U.S. patent application number 16/999695 was filed with the patent office on 2021-07-22 for antisense nucleic acids.
This patent application is currently assigned to NIPPON SHINYAKU CO., LTD.. The applicant listed for this patent is NATIONAL CENTER OF NEUROLOGY AND PSYCHIATRY, NIPPON SHINYAKU CO., LTD.. Invention is credited to Tetsuya NAGATA, Haruna SEO, Shin'ichi TAKEDA, Naoki WATANABE.
Application Number | 20210222169 16/999695 |
Document ID | / |
Family ID | 1000005492760 |
Filed Date | 2021-07-22 |
United States Patent
Application |
20210222169 |
Kind Code |
A1 |
WATANABE; Naoki ; et
al. |
July 22, 2021 |
ANTISENSE NUCLEIC ACIDS
Abstract
The present invention provides a pharmaceutical agent which
causes skipping of the 55th, 45th, 50th or 44th exon in the human
dystrophin gene with a high efficiency. The present invention
provides an oligomer which efficiently enables to cause skipping of
the 55th, 45th, 50th or 44th exon in the human dystrophin gene.
Inventors: |
WATANABE; Naoki; (Ibaraki,
JP) ; SEO; Haruna; (Tokyo, JP) ; TAKEDA;
Shin'ichi; (Tokyo, JP) ; NAGATA; Tetsuya;
(Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NIPPON SHINYAKU CO., LTD.
NATIONAL CENTER OF NEUROLOGY AND PSYCHIATRY |
Kyoto-shi
Tokyo |
|
JP
JP |
|
|
Assignee: |
NIPPON SHINYAKU CO., LTD.
Kyoto-shi
JP
NATIONAL CENTER OF NEUROLOGY AND PSYCHIATRY
Tokyo
JP
|
Family ID: |
1000005492760 |
Appl. No.: |
16/999695 |
Filed: |
August 21, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15870308 |
Jan 12, 2018 |
10781448 |
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16999695 |
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15339069 |
Oct 31, 2016 |
9890381 |
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15870308 |
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14368307 |
Jun 24, 2014 |
9512424 |
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PCT/JP2012/084295 |
Dec 17, 2012 |
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15339069 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/113 20130101;
C12N 15/111 20130101; C12N 2310/315 20130101; C12N 2310/321
20130101; C12N 2310/314 20130101; C12N 2310/322 20130101; C12N
2310/11 20130101; C12N 2310/3233 20130101; C12N 2320/33
20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; C12N 15/11 20060101 C12N015/11 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 28, 2011 |
JP |
2011-288040 |
Feb 29, 2012 |
JP |
2012-043092 |
Claims
[0560] 1-20. (canceled)
21. An antisense oligomer which causes skipping of the 50th exon in
a human dystrophin gene, consisting of a nucleotide sequence
complementary to any one of the nucleotide sequences consisting of
the 106th to the 126th, the 107th to the 127th, the 108th to the
127th, the 108th to the 128th, or the 109th to the 129th
nucleotides, from the 5' end of the human dystrophin gene's 50th
exon, wherein the 50th exon of the human dystrophin gene consists
of position 1 to 109 of the nucleotide sequence SEQ ID NO: 3, and
wherein the antisense oligomer is a morpholino oligomer, a peptide
nucleic acid (PNA), or an oligonucleotide comprising at least one
nucleotide having a modified sugar moiety and/or a modified
phosphate-binding region.
22. A pharmaceutical composition for the treatment of muscular
dystrophy, comprising as an active ingredient the antisense
oligomer of claim 21, or a pharmaceutically acceptable salt or
hydrate thereof.
23. A method of treating muscular dystrophy, comprising
administering to a patient in need thereof a therapeutically
effective amount of the antisense oligomer of claim 21.
24. The method of claim 23, wherein the antisense oligomer consists
of a complementary sequence to the nucleotide sequences consisting
of the 106th to the 126th or the 107th to the 127th nucleotides,
from the 5' end of the human dystrophin gene's 50th exon.
25. The method of claim 23, wherein the antisense oligomer consists
of the nucleotide sequence selected from the group consisting of
the 4th to the 24th and the 3rd to the 23rd nucleotides of SEQ ID
NO: 7.
26. The method of claim 23, wherein the antisense oligomer consists
of a complementary sequence to the nucleotide sequences consisting
of the 108th to the 127th, the 108th to the 128th, or the 109th to
the 129th nucleotides, from the 5' end of the human dystrophin
gene's 50th exon.
27. The method of claim 23, wherein the antisense oligomer consists
of the nucleotide sequence selected from the group consisting of
the 3rd to the 22nd, the 2nd to the 22nd, or the 1st to the 21st
nucleotides of SEQ ID NO: 7.
28. The method of claim 23, wherein the modified sugar moiety is a
ribose in which the 2'--OH group is replaced by any one selected
from the group consisting of OR, R, R'OR, SH, SR, NH.sub.2, NHR,
NR.sub.2, N.sub.3, CN, F, Cl, Br and I (wherein R is an alkyl or an
aryl and R' is an alkylene).
29. The method of claim 23, wherein the modified phosphate-binding
region is any one selected from the group consisting of a
phosphorothioate bond, a phosphorodithioate bond, an
alkylphosphonate bond, a phosphoramidate bond and a boranophosphate
bond.
30. The method of claim 23, wherein the antisense oligomer is a
morpholino oligomer.
31. The method of claim 30, wherein the antisense oligomer is a
phosphorodiamidate morpholino oligomer.
32. The method of claim 30, wherein the 5' end of the morpholino
oligomer is any one of the groups of chemical formulae (1) to (3)
below: ##STR00024##
33. The method of claim 23, wherein the antisense oligomer is a
PNA.
34. The method of claim 23, wherein the antisense oligomer is
administered intravenously.
35. A method of treating muscular dystrophy, comprising
administering to a patient in need thereof a therapeutically
effective amount of the pharmaceutical composition of claim 22.
36. The method of claim 35, wherein the pharmaceutical composition
is administered intravenously.
37. An antisense oligomer which causes skipping of the 55th exon in
a human dystrophin gene, wherein the base sequence of the antisense
oligomer consists of the base sequence of the 157th to the 177th,
the 157th to the 176th, the 160th to the 181st, the 159th to the
181st, the 159th to the 180th, the 157th to the 178th, the 156th to
the 178th, the 155th to the 178th, the 156th to the 177th, the
155th to the 177th, the 155th to the 176th, the 157th to the 175th,
or the 156th to the 175th nucleotides of SEQ ID NO: 5, and wherein
the antisense oligomer is a morpholino oligomer, a peptide nucleic
acid (PNA), or an oligonucleotide comprising at one nucleotide
having a modified sugar moiety and/or a modified phosphate-binding
region.
38. A pharmaceutical composition for the treatment of muscular
dystrophy, comprising as an active ingredient the antisense
oligomer of claim 37, or a pharmaceutically acceptable salt or
hydrate thereof.
39. A method of treating muscular dystrophy, comprising
administering to a patient in need thereof a therapeutically
effective amount of the antisense oligomer of claim 37.
40. The method of claim 39, wherein the modified sugar moiety is a
ribose in which the 2'--OH group is replaced by any one selected
from the group consisting of OR, R, R'OR, SH, SR, NH.sub.2, NHR,
NR.sub.2, N.sub.3, CN, F, Cl, Br and I (wherein R is an alkyl or an
aryl and R' is an alkylene).
41. The method of claim 39, wherein the modified phosphate-binding
region is any one selected from the group consisting of a
phosphorothioate bond, a phosphorodithioate bond, an
alkylphosphonate bond, a phosphoramidate bond and a boranophosphate
bond.
42. The method of claim 39, wherein the antisense oligomer is a
morpholino oligomer.
43. The method of claim 42, wherein the antisense oligomer is a
phosphorodiamidate morpholino oligomer.
44. The method of claim 42, wherein the 5' end of the morpholino
oligomer is any one of the groups of chemical formulae (1) to (3)
below: ##STR00025##
45. The method of claim 39, wherein the antisense oligomer is a
PNA.
46. The method of claim 39, wherein the antisense oligomer is
administered intravenously.
47. A method of treating muscular dystrophy, comprising
administering to a patient in need thereof a therapeutically
effective amount of the pharmaceutical composition of claim 38.
48. The method of claim 47, wherein the pharmaceutical composition
is administered intravenously.
Description
TECHNICAL FIELD
[0001] The present invention relates to an antisense oligomer which
causes skipping of exon 55, 45, 50 or 44 in the human dystrophin
gene, and a pharmaceutical composition comprising the oligomer.
BACKGROUND ART
[0002] Duchenne muscular dystrophy (DMD) is the most frequent 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 weakness 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.
[0003] 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 introns are removed by
splicing to synthesize 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 cells. Consequently, an inflammation-like
response occurs to promote fibrosis so that muscle cells can be
regenerated only with difficulty.
[0004] Becker muscular dystrophy (BMD) is also caused by a 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 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 specifically, in DMD, the presence of
mutation shifts the amino acid reading frame so that the expression
of functional dystrophin protein is abolished, whereas in BMD the
dystrophin protein that functions, though imperfectly, is produced
because the amino acid reading frame is preserved, while a part of
the exons are deleted by the mutation.
[0005] 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 skipping, will be lost. For this reason, the
dystrophin protein expressed by this treatment becomes shorter than
normal one but since the amino acid reading frame is maintained,
the function to stabilize muscle cells is partially retained.
Consequently, it is expected that exon skipping will lead DMD to
the similar symptoms to that of BMD which is milder. The exon
skipping approach has passed the animal tests using mice or dogs
and now is currently assessed in clinical trials on human DMD
patients.
[0006] The skipping of an exon can be induced by binding of
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 skipping can be induced by
targeting the splice sites with 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.
[0007] Since a mutation of the dystrophin gene may vary depending
on DMD patients, antisense nucleic acids need to be designed based
on the site or type of respective genetic mutation. 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 exon skipping for 39 exons were produced by Annemieke
Aartsma-Rus, et al., Netherlands (Non-Patent Document 4).
[0008] It is considered that approximately 20% of all DMD patients
may be treated by skipping the 55th, the 45th, the 50th and the
44th exons (hereinafter referred to as "exon 55", "exon 45", "exon
50" and "exon 44", respectively). In recent years, several research
organizations reported on the studies where exon 55, 45, 50 or 44
in the dystrophin gene was targeted for exon skipping (Patent
Documents 1 to 8). However, a technique for skipping exon 55, 45,
50 or 44 with a high efficiency has not yet been established.
[0009] Patent Document 1: International Publication WO 2006/000057
[0010] Patent Document 2: International Publication WO 2004/048570
[0011] Patent Document 3: US Unexamined Patent Application
Publication US 2010/0168212 [0012] Patent Document 4: International
Publication WO2010/048586 [0013] Patent Document 5: International
Publication WO 2004/083446 [0014] Patent Document 6: International
Publication WO 2010/050801 [0015] Patent Document 7: International
Publication WO 2009/139630 [0016] Non-Patent Document 1: Monaco A.
P. et al., Genomics 1988; 2: p. 90-95 [0017] Non-Patent Document 2:
Matsuo M., Brain Dev 1996; 18: p. 167-172 [0018] Non-Patent
Document 3: Wilton S. D., et al., Molecular Therapy 2007: 15: p.
1288-96 [0019] Non-Patent Document 4: Annemieke Aartsma-Rus et al.
(2002) Neuromuscular Disorders 12: S71-S77 [0020] Non-Patent
Document 5: Linda J. Popplewell et al., (2010) Neuromuscular
Disorders, vol. 20, no. 2, p. 102-10
DISCLOSURE OF THE INVENTION
[0021] Under the foregoing circumstances, antisense oligomers that
strongly induce skipping of exon 55, exon 45, exon 50 or exon 44 in
the dystrophin gene and muscular dystrophy therapeutics comprising
oligomers thereof have been desired.
[0022] As a result of detailed studies of the structure of the
dystrophin gene, the present inventors have found that exon 55
skipping can be induced with a high efficiency by antisense
oligomers which target the sequence consisting of around the 1st to
the 21st, the 11th to the 31st, and the 14th to the 34th
nucleotides from the 5' end of exon 55 in the mRNA precursor
(hereinafter referred to as "pre-mRNA") in the dystrophin gene with
antisense oligomers.
[0023] The present inventors have also found that exon 45 skipping
can be induced with a high efficiency by antisense oligomers which
target the sequence consisting of around the 1st to the 25th and
the 6th to the 30th nucleotides from the 5' end of exon 45 in the
pre-mRNA in the dystrophin gene with antisense oligomers.
[0024] Furthermore, the present inventors have found that exon 50
skipping can be induced with a high efficiency by antisense
oligomers which target the sequence consisting of around the 107th
to the 127th nucleotides from the 5' end of exon 50 in the pre-mRNA
in the dystrophin gene with antisense oligomers.
[0025] Additionally, the present inventors have also found that
exon 44 skipping can be induced with a high efficiency by antisense
oligomers which target the sequence consisting of around the 11th
to the 32nd and the 26th to the 47th nucleotides from the 5' end of
exon 44 in the pre-mRNA in the dystrophin gene with antisense
oligomers.
[0026] Based on this finding, the present inventors have
accomplished the present invention.
[0027] That is, the present invention is as follows.
[0028] [1] An antisense oligomer which causes skipping of the 55th
exon in the human dystrophin gene, consisting of a nucleotide
sequence complementary to any one of the nucleotide sequences
consisting of the -2nd to the 19th, the -2nd to the 20th, the -2nd
to the 21st, the -2nd to the 22nd, the -2nd to the 23rd, the -1st
to the 19th, the -1st to the 20th, the -1st to the 21st, the -1st
to the 22nd, the -1st to the 23rd, the 1st to the 19th, the 1st to
the 20th, the 1st to the 21st, the 1st to the 22nd, the 1st to the
23rd, the 2nd to the 19th, the 2nd to the 20th, the 2nd to the
21st, the 2nd to the 22nd, the 2nd to the 23rd, the 3rd to the
19th, the 3rd to the 20th, the 3rd to the 21st, the 3rd to the
22nd, the 3rd to the 23rd, the 9th to the 29th, the 9th to the
30th, the 9th to the 31st, the 9th to the 32nd, the 9th to the
33rd, the 10th to the 29th, the 10th to the 30th, the 10th to the
31st, the 10th to the 32nd, the 10th to the 33rd, the 11th to the
29th, the 11th to the 30th, the 11th to the 31st, the 11th to the
32nd, the 11th to the 33rd, the 12th to the 29th, the 12th to the
30th, the 12th to the 31st, the 12th to the 32nd, the 12th to the
33rd, the 13th to the 29th, the 13th to the 30th, the 13th to the
31st, the 13th to the 32nd, the 13th to the 33rd, the 12th to the
34th, the 12th to the 35th, the 12th to the 36th, the 13th to the
34th, the 13th to the 35th, the 13th to the 36th, the 14th to the
32nd, the 14th to the 33rd, the 14th to the 34th, 14th to the 35th,
the 14th to the 36th, the 15th to the 32nd, the 15th to the 33rd,
the 15th to the 34th, the 15th to the 35th, the 15th to the 36th,
the 16th to the 32nd, the 16th to the 33rd, the 16th to the 34th,
the 16th to the 35th, or the 16th to the 36th nucleotides, from the
5' end of the 55th exon in the human dystrophin gene.
[0029] [2] An antisense oligomer which causes skipping of the 45th
exon in the human dystrophin gene, consisting of a nucleotide
sequence complementary to any one of the nucleotide sequences
consisting of the -3rd to the 19th, the -3rd to the 20th, the -3rd
to the 21st, the -3 rd to the 22nd, the -3rd to the 23rd, the -2nd
to the 19th, the -2nd to the 20th, the -2nd to the 21st, the -2nd
to the 22nd, the -2nd to the 23rd, the -1st to the 19th, the -1st
to the 20th, the -1 st to the 21st, the -1st to the 22nd, the -1st
to the 23rd, the 1st to the 19th, the 1st to the 20th, the 1st to
the 21st, the 1st to the 22nd, the 1st to the 23rd, the 2nd to the
19th, the 2nd to the 20th, the 2nd to the 21st, the 2nd to the
22nd, the 2nd to the 23rd, the -2nd to the 24th, the -2nd to the
25th, the -2nd to the 26th, the -2 nd to the 27th, the -1st to the
24th, the -1st to the 25th, the -1st to the 26th, the -1st to the
27th, the 1st to the 24th, the 1st to the 25th, the 1st to the
26th, the 1st to the 27th, the 2nd to the 24th, the 2nd to the
25th, the 2nd to the 26th, the 2nd to the 27th, the 3rd to the
23rd, the 3rd to the 24th, the 3rd to the 25th, the 3rd, to the
26th, the 3rd to the 27th, the 4th to the 28th, the 4th to the
29th, the 4th to the 30th, the 4th to the 31st, the 4th to the
32nd, the 5th to the 28th, the 5th to the 29th, the 5th to the
30th, the 5th to the 31st, the 5th to the 32nd, the 6th to the
28th, the 6th to the 29th, the 6th to the 30th, the 6th to the
31st, the 6th to the 32nd, the 7th to the 28th, the 7th to the
29th, the 7th to the 30th, the 7th to the 31st, the 7th to the
32nd, the 8th to the 28th, the 8th to the 29th, the 8th to the
30th, the 8th to the 31st, or the 8th to the 32nd nucleotides, from
the 5' end of the 45th exon in the human dystrophin gene.
[0030] [3] An antisense oligomer which causes skipping of the 50th
exon in the human dystrophin gene, consisting of a nucleotide
sequence complementary to any one of the nucleotide sequences
consisting of the 105th to the 125th, the 105th to the 126th, the
105th to the 127th, the 105th to the 128th, the 105th to the 129th,
the 106th to the 125th, the 106th to the 126th, the 106th to the
127th, the 106th to the 128th, the 106th to the 129th, the 107th to
the 125th, the 107th to the 126th, the 107th to the 127th, the
107th to the 128th, the 107th to the 129th, the 108th to the 125th,
the 108th to the 126th, the 108th to the 127th, the 108th to the
128th, the 108th to the 129th, the 109th to the 125th, the 109th to
the 126th, the 109th to the 127th, the 109th to the 128th, or the
109th to the 129th nucleotides, from the 5' end of the 50th exon in
the human dystrophin gene.
[0031] [4] An antisense oligomer which causes skipping of the 44th
exon in the human dystrophin gene, consisting of a nucleotide
sequence complementary to any one of the nucleotide sequences
consisting of the 9th to the 30th, 9th to the 31st, the 9th to the
32nd, the 9th to the 33rd, the 9th to the 34th, the 10th to the
30th, the 10th to the 31st, the 10th to the 32nd, the 10the to the
33rd, the 10th to the 34th, the 11th to the 30th, the 11th to the
31st, the 11th to the 32nd, the 11th to the 33rd, the 11th to the
34th, the 12th to the 30th, the 12th to the 31st, the 12th to the
32nd, the 12th to the 33rd, the 12th to the 34th, the 13th to the
30th, the 13th to the 31st, the 13th to the 32nd, the 13th to the
33rd, the 13th to the 34th, the 24th to the 45th, the 24th to the
46th, the 24th to the 47th, the 24th to the 48th, the 24th to the
49th, the 25th to the 45th, the 25th to the 46th, the 25th to the
47th, the 25th to the 48th, the 25th to the 49th, the 26th to the
45th, the 26th to the 46th, the 26th to the 47th, the 26th to the
48th, the 26th to the 49th, the 27th to the 45th, the 27th to the
46th, the 27th to the 47th, the 27th to the 48th, the 27th to the
49th, the 28th to the 45th, the 28th to the 46th, the 28th to the
47th, the 28th to the 48th, the 28th to the 49th, the 29th to the
45th, the 29th to the 46th, the 29th to the 47th, the the 29th to
the 48th, or the 29th to the 49th nucleotides, from the 5' end of
the 44th exon in the human dystrophin gene.
