ex4_437-466
GTCCACGTTCACTTCACAATTCTGACCCTC

5. Method for Enhancing Efficiency of Multi-Exon Skipping

The present invention provides a method for enhancing the efficiency of skipping of two or more numerically consecutive exons, which comprises

In the enhancement method of the present invention, the skipping of two or more numerically consecutive exons in the pre-mRNA of interest can be performed using an antisense oligomer that induces multi-exon skipping, for example, the antisense oligomer of the present invention or the pharmaceutically acceptable salt thereof, or hydrate thereof.

The enhancement method of the present invention can enhance the efficiency of multi-exon skipping by inhibiting a splicing silencer sequence, a splice site sequence, or a branch site sequence in pre-mRNA of interest, and thereby suppressing single skipping. The enhancement method of the present invention comprises inhibiting a function of the splicing silencer sequence, the splice site sequence, or the branch site sequence in the pre-mRNA of interest, and more specifically, comprises suppressing single exon skipping by targeting these sequences using a suppressor antisense oligomer.

In the enhancement method of the present invention, the inhibition of the splicing silencer sequence, the splice site, or the branch site sequence can be performed using a suppressor antisense oligomer such as the suppressor antisense oligomer of the present invention, or a pharmaceutically acceptable salt thereof, or hydrate thereof.

In the enhancement method of the present invention, an oligomer comprising a base sequence complementary to the splicing silencer sequence, the branch site sequence, or the splice site sequence in the pre-mRNA of interest can be used as the suppressor antisense oligomer in the inhibition of the splicing silencer sequence, the branch site sequence, or the splice site sequence.

The splicing silencer sequence, the branch site sequence, and the splice site sequence are as mentioned above. In a certain embodiment of the enhancement method of the present invention, the splicing silencer sequence may be a recognition sequence of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). Examples of the recognition sequence of human hnRNP A1 are shown in SEQ ID NOs: 370 to 379.

The term “using” or “used” in relation to the antisense oligomer and the suppressor antisense oligomer in the enhancement method of the present invention means that cells expressing the pre-mRNA of interest are allowed to incorporate the antisense oligomer and the suppressor antisense oligomer so that exon skipping is caused in the pre-mRNA of interest. Examples of the method for allowing the cells to incorporate the antisense oligomer and the suppressor antisense oligomer include introduction methods using cationic carriers such as cationic liposomes, cationic polymers, etc., or carriers using viral envelope. The cationic liposomes are, for example, liposomes composed of 2-O-(2-diethylaminoethyl)carbamoyl-1,3-O-dioleoylglycerol and phospholipids as the essential constituents (hereinafter referred to as “liposome A”), Oligofectamine (registered trademark) (manufactured by Invitrogen Corp.), Lipofectin (registered trademark) (manufactured by Invitrogen Corp.), Lipofectamine (registered trademark) (manufactured by Invitrogen Corp.), Lipofectamine 2000 (registered trademark) (manufactured by Invitrogen Corp.), DMRIE-C(registered trademark) (manufactured by Invitrogen Corp.), GeneSilencer (registered trademark) (manufactured by Gene Therapy Systems), TransMessenger (registered trademark) (manufactured by QIAGEN, Inc.), TransIT TKO (registered trademark) (manufactured by Mirus) and Nucleofector II (Lonza). Among others, liposome A is preferred. Examples of cationic polymers are JetSI (registered trademark) (manufactured by Qbiogene, Inc.) and Jet-PEI (registered trademark) (polyethylenimine, manufactured by Qbiogene, Inc.). An example of carriers using viral envelop is GenomeOne (registered trademark) (HVJ-E liposome, manufactured by Ishihara Sangyo). Alternatively, the medical devices described in Japanese Patent Nos. 2924179 and the cationic carriers described in Japanese Domestic Re-Publication PCT Nos. 2006/129594 and 2008/096690 may be used as well.

For the method for allowing cells expressing pre-mRNA of interest in the body of a patient to incorporate the antisense oligomer and the suppressor antisense oligomer, the administration method described in the section “3. Medical application” can be referred.

In the enhancement method of the present invention, the inhibition of the splicing silencer sequence, the splice site sequence, or the branch site sequence in the pre-mRNA of interest enhances the efficiency of multi-exon skipping by 2% or higher, 4% or higher, 6% or higher, 8% or higher, 10% or higher, 12% or higher, 14% or higher, 16% or higher, 18% or higher, 20% or higher, 22% or higher, 24% or higher, 26% or higher, 28% or higher, 30% or higher, 32% or higher, 34% or higher, 36% or higher, 38% or higher, 40% or higher, 42% or higher, 44% or higher, 46% or higher, 48% or higher, 50% or higher, 52% or higher, 54% or higher, 56% or higher, 58% or higher, 60% or higher, 62% or higher, 64% or higher, 66% or higher, 68% or higher, 70% or higher, 72% or higher, 74% or higher, 76% or higher, 78% or higher, 80% or higher, 82% or higher, 84% or higher, 86% or higher, 88% or higher, 90% or higher, 92% or higher, 94% or higher, 96% or higher, 98% or higher, or 100% or higher when the efficiency obtained without the inhibition is defined as 100.

In the enhancement method of the present invention, examples of the pre-mRNA of interest include human γ-sarcoglycan (SGCG) pre-mRNA, human Notch3 pre-mRNA, and human dystrophin pre-mRNA. In a certain embodiment of the present invention, the pre-mRNA of interest is human dystrophin pre-mRNA. When the pre-mRNA of interest is human dystrophin pre-mRNA, the two or more numerically consecutive exons are exons selected from the group consisting of the 45th exon to the 55th exon in the human dystrophin pre-mRNA.

EXAMPLES

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples below, but is not deemed to be limited thereto.

Example 1: Production of Antisense Oligomer
Production of 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid supported on amino polystyrene resin (Compound 1)
Step 1: Production of 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid

Under argon atmosphere, 3.44 g of N-{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide and 1.1 g of 4-dimethylaminopyridine (4-DMAP) were suspended in 50 mL of dichloromethane, and 0.90 g of succinic anhydride was added to the suspension, followed by stirring at room temperature for 3 hours. To the reaction mixture, 10 mL of methanol was added, and the mixture was concentrated under reduced pressure. The residue was extracted using ethyl acetate and 0.5 M aqueous potassium dihydrogenphosphate solution. The resulting organic layer was washed sequentially with 0.5 M aqueous potassium dihydrogenphosphate solution, water and brine in the order mentioned. The resulting organic layer was dried over sodium sulfate and concentrated under reduced pressure to give 4.0 g of the product.

Step 2; Production of 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid supported on amino polystyrene resin

After 4.0 g of 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid was dissolved in 200 mL of pyridine (dehydrated), 0.73 g of 4-DMAP and 11.5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to the solution. Then, 25.0 g of amino polystyrene resin Primer support 200 amino (manufactured by GE Healthcare Japan Co., Ltd., 17-5214-97) and 8.5 mL of triethylamine were added to the mixture, followed by shaking at room temperature for 4 days. After completion of the reaction, the resin was taken out by filtration. The resulting resin was washed sequentially with pyridine, methanol and dichloromethane in the order mentioned, and dried under reduced pressure. To the resulting resin, 200 mL of tetrahydrofuran (dehydrate), 15 mL of acetic anhydride and 15 mL of 2,6-lutidine were added, and the mixture was shaken at room temperature for 2 hours. The resin was taken out by filtration, washed sequentially with pyridine, methanol and dichloromethane in the order mentioned and dried under reduced pressure to give 26.7 g of the product of interest.

The loading amount of the product was determined from the molar amount of the trityl per g resin by measuring UV absorbance at 409 nm using a known method. The loading amount of the resin was 129.2 μmol/g.

Conditions of UV Measurement

Apparatus: U-2910 (Hitachi, Ltd.)

Solvent: methanesulfonic acid

Wavelength: 409 nm

ε value: 45000

Production of 4-{[(2S,6R)-6-(5-methyl-2,4-dioxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid supported on amino polystyrene resin (Compound 2)

The title compound was produced in the same manner as in Compound 1 except that 1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-5-methylpyrimidine-2,4(1H,3H)-dione was used in this step instead of N-{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide used in Step 1 in the production of Compound 1.

Production of 4-{[(2S,6R)-6-(6-benzamidopurin-9-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid supported on amino polystyrene resin (Compound 3)

The title compound was produced in the same manner as in Compound 1 except that N-{9-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]purin-6-yl}benzamide was used in this step instead of N-{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide used in Step 1 in the production of Compound 1.

Production of 4-{{(2S,6R)-6-{6-(2-cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purin-9-yl}-4-tritylmorpholin-2-yl}methoxy}-4-oxobutanoic acid supported on amino polystyrene resin (Compound 4)

The title compound was produced in the same manner as in Compound 1 except that N-{6-(2-cyanoethoxy)-9-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]purin-2-yl}-2-phenoxyacetamide was used in this step instead of N-{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide used in Step 1 in the production of Compound 1.

Production of Antisense PMO Targeting Region in Vicinity of Donor or Acceptor of Intron in Human Dystrophin Gene

According to the description given below, PMOs having the base sequences of PMO Nos. 1 to 232 (SEQ ID NOs: 1 to 232) shown in Table 11 which targeted regions in the vicinity of donors or acceptors of introns 44 to 55 in human dystrophin pre-mRNA shown in Table 10 were synthesized. The 5′ end of each PMO is Group (3) below. The synthesized PMO was dissolved in water for injection (manufactured by Otsuka Pharmaceutical Factory, Inc.).

