I 1111111111111111 1111111111 1111111111 111111111111111 IIIII IIIIII IIII IIII IIII
`USO 10407 461 B2
`
`c12) United States Patent
`Watanabe et al.
`
`(IO) Patent No.: US 10,407,461 B2
`*Sep.10,2019
`(45) Date of Patent:
`
`(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, Tsuknba (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, Kodaira-shi, 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.
`
`This patent is subject to a terminal dis(cid:173)
`claimer.
`
`(21) Appl. No.: 16/364,451
`
`(22) Filed:
`
`Mar. 26, 2019
`
`(65)
`
`Prior Publication Data
`
`US 2019/0211050 Al
`
`Jul. 11, 2019
`
`Related U.S. Application Data
`
`(63) Continuation of application No. 15/619,996, filed on
`Jun. 12, 2017, which is a continuation of application
`No. 14/615,504, filed on Feb. 6, 2015, now Pat. No.
`9,708,361, which is a 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)
`(2010.01)
`(2006.01)
`(2006.01)
`
`Int. Cl.
`C07H 21104
`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
`6,727,355 B2
`8,084,601 B2
`8,455,636 B2
`8,871,918 B2
`9,024,007 B2
`9,994,851 B2
`10,227,590 B2
`10,266,827 B2
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`2010/0168212 Al
`2012/0190728 Al
`2013/0072541 Al
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`
`FOREIGN PATENT DOCUMENTS
`
`CA
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`JP
`JP
`JP
`JP
`WO
`
`6/2004
`2507125 Al
`1054058 Al
`11/2000
`1160318 A2
`12/2001
`1191097 Al
`3/2002
`1191098 A2
`3/2002
`1568769 Al
`8/2005
`2206781 A2
`7/2010
`2602322 Al
`6/2013
`2594640 Bl
`12/2015
`2602322 Bl
`3/2016
`3404100 Al
`11/2018
`2000-325085 A
`11/2000
`2002-10790 A
`1/2002
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`11/2002
`6406782 B2
`10/2018
`WO-02/24906 Al
`3/2002
`(Continued)
`
`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
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`(Continued)
`
`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.
`2 Claims, 19 Drawing Sheets
`Specification includes a Sequence Listing.
`
`

`

`US 10,407,461 B2
`Page 2
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`(56)
`
`References Cited
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`FOREIGN PATENT DOCUMENTS
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`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`WO-03/095647 A2
`WO-2004/048570 Al
`WO-2004/083432 Al
`WO-2004/083446 A2
`WO-2006/000057 Al
`WO-2006/017522 A2
`WO-2006/112705 A2
`WO-2007/135105 Al
`WO-2008/036127 A2
`WO-2009/054725 A2
`WO-2009/139630 A2
`WO-2010/048586 Al
`WO-2010/050801 Al
`WO-2010/050802 A2
`WO-2010/123369 Al
`WO-2011/057350 Al
`WO-2012/109296 Al
`WO-2012/150960 Al
`WO-2013/112053 Al
`WO-2014/007620 A2
`WO-2014/100714 Al
`WO-2014/144978 A2
`WO-2014/153220 A2
`WO-2014/153240 A2
`WO-2016/025339 A2
`WO-2017/059131 Al
`WO-2017 /062835 A2
`WO-20 l 7 /205496 Al
`WO-2017/205513 Al
`WO-2017/205879 A2
`WO-2017/205880 Al
`WO-2017/213854 Al
`WO-2017/205879 A3
`WO-2018/005805 Al
`WO-2018/091544 Al
`WO-2018/118599 Al
`WO-2018/118627 Al
`WO-2018/118662 Al
`WO-2019/046755 Al
`WO-2019/067975 Al
`WO-2019/067979 Al
`WO-2019/067981 Al
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`11/2003
`6/2004
`9/2004
`9/2004
`1/2006
`2/2006
`10/2006
`11/2007
`3/2008
`4/2009
`11/2009
`4/2010
`5/2010
`5/2010
`10/2010
`5/2011
`8/2012
`11/2012
`8/2013
`1/2014
`6/2014
`9/2014
`9/2014
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`4/2017
`4/2017
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`11/2017
`11/2017
`11/2017
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`

`

`US 10,407,461 B2
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`
`

`

`U.S. Patent
`U.S Patent
`
`Sep.10,2019
`Sep. 10, 2019
`
`Sheet 1 of 19
`Sheet 1 of 19
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`US 10,407,461 B2
`US 10,407,461 132
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`U.S. Patent
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`Sep.10,2019
`
`Sheet 2 of 19
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`US 10,407,461 B2
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`Figure 2
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`

