(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION
`TREATY (PCT)
`
`PCT/JP2011/070318
`31 August 2011 (31.08.2011)
`Japanese
`Japanese
`
`
`Publication
`International
`(10)
`Number
`WO 2012/029986 A1
`
`Intellectual
`World
`(19)
`Property Organization
`(43) International
`Publication Date
`8 Mar. 2012 (08.03.2012)
`
`(51) International Patent Classification:
`C12N 15/113 (2010.01) A61P 21/04 (2006.01)
`
`A61K 31/7125 (2006.01) C07H 21/04 (2006.01)
`
`(21) International Application Number:
`(22) International Filing Date:
`(25) International Filing Language:
`(26) International Publication Language:
`(30) Priority Data:
`JP
`
`Application 2010-196032 1 Sep. 2010 (01.09.2010)
`(71) Applicant (for all designated States except US): NIPPON SHINYAKU CO.,
`LTD. [JP/JP]; 14, Nishinosho-Monguchi-cho, Kisshoin, Minami-ku, Kyoto
`601-8550 (JP). NATIONAL CENTER OF NEUROLOGY AND PSYCHIATRY [JP/JP];
`4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8551 (JP).
`(72) Inventor; and
`(75) Inventor/Applicant (USA only): WATANABE, Naoki [JP/JP]; Rubio II-402,
`Sakura 1-chome, Tsukuba-shi, Ibaraki-ken 305-0003 (JP). SATOU, Youhei
`[JP/JP]; 1-13-5 Minamihibarigaoka, Takarazuka-shi, Hyogo 665-0811
`(JP); TAKEDA, Shin'ichi [JP/JP]; National Center of Neurology and
`Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8551 (JP). NAGATA,
`Tetsuya [JP/JP]; National Center of Neurology and Psychiatry, 4-1-1
`Ogawa-Higashi, Kodaira, Tokyo 187-8551 (JP).
`(74) Agent: KOBAYASHI, Hiroshi et al.; Abe, Ikubo & Katayama. 9F Fukuoka
`Building, 2-8-7 Yaesu, Chuo-ku, Tokyo 104-0028 (JP).
`(81) Designated States (unless otherwise indicated, for every kind of
`national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA,
`BB, BG, BH, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK,
`DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU,
`ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS,
`LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO,
`NZ, OM, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC, SD, SE, SG, SK,
`SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC,
`VN, ZA, ZM, ZW.
`(84) Designated States (unless otherwise indicated, for every kind of
`regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ,
`NA, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasia (AM, AZ, BY, KG, KZ, MD, RU,
`TJ, TM), Europe (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR,
`GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT,
`RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, ML, MR, NE, SN, TD, TG).
`Additional Published Documents
`-International Search Report (PCT Article 21 (3))
`-Sequence listings presented as separate part of the description
` (Rule 5.2(a))
`
`

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`(54) Title: ANTISENSE NUCLEIC ACID
`
` Figure 1
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`(57) Abstract: The object of the present invention is to offer
`drugs which bring about highly efficient skipping of the 53rd
`exon of the human dystrophin gene. The present invention
`offers oligomers which enable skipping of the 53rd exon of the
`human dystrophin gene.
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`SPECIFICATION
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`Title of the Invention
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`Antisense nucleic acid
`
`Technical Field
`
`The present invention relates to antisense oligomers
`which enable skipping of exon 53 of the human dystrophin gene,
`and to pharmaceutical compositions which include said
`oligomers.
`
`Background Art
`
`Duchenne muscular dystrophy (DMD) is the most frequent
`form of hereditary progressive muscle atrophy, affecting one
`in ca 3500 newborn males. Although motor functions are
`substantially 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 therapeutic agent is strongly desired.
`
`DMD is known to be caused by a mutation in 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 synthesized by
`transcription from DNA to an mRNA precursor, and then removal
`of introns by splicing. 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
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`response occurs, promoting fibrosis so that the muscle cells
`cannot readily be regenerated.
`
`Becker muscular dystrophy (BMD) is also caused by a
`mutation in 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 dystrophin 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.