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The target base sequence of the antisense oligomer of the present invention or the suppressor antisense oligomer of the present invention was described as “Ha_{1_}b₁-c₁”, “Ha_{2_}b₂-c_{2_}Ha_{3_}b₃-c₃”, or “Ha_{4_}b₄-c_{4_}Ha_{5_}b₅-c₅_Ha_{6_}b₆-c₆”.

“Ha_{1_}b₁-c₁” is the target base sequence of the antisense oligomer of the first embodiment used herein. “Ha₁” represents the ath exon of the human dystrophin gene, “b₁” represents the 5′-terminal base of the target base sequence, and “c₁” represents the 3′-terminal base of the target base sequence. “Ha_{1_}_b-c₁” may have one additional base at its 3′ end, and such a target base sequence is represented by “Ha_{1_}b₁-c_{1_}N” (“N” represents a given base).

When “b₁” and “c₁” are positive integers, “b₁” and “c₁” each represent a base number in the downstream direction when the 5′-terminal base of the ath exon is counted as the 1st base. On the other hand, when “b₁” and “c₁” are negative numbers, “b₁” and “c₁” each represent a base number in the upstream direction when the 3′-terminal base of the (a-1)th intron is counted as the 1st base.

For example, “H45_(−10)-15” means a base sequence in which the 5′ end of the target base sequence is the 10th base in the upstream direction from the 3′ end of the 44th intron and the 3′ end of the target base sequence is the 15th base in the downstream direction from the 5′ end of the 45th exon.

“Ha_{2_}b₂-c_{2_}Ha_{3_}b₃-c₃” is the target base sequence of the antisense oligomer of the second embodiment used herein. “Ha₂_b₂-c₂” which is the first part of “Ha_{2_}b₂-c_{2_}Ha_{3_}b₃-c₃” means the target base sequence of a 3′ unit oligomer constituting the antisense oligomer, and the second part “Ha_{3_}b₃-c₃” means the target base sequence of a 5′ unit oligomer constituting the antisense oligomer. “Ha_{3_}b₃-c₃” may have one additional base at its 3′ end, and such a target base sequence is represented by “Ha_{2_}b₂-c_{2_}Ha_{3_}b₃-c_{3_}N” (“N” represents a given base). Each of “Ha_{2_}b₂-c₂” and “Ha_{3_}b_{3_}-c₃” abides by the same notation as that of “Ha_{1_}b₁-c₁”.

When “Ha₂” and “Ha₃” are the same, the “_Ha₃” part may be omitted.

For example, “H45_(−5)-5_25-35” or “H45_(−5)-5_H45_25-35” means a base sequence in which the target base sequence of the 3′ unit oligomer constituting the antisense oligomer is “H45_(−5)-5” and the target base sequence of the 5′ unit oligomer constituting the antisense oligomer is “H45_25-35”.

“Ha_{4_}b₄-c_{4_}Ha_{5_}b₅-c_{5_}Ha_{6_}b₆-c₆” is the target base sequence of the antisense oligomer of the third embodiment used herein. “Ha_{4_}b₄-c₄” which is the first part of “Ha_{4_}b₄-c_{4_}Ha_{5_}b₅-c_{5_}Ha_{6_}b₆-c₆” means the target base sequence of a 3′ unit oligomer constituting the antisense oligomer, the second part “Ha_{5_}b₅-c₅” means the target base sequence of an intermediate unit oligomer constituting the antisense oligomer, and the third part “Ha_{6_}b₆-c₆” means the target base sequence of a 5′ unit oligomer constituting the antisense oligomer. “Ha_{6_}b₆-c₆” may have one additional base at its 3′ end, and such a target base sequence is represented by “Ha_{4_}b₄-c_{4_}Ha_{5_}b₅-c_{5_}Ha_{6_}b₆-c_{6_}N” (“N” represents a given base). Each of “Ha_{4_}b₄-c₄”, “Ha_{5_}b_{5_}-c₅”, and “Ha_{6_}b_{6_}-c₆” abides by the same notation as that of “Ha_{1_}b₁-c₁”.

When “Ha₄” is the same with “Ha₅” and “Ha₆”, the “_Ha₅” and “_Ha₆” parts may be omitted.

For example, “H45_(−5)-5_25-35_60-70” or “H45_(−5)-5_H45_25-35_H45_60-70” means a base sequence in which the target base sequence of the 3′ unit oligomer constituting the antisense oligomer is “H45_(−5)-5”, the target base sequence of the intermediate unit oligomer is “H45_25-35”, and the target base sequence of the 5′ unit oligomer is “H45_60-70”.

