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`1 September 2010
`JP 2010-196032
`JP 2010-196032
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`NIPPON SHINYAKU CO., LTD.
`NATIONAL CENTER OF NEUROLOGY
`AND PSYCHIATRY
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`20 September 2011
`Iwai Yoshiyuki
`Commissioner, Japan Patent Office
`
`[Official Seal]
`
`

`

`
`[Document]
`[Reference No.]
`[Date Submitted]
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`[Inventor]
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`[Name]
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`[Inventor]
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`[Address or Residence]
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`[Name]
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`[Inventor]
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`Patent Application
`P10-0121
`1 September 2010
`Commissioner, Patent Office
`C12N 15/00
`A61K 31/7105
`A61K 31/711
`A61K 31/7115
`A61K 31/712
`A61K 31/7125
`C07H 21/00
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`NIPPON SHINYAKU CO., LTD. 3-
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`Naoki WATANABE
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`NIPPON SHINYAKU CO., LTD., 14,
`Nishinosho-Monguchi-cho,
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`shi, Kyoto-fu
`Yohei SATO
`
`NATIONAL CENTER OF NEUROLOGY
`AND PSYCHIATRY, 4-1-1 Ogawa-
`Higashi, Kodaira, Tokyo-to
`Shin'ichi TAKEDA
`
`NATIONAL CENTER OF NEUROLOGY
`AND PSYCHIATRY, 4-1-1 Ogawa-
`Higashi, Kodaira, Tokyo-to
`Tetsuya NAGATA
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`000004156
`NIPPON SHINYAKU CO., LTD.
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`510147776
`NATIONAL CENTER OF NEUROLOGY
`AND PSYCHIATRY
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`[Item]
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`100092783
`
`Hiroshi KOBAYASHI
`03-3273-2611
`
`100095360
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`Eiji KATAYAMA
`
`100120134
`
`Norio OMORI
`
`100147131
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`Takayuki IMASATO
`
`100104282
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`Yasuhito SUZUKI
`
`157061
`15,000 yen
`
`Claims 1
`Specification 1
`Abstract 1
`Drawings 1
`
`

`

`[Document Name] Specification
`[Title of the invention] Antisense nucleic acid
`[Technical Field]
`
`[0001]
`
`The present invention relates to antisense oligomers
`which enable skipping of exon 53 in the human dystrophin gene,
`and to pharmaceutical compositions which include said
`oligomers.
`[Background Art]
`
`[0002]
`
`Duchenne muscular dystrophy (DMD) is the most frequent
`form of muscle atrophy, affecting one in ca 3500 newborn
`males. Although motor functions are subtantially unchanged
`from those of healthy humans in infancy, muscle weakness is
`observed from around 4-5 years old. Muscle weakness
`subsequently progresses, with inability to walk by about 12
`years old, and death due to cardiac or respiratory
`insufficiency in the twenties; it is a serious disorder. There
`is currently no effective therapy for DMD, and the development
`of a new therapy is strongly desired.
`
`[0003]
`
`DMD is known to be caused by a mutation of the dystrophin
`gene. The dystrophin gene is located on the X chromosome and
`is a gigantic gene consisting of DNA of 2.2 million nucleotide
`pairs. mRNA with 79 linked exons is formed by transcription
`from DNA to an mRNA precursor, and then removal of introns by
`splicing comprises 11,058 nucleotides. This mRNA is translated
`into 3685 amino acids, to produce the dystrophin protein. The
`dystrophin protein contributes to the maintenance of membrane
`stability in muscle cells and is necessary to make muscle
`cells less fragile. Because the dystrophin gene from patients
`with DMD contains a mutation, there is hardly any expression
`of functional dystrophin protein in muscle cells. Therefore,
`in the bodies of DMD patients the structure of muscle cells
`cannot be maintained, and a large quantity of calcium ions
`flows into the muscle cells. As a result, an inflammation-like
`response occurs, promoting fibrosis so that the muscle cells
`cannot readily be regenerated.
`
`[0004]
`
`