`

`U.S. Patent
`U.S. Patent
`
`Sep.10,2019
`Sep. 10, 2019
`
`Sheet 3 of 19
`Sheet 3 of 19
`
`US 10,407,461 B2
`US 10,407,461 132
`
`Figure 3
`Figure 3
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`No.12
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`Sarepta Exhibit 1001, Page 6 of 65
`
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`Sep.10,2019
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`

`U.S. Patent
`
`Sep.10,2019
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`US 10,407,461 B2
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`

`

`US 10,407,461 B2
`
`1
`ANTISENSE NUCLEIC ACIDS
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`This is a Continuation of copending application Ser. No.
`15/619,996, filed Jun. 12, 2017, which is a Continuation of
`application Ser. No. 14/615,504, filed Feb. 6, 2015 (now
`U.S. Pat. No. 9,708,361 issued Jul. 18, 2017), which is a
`Continuation of application Ser. No. 13/819,520, filed Apr.
`10, 2013 (now U.S. Pat. No. 9,079,934 issued Jul. 14, 2015),
`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, all of which are incorpo(cid:173)
`rated by reference in their entireties.
`
`SEQUENCE LISTING
`
`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
`
`2
`when compared to DMD. In many cases, its onset 1s m
`adulthood. Differences in clinical symptoms between DMD
`and BMD are considered to reside in whether the reading
`frame for amino acids on the translation of dystrophin
`5 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
`acid reading frame so that the expression of functional
`dystrophin protein is abolished, whereas in BMD the dys-
`lO 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
`15 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-
`The instant application contains a Sequence Listing which
`has been submitted in ASCII format via EFS-Web and is 20 ping, will be lost. For this reason, the dystrophin protein
`expressed by this treatment becomes shorter than normal
`hereby incorporated by reference in its entirety. Said ASCII
`one but since the amino acid reading frame is maintained,
`on Apr.
`4,
`2019
`is
`named
`copy,
`created
`the function to stabilize muscle cells is partially retained.
`sub209658_0001_04_US_586696_ST25.txt and is 25,034
`Consequently, it is expected that exon skipping will lead
`bytes in size.
`25 DMD to the similar symptoms to that of BMD which is
`milder. The exon skipping approach has passed the animal
`tests using mice or dogs and now is currently assessed in
`clinical trials on human DMD patients.
`The skipping of an exon can be induced by binding of
`30 antisense nucleic acids targeting either 5' or 3' splice site or
`both sites, or exon-internal sites. An exon will only be
`included in the mRNA when both splice sites thereof are
`recognized by the spliceosome complex. Thus, exon skip(cid:173)
`ping can be induced by targeting the splice sites with
`35 antisense nucleic acids. Furthermore, the binding of an SR
`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-
`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
`45 Wilton, et al., University of Western Australia (Non-Patent
`Document 3), and the antisense nucleic acids which induce
`exon skipping for 39 exons were produced by Annemieke
`Aartsma-Rus, et al., Netherlands (Non-Patent Document 4).
`It is considered that approximately 8% of all DMD
`50 patients may be treated by skipping the 53rd exon (herein(cid:173)
`after referred to as "exon 53"). In recent years, a plurality of
`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-
`55 ever, a technique for skipping exon 53 with a high efficiency
`has not yet been established.
`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
`
`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- 40
`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
`DMD, leading to a large influx of calcium ions into muscle 60
`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
`mutation in the dystrophin gene. The symptoms involve 65
`muscle weakness accompanied by atrophy of muscle but are
`typically mild and slow in the progress of muscle weakness,
`
`

`

`US 10,407,461 B2
`
`3
`Non-Patent Document 3: Wilton S. D., e t 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) 5
`Neuromuscular Disorders, vol. 20, no. 2, p. 102-10
`
`DISCLOSURE OF THE INVENTION
`
`Under the foregoing circumstances, antisense oligomers 10
`that strongly induce exon 53 skipping in the dystrophin gene
`and muscular dystrophy therapeutics comprising oligomers
`thereof have been desired.
`As a result of detailed studies of the structure of the
`dystrophin gene, the present inventors have found that exon 15
`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
`precursor (hereinafter referred to as "pre-mRNA") in the
`dystrophin gene with antisense oligomers. Based on this 20
`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 25
`nucleotide sequence complementary to any one of the
`sequences consisting of the 31st to the 53rd, the 31st 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
`the 54th, the 32nd to the 55th, the 32nd to the 56th, the 32nd 30
`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
`the 56th, the 34th to the 57th, the 34th to the 58th, the 35th 35
`to the 53rd, the 35th to the 54th, the 35th to the 55th, the 35th
`to the 56th, the 35th to the