TABLE 10

Target region in human dystrophin pre-mRNA

SEQ

Target region
Range of target region
ID NO

(R1) Region in vicinity of
Region indicated by range of −20
233

donor of intron 44
bases to +400 bases with 5′ end of

intron 44 defined as basing point 0

(R2) Region in vicinity of
Region indicated by range of −600
234

acceptor of intron 44
bases to +50 bases with 3′ end of

intron 44 defined as basing point 0

(R3) Region in vicinity of
Region indicated by range of −20
235

donor of intron 45
bases to +400 bases with 5′ end of

intron 45 defined as basing point 0

(R4) Region in vicinity of
Region indicated by range of −400
236

acceptor of intron 45
bases to +50 bases with 3′ end of

intron 45 defined as basing point 0

(R5) Region in vicinity of
Region indicated by range of −20
237

donor of intron 46
bases to +400 bases with 5′ end of

intron 46 defined as basing point 0

(R6) Region in vicinity of
Region indicated by range of −400
238

acceptor of intron 46
bases to +50 bases with 3′ end of

intron 46 defined as basing point 0

(R7) Region in vicinity of
Region indicated by range of −20
239

donor of intron 47
bases to +400 bases with 5′ end of

intron 47 defined as basing point 0

(R8) Region in vicinity of
Region indicated by range of −400
240

acceptor of intron 47
bases to +50 bases with 3′ end of

intron 47 defined as basing point 0

(R9) Region in vicinity of
Region indicated by range of −20
241

donor of intron 48
bases to +400 bases with 5′ end of

intron 48 defined as basing point 0

(R10) Region in vicinity of
Region indicated by range of −400
242

acceptor of intron 48
bases to +50 bases with 3′ end of

intron 48 defined as basing point 0

(R11) Region in vicinity of
Region indicated by range of −20
243

donor of intron 49
bases to +400 bases with 5′ end of

intron 49 defined as basing point 0

(R12) Region in vicinity of
Region indicated by range of −400
244

acceptor of intron 49
bases to +50 bases with 3′ end of

intron 49 defined as basing point 0

(R13) Region in vicinity of
Region indicated by range of −20
245

donor of intron 50
bases to +400 bases with 5′ end of

intron 50 defined as basing point 0

(R14) Region in vicinity of
Region indicated by range of −400
246

acceptor of intron 50
bases to +50 bases with 3′ end of

intron 50 defined as basing point 0

(R15) Region in vicinity of
Region indicated by range of −20
247

donor of intron 51
bases to +400 bases with 5′ end of

intron 51 defined as basing point 0

(R16) Region in vicinity of
Region indicated by range of −400
248

acceptor of intron 51
bases to +50 bases with 3′ end of

intron 51 defined as basing point 0

(R17) Region in vicinity of
Region indicated by range of −20
249

donor of intron 52
bases to +400 bases with 5′ end of

intron 52 defined as basing point 0

(R18) Region in vicinity of
Region indicated by range of −400
250

acceptor of intron 52
bases to +50 bases with 3′ end of

intron 52 defined as basing point 0

(R19) Region in vicinity of
Region indicated by range of −20
251

donor of intron 53
bases to +400 bases with 5′ end of

intron 53 defined as basing point 0

(R20) Region in vicinity of
Region indicated by range of −400
252

acceptor of intron 53
bases to +50 bases with 3′ end of

intron 53 defined as basing point 0

(R21) Region in vicinity of
Region indicated by range of −20
253

donor of intron 54
bases to +400 bases with 5′ end of

intron 54 defined as basing point 0

(R22) Region in vicinity of
Region indicated by range of −400
254

acceptor of intron 54
bases to +50 bases with 3′ end of

intron 54 defined as basing point 0

(R23) Region in vicinity of
Region indicated by range of −20
255

donor of intron 55
bases to +400 bases with 5′ end of

intron 55 defined as basing point 0

(R24) Region in vicinity of
Region indicated by range of −400
256

acceptor of intron 55
bases to +50 bases with 3′ end of

intron 55 defined as basing point 0

TABLE 11

PMO
Target

SEQ

No.
region
Target base sequence
Base sequence of PMO
ID NO

1
R1
H44150-175
TTTCGAAAAAACAAATCAAAGACTTA
1

2

H44_157-196
ATGTGCTGAAGATAAATACAATTTCGAAAA
2

AACAAATCAA

3

H44_l59-193
TGCTGAAGATAAATACAATTTCGAAAAAAC
3

AAATC

4

H44_170-199
CAGATGTGCTGAAGATAAATACAATTTCGA
4

5

H44_l79-206
AAGAGTCCAGATGTGCTGAAGATAAATA
5

6

H44_209-236
ACCCTTCAGAACCTGATCTTTAAGAAGT
6

7

H44_239-266
TGACAACAACAGTCAAAAGTAATTTCCA
7

8

H44_269-296
ATGATAATTTTCTTTCTAGTAATATAAT
8

9

H44_299-326
TCCATAGCACCGTGCTCTAATATTATCA
9

10

H44_330-355
GGCAAACTCTCTCATCCTGACACAAA
10

11

H44_354-381
TTTATCAGATAAACCAGCTCCGTCCAGG
11

12

H44_389-416
CTTCCCTCTGTCACAGATTCAATTATAT
12

13

H44_413-438
AAAACACCTTGCTGTTACGATGCTTC
13

14

H44_423-452
TGCCCCAAAGCCACAAAACACCTTGCTGTT
14

15

H44_455-480
GGTTCCAACATAAAGCCGAAATACAC
15

16

H44_479-506
TATGCCACAAGTTCTCCTTCTGGAAAGG
16

17

H44_509-536
GATATTTCTAGCAACTTCATTTTAGCTA
17

18

H44_150-164_179-193
TGCTGAAGATAAATACAAATCAAAGACTTA
18

19

H44_150-164_192-206
AAGAGTCCAGATGTGCAAATCAAAGACTTA
19

20

H44_161-175_179-193
TGCTGAAGATAAATATTTCGAAAAAACAAA
20

21

H44_161-175_192-206
AAGAGTCCAGATGTGTTTCGAAAAAACAAA
21

22
R2
H45_(−598)-(−571)
TTAATTGCTTTCAGGAGCATCCCATCAA
22

23

H45_(−568)-(−541)
TTTGCATTAGAAGCCACAAAAAACTGAG
23

24

H45_(−538)-(−511)
TCATTTCAAATTCTGTCTGCGTCAATGT
24

25

H45_(−518)-(−491)
TTGCTATATTAGAAGAAAATTCATTTCA
25

26

H45_(−515)-(−488)
TAATTGCTATATTAGAAGAAAATTCATT
26

27

H45_(−508)-(−479)
AAATAAAATTAATTGCTATATTAGAAGAAA
27

28

H45_(−478)-(−449)
CAGTATTAAAAAAAAACTCTAGAGATATTT
28

29

H45_(−470)-(−443)
TAGTCACAGTATTAAAAAAAAACTCTAG
29

30

H45_(−448)-(−421)
GTGAAAAAGAACAAACATAGGTTAGTCA
30

31

H45_(−439)-(−412)
ATACGAGAGGTGAAAAAGAACAAACATA
31

32

H45_(−436)-(−409)
TGGATACGAGAGGTGAAAAAGAACAAAC
32

33

H45_(−422)-(−395)
TTTCTTAGTGATCGTGGATACGAGAGGT
33

34

H45_(−418)-(−394)
GTTTCTTAGTGATCGTGGATACGAG
34

35

H45_(−418)-(−391)
TGGGTTTCTTAGTGATCGTGGATACGAG
35

36

H45_(−415)-(−391)
TGGGTTTCTTAGTGATCGTGGATAC
36

37

H45_(−413)-(−386)
GTATTTGGGTTTCTTAGTGATCGTGGAT
37

38

H45_(−405)-(−378)
TGAACAAAGTATTTGGGTTTCTTAGTGA
38

39

H45_(−389)-(−362)
TTGTAAAATTTAAACATGAACAAAGTAT
39

40

H45_(−349)-(−322)
TCCCCACAAGGATGTTCCATGTTTAATA
40

41

H45_(−320)-(−293)
ACCTTTTCAAGAGCAAATTCGATTTCTT
41

42

H45_(−305)-(−280)
CAATTAGTTGGAAACCTTTTCAAGAG
42

43

H45_(−290)-(−263)
TATAATGTCCTACAAATCAATTAGTTGG
43

44

H45_(−275)-(−250)
AGCTAGAGGATGTTATAATGTCCTAC
44

45

H45_(−260)-(−233)
ATTTTTGTAAGCTTGTCAGCTAGAGGAT
45

46

H45_(−230)-(−203)
AAAGCACCCTCTCGGTTAGCTCCAGTTT
46

47

H45_(−200)-(−173)
CAGAAAGACACCTTTTATGTGTCAGGGA
47

48

H45_(−190)-(−163)
GGATACAAGACAGAAAGACACCTTTTAT
48

49

145_(−185)-(−160)
AAAGGATACAAGACAGAAAGACACCT
49

50

H45_(−l7l)-(−144)
TGACATGCCCATATCCAAAGGATACAAG
50

51

H45_(−168)-(−141)
AACTGACATGCCCATATCCAAAGGATAC
51

52

H45_(−142)-(−115)
AGCTCCATGTGAAAATTTCCCTATGAAA
52

53

H45_(−110)-(−83)
TGCAGTTGTACTGGCAAAGAAAGAAATA
53

54

H45_(−80)-(−53)
TGAGAAAAGATTAAACAGTGTGCTACCA
54

55

H45_(−78)-(−51)
TTTGACAAAAGATTAAACAGTGTGCTAC
55

56

H45_(−70)-(−43)
TCTTTTTATTTGAGAAAAGATTAAACAG
56

57

H45_(−60)-(−33)
AAGCCCCATGTCTTTTTATTTGAGAAAA
57

58

H45_(−50)-(−23)
AACAAAAATGAAGCCCCATGTCTTTTTA
58

59

H45_(−44)-(−17)
AGGCAAAACAAAAATGAAGCCCCATGTC
59

60

H45_(−25)-3
TTCCTGTAAGATACCAAAAAGGCAAAAC
60

61

H45_(−23)-5
AGTTCCTGTAAGATACCAAAAAGGCAAA
61

62

H45_(−22)-3
TTCCTGTAAGATACCAAAAAGGCAA
62

63

H45_(−21)-7
GGAGTTCCTGTAAGATACCAAAAAGGCA
63

64

H45_(−20)-5
AGTTCCTGTAAGATACCAAAAAGGC
64

65

H45_(−20)-8
TGGAGTTCCTGTAAGATACCAAAAAGGC
65

66

H45_(−18)-10
CCTGGAGTTCCTGTAAGATACCAAAAAG
66

67

H45_(−17)-8
TGGAGTTCCTGTAAGATACCAAAAA
67

68

H45_(−16)-12
ATCCTGGAGTTCCTGTAAGATACCAAAA
68

69

H45_(−15)-10
CCTGGAGTTCCTGTAAGATACCAAA
69

70

H45_(−l4)-14
CCATCCTGGAGTTCCTGTAAGATACCAA
70

71

H45_(−10)-16
TGCCATCCTGGAGTTCCTGTAAGATA
71

72

H45_(−5)-20
CCAATCCCATCCTGGAGTTCCTGTA
72

73

H45_l-25
GCTGCCCAATGCCATCCTGGAGTTC
73

74

H45_6-30
TTGCCGCTGCCCAATGCCATCCTGG
74

75

H45_11-35
ACAGTTTGCCGCTGCCCAATGCCAT
75

76

H45_l1-40
TGACAACAGTTTGCCGCTGCCCAATGCCAT
76

77

H45_16-35
ACAGTTTGCCGCTGCCCAAT
77

78

H45_l6-40
TGACAACAGTTTGCCGCTGCCCAAT
78

79

H45_21-45
TGTTCTGACAACAGTTTGCCGCTGC
79

80

H45_(−423)-(−412)_16-
AGTTTGCCGCTGCCCAATATACGAGAGGTG
80

33

81

H45_(−416)-(−402)_16-
TTCCCGCTGCCCAATGTGATCGTGGATACG
81

30

82

H45_(−411)-(−400)_16-
AGTTTGCCCCTGCCCAATTAGTGATCGTGG
82

33

83

H45_(−405)-(−391)_16-
TTGCCGCTGCCCAATTGGGTTTCTTAGTGA
83

30

84

H45_(−402)-(−391)_16-
AGTTTGCCGCTGCCCAATTGGGTTTCTTAG
84

33

85

H45_(−156)-(−143)-
AATTTCCCTATGAACTGACATGCCCATA
85

(−141)-(−128)

86

H45_(−78)-(−65)_(−44)-
TGAAGCCCCATGTCAAACAGTGTGCTAC
86

(−31)

87

H45_(−78)-(−65)_(−34)-
AAAACAAAAATGAAGAACAGTGTGCTAC
87

(−21)

88

H45_(−78)-(−65)_16-29
TGCCGCTGCCCAATAAACAGTGTGCTAC
88

89

B45_(−78)-(−65)_21-34
CAGTTTGCCGCTGCAAACAGTGTGCTAC
89

90

H45_(−78)-(−65)_27-40
TGACAACAGTTTGCAAACAGTGTGCTAC
90

91

H45_(−74)-(−62)_(−40)-
AAATGAAGCCCCAATTAAACAGTGTG
91

(−28)

92

H45_(−72)-(−58)_(−38)-
AAAAATGAAGCCCAAAGATTAAACAGTG
92

(−26)

93

H45_(−71)-(−58)_(−38)-
CAAAAATGAAGCCCAAAGATTAAACAGT
93

(−25)

94

H45_(−71)-(−58)_16-29
TGCCCCTGCCCAATAAAGATTAAACAGT
94

95

H45_(−71)-(−58)_21-34
CAGTTTGCCGCTGCAAAGATTAAACAGT
95

96

H45_(−71)-(−58)_27-40
TGACAACAGTTTGCAAAGATTAAACAGT
96

97

H45_(−68)-(−60)_(−36)-
GCCGCTGCCC_AAATGAAGCAGATTAAAC
97

(−28)_19-28

98

H45_(−64)-(−51)_(−44)-
TGAAGCCCCATGTCTTTGAGAAAAGATT
98

(−31)

99

H45_(−64)-(−51)_(−35)-
AAACAAAAATGAAGTTTGAGAAAAGATT
99

(−22)

