I 1111111111111111 11111 1111111111 11111 111111111111111 IIIII IIIIII IIII IIII IIII
`US009708361B2
`
`c12) United States Patent
`Watanabe et al.
`
`(IO) Patent No.:
`(45) Date of Patent:
`
`US 9,708,361 B2
`Jul. 18, 2017
`
`(54) ANTISENSE NUCLEIC ACIDS
`
`(56)
`
`References Cited
`
`(71) Applicants:NIPPON SHINYAKU CO., LTD.,
`Kyoto-shi, Kyoto (JP); NATIONAL
`CENTER OF NEUROLOGY AND
`PSYCHIATRY, Kodaira-shi, Tokyo
`(JP)
`
`(72)
`
`Inventors: Naoki Watanabe, Tsuknba (JP); Youhei
`Satou, Tsuknbu (JP); Shin'ichi Takeda,
`Kodaira (JP); Tetsuya Nagata, Kodaira
`(JP)
`
`(73) Assignees: NIPPON SHINYAKU CO., LTD.,
`Kyoto-shi, Kyoto (JP); NATIONAL
`CENTER OF NEUROLOGY AND
`PSYCHIATRY, Tokyo (JP)
`
`( *) Notice:
`
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by O days.
`
`(21) Appl. No.: 14/615,504
`
`(22) Filed:
`
`Feb. 6, 2015
`
`(65)
`
`Prior Publication Data
`
`US 2015/0166995 Al
`
`Jun. 18, 2015
`
`Related U.S. Application Data
`
`(63) Continuation of application No. 13/819,520, filed as
`application No. PCT/JP2011/070318 on Aug. 31,
`2011, now Pat. No. 9,079,934.
`
`(30)
`
`Foreign Application Priority Data
`
`Sep. 1, 2010
`
`(JP) ................................. 2010-196032
`
`(51)
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2010.01)
`(2006.01)
`(2006.01)
`
`Int. Cl.
`C07H 21102
`C07H 21104
`A61K 31170
`C12N 15111
`C12N 151113
`C07H 21100
`Cl2N 5100
`(52) U.S. Cl.
`CPC ............. C07H 21104 (2013.01); C07H 21100
`(2013.01); C12N 151111 (2013.01); C12N
`151113 (2013.01); Cl2N 2310111 (2013.01);
`Cl2N 2310/315 (2013.01); Cl2N 2310/3145
`(2013.01); Cl2N 2310/321 (2013.01); Cl2N
`2310/3525 (2013.01); Cl2N 2320/33 (2013.01)
`( 58) Field of Classification Search
`None
`See application file for complete search history.
`
`U.S. PATENT DOCUMENTS
`
`6,653,467 Bl
`2010/0130591 Al
`2010/0168212 Al
`2012/0190728 Al
`2013/0109091 Al
`
`11/2003 Matsuo et al.
`5/2010 Sazani et al.
`7/2010 Popplewell et al.
`7/2012 Bennett et al.
`5/2013 Baker et al.
`
`FOREIGN PATENT DOCUMENTS
`
`JP
`WO
`WO
`WO
`WO
`WO
`
`2002-10790
`WO-2004/048570 Al
`WO-2006/000057 Al
`WO-2008/036127 A2
`WO-2010/048586 Al
`WO-2011/057350 Al
`
`1/2002
`6/2004
`1/2006
`3/2008
`4/2010
`5/2011
`
`OTHER PUBLICATIONS
`
`Linda J. Popplewell et al., "Design of Phosphorodiamidate
`Morpholino Oligomers (PMOs) for the Induction of Exon Skipping
`of the Human DMD Gene," Mo!. Ther., vol. 17, No. 3, Mar. 2009,
`pp. 554-561.
`Linda J. Popplewell et al., "Comparative analysis of antisense
`oligonucleotide sequences targeting exon 53 of the human DMD
`gene: Implications for future clinical trials," Neuromuscular Disor(cid:173)
`ders, vol. 20, No. 2, Feb. 2010, pp. 102-110.
`Annemieke Aartsma-Rus et al., "Targeted exon skipping as a
`potential gene correction therapy for Duchenne muscular dystro(cid:173)
`phy," Neuromuscular Disorders, vol. 12, 2002, pp. S71-S77.
`Steve D. Wilton et al., "Antisense Oligonucleotide-induced Exon
`Skipping Across the Human Dystrophin Gene Transcript," Mo!
`Ther., vol. 15, No. 7, Jul. 2007, pp. 1288-1296.
`Anthony P. Monaco et al., "An Explanation for the Phenotypic
`Differences between Patients Bearing Partial Deletions of the DMD
`Locus," Genomics, 1988; 2, pp. 90-95.
`Masafumi Matsuo, "Duchenne / Becker muscular dystrophy: from
`molecular diagnosis to gene therapy," Brian & Development, 1996;
`18, pp. 167-172.
`International Search Report dated Oct. 11, 2011 in PCT/JP2011/
`070318 filed Aug. 31, 2011.
`Mitrpant, et al., "By-passing the nonsense mutation in the 4cv
`mouse model of muscular dystrophy by induced exon skipping",
`The Journal of Gene Medicine, Jan. 2009, vol. 11, No. 1, pp. 46-56.
`
`Primary Examiner - Sean McGarry
`(74) Attorney, Agent, or Firm - Drinker Biddle & Reath
`LLP
`
`ABSTRACT
`(57)
`The present invention provides an oligomer which effi(cid:173)
`ciently enables to cause skipping of the 53rd exon in the
`human dystrophin gene. Also provided is a pharmaceutical
`composition which causes skipping of the 53rd exon in the
`human dystrophin gene with a high efficiency.
`
`7 Claims, 19 Drawing Sheets
`
`