`

`Becker muscular dystrophy (BMD) is also caused by a
`
`mutation of the dystrophin gene, but although the condition
`presents muscle weakness due to muscle atrophy, it is
`generally milder than DMD and muscle weakness also progresses
`more slowly; and in many cases, onset is in adulthood. It is
`thought that the differences in clinical symptoms between DMD
`and BMD depend on whether the reading frame for amino acids
`when dystrophin mRNA is translated into protein is disrupted
`due to the mutation, or is maintained (Non-Patent Document 1).
`Thus, in DMD there is hardly any expression of functional
`dystrophin protein because there is a mutation which shifts
`the amino acid reading frame; but in BMD, although some exons
`are lost due to the mutation, incomplete but functional
`dystrophin protein is produced, because the amino acid reading
`frame is maintained.
`
`[0005]
`
`Exon skipping offers expectations as a method for
`treating DMD. This method restores the amino acid reading
`frame of dystrophin mRNA by modifying splicing, and induces
`expression of protein with partially restored function (Non-
`Patent Document 2). The mutation and the partial amino acid
`sequence which is a target for exon skipping are lost.
`Consequently, dystrophin protein expressed by this treatment
`is shorter than the normal protein, but because the amino acid
`reading frame is maintained, it partially retains the function
`of stabilizing muscle cells. Therefore, it is expected that
`exon skipping will give DMD which presents similar symptoms to
`that of the milder BMD. The exon skipping approach has passed
`through animal experiments using mice and dogs, and clinical
`trials on human DMD patients are in progress.
`
`[0006]
`
`Exon skipping can be induced by binding of antisense
`nucleic acid targeting either the 5’ or 3’ splice site or
`both, or an exon-internal sequence. An exon will only be
`included in the mRNA when both splice sites are recognized by
`the spliceosome complex. Therefore, exon skipping can be
`induced by targeting a splice site with antisense nucleic
`acid. Binding of an SR protein to an exon splicing enhancer
`(ESE) is also thought to be necessary for recognition of an
`
`

`

`exon in the splicing mechanism, and exon skipping can also be
`induced by targeting the ESE.
`
`[0007]
`
`Because mutations of the dystrophin gene differ among DMD
`patients, antisense nucleic acid suited to the site or type of
`gene mutation is needed. So far, antisense nucleic acids that
`induce exon skipping have been produced by Steve Wilton et al.
`of the University of Western Australia for all 79 exons (Non-
`Patent Document 3); and antisense nucleic acids which induce
`exon skipping have been produced by Annemieke Aartsma-Rus et
`al. in the Netherlands for 39 exons (Non-Patent Document 4).
`
`[0008]
`
`It is thought that about 8% of all DMD patients could be
`treated by skipping the 53rd exon (hereinafter referred to as
`"exon 53"). In recent years, several research organizations
`have reported studies on exon 53 of the dystrophin gene as a
`target for exon skipping (Patent Documents 1-3; Non-Patent
`Document 5). However, a technique for highly efficient
`skipping of exon 53 has yet to be established.
`[Prior Art Documents]
`[Patent Documents]
`
`[0009]
`
`Patent Document 1: WO 2006/000057 A1
`
`Patent Document 2: WO 2004/048570 A1
`
`Patent Document 3: US 2010/0168212 A1
`[Non-Patent Documents]
`[0010]
`Non-Patent Document 1: Monaco A. P. et al., Genomics
`1988; 2: p. 90-95
`Non-Patent Document 2: Matsuo M., Brain Dev 1996; 18: p.
`167-172
`Non-Patent Document 3: Wilton S. D., et al., Molecular
`Therapy 2007: 15: p. 1288-96
`Non-Patent Document 4: Annemieke Aartsma-Rus et al.,
`(2002) Neuromuscular Disorders 12: S71-S77
`Non-Patent Document 5: Linda J. Popplewell et al.,
`(2010) Neuromuscular Disorders , vol. 20, no. 2, p.
`102-10
`[Synopsis of the Invention]
`
`

`

`[Problem which the invention is intended to solve]
`
`[0011]
`
`Given the situation described above, an antisense
`oligomer that strongly induces skipping of exon 53 of the
`dystrophin gene and treatments for muscular dystrophy which
`include such an oligomer are desired.
`[Means for solving the problem]
`
`[0012]
`
`As a result of detailed studies of the structure of
`dystrophin mutant genes having a mutation in exon 53, the
`present inventors have found that skipping of exon 53 can be
`induced with high efficiency by targeting the sequence
`comprising peripheral nucleotides 32-56 from the 5' end of
`exon 53 in the mRNA precursor (hereinafter referred to as
`"pre-mRNA") of the dystrophin gene, by using an antisense
`oligomer. The present inventors have perfected the present
`invention based on this insight.
`
`[0013]
`
`Thus, the present invention is as follows.
`[1] An antisense oligomer, which is an antisense oligomer
`which enables skipping of exon 53 of the human dystrophin
`gene, comprising a nucleotide sequence complementary to any
`one of the sequences comprising nucleotides 31-53, 31-54, 31-
`55, 31-56, 31-57, 31-58, 32-53, 32-54, 32-55, 32-56, 32-57,
`32-58, 33-53, 33-54, 33-55, 33-56, 33-57, 33-58, 34-53, 34-54,
`34-55, 34-56, 34-57, 34-58, 35-53, 35-54, 35-55, 35-56, 35-57,
`35-58, 36-53, 36-54, 36-55, 36-56, 36-57 or 36-58 from the 5'
`end of exon 53 in the human dystrophin gene.
`[2] An antisense oligomer according to [1] above, which is an
`oligonucleotide.
`[3] An antisense oligomer according to [2] above, wherein the
`sugar moiety and/or the phosphate-binding moiety of at least
`one nucleotide constituting the oligonucleotide is modified.
`[4] An antisense oligomer according to [3] above, wherein the
`sugar moiety of at least one nucleotide constituting the
`oligonucleotide is ribose in which the 2’-OH group is replaced
`by any group selected from a set comprising OR, R, R’ OR, SH,
`SR, NH2, NHR, NR2, N3, CN, F, Cl, Br and I. (Where R
`
`