100

H45_(−64)-(−51)-16-29
TGCCGCTGCCCAATTTTGAGAAAAGATT
100

101

H45_(−64)-(−51)_21-34
CAGTTTGCCGCTGCTTTGAGAAAAGATT
101

102

H45_(−64)-(−51)-27-40
TGACAACAGTTTGCTTTGAGAAAAGATT
102

103

H45_(−44)-(−3l)_l6-29
TGCCGCTGCCCAATTGAAGCCCCATGTC
103

104

H45_(−44)-(−31)_27-40
TGACAACAGTTTGCTGAAGCCCCATGTC
104

105

H45_(−44)-(−31)_21-
TAGTTTGCCGCTGCTGAAGCCCCATGTC
105

33_T

106

H45_(−38)-(−25)_16-29
TGCCGCTGCCCAATCAAAAATGAAGCCC
106

107

H45_(−38)-(−25)_21-34
CAGTTTGCCGCTGCCAAAAATGAAGCCC
107

108

H45_(−38)-(−25)_27-40
TGACAACAGTTTGCCAAAAATGAAGCCC
108

109

H45_(−34)-(−21)_16-29
TGCCGCTGCCCAATAAAACAAAAATGAA
109

110

H45_(−34)-(−21)_21-34
CAGTTTGCCGCTGCAAAACAAAAATGAA
110

111

H45_(−34)-(−21)_27-40
TGACAACAGTTTGCAAAACAAAAATGAA
111

112

H45-1-21
CCCAATGCCATCCTGGAGTTC
112

144
R3
H45_167-194
TTAGATCTGTCGCCCTACCTCTTTTTTC
144

145

H45_227-254
TCTCATGAAATATTCTTCTAAAGAAAGC
145

146

H45_257-284
ATGTTAGTGCCTTTCACCCTGCTTATAA
146

147

H45_287-314
GCTGTTGATTAATGGTTGATAGGTTCTT
147

148

H45_317-344
TGAAAAAAAGAAATAAAAAATTTCTTTA
148

149

H45_347-374
TAACTAGCCACAAGTATATATTTTAGTA
149

150
R4
H46_(−200)-(−173)
TCAAGAATCTCTAAATGATAAGAGATTA
150

151

H46_(−171)-(−144)
TTCACTTTGAACAAAGTAATTTCAATAT
151

152

H46_(−146)-(−119)
ACCATACATAATTTAAGAAAATTCATTC
152

153

H46_(−141)-(−114)
TGTTAACCATACATAATTTAAGAAAATT
153

154

H46_(−136)-(−109)
AAAGATGTTAACCATACATAATTTAAGA
154

155

H46_(−126)-(−99)
AAGCAATTTAAAAGATGTTAACCATACA
155

156

H46_(−116)-(−89)
ATTTAAAAATAAGCAATTTAAAAGATGT
156

157

H46_(−111)-(−84)
TGGCAATTTAAAAATAAGCAATTTAAAA
157

158

H46_(−106)-(−79)
AAACATGGCAATTTAAAAATAAGCAATT
158

159

H46_(−81)-(−54)
TATTTGTTAATGCAAACTGGGACACAAA
159

160

H46_(−51)-(−24)
TTATTTTTTTTTCCAACATAGTTCTCAA
160

161

H46_(−18)-6
TCTAGCCTGGAGAAAGAAGAATAA
161

162

H46_(−15)-9
TCTTCTAGCCTGGAGAAAGAAGAA
162

163

H46_(−9)-15
TTTTGTTCTTCTAGCCTGGAGAAA
163

164
R5
H46_139-166
TTGAGAAAATAAAATTACCTTGACTTGC
164

165

H46_l69-196
ACTTCTTTATGCAAGCAGGCCCTGGGGG
165

166

H46_199-226
CAATGATTGAATTAAAAAATAGATTCAT
166

167

H46_229-256
GAACTATGAATAACCTAATGGGCAGAAA
167

168

H46_259-286
ATAAAGTTGTGAGAAAAACACTTTAGCA
168

169

H46_289-316
ACTGGTTCAGAACTGCAGGGTTAAGAAG
169

170

H46_319-346
ACACACATATATACATATGTTCTTATGT
170

171
R6
H47_(−200)-(−173)
CTCACCCCCTCAGTAGATAAATCTCTGT
171

172

H47_(−171)-(−144)
TCTGAGCACAGAGCTGATTGACTGAAAC
172

173

H47_(−141)-(−114)
AGTGGTACCTCAAATACCAACAGTTTTC
173

174

H47_(−111)-(−84)
ATCAAAATGAAGCGACTTGACCGAGGGC
174

175

H47_(−81)-(−54)
TACCTTGTCTTTGCTTCTATTGATTAGT
175

176

H47_(−51)-(−24)
GTTTAAAATGAATTACAGCACAATTCCA
176

177

H47_(−16)-12
TTCCACCAGTAACTGAAACAGACAAATG
177

178

H47_l-29
TGGCGCAGGGGCAACTCTTCCACCAGTAA
178

179
R7
H47_135-162
TTAATGTCTAACCTTTATCCACTGGAGA
179

180
R8
H48_(−21)-7
TGGAAACCTGAAAGGAAAATACATTTTA
180

181

H48_1-28
CTTGTTTCTCAGGTAAAGCTCTGGAAAC
181

182
R9
H48_177-204
AAGCAAAAAGTTCCCTACCTGAACGTCA
182

183
R10
H49_(−21)-7
CAGTTTCCTGGGGAAAAGAACCCATATA
183

184

H49_22-47
ATCTCTTCCACATCCGGTTGTTTAGC
184

185
R11
H49_93-l20
TAGAGGTTGCTTCATTACCTTCACTGGC
185

186
R12
H50_(−200)-(−173)
ACCCTACAAATATTTATCAATTGCTCCA
186

187

H50_(−171)-(−144)
TAAAGGAATTATAATTATTTTAGCCAAC
187

188

H50_(-141)-(−114)
TAAATTAACTTTAGTGGGTAGAATTTCT
188

189

H50_(−111)-(−84)
TATTATTGGATTTCTATTATATTTTACT
189

190

H50_(−81)-(−54)
TCATGAACATCTTAATCCATTTGGTGAA
190

191

H50_(−51)-(−24)
TACTTATTCGATTAACACTTTGAAGATA
191

192

H50_(−21)-7
ACTTCCTCTTTAACAGAAAAGCATACAC
192

193
R13
H50_l00-127
GGGATCCAGTATACTTACAGGCTCCAAT
193

194

H50_90-114
CTTACAGGCTCCAATAGTGGTCAGT
194

195
R14
H51_(−181-10
TGAGTAGGAGCTAAAATATTTTGGGTTT
195

196
R15
H51_224-251
TATCATTTTTTCTCATACCTTCTGCTTG
196

197
R16
H52_(−47)-(−20)
TTAGTATCAGGGTTCTTCAGCGTTGTGT
197

198

H52_(−18)-10
GCATTGTTGCCTGTAAGAACAAATATCC
198

199

H52_9-38
AACTGGGGACGCCTCTGTTCCAAATCCTGC
199

200
R17
H52_109-136
GCTTGTTAAAAAACTTACTTCGATCCGT
200

201
R18
H53_(−197)-(−170)
AATATTAGTTTCTGTTAAATTATTTTCC
201

202

H53_(−167)-(−140)
ACAACAGGATTCTTTGCTTTTTTGATGG
202

203

H53_(−137)-(−110)
TTATTCATTGTGTTATGGCTAGGATGAT
203

204

H53_(−107)-(−80)
ATCTCACATTTATGTTGCTTATTTAAAA
204

205

H53_(−77)-(−50)
GAGACATTTTAAATGTAACTTCCAAACG
205

206

H53_(−47)-(−20)
AAATATATAGTAGTAAATGCTAGTCTGG
206

207

H53_(−18)-10
ATTCTTTCAACTAGAATAAAAGGAAAAA
207

208
R19
H53_203-230
GGTATCTTTGATACTAACCTTGGTTTCT
208

209
R20
H54_(−18)-10
TGGCCAACTGCTATAGATTTTTATGAGA
209

210
R21
H54_146-173
AAATAATGTAATTCATACCTTTTATGAA
210

211

H54_176-203
CCCCATTATTACAGCCAACAGTAGTTTT
211

212

H54_206-233
CAAATCCTCATGGTCCATCCAGTTTCAC
212

213

H54_236-263
CCAAGCTCCAGTTTAGCTGGATTGGAAA
213

214

H54_266-293
TTTAGTTGGTATTTATCGTCTTGAACCC
214

215

H54_296-323
TAAAATAGAAGTCTGAGCCAAGTCCGTG
215

216

H54_326-353
GGGCAAATGAGATCTTATGTTCCTCGTT
216

217
R22
H55_(−197)-(−170)
TTTACTTTCATTTTTATTTAACTTAAAG
217

218

H55_(−167)-(−140)
TAAGCAACAACTATAATATTGTGCAGTC
218

219

H55_(−137)-(−110)
TGGGGTGAGTTGTTGCTACAGCTCTTCC
219

220

H55_(−107)-(−80)
TGGAGGAACTAAATTGTAATATACCAAC
220

221

H55_(−77)-(−50)
CACCTAGTGAACTCCATAAAAAGAGAAA
221

222

H55_(−47)-(−20)
CAGATGCAATTATTAAATATCAGAATGG
222

223

H55_(−18)-10
CTCACTCACCCTGCAAAGGACCAAATGT
223

224

H55_12-34
AGTTTCTTCCAAAGCAGCCTCTC
224

113
R23
H55_177-204
AATGCCTGACTTACTTGCCATTGTTTCA
113

114

H55_191-218
AGTGCTAAAGCGGAAATGCCTGACTTAC
114

115

H55_221-248
TGCTGAGAATTGTTCAATTGCATCCACA
115

116

H55_251-278
TGTCCCTGGCTTGTCAGTTACAAGTACA
116

117

H55_281-308
TCAGGCTGTATAAAAGCAACTATTTTGI
117

118

H55_311-338
CTCTCCTCCTTGTCCAAATACCGAAATA
118

119

H55_334-361
GATGTTTCCTTCTCCCTCTGCCTCTCTC
119

120

H55_341-368
TATAAATGATGTTTCCTTCTCCCTCTGC
120

121

H55_371-398
TTTTTCTAAGACGAGGGTGTTAAGTGGA
121

122

H55_401-428
TGCAAATGTTTTCCTGGTCAGAGCATGT
122

123

H55_431-458
TTTCTCCTTGACCGAAGCTCTGGTTTTA
123

124

H55_461-488
CCAATCCCTAAGTTATTTCTCTGAGCAA
124

125

H55_491-518
CAAAAATGTCAACTTTTAAAATTTAATA
125

126

H55_521-548
GCTTTCCCTGTAAAATATTAAATAAACA
126

127

H55_548-575
TAAATAAAACAGACAATTCATACAGATG
127

128
R24
H56_(−400)-(−373)
AATAATTTCATTACTATATGGATCAAGT
128

129

H56_(−380)-(−355)
AAAGTGTACCCCAGTGCCAATAATTT
129

130

H56_(−345)-(−318)
TATTTTATGGAAGTGATAGGCAATAAAA
130

131

H56_(−315)-(−288)
TATATCTTCTAGTCTATGGACAAAATGT
131

132

H56_(−285)-(−258)
TGTATTTATAACTTTATAAAGTTCACAA
132

133

H56_(−255)-(−228)
TATTCCTTGCCATTATGAGTTGGAAAGT
133

134

H56_(−225)-(−198)
AAATGGGCATCTTATTAGTTGTAATAGA
134

135

H56_(−195)-(−168)
ATTGCCTTCTCCTGTTATTATGTAGATT
135

136

H56_(−165)-(−138)
AGATACATCTCAAATCCCTTTTCTTGGC
136

137

H56_(−135)-(−108)
AGACATTTCAATCAGGCTAAACTAACAA
137

138

H56_(−105)-(−78)
TTGCAAAATATAAATAATTATTAGTTCA
138

139

H56_(−76)-(−51)
ACAAGCGATGAATGTGAATTTGGAGA
139

140

H56_(−59)-(−34)
ATTACCAAACAAAAGAAACAAGCGAT
140

141

H56_(−39)-(−14)
GGAAGAAGAATATGTGCAGAATTACC
141

142