[0032] [5] The antisense oligomer according to [1], which consists
of a complementary sequence to the nucleotide sequences consisting
of the 1st to the 21st, the 11th to the 31st, or the 14th to the
34th nucleotides, from the 5' end of the 55th exon in the human
dystrophin gene.
[0033] [6] The antisense oligomer according to [1], consisting of
the nucleotide sequence shown by any one selected from the group
consisting of the 170th to the 190th, the 160th to the 180th, and
the 157th to the 177th nucleotides of SEQ ID NO: 5.
[0034] [7] The antisense oligomer according to [2], which consists
of a complementary sequence to the nucleotide sequences consisting
of the -2nd to the 19th, the 1st to the 21st, the 1st to the 25th,
or the 6th to the 30th nucleotides, from the 5' end of the 45th
exon in the human dystrophin gene.
[0035] [8] The antisense oligomer according to [2], consisting of
the nucleotide sequence shown by any one selected from the group
consisting of the 158th to the 178th, the 156th to the 176th, the
152nd to the 176th, and the 147th to the 171st nucleotides of SEQ
ID NO: 6.
[0036] [9] The antisense oligomer according to [3], which consists
of a complementary sequence to the nucleotide sequences consisting
of the 106th to the 126th or the 107th to the 127th nucleotides,
from the 5' end of the 50th exon in the human dystrophin gene.
[0037] [10] The antisense oligomer according to [3], consisting of
the nucleotide sequence shown by any one selected from the group
consisting of the 4th to the 24th and the 3rd to the 23rd
nucleotides of SEQ ID NO: 7.
[0038] [11] The antisense oligomer according to [4], which consists
of a complementary sequence to the nucleotide sequences consisting
of the 11th to the 32nd, the 25th to the 45th, the 26th to the
46th, the 26th to the 47th or the 27th to the 47th nucleotides,
from the 5' end of the 44th exon in the human dystrophin gene.
[0039] [12] The antisense oligomer according to [4], consisting of
the nucleotide sequence shown by any one selected from the group
consisting of the 117th to the 138th, the 104th to the 124th, the
103rd to the 123rd, the 102nd to the 123rd and the 102nd to the
122nd nucleotides of SEQ ID NO: 8.
[0040] [13] The antisense oligomer according to any one of [1] to
[12], which is an oligonucleotide.
[0041] [14] The antisense oligomer according to [13], wherein the
sugar moiety and/or the phosphate-binding region of at least one
nucleotide constituting the oligonucleotide is modified.
[0042] [15] The antisense oligomer according to [14], wherein the
sugar moiety of at least one nucleotide constituting the
oligonucleotide is a ribose in which the 2'--OH group is replaced
by any one selected from the group consisting of OR, R, R'OR, SH,
SR, NH.sub.2, NHR, NR.sub.2, N.sub.3, CN, F, Cl, Br and I (wherein
R is an alkyl or an aryl and R' is an alkylene).
[0043] [16] The antisense oligomer according to [14] or [15],
wherein the phosphate-binding region of at least one nucleotide
constituting the oligonucleotide is any one selected from the group
consisting of a phosphorothioate bond, a phosphorodithioate bond,
an alkylphosphonate bond, a phosphoramidate bond and a
boranophosphate bond.
[0044] [17] The antisense oligomer according to any one of [1] to
[12], which is a morpholino oligomer.
[0045] [18] The antisense oligomer according to [17], which is a
phosphorodiamidate morpholino oligomer.
[0046] [19] The antisense oligomer according to [17] or [18],
wherein the 5' end is any one of the groups of chemical formulae
(1) to (3) below:
##STR00001##
[0047] [20] A pharmaceutical composition for the treatment of
muscular dystrophy, comprising as an active ingredient the
antisense oligomer according to any one of [1] to [19], or a
pharmaceutically acceptable salt or hydrate thereof.
[0048] The antisense oligomer of the present invention can induce
skipping of exon 55, exon 45, exon 50 or exon 44 in the human
dystrophin gene with high efficiencies. Also, the symptoms of
Duchenne muscular dystrophy can be effectively alleviated by
administering the pharmaceutical composition of the present
invention. In addition, since the antisense oligomer of the present
invention targets only exon sequences in patients, the target
sequences are conserved among individuals compared to the cases
with those targeting sequences in introns. Therefore, the antisense
oligomer of the present invention is capable of achieving excellent
skipping efficiencies regardless of individual varieties (personal
differences). Furthermore, the antisense oligomer of the present
invention has short length of 20 bp or around and has less
probability of containing mutations raised from individual
varieties (interpersonality) e.g. SNP (Single Nucleotide
Polymorphism) in the target sequences compared to conventional
antisense oligomers for DMD treatment having lengths of 25 bp or
so. This feature also helps the antisense oligomer of the present
invention in achieving excellent skipping efficiencies regardless
of individual variety (personal differences). Moreover, the
antisense oligomer of the present invention have less side effects
raised by the induction of cytokines and so on, since antisense
oligomers having shorter chains have less tendency to induce
immunity in general.
[0049] Also, since the antisense oligomer of the present invention
is rather short, the cost of manufacturing is relatively small.
BRIEF DESCRIPTION OF DRAWINGS
[0050] FIG. 1 shows the efficiency of exon 45 skipping by
2'-OMe-S-RNA oligomer in the human dystrophin gene in human
rhabdomyosarcoma cell line (RD cells).
[0051] FIG. 2 shows the efficiency of exon 45 skipping by
2'-OMe-S-RNA oligomer in the human dystrophin gene in human
rhabdomyosarcoma cell line (RD cells).
[0052] FIG. 3 shows the efficiency of exon 45 skipping by PMO in
the human dystrophin gene in human rhabdomyosarcoma cell line (RD
cells).
[0053] FIG. 4 shows the efficiency of exon 45 skipping by PMO in
the human dystrophin gene in human rhabdomyosarcoma cell line (RD
cells).
[0054] FIG. 5 shows the efficiency of exon 45 skipping by PMO in
the human dystrophin gene in the cells where human MyoD gene is
induced into fibroblasts from human DMD patient (GM05017 cells) to
induce differentiation into muscle cells.
[0055] FIG. 6 shows the efficiency of exon 55 skipping by
2'-OMe-S-RNA oligomer in the human dystrophin gene in human
rhabdomyosarcoma cell line (RD cells).
[0056] FIG. 7 shows the efficiency of exon 55 skipping by
2'-OMe-S-RNA oligomer in the human dystrophin gene in human
rhabdomyosarcoma cell line (RD cells).
[0057] FIG. 8 shows the efficiency of exon 55 skipping by PMO in
the human dystrophin gene in human rhabdomyosarcoma cell line (RD
cells).
[0058] FIG. 9 shows the efficiency of exon 44 skipping by
2'-OMe-S-RNA oligomer in the human dystrophin gene in human
rhabdomyosarcoma cell line (RD cells).
[0059] FIG. 10 shows the efficiency of exon 44 skipping by
2'-OMe-S-RNA oligomer in the human dystrophin gene in human
rhabdomyosarcoma cell line (RD cells).
[0060] FIG. 11 shows the efficiency of exon 44 skipping by PMO in
the human dystrophin gene in human rhabdomyosarcoma cell line (RD
cells).
[0061] FIG. 12 shows the efficiency of exon 44 skipping by PMO in
the human dystrophin gene in human rhabdomyosarcoma cell line (RD
cells).
[0062] FIG. 13 shows the efficiency of exon 50 skipping by PMO in
the human dystrophin gene in human rhabdomyosarcoma cell line (RD
cells).
[0063] FIG. 14 shows the efficiency of exon 45 skipping by PMO in
the human dystrophin gene in human rhabdomyosarcoma cell line (RD
cells).
[0064] FIG. 15 shows the efficiency of exon 45 skipping by PMO in
the human dystrophin gene in human rhabdomyosarcoma cell line (RD
cells).
[0065] FIG. 16 shows the efficiency of exon 55 skipping by PMO in
the human dystrophin gene in human rhabdomyosarcoma cell line (RD
cells).
[0066] FIG. 17 shows the efficiency of exon 55 skipping by PMO in
the human dystrophin gene in human rhabdomyosarcoma cell line (RD
cells).
[0067] FIG. 18 shows the efficiency of exon 44 skipping by PMO in
the human dystrophin gene in human rhabdomyosarcoma cell line (RD
cells).
[0068] FIG. 19 shows the efficiency of exon 50 skipping by PMO in
the human dystrophin gene in human rhabdomyosarcoma cell line (RD
cells).
[0069] FIG. 20 shows the efficiency of exon 44 skipping by PMO in
the human dystrophin gene in the fibroblasts from human DMD patient
with deletion of exon 45 (GM05112
[0070] FIG. 21 shows the effect (Western Blotting) of exon 44
skipping by PMO in the human dystrophin gene in the fibroblasts
from human DMD patient with deletion of exon 45 (GM05112
cells).
[0071] FIG. 22 shows the effect (RT-PCR) of exon 50 skipping by PMO
in the human dystrophin gene in the fibroblasts from human DMD
patient with deletion of exon 45 (GM05112 cells).
[0072] FIG. 23 shows the efficiency of exon 50 skipping by PMO in
the human dystrophin gene in the fibroblasts from human DMD patient
with deletion of exon 45 (GM05112 FIG. 24 shows the effect (RT-PCR)
of exon 55 skipping by PMO in the human dystrophin gene in the
fibroblasts from human DMD patient with deletion of exon 45
(GM05112 cells).
[0073] FIG. 25 shows the efficiency of exon 55 skipping by PMO in
the human dystrophin gene in the fibroblasts from human DMD patient
with deletion of exon 45 (GM05112 cells).
[0074] FIG. 26 shows the effect (RT-PCR) of exon 50 skipping by PMO
in the human dystrophin gene in the fibroblasts from human DMD
patient with duplication of exons 8-9 (11-0627 cells).
[0075] FIG. 27 shows the efficiency of exon 50 skipping by PMO in
the human dystrophin gene in the fibroblasts from human DMD patient
with duplication of exons 8-9 (11-0627
[0076] FIG. 28 shows the effect (RT-PCR) of exon 50 skipping by PMO
in the human dystrophin gene in the fibroblasts from human DMD
patient with deletion of exons 51-55 (GM04364 cells).
[0077] FIG. 29 shows the efficiency of exon 50 skipping by PMO in
the human dystrophin gene in the fibroblasts from human DMD patient
with deletion of exons 51-55 (GM04364 cells).
[0078] FIG. 30 shows the effect (RT-PCR) of exon 55 skipping by PMO
in the human dystrophin gene in the fibroblasts from human DMD
patient with deletion of exon 54 (04-035 cells).
[0079] FIG. 31 shows the efficiency of exon 55 skipping by PMO in
the human dystrophin gene in the fibroblasts from human DMD patient
with deletion of exon 54 (04-035 cells).
BEST MODE FOR CARRYING OUT THE INVENTION
[0080] Hereinafter, the present invention is described in detail.
The embodiments described below are intended to be presented by way
of example merely to describe the invention but not limited only to
the following embodiments. The present invention may be implemented
in various ways without departing from the gist of the
invention.
[0081] 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 Application (No.
2011-288040) filed Dec. 28, 2011 and the Japanese Patent
Application (No. 2012-043092) filed Feb. 29, 2012, from which the
priority was claimed.
[0082] Hereinafter, the present invention is described in detail.
The embodiments described below are intended to be presented by way
of example merely to describe the invention but not limited only to
the following embodiments. The present invention may be implemented
in various ways without departing from the gist of the
invention.
[0083] Without description in particular, the amino acid sequence
represents the amino terminus as left and carboxyl terminus as
right, and the base sequence represents the 5' end as left and the
3' end as right.
1. Antisense Oligomer
[0084] The present invention provides the antisense oligomer
(hereinafter referred to as the "exon 55 skipping oligomer of the
present invention") which causes skipping of exon 55 in the human
dystrophin gene, consisting of a nucleotide sequence complementary
to any one of the nucleotide sequences (hereinafter also referred
to as the "exon 55 target sequence") consisting of the -2nd to the
19th, the -2nd to the 20th, the -2nd to the 21st, the -2nd to the
22nd, the 2nd to the 23rd, the -1st to the 19th, the -1st to the
20th, the -1st to the 21st, the -1st to the 22nd, the -1st to the
23rd, the 1st to the 19th, the 1st to the 20th, the 1st to the
21st, the 1st to the 22nd, the 1st to the 23rd, the 2nd to the
19th, the 2nd to the 20th, the 2nd to the 21st, the 2nd to the
22nd, the 2nd to the 23rd, the 3rd to the 19th, the 3rd to the
20th, the 3rd to the 21st, the 3rd to the 22nd, the 3rd to the
23rd, the 9th to the 29th, the 9th to the 30th, the 9th to the
31st, the 9th to the 32nd, the 9the to the 33rd, the 10th to the
29th, the 10th to the 30th, the 10th to the 31st, the 10th to the
32nd, the 10th to the 33rd, the 11th to the 29th, the 11th to the
30th, the 11th to the 31st, the 11th to the 32nd, the 11th to the
33rd, the 12th to the 29th, the 12th to the 30th, the 12th to the
31st, the 12th to the 32nd, the 12th to the 33rd, the 13th to the
29th, the 13th to the 30th, the 13th to the 31st, the 13th to the
32nd, the 13th to the 33rd, the 12th to the 34th, the 12th to the
35th, the 12th to the 36th, the 13th to the 34th, the 13th to the
35th, the 13th to the 36th, the 14th to the 32nd, the 14th to the
33rd, the 14th to the 34th, the 14th to the 35th, the 14th to the
36th, the 15th to the 32nd, the 15th to the 33rd, the 15th to the
34th, the 15th to the 35th, the 15th to the 36th, the 16th to the
32nd, the 16th to the 33rd, the 16th to the 34th, the 16th to the
35th, or the 16th to the 36th nucleotides, from the 5' end of exon
55 in the human dystrophin gene.
[0085] The present invention also provides the antisense oligomer
(hereinafter referred to as the "exon 45 skipping oligomer of the
present invention") which causes skipping of exon 45 in the human
dystrophin gene, consisting of a nucleotide sequence complementary
to any one of the nucleotide sequences (hereinafter also referred
to as the "exon 45 target sequence") consisting of the -3rd to the
19th, the -3rd to the 20th, the -3rd to the 21st, the -3rd to the
22nd, the -3rd to the 23rd, the -2nd to the 19th, the -2nd to the
20th, the -2nd to the 21st, the -2nd to the 22nd, the -2nd to the
23rd, the -1st to the 19th, the -1st to the 20th, the -1st to the
21st, the -1st to the 22nd, the -1st to the 23rd, the 1st to the
19th, the 1st to the 20th, the 1st to the 21st, the 1st to the
22th, the 1st to the 23rd, the 2nd to the 19th, the 2nd to the
20th, the 2nd to the 21st, the 2nd to the 22nd, the 2nd to the
23rd, the -2nd to the 24th, the -2nd to the 25th, the -2nd to the
26th, the -2nd to the 27th, the -1st to the 24th, the -1st to the
25th, the -1st to the 26th, the -1st to the 27th, the 1st to the
24th, the 1st to the 25th, the 1st to the 26th, the 1st to the
27th, the 2nd to the 24th, the 2nd to the 25th, the 2nd to the
26th, the 2nd to the 27th, the 3rd to the 23rd, the 3rd to the
24th, the 3rd to the 25th, the 3rd to the 26th, the 3rd to the
27th, the 4th to the 28th, the 4th to the 29th, the 4th to the
30th, the 4th to the 31th, the 4th to the 32nd, the 5th to the
28th, the 5th to the 29th, the 5th to the 30th, the 5th to the
31st, the 5th to the 32nd, the 6th to the 28th, the 6th to the
29th, the 6th to the 30th, the 6th to the 31st, the 6th to the
32nd, the 7th to the 28th, the 7th to the 29th, the 7th to the
30th, the 7th to the 31st, the 7th to the 32nd, the 8th to the
28th, the 8th to the 29th, the 8th to the 30th, the 8th to the
31st, or the 8th to the 32nd nucleotides, from the 5' end of exon
45 in the human dystrophin gene.
[0086] Additionally, the present invention provides the antisense
oligomer (hereinafter referred to as the "exon 50 skipping oligomer
of the present invention") which causes skipping of exon 50 in the
human dystrophin gene, consisting of a nucleotide sequence
complementary to any one of the nucleotide sequences (hereinafter
also referred to as the "exon 50 target sequence") consisting of
the 105th to the 125th, the 105th to the 126th, the 105th to the
127th, the 105th to the 128th, the 105th to the 129th, the 106th to
the 125th, the 106th to the 126th, the 106th o the 127th, the 106th
to the 128th, the 106th to the 129th, the 107th to the 125th, the
107th to the 126th, the 107th to the 127th, the 107th to the 128th,
the 107th to the 129th, the 108th to the 125th, the 108th to the
126th, the 108th to the 127th, the 108th to the 128th, the 108th to
the 129th, the 109th to the 125th, the 109th to the 126th, the
109th to the 127th, the 109th to the 128th, or the 109th to the
129th nucleotides, from the 5' end of exon 50 in the human
dystrophin gene.
[0087] Furthermore, the present invention provides the antisense
oligomer (hereinafter referred to as the "exon 44 skipping oligomer
of the present invention") which causes skipping of exon 44 in the
human dystrophin gene, consisting of a nucleotide sequence
complementary to any one of the nucleotide sequences (hereinafter
also referred to as the "exon 44 target sequence") consisting of
the 9th to the 30th, the 9th to the 31st, the 9th to the 32nd, the
9th to the 33rd, the 9th to the 34th, the 10th to the 30th, the
10th to the 31st, the 10th to the 32nd, the 10th to the 33rd, the
10th to the 34th, the 11th to the 30th, the 11th to the 31st, the
11th to the 32nd, the 11th to the 33rd, the 11th to the 34th, the
12th to the 30th, the 12th to the 31st, the 12th to the 32nd, the
12th to the 33rd, the 12th to the 34th, the 13th to the 30th, the
13th to the 31st, the 13th to the 32nd, the 13th to the 33rd, the
13th to the 34th, the 24th to the 45th, the 24th to the 46th, the
24th to the 47th, the 24th to the 48th, the 24th to the 49th, the
25th to the 45th, the 25th to the 46th, the 25th to the 47th, the
25th to the 48th, the 25th to the 49th, the 26th to the 45th, the
26th to the 46th, the 26th to the 47th, the 26th to the 48th, the
26th to the 49th, the 27th to the 45th, the 27th to the 46th, the
27th to the 47th, the 27th to the 48th, the 27th to the 49th, the
28th to the 45th, the 28th to the 46th, the 28th to the 47th, the
27th to the 48th, the 27th to the 49th, the 28th to the 45th, the
28th to the 46th, the 28th to the 47th, the 28th to the 48th, the
28th to the 49th, the 29th to the 45th, the 29th to the 46th, the
29th to the 47th, the 29th to the 48th, or the 29th to the 49th
nucleotides, from the 5' end of exon 44 in the human dystrophin
gene.