`

`indicates a C1-6 alkyl or C1-6 aryl and R’ indicates a C1-6
`alkylene.)
`[5] An antisense oligomer according to [3] or [4] above,
`wherein the phosphate constituting the oligonucleotide is any
`one selected from a set comprising a phosphorothioate bond, a
`phosphorodithioate bond, an alkylphosphonate bond and a
`phosphoroamidate bond.
`[6] An antisense oligomer according to [1] above, which is a
`morpholino oligomer.
`[7] An antisense oligomer according to [6] above which is a
`phosphorodiamidate morpholino oligomer.
`[8] An antisense oligomer according to any one of [1]-[7]
`above, wherein the 5'-methylene bound to the ribose or
`morpholino ring of the 5' terminal nucleic acid residue is
`modified with any one of the groups below.
`
`[Formula 1]
`
`
`
`[Formula 2]
`
`
`
`
`
`[Formula 3]
`
`
`
`or
`
`
`
`

`

`[9] An antisense oligomer according to any one of [1]-[8]
`above, comprising a nucleotide sequence complementary to a
`sequence comprising nucleotides 32-56 or 36-56 from the 5' end
`of exon 53 of the human dystrophin gene.
`[10] An antisense oligomer according to any one of [1]-[8]
`above, comprising any one nucleotide sequence selected from a
`set comprising SEQ ID NO: 2-37.
`[11] An antisense oligomer according to any one of [1]-[8]
`above, comprising any one nucleotide sequence selected from a
`set comprising SEQ ID NO: 11, 17, 23, 29 and 35.
`[12] An antisense oligomer according to any one of [1]-[8]
`above, comprising the nucleotide sequence of either SEQ ID NO:
`11 or 35.
`[13] A pharmaceutical composition for treating muscular
`dystrophy, in which an active ingredient is an antisense
`oligomer according to any one of [1] to [12] above, or a
`pharmaceutically acceptable salt or hydrate thereof.
`[Effects of the invention]
`
`[0014]
`
`An antisense oligomer of the present invention can induce
`skipping of exon 53 of the dystrophin gene with high
`efficiency. In addition, the symptoms of Duchenne muscular
`dystrophy can be effectively alleviated by administering a
`pharmaceutical composition of the present invention.
`[Brief Description of the Drawings]
`
`[0015]
`[Figure 1] is a graph showing the efficiency of skipping
`
`of exon 53 of the dystrophin gene in human rhabdomyosarcoma
`cell line (RD cells).
`[Figure 2] is a graph showing the efficiency of skipping
`
`of exon 53 of the dystrophin gene in fibroblasts from normal
`human tissue (TIG-119 cells), induced to differentiate into
`muscle cells by introducing the human myoD gene.
`[Figure 3] is a graph showing the efficiency of skipping
`
`of exon 53 of the dystrophin gene in fibroblasts from a human
`DMD patient (5017 cells), induced to differentiate into muscle
`cells by introducing the human myoD gene.
`[Mode for Carrying Out the Invention]
`
`[0016]
`
`