H56_(−31)-(−6)
GGACAGCAGGAAGAAGAATATGTGCA
142

143

H56_(−14)-13
TCACCTTGGAGGTCCTACAGGACAGCAG
143

225
R1 +
H44_188-2D2_H56_
ACATCTCAAATCCCTGTCCAGATGTGCTGA
225

R24
(−156)-(−142)

226

H44_188-202_H56_
ACATTTCAATCAGGCGTCCAGATGTGCTGA
226

(−124)-(−110)

227

H44_188-202_H56_(−85)-
TGGAGAATTGCAAAAGTCCAGATGTGCTGA
227

(−71)

228

H56_(−156)-
GTCCAGATGTGCTGAACATCTCAAATCCCT
228

(−142)_H44_188-202

229

H56_(−124)-
GTCCAGATGTGCTGAACATTTCAATCAGGC
229

(−110)_H44_188-202

230

H56_(−85)-
GTCCAGATGTGCTGATGGAGAATTGCAAAA
230

(−71)_H44_188-202

231
R2 +
H45_16-35_H46_(−134)-
GTTAACCATACATAATTTAAACAGTTTGCC
231

R4
(−115)
GCTGCCCAAT

232

H45_16-35_H46_(−103)-
TGGCAATTTAAAAATAAGCAACAGTTTGCC
232

(−84)
GCTGCCCAAT

In the PMO of Table 11,

PMO for the target region R2 includes PMO Nos. 22 to 71, 91 to 93, 98 and 99 in another embodiment, and PMO Nos. 22 to 59, 91 to 93, 98 and 99 in still another embodiment;

PMO for the target region R4 includes PMO Nos. 150 to 163 in another embodiment, and PMO Nos. 150 to 160 in still another embodiment;

PMO for the target region R6 includes PMO Nos. 171 to 177 in another embodiment, and PMO Nos. 171 to 176 in still another embodiment;

PMO for the target region R8 includes PMO No. 180 in another embodiment; PMO for the target region R10 includes PMO No. 183 in another embodiment; PMO for the target region R12 includes PMO Nos. 186 to 191 in another embodiment PMO for the target region R16 includes PMO Nos. 197 and 198 in another embodiment, and PMO No. 197 in still another embodiment;

PMO for the target region R18 includes PMO Nos. 201 to 206 in another embodiment;

PMO for the target region R22 includes PMO Nos. 217 to 223 in another embodiment, and PMO Nos. 217 to 222 in still another embodiment; and

PMO for the target region R24 includes PMO Nos. 128 to 142 in another embodiment.

0.2 g of 4-{[(2S,6R)-6-(4-benzamide-2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid supported on an aminopolystyrene resin (Compound 1), 4-{[(2S,6R)-6-(5-methyl-2,4-dioxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid supported on amino polystyrene resin (Compound 2), 4-{[(2S,6R)-6-(6-benzamidopurin-9-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid supported on amino polystyrene resin (Compound 3), or 4-{{(2S,6R)-6-{6-(2-cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purin-9-yl}-4-tritylmorpholin-2-yl}methoxy}-4-oxobutanoic acid supported on amino polystyrene resin (Compound 4) appropriate for the 5′-terminal base was filled in a column with a filter tip. Then, the synthetic cycle shown in Table 12 was started using a nucleic acid synthesizing machine (AKTA Oligopilot 10 plus). The desired morpholino monomer compound was added in each coupling cycle to give the base sequence of each PMO described in Table 11 (see Table 12).

TABLE 12

Step
Reagent
Volume (mL)
Time (min)

1
deblocking solution
18-32
1.8-3.2

2
neutralizing and washing
30
1.5

solution

3
coupling solution B
5
0.5

4
coupling solution A
1.3
0.25

5
coupling reaction by the

120-300

reagents added in the steps

3 and 4

6
acetonitrile
20
1.0

7
capping solution
9
2.0

8
acetonitrile
30
2.0

Note that only for 3′-terminal acetylation, only the steps 1, 2, 7, and 8 were carried out again after the final cycle.

The deblocking solution used was dichloromethane solution containing 3% (w/v) trifluoroacetic acid. The neutralizing and washing solution used was a solution obtained by dissolving N,N-diisopropylethylamine to be 10% (v/v) and tetrahydrofuran to be 5% (v/v) in dichloromethane containing 35% (v/v) acetonitrile. The coupling solution A used was a solution obtained by dissolving the morpholino monomer compound in tetrahydrofuran to be 0.10 M. The coupling solution B used was a solution obtained by dissolving N,N-diisopropylethylamine to be 20% (v/v) and tetrahydrofuran to be 10% (v/v) in acetonitrile. The capping solution used was a solution obtained by dissolving 20% (v/v) acetic anhydride and 30% (v/v) 2,6-lutidine in acetonitrile.

The morpholino monomer compound (A), the morpholino monomer compound (C), the morpholino monomer compound (T), and the morpholino monomer compound (G) described in Table 13 were used as the morpholino monomer compound.

TABLE 13

Morpholino monomer
Morpholino monomer

compound
compound

(A)
(C)

embedded image

Morpholino monomer
Morpholino monomer

compound
compound

(T)
(G)

embedded image

The aminopolystyrene resin loaded with the PMO synthesized above was recovered from the reaction vessel and dried at room temperature for at least 2 hours under reduced pressure. The dried PMO loaded onto aminopolystyrene resin was charged in a reaction vessel, and 5 mL of 28% ammonia water-ethanol (1/4) was added thereto. The mixture was stirred at 55° C. for 15 hours. The aminopolystyrene resin was separated by filtration and washed with 1 mL of water-ethanol (1/4). The resulting filtrate was concentrated under reduced pressure. The resulting residue was dissolved in 10 mL of a solvent mixture of 20 mM of acetic acid-triethylamine buffer (TEAA buffer) and acetonitrile (4/1) and filtered through a membrane filter. The filtrate obtained was purified by reversed phase HPLC. The conditions used are shown in Table 14.

TABLE 14

Column
XBridge 5 μm C18 (Waters, ϕ19 × 50 mm,

1 CV = 14 mL)

Flow rate
10 mL/min

Column
room temperature

temperature

Solution A
20 mM TEAA buffer

Solution B
acetonitrile (CH₃CN)

Gradient
Solution B: (10% → 70%)/15 CV

CV: column volume

Each fraction was analyzed, and the product of interest was recovered and concentrated under reduced pressure. To the concentrated residue, 0.5 mL of 2 M phosphoric acid aqueous solution was added, and the mixture was stirred for 15 minutes. Furthermore, 2 mL of 2 M sodium hydroxide aqueous solution was added to make the mixture alkaline, followed by filtration through a membrane filter (0.45 μm).

The resulting aqueous solution containing the product of interest was purified by an anionic exchange resin column. The conditions used are shown in Table 15.

TABLE 15

Column
Source 15Q (GE Healthcare, ϕ10 × 108 mm,

1 CV = 8.5 mL)

Flow rate
8.5 mL/min

Column
room temperature

temperature

Solution A
10 mM sodium hydroxide aqueous solution

Solution B
10 mM sodium hydroxide aqueous solution,

1M sodium chloride aqueous solution

Gradient
Solution B: (1% → 50%)/40 CV

Each fraction was analyzed (on HPLC) and the product of interest was obtained as an aqueous solution. To the resulting aqueous solution was added 0.1 M phosphate buffer (pH 6.0) for neutralization. Next, the mixture obtained was demineralized by reversed phase HPLC under the conditions described in Table 16.