`

`U.S. Patent
`U.S. Patent
`
`Jul. 18, 2017
`Jul. 18, 2017
`
`Sheet 1 of 19
`Sheet 1 of 19
`
`US 9,708,361 B2
`US 9,708,361 B2
`
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`Sarepta Exhibit 1001, Page 2 of 64
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`

`

`U.S. Patent
`
`Jul. 18, 2017
`
`Sheet 2 of 19
`
`US 9,708,361 B2
`
`Figure 2
`
`1()\
`
`3%
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`1-: l
`
`

`

`U.S. Patent
`
`Jul. 18, 2017
`
`Sheet 3 of 19
`
`US 9,708,361 B2
`
`Figure 3
`
`...................... _____________
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`
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`
`

`

`U.S. Patent
`U.S. Patent
`
`Jul. 18, 2017
`Jul. 18, 2017
`
`Sheet 4 of 19
`Sheet 4 of 19
`
`US 9,708,361 B2
`US 9,708,361 B2
`
`
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`

`

`U.S. Patent
`
`Jul. 18, 2017
`
`Sheet 5 of 19
`
`US 9,708,361 B2
`
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`

`

`U.S. Patent
`
`Jul. 18, 2017
`
`Sheet 6 of 19
`
`US 9,708,361 B2
`
`PMONo,8
`10µM
`
`Dystrophin
`250kDa
`
`

`

`"'""' = N
`-....l = 00 w 0--,
`d r.,;_
`
`_."-0
`
`(PMO No. 3)
`Patient ivith Exon 48-52 Deletion Patient with Exon 48-52 Deletion
`
`(PMO No. 8)
`
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`0 ....
`-....J
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`
`(PMO No. 8)
`Patient with Exon 48-52 Deletion Patient with Exon 45-52 Deletion
`
`(No PMO)
`
`Figure 7
`
`

`

`U.S. Patent
`
`Jul. 18, 2017
`
`Sheet 8 of 19
`
`US 9,708,361 B2
`
`Figure 8
`
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`
`

`

`U.S. Patent
`
`Jul. 18, 2017
`
`Sheet 18 of 19
`
`US 9,708,361 B2
`
`100
`
`i
`
`<'"
`
`'"
`
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`
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`
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`
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`
`Concentration (~tM)
`
`Figure 18
`
`