`

`1. Antisense oligomers
`
` The present invention offers antisense oligomers
`(hereinafter referred to as "oligomers of the present
`invention") which enable skipping of exon 53 in the human
`dystrophin
`gene,
`comprising
`a
`nucleotide
`sequence
`complementary to any one of the sequences (hereinafter also
`referred to as "target sequences") consisting of nucleotides
`31-53, 31-54, 31-55, 31-56, 31-57, 31-58, 32-53, 32-54, 32-55,
`32-56, 32-57, 32-58, 33-53, 33-54, 33-55, 33-56, 33-57, 33-58,
`34-53, 34-54, 34-55, 34-56, 34-57, 34-58, 35-53, 35-54, 35-55,
`35-56, 35-57, 35-58, 36-53, 36-54, 36-55, 36-56, 36-57, or
`36-58 from the 5' end of exon 53 of the human dystrophin gene.
`
`[0017]
`[Exon 53 of the human dystrophin gene]
`
`In the present invention, the term "gene", in addition to
`genomic genes, also includes cDNA, mRNA precursors and mRNA.
`Preferably, the gene is an mRNA precursor, i.e., pre-mRNA.
`
`In the human genome, the human dystrophin gene is located
`at locus Xp21.2. The human dystrophin gene has a size of 3.0
`Mbp and is the largest of the known human genes. However, the
`coding region of the human dystrophin gene is a mere 14 kb,
`and said coding region is dispersed within the dystrophin gene
`as 79 exons (Roberts, RG., et al., Genomics, 16: 536-538
`(1993)). Pre-mRNA, which is the transcript of the human
`dystrophin gene, undergoes splicing to produce mature mRNA of
`14 kb. The nucleotide sequence of the human wild-type
`dystrophin gene is known (GenBank Accession No. NM_004006).
`
`The nucleotide sequence of exon 53 in the human wild-type
`dystrophin gene is shown in SEQ ID NO: 1.
`
`[0018]
`
`The oligomers of the present invention are created in
`order to modify the protein encoded by a DMD dystrophin gene
`into a BMD dystrophin by skipping of exon 53. Therefore, exon
`53 of the dystrophin gene, which is the target of exon
`skipping by an oligomer of the present invention, includes
`mutant forms as well as the wild type.
`
`Specifically, mutant exon 53 of human dystrophin genes is
`a polynucleotide described in (a) or (b) below.
`
`

`

` (a) A polynucleotide that hybridizes under stringent
`conditions with a polynucleotide comprising a nucleotide
`sequence complementary to the nucleotide sequence of SEQ ID
`NO: 1; and
` (b) a polynucleotide comprising a nucleotide sequence having
`at least 90% homology with the nucleotide sequence of SEQ ID
`NO: 1.
`
`[0019]
`
`In the present description, "polynucleotide" means DNA or
`RNA, but is preferably RNA.
`
`In the present description, the term "polynucleotide that
`hybridizes under stringent conditions" means, for example, a
`polynucleotide obtained by colony hybridization, plaque
`hybridization or Southern hybridization, etc., using as a
`probe all or part of a polynucleotide consisting of a
`nucleotide sequence complementary to the nucleotide sequence
`of SEQ ID NO: 1, for example. As the hybridization method, a
`method described, for example, in "Sambrook & Russell,
`Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring
`Harbor, Laboratory Press 2001" or "Ausubel, Current Protocols
`in Molecular Biology, John Wiley & Sons 1987-1997", etc., can
`be employed.
`
`[0020]
`
`In this description, the term "stringent conditions" may
`be any of conditions of low stringency, moderately stringent
`conditions or highly stringent conditions. "Conditions of low
`stringency" are, for example, conditions of 5×SSC, 5×Denhardts
`solution, 0.5% SDS and 50% formamide, at 32°C. "Moderately
`stringent conditions" are, for example, conditions of 5×SSC,
`5×Denhardts solution, 0.5% SDS and 50% formamide, at 42°C, or
`5×SSC, 1% SDS, 50 mM Tris-HCl (pH 7.5) and 50% formamide, at
`42°C. "Highly stringent conditions" are, for example, 5×SSC,
`5×Denhardts solution, 0.5% SDS and 50% formamide, at 50°C, or
`0.2×SSC and 0.1% SDS, at 65°C. Under these conditions,
`polynucleotides with high homology can be expected to be
`obtained more efficiently at higher temperatures. However,
`multiple factors, such as temperature, probe concentration,
`probe length, ionic strength, time and salt concentration, can
`be expected to affect hybridization stringency, and those
`
`