TABLE 16

Column
XBridge 5 μm C18 (Waters, ϕ10 × 50 mm,

1 CV = 4 mL)

Flow rate
4 mL/min

Column
60° C.

temperature

Solution A
water

Solution B
CH₃CN

Gradient
Solution B: (0% → 50%)/20 CV

The product of interest was recovered and the mixture was concentrated under reduced pressure. The resulting residue was dissolved in water. The aqueous solution obtained was freeze-dried to give each PMO as the compound of interest as a white cotton-like solid. The calculated and measured values of ESI-TOF-MS are shown in Table 17 below.

TABLE 17

PMO No.
Target base sequence
Calculated
Measured

1
H44_150-175
8610.02
8610.48

2
H44_157-196
13354.66
13354.45

3
H44_159-193
11652.07
11652.09

4
H44_170-199
10012.48
10013.39

5
H44_179-206
9392.27
9392.54

6
H44_209-236
9230.21
9230.92

7
H44_239-266
9256.24
9256.17

8
H44_269-296
9264.2
9263.99

9
H44_299-326
9181.19
9181.46

10
H44_330-355
8514.98
8514.36

11
H44_354-381
9231.21
9231.24

12
H44_389-416
9147.17
9147.4

13
H44_413-438
8551.97
8552.71

14
H44_423-452
9830.43
9830.2

15
H44_455-480
8588.01
8587.53

16
H44_479-506
9262.21
9262.15

17
H44_509-536
9226.19
9225.7

18
H44_150-164_l79-193
9989.49
9989.23

19
H44_150-164_l92-206
10006.49
10006.81

20
H44_161-175_179-193
10013.5
10013.95

21
H44_161-175_192-206
10030.5
10031.48

22
H45_(−598)-(−571)
9221.2
9221.27

23
H45_(−568)-(−541)
9312.25
9311.93

24
H45_(−538)-(−511)
9218.18
9218.33

25
H45_(−518)-(−491)
9283.22
9283.26

26
H45_(−515)-(−488)
9307.23
9307.34

27
H45_(−508)-(−479)
10027.5
10027.98

28
H45_(−478)-(−449)
9979.48
9980.37

29
H45_(−470)-(−443)
9295.25
9296.02

30
H45_(−448)-(−421)
9385.28
9385.45

31
H45_(−439)-(−412)
9403.3
9403.98

32
H45_(−436)-(−409)
9435.3
9435.78

33
H45_(−422)-(−395)
9388.23
9388.34

34
H45_(−418)-(−394)
8363.88
8363.44

35
H45_(−418)-(−391)
9404.23
9404.6

36
H45_(−415)-(−391)
8354.87
8354.48

37
H45_(−413)-(−386)
9385.21
9385.07

38
H45_(−405)-(−378)
9371.23
9371.33

39
H45_(−389)-(−362)
9325.25
9325.6

40
H45_(−349)-(−322)
9221.2
9221.14

41
H45_(−320)-(−293)
9211.19
9211.14

42
H45_(−305)-(−280)
8640
8640.11

43
H45_(−290)-(−263)
9284.22
9284.05

44
H45_(−275)-(−250)
8656
8656.36

45
H45_(−260)-(−233)
9347.22
9347

46
H45_(−230)-(−203)
9198.19
9198.43

47
H45_(−200)-(−173)
9335.24
9335.21

48
H45_(−190)-(−163)
9312.25
9312.03

49
H45_(−185)-(−160)
8670.05
8670.14

50
H45_(−171)-(−144)
9273.24
9273.53

51
H45_(−188)-(−141)
9233.23
9233.36

52
H45_(−142)-(−115)
9254.22
9254.39

53
H45_(−110)-(−83)
9392.27
9392.55

54
H45_(−80)-(−53)
9352.26
9352.54

55
H45_(−78)-(−51)
9358.25
9358.63

56
H45_(−70)-(−43)
9323.23
9323.51

57
H45_(−60)-(−33)
9260.21
9260.39

58
H45_(−50)-(−23)
9238.22
9238.79

59
H45_(−44)-(−17)
9291.26
9291.37

60
H45_(−25)-3
9281.25
9281.54

61
H45_(−23)-5
9321.26
9321.92

62
H45_(−22)-3
8287.9
8287.54

63
H45_(−21)-7
9353.26
9353.35

64
E45_(−20)-5
8303.9
8303.52

65
H45_(−20)-8
9344.25
9344.57

66
H45_(−18)-10
9304.24
9304.44

67
H45_(−17)-8
8318.9
8318.93

68
H45_(−16)-12
9279.23
9278.95

69
H45_(−15)-10
8270.88
8270.96

70
H45_(−14)-14
9231.21
9231.28

71
H45_(−10)-16
8607.98
8607.59

72
H45_(−5)-20
8213.85
8213.45

73
H45_l-25
8214.85
8214.52

74
H45_6-30
8190.84
8190.04

75
H45_11-35
8198.85
8198.45

76
H45_11-40
9877.43
9877.12

77
H45_16-35
6544.28
6544.07

78
H45_16-40
8222.86
8222.85

79
H45_21-45
8244.85
8245.01

80
H45_(−423)-(−412)_16-33
9997.46
9997.49

81
H45_(−416)-(−402)_16-30
9964.44
9964.38

82
H45_(−411)-(−400)_16-33
9979.44
9979.18

83
H45_(−405)-(−391)_16-30
9945.42
9945.04

84
H45_(−402)-(−391)_16-33
9945.42
9945.07

85
H45_(−156)-(−143)-(−141)-(−128)
9190.2
9189.84

86
H45_(−78)-(−65)_(−44)-(−31)
9256.22
9256.49

87
H45_(−78)-(−65)_(−34)-(−21)
9354.28
9354.26

88
H45_(−78)-(−65)-16-29
9232.21
9233.24

89
H45_(−78)-(−65)_21-34
9263.21
9263.82

90
H45_(−78)-(−65)_27-40
9295.23
9295.5

91
H45_(−74)-(−62)_(−40)-(−28)
8643.02
8643.41

92
H45_(−72)-(−58)_(−38)-(−26)
9354.28
9354.28

93
H45_(−71)-(−58)_(−38)-(−25)
9314.27
9314.5

94
H45_(−71)-(−58)_16-29
9264.23
9262.81

95
H45_(−71)-(−58)_21-34
9295.23
9295.83

96
H45_(−71)-(−58)_27-40
9327.25
9327.61

97
H45_(−68)-(−60)_(−36)-(−28)_19-28
9274.24
9274.8

98
H45_(−64)-(−51)_(−44)-(−31)
9310.23
9310.1

99
145_(−64)-(−51)_(−35)-(−22)
9408.29
9408.3

100
H45_(−64)-(−51)_16-29
9286.22
9286.47

101
H45_(−64)-(−51)_21-34
9317.22
9317.12

102
H45_(−64)-(−51)_27-40
9349.24
9349.37

103
H45_(−44)-(−31)_16-29
9184.19
9183.98

104
H45_(−44)-(−31)_27-40
9247.21
9247.14

105
H45_(−44)-(−31)_21-33_T
9230.19
9230.7

106
H45_(−38)-(−25)_16-29
9210.22
9210.11

107
H45_(−38)-(−25)_21-34
9241.22
9241.87

108
H45_(−38)-(−25)_27-40
9273.24
9273.24

109
H45_(−34)-(−21)_16-29
9266.25
9266.72

110
H45_(−34)-(−21)_21-34
9297.25
9297.85

111
H45_(−34)-(−21)_27-40
9329.27
9329.78

112
H45_1-21
6859.387
6859.02

144
H45_167-194
9121.14
9121.27

145
H45_227-254
9253.22
9253.57

146
H45_257-284
9203.18
9203.09

147
H45_287-314
9369.21
9369.79

148
H45_317-344
9342.27
9342.18

149
H45_347-374
9268.22
9268.72

150
H46_(−200)-(−173)
9326.25
9325.78

151
H46_(−171)-(−144)
9243.21
9243.17

152
H46_(−146)-(−119)
9221.22
9221.07

153
H46_(−141)-(−114)
9276.23
9275.87

154
H46_(−136)-(−109)
9310.25
9310.07

155
H46_(−126)-(−99)
9295.25
9295.56

156
H46_(−116)-(−89)
9349.26
9349.16

157
H46_(−111)-(−84)
9334.26
9333.8

158
H46_(−106)-(−79)
9319.26
9319.07

159
H46_(−81)-(−54)
9318.24
9317.86

160
H46_(−51)-(−24)
9167.16
9167.63

161
H46_(−18)-6
8037.81
8037.34

162
H46_(−15)-9
8004.79
8004.24

163
H46_(−9)-15
7943.74
7944.19

164
H46_139-l66
9293.23
9292.5

165
H46_169-196
9319.22
9319.52

166
H46_199-226
9325.25
9325.91

167
H46_229-256
9361.27
9361.58

168
H46_259-286
9351.26
9351.88

169
H46_289-316
9400.26
9400.38

170
H46_319-346
9219.2
9219.51

171
H47_(−200)-(−173)
9142.18
9142.19

172
H47_(−171)-(−144)
9320.24
9320.43

173
H47_(−141)-(−114}
9230.21
9230.7

174
H47_(−111)-(−84)
9354.26
9354.05

175
H47_(−81)-(−54)
9215.16
9215.57

176
H47_(−51)-(−24)
9262.23
9262.28

177
H47_(−16)-12
9257.24
9257.23

178
H47_l-29
9612.33
9613.79

179
H47_135-162
9236.2
9236.32

180
H48_(−21)-7
9351.26
9350.96

181
H48_l-28
9277.21
9277.18

182
H48_177-204
9233.23
9232.72

183
H49_(−21)-7
9304.24
9304.25

184
H49_22-47
8509.94
8509.66

185
H49_93-120
9244.19
9244.51

186
H50_(−200)-(−173)
9149.19
9148.87

187
H50_(−171)-(−144)
9292.23
9291.93

188
H50_(−141)-(−114)
9330.22
9330.33

189
H50_(−111)-(−84)
9237.17
9236.8

190
H50_(−81)-(−54)
9260.21
9259.85

191
H50_(−51)-(−24)
9259.21
9258.85

192
H50_(−21)-7
9183.21
9183.31

193
H50_l00-127
9271.22
9271.25

194
H50_90-114
8277.87
8278.28

195
H51_(−18)-10
9411.24
9411.6

196
H51_224-251
9135.14
9135.51

197
H52_(−47)-(−20)
9330.2
9330.03

198
H52_(−18)-10
9270.22
9270.37

199
H52_9-38
9893.42