`

`U.S. Patent
`
`Jul. 18, 2017
`
`Sheet 19 of 19
`
`US 9,708,361 B2
`
`100
`
`....,,....PMO No.8
`....-pMo No.14
`
`......-pMo No.3
`
`0
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`
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`
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`
`Concentration (p.l\i)
`
`]?igu:re 19
`
`

`

`US 9,708,361 B2
`
`1
`ANTISENSE NUCLEIC ACIDS
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`This is a Continuation of copending application Ser. No.
`13/819,520, filed Apr. 10, 2013, which is a PCT National
`Stage of PCT/JP2011/070318 filed Aug. 31, 2011, which
`claims priority to JP Application No, 2010-196032 filed Sep.
`1, 2010.
`
`SEQUENCE LISTING
`
`A Sequence Listing containing SEQ ID NO: 1-123 1s
`incorporated herein by reference.
`
`TECHNICAL FIELD
`
`The present invention relates to an antisense oligomer
`which causes skipping of exon 53 in the human dystrophin
`gene, and a pharmaceutical composition comprising the
`oligomer.
`
`BACKGROUND ART
`
`5
`
`2
`trophin protein that functions, though imperfectly, is pro(cid:173)
`duced because the amino acid reading frame is preserved,
`while a 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 Document 2). The
`amino acid sequence part, which is a target for exon skip-
`10 ping, 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
`15 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 either 5' or 3' splice site or
`20 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 skip(cid:173)
`ping can be induced by targeting the splice sites with
`antisense nucleic acids. Furthermore, the binding of an SR
`25 protein 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 depend-
`30 ing on DMD patients, antisense nucleic acids need to be
`desined based on the site or type of respective genetic
`mutation. In the past, antisense nucleic acids that induce
`exon skipping for all 79 exons were produced by Steve
`Wilton, et al., University of Western Australia (Non-Patent
`Document 3), and the antisense nucleic acids which induce
`35 exon skipping for 39 exons were produced by Amlemieke
`Aartsma-Rus, et al., Netherlands (Non-Patent Document 4).
`It is considered that approximately 8% of all DMD
`patients may be treated by skipping the 53rd exon (herein(cid:173)
`after referred to as "exon 53"). In recent years, a plurality of
`40 research organizations reported on the studies where exon
`53 in the dystrophin gene was targeted for exon skipping
`(Patent Documents 1 to 4; Non-Patent Document 5). How(cid:173)
`ever, a technique for skipping exon 53 with a high efficiency
`has not yet been established.
`45 Patent Document 1: International Publication WO 2006/
`000057
`Patent Document 2: International Publication WO 2004/
`048570
`Patent Document 3: US 2010/0168212
`Patent Document 4: International Publication WO 2010/
`048586
`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: Amlemieke 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
`
`Duchenne muscular dystrophy (DMD) is the most fre(cid:173)
`quent form of hereditary progressive muscular dystrophy
`that affects one in about 3,500 newborn boys. Although the
`motor functions are rarely different from healthy humans in
`infancy and childhood, muscle weakness is observed in
`children from around 4 to 5 years old. Then, muscle weak(cid:173)
`ness progresses to the loss of ambulation by about 12 years
`old and death due to cardiac or respiratory insufficiency in
`the twenties. DMD is such a severe disorder. At present,
`there is no effective therapy for DMD available, and it has
`been strongly desired to develop a novel 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 nucleotide pairs. DNA is transcribed into mRNA
`precursors, and intrans are removed by splicing to synthe(cid:173)
`size mRNA 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. The dystrophin
`gene from patients with DMD contains a mutation and
`hence, the dystrophin protein, which is functional in muscle
`cells, is rarely expressed. Therefore, the structure of muscle
`cells cannot be maintained in the body of the patients with 50
`DMD, leading to a large influx of calcium ions into muscle
`cells. Consequently, an inflammation-like response occurs to
`promote fibrosis so that muscle cells can be regenerated only
`with difficulty.
`Becker muscular dystrophy (BMD) is also caused by a 55
`mutation in the dystrophin gene. The symptoms involve
`muscle weakness accompanied by atrophy of muscle 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 60
`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 Document 1). More specifi(cid:173)
`cally, in DMD, the presence of mutation shifts the amino 65
`acid reading frame so that the expression of functional
`dystrophin protein is abolished, whereas in BMD the dys-
`
`DISCLOSURE OF THE INVENTION
`
`Under the foregoing circumstances, antisense oligomers
`that strongly induce exon 53 skipping in the dystrophin gene
`and muscular dystrophy therapeutics comprising oligomers
`thereof have been desired.
`
`