[0088] Hereinafter, the skipping oligomers of exon 55, 45, 50 and
44 may be collectively referred to as the "oligomers of the present
invention".
[Exon 55, 45, 50 and 44 in Human Dystrophin Gene]
[0089] In the present invention, the term "gene" is intended to
mean a genomic gene and also include cDNA, mRNA precursor and mRNA.
Preferably, the gene is mRNA precursor, i.e. pre-mRNA.
[0090] 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. 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 mature mRNA of 14 kb. The nucleotide sequence of human
wild-type dystrophin gene is known (GeneBank Accession No.
NM_004006).
[0091] The nucleotide sequence consisting of the -2nd to the 190th
nucleotides, from the 5' end of exon 55 in the human wild-type
dystrophin gene is represented by SEQ ID NO: 1. The nucleotide
sequence consisting of the -3rd to the 176th nucleotides, from the
5' end of exon 45 in the human wild-type dystrophin gene is
represented by SEQ ID NO: 2. The nucleotide sequence consisting of
the 1st to the 109th nucleotides, from the 5' end of exon 50 and
the 1st to the 20th nucleotides, from the 5' end of intron 50 in
the human wild-type dystrophin gene is represented by SEQ ID NO:
3.
[0092] The nucleotide sequence consisting of the 1st to the 148th
nucleotides, from the 5' end of exon 44 in the human wild-type
dystrophin gene is represented by SEQ ID NO: 4.
[0093] The oligomer of the present invention is designed to cause
skipping of exon 55, 45, 50 or 44 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 55,
45, 50 and 44 in the dystrophin gene that are the targets of exon
skipping by the oligomer of the present invention include both wild
and mutant types.
[0094] Specifically, exon 55, 45, 50 and 44 mutants of the human
dystrophin gene are the polynucleotides defined in (a) or (b)
below.
[0095] (a) A polynucleotide that hybridizes under stringent
conditions to a polynucleotide consisting of a nucleotide sequence
complementary to the nucleotide sequence of SEQ ID NO: 1 (or a
nucleotide sequence consisting of the 3rd to the 192nd nucleotides
of SEQ ID NO:1), SEQ ID NO: 2 (or a nucleotide sequence consisting
of the 4th to the 179th nucleotides of SEQ ID NO:2), SEQ ID NO: 3
(or a nucleotide sequence consisting of the 1st to the 109th
nucleotides of SEQ ID NO:3), or SEQ ID NO: 4;
[0096] (b) A polynucleotide consisting of a nucleotide sequence
having at least 90% homology with the nucleotide sequence of SEQ ID
NO:1 (or a nucleotide sequence consisting of the 3rd to the 192nd
nucleotides of SEQ ID NO:1), SEQ ID NO: 2 (or a nucleotide sequence
consisting of the 4th to the 179th nucleotides of SEQ ID NO:2), SEQ
ID NO: 3 (or a nucleotide sequence consisting of the 1st to the
109th nucleotides of SEQ ID NO:3), or SEQ ID NO: 4.
[0097] As used herein, the term "polynucleotide" is intended to
mean DNA or RNA.
[0098] As used herein, the term "polynucleotide that hybridizes
under stringent conditions" refers to, for example, a
polynucleotide obtained by colony hybridization, plaque
hybridization, 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 SEQ ID NO: 1 (or a
nucleotide sequence consisting of the 3rd to the 192nd nucleotides
of SEQ ID NO:1), SEQ ID NO: 2 (or a nucleotide sequence consisting
of the 4th to the 179th nucleotides of SEQ ID NO:2), SEQ ID NO: 3
(or a nucleotide sequence consisting of the 1st to the 109th
nucleotides of SEQ ID NO:3), or SEQ ID NO: 4. The hybridization
method which may be used includes methods described in, for
example, "Sambrook & Russell, Molecular Cloning: A Laboratory
Manual Vol. 3, Cold Spring Harbor, Laboratory Press 2001,"
"Ausubel, Current Protocols in Molecular Biology, John Wiley &
Sons 1987-1997," etc.
[0099] 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-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 to 2, or one nucleotide non-complementary to the
target nucleotide sequence.
[0100] As used herein, the term "stringent conditions" may be any
of low stringent conditions, moderate stringent conditions or high
stringent conditions. The term "low stringent condition" is, for
example, 5.times.SSC, 5.times.Denhardt's solution, 0.5% SDS, 50%
formamide at 32.degree. C. The term "moderate stringent condition"
is, for example, 5.times.SSC, 5.times.Denhardt's solution, 0.5%
SDS, 50% formamide at 42.degree. C., or 5.times.SSC, 1% SDS, 50 mM
Tris-HCl (pH 7.5), 50% formamide at 42.degree. C. The term "high
stringent condition" is, for example, 5.times.SSC,
5.times.Denhardt's solution, 0.5% SDS, 50% formamide at 50.degree.
C. or 0.2.times.SSC, 0.1% SDS at 65.degree. C. Under these
conditions, polynucleotides with higher homology are expected to be
obtained efficiently at higher temperatures, although multiple
factors are involved in hybridization stringency including
temperature, probe concentration, probe length, ionic strength,
time, salt concentration and others, and those skilled in the art
may approximately select these factors to achieve similar
stringency.
[0101] When commercially available kits are used for hybridization,
for example, an Alkphos Direct Labelling and Detection System (GE
Healthcare) may be used. In this case, according to the attached
protocol, after cultivation with a labeled probe overnight, the
membrane is washed with a primary wash buffer containing 0.1% (w/v)
SDS at 55.degree. C., thereby detecting hybridized polynucleotides.
Alternatively, when the probe is labeled with digoxigenin (DIG)
using a commercially available reagent (e.g., a PCR Labelling Mix
(Roche Diagnostics), etc.) in producing a probe based on all or
part of the complementary sequence to the nucleotide sequence of
SEQ ID NO: 1 (or a nucleotide sequence consisting of the 3rd to the
192nd nucleotides of SEQ ID NO:1), SEQ ID NO: 2 (or a nucleotide
sequence consisting of the 4th to the 179th nucleotides of SEQ ID
NO:2), SEQ ID NO: 3 (or a nucleotide sequence consisting of the 1st
to the 109th nucleotides of SEQ ID NO:3), or SEQ ID NO: 4,
hybridization can be detected with a DIG Nucleic Acid Detection Kit
(Roche Diagnostics).
[0102] In addition to the polynucleotides described above, other
polynucleotides that can be hybridized include polynucleotides
having 90% or higher, 91% or higher, 92% or higher, 93% or higher,
94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or
higher, 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or
higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or
higher, 99.8% or higher, 99.9% or higher identity with the
polynucleotide of SEQ ID NO: 1 (or a nucleotide sequence consisting
of the 3rd to the 192nd nucleotides of SEQ ID NO:1), SEQ ID NO: 2
(or a nucleotide sequence consisting of the 4th to the 179th
nucleotides of SEQ ID NO:2), SEQ ID NO: 3 (or a nucleotide sequence
consisting of the 1st to the 109th nucleotides of SEQ ID NO:3), or
SEQ ID NO: 4, as calculated by homology search software BLAST using
the default parameters.
[0103] The identity between nucleotide sequences may be determined
using algorithm BLAST (Basic Local Alignment Search Tool) by Karlin
and Altschul (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc.
Natl. Acad. Sci. USA 90: 5873, 1993). Programs called BLASTN and
BLASTX based on the BLAST algorithm have been developed (Altschul S
F, et al: J. Mol. Biol. 215: 403, 1990). When a nucleotide sequence
is sequenced using BLASTN, the parameters are, for example,
score-100 and wordlength=12. When BLAST and Gapped BLAST programs
are used, the default parameters for each program are employed.
[0104] The sequence complementary to the nucleotide sequence
consisting of the -2nd to the 190th nucleotides from the 5' end of
exon 55 is represented by SEQ ID NO: 5. Herein, the nucleotide
sequence consisting of the -2nd to the -1st nucleotides, from the
5' end of exon 55 (the nucleotide sequence consisting of the 1st to
the 2nd nucleotides of SEQ ID NO: 1) represents nucleotide sequence
consisting of 2 nucleotides at the most 3' end downstream of intron
54 which is located between exon 54 and exon 55. More specifically,
the nucleotide sequence of exon 55 is one consisting of the 3rd to
the 192nd nucleotides of SEQ ID NO: 1 and the sequence
complementary to exon 55 is one consisting of the 1st to the 190th
nucleotides of SEQ ID NO: 5.
[0105] Herein, the complementary sequence to the nucleotide
sequences consisting of the -2nd to the 19th, the -2nd to the 20th,
the -2nd to the 21st, the -2nd to the 22nd, the -2nd to the 23rd,
the -1st to the 19th, the -1st to the 20th, the -1 st to the 21st,
the -1st to the 22nd, the -1st to the 23rd, the 1st to the 19th,
the 1st to the 20th, the 1st to the 21st, the 1st to the 22nd, the
1st to the 23rd, the 2nd to the 19th, the 2nd to the 20th, the 2nd
to the 21st, the 2nd to the 22nd, the 2nd to the 23rd, the 3rd to
the 19th, the 3rd to the 20th, the 3rd to the 21st, the 3rd to the
22nd, the 3rd to the 23rd, the 9th to the 29th, the 9th to the
30th, the 9th to the 31st, the 9the to the 32nd, the 9th to the
33rd, the 10th to the 29th, the 10th to the 30th, the 10th to the
31st, the 10th to the 32nd, the 10th to the 33rd, the 11th to the
29th, the 11th to the 30th, the 11th to the 31st, the 11th to the
32nd, the 11th to the 33rd, the 12th to the 29th, the 12th to the
30th, the 12th to the 31st, the 12th to the 32nd, the 12th to the
33rd, the 13th to the 29th, the 13th to the 30th, the 13th to the
31st, the 13th to the 32nd, the 13th to the 33rd, the 12th to the
34th, the 12th to the 35th, the 12th to the 36th, the 13th to the
34th, the 13th to the 35th, the 13th to the 36th, the 14th to the
32nd, the 14th to the 33rd, the 14th to the 34th, the 14th to the
35th, the 14th to the 36th, the 15th to the 32nd, the 15th to the
33rd, the 15th to the 34th, the 15th to the 35th, the 15th to the
36th, the 16th to the 32nd, the 16th to the 33rd, the 16th to the
34th, the 16th to the 35th, or the 16th to the 36th nucleotides,
from the 5' end of the 55th exon in the human dystrophin gene is
respectively identical to the nucleotide sequence consisting of the
172nd to the 192nd, the 171st to the 192nd, the 170th to the 192nd,
the 169th to the 192nd, the 168th to the 192nd, the 172nd to the
191st, the 171st to the 191st, the 170th to the 191st, the 169th to
the 191st, the 168th to the 191st, the 172nd to the 190th, the
171st to the 190th, the 170th to the 190th, the 169th to the 190th,
the 168th to the 190th, the 172nd to the 189th, the 171st to the
189th, the 170th to the 189th, the 169th to the 189th, the 168th to
the 189th, the 172nd to the 188th, the 171st to the 188th, the
170th to the 188th, the 169th to the 188th, the 168th to the 188th,
the 162nd to the 182nd, the 161st to the 182nd, the 160th to the
182nd, the 159th to the 182nd, the 158th to the 182nd, the 162nd to
the 181st, the 161st to the 181st, the 160th to the 181st, the
159th to the 181st, the 158th to the 181st, the 162nd to the 180th,
the 161st to the 180th, the 160th to the 180th, the 159th to the
180th, the 158th to the 180th, the 162nd to the 179th, the 161st to
the 179th, the 160th to the 179th, the 159th to the 179th, the
158th to the 179th, the 162nd to the 178th, the 161st to the 178th,
the 160th to the 178th, the 159th to the 178th, the 158th to the
178th, the 157th to the 179th, the 156th to the 179th, the 155th to
179th, the 157th to the 178th, the 156th to the 178th, the 155th to
the 178th, the 159th to the 177th, the 158th to the 177th, the
157th to the 177th, the 156th to the 177th, the 155th to the 177th,
the 159th to the 176th, the 158th to the 176th, the 157th to the
176th, the 156th to the 176th, the 155th to the 176th, the 159th to
the 175th, the 158th to the 175th, the 157th to the 175th, the
156th to the 175th, or the 155th to the 175th nucleotides of SEQ ID
NO: 5.
[0106] The complementary sequence to the nucleotide sequence
consisting of the -3rd to the 176th nucleotides, from the 5' end of
exon 45 is represented by SEQ ID NO: 6. Herein, the nucleotide
sequence consisting of the -3rd to the -1st nucleotides, from the
5' end of exon 45 (the nucleotide sequence consisting of the 1st to
the 3rd nucleotides of SEQ ID NO: 2) represents the nucleotide
sequence consisting of 3 nucleotides at the most 3' end downstream
of intron 44 which is located between exon 44 and exon 45. More
specifically, the nucleotide sequence of exon 45 is the nucleotide
sequence consisting of the 4th to the 179th nucleotides of SEQ ID
NO: 2 and the complementary sequence to exon 45 is the nucleotide
sequence consisting of the 1st to the 176th nucleotides of SEQ ID
NO: 6.
[0107] Herein, the complementary sequence to the nucleotide
sequences consisting of the -3rd to the 19th, the -3rd to the 20th,
the -3rd to the 21st, the -3 rd to the 22nd, the -3rd to the 23rd,
the -2nd to the 19th, the -2nd to the 20th, the -2nd to the 21st,
the -2nd to the 22nd, the -2nd to the 23rd, the -1 st to the 19th,
the -1st to the 20th, the -1 st to the 21st, the -1st to the 22nd,
-1st to the 23rd, the 1st to the 19th, the 1st to the 20th, the 1st
to the 21st, the 1st to the 22nd, the 1st to the 23rd, the 2nd to
the 19th, the 2nd to the 20th, the 2nd to the 21st, the 2nd to the
22nd, the 2nd to the 23rd, the -2nd to the 24th, the -2nd to the
25th, the -2nd to the 26th, the -2nd to the 27th, the -1st to the
24th, the -1st to the 25th, the -1st to the 26th, the -1st to the
27th, the 1st to the 24th, the 1st to the 25th, the 1st to the
26th, the 1st to the 27th, the 2nd to the 24th, the 2nd to the
25th, the 2nd to the 26th, the 2nd to the 27th, the 3rd to the
23rd, the 3rd to the 24th, the 3rd to the 25th, the 3rd to the
26th, the 3rd to the 27th, the 4th to the 28th, the 4th to the
29th, the 4th to the 30th, the 4th to the 31st, the 4th to the
32nd, the 5th to the 28th, the 5th to the 29th, the 5th to the
30th, the 5th to the 31st, the 5th to the 32nd, the 6th to the
28th, the 6th to the 29th, the 6th to the 30th, the 6th to the
31st, the 6th to the 32nd, the 7th to the 28th, the 7th to the
29th, the 7th to the 30th, the 7th to the 31st, the 7th to the
32nd, the 8th to the 28th, the 8th to the 29th, the 8th to the
30th, the 8th to the 31st, or the 8th to the 32nd nucleotides, from
the 5' end of the 45th exon in the human dystrophin gene is
respectively identical to the nucleotide sequence consisting of the
158th to the 179th, the 157th to the 179th, the 156th to the 179th,
the 155th to the 179th, the 154th to the 179th, the 158th to the
178th, the 157th to the 178th, the 156th to the 178th, the 155th to
the 178th, the 154th to the 178th, the 158th to the 177th, the
157th to the 177th, the 156th to the 177th, the 155th to the 177th,
the 154th to the 177th, the 158th to the 176th, the 157th to the
176th, the 156th to the 176th, the 155th to the 176th, the 154th to
the 176th, the 158th to the 175th, the 157th to the 175th, the
156th to the 175th, the 155th to the 175th, the 154th to the 175th,
the 153rd to the 178th, the 152nd to the 178th, the 151st to the
178th, the 150th to the 178th, the 153rd to the 177th, the 152nd to
the 177th, the 151st to the 177th, the 150th to the 177th, the
153rd to the 176th, the 152nd to the 176th, the 151st to the 176th,
the 150th to the 176th, the 153rd to the 175th, the 152nd to the
175th, the 151st to the 175th, the 150th to the 175th, the 154th to
the 174th, the 153rd to the 174th, the 152nd to the 174th, the
151st to the 174th, the 150th to the 174th, the 149th to the 173rd,
the 148th to the 173rd, the 147th to the 173rd, the 146th to the
173rd, the 147th to the 173rd, the 149th to the 172nd, the 148th to
the 172nd, the 147th to the 172nd, the 146th to the 172nd, the
145th to the 172nd, the 149th to the 171st, the 148th to the 171st,
the 147th to the 171st, the 146th to the 171st, the 145th to the
171st, the 149th to the 170th, the 148th to the 170th, the 147th to
the 170th, the 146th to the 170th, the 145th to the 170th, the
149th to the 169th, the 148th to the 169th, the 147th to the 169th,
the 146th to the 169th or the 145th to the 169th nucleotides of SEQ
ID NO: 6.
[0108] The complementary sequence to the nucleotide sequence
consisting of the 1st to the 109th nucleotides, from the 5' end of
exon 50, and the 1st to the 20th nucleotides, from the 5' end of
intron 50, is represented by SEQ ID NO: 7. Herein, the nucleotide
sequence consisting of the 1st to the 20th nucleotides, from the 5'
end of intron 50 (the nucleotide sequence consisting of the 110th
to the 129th nucleotides of SEQ ID NO: 3) is the nucleotide
sequence consisting of 20 nucleotides at the most 5' end upstream
of intron 50 which is located between exon 50 and exon 51. More
specifically, the nucleotide sequence of exon 50 is the nucleotide
sequence consisting of the 1st to the 109th nucleotides of SEQ ID
NO: 3 and the complementary sequence to exon 50 is the nucleotide
sequence consisting of the 21st to the 129th nucleotides of SEQ ID
NO: 7.
[0109] Herein, the complementary sequence to the nucleotide
sequences consisting of the 105th to the 125th, the 105th to the
126th, the 105th to the 127th, the 105th to the 128th, the 105th to
the 129th, the 106th to the 125th, the 106th to the 126th, the
106th to the 127th, the 106th to the 128th, the 106th to the 129th,
the 107th to the 125th, the 107th to the 126th, the 107th to the
127th, the 107th to the 128th, the 107th to the 129th, the 108th to
the 125th, the 108th to the 126th, the 108th to the 127th, the
108th to the 128th, the 108th to the 129th, the 109th to the 125th,
the 109th to the 126th, the 109th to the 127th, the 109th to the
128th or the 109th to the 129th nucleotides, from the 5' end of the
50th exon in the human dystrophin gene is respectively identical to
the nucleotide sequence consisting of the 5th to the 25th, the 4th
to the 25th, the 3rd to the 25th, the 2nd to the 25th, the 1st to
the 25th, the 5th to the 24th, the 4th to the 24th, the 3rd to the
24th, the 2nd to the 24th, the 1st to the 24th, the 5th to the
23rd, the 4th to the 23rd, the 3rd to the 23rd, the 2nd to the
23rd, the 1st to the 23rd, the 5th to the 22nd, the 4th to the
22nd, the 3rd to the 22nd, the 2nd to the 22nd, the 1st to the
22nd, the 5th to the 21st, the 4th to the 21st, the 3rd to the
21st, the 2nd to the 21st or the 1st to the 21st nucleotides of SEQ
ID NO: 7.
[0110] The complementary sequence to the nucleotide sequence
consisting of the 1st to the 148th nucleotides, from the 5' end of
exon 44 is represented by SEQ ID NO: 8.