`
`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 dystrophin protein with partially restored
`function (Non-Patent Document 2). The partial amino acid
`sequence which is a target for exon skipping is 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
`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.
`
`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
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`splicing mechanism, so that exon skipping can also be induced
`by targeting the ESE.
`
`Because mutations in 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).
`
` 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 1-4; Non-Patent
`Document 5). However, a technique for highly efficient
`skipping of exon 53 has yet to be established.
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`Patent Document 1: WO 2006/000057 A1
`Patent Document 2: WO 2004/048570 A1
`Patent Document 3: US 2010/0168212 A1
`Patent Document 4: WO 2010/048586 A1
`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
`
`
`Disclosure of the Invention
`
`Given the situation described above, an antisense
`oligomer that strongly induces skipping of exon 53 of the
`dystrophin gene, and therapeutic agents for muscular dystrophy
`which include such an oligomer are desired.
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` As a result of detailed studies of the structure of the
`
`dystrophin gene, 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.
`
`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 of 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 bond 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 an alkyl or aryl and R’ indicates an
`alkylene).
`[5] An antisense oligomer according to [3] or [4] above,
`wherein the phosphate bond of at least one nucleotide
`constituting the oligonucleotide is any one selected from a
`set comprising a phosphorothioate bond, a phosphorodithioate
`bond, an alkylphosphonate bond, a phosphoroamidate bond and a
`boranophosphate bond.
`[6] An antisense oligomer according to [1] above, which is a
`morpholino oligomer.
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`[7] An antisense oligomer according to [6] above which is a
`phosphoroamidate morpholino oligomer.
`[8] An antisense oligomer according to any one of [1]-[7]
`above, wherein the 5' end is any one of the groups in chemical
`formulae (1) to (3) below:
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`[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 permissible salt or hydrate thereof.
`
`An antisense oligomer of the present invention can induce
`
`skipping of exon 53 of the human 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.
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`Brief Description of the Drawings
`
`Figure 1: is a graph showing the efficiency of skipping
`of exon 53 of the human dystrophin gene in human rhabdo-
`myosarcoma cell line (RD cells).
`
`Figure 2: is a graph showing the efficiency of skipping
`of exon 53 of the human 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 human dystrophin gene in fibroblasts from a
`human DMD patient (5017 cells), induced to differentiate into
`muscle cells by introducing the human myoD gene.
`
`Figure 4: is a graph showing the efficiency of skipping
`of exon 53 of the human dystrophin gene in fibroblasts from a
`human DMD patient (with deletion of exons 45-52), induced to
`differentiate into muscle cells by introducing the human myoD
`gene.
`
`Figure 5: is a graph showing the efficiency of skipping
`of exon 53 of the human dystrophin gene in fibroblasts from a
`human DMD patient (with deletion of exons 48-52), induced to
`differentiate into muscle cells by introducing the human myoD
`gene.
`
`Figure 6: is a graph showing the efficiency of skipping
`of exon 53 of the human dystrophin gene in fibroblasts from a
`human DMD patient (with deletion of exons 48-52), induced to
`differentiate into muscle cells by introducing the human myoD
`gene.
`
`Figure 7: is a graph showing the efficiency of skipping
`of exon 53 of the human dystrophin gene in fibroblasts from a
`human DMD patient (with deletion of exons 45-52 or deletion of
`exons 48-52), induced to differentiate into muscle cells by
`introducing the human myoD gene.
`
`Figure 8: is a graph showing the efficiency of skipping
`of exon 53 of the human dystrophin gene in fibroblasts from a
`human DMD patient (with deletion of exons 45-52), induced to
`differentiate into muscle cells by introducing the human myoD
`gene.
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`Figure 9: is a graph showing the efficiency of skipping
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`of exon 53 (2'-OMe-S-RNA) of the human dystrophin gene in
`human rhabdomyosarcoma cells (RD cells).
`
`Figure 10: is a graph showing the efficiency of skipping
`of exon 53 (2'-OMe-S-RNA) of the human dystrophin gene in
`human rhabdomyosarcoma cells (RD cells).