`

`US 9,708,361 B2
`
`10
`
`4
`-continued
`
`OH
`~
`
`(2)
`
`(3)
`
`[9] The antisense oligomer according to any one of [1] to
`[8] above, consisting of a nucleotide sequence complemen(cid:173)
`tary to the sequences consisting of the 32nd to the 56th or the
`36th to the 56th nucleotides from the 5' end of the 53rd exon
`in the human dystrophin gene.
`[10] The antisense oligomer according to any one of [1]
`to [8] above, consisting of the nucleotide sequence shown by
`any one selected from the group consisting of SEQ ID NOS:
`2 to 37.
`[11] The antisense oligomer according to any one of [1]
`to [8] above, consisting of the nucleotide sequence shown by
`any one selected from the group consisting of SEQ ID NOS:
`11, 17, 23, 29 and 35.
`[12] The antisense oligomer according to any one of [1]
`to [8] above, consisting of the nucleotide sequence shown by
`SEQ ID NO: 11 or 35.
`[13] A pharmaceutical composition for the treatment of
`muscular dystrophy, comprising as an active ingredient the
`antisense oligomer according to any one of [1] to [12] above,
`or a pharmaceutically acceptable salt or hydrate thereof.
`The antisense oligomer of the present invention can
`induce exon 53 skipping in the human dystrophin gene with
`a high efficiency. In addition, the symptoms of Duchenne
`muscular dystrophy can be effectively alleviated by admin(cid:173)
`istering the pharmaceutical composition of the present
`invention.
`
`BRIEF DESCRIPTION OF DRAWINGS
`
`3
`As a result of detailed studies of the structure of the
`dystrophin gene, the present inventors have found that exon
`53 skipping can be induced with a high efficiency by
`targeting the sequence consisting of the 32nd to the 56th
`nucleotides from the 5' end of exon 53 in the mRNA 5
`precursor (hereinafter referred to as "pre-mRNA") in the
`dystrophin gene with antisense oligomers. Based on this
`finding, the present inventors have accomplished the present
`invention.
`That is, the present invention is as follows.
`[1] An antisense oligomer which causes skipping of the
`53rd exon in the human dystrophin gene, consisting of a
`nucleotide sequence complementary to any one of the
`sequences consisting of the 31st to the 53rd, the 31 st to the
`54th, the 31st to the 55th, the 31st to the 56th, the 31st to the
`57th, the 31st to the 58th, the 32nd to the 53rd, the 32nd to 15
`the 54th, the 32nd to the 55th, the 32nd to the 56th, the 32nd
`to the 57th, the 32nd to the 58th, the 33rd to the 53rd, the
`33rd to the 54th, the 33rd to the 55th, the 33rd to the 56th,
`the 33rd to the 57th, the 33rd to the 58th, the 34th to the
`53rd, the 34th to the 54th, the 34th to the 55th, the 34th to 20
`the 56th, the 34th to the 57th, the 34th to the 58th, the 35th
`to the 53rd, the 35th to the 54th, the 35th to the 55th, the 35th
`to the 56th, the 35th to the 57th, the 35th to the 58th, the 36th
`to the 53rd, the 36th to the 54th, the 36th to the 55th, the 36th
`to the 56th, the 36th to the 57th, or the 36th to the 58th 25
`nucleotides, from the 5' end of the 53rd exon in the human
`dystrophin gene.
`[2] The antisense oligomer according to [1] above, which
`is an oligonucleotide.
`[3] The antisense oligomer according to [2] above,
`wherein the sugar moiety and/or the phosphate-binding
`region of at least one nucleotide constituting the oligonucle(cid:173)
`otide is modified.
`[4] The antisense oligomer according to [3] above,
`wherein the sugar moiety of at least one nucleotide consti(cid:173)
`tuting the oligonucleotide is a ribose in which the 2'-OH 35
`group is replaced by any one selected from the group
`consisting of OR, R, R'OR, SiH, SR, NH2 , NHR, NR2 , N3 ,
`CN, F, Cl, Br and I (wherein R is an alkyl or an aryl and R'
`is an alkylene).
`[5] The antisense oligomer according to [3] or [4] above, 40
`wherein the phosphate-binding region of at least one nucleo(cid:173)
`tide constituting the oligonucleotide is any one selected from
`the group consisting of a phosphorothioate bond, a phos(cid:173)
`phorodithioate bond, an alkylphosphonate bond, a phospho-
`ramidate bond and a boranophosphate bond.
`[6] The antisense oligomer according to [1] above, which
`is a morpholino oligomer.
`[7] The antisense oligomer according to [ 6] above, which
`is a phosphorodiamidate morpholino oligomer.
`[8] The antisense oligomer according to any one of [1] to 50
`[7] above, wherein the 5' end is any one of the groups of
`chemical formulae (1) to (3) below:
`
`30
`
`45
`
`FIG. 1 shows the efficiency of exon 53 skipping in the
`human dystrophin gene in human rhabdomyosarcoma cell
`line (RD cells).
`FIG. 2 shows the efficiency of exon 53 skipping in the
`human dystrophin gene in the cells where human myoD
`gene is introduced into human normal tissue-derived fibro(cid:173)
`blasts (TIG-119 cells) to induce differentiation into muscle
`cells.
`FIG. 3 shows the efficiency of exon 53 skipping in the
`(1) 55 human dystrophin gene in the cells where human myoD
`gene is introduced into human DMD patient-derived fibro(cid:173)
`blasts (5017 cells) to induce differentiation into muscle cells.
`FIG. 4 shows the efficiency of exon 53 skipping in the
`human dystrophin gene in the cells where human myoD
`60 gene is introduced into fibroblasts from human DMD patient
`(with deletion of exons 45-52) to induce differentiation into
`muscle cells.
`FIG. 5 shows the efficiency of exon 53 skipping in the
`human dystrophin gene in the cells where human myoD
`65 gene is introduced into fibroblasts from human DMD patient
`(with deletion of exons 48-52) to induce differentiation into
`muscle cells.
`
`oy o~o~o~oH
`
`() I /H3
`
`O=P-N
`I
`\
`o
`CH3
`~
`
`