`

`skilled in the art can achieve similar stringency by
`appropriate selection of these factors.
`
`[0021]
`
`It should be noted that when using a commercially
`available kit for hybridization, the Alkphos Direct Labeling
`and Detection System (GE Healthcare), for example, can be
`used. In this case, hybridized polynucleotides can be detected
`after incubation overnight with the labeled probe, and then
`washing the membrane with a primary wash buffer containing
`0.1% (w/v) SDS at 55°C, in accordance with the protocol
`included in the kit. Alternatively, when creating a probe
`based on an entire or partial nucleotide sequence
`complementary to the nucleotide sequence of SEQ ID NO: 1, if
`the probe is labeled with digoxigenin (DIG) using a
`commercially available reagent (for example, PCR Labeling Mix
`(Roche Diagnostics), etc.), hybridization can be detected by
`using a DIG Nucleic Acid Detection Kit (Roche Diagnostics).
`
`[0022]
`
`Polynucleotides other than the hybridizable poly-
`nucleotides described above include polynucleotides having
`≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%,
`≥99.1%, ≥99.2%, ≥99.3%, ≥99.4%, ≥99.5%, ≥99.6%, ≥99.7%, ≥99.8%
`or ≥99.9% homology with the polynucleotide of SEQ ID NO: 1, as
`calculated by the homology search software BLAST, using the
`default parameters.
`
`[0023]
`
`Homology between nucleotide sequences can be determined
`using the 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). BLASTN
`and BLASTX programs have been developed based on the BLAST
`algorithm (Altschul SF, et al: J. Mol. Biol. 215: 403, 1990).
`When a nucleotide sequence is analyzed by using BLASTN, the
`parameters should be, for example, score = 100 and wordlength
`= 12. When BLAST and Gapped BLAST programs are used, the
`default parameters for each program are used.
`
`[0024]
`
`Examples of nucleotide sequences complementary to
`sequences comprising nucleotides 31-53, 31-54, 31-55, 31-56,
`
`

`

`31-57, 31-58, 32-53, 32-54, 32-55, 32-56, 32-57, 32-58, 33-53,
`33-54, 33-55, 33-56, 33-57, 33-58, 34-53, 34-54, 34-55, 34-56,
`34-57, 34-58, 35-53, 35-54, 35-55, 35-56, 35-57, 35-58, 36-53,
`36-54, 36-55, 36-56, 36-57 and 36-58 from 5' of exon 53, are
`shown in the table below.
`[Table 1]
`
`Nucleotides of exon 53
`31-53
`31-54
`31-55
`31-56
`31-57
`31-58
`32-53
`32-54
`32-55
`32-56
`32-57
`32-58
`33-53
`33-54
`33-55
`33-56
`33-57
`33-58
`34-53
`34-54
`34-55
`34-56
`34-57
`34-58
`35-53
`35-54
`35-55
`35-56
`35-57
`35-58
`36-53
`36-54
`36-55
`
`Complementary nucleotide sequence
`5'-CCGGTTCTGAAGGTGTTCTTGTA-3'
`5'-TCCGGTTCTGAAGGTGTTCTTGTA-3'
`5'-CTCCGGTTCTGAAGGTGTTCTTGTA-3'
`5'-CCTCCGGTTCTGAAGGTGTTCTTGTA-3'
`5'-GCCTCCGGTTCTGAAGGTGTTCTTGTA-3'
`5'-TGCCTCCGGTTCTGAAGGTGTTCTTGTA-3'
`5'-CCGGTTCTGAAGGTGTTCTTGT-3'
`5'-TCCGGTTCTGAAGGTGTTCTTGT-3'
`5'-CTCCGGTTCTGAAGGTGTTCTTGT 3'
`5'-CCTCCGGTTCTGAAGGTGTTCTTGT-3'
`5'-GCCTCCGGTTCTGAAGGTGTTCTTGT-3'
`5'-TGCCTCCGGTTCTGAAGGTGTTCTTGT-3'
`5'-CCGGTTCTGAAGGTGTTCTTG-3'
`5'-TCCGGTTCTGAAGGTGTTCTTG-3'
`5'-CTCCGGTTCTGAAGGTGTTCTTG-3'
`5'-CCTCCGGTTCTGAAGGTGTTCTTG-3'
`5'-GCCTCCGGTTCTGAAGGTGTTCTTG-3'
`5'-TGCCTCCGGTTCTGAAGGTGTTCTTG-3'
`5'-CCGGTTCTGAAGGTGTTCTT-3'
`5'-TCCGGTTCTGAAGGTGTTCTT-3'
`5'-CTCCGGTTCTGAAGGTGTTCTT-3'
`5'-CCTCCGGTTCTGAAGGTGTTCTT-3'
`5'-GCCTCCGGTTCTGAAGGTGTTCTT-3'
`5'-TGCCTCCGGTTCTGAAGGTGTTCTT-3'
`5'-CCGGTTCTGAAGGTGTTCT-3'
`5'-TCCGGTTCTGAAGGTGTTCT-3'
`5'-CTCCGGTTCTGAAGGTGTTCT-3'
`5'-CCTCCGGTTCTGAAGGTGTTCT-3'
`5'-GCCTCCGGTTCTGAAGGTGTTCT-3'
`5'-TGCCTCCGGTTCTGAAGGTGTTCT-3'
`5'-CCGGTTCTGAAGGTGTTC-3'
`5'-TCCGGTTCTGAAGGTGTTC-3'
`5'-CTCCGGTTCTGAAGGTGTTC-3'
`
`SEQ ID NO:
`SEQ ID NO: 2
`SEQ ID NO: 3
`SEQ ID NO: 4
`SEQ ID NO: 5
`SEQ ID NO: 6
`SEQ ID NO: 7
`SEQ ID NO: 8
`SEQ ID NO: 9
`SEQ ID NO: 10
`SEQ ID NO: 11
`SEQ ID NO: 12
`SEQ ID NO: 13
`SEQ ID NO: 14
`SEQ ID NO: 15
`SEQ ID NO: 16
`SEQ ID NO: 17
`SEQ ID NO: 18
`SEQ ID NO: 19
`SEQ ID NO: 20
`SEQ ID NO: 21
`SEQ ID NO: 22
`SEQ ID NO: 23
`SEQ ID NO: 24
`SEQ ID NO: 25
`SEQ ID NO: 26
`SEQ ID NO: 27
`SEQ ID NO: 28
`SEQ ID NO: 29
`SEQ ID NO: 30
`SEQ ID NO: 31
`SEQ ID NO: 32
`SEQ ID NO: 33
`SEQ ID NO: 34
`
`