`
`Figure 11: is a graph showing the efficiency of skipping
`of exon 53 (2'-OMe-S-RNA) of the human dystrophin gene in
`human rhabdomyosarcoma cells (RD cells).
`
`Figure 12: is a graph showing the efficiency of skipping
`of exon 53 (2'-OMe-S-RNA) of the human dystrophin gene in
`human rhabdomyosarcoma cells (RD cells).
`
`Figure 13: is a graph showing the efficiency of skipping
`of exon 53 (2,-OMe-S-RNA) of the human dystrophin gene in
`human rhabdomyosarcoma cells (RD cells).
`
`Figure 14: is a graph showing the efficiency of skipping
`of exon 53 (2,-OMe-S-RNA) of the human dystrophin gene in
`human rhabdomyosarcoma cells (RD cells).
`
`Figure 15: is a graph showing the efficiency of skipping
`of exon 53 (2'-OMe-S-RNA) of the human dystrophin gene in
`human rhabdomyosarcoma cells (RD cells).
`
`Figure 16: is a graph showing the efficiency of skipping
`of exon 53 (2'-OMe-S-RNA) of the human dystrophin gene in
`human rhabdomyosarcoma cells (RD cells).
`
`Figure 17: is a graph showing the efficiency of skipping
`of exon 53 (2'-OMe-S-RNA) of the human dystrophin gene in
`human rhabdomyosarcoma cells (RD cells).
`
`Figure 18: is a graph showing the efficiency of skipping
`of exon 53 of the human dystrophin gene in human
`rhabdomyosarcoma cells (RD cells) at different oligomer
`concentrations.
`
`Figure 19: is a graph showing the efficiency of skipping
`of exon 53 of the human dystrophin gene in human
`rhabdomyosarcoma cells (RD cells) at different oligomer
`concentrations.
`
`Best Mode for Carrying Out the Invention
`
`The present invention is described in detail below. The
`embodiments described below are presented as examples in order
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`to describe the present invention, and do not imply that the
`present invention is limited these embodiments. The present
`invention may be carried out in various ways provided that
`they do not depart from the gist of the invention.
`
`It should be noted that all of the literature, laid-open
`patent applications, patent documents and other patent
`literature cited in the present description are included in
`this description for reference. The present description also
`embraces the contents of the description and drawings in the
`Japanese Patent Application (No. 2010-196032) filed 1
`September 2010, which serves as a basis for claiming a right
`of priority.
`
`1. Antisense oligomers
`
` The present invention offers antisense oligomers
`(hereinafter referred to as "oligomers of the present
`invention") which enable skipping of exon 53 of the human
`dystrophin
`gene,
`comprising
`a
`nucleotide
`sequence
`complementary to any one of the sequences (hereinafter also
`referred to as "target 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 of the human dystrophin gene.
`
`[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 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 dystrophin gene is a mere 14 kb, and said
`coding region is dispersed within the human 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
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`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.
`
`The oligomers of the present invention are created in
`
`order to modify protein encoded by a DMD dystrophin gene into
`a BMD dystrophin protein by skipping of exon 53 of the human
`dystrophin gene. 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;
` (b) a polynucleotide comprising a nucleotide sequence having
`at least 90% homology with the nucleotide sequence of SEQ ID
`NO: 1.
`
`
`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.
`
`In this description, the term "complementary nucleotide
`
`sequence" is not limited to nucleotide sequences that form
`
`In the present description, "polynucleotide" means DNA or
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`Watson-Crick pairs with the nucleotide sequence in question,
`and includes nucleotide sequences which form wobble base pairs.
`In this connection, Watson-Crick pair are base pairs in which
`hydrogen bonds are formed between adenine-thymine, adenine-
`uracil and guanine-cytosine; and Wobble base pairs are base
`pairs
`in
`which
`hydrogen
`bonds
`are
`formed
`between
`guanine-uracil, inosine-uracil, inosine-adenine and inosine-
`cytosine. In addition, a "complementary nucleotide sequence"
`does not have to have 100% complementarity with the target
`nucleotide sequence, and can include, for example, 1-3, 1-2,
`or 1 non-complementary nucleotide.