[0111] Herein, the complementary sequence to the nucleotide
sequences consisting of the 9th to the 30th, the 9th to the 31st,
the 9th to the 32nd, the 9th to the 33rd, the 9the to the 34th, the
10th to the 30th, the 10th to the 31st, the 10the to the 32nd, the
10th to the 33rd, the 10th to the 34th, the 11th to the 30th, the
11th to the 31st, the 11th to the 32nd, the 11th to the 33rd, the
11th to the 34th, the 12th to the 30th, the 12th to the 31st, the
12th to the 32nd, the 12th to the 33rd, the 12th to the 34th, the
13th to the 30th, the 13th to the 31st, the 13th to the 32nd, the
13th to the 33rd, the 13th to the 34th, the 24th to the 45th, the
24th to the 46th, the 24th to the 47th, the 24th to the 48th, the
24th to the 49th, the 25th to the 45th, the 25th to the 46th, the
25th to the 47th, the 25th to the 48th, the 25th to the 49th, the
26th to the 45th, the 26th to the 46th, the 26th to the 47th, the
26th to the 48th, the 26th to the 49th, the 27th to the 45th, the
27th to the 46th, the 27th to the 47th, the 27th to the 48th, the
27th to the 49th, the 28th to the 45th, the 28th to the 46th, the
28th to the 47th, the 28th to the 48th, the 28th to the 49th, the
29th to the 45th, the 29th to the 46th, the 29th to the 47th, the
29th to the 48th or the 29th to the 49th nucleotides, from the 5'
end of the 44th exon in the human dystrophin gene is respectively
identical to the nucleotide sequence consisting of the 119th to the
140th, the 118th to the 140th, the 117th to the 140th, the 116th to
the 140th, the 115th to the 140th, 119th to the 139th, the 118th to
the 139th, the 117th to the 139th, the 116th to the 139th, the
115th to the 139th, 119th to the 138th, the 118th to the 138th, the
117th to the 138th, the 116th to the 138th, the 115th to the 138th,
119th to the 137th, the 118th to the 137th, the 117th to the 137th,
the 116th to the 137th, the 115th to the 137th, 119th to the 136th,
the 118th to the 136th, the 117th to the 136th, the 116th to the
136th, the 115th to the 136th, the 104th to the 125th, the 103rd to
the 125th, the 102nd to the 125th, the 101th to the 125th, the
100th to the 125th, the 104th to the 124th, the 103rd to the 124th,
the 102nd to the 124th, the 101st to the 124th, the 100th to the
124th, the 104th to the 123rd, the 103rd to the 123rd, the 102nd to
the 123rd, the 101st to the 123rd, the 100th to the 123rd, the
104th to the 122nd, the 103rd to the 122nd, the 102nd to the 122nd,
the 101st to the 122nd, the 100th to the 122nd, the 104th to the
121st, the 103rd to the 121st, the 102nd to the 121st, the 101st to
the 121st, the 100th to the 121st, the 104th to the 120th, the
103rd to the 120th, the 102nd to the 120th, the 101st to the 120th
or the 100th to the 120th nucleotides of SEQ ID NO: 8.
[0112] The relationship between the location in the nucleotide
sequence from the 5' end of the 55th, the 45th, the 50th, and the
44th exon and the location in the nucleotide sequence of SEQ ID NO:
5-8 is represented as the tables below.
TABLE-US-00001 TABLE 1 the location of nucleotides the location of
corresponding from 5'end of exon 55 nucleotides in the nucleotide
nucleotide sequences sequences of SEQ ID NO. 5 -2nd~19th
172nd~192nd -2nd~20th 171st~192nd -2nd~21st 170th~192nd -2nd~22nd
169th~192nd -2nd~23rd 168th~192nd -1st~19th 172nd~191st -1st~20th
171st~191st -1st~21st 170th~191st -1st~22nd 169th~191st -1st~23rd
168th~191st 1st~19th 172nd~190th 1st~20th 171st~190th 1st~21st
170th~190th 1st~22nd 169th~190th 1st~23rd 168th~190th 2nd~19th
172nd~189th 2nd~20th 171st~189th 2nd~21st 170th~189th 2nd~22nd
169th~189th 2nd~23rd 168th~189th 3rd~19th 172nd~188th 3rd~20th
171st~188th 3rd~21st 170th~188th 3rd~22nd 169th~188th 3rd~23rd
168th~188th 9th~29th 162nd~182nd 9th~30th 161st~182nd 9th~31st
160th~182nd 9th~32nd 159th~182nd 9th~33rd 158th~182nd 10th~29th
162nd~181st 10th~30th 161st~181st 10th~31st 160th~181st 10th~32nd
159th~181st 10th~33rd 158th~181st 11th~29th 162nd~180th 11th~30th
161st~180th 11th~31st 160th~180th 11th~32nd 159th~180th 11th~33rd
158th~180th 12th~29th 162nd~179th 12th~30th 161st~179th 12th~31st
160th~179th 12th~32nd 159th~179th 12th~33rd 158th~179th 13th~29th
162nd~178th 13th~30th 161st~178th 13th~31st 160th~178th 13th~32nd
159th~178th 13th~33rd 158th~178th 12th~34th 157th~179th 12th~35th
156th~179th 12th~36th 155th~179th 13th~34th 157th~178th 13th~35th
156th~178th 13th~36th 155th~178th 14th~32nd 159th~177th 14th~33rd
158th~177th 14th~34th 157th~177th 14th~35th 156th~177th 14th~36th
155th~177th 15th~32nd 159th~176th 15th~33rd 158th~176th 15th~34th
157th~176th 15th~35th 156th~176th 15th~36th 155th~176th 16th~32nd
159th~175th 16th~33rd 158th~175th 16th~34th 157th~175th 16th~35th
156th~175th 16th~36th 155th~175th
TABLE-US-00002 TABLE 2 the location of nucleotides the location of
corresponding from 5'end of exon 45 nucleotides in the nucleotide
nucleotide sequences sequences in SEQ ID NO. 6 -3rd~19th
158th~179th -3rd~20th 157th~179th -3rd~21st 156th~179th -3rd~22nd
155th~179th -3rd~23rd 154th~179th -2nd~19th 158th~178th -2nd~20th
157th~178th -2nd~21st 156th~178th -2nd~22nd 155th~178th -2nd~23rd
154th~178th -1st~19th 158th~177th -1st~20th 157th~177th -1st~21st
156th~177th -1st~22nd 155th~177th -1st~23rd 154th~177th 1st~19th
158th~176th 1st~20th 157th~176th 1st~21st 156th~176th 1st~22nd
155th~176th 1st~23rd 154th~176th 2nd~19th 158th~175th 2nd~20th
157th~175th 2nd~21st 156th~175th 2nd~22nd 155th~175th 2nd~23rd
154th~175th -2nd~24th 153rd~178th -2nd~25th 152nd~178th -2nd~26th
151st~178th -2nd~27th 150th~178th -1st~24th 153rd~177th -1st~25th
152nd~177th -1st~26th 151st~177th -1st~27th 150th~177th 1st~24th
153rd~176th 1st~25th 152nd~176th 1st~26th 151st~176th 1st~27th
150th~176th 2nd~24th 153rd~175th 2nd~25th 152nd~175th 2nd~26th
151st~175h 2nd~27th 150th~175th 3rd~23rd 154th~174th 3rd~24th
153rd~174th 3rd~25th 152nd~174th 3rd~26th 151st~174th 3rd~27th
150th~174th 4th~28th 149th~173rd 4th~29th 148th~173rd 4th~30th
147th~173rd 4th~31st 146th~173rd 4th~32nd 147th~173rd 5th~28th
149th~172nd 5th~29th 148th~172nd 5th~30th 147th~172nd 5th~31st
146th~172nd 5th~32nd 145th~172nd 6th~28th 149th~171st 6th~29th
148th~171st 6th~30th 147th~171st 6th~31st 146th~171st 6th~32nd
145th~171st 7th~28th 149th~170th 7th~29th 148th~170th 7th~30th
147th~170th 7th~31st 146th~170th 7th~32nd 145th~170th 8th~28th
149th~169th 8th~29th 148th~169th 8th~30th 147th~169th 8th~31st
146th~169th 8th~32nd 145th~169th
TABLE-US-00003 TABLE 3 the location of nucleotides the location of
corresponding from 5'end of exon 50 nucleotides in the nucleotide
nucleotide sequences sequences in SEQ ID NO. 7 105th~125th 5th~25th
105th~126th 4th~25th 105th~127th 3rd~25th 105th~128th 2nd~25th
105th~129th 1st~25th 106th~125th 5th~24th 106th~126th 4th~24h
106th~127th 3rd~24th 106th~128h 2nd~24th 106th~129th 1st~24th
107th~125th 5th~23rd 107th~126h 4th~23rd 107th~127th 3rd~23rd
107th~128th 2nd~23rd 107th~129th 1st~23rd 108th~125th 5th~22nd
108th~126th 4th~22nd 108th~127h 3rd~22nd 108th~128th 2nd~22nd
108th~129th 1st~22nd 109th~125th 5th~21st 109th~126th 4th~21st
109th~127th 3rd~21st 109th~128th 2nd~21st 109th~129h 1st~21st
TABLE-US-00004 TABLE 4 the location of nucleotides the location of
corresponding from 5'end of exon 44 nucleotides in the nucleotide
nucleotide sequences sequences in SEQ ID NO. 8 9th~30th 119th~140th
9th~31st 118th~140th 9th~32nd 117th~140th 9th~33rd 116th~140th
9th~34th 115th~140th 10th~30th 119th~139th 10th~31st 118th~139th
10th~32nd 117th~139th 10th~33rd 116th~139th 10th~34th 115th~139th
11th~30th 119th~138th 11th~31st 118th~138th 11th~32nd 117th~138th
11th~33rd 116th~138th 11th~34th 115th~138th 12th~30th 119th~137th
12th~31st 118th~137th 12th~32nd 117th~137th 12th~33rd 116th~137th
12th~34th 115th~137th 13th~30th 119th~136th 13th~31st 118th~136th
13th~32nd 117th~136th 13th~33rd 116th~136th 13th~34th 115th~136th
24th~45th 104th~125th 24th~46th 103rd~125th 24th~47th 102nd~125th
24th~48th 101st~125th 24th~49th 100th~125h 25th~45th 104th~124th
25th~46th 103rd~124th 25th~47th 102nd~124th 25th~48th 101st~124th
25th~49th 100th~124th 26th~45th 104th~123rd 26th~46th 103rd~123rd
26th~47th 102nd~123rd 26th~48th 101st~123rd 26th~49th 100th~123rd
27th~45th 104th~122nd 27th~46th 103rd~122nd 27th~47th 102nd~122nd
27th~48th 101st~122nd 27th~49th 100th~122nd 28th~45th 104th~121st
28th~46th 103rd~121st 28th~47th 102nd~121st 28th~48th 101st~121st
28th~49th 100th~121st 29th~45th 104th~120th 29th~46th 103rd~120th
29th~47th 102nd~120th 29th~48th 101st~120th 29th~49th
100th~120th
[0113] It is preferred that the exon 55 skipping oligomer of the
present invention consists of a complementary sequence to any one
of the nucleotide sequences consisting of the 1st to the 21st, the
11th to the 31st or the 14th to the 34th nucleotides, from the 5'
end of the 55th exon in the human dystrophin gene (e.g., any one of
the sequences consisting of the 170th to the 190th, the 160th to
the 180th or the 157th to the 177th of SEQ ID NO: 5).
[0114] It is preferred that the exon 45 skipping oligomer of the
present invention consists of a complementary sequence to any one
of the nucleotide sequences consisting of the -2nd to the 19th, the
1st to the 21st, the 1st to the 25th or the 6th to the 30th
nucleotides, from the 5' end of the 45th exon in the human
dystrophin gene (e.g., any one of the sequences consisting of the
158th to the 178th, 156th to the 176th, the 152nd to the 176th or
the 147th to the 171st nucleotides of SEQ ID NO: 6).
[0115] It is preferred that the exon 50 skipping oligomer of the
present invention consists of a complementary sequence to any one
of the nucleotide sequences consisting of the 106th to the 126th or
the 107th to the 127th nucleotides, from the 5' end of the 50th
exon in the human dystrophin gene (e.g., any one of the sequences
consisting of the 4th to the 24th or the 3rd to the 23rd
nucleotides of SEQ ID NO: 7).
[0116] It is preferred that the exon 44 skipping oligomer of the
present invention consists of a complementary sequence to any one
of the nucleotide sequences consisting of the 11th to the 32nd, the
25th to the 45th, the 26th to the 46th, the 26th to the 47th or the
27th to the 47th nucleotides, from the 5' end of the 44th exon in
the human dystrophin gene (e.g., any one of the sequences
consisting of the 117th to the 138th, the 104th to the 124th, the
103rd to the 123rd, the 102nd to the 123rd or the 102nd to the
122nd nucleotides of SEQ ID NO: 8).
[0117] The term "cause skipping of the 55th exon in the human
dystrophin gene" is intended to mean that by binding of the
oligomer of the present invention to the site corresponding to exon
55 of the transcript (e.g., pre-mRNA) of the human dystrophin gene,
for example, the nucleotide sequence corresponding to the 5' end of
exon 56 is ligated to the 3' side of the nucleotide sequence
corresponding to the 3' end of exon 53 in DMD patients with
deletion of exon 54 when the transcript is spliced, thus resulting
in formation of mature mRNA which is free of codon frame shift.
[0118] The term "cause skipping of the 45th exon in the human
dystrophin gene" is intended to mean that by binding of the
oligomer of the present invention to the site corresponding to exon
45 of the transcript (e.g., pre-mRNA) of the human dystrophin gene,
for example, the nucleotide sequence corresponding to the 5' end of
exon 46 is ligated to the 3' side of the nucleotide sequence
corresponding to the 3' end of exon 43 in DMD patients with
deletion of exon 44 when the transcript is spliced, thus resulting
in formation of mature mRNA which is free of codon frame shift.
[0119] The term "cause skipping of the 50th exon in the human
dystrophin gene" is intended to mean that by binding of the
oligomer of the present invention to the site corresponding to exon
50 of the transcript (e.g., pre-mRNA) of the human dystrophin gene,
for example, the nucleotide sequence corresponding to the 5' end of
exon 52 is ligated to the 3' side of the nucleotide sequence
corresponding to the 3' end of exon 49 in DMD patients with
deletion of exon 51 when the transcript is spliced, thus resulting
in formation of mature mRNA which is free of codon frame shift.
[0120] The term "cause skipping of the 44th exon in the human
dystrophin gene" is intended to mean that by binding of the
oligomer of the present invention to the site corresponding to exon
44 of the transcript (e.g., pre-mRNA) of the human dystrophin gene,
for example, the nucleotide sequence corresponding to the 5' end of
exon46 is ligated to the 3' side of the nucleotide sequence
corresponding to the 3' end of exon 43 in DMD patients with
deletion of, exon 45 when the transcript is spliced, thus resulting
in formation of mature mRNA which is free of codon frame shift.
[0121] Accordingly, it is not required for the oligomer of the
present invention to have a nucleotide sequence 100% complementary
to each target sequence, as far as it causes exon 55, 45, 50 or 44
skipping in the human dystrophin gene. The oligomer of the present
invention may include, for example, 1 to 3, 1 or 2, or one
nucleotide non-complementary to the target sequence.
[0122] Herein, the term "binding" described above is intended to
mean that when the oligomer of the present invention is mixed with
the transcript of human dystrophin gene, both are hybridized under
physiological conditions to form a double strand. The term "under
physiological conditions" refers to conditions set to mimic the in
vivo environment in terms of pH, salt composition and temperature.
The conditions are, for example, 25 to 40.degree. C., preferably
37.degree. C., pH 5 to 8, preferably pH 7.4 and 150 mM of sodium
chloride concentration.
[0123] Whether the skipping of exon 55, 45, 50 or 44 in the human
dystrophin gene is caused or not can be confirmed by introducing
the oligomer of the present invention into a dystrophin expressing
cell (e.g., human rhabdomyosarcoma cells), amplifying the region
surrounding exon 55, 45, 50 or 44 of mRNA of the human dystrophin
gene by RT-PCR from the total RNA of the dystrophin expressing cell
and performing nested PCR or sequence analysis on the PCR amplified
product.
[0124] The skipping efficiency can be determined as follows. The
mRNA for the human dystrophin gene is collected from test cells; in
the mRNA, the polynucleotide level "A" of the band where exon 55,
45, 50 or 44 is skipped and the polynucleotide level "B" of the
band where exon 55, 45, 50 or 44 is not skipped are measured. Using
these measurement values of "A" and "B," the efficiency is
calculated by the following equation:
Skipping Efficiency (%)=A/(A+B).times.100
[0125] The oligomer of the present invention includes, for example,
an oligonucleotide, morpholino oligomer or peptide nucleic acid
(PNA), having a length of 18 to 28 nucleotides. The length is
preferably from 15 to 30 nucleotides or 20 to 25 nucleotides and
morpholino oligomers are preferred.
[0126] The oligonucleotide described above (hereinafter referred to
as "the oligonucleotide of the present invention") is the oligomer
of the present invention composed of nucleotides as constituent
units. Such nucleotides may be any of ribonucleotides,
deoxyribonucleotides and modified nucleotides.
[0127] The modified nucleotide refers to one having fully or partly
modified nucleobases, sugar moieties and/or phosphate-binding
regions, which constitute the ribonucleotide or
deoxyribonucleotide.
[0128] The nucleobase includes, for example, adenine, guanine,
hypoxanthine, cytosine, thymine, uracil, and modified bases
thereof. Examples of such modified nucleobases include, but not
limited to, pseudouracil, 3-methyluracil, dihydrouracil,
5-alkylcytosines (e.g., 5-methylcytosine), 5-alkyluracils (e.g.,
5-ethyluracil), 5-halouracils (5-bromouracil), 6-azapyrimidine,
6-alkylpyrimiclines (6-methyluracil), 2-thiouracil, 4-thiouracil,
4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil,
5'-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, 1-methyladenine,
1-methylhypoxanthine, 2,2-dimethylguanine, 3-methylcytosine,
2-methyladenine, 2-methylguanine, N6-methyladenine,
7-methylguanine, 5-methoxyaminomethyl-2-thiouracil,
5-methylaminomethyluracil, 5-methylcarbonylmethyluracil,
5-methyloxyuracil, 5-methyl-2-thiouracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid,
2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine,
isoguanine, indole, imidazole, xanthine, etc.
[0129] Modification of the sugar moiety may include, for example,
modifications at the 2'-position of ribose and modifications of the
other positions of the sugar. The modification at the 2'-position
of ribose includes replacement of the 2'-OH of ribose with OR, R,
R'OR, SH, SR, NH.sub.2, NHR, NR.sub.2, N.sub.3, CN, F, Cl, Br or I,
wherein R represents an alkyl or an aryl and R' represents an
alkylene.
[0130] The modification for the other positions of the sugar
includes, for example, replacement of O at the 4' position of
ribose or deoxyribose with S, bridging between 2' and 4' positions
of the sugar, e.g., LNA (Locked Nucleic Acid) or ENA
(2'-0,4'-C-Ethylene-bridged Nucleic Acids), but is not limited
thereto.
[0131] A modification of the phosphate-binding region includes, for
example, a modification of replacing phosphodiester bond with
phosphorothioate bond, phosphorodithioate bond, alkyl phosphonate
bond, phosphoroamidate bond or boranophosphate bond (Enya et al:
Bioorganic & Medicinal Chemistry, 2008, 18, 9154-9160) (cf.,
e.g., Japan Domestic Re-Publications of PCT Application Nos.
2006/129594 and 2006/038608).
[0132] The alkyl is preferably a straight or branched alkyl having
1 to 6 carbon atoms. Specific examples include methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, sec butyl, tort-butyl,
n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl and isohexyl.
The alkyl may optionally be substituted. Examples of such
substituents are a halogen, an alkoxy, cyano and nitro. The alkyl
may be substituted with 1 to 3 substituents.
[0133] The cycloalkyl is preferably a cycloalkyl having 5 to 12
carbon atoms. Specific examples include cyclopentyl, cyclohexyl,
cycloheptyl, cyclooctyl, cyclodecyl and cyclododecyl.
[0134] The halogen includes fluorine, chlorine, bromine and
iodine.
[0135] The alkoxy is a straight or branched alkoxy having 1 to 6
carbon atoms such as methoxy, ethoxy, n-propoxy, isopropoxy,
n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy,
isopentyloxy, n-hexyloxy, isohexyloxy, etc. Among others, an alkoxy
having 1 to 3 carbon atoms is preferred.
[0136] The aryl is preferably an aryl having 6 to 10 carbon atoms.
Specific examples include phenyl, .alpha.-naphthyl and -naphthyl.
Among others, phenyl is preferred. The aryl may optionally be
substituted. Examples of such substituents are an alkyl, a halogen,
an alkoxy, cyano and nitro. The aryl may be substituted with one to
three of such substituents.
[0137] The alkylene is preferably a straight or branched alkylene
having 1 to 6 carbon atoms. Specific examples include methylene,
ethylene, trimethylene, tetramethylene, pentamethylene,
hexamethylene, 2-(ethyl) trimethylene and 1-(methyl)
tetramethylene.
[0138] The acyl includes a straight or branched alkanoyl or aroyl.
Examples of the alkanoyl include formyl, acetyl, 2-methylacetyl,
2,2-dimethylacetyl, propionyl, butyryl, isobutyryl, pentanoyl,
2,2-dimethylpropionyl, hexanoyl, etc. Examples of the aroyl include
benzoyl, toluoyl and naphthoyl. The aroyl may optionally be
substituted at substitutable positions and may be substituted with
an alkyl(s).
[0139] Preferably, the oligonucleotide of the present invention is
the oligomer of the present invention containing a constituent unit
represented by general formula below wherein the --OH group at
position 2' of ribose is substituted with methoxy and the
phosphate-binding region is a phosphorothioate bond:
##STR00002##
wherein Base represents a nucleobase.
[0140] The oligonucleotide of the present invention may be easily
synthesized using various automated synthesizer (e.g., AKTA
oligopilot plus 10/100 (GE Healthcare)). Alternatively, the
synthesis may also be entrusted to a third-party organization
(e.g., Promega Inc., or Takara Co.), etc.
[0141] The morpholino oligomer of the present invention is the
oligomer of the present invention comprising the constituent unit
represented by general formula below:
##STR00003##
wherein Base has the same significance as defined above, and, W
represents a group shown by any one of the following groups:
##STR00004##
wherein X represents --CH.sub.2R.sup.1, --O--CH.sub.2R.sup.1,
--S--CH.sub.2R.sup.1, --NR.sup.2R.sup.3 or F;
[0142] R.sup.1 represents H or an alkyl;
[0143] R.sup.2 and R.sup.3, which may be the same or different,
each represents H, an alkyl, a cycloalkyl or an aryl;
[0144] Y.sub.1 represents O, S, CH.sub.2 or NR.sup.1;
[0145] Y.sub.2 represents O, S or NRS;
[0146] Z represents O or S.
[0147] Preferably, the morpholino oligomer is an oligomer
comprising a constituent unit represented by general formula below
(phosphorodiamidate morpholino oligomer (hereinafter referred to as
"PMO")).
##STR00005##
wherein Base, R.sup.2 and R.sup.3 have the same significance as
defined above.
[0148] The morpholino oligomer may be produced in accordance with,
e.g., WO 1991/009033 or WO 2009/064471. In particular, PMO can be
produced by the procedure described in WO 2009/064471 or produced
by the process shown below.
[Method for Producing PMO]
[0149] An embodiment of PMO is, for example, the compound
represented by general formula (I) below (hereinafter PMO
##STR00006##
wherein Base, R.sup.2 and R.sup.3 have the same significance as
defined above; and, n is a given integer of 1 to 99, preferably a
given integer of 18 to 28.
[0150] PMO (I) can be produced in accordance with a known method,
for example, can be produced by performing the procedures in the
following steps.
[0151] The compounds and reagents used in the steps below are not
particularly limited so long as they are commonly used to prepare
PMO.
[0152] Also, the following steps can all be carried out by the
liquid phase method or the solid phase method (using manuals or
commercially available solid phase automated synthesizers). In
producing PMO by the solid phase method, it is desired to use
automated synthesizers in view of simple operation procedures and
accurate synthesis.
(1) Step A:
[0153] The compound represented by general formula (II) below
(hereinafter referred to as Compound (II)) is reacted with an acid
to prepare the compound represented by general formula (III) below
(hereinafter referred to as Compound (III)):
##STR00007##
wherein n, R.sup.2 and R.sup.3 have the same significance as
defined above; each B.sup.P independently represents a nucleobase
which may optionally be protected; T represents trityl,
monomethoxytrityl or dimethoxytrityl; and, L represents hydrogen,
an acyl or a group represented by general formula (W) below
(hereinafter referred to as group (IV)).
##STR00008##
[0154] The "nucleobase" for B.sup.P includes the same "nucleobase"
as in Base, provided that the amino or hydroxy group in the
nucleobase shown by B.sup.P may be protected.
[0155] Such protective group for amino is not particularly limited
so long as it is used as a protective group for nucleic acids.
Specific examples include benzoyl, 4-methoxybenzoyl, acetyl,
propionyl, butyryl, isobutyryl, phenylacetyl, phenoxyacetyl,
4-tert-butylphenoxyacetyl, 4-isopropylphenoxyacetyl and
(dimethylamino)methylene. Specific examples of the protective group
for the hydroxy group include 2-cyanoethyl, 4-nitrophenethyl,
phenylsulfonylethyl, methylsulfonylethyl and trimethylsilylethyl,
and phenyl, which may be substituted by 1 to 5 electron-withdrawing
group at optional substitutable positions, diphenylcarbamoyl,
dimethylcarbamoyl, diethylcarbamoyl, methylphenylcarbamoyl,
1-pyrolidinylcarbamoyl, morpholinocarbamoyl, 4-(tert-butylcarboxy)
benzyl, 4-[(dimethylamino)carboxy]benzyl and
4-(phenylcarboxy)benzyl, (cf., e.g., WO 2009/064471).
[0156] The "solid carrier" is not particularly limited so long as
it is a carrier usable for the solid phase reaction of nucleic
acids. It is desired for the solid carrier to have the following
properties: e.g., (i) it is sparingly soluble in reagents that can
be used for the synthesis of morpholino nucleic acid derivatives
(e.g., dichloromethane, acetonitrile, tetrazole, Nmethylimidazole,
pyridine, acetic anhydride, lutidine, trifluoroacetic acid); (ii)
it is chemically stable to the reagents usable for the synthesis of
morpholino nucleic acid derivatives; (iii) it can be chemically
modified; (iv) it can be charged with desired morpholino nucleic
acid derivatives; (v) it has a strength sufficient to withstand
high pressure through treatments; and (vi) it has a uniform
particle diameter range and distribution. Specifically, swellable
polystyrene (e.g., aminomethyl polystyrene resin 1% dibenzylbenzene
crosslinked (200-400 mesh) (2.4-3.0 mmol/g) (manufactured by Tokyo
Chemical industry), Aminomethylated Polystyrene Resin
[dibenzylbenzene 1%, 100-200 mesh] (manufactured by Peptide
Institute, Inc.)), non-swellable polystyrene (e.g., Primer Support
(manufactured by GE Healthcare)), PEG chain-attached polystyrene
(e.g., NH.sub.2-PEG resin (manufactured by Watanabe Chemical Co.),
TentaGel resin), controlled pore glass (controlled pore glass; CPG)
(manufactured by, e.g., CPG), oxalyl-controlled pore glass (cf.,
Alul et al., Nucleic Acids Research, Vol. 19, 1527 (1991)),
TentaGel support-aminopolyethylene glycol-derivatized support (cf.,
e.g., Wright et al., Tetrahedron Letters, Vol. 34, 3373 (1993)),
and a copolymer of Poros-polystyrene/divinylbenzene.
[0157] A "linker" which can be used is a known linker generally
used to connect nucleic acids or morpholino nucleic acid
derivatives. Examples include 3-aminopropyl, succinyl,
2,2'-diethanolsulfonyl and a long chain alkyl amino (LCAA).
[0158] This step can be performed by reacting Compound (II) with an
acid.
[0159] The "acid" which can be used in this step includes, for
example, trifluoroacetic acid, dichloroacetic acid and
trichloroacetic acid. The acid used is appropriately in a range of,
for example, 0.1 mol equivalent to 1000 mol equivalents based on 1
mol of Compound (II), preferably in a range of 1 mol equivalent to
100 mol equivalents based on 1 mol of Compound (II).
[0160] An organic amine can be used in combination with the acid
described above. The organic amine is not particularly limited and
includes, for example, triethylamine. The amount of the organic
amine used is appropriately in a range of, e.g., 0.01 mol
equivalent to 10 mol equivalents, and preferably in a range of 0.1
mol equivalent to 2 mol equivalents, based on 1 mol of the
acid.
[0161] When a salt or mixture of the acid and the organic amine is
used in this step, the salt or mixture includes, for example, a
salt or mixture of trifluoroacetic acid and triethylamine, and more
specifically, a mixture of 1 equivalent of triethylamine and 2
equivalents of trifluoroacetic acid.
[0162] The acid which can be used in this step may also be used in
the form of a dilution with an appropriate solvent in a
concentration of 0.1% to 30%. The solvent is not particularly
limited as far as it is inert to the reaction, and includes, for
example, dichloromethane, acetonitrile, an alcohol (ethanol,
isopropanol, trifluoroethanol, etc.), water, or a mixture
thereof.
[0163] The reaction temperature in the reaction described above is
preferably in a range of, e.g., 10.degree. C. to 50.degree. C.,
more preferably, in a range of 20.degree. C. to 40.degree. C., and
most preferably, in a range of 25.degree. C. to 35.degree. C.
[0164] The reaction time may vary depending upon kind of the acid
used and reaction temperature, and is appropriately in a range of
0.1 minute to 24 hours in general, and preferably in a range of 1
minute to 5 hours.
[0165] After completion of this step, a base may be added, if
necessary, to neutralize the acid remained in the system. The
"base" is not particularly limited and includes, for example,
diisopropylamine. The base may also be used in the form of a
dilution with an appropriate solvent in a concentration of 0.1%
(v/v) to 30% (v/v).
[0166] The solvent used in this step is not particularly limited so
long as it is inert to the reaction, and includes dichloromethane,
acetonitrile, an alcohol (ethanol, isopropanol, trifluoroethanol,
etc.), water, and a mixture thereof. The reaction temperature is
preferably in a range of, e.g., 10.degree. C. to 50.degree. C.,
more preferably, in a range of 20.degree. C. to 40.degree. C., and
most preferably; in a range of 25.degree. C. to 35.degree. C.
[0167] The reaction time may vary depending upon kind of the base
used and reaction temperature, and is appropriately in a range of
0.1 minute to 24 hours in general, and preferably in a range of 1
minute to 5 hours.
[0168] In Compound (II), the compound of general formula (IIa)
below (hereinafter Compound (IIa)), wherein n is 1 and L is a group
(IV), can be produced by the following procedure.
##STR00009##
wherein B.sup.P, T, linker and solid carrier have the same
significance as defined above.
Step 1:
[0169] The compound represented by general formula (V) below is
reacted with an acylating agent to prepare the compound represented
by general formula. (VI) below (hereinafter referred to as Compound
(VI)).
##STR00010##
wherein B.sup.P, T and linker have the same significance as defined
above; and, R.sup.4 represents hydroxy, a halogen or amino.
[0170] This step can be carried out by known procedures for
introducing linkers, using Compound (V) as the starting
material.
[0171] In particular, the compound represented by general formula
(VIa) below can be produced by performing the method known as
esterification, using Compound (V) and succinic anhydride.
##STR00011##
wherein B.sup.P and T have the same significance as defined
above.
Step 2:
[0172] Compound (VI) is reacted with a solid career by a condensing
agent to prepare Compound (IIa).
##STR00012##
wherein B.sup.P, R.sup.4, T, linker and solid carrier have the same
significance as defined above.
[0173] This step can be performed using Compound (VI) and a solid
carrier in accordance with a process known as condensation
reaction.
[0174] In Compound (II), the compound represented by general
formula (IIa2) below wherein n is 2 to 99 and L is a group
represented by general formula (IV) can be produced by using
Compound (IIa) as the starting material and repeating step A and
step B of the PMO production method described in the specification
for a desired number of times.
##STR00013##
wherein B.sup.P, R.sup.2, R.sup.3, T, linker and solid carrier have
the same significance as defined above; and, n' represents 1 to
98.
[0175] In Compound (II), the compound of general formula (IIb)
below wherein n is 1 and L is hydrogen can be produced by the
procedure described in, e.g., WO 1991/009033.
##STR00014##
wherein B.sup.P and T have the same significance as defined
above.
[0176] In Compound (II), the compound represented by general
formula (IIb2) below wherein n is 2 to 99 and L is hydrogen can be
produced by using Compound (IIb) as the starting material and
repeating step A and step B of the PMO production method described
in the specification for a desired number of times.
##STR00015##
wherein B.sup.P, n', R.sup.2, R.sup.3 and T have the same
significance as defined above.
[0177] In Compound (II), the compound represented by general
formula (IIc) below wherein n is 1 and L is an acyl can be produced
by performing the procedure known as acylation reaction, using
Compound (Jib).
##STR00016##
wherein B.sup.P and T have the same significance as defined above;
and, R.sup.5 represents an acyl.
[0178] In Compound (II), the compound represented by general
formula (IIc2) below wherein n is 2 to 99 and L is an acyl can be
produced by using Compound (IIc) as the starting material and
repeating step A and step B of the PMO production method described
in the specification for a desired number of times.
##STR00017##
wherein B.sup.P, n', R.sup.2, R.sup.3, R.sup.5 and T have the same
significance as defined above.
(2) Step B
[0179] Compound (III) is reacted with a morpholino monomer compound
in the presence of a base to prepare the compound represented by
general formula (VII) below (hereinafter referred to as Compound
(VII)):
##STR00018##
wherein B.sup.P, L, n, R.sup.2, R.sup.3 and T have the same
significance as defined above.
[0180] This step can be performed by reacting Compound (III) with
the morpholino monomer compound in the presence of a base.
[0181] The morpholino monomer compound includes, for example,
compounds represented by general formula (VIII) below:
##STR00019##
wherein B.sup.P, R.sup.2, R.sup.3 and T have the same significance
as defined above.
[0182] The "base" which can be used in this step includes, for
example, diisopropylamine, triethylamine and N-ethylmorpholine. The
amount of the base used is appropriately in a range of 1 mol
equivalent to 1000 mol equivalents based on 1 mol of Compound
preferably, 10 mol equivalents to 100 mol equivalents based on 1
mol of Compound (III).
[0183] The morpholino monomer compound and base which can be used
in this step may also be used as a dilution with an appropriate
solvent in a concentration of 0.1% to 30%. The solvent is not
particularly limited as far as it is inert to the reaction, and
includes, for example, N,N-dimethylimidazolidone,
N-methylpiperidone, DMF, dichloromethane, acetonitrile,
tetrahydrofuran, or a mixture thereof.
[0184] The reaction temperature is preferably in a range of, e.g.,
0.degree. C. to 100.degree. C., and more preferably, in a range of
10.degree. C. to 50.degree. C.
[0185] The reaction time may vary depending upon kind of the base
used and reaction temperature, and is appropriately in a range of 1
minute to 48 hours in general, and preferably in a range of 30
minutes to 24 hours.
[0186] Furthermore, after completion of this step, an acylating
agent can be added, if necessary. The "acylating agent" includes,
for example, acetic anhydride, acetyl chloride and phenoxyacetic
anhydride. The acylating agent may also be used as a dilution with
an appropriate solvent in a concentration of 0.1% to 30%. The
solvent is not particularly limited as far as it is inert to the
reaction, and includes, for example, dichloromethane, acetonitrile,
an alcohol(s) (ethanol, isopropanol, trifluoroethanol, etc.),
water, or a mixture thereof.
[0187] If necessary, a base such as pyridine, lutidine, collidine,
triethylamine, diisopropylethylamine, N-ethylmorpholine, etc. may
also be used in combination with the acylating agent. The amount of
the acylating agent is appropriately in a range of 0.1 mol
equivalent to 10000 mol equivalents, and preferably in a range of 1
mol equivalent to 1000 mol equivalents. The amount of the base is
appropriately in a range of, e.g., 0.1 mol equivalent to 100 mol
equivalents, and preferably in a range of 1 mol equivalent to 10
mol equivalents, based on 1 mol of the acylating agent.
[0188] The reaction temperature in this reaction is preferably in a
range of 10.degree. C. to 50.degree. C., more preferably, in a
range of 10.degree. C. to 50.degree. C., much more preferably, in a
range of 20.degree. C. to 40.degree. C., and most preferably, in a
range of 25.degree. C. to 35.degree. C. The reaction time may vary
depending upon kind of the acylating agent used and reaction
temperature, and is appropriately in a range of 0.1 minute to 24
hours in general, and preferably in a range of 1 minute to 5
hours.
(3) Step C:
[0189] In Compound (VII) produced in Step B, the protective group
is removed using a deprotecting agent to prepare the compound
represented by general formula (IX).
##STR00020##
wherein Base, B.sup.P, L, n, R.sup.2, R.sup.3 and T have the same
significance as defined above.
[0190] This step can be performed by reacting Compound (VII) with a
deprotecting agent.
[0191] The "deprotecting agent" includes, e.g., conc. ammonia water
and methylamine. The "deprotecting agent" used in this step may
also be used as a dilution with, e.g., water, methanol, ethanol,
isopropyl alcohol, acetonitrile, tetrahydrofuran, DMF,
N,N-dimethylimidazolidone, N-methylpiperidone, or a mixture of
these solvents. Among others, ethanol is preferred. The amount of
the deprotecting agent used is appropriately in a range of, e.g., 1
mol equivalent to 100000 mol equivalents, and preferably in a range
of 10 mol equivalents to 1000 mol equivalents, based on 1 mol of
Compound (VII).
[0192] The reaction temperature is appropriately in a range of
15.degree. C. to 75.degree. C., preferably, in a range of
40.degree. C. to 70.degree. C., and more preferably, in a range of
50.degree. C. to 60.degree. C. The reaction time for deprotection
may vary depending upon kind of Compound (VII), reaction
temperature, etc., and is appropriately in a range of 10 minutes to
30 hours, preferably 30 minutes to 24 hours, and more preferably in
a range of 5 hours to 20 hours.
(4) Step D:
[0193] PMO (I) is produced by reacting Compound (Ix) produced in
step C with an acid:
##STR00021##
wherein Base, n, R.sup.2, R.sup.3 and T have the same significance
as defined above.
[0194] This step can be performed by adding an acid to Compound
(IX).
[0195] The "acid" which can be used in this step includes, for
example, trichloroacetic acid, dichloroacetic acid, acetic acid,
phosphoric acid, hydrochloric acid, etc. The acid used is
appropriately used to allow the solution to have a pH range of 0.1
to 4.0, and more preferably, in a range of pH 1.0 to 3.0. The
solvent is not particularly limited so long as it is inert to the
reaction, and includes, for example, acetonitrile, water, or a
mixture of these solvents thereof.
[0196] The reaction temperature is appropriately in a range of
10.degree. C. to 50.degree. C., preferably, in a range of
20.degree. C. to 40.degree. C., and more preferably, in a range of
25.degree. C. to 35.degree. C. The reaction time for
&protection may vary depending upon kind of Compound (IX),
reaction temperature, etc., and is appropriately in a range of 0.1
minute to 5 hours, preferably 1 minute to 1 hour, and more
preferably in a range of 1 minute to 30 minutes.
[0197] PMO (I) can be obtained by subjecting the reaction mixture
obtained in this step to conventional means of separation and
purification such as extraction, concentration, neutralization,
filtration, centrifugal separation, recrystallization, reversed
phase column chromatography C8 to C18, cation exchange column
chromatography, anion exchange column chromatography, gel
filtration column chromatography, high performance liquid
chromatography, dialysis, ultrafiltration, etc., alone or in
combination thereof. Thus, the desired PMO (I) can be isolated and
purified (cf., e.g., WO 1991/09033).
[0198] In purification of PMO (I) using reversed phase
chromatography, e.g., a solution mixture of 20 mM
triethylamine/acetate buffer and acetonitrile can be used as an
elution solvent.
[0199] In purification of PMO (I) using ion exchange
chromatography, e.g., a solution mixture of 1 M saline solution and
10 mM sodium hydroxide aqueous solution can be used as an elution
solvent.
[0200] A peptide nucleic acid is the oligomer of the present
invention having a group represented by the following general
formula as the constituent unit:
##STR00022##
wherein Base has the same significance as defined above.
[0201] Peptide nucleic acids can be prepared by referring to, e.g.,
the following literatures. [0202] 1) P. E. Nielsen, M. Egholm, R.
H. Berg, O. Buchardt, Science, 254, 1497 (1991) [0203] 2) M.
Egholm, O. Buchardt, P. E. Nielsen, R. H. Berg, Jacs., 114, 1895
(1992) [0204] 3) K. L. Dueholm, M. Egholm, C. Behrens, L.
Christensen, H. F. Hansen, T. Vulpius, K. H. Petersen, R. H. Berg,
P. E. Nielsen, O. Buchardt, J. Org. Chem., 59, 5767 (1994) [0205]
4) L. Christensen, R. Fitzpatrick, B. Gildea, K. H. Petersen, H. F.
Hansen, T. Koch, M. Egholm, O. Buchardt, P. E. Nielsen, J. Colin,
R. H. Berg, J. Pept. Sci., 1, 175 (1995) [0206] 5) T. Koch, H. F.
Hansen, P. Andersen, T. Larsen, H. G. Batz, K. Otteson, H. Orum, J.
Pept. Res., 49, 80 (1997)
[0207] In the oligomer of the present invention, the 5' end may be
any of chemical structures (1) to (3) below, and preferably is (3)
--OH.
##STR00023##
[0208] Hereinafter, the groups shown by (1), (2) and (3) above are
referred to as "Group (1)," "Group (2)" and "Group (3),"
respectively.
2. Pharmaceutical Composition
[0209] The oligomer of the present invention causes exon 55, 45, 50
and 44 skipping with a higher efficiency as compared to the prior
art antisense oligomers. It is thus expected that conditions of
muscular dystrophy can be relieved with high efficiency by
administering the pharmaceutical composition comprising the
oligomer of the present invention to DMD patients. For example,
when the pharmaceutical composition comprising the oligomer of the
present invention is used, the same therapeutic effects can be
achieved even in a smaller dose than that of the oligomers of the
prior art. Accordingly, side effects can be alleviated and such is
economical.
[0210] In another embodiment, the present invention provides the
pharmaceutical composition for the treatment of muscular dystrophy,
comprising as an active ingredient the oligomer of the present
invention, a pharmaceutically acceptable salt or hydrate thereof
(hereinafter referred to as "the composition of the present
invention").
[0211] Examples of the pharmaceutically acceptable salt of the
oligomer of the present invention contained in the composition of
the present invention are alkali metal salts such as salts of
sodium, potassium and lithium; alkaline earth metal salts such as
salts of calcium and magnesium; metal salts such as salts of
aluminum, iron, zinc, copper, nickel, cobalt, etc.; ammonium salts;
organic amine salts such as salts of t-octylamine, dibenzylamine,
morpholine, glucosamine, phenylglycine alkyl ester,
ethylenediamine, N-methylglucamine, guanidine, diethylamine,
triethylamine, dicyclohexylamine, N N'-dibenzylethylenediamine,
chloroprocaine, procaine, diethanolamine, N-benzylphenethylamine,
piperazine, tetramethylammonium, tris(hydroxymethyl)aminomethane;
hydrohalide salts such as salts of hydrofluorates, hydrochlorides,
hydrobromides and hydroiodides; inorganic acid salts such as
nitrates, perchlorates, sulfates, phosphates, etc.; lower alkane
sulfonates such as methanesulfonates, trifluoromethanesulfonates
and ethanesulfonates; arylsulfonates such as benzenesulfonates and
p-toluenesulfonates; organic acid salts such as acetates, malates,
fumarates, succinates, citrates, tartarates, oxalates, maleates,
etc.; and, amino acid salts such as salts of glycine, lysine,
arginine, ornithine, glutamic acid and aspartic acid. These salts
may be produced by known methods. Alternatively, the oligomer of
the present invention contained in the composition of the present
invention may be in the form of a hydrate thereof.
[0212] Administration route for the composition of the present
invention is not particularly limited so long as it is
pharmaceutically acceptable route for administration, and can be
chosen depending upon method of treatment. In view of easiness in
delivery to muscle tissues, preferred are intravenous
administration, intraarterial administration, intramuscular
administration, subcutaneous administration, oral administration,
tissue administration, transdermal administration, etc. Also,
dosage forms which are available for the composition of the present
invention are not particularly limited, and include, for example,
various injections, oral agents, drips, inhalations, ointments,
lotions, etc.
[0213] In administration of the oligomer of the present invention
to patients with muscular dystrophy, the composition of the present
invention preferably contains a carrier to promote delivery of the
oligomer to muscle tissues. Such a carrier is not particularly
limited as far as it is pharmaceutically acceptable, and examples
include cationic carriers such as cationic liposomes, cationic
polymers, etc., or carriers using viral envelope. The cationic
liposomes are, for example, liposomes composed of
2-O-(2-diethylaminoethyl)carabamoyl-1,3-O-dioleoylglycerol and
phospholipids as the essential constituents (hereinafter referred
to as "liposome A"), Oligofectamine (registered trademark)
(manufactured by Invitrogen Corp.), Lipofectin (registered
trademark) (manufactured by Invitrogen Corp.), Lipofectamine
(registered trademark) (manufactured by Invitrogen Corp.),
Lipofectamine 2000 (registered trademark) (manufactured by
Invitrogen Corp.), DMRIE-C (registered trademark) (manufactured by
Invitrogen Corp.), GeneSilencer (registered trademark)
(manufactured by Gene Therapy Systems), TransMessenger (registered
trademark) (manufactured by QIAGEN, Inc.), TransIT TKO (registered
trademark) (manufactured by Minis) and Nucleofector II (Lonza).
Among others, liposome A is preferred. Examples of cationic
polymers are JetSI (registered trademark) (manufactured by
Qbiogene, Inc.) and Jet-PEI (registered trademark)
(polyethylenimine, manufactured by Qbiogene, Inc.). An example of
carriers using viral envelop is GenomeOne (registered trademark)
(HVJ-E liposome, manufactured by Ishihara Sangyo). Alternatively,
the medical devices described in Japanese Patent No. 2924179 and
the cationic carriers described in Japanese Domestic Re-Publication
PCT Nos. 2006/129594 and 2008/096690 may be used as well.
[0214] A concentration of the oligomer of the present invention
contained in the composition of the present invention may vary
depending on kind of the carrier, etc., and is appropriately in a
range of 0.1 nM to 100 .mu.M, preferably in a range of 1 nM to 10
.mu.M, and more preferably in a range of 10 nM to 1 .mu.M. A weight
ratio of the oligomer of the present invention contained in the
composition of the present invention and the carrier
(carrier/oligomer of the present invention) may vary depending on
property of the oligomer, type of the carrier, etc., and is
appropriately in a range of 0.1 to 100, preferably in a range of 1
to 50, and more preferably in a range of 10 to 20.
[0215] In addition to the oligomer of the present invention and the
carrier described above, pharmaceutically acceptable additives may
also be optionally formulated in the composition of the present
invention. Examples of such additives are emulsification aids
(e.g., fatty acids having 6 to 22 carbon atoms and their
pharmaceutically acceptable salts, albumin and dextran),
stabilizers (e.g., cholesterol and phosphatidic acid), isotonizing
agents (e.g., sodium chloride, glucose, maltose, lactose, sucrose,
trehalose), and pH controlling agents (e.g., hydrochloric acid,
sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide,
potassium hydroxide and triethanolamine). One or more of these
additives can be used. The content of the additive in the
composition of the present invention is appropriately 90 wt % or
less, preferably 70 wt % or less and more preferably, 50 wt % or
less.
[0216] The composition of the present invention can be prepared by
adding the oligomer of the present invention to a carrier
dispersion and adequately stirring the mixture. Additives may be
added at an appropriate step either before or after addition of the
oligomer of the present invention. An aqueous solvent that can be
used in adding the oligomer of the present invention is not
particularly limited as far as it is pharmaceutically acceptable,
and examples are injectable water or injectable distilled water,
electrolyte fluid such as physiological saline, etc., and sugar
fluid such as glucose fluid, maltose fluid, etc. A person skilled
in the art can appropriately choose conditions for pH and
temperature for such matter.
[0217] The composition of the present invention may be prepared
into, e.g., a liquid form and its lyophilized preparation. The
lyophilized preparation can be prepared by lyophilizing the
composition of the present invention in a liquid form in a
conventional manner. The lyophilization can be performed, for
example, by appropriately sterilizing the composition of the
present invention in a liquid form, dispensing an aliquot into a
vial container, performing preliminary freezing for 2 hours at
conditions of about -40 to -20.degree. C., performing a primary
drying at 0 to 10.degree. C. under reduced pressure, and then
performing a secondary drying at about 15 to 25.degree. C. under
reduced pressure. In general, the lyophilized preparation of the
composition of the present invention can be obtained by replacing
the content of the vial with nitrogen gas and capping.
[0218] The lyophilized preparation of the composition of the
present invention can be used in general upon reconstitution by
adding an optional suitable solution (reconstitution liquid) and
redissolving the preparation. Such a reconstitution liquid includes
injectable water, physiological saline and other infusion fluids. A
volume of the reconstitution liquid may vary depending on the
intended use, etc., is not particularly limited, and is suitably
0.5 to 2-fold greater than the volume prior to lyophilization or no
more than 500 mL.
[0219] It is desired to control a dose of the composition of the
present invention to be administered, by taking the following
factors into account: the type and dosage form of the oligomer of
the present invention contained; patients' conditions including
age, body weight, etc.; administration route; and the
characteristics and extent of the disease. A daily dose calculated
as the amount of the oligomer of the present invention is generally
in a range of 0.1 mg to 10 g/human, and preferably 1 mg to 1
g/human. This numerical range may vary occasionally depending on
type of the target disease, administration route and target
molecule. Therefore, a dose lower than the range may be sufficient
in some occasion and conversely, a dose higher than the range may
be required occasionally. The composition can be administered from
once to several times daily or at intervals from one day to several
days.
[0220] In still another embodiment of the composition of the
present invention, there is provided a pharmaceutical composition
comprising a vector capable of expressing the oligonucleotide of
the present invention and the carrier described above. Such an
expression vector may be a vector capable of expressing a plurality
of the oligonucleotides of the present invention. The composition
may be formulated with pharmaceutically acceptable additives as in
the case with the composition of the present invention containing
the oligomer of the present invention. A concentration of the
expression vector contained in the composition may vary depending
upon type of the career, etc., and is appropriately in a range of
0.1 nM to 100 .mu.M, preferably in a range of 1 nM to 10 .mu.M, and
more preferably in a range of 10 nM to 1 .mu.M. A weight ratio of
the expression vector contained in the composition and the carrier
(carrier/expression vector) may vary depending on property of the
expression vector, type of the carrier, etc., and is appropriately
in a range of 0.1 to 100, preferably in a range of 1 to 50, and
more preferably in a range of 10 to 20. The content of the carrier
contained in the composition is the same as in the case with the
composition of the present invention containing the oligomer of the
present invention, and a method for producing the same is also the
same as in the case with the composition of the present
invention.
EXAMPLES
[0221] Hereinafter, the present invention will be described in more
detail with reference to EXAMPLES and TEST EXAMPLES below, but is
not deemed to be limited thereto.
Reference Example 1
4-{[(2S,
6R)-6-(4-Benzamido-2-oxopyrimidin-1-yl)-4-tritylmorpholine-2-yl]
methoxy}-4-oxobutanoic acid loaded onto amino polystyrene resin
Step 1: Production of 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1
(2H)--O-4-tritylmorpholine-2-yl]methoxy}-4-oxobutanoic acid
[0222] Under argon atmosphere, 3.44 g of N-{1-[(2R,
6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-oxo-1,2-dihydropyrimidin--
4-yl}benzamide and 1.1 g of 4-dimethylaminopyridine (4-DMAP) were
suspended in 50 mL of dichloromethane, and 0.90 g of succinic
anhydride was added to the suspension, followed by stirring at room
temperature for 3 hours. To the reaction mixture was added 10 mL of
methanol, and the mixture was concentrated under reduced pressure.
The residue was extracted using ethyl acetate and 0.5 M aqueous
potassium dihydrogenphosphate solution. The resulting organic layer
was washed sequentially with 0.5 M aqueous potassium
dihydrogenphosphate solution, water and brine in the order
mentioned. The resulting organic layer was dried over sodium
sulfate and concentrated under reduced pressure to give 4.0 g of
the product.
Step 2; Production of
4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]me-
thoxy}-4-oxobutanoic Acid Loaded onto Amino Polystyrene Resin
[0223] After 4.0 g of 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1
(2H)-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid was
dissolved in 200 mL of pyridine (dehydrated), 0.73 g of 4-DMAP and
11.5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride were added to the solution. Then, 25.0 g of amino
polystyrene resin Primer support 200 amino (manufactured GE
Healthcare Japan Co., Ltd., 17-5214-97) and 8.5 mL of triethylamine
were added to the mixture, followed by shaking at room temperature
for 4 days. After completion of the reaction, the resin was taken
out by filtration. The resulting resin was washed sequentially with
pyridine, methanol and dichloromethane in the order mentioned, and
dried under reduced pressure. To the resulting resin were added 200
mL of tetrahydrofuran (dehydrate), 15 mL of acetic anhydride and 15
mL of 2,6-lutidine, and the mixture was shaken at room temperature
for 2 hours. The resin was taken out by filtration, washed
sequentially with pyridine, methanol and dichloromethane in the
order mentioned and dried under reduced pressure to give 26.7 g of
the product.
[0224] The loading amount of the product was determined from the
molar amount of the trityl per g resin by measuring UV absorbance
at 409 nm using a known method. The loading amount of the resin was
192.2 .mu.mol/g.
Conditions of UV Measurement
[0225] Apparatus: U-2910 (Hitachi, Ltd.)
[0226] Solvent: methanesulfonic acid
[0227] Wavelength: 265 nm
[0228] Wavelength: 26
Reference Example 2
4-[[(2S,
6R)-6-[6-(2-Cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purine-9-yl]-4-
-tritylmorpholin-2-yl]methoxy]-4-oxo-butanoic acid loaded onto
aminopolystyrene resin
Step 1: Production of N.sup.2-(phenoxyacetyl) guanosine
[0229] Guanosine, 100 g, was dried at 80.degree. C. under reduced
pressure for 24 hours. After 500 mL of pyridine (anhydrous) and 500
mL of dichloromethane (anhydrous) were added thereto, 401 mL of
chlorotrimethylsilane was dropwise added to the mixture under an
argon atmosphere at 0.degree. C., followed by stirring at room
temperature for 3 hours. The mixture was again ice-cooled and 66.3
g of phenoxyacetyl chloride was dropwise added thereto. Under ice
cooling, the mixture was stirred for further 3 hours. To the
reaction solution was added 500 mL of methanol, and the mixture was
stirred at room temperature overnight. The solvent was then removed
by distillation under reduced pressure. The residue was added with
500 mL of methanol and concentrated under reduced pressure, the
process was performed 3 times. To the residue was added 4 L of
water, and the mixture was stirred for an hour under ice cooling.
The precipitates formed were taken out by filtration, washed
sequentially with water and cold methanol and then dried to give
150.2 g of the objective compound (yield 102%)(cf.: Org. Lett.
(2004), Vol. 6, No. 15, 2555-2557).
Step 2:
N.sup.9-{[(2R,6S)-6-(hydroxymethyl)-4-morpholin-2-yl]-6-oxo-6,9-di-
hydro-1H-purin-2-yl}-2-phenoxyacetamide p-toluenesulfonate
[0230] In 480 mL of methanol was suspended 30 g of the compound
obtained in Step 1, and 130 mL of 2N hydrochloric acid was added to
the suspension under ice cooling. Subsequently, 56.8 g of ammonium
tetraborate tetrahydrate and 16.2 g of sodium periodate were added
to the mixture in the order mentioned and the mixture was stirred
at room temperature for 3 hours. The reaction mixture was ice
cooled and the insoluble matters were removed off by filtration,
followed by washing with 100 mL of methanol. The filtrate and
washing liquid were combined and the mixture was ice cooled. To the
mixture was added 11.52 g of 2-picoline borane. After stirring for
20 minutes, 54.6 g of p-toluenesulfonic acid monohydrate was slowly
added to the mixture, followed by stirring at 4.degree. C.
overnight. The precipitates formed were taken out by filtration and
washed with 500 mL of cold methanol and dried to give 17.7 g of the
objective compound (yield: 43.3%).
[0231] .sup.1H NMR (ht. The precipitates were taken o35 (1H, s),
7.55 (2H, m), 7.35 (2H, m), 7.10 (2H, d, J=7.82 Hz), 7.00 (3H, m),
5.95 (1H, dd, J=10.64, 2.42 Hz), 4.85 (2H, s), 4.00 (1H, m),
3.90-3.60 (2H, m), 3.50-3.20 (5H, m), 2.90 (1H, m), 2.25 (3H,
s)
Step 3: Production of N.sup.9-{(2R,
6S)-6-hydroxymethyl-4-tritylmorpholine-2-yl}-N.sup.2-(phenoxyacetyl)
guanine
[0232] In dichloromethane (30 mL) was suspended 2.0 g of the
compound (2.0 g) obtained by Step 2, and triethylamine (13.9 g) and
trityl chloride (18.3 g) were added to the suspension under ice
cooling. The mixture was stirred at room temperature for an hour.
The reaction mixture was washed with saturated sodium bicarbonate
aqueous solution and then with water. The organic layer was
collected, dried over magnesium sulfate and concentrated under
reduced pressure. To the residue was added 0.2 M sodium citrate
buffer (pH 3)/methanol (1:4 (v/v), 40 mL), and the mixture was
stirred. Subsequently, water (40 mL) was added and the suspension
mixture was stirred for an hour under ice cooling. The precipitates
were taken out by filtration, washed with cold methanol and dried
to give 1.84 g of the objective compound (yield: 82.0%).
Step 4: Production of N.sup.9-[(2R,
6S)-6-{(tert-butyldimethylsilyloxy)methyl}-4-tritylmorpholin-2-yl]-N.sup.-
2-(phenoxyacetyl) guanine
[0233] In dichloromethane (300 mL) was dissolved the compound (38.3
g) obtained by Step 3, and imidazole (4.64 g) and
t-butyldimethylsilyl chloride (9.47 g) were added to the solution
in this order mentioned under ice cooling. The reaction solution
was stirred at room temperature for an hour. The reaction solution
was washed with 0.2 M sodium citrate buffer (pH 3) and then with
brine. The organic layer was collected, dried over magnesium
sulfate and concentrated under reduced pressure to give 44.1 g of
the objective compound as a crude product.
Step 5: Production of N.sup.9-[(2R,
65)-6-{(tert-butyldimethylsilyloxy)methyl}-4-tritylmorpholin-2-yl]-N.sup.-
2-(phenoxyacetyl)-O.sup.6-triisopropylbenzenesulfonyl guanine
[0234] In dichloromethane (300 mL) was dissolved the compound (44.1
g) obtained by Step 4, and 4-dimethylaminopyridine (0.64 g),
triethylamine (29.2 mL) and triisopropylbenzensulfonyl chloride
(19.0 g) were added to the solution under ice cooling. The reaction
solution was stirred at room temperature for an hour. The reaction
solution was washed with 1 M aqueous sodium dihydrogenphosphate
solution. The organic layer was collected, dried over magnesium
sulfate and concentrated under reduced pressure to give 60.5 g of
the objective compound as a crude product.
Step 6: Production of N.sup.9-[(2R,
6S)-6-{(tert-butyldimethylsilyloxy)
methyl}-4-tritylmorpholin-2-yl]-N.sup.2-(phenoxyacetyl)-O.sup.6-(2-cyanoe-
thyl) guanine
[0235] In dichloromethane (300 mL) was dissolved the compound (60.5
g) obtained by Step 5, and N-methylpyrrolidine (54.5 mL) was added
to the solution under ice cooling. The reaction solution was
stirred under ice cooling for an hour. Then, ethylene cyanohydrin
(37.2 g), and 1,8-diazabicyclo [5.4.0] undec-7-ene (11.96 g) were
added to the solution, and the solution was stirred under ice
cooling for 2 hours. The reaction solution was washed with 1 M
sodium dihydrogenphosphate solution and then with water. The
organic layer was collected, dried over magnesium sulfate and
concentrated under reduced pressure to give 72.4 g of the objective
compound as a crude product.
Step 7: Production of N.sup.9-[(2R,
6S)-6-hydroxymethyl-4-tritylmorpholin-2-yl]-N.sup.2-(phenoxyacetyl)-O.sup-
.6-(2-cyanoethyl) guanine
[0236] In dichloromethane (300 mL) was dissolved the compound (72.4
g) obtained in Step 6, and triethylaminetrihydrofluoride (21.1 g)
was added to the solution. The reaction solution was stirred at
room temperature for 17 hours. The reaction solution was poured
into cold saturated sodiumbicarbonate aqueous solution to
neutralize the reaction solution. Then, the dichloromethane layer
was collected, dried over magnesium sulfate and concentrated under
reduced pressure. The residue was purified by a silica gel column
chromatography (PSQ100B (manufactured by FUJI SILYSIA CHEMICAL LTD.
The same shall apply hereinafter.)) to give 14.3 g of the objective
compound (yield from Step 4: 39.2%).
Step 8: Production of 4-[[(2S,
6S)-6-[6-(2-cyanoethoxy)-2-(2-phenoxyacetyl)
amino]purin-9-yl]-4-tritylmorpholin-2-yl] methoxy]-4-oxo-butanoic
acid loaded onto amino polystyrene resin
[0237] The title compound was produced in a manner similar to
REFERENCE EXAMPLE 1, except that
N.sup.9-[(2R,6S)-6-hydroxymethyl-4-tritylmorpholin-2-yl]-N.sup.2-(phenoxy-
acetyl)-O.sup.6-(2-cyanoeth yl) guanine was used in this step,
instead of N-{1-[(2R,
6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-oxo-1,2-dihydropyrimidin--
4-yl}benzamide used in Step 1 of REFERENCE EXAMPLE 1.
Reference Example 3
4-{[(2S,6R)-6-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1-yl)-4-tritylmorph-
olin-2-yl]methoxy}-4-oxobutanoic acid loaded onto aminopolystyrene
resin
[0238] The title compound was produced in a manner similar to
REFERENCE EXAMPLE 1, except that 1-[(2R,
6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-5-methylpyrimidine-2,4
(1H, 3H)-dione was used in this step, instead of
N-{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-oxo-1,2-dihydro-
pyrimidin-4-yl}benzamide used in Step 1 of REFERENCE EXAMPLE 1.
Reference Example 4
4-{[(2S,6R)-6-(6-benzamidepurine-9-yl)-4-tritylmorpholin-2-yl]methoxy}-4-o-
xobutanoic Acid Loaded onto Aminopolystyrene Resin
[0239] The title compound was produced in a manner similar to
REFERENCE EXAMPLE 1, except that N-{9-[(2R,
6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]purine-6-yl} benzamide
was used in this step, instead of N-{1-[(2R,
6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-oxo-1,2-dihydropyrimidin--
4-yl} benzamide used in Step 1 of REFERENCE EXAMPLE 1.
Reference Example 5
1,12-Dioxo-1-(4-tritylpiperazin-1-yl)-2,5,8,11-tetraoxa-15-pentadecanoic
acid loaded onto aminopolystyrene resin
[0240] The title compound was produced in a manner similar to
REFERENCE EXAMPLE 1, except that 2-[2-(2-hydroxyethoxy)ethoxy]ethyl
4-tritylpiperazine-1-carboxylic acid (the compound described in
WO2009/064471) was used in this step, instead of
N-{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-oxo-1,2-dihydro-
pyrimidin-4-yl} benzamide used in Step 1 of REFERENCE EXAMPLE
1.
EXON 45
[0241] According to the descriptions in EXAMPLES 1 to 8 and
REFERENCE EXAMPLE 1 below, various types of PMO shown by PMO Nos.
1-6 and 8-10 in TABLE 5 were synthesized. The PMO synthesized was
dissolved in water for injection (manufactured by Otsuka
Pharmaceutical Factory, Inc.). PMO No. 7 was purchased from Gene
Tools, LLC.
TABLE-US-00005 TABLE 5 SEQ PMO ID No. Sequence name Note NO: 1
H45_-2-19(OH) 5' end: group (3) 9 2 H45_-1-20(OH) 5' end: group (3)
10 3 H45_1-21(OH) 5' end: group (3) 11 4 H45_2-22(OH) 5' end: group
(3) 12 5 H45_3-23(OH) 5' end: group (3) 13 6 H45_-421(OH) Sequence
corresponding 14 to SEQ ID NO: 30 in Patent Document 4, 5' end:
group (3) 7 H45_5-34(GT) Sequence corresponding 15 to SEQ ID NO: 4
in Patent Document 3, 5' end: group (2) 8 H45_1-20(OH) 5' end:
group (3) 16 9 H45_2-21(OH) 5' end: group (3) 17 10 H45 1-21(TEG)
5' end: group (1) 18
EXAMPLE
PMO No. 1
[0242] 0.2 g 4-{[(2S,
6R)-6-(4-benzamide-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl]methox-
y}4-oxobutanoic acid supported on an aminopolystyrene resin
(Reference Example 1) (26 .mu.mol) was filled in a column with a
filter tip. Then, the synthetic cycle shown below was started using
an nucleic acid synthesizing machine (AKTA Oligopilot 10 plus). The
desired morpholino monomer compound was added in each coupling
cycle to give the nucleotide sequence of the title compound.
TABLE-US-00006 TABLE 6 Volume Time Step Reagent (mL) (min) 1
deblocking solution 18-32 1.8-3.2 2 neutralizing and 30 1.5 washing
solution 3 coupling solution B 5 0.5 4 coupling solution A 1.3 0.25
5 mixture of step 3 and 6.3 120-300 step 4 reagents mixture 6
acetonitrile 20 1.0 7 capping solution 9 2.0 8 acetonitrile 30
2.0
[0243] The deblocking solution used was dichloromethane containing
3% (w/v) trifluoroacetic acid. The neutralizing and washing
solution used was a solution obtained by dissolving
N,N-diisopropylethylamine to be 10% (v/v) and tetrahydrofuran to be
5% (v/v) in dichloromethane containing 35% (v/v) acetonitrile. The
coupling solution A used was a solution obtained by dissolving the
morpholino monomer compound in tetrahydrofuran to be 0.10 M. The
coupling solution B used was a solution obtained by dissolving
N,N-diisopropylethylamine to be 20% (v/v) and tetrahydrofuran to be
10% (v/v) in acetonitrile. The capping solution used was a solution
obtained by dissolving 20% (v/v) acetic anhydride and 30% (v/v)
2,6-lutidine in acetonitrile.
[0244] The aminopolystyrene resin loaded with the PMO synthesized
above was recovered from the reaction vessel and dried at room
temperature for at least 2 hours under reduced pressure. The dried
PMO loaded onto aminopolystyrene resin was charged in a reaction
vessel, and 5 mL of 28% ammonia water-ethanol (1/4) was added
thereto. The mixture was stirred at 55.degree. C. for 15 hours. The
aminopolystyrene resin was separated by filtration and washed with
1 mL of water-ethanol (1/4). The resulting filtrate was
concentrated under reduced pressure. The resulting residue was
dissolved in 10 mL of a solvent mixture of 20 mM of acetic
acid--triethylamine buffer (TEAA buffer) and 10 ml of acetonitrile
(4/1) and filtered through a membrane filter. The filtrate obtained
was purified by reversed phase HPLC. The conditions used are as
follows.
TABLE-US-00007 TABLE 7 Column XBridge 5 .mu.m C18 (Waters, .PHI. 19
.times. 50 mm, 1 CV = 14 mL) Flow rate 10 mL/min Column room
temperature temperature Solution A 20 mM TEAA buffer Solution B
CH.sub.3CN Gradient (B) conc. 10.fwdarw.70%/15 CV
[0245] Each fraction was analyzed, and the objective product was
recovered and concentrated under reduced pressure. To the
concentrated residue was added 0.5 mL of 2 M phosphoric acid
aqueous solution, and the mixture was stirred for 15 minutes.
Furthermore, 2 mL of 2 M sodium hydroxide aqueous solution was
added to make the mixture alkaline, followed by filtration through
a membrane filter (0.45 .mu.m).
[0246] The resulting aqueous solution containing the objective
product was purified by an anionic exchange resin column. The
conditions used are as follows.
TABLE-US-00008 TABLE 8 Column Source 15Q (GE Healthcare, .PHI. 10
.times. 108 mm, 1 CV = 8.5 mL) Flow rate 8.5 mL/min Column
temperature room temperature Solution A 10 mM sodium hydroxide
aqueous solution Solution B 10 mM sodium hydroxide aqueous
solution, 1M sodium chloride aqueous solution Gradient (B) conc.
1.fwdarw.50%/40 CV
[0247] Each fraction was analyzed (on HPLC) and the objective
product was obtained as an aqueous solution. To the resulting
aqueous solution was added 0.1 M phosphate buffer (pH 6.0) for
neutralization. Next, the mixture obtained was demineralized by
reversed phase HPLC under the conditions described below.
TABLE-US-00009 TABLE 9 Column XBridge 5 .mu.m C8 (Waters, .PHI. 10
.times. 50 mm, 1 CV = 4 mL) Flow rate 4 mL/min Column temperature
60.degree. C. Solution A water Solution B CH.sub.3CN Gradient (B)
conc. 0.fwdarw.50%/20 CV
[0248] The objective product was recovered and the mixture was
concentrated under reduced pressure. The resulting residue was
dissolved in water. The aqueous solution obtained was freeze-dried
to give 1.5 mg of the objective compound as a white cotton-like
solid.
[0249] ESI-TOF-MS Clcd.: 6877.8
[0250] Found: 6877.4
Example 2
PMO. No. 3
[0251] The title compound was produced in accordance with the
procedure of EXAMPLE 1.
[0252] ESI-TOF-MS Clcd.: 6862.8
[0253] Found: 6862.5
Example 31
PMO. No. 2
[0254] The title compound was produced in accordance with the
procedure of EXAMPLE 1.
[0255] ESI-TOF-MS Clcd.: 6862.8
[0256] Found: 6862.3
Example 4
PMO. No. 4
[0257] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-[[(2S,
6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purin-9-yl]-4-tritylmo-
rpholin-2-yl]methoxy]-4-oxo-butanoic acid loaded onto
aminopolystyrene resin (REFERENCE EXAMPLE 2) was used as the
starting material.
[0258] ESI-TOF-MS Clcd.: 6902.8
[0259] Found: 6902.3
Example 5
PMO. No. 5
[0260] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-(((2S,
6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorphol-
in-2-yl)methoxy)-4-oxobutanoic acid loaded onto aminopolystyrene
resin (REFERENCE EXAMPLE 3) was used as the starting material.
[0261] ESI-TOF-MS Clcd.: 6902.8
[0262] Found: 6902.4
Example 6
PMO. No. 8
[0263] The title compound was produced in accordance with the
procedure of EXAMPLE 1.
[0264] ESI-TOF-MS Clcd.: 6547.5
[0265] Found: 6547.2
Example 7
PMO. No. 9
[0266] The title compound was produced in accordance with the
procedure of EXAMPLE 1.
[0267] ESI-TOF-MS Clcd.: 6547.5
[0268] Found: 6547.2
Example 8
[0269] PMO. No. 10
[0270] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 1,
12-Dioxo-1-(4-tritylpiperazin-1-yl)-2,5,8,11-tetraoxa-15-pentadecanoic
acid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 5) was
used as the starting material.
[0271] ESI-TOF-MS Clcd.: 7214.1
[0272] Found: 7213.7
Comparative Example 1
PMO. No. 6
[0273] The title compound was produced in accordance with the
procedure of EXAMPLE 1.
[0274] ESI-TOF-MS Clcd.: 8193.9
[0275] Found: 8195.3
Test Example 1
In Vitro Assay
[0276] Experiments were performed using the antisense oligomers of
2'-O-methoxy-phosphorothioates (2'-OMe-S-RNA) shown by SEQ ID NO:
19 to SEQ ID NO: 35. Various antisense oligomers used for the assay
were purchased from Japan Bio Services. The sequences of various
antisense oligomers are given below.
TABLE-US-00010 TABLE 10 Antisense SEQ ID oligomer Nucleotide
sequence NO: H45_1-25 GCUGCCCAAUGCCAUCCUGGAGUUC 19 H45_6-30
UUGCCGCUGCCCAAUGCCAUCCUGG 20 H45_11-35 ACAGUUUGCCGCUGCCCAAUGCCAU 21
H45_16-40 UGACAACAGUUUGCCGCUGCCCAAU 22 H45_21-45
UGUUCUGACAACAGUUUGCCGCUGC 23 H45_26-50 UUCAAUGUUCUGACAACAGUUUGCC 24
H45_31-55 UUGCAUUCAAUGUUCUGACAACAGU 25 H45_36-60
CCCAGUUGCAUUCAAUGUUCUGACA 26 H45_41-65 UCUUCCCCAGUUGCAUUCAAUGUUC 27
H45_46-70 UUAUUUCUUCCCCAGUUGCAUUCAA 28 H45_51-75
CUGAAUUAUUUCUUCCCCAGUUGCA 29 H45_56-80 GAUUGCUGAAUUAUUUCUUCCCCAG 30
H45_61-85 UUGAGGAUUGCUGAAUUAUUUCUUC 31 H45_66-90
UGUUUUUGAGGAUUGCUGAAUUAUU 32 H45_71-95 GCAUCUGUUUUUGAGGAUUGCUGAA 33
H45_76-100 UACUGGCAUCUGUUUUUGAGGAUUG 34 H45_7-31
UUUGCCGCUGCCCAAUGCCAUCCUG 35
[0277] RD cells (human rhabdomyosarcoma cell line) were plated at
1.times.10.sup.5 in a 12-well plate and cultured in 1 mL of Eagle's
minimal essential medium (EMEM) (manufactured by Sigma, Inc.,
hereinafter the same) containing 10% fetal calf serum (FCS)
(manufactured by Invitrogen Corp.) under conditions of 37.degree.
C. and 5% CO2 overnight. Complexes of various antisense oligomers
(Japan Bio Services) (0.3 or 1 .mu.M) for exon 45 skipping and
Lipofectamine 2000 (manufactured by Invitrogen Corp.) were prepared
and 1004 of the complex was added to RD cells where 0.9 mL of the
medium was exchanged, to reach the final concentration of 30 or 100
nM.
[0278] After completion of the addition, the cells were cultured
overnight. The cells were washed twice with PBS (manufactured by
Nissui, hereafter the same) and then 250 .mu.L of ISOGEN
(manufactured by Nippon Gene) was added to the cells. After the
cells were allowed to stand at room temperature for a few minutes
for cell lysis, the lysate was collected in an Eppendorf tube. The
total RNA was extracted according th the protocol attached to
ISOGEN. The concentration of the total RNA extracted was determined
using a NanoDrop ND-1000 (manufactured by LMS).
[0279] RT-PCR was performed with 400 ng of the extracted total RNA
using a QIAGEN OneStep RT-PCR Kit. A reaction solution was prepared
in accordance with the protocol attached to the kit. A PTC-100
(manufactured by MJ Research) was used as a thermal cycler. The
RT-PCR program used is as follows.
[0280] 50.degree. C., 30 mins: reverse transcription
[0281] 94.degree. C., 15 mins: thermal denaturation
[0282] [94.degree. C., 30 seconds; 60.degree. C., 30 seconds;
72.degree. C., 1 min].times.35 cycles: PCR amplification
[0283] 72.degree. C., 10 mins:
[0284] The nucleotide sequences of the forward primer and reverse
primer used for RT-PCR are given below.
TABLE-US-00011 Forward primer: (SEQ ID NO: 36)
5'-GCTCAGGTCGGATTGACATT-3' Reverse primer: (SEQ ID NO: 37)
5'-GGGCAACTCTTCCACCAGTA-3'
[0285] The reaction product, 1 .mu.L of the PCR above was analyzed
using a Bioanalyzer (manufactured by Agilent Technologies, Inc.).
The polynucleotide level "A" of the band with exon 45 skipping and
the polynucleotide level "B" of the band without exon 45 skipping
were measured. Based on these measurement values of "A" and "B",
the skipping efficiency was determined by the following
equation:
Skipping efficiency (%)=A/(A+B).times.100
Experimental Results
[0286] The results are shown in FIGS. 1 and 2. These experiments
revealed that, when the antisense oligomers were designed at the
1st to the 25th, or the 6th to the 30th nucleotides from the 5' end
of exon 45 in the human dystrophin gene, exon 45 skipping could be
caused with a higher efficiency than that of the antisense oligomer
which is designed at the 7th to the 31st nucleotides from the 5'
end of exon 45.
Test Example 2
In Vitro Assay
[0287] Using an Amaxa Cell Line Nucleofector Kit L on Nucleofector
II (Lonza), 1, 3, or 10 .mu.M of the oligomers PMO Nos. 1 to 5 and
8 to 10 of the present invention and the antisense oligomers PMO
Nos. 6 and 7 were transfected with 3.5.times.10.sup.5 of RD cells
(human rhabdomyosarcoma cell line). The Program T-030 was used.
[0288] After transfection, the cells were cultured for 3 days in 2
mL of Eagle's minimal essential medium (EMEM) (manufactured by
Sigma, hereinafter the same) containing 10% fetal calf serum (FCS)
(manufactured by Invitrogen) under conditions of 37.degree. C. and
5% CO.sub.2. The cells were washed twice with PBS (manufactured by
Nissui, hereinafter the same) and 500 .mu.L of ISOGEN (manufactured
by Nippon Gene) was added to the cells. After the cells were
allowed to stand at room temperature for a few minutes to lyse the
cells, the lysate was collected in an Eppendorf tube. The total RNA
was extracted according to the protocol attached to ISOGEN. The
concentration of the total RNA extracted was determined using a
NanoDrop ND-1000 (manufactured by LMS).
[0289] RT-PCR was performed with 400 ng of the extracted total RNA
using a QIAGEN OneStep RT-PCR Kit (manufactured by QIAGEN). A
reaction solution was prepared in accordance with the protocol
attached to the kit. A PTC-100 (manufactured by MJ Research) was
used as a thermal cycler. The RT-PCR program used is as
follows.
[0290] 50.degree. C., 30 mins: reverse transcription
[0291] 95.degree. C., 15 mins; thermal denaturation
[0292] [94.degree. C., 30 seconds; 60.degree. C., 30 seconds;
72.degree. C., 1 min].times.35 cycles: PCR amplification
[0293] 72.degree. C., 10 mins
[0294] The nucleotide sequences of the forward primer and reverse
primer used for RT-PCR are given below.
TABLE-US-00012 Forward primer: (SEQ ID NO: 36)
5'-GCTCAGGTCGGATTGACATT-3' Reverse primer: (SEQ ID NO: 37)
5'-GGGCAACTCTTCCACCAGTA-3'
The reaction product, 1 .mu.L, of the PCR above was analyzed using
a Bioanalyzer (manufactured by Agilent Technologies, Inc.).
[0295] The polynucleotide level "A" of the band with exon 45
skipping and the polynucleotide level "B" of the band without exon
45 skipping were measured. Based on these measurement values of "A"
and "B", the skipping efficiency was determined by the following
equation:
Skipping efficiency (%)=A/(A+B).times.100
Experimental Results
[0296] The results are shown in FIGS. 3, 4, 14 and 15. These
experiments revealed that the oligomers PMO Nos. 1 and 3 of the
present invention caused exon 45 skipping with a equivalent
efficiency to the antisense oligomer PMO No. 6 in RD cells (FIG. 3,
4). In addition, the experiments revealed that the oligomers PMO
Nos. 1, 2 and 3 of the present invention caused exon 45 skipping
with a higher efficiency than the antisense oligomer PMO No. 7
(FIG. 14). Furthermore, the experiments revealed that the oligomer
PMO No. 3 caused exon 45 skipping with a higher efficiency than the
antisense oligomer PMO No. 10 whose end structure is different from
that of PMO No. 3 (FIG. 15).
Test Example 3
In Vitro Assay Using Human Fibroblasts
[0297] Human myoD gene (SEQ ID NO: 38) was introduced into the
GM05017 cells (human DMD-patient derived fibroblasts, Coriell
Institute for Medical Research) using a ZsGreen1 coexpression
retroviral vector.
[0298] After incubation for 4 to 5 days, ZsGreen-positive
MyoD-transformed fibroblasts were collected by FACS and plated at
5.times.10.sup.4/cm.sup.2 into a 12-well plate. As a growth medium,
there was used 1 mL of Dulbecco's Modified Eagle Medium:Nutrient
Mixture F-12 (DMEM .cndot. F-12) (Invitrogen Corp.) containing 10%
FCS and 1% Penicillin/Streptomycin (P/S) (Sigma-Aldrich, Inc).
[0299] The medium was replaced 24 hours later by a differentiation
medium (DMEM/F-12 containing 2% equine serum (Invitrogen Corp.), 1%
P/S and ITS Liquid Media Supplement (Sigma, Inc.)). The medium was
exchanged every 2 to 3 days and incubation was continued for 12 to
14 days to differentiate into myotubes.
[0300] Subsequently, the differentiation medium was replaced by a
differentiation medium containing 6 .mu.M Endo-Porter (Gene Tools),
and a morpholino oligomer was added thereto at a final
concentration of 10 .mu.M. After incubation for 48 hours, total RNA
was extracted from the cells using a TRIzol (manufactured by
Invitrogen Corp.). RT-PCR was performed with 50 ng of the extracted
total RNA using a QIAGEN OneStep RT-PCR Kit. A reaction solution
was prepared in accordance with the protocol attached to the kit.
An iCycler (manufactured by Bio-Rad) was used as a thermal cycler.
The RT-PCR program used is as follows.
[0301] 50.degree. C., 30 mins: reverse transcription
[0302] 95.degree. C., 15 mins: thermal denaturation
[0303] [94.degree. C., 1 mins; 60.degree. C., 1 mins; 72.degree.
C., 1 mins].times..times.35 cycles: PCR amplification
[0304] 72.degree. C., 7 mins: thermal inactivation of
polymerase
[0305] The primers used were hDMD44F and hDMD46R.
TABLE-US-00013 hDMD44F: (SEQ ID NO: 39) 5'-CCTGAGAATTGGGAACATGC-3'
hDMD46R: (SEQ ID NO: 40) 5'-TTGCTGCTCTTTTCCAGGTT-3'
[0306] The reaction product of RT-PCR above was separated by 2%
agarose gel electrophoresis and gel images were captured with a
GeneFlash (Syngene). The polynucleotide level "A" of the band with
exon 45 skipping and the polynucleotide level "B" of the band
without exon 45 skipping were measured using an Image J
(manufactured by National Institutes of Health). Based on these
measurement values of "A" and "B," the skipping efficiency was
determined by the following equation.
Skipping efficiency (%)=A/(A+B).times.100
Experimental Results
[0307] The results are shown in FIG. 5. This experiment revealed
that the oligomer PMO Nos. 3 of the present invention caused exon
45 skipping with a high efficiency in GM05017 cells.
EXON 55
[0308] According to the descriptions in EXAMPLES 9 to 19 below,
various types of PMO shown by PMO Nos. 11-14 and 16-22 in TABLE 11
were synthesized. The PMO synthesized was dissolved in water for
injection (manufactured by Otsuka Pharmaceutical Factory, Inc.).
PMO No. 15 was purchased from Gene Tools, LLC.
TABLE-US-00014 TABLE 11 SEQ PMO ID No. Sequence name Note NO: 11
H55_2-22(OH) 5' end: group (3) 41 12 H55_8-28(OH) 5' end: group (3)
42 13 H55_11-31(OH) 5' end: group (3) 43 14 H55_14-34(OH) 5' end:
group (3) 44 15 H55_139-156(GT) Sequence corresponding to 115
h55AON6 in Patent Docu- ment 5, 5' end: group (2) 16 H55_12-32(OH)
5' end: group (3) 45 17 H55_13-33(OH) 5' end: group (3) 46 18
H55_15-35(0E) 5' end: group (3) 47 19 H55_16-36(OH) 5' end: group
(3) 48 20 H55_14-33(OH) 5' end: group (3) 116 21 H55_15-34(OH) 5'
end: group (3) 117 22 H55_14-34(TEG) 5' end: group (3) 118
Example 91
PMO. No. 11
[0309] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-{[(2S, 6R)-6-(6-benzamide
prine-9-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid
loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 4) was used
as the starting material.
[0310] ESI-TOF-MS Clcd.: 6807.8
[0311] Found: 6807.0
Example 10
PMO. No. 12
[0312] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-(((2S,
6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorphol-
in-2-yl) methoxy)-4-oxobutanoic acid loaded onto aminopolystyrene
resin (REFERENCE EXAMPLE 3) was used as the starting material.
[0313] ESI-TOF-MS Clcd.: 6822.8
[0314] Found: 6822.5
Example 11
PMO. No. 13
[0315] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-(((2S,
6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)--O-4-tritylmorpholi-
n-2-yl) methoxy)-4-oxobutanoic acid loaded onto aminopolystyrene
resin (REFERENCE EXAMPLE 3) was used as the starting material.
[0316] ESI-TOF-MS Clcd.: 6837.8
[0317] Found: 6837.3
Example 12
PMO. No. 14
[0318] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-[[(2S, 6R)-6-(6-benzamide
prine-9-yl)-4-tritylmorpholin-2-yl]methoxy]-4-oxobutanoic acid
loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 4) was used
as the starting material.
[0319] ESI-TOF-MS Clcd.: 6861.8
[0320] Found: 6861.4
Example 13
PMO. No. 16
[0321] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-(((2S,
6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorphol-
in-2-yl)methoxy)-4-oxobutanoic acid loaded onto aminopolystyrene
resin (REFERENCE EXAMPLE 3) was used as the starting material.
[0322] ESI-TOF-MS Clcd.: 6812.8
[0323] Found: 6812.7
Example 14
PMO. No. 17
[0324] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-[[(2S,
6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purine-9-yl]-4-tritylm-
orpholin-2-yl]methoxy]-4-oxo-butanoic acid loaded onto
aminopolystyrene resin (REFERENCE EXAMPLE 2) was used as the
starting material.
[0325] ESI-TOF-MS Clcd.: 6852.8
[0326] Found: 6852.7
Example 15
PMO. No. 18
[0327] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that
4-[[(2S,6R)-6-[6-(2-cyanoethodxy)-2-[(2-phenoxyacetyl)
amino]purine-9-yl]-4-tritylmorpholin-2-yl]methoxy]-1-4-oxo-butanoic
acid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 2) was
used as the starting material.
[0328] ESI-TOF-MS Clcd.: 6901.8
[0329] Found: 6901.5
Example 16
PMO. No. 19
[0330] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-(((2S,
6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1
(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)-4-oxobutanoic acid loaded
onto aminopolystyrene resin (REFERENCE EXAMPLE 3) was used as the
starting material.
[0331] ESI-TOF-MS Clcd.: 6901.8
[0332] Found: 6901.7
Example 17
PMO. No. 20
[0333] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-[[(2S,
6R)-6-[6-(2-Cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purine-9-yl]-4-tritylm-
orpholin-2-yl]methoxy]-4-oxo-butanoic acid loaded onto
aminopolystyrene resin (REFERENCE EXAMPLE 2) was used as the
starting material.
[0334] ESI-TOF-MS Clcd.: 6522.5
[0335] Found: 6522.0
Example 18
PMO. No. 21
[0336] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-{[(2S, 6R)-6-(6-benzamide
prine-9-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid
loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 4) was used
as the starting material.
[0337] ESI-TOF-MS Clcd.: 6546.5
[0338] Found: 6546.0
Example 19
PMO. No. 22
[0339] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 1,
12-dioxo-1-(4-tritylpiperazin-1-yl)-2,5,8,
11-tetraoxa-15-pentadecanoic acid (REFERENCE EXAMPLE 5) loaded onto
aminopolystyrene resin was used as the starting material.
[0340] ESI-TOF-MS Clcd.: 7213.1
[0341] Found: 7212.5
Test Example 41
In Vitro Assay
[0342] Experiments were performed using the antisense oligomers of
2'-O-methoxy-phosphorothioates (2'-OMe-S-RNA) shown by SEQ ID NO:
49 to SEQ ID NO: 68. Various antisense oligomers used for the assay
were purchased from Japan Bio Services. The sequences of various
antisense oligomers are given below.
TABLE-US-00015 TABLE 12 Antisense SEQ ID oligomer Nucleotide
sequence NO: H55_1-21 GCAGCCUCUCGCUCACUCACC 49 H55_6-26
CCAAAGCAGCCUCUCGCUCAC 50 H55_11-31 UUCUUCCAAAGCAGCCUCUCG 51
H55_21-41 AUCUAUGAGUUUCUUCCAAAG 52 H55_31-51 UGUUGCAGUAAUCUAUGAGUU
53 H55_41-61 CAGGGGGAACUGUUGCAGUAA 54 H55_51-71
UUUCCAGGUCCAGGGGGAACU 55 H55_61-81 GCAAGAAACUUUUCCAGGUCC 56
H55_71-91 UGUAAGCCAGGCAAGAAACUU 57 H55_81-101 UUUCAGCUUCUGUAAGCCAGG
58 H55_91-111 UUGGCAGUUGUUUCAGCUUCU 59 H55_101-121
CUGUAGGACAUUGGCAGUUGU 60 H55_111-131 GGGUAGCAUCCUGUAGGACAU 61
H55_121-141 CUUUCCUUACGGGUAGCAUCC 62 H55_131-151
UUCUAGGAGCCUUUCCUUACG 63 H55_141-161 CCUUGGAGUCUUCUAGGAGCC 64
H55_151-171 UCUUUUACUCCCUUGGAGUCU 65 H55_161-181
UUUCAUCAGCUCUUUUACUCC 66 H55_171-190 UUGCCAUUGUUUCAUCAGCU 67
H55_104-123 UCCUGUAGGACAUUGGCAGU 68
[0343] Experiments were performed in accordance with the condition
and the procedure of Exon 45 (TEST EXAMPLE 1), except that the
RT-PCR was performed using the primers below.
TABLE-US-00016 Forward primer: (SEQ ID NO: 69)
5'-CATGGAAGGAGGGTCCCTAT-3' Reverse primer: (SEQ ID NO: 70)
5'-CTGCCGGCTTAATTCATCAT-3'
Experimental Results
[0344] The results are shown in FIGS. 6 and 7. These experiments
revealed that, when the antisense oligomers were designed at the
1st to the 21st, or the 11th to the 31st nucleotides from the 5'
end of exon 55 in the human dystrophin gene, exon 55 skipping of
these antisense oligomers could be caused with a higher efficiency
than that of the antisense oligomer which is designed at the 104th
to the 123rd nucleotides from the 5' end of exon 55.
Test Example 5
In Vitro Assay
[0345] Experiments were performed in accordance with the condition
and the procedure of exon 45 (TEST EXAMPLE 2), except that the
RT-PCR was performed using the primers below.
TABLE-US-00017 Forward primer: (SEQ ID NO: 69)
5'-CATGGAAGGAGGGTCCCTAT-3' Reverse primer: (SEQ ID NO: 70)
5'-CTGCCGGCTTAATTCATCAT-3'
Experimental Results
[0346] The results are shown in FIGS. 8, 16 and 17. These
experiments revealed that in RD cells, the oligomers PMO Nos. 12,
13 and 14 (H55_8-28 (OH), H55_11-31 (OH) and H55_14-34 (OH)) of the
present invention all caused exon 55 skipping with a high
efficiency (FIG. 8). Also, the oligomers PMO Nos. 14, 16, 17, 18
and 19 (H55_14-34 (OH), H55_12-32 (OH), H55_13-33 (OH), H55_15-35
(OH) and H55_16-36 (OH)) of the present invention were found to
cause exon 55 skipping with a notably higher efficiency than that
of the antisense oligomer PMO No. 15 (H55_139-156 (GT)) in RD cells
(FIG. 16). The oligomer PMO No. 14 of the present invention and the
oligomer PMO No. 21 (H55_15-34(OH)), which is one base shorter than
the oligomer PMO No. 14, were found to cause exon 55 skipping with
the same efficiency (FIG. 17). Furthermore, the experiments
revealed that the oligomer PMO No. 14 of the present invention
caused exon 55 skipping with the same efficiency as the oligomer
PMO No. 22 (H55_14-34 (TEG)), which has a different end structure
from that of the oligomer PMO No. 14 (FIG. 17).
EXON 44
[0347] According to the descriptions in EXAMPLES 20 to 29 below,
various types of PMO shown by PMO Nos. 23-29 and 31-33 in TABLE
below were synthesized. The PMO synthesized was dissolved in water
for injection (manufactured by Otsuka Pharmaceutical Factory,
Inc.). PMO No. 30 was purchased from Gene Tools, LLC.
TABLE-US-00018 TABLE 13 SEQ PMO ID No. Sequence name Note NO: 23
H44_23-43(OH) 5' end: group (3) 71 24 H44_25-45(OH) 5' end: group
(3) 72 25 H44_26-46(OH) 5' end: group (3) 73 26 H44_27-47(OH) 5'
end: group (3) 74 27 H44_28-48(OH) 5' end: group (3) 75 28
H44_29-49(OH) 5' end: group (3) 76 29 H44_30-50(OH) 5' end: group
(3) 77 30 H44_10-39(GT) Sequence corresponding to 78 SEQ ID NO: 1
in Patent Document 3, 5' end: group (2) 31 H44_27-46(OH) 5' end:
group (3) 79 32 H44_28-47(OH) 5' end: group (3) 80 33
H44_27-47(TEG) 5' end: group (1) 81
Example 20
PMO. No. 23
[0348] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-(((2S,
6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1
(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)-4-oxobutanoic acid loaded
onto aminopolystyrene resin (REFERENCE EXAMPLE 3) was used as the
starting material.
[0349] ESI-TOF-MS Clcd.: 6918.9
[0350] Found: 6918.3
Example 21
[0351] PMO. No. 24
[0352] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-(((2S,
6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorphom-
ethoxy)-4-oxobutanoic acid loaded onto aminopolystyrene resin
(REFERENCE EXAMPLE 3) was used as the starting material.
[0353] ESI-TOF-MS Clcd.: 6903.9
[0354] Found: 6904.2
Example 22
PMO. No. 25
[0355] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-{[(2S, 6R)-6-(6-benzamide
prine-9-yl)-4-tritylmorpholin-2-yl}methoxy]-4-oxobutanoic acid
loaded, onto aminopolystyrene resin (REFERENCE EXAMPLE 4) was used
as the starting material.
[0356] ESI-TOF-MS Clcd.: 6912.9
[0357] Found: 6912A
Example 23
PMO. No. 26
[0358] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-(((2S,
6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorphol-
in-2-yl)methoxy)-4-oxobutanoic acid loaded onto aminopolystyrene
resin (REFERENCE EXAMPLE 3) was used as the starting material.
[0359] ESI-TOF-MS Clcd.: 6903.9
[0360] Found: 6904.2
Example 24
PMO. No. 27
[0361] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-{[(2S, 6R)-6-(6-benzamide
prine-9-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid
loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 4) was used
as the starting material.
[0362] ESI-TOF-MS Clod.: 6927.9
[0363] Found: 6927.4
Example 25
PMO. No. 28
[0364] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-(((2S,
6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorphom-
ethoxy)-4-oxobutanoic acid loaded onto aminopolystyrene resin
(REFERENCE EXAMPLE 3) was used as the starting material.
[0365] ESI-TOF-MS Clcd.: 6942.9
[0366] Found: 6942.3
Example 26
PMO. No. 29
[0367] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-(((2S,
6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorphol-
in-2-yl) methoxy)-4-oxobutanoic acid loaded onto aminopolystyrene
resin (REFERENCE EXAMPLE 3) was used as the starting material.
[0368] ESI-TOF-MS Clcd.: 6917.9
[0369] Found: 6918.3
Example 27
PMO. No. 31
[0370] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-{[(2S, 6R)-6-(6-benzamide
prine-9-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid
loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 4) was used
as the starting material.
[0371] ESI-TOF-MS Clcd.: 