`

`SEQ ID NO: 35
`SEQ ID NO: 36
`SEQ ID NO: 37
`
`5'-CCTCCGGTTCTGAAGGTGTTC-3'
`5'-GCCTCCGGTTCTGAAGGTGTTC-3'
`5'-TGCCTCCGGTTCTGAAGGTGTTC-3'
`
`36-56
`36-57
`36-58
`[0025]
`
`An oligomer of the present invention preferably comprises
`
`a nucleotide sequence complementary to any one of the
`sequences consisting of nucleotides 32-56 (SEQ ID NO: 11), 33-
`56 (SEQ ID NO: 17), 34-56 (SEQ ID NO: 23), 35-56 (SEQ ID NO:
`29) or 36-56 (SEQ ID NO: 35) from the 5' end of exon 53 of the
`human dystrophin gene.
`
`Preferably, an oligomer of the present invention
`comprises a nucleotide sequence complementary to either of the
`sequences comprising nucleotides 32-56 (SEQ ID NO: 11) or
`36-56 (SEQ ID NO: 35) from the 5' end of exon 53 in the human
`dystrophin gene.
`
`[0026]
`
`The term "enables skipping of exon 53 in the human
`dystrophin gene" means that, in the case of a DMD patient with
`deletion of exon 52 for example, by binding an oligomer of the
`present invention to a site corresponding to exon 53 of the
`transcript (for example, pre-mRNA) of the human dystrophin
`gene, when said transcript is spliced the nucleotide sequence
`corresponding to the 5' end of exon 54 is spliced to the 3’
`side of the nucleotide sequence corresponding to the 3’ end of
`exon 51, so that no codon frame-shift occurs and mature mRNA
`is formed.
`
`Therefore, as long as an oligomer of the present
`invention enables skipping of exon 53 of the dystrophin gene,
`it does not need to have a nucleotide sequence 100%
`complementary to the target sequence. For example, an oligomer
`of the present invention may include 1-3, 1-2, or 1 nucleotide
`non-complementary to the target sequence.
`
`[0027]
`
`In this connection, "binding" above means that when an
`oligomer of the present invention is mixed with a transcript
`of the human dystrophin gene, the two hybridize under
`physiological conditions to form double-stranded nucleic acid.
`Here, "under physiological conditions" means conditions of pH,
`salt composition and temperature adjusted to mimic the in vivo
`
`