`

`US 9,708,361 B2
`
`5
`FIG. 6 shows the efficiency of exon 53 skipping in the
`human dystrophin gene in the cells where human myoD
`gene is introduced into fibroblasts from human DMD patient
`(with deletion of exons 48-52) to induce differentiation into
`muscle cells.
`FIG. 7 shows the efficiency of exon 53 skipping in the
`human dystrophin gene in the cells where human myoD
`gene is introduced into fibroblasts from human DMD patient
`(with deletion of exons 45-52 or deletion of exons 48-52) to
`induce differentiation into muscle cells.
`FIG. 8 shows the efficiency of exon 53 skipping in the
`human dystrophin gene in the cells where human myoD
`gene is introduced into fibroblasts from human DMD patient
`(with deletion of exons 45-52) to induce differentiation into
`muscle cells.
`FIG. 9 shows the efficiency of exon 53 skipping (2'-OMe(cid:173)
`S-RNA) in the human dystrophin gene in human rhab(cid:173)
`domyosarcoma cells (RD cells).
`FIG. 10 shows the efficiency of exon 53 skipping (2'(cid:173)
`OMe-S-RNA) in the human dystrophin gene in human
`rhabdomyosarcoma cells (RD cells).
`FIG. 11 shows the efficiency of exon 53 skipping (2'(cid:173)
`OMe-S-RNA) in the human dystrophin gene in human
`rhabdomyosarcoma cells (RD cells).
`FIG. 12 shows the efficiency of exon 53 skipping (2'(cid:173)
`OMe-S-RNA) in the human dystrophin gene in human
`rhabdomyosarcoma cells (RD cells).
`FIG. 13 shows the efficiency of exon 53 skipping (2'(cid:173)
`OMe-S-RNA) in the human dystrophin gene in human
`rhabdomyosarcoma cells (RD cells).
`FIG. 14 shows the efficiency of exon 53 skipping (2'(cid:173)
`OMe-S-RNA) in the human dystrophin gene in human
`rhabdomyosarcoma cells (RD cells).
`FIG. 15 shows the efficiency of exon 53 skipping (2'(cid:173)
`OMe-S-RNA) in the human dystrophin gene in human
`rhabdomyosarcoma cells (RD cells).
`FIG. 16 shows the efficiency of exon 53 skipping (2'(cid:173)
`OMe-S-RNA) in the human dystrophin gene in human 40
`rhabdomyosarcoma cells (RD cells).
`FIG. 17 shows the efficiency of exon 53 skipping (2'(cid:173)
`OMe-S-RNA) in the human dystrophin gene in human
`rhabdomyosarcoma cells (RD cells).
`FIG. 18 shows the efficiency of exon 53 skipping in the
`human dystrophin gene in human rhabdomyosarcoma cells
`(RD cells) at the respective concentrations of the oligomers.
`FIG. 19 shows the efficiency of exon 53 skipping in the
`human dystrophin gene in human rhabdomyosarcoma cells
`(RD cells) at the respective concentrations of the oligomers.
`
`BEST MODE FOR CARRYING OUT THE
`INVENTION
`
`6
`
`1. Antisense Oligomer
`The present invention provides the antisense oligomer
`(hereinafter referred to as the "oligomer of the present
`invention") which causes skipping of the 53rd exon in the
`5 human dystrophin gene, consisting of a nucleotide sequence
`complementary to any one of the sequences (hereinafter also
`referred to as "target sequences") consisting of the 31 st to
`the 53rd, the 31 st to the 54th, the 31 st to the 55th, the 31
`st to the 56th, the 31 st to the 57th, the 31st to the 58th, the
`10 32nd to the 53rd, the 32nd to the 54th, the 32nd to the 55th,
`the 32nd to the 56th, the 32nd to the 57th, the 32nd to the
`58th, the 33rd to the 53rd, the 33rd to the 54th, the 33rd to
`the 55th, the 33rd to the 56th, the 33rd to the 57th, the 33rd
`to the 58th, the 34th to the 53rd, the 34th to the 54th, the 34th