9893.2

200
H52_109-136
9236.2
9235.9

201
H53_(−197)-(−170)
9231.18
9231.18

202
H53_(−167)-(−140)
9298.2
9298.58

203
H53_(−137)-(−110)
9353.21
9353.6

204
H53_(−107)-(−80)
9225.19
9224.55

205
H53_(−77)-(−50)
9278.23
9278.35

206
H53_(−47)-(−20)
9373.25
9373.48

207
H53_(−18)-10
9319.26
9319

208
H53_203-230
9249.18
9249.06

209
H54_(−18)-10
9316.22
9316.33

210
H54_146-173
9267.22
9267.45

211
H54_176-203
9181.19
9181.18

212
H54_206-233
9142.18
9142.41

213
H54_236-263
9311.23
9311.57

214
H54_266-293
9249.18
9248.66

215
H54_296-323
9344.25
9344.57

216
H54_326-353
9308.21
9307.9

217
H55_(−197)-(−170)
9206.17
9206.95

218
H55_(−167)-(−140)
9278.23
9278.65

219
H55_(−137)-(−110)
9316.2
9316.05

220
H55_(−107)-(−80)
9327.25
9326.87

221
H55_(−77)-(−50)
9290.26
9290.43

222
II55_(−47)-(−20)
9342.25
9341.86

223
H55_(−18)-10
9185.21
9185.78

224
H55_12-34
6858.387
6858.97

113
H55_177-204
9203.18
9203.35

114
H55_191-218
9320.24
9320.46

115
H55_221-248
9301.22
9301.7

116
H55_251-278
9253.2
9253.12

117
H55_281-308
9300.22
9300.57

118
H55_311-338
9126.18
9126.01

119
H55_334-361
9058.12
9057.77

120
H55_341-368
9154.16
9154.22

121
H55_371-398
9412.24
9412.07

122
H55_401-428
9308.21
9308.06

123
H55_431-458
9210.17
9209.71

124
H55_461-488
9181.19
9181.31

125
H55_491-518
9260.23
9260.72

126
H55_521-548
9237.22
9235.93

127
H55_548-575
9304.26
9304.44

128
H56_(−400)-(−373)
9283.22
9282.84

129
H56_(−380)-(−355)
8584.99
8585.11

130
H56_(−345)-(−318)
9397.26
9397.16

131
H56_(−315)-(−288)
9275.21
9275.43

132
H56_(−285)-(−258)
9258.21
9257.72

133
H56_(−255)-(−228)
9307.21
9307.98

134
H56_(−225)-(−198)
9364.24
9364.79

135
H56_(−195)-(−168)
9224.17
9224.08

136
H56_(−165)-(−138)
9172.18
9172.28

137
H56_(−135)-(−108)
9256.24
9256.9

138
H56_(−105)-(−78)
9291.23
9291.37

139
H56_(−76)-(−51)
8754.04
8754.09

140
H56_(−59)-(−34)
8629.04
8629.59

141
H56_(−39)-(−14)
8723.04
8723.84

142
H56_(−31)-(−6)
8773.06
8773.68

143
H56_(−14)-13
9297.23
9296.6

225
H44_188-202_H56_(−156)-(−142)
9876.43
9876.87

226
H44_188-202_H56_(−124)-(−110)
9956.45
9956.24

227
H44_188-202_H56_(−85)-(−71)
10069.49
10070.12

228
H56_(−156)-(−142)_H44_188-202
9876.43
9876.63

229
H56_(−124)-(−110)_H44_188-202
9956.45
9956.72

230
H56_(−85)-(−71)_H44_188-202
10069.49
10069.93

231
H45_16-35_H46_(−134)-(−115)
13207.58
13207.56

232
H45_16-35_H46_(−103)-(−84)
13281.61
13281.25

Production of Antisense PMO Targeting Splicing Silencer in Intron of Human Dystrophin Gene or Splice Site Involved in Single Skipping of Human Dystrophin Gene

(i) PMO having the base sequence of PMO No. 233 (SEQ ID NO: 257) targeting a splicing silencer sequence of intron 44 or a splice site involved in single skipping, (ii) PMOs having the base sequences of PMO Nos. 234 to 244 (SEQ ID NOs: 258 to 268) targeting a splicing silencer sequence of intron 45 or a splice site involved in single skipping, (iii) PMOs (linked type) having the base sequences of PMO Nos. 245 to 249 (SEQ ID NOs: 269 to 273) targeting a region (R2) in the vicinity of an acceptor of intron 44 and a splicing silencer sequence of intron 45 or a splice site involved in single skipping, and (iv) PMOs having the base sequences of PMO Nos. 250 to 251 (SEQ ID NOs: 274 to 275) targeting a splicing silencer sequence of exon 45 in the human dystrophin gene were synthesized in the same manner as in the PMO targeting a region in the vicinity of a donor or an acceptor of an intron. The 5′ end of each PMO is Group (3), as in the PMO targeting a region in the vicinity of a donor or an acceptor of an intron. Each PMO is shown in Table 18, and the calculated (Clcd) and measured (Found) values of ESI-TOF-MS are shown in Table 19.

TABLE 18

PMO

SEQ

No.
Target
Target base sequence
Base sequence of PMO
ID NO

233
(i)
H45_(−93)-(−70)
GTGTGCTACCACATGCAGTTGTAC
257

234
(ii)
H45_166-189
TCTGTCGCCCTACCTCTTTTTTCT
258

235

H45_178-201
ATTCCTATTAGATCTGTCGCCCTA
259

236

H45_183-206
TTTTCATTCCTATTAGATCTGTCG
260

237

H45_188-211
AAATGTTTTCATTCCTATTAGATC
261

238

H45_191-214
CTAAAATGTTTTCATTCCTATTAG
262

239

H45_197-220
AGTCTGCTAAAATGTTTTCATTCC
263

240

H46_(−35)-(−12)
AGAATAAAATTGTTATTTTTTTTT
264

241

H46_(−22)_2
GCCTGGAGAAAGAAGAATAAAATT
265

242

H46_(−12)_12
TGTTCTTCTAGCCTGGAGAAAGAA
266

243

H46_(−2)_22
GATATTCTTTTGTTCTTCTAGCCT
267

244

H45_20236-20259
ATCCATGCATTCTTCCATGCACAG
268

245
(iii)
H45_16-30_191-205
TTTCATTCCTATTAGTTGCCGCTGCCCAAT
269

246

H45_16-30_197-211
AAATGTTTTCATTCCTTGCCGCTGCCCAAT
270

247

H45_16-33_197-208
TGTTTTCATTCCAGTTTGCCGCTGCCCAAT
271

248

H45_16-35_183-192
AGATCTGTCGACAGTTTGCCGCTGCCCAAT
272

249

H45 18-35_191-202
CATTCCTATTAGACAGTTTGCCGCTGCCCA
273

250
(iv)
H45_31-54
TGCATTCAATGTTCTGACAACAGT
274

251

H45_131-154
GTTTGCAGACCTCCTGCCACCGCA
275

A base sequence presumed to correspond to the splicing silencer sequence in the intron or the splice site involved in single skipping is underlined.

A base sequence presumed to correspond to the splicing silencer sequence in the intron or the splice site involved in single skipping is underlined.

TABLE 19

PMO No.
Target base sequence
Calculated
Measured

233
H45_(−93)-(−70)
7938.75
7938.95

234
H45_166-189
7757.67
7757.89

235
H45_178-201
7848.72
7848.99

236
H45_183-206
7869.71
7869.94

237
H45_188-211
7886.73
7886.24

238
H45_191-214
7886.73
7886.37

239
H45_197-220
7887.73
7887.49

240
H46_(−35)-(−12)
7955.74
7955.61

241
H46_(−22)_2
8061.82
8062.19

242
H46_(−12)_12
7986.77
7986.42

243
H46_(−2)_22
7860.7
7860.83

244
H45_20236-20259
7842.73
7842.9

245
H45_16-30_l91-205
9824.39
9824.66

246
H45_16-30_l97-211
9833.4
9833.32

247
H45_16-33_197-208
9840.39
9840.1

248
H45_16-35_183-192
9908.43
9908.14

249
H45_18-35_l91-202
9843.41
9843.08

250
H45_31-54
7848.72
7848.99

251
H45_131-154
7844.73
7844.85

Example 2: Test on Multi-Exon Skipping Activity of Antisense Oligomer
Test Example 1
In Vitro Assay of Multi-Exon Skipping in Human Dystrophin Gene—(1): Induction of Multi-Exon Skipping
Procedures

Each antisense oligomer (PMO Nos. 55, 59, 78, 65, 75, 77, 102, 35, 52, 47, 50, 46, 43, 40, 139, 119, 142, 113, 51, 11, 45, 54, 41, 74, 66, 64, 63, 68, 76, 28, 27, 225, 228, 1, 5, 132, 131, 128, 163, 162, and 161 (SEQ ID NO: 55, 59, 78, 65, 75, 77, 102, 35, 52, 47, 50, 46, 43, 40, 139, 119, 142, 113, 51, 11, 45, 54, 41, 74, 66, 64, 63, 68, 76, 28, 27, 225, 228, 1, 5, 132, 131, 128, 163, 162, and 161)) of Table 11 was transfected in a concentration of 25 to 50 μM either singly or in combination of two or three oligomers with 3.5×10⁵of RD cells (human rhabdomyosarcoma cell line, CCL-136, purchased from ATCC). The reagent used for the transfection was an Amaxa Cell Line Nucleofector Kit L on Nucleofector II (Lonza). The Program T-030 was used.