`

`environment. For example, conditions of 25-40°C, and
`preferably 37°C, pH 5-8, and preferably pH 7.4, and sodium
`chloride concentration 150 mM.
`
`[0028]
`
`It is possible to confirm whether skipping of exon 53 in
`the human dystrophin gene is produced by introducing the
`oligomer of the present invention into dystrophin-expressing
`cells (for example, human rhabdomyosarcoma cells), amplifying
`the region surrounding exon 53 of mRNA of the dystrophin gene
`from the total RNA of the dystrophin-expressing cells by RT-
`PCR and performing nested PCR or sequence analysis on said
`PCR-amplified product. By recovering human dystrophin gene
`mRNA from the test cells, measuring the quantity of
`polynucleotide "A" in the band for said mRNA in which exon 53
`is skipped, and the quantity of polynucleotide "B" in the
`bands in which exon 53 has not been skipped, skipping
`efficiency can be calculated from the measured values for "A"
`and "B" by the following equation.
`
`
`Skipping efficiency (%) = A/(A + B) × 100
`
`
`[0029]
`
`Oligomers of the present invention include oligomers in
`
`which nucleotides are the monomers, having a length of 18-28
`nucleotides, with the nucleic acid monomers being linked by
`phosphate ester bonds, in other words oligonucleotides
`(hereinafter referred to as "oligonucleotides of the present
`invention"). These nucleotides can be either ribonucleotides
`or deoxyribonucleotide, and are preferably ribonucleotides. An
`oligonucleotide of the present invention can be synthesized
`easily by using different types of automated synthesizer (for
`example, AKTA oligopilot plus 10/100 (GE Healthcare)).
`Alternatively, the synthesis can also be entrusted to a third-
`party organization (for example, Promega Inc., or Takara Co.),
`etc.
`[0030]
`
`In addition, in order to heighten nuclease-resistance,
`
`etc., or stability in the body, an oligonucleotide of the
`present invention can have at least one modification of the
`
`

`

`ribose or phosphate backbone constituting the nucleotides
`thereof.
`Such
`modifications
`include,
`for
`example,
`modifications of the ribose 2' position and modifications of
`other sites on the sugar, and modifications of the phosphate
`backbone. Modification of the ribose 2' position includes, for
`example, replacement of the 2'-OH of ribose by OR, R, R' OR,
`SH, SR, NH2, NHR, NR2, N3, CN, F, Cl, Br or I. R here
`represents an alkyl or aryl. R as an alkyl is preferably a
`straight-chain or branched-chain C1-6 alkyl. 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. This alkyl may optionally be
`substituted; examples of substituents here include halogens,
`alkoxy groups, cyano and nitro. There may be 1-3 of these
`substituents. The halogen(s) here can be fluorine, chlorine,
`bromine or iodine. The alkyl can be an alkyl mentioned above.
`The alkoxy can be a straight-chain or branched-chain C1-6
`alkoxy,
`examples
`include
`methoxy,
`ethoxy,
`n-propoxy,
`isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-
`pentyloxy, isopentyloxy, n-hexyloxy and isohexyloxy, etc. A
`C1-3 alkoxy is particularly preferred. R as aryl is preferably
`a C6-10 aryl. Specific examples include phenyl, α-naphthyl and
`β-naphthyl. Phenyl is particularly preferred. R' represents an
`alkylene. R' as an alkylene is preferably a straight-chain or
`branched-chain C1-6 alkylene. Specific examples include
`methylene,
`ethylene,
`trimethylene,
`tetramethylene,
`pentamethylene,
`hexamethylene,
`2-(ethyl)trimethylene
`and
`1-(methyl)tetramethylene. The modifications of other sites on
`ribose include, for example, replacement of O at the 4'
`position by S, and constitution of artificial nucleic acid
`with a fixed configuration by bridging the 2' and 4' positions
`with -O-CH2-. Examples of such artificial nucleic acids include
`LNA (locked nucleic acid) or ENA (2'-O,4'-C-ethylene-bridged
`nucleic acids), but are not limited to these.
`
`[0031]
`
`Modifications of the phosphate backbone includes, for
`example, modification by replacing the phosphodiester bond
`with a phosphorothioate bond, a phosphorodithioate bond, alkyl
`phosphonate bond, a phosphoroamidate bond or a boranophosphate
`
`

`

`bond (Enya et al.: Bioorganic & Medicinal Chemistry, 2008, 18,
`9154-9160) (see for example, Japan Domestic Re-Publications of
`WO2006/129594 A1 and WO2006/038608 A1).
`
`[0032]
`
`Preferably, an oligonucleotide of the present invention
`is an oligomer in which the monomer is -OMe-S-RNA, with the
`2'-OH group of ribose replaced by -OMe (Me: methyl), and -O-
`in the phosphate group replaced by -S.
`
`[Formula 4]
`
`
`(In the formula, Me indicates methyl, and Base indicates any
`base or modified base of adenine, guanine, hypoxanthine,
`cytosine or uracil.)
`
`[0033]
`
`Alternatively, an oligomer of the present invention can
`be an oligomer in which the monomers are nucleotide analogues.
`Nucleotide
`monomers
`include,
`for
`example,
`morpholino
`(compounds described in WO91/09033 A1) and peptide nucleic
`acids (PNA).
`
`[0034]
`
`A morpholino has the partial structure below. A
`morpholino differs from a nucleotide in that there is a
`morpholino ring rather than ribose. Methylene is bound to the
`4' position on the morpholino ring. The 5' position is linked
`with a neighboring morpholino nitrogen atom via a group the
`main chain of which is constituted of 1-3 atoms.
`
`[Formula 5]
`
`

`

`
`guanine,
`adenine,
`indicates
`Base
`formula,
`the
`(In
`hypoxanthine, cytosine, thymine or uracil, or a modified
`base.)
`
`[0035]
`
`The group, the main chain of which is constituted of 1-3
`atoms, is represented by any of the formulae below.
`
`[Formula 6]
`
`
`
`[Formula 7]
`
`
`
`[Formula 8]
`
`
`
`[Formula 9]
`
`
`
`
`
`
`
`

`

`
`
`[Formula 10]
`
`
`
`
`-CH2R1,
`represents
`X
`groups,
`bound
`the
`In
`
`O-CH2R1, -S-CH2R1, -NR2R3 or F; R1 represents H or methyl, or an
`atom group which does not affect binding with the target
`sequence R2 and R3, can be mutually different, and are each R1
`or an alicyclic group or aromatic group; Y1 is O, S, CH2 or
`NR1; Y2 represents O, S or CH2; Y3 is O, S or NR1 and Z is O or
`S.
`
`A1.
`[0036]
`
`In some embodiments, the oligomer of the present
`
`invention is a phosphorodiamidate represented by the formula
`below.
`
`[Formula 11]
`
`For the details of morpholino structure see WO1992/009033
`
`

`

`
`(In the formula, Me indicates methyl, and Base indicates
`adenine, guanine, hypoxanthine, cytosine, thymine or uracil,
`or a modified base.)
`
`[0037]
`
`Because the monomer in phosphorodiamidate morpholino
`oligomers (PMO) has the structure above, PMOs can form Watson-
`Crick base-pairs with other phosphorodiamidate morpholino
`chains or natural nucleotides (Corey, D. R. and Abrams, J. M.
`(2001) Genome Biol, 2, reviews 1015.1-1015.3). PMOs can
`maintain an antisense effect in cells for a long time, and
`because -O- in the phosphate group is replaced by NMe2, PMOs
`are electrically neutral, and therefore, they have the
`advantage that they are not prone to non-specific binding with
`biomolecules other than the target gene. For processes for
`synthesizing PMOs see the following document: WP2009/064471.
`
`[0038]
`In an oligomer of the present invention the bases can be
`
`natural bases (adenine, guanine, hypoxanthine, cytosine,
`thymine or uracil), or they can be modified bases. Modified
`bases include, for example, pyridin-4-one, pyridin-2-one,
`phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil,
`dihydrouridine, naphthyl, aminophenyl, 5-alkycytidine (for
`example, 5-methylcytidine), 5-alkyuridine (for example,
`ribothymidine),
`5-halouridine
`(5-bomouridine),
`6-aza-
`pyrimidine, 6-alkylpyrimidine, (6-methyluridine), propene,
`queosine,
`2-thiouridine,
`4-thiouridine,
`wybutosine,
`wybutoxosine, 4-acetyltidine, 5-(carboxyhydroxymethyl)uridine,
`5-carboxymethylaminomethyl-2-thiouridine 5-carboxymethylamino-
`methyluridine,
`β-D-galactosylqueosine,
`1-methyladenosine,
`1-methylhypoxanthine, 2,2-dimethylguanosine, 3-methylcytosine,
`2-methyladenosine,
`2-methylguanosine,
`N6-methyladenosine,
`
`

`

`5-methoxyaminomethyl-2-thiouridine,
`7-methylguanosine,
`5-methylaminomethyluridine,
`5-methylcarbonylmethyluridine,
`5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-
`isopentenyladenosine, β-D-galactmannosylqueosine, uridine-5-
`oxyacetic acid, 2-thiocytidine, threonine derivatives, purine,
`2,6-diaminopurine,
`2-diaminopurine,
`isoguanine,
`indole,
`imidazole, andxanthine, but are not limited to these.
`
`[0039]
`In addition, the 5'-methylene bound to ribose or the
`
`morpholino ring in the 5'-terminal nucleic acid residue of an
`oligomer of the present invention can be modified with any of
`the groups below.
`
`[Formula 12]
`
`
`
`[Formula 13]
`
`
`
`
`
`[Formula 14]
`
`
`
`or
`
`[0040]
`
`2. Pharmaceutical composition
`
`Oligomers of the present invention enable skipping of
`exon 53 with higher efficiency than antisense oligomers in the
`
`
`
`

`

`prior art. Therefore, it is predicted that administration to
`DMD patients of a pharmaceutical composition which includes an
`oligomer of the present invention will enable highly efficient
`alleviation of the signs and symptoms of muscular dystrophy.
`For