`15 to the 55th, the 34th to the 56th, the 34th to the 57th, the 34th
`to the 58th, the 35th to the 53rd, the 35th to the 54th, the 35th
`to the 55th, the 35th to the 56th, the 35th to the 57th, the 35th
`to the 58th, the 36th to the 53rd, the 36th to the 54th, the 36th
`to the 55th, the 36th to the 56th, the 36th to the 57th, or the
`20 36th to the 58thnucleotides, from the 5' end of the 53rd exon
`in the human dystrophin gene.
`[Exon 53 in Human Dystrophin Gene]
`In the present invention, the term "gene" is intended to
`mean a genomic gene and also include cDNA, mRNA
`25 precursor and mRNA. Preferably, the gene is mRNA pre(cid:173)
`cursor, i.e., pre-mRNA.
`In the human genome, the human dystrophin gene locates
`at locus Xp21.2. The human dystrophin gene has a size of
`3.0 Mbp and is the largest gene among known human genes.
`30 However, the coding regions of the human dystrophin gene
`are only 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 generate
`35 mature mRNA of 14 kb. The nucleotide sequence of human
`wild-type dystrophin gene is known (GenBank Accession
`No. NM_004006).
`The nucleotide sequence of exon 53 in the human wild(cid:173)
`type dystrophin gene is represented by SEQ ID NO: 1.
`The oligomer of the present invention is designed to cause
`skipping of exon 53 in the human dystrophin gene, thereby
`modifying the protein encoded by DMD type of dystrophin
`gene into the BMD type of dystrophin protein. Accordingly,
`exon 53 in the dystrophin gene that is the target of exon
`45 skipping by the oligomer of the present invention includes
`both wild and mutant types.
`Specifically, exon 53 mutants of the human dystrophin
`gene include the polynucleotides defined in (a) or (b) below.
`(a) A polynucleotide that hybridizes under stringent con-
`50 ditions to a polynucleotide consisting of a nucleotide
`sequence complementary to the nucleotide sequence of SEQ
`ID NO: 1; and,
`(b) A polynucleotide consisting of a nucleotide sequence
`having at least 90% identity with the nucleotide sequence of
`55 SEQ ID NO: 1.
`As used herein, the term "polynucleotide" is intended to
`mean DNA or RNA.
`As used herein, the term "polynucleotide that hybridizes
`under stringent conditions" refers to, for example, a poly-
`60 nucleotide obtained by colony hybridization, plaque hybrid(cid:173)
`ization, Southern hybridization or the like, using as a probe
`all or part of a polynucleotide consisting of a nucleotide
`sequence complementary to the nucleotide sequence of, e.g.,
`SEQ ID NO: 1. The hybridization method which may be
`used includes methods described in, for example, "Sam(cid:173)
`brook & Russell, Molecular Cloning: A Laboratory Manual
`Vol. 3, Cold Spring Harbor, Laboratory Press 2001,"
`
`Hereinafter, the present invention is described in detail.
`The embodiments described below are intended to be pre(cid:173)
`sented 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.
`All of the publications, published patent applications,
`patents and other patent documents cited in the specification
`are herein incorporated by reference in their entirety. The
`specification hereby incorporates by reference the contents
`of the specification and drawings in the Japanese Patent 65
`Application (No. 2010-196032) filed Sep. 1, 2010, from
`which the priority was claimed.
`
`

`

`US 9,708,361 B2
`
`7
`"Ausubel, Current Protocols in Molecular Biology, John
`Wiley & Sons 1987-1997," etc.
`As used herein, the term "complementary nucleotide
`sequence" is not limited only to nucleotide sequences that
`form Watson-Crick pairs with target nucleotide sequences,
`but is intended to also include nucleotide sequences which
`form Wobble base pairs. As used herein, the term Watson(cid:173)
`Crick pair refers to a pair of nucleobases in which hydrogen
`bonds are formed between adenine-thymine, adenine-uracil
`or guanine-cytosine, and the term Wobble base pair refers to
`a pair of nucleobases in which hydrogen bonds are formed
`between guanine-uracil, inosine-uracil, inosine-adenine or
`inosine-cytosine. As used herein, the term "complementary
`nucleotide sequence" does not only refers to a nucleotide
`sequence 100% complementary to the target nucleotide
`sequence but also refers to a complementary nucleotide
`sequence that may contain, for example, 1 to 3, 1 or 2, or one
`nucleotide non-complementary to the target nucleotide
`sequence.
`As used herein, the term "stringent conditions" may be
`any of low stringent conditions, moderate stringent condi(cid:173)
`tions or high stringent conditions. The term "low stringent
`conditions" are, for example, 5xSSC, 5xDenhardt's solu(cid:173)
`tion, 0.5% SDS, 50% formamide at 32° C. The term "mod(cid:173)
`erate stringent conditions" are, for example, 5xSSC, 5xDen(cid:173)
`hardt's solution, 0.5% SDS, 50% formamide at 42° C., or
`5xSSC, 1 % SDS, 50 mM Tris-HCl (pH 7 .5), 50% forma(cid:173)
`mide at 42° C. The term "high stringent conditions" are, for
`example, 5xSSC, 5xDenhardt's solution, 0.5% SDS, 50%
`formamide at 50° C. or 0.2xSSC, 0.1 % SDS at 65° C. Under
`these conditions, polynucleotides with higher homology are
`expected to be obtained efficiently at higher temperatures,
`although multiple factors are involved in hybridization strin(cid:173)
`gency 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 hybrid(cid:173)
`ization, for example, anAlkphos Direct Labeling and Detec(cid:173)
`tion System (GE Healthcare) may b