After transfection, the cells were cultured for three nights in 2 mL of Eagle's minimal essential medium (EMEM) (manufactured by Sigma, hereinafter the same) containing 10% fetal bovine serum (FBS) (manufactured by Invitrogen) under conditions of 37° C. and 5% CO₂.

The RD cells were washed once with PBS (manufactured by Nissui, hereinafter the same) and 350 μL of Buffer RLT (manufactured by Qiagen) containing 1% 2-mercaptoethanol (manufactured by Nacalai Tesque, Inc.) was added to the cells. After the cells were allowed to stand at room temperature for a few minutes to lyse the cells, the lysate was collected into a QIAshredder homogenizer (manufactured by Qiagen). A homogenate was produced by centrifugation at 15,000 rpm for 2 minutes. The total RNA was extracted according to the protocol attached to RNeasy Mini Kit (manufactured by Qiagen). The concentration of the total RNA extracted was determined using a NanoDrop ND-1000 (manufactured by LMS Co., Ltd.).

One-Step RT-PCR was performed with 800 ng of the extracted total RNA using a QIAGEN One Step RT-PCR Kit (manufactured by Qiagen). A reaction solution was prepared in accordance with the protocol attached to the kit. TaKaRa PCR Thermal Cycler Dice Touch (manufactured by Takara Bio Inc.) was used as the thermal cycler. The RT-PCR program used is as follows.

50° C., 30 mins: reverse transcription reaction

95° C., 15 mins: polymerase activation, reverse transcriptase inactivation, cDNA thermal denaturation

[94° C., 10 seconds; 57° C., 30 seconds; 72° C., 1 minute]×33 cycles: PCR amplification

72° C., 10 mins: final extension

The base sequences of the forward primer and reverse primer used for RT-PCR are given below.

Forward primer:

(SEQ ID NO: 276)

5′-ATTTGACAGATCTGTTGAGAAATGG-3′

Reverse primer 1:

(SEQ ID NO: 277)

5′-GGCTGTTTTCATCCAGGTTGTG-3′

Reverse primer 2:

(SEQ ID NO: 278)

5′-AGTTGCTGCTCTTTTCCAGGT-3′

Transcripts having multi-exon skipping of exons 45 to 55 can be detected by a combination of the forward primer and the reverse primer 2. Transcripts having no skipping and transcripts having single exon skipping of exon 45 can be detected by a combination of the forward primer and the reverse primer 1.

The reaction product of the PCR above was analyzed using a Bioanalyzer (manufactured by Agilent Technologies, Inc.) and MultiNA (manufactured by Shimadzu Corp.).

The polynucleotide level “A” of the band with skipping of any two or more numerically consecutive exons among exons 45 to 55, the polynucleotide level “B” of the band with skipping of any one exon among exons 45 to 55, and the polynucleotide level “C” of the band without skipping were measured. Based on these measurement values of “A”, “B”, and “C”, the skipping efficiency of multi-exon skipping was determined by the following equation.

Skipping efficiency (%)=A/(A+B+C)×100

Results

The results are shown in FIGS. 1 to 16. The introduction of PMO Nos. 55, 59, 78, 65, 75, 77, 102, 35, 52, 47, 50, 46, 43, 40, 139, 119, 142, 113, 51, 11, 45, 54, 41, 74, 66, 64, 63, 68, 76, 28, 27, 225, 228, 1, 5, 132, 131, 128, 163, 162, and 161 (SEQ ID NO: 55, 59, 78, 65, 75, 77, 102, 35, 52, 47, 50, 46, 43, 40, 139, 119, 142, 113, 51, 11, 45, 54, 41, 74, 66, 64, 63, 68, 76, 28, 27, 225, 228, 1, 5, 132, 131, 128, 163, 162, and 161) singly or in combination induced multi-exon skipping of exons 45 to 55.

These results revealed that the antisense oligomers of the present invention described in Table 11 effectively induce or would effectively induce multi-exon skipping when used singly or in combination.

It was also confirmed that the introduction of PMO Nos. 6, 7, 8, 10, 14, 26, 29, 38, 39, 53, 58, 67, 80, 82, 86, 92, 97, 98, 100, 121, 122, 124, 125, 126, 130, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 231, 232 (SEQ ID NO: 6, 7, 8, 10, 14, 26, 29, 38, 39, 53, 58, 67, 80, 82, 86, 92, 97, 98, 100, 121, 122, 124, 125, 126, 130, 144, 146, 147, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 231, 232) also induce multi-exon skipping of exons 45 to 55.

Test Example 2
In Vitro Assay of Multi-Exon Skipping in Human Dystrophin Gene—(2): Promotion of Multi-Exon Skipping
Procedures

The antisense oligomer (PMO No. 75) of Table 11 was added in a concentration of 25 to 50 μM to 3.5×10⁵of RD cells (human rhabdomyosarcoma cell line, CCL-136, purchased from ATCC), and each antisense oligomer (PMO No. 236, 237, or 239) of Table 18 was introduced in combination therewith in a concentration of 25 to 50 μM. The assay was conducted by the same procedures as in Test Example 1.

Results

The results are shown in FIGS. 17 and 18. The introduction of PMO No. 75 (SEQ ID NO: 75) in combination with PMO No. 236, 237, or 239 (SEQ ID NO: 260, 261, or 263) enhanced the skipping efficiency of multi-exon skipping of exons 45 to 55 as compared to the case of PMO No. 75 singly. On the other hand, this approach decreased the skipping efficiency of single exon skipping of exon 45.

These results revealed that the antisense oligomer of the present invention described in Table 11 promote or would promote multi-exon skipping when used in combination with the suppressor antisense oligomer of the present invention described in Table 18.

Test Example 3
In Vitro Assay of Multi-Exon Skipping in Human Dystrophin Gene—(3): Promotion of Overall Multi-Exon Skipping
Procedures

Test Example 3 was conducted by the same procedures as in Test Example 2 except that One-Step RT-PCR was performed as follows.

50° C., 30 mins: reverse transcription reaction

95° C., 15 mins: polymerase activation, reverse transcriptase inactivation, cDNA thermal denaturation

[94° C., 10 seconds; 60° C., 30 seconds; 72° C., 2 min]×40 cycles: PCR amplification

72° C., 10 mins: final extension

The base sequences of the forward primer and reverse primer used for RT-PCR are given below.

Forward primer 2:

(SEQ ID NO: 279)

5′-ATTTGACAGATCTGTTGAGAAATGG-3′

Reverse primer 3:

(SEQ ID NO: 280)

5′-GGCTCCAATAGTGGTCAGTCC-3′

Forward primer 3:

(SEQ ID NO: 281)

5′-CCTGAGAATTGGGAACATGC-3′

Reverse primer 4:

(SEQ ID NO: 282)

5′-CCTCCTTCCATGACTCAAGC-3′

Transcripts having multi-exon skipping of exons 45 and 46, exons 45 to 47, exons 45 to 48, or exons 45 to 49 in the human dystrophin gene can be detected by a combination of the forward primer 2 and the reverse primer 3. On the other hand, transcripts having multi-exon skipping of exons 45 and 46, exons 45 to 47, exons 45 to 48, exons 45 to 49, exons 45 to 50, exons 45 to 51, or exons 45 to 52 are detected by a combination of the forward primer 3 and the reverse primer 4.

The reaction product of the PCR above was analyzed using a Bioanalyzer (manufactured by Agilent Technologies, Inc.) and MultiNA (manufactured by Shimadzu Corp.) to quantify the amount of the PCR product of the transcript having each exon skipping.

Results

The results are shown in FIGS. 19 to 22. The introduction of PMO No. 75 (SEQ ID NO: 75) in combination with PMO No. 236 or 239 (SEQ ID NO: 260 or 263) enhanced the skipping efficiency of multi-exon skipping of exons 45 to 55 (Test Example 2) as well as multi-exon skipping of exons 45 and 46, exons 45 to 47, exons 45 to 48, exons 45 to 49, exons 45 to 50, exons 45 to 51, and exons 45 to 52 as compared to the case of PMO No. 75 singly.

These results revealed that the antisense oligomer of the present invention described in Table 11 promote or would promote overall multi-exon skipping when used in combination with the suppressor antisense oligomer of the present invention described in Table 18.

INDUSTRIAL APPLICABILITY

The present invention provides an antisense oligomer and a suppressor antisense oligomer that cause multi-exon skipping in human dystrophin. Use of the antisense oligomer and the suppressor antisense oligomer of the present invention provides a novel therapeutic agent for DMD and method for treatment of DMD.

ANTISENSE NUCLEIC ACID ENABLING EXON SKIPPING

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