`
`In this description, the term "stringent conditions" can
`
`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-HCI (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.
`
`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
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`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).
`
`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 homology search software BLAST, using the
`default parameters.
`
`Homology between nucleotide sequences can 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). 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 analysed 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.
`
`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
`Target sequence in
`exon 53
`31-53
`31-54
`
`Complementary nucleotide sequence
`5'-CCGGTTCTGAAGGTGTTCTTGTA-3'
`5'-TCCGGTTCTGAAGGTGTTCTTGTA-3'
`
`SEQ ID NO:
`SEQ ID NO: 2
`SEQ ID NO: 3
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`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
`36-58
`
`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'
`5'-CCTCCGGTTCTGAAGGTGTTC-3'
`5'-GCCTCCGGTTCTGAAGGTGTTC-3'
`5'-TGCCTCCGGTTCTGAAGGTGTTC-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
`
`
`The oligomer of the present invention preferably
`
`comprises a nucleotide sequence complementary to any one of
`the sequences consisting of nucleotides 32-56, 33-56, 34-56,
`35-56 or 36-56 from the 5' end of exon 53 of the human
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`dystrophin gene (for example, SEQ ID NO: 11, SEQ ID NO: 17,
`SEQ ID NO: 23, SEQ ID NO: 29 or SEQ ID NO: 35).
`
`Preferably, the oligomer of the present invention
`comprises a nucleotide sequence complementary to either of the
`sequences comprising nucleotides 32-56 or 36-56 from the 5'
`end of exon 53 of the human dystrophin gene, (for example, SEQ
`ID NO: 11 or SEQ ID NO: 35).
`
`The term "enables skipping of exon 53 of 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.
`
`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.
`
`Whether or not skipping of exon 53 of the human
`
`dystrophin gene is produced can be confirmed 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 human
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`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
`
`
`Oligomers of the present invention include, for example,
`
`oligonucleotides, morpholino oligomers, or peptide nucleic
`acid (PNA) oligomers, having a length of 18-28 nucleotides. A
`length of 21-25 nucleotides is preferred, and morpholino
`oligomers are preferred.
`
`Aforementioned oligonucleotides (hereinafter referred to
`
`as "oligonucleotides of the present invention") are oligomers
`of the present invention having nucleotides as constituent
`units; these nucleotides can be ribonucleotides, deoxyribo-
`nucleotides or modified nucleotides.
`
`A modified nucleotide is a ribonucleotide or deoxyribo-
`nucleotide in which the constituent nucleobase, sugar moiety
`and/or phosphate bond is/are modified.
`
`Nucleobases include, for example, adenine, guanine,
`hypoxanthine, cytosine, thymine, uracil and modified such
`bases. Examples of modified such bases include, but are not
`limited to, pseudouracil, 3-methyluracil, dihydrouracil, 5-
`alkylcytosines (for example, 5-methylcytosine), 5-alkyluracils
`(for example, 5-ethyluracil), 5-halouracils (5-bromouracil),
`6-azapyrimidine, 6-alkylpyrimidines (6-methyluracil), 2-thio-
`uracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydroxy-
`methyl)uracil, 5’-carboxymethylaminomethyl-2-thiouracil, 5-
`carboxymethylaminomethyluracil,
`1-methyladenine,
`1-methyl-
`hypoxanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyl-
`adenine, 2-methylguanine, N6-methyladenine, 7-methylguanine,
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`5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil,
`5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-
`thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxy-
`acetic acid, 2-thiocytosine, purine, 2,6-diaminopurine,
`2-aminopurine, isoguanine, indole, imidazole and xanthine, etc.
`
`Modification of the sugar moiety includes, for example,
`modifications of the ribose 2' position and modifications of
`other sites on the sugar. Modification of the ribose 2'
`position includes 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' represents an alkylene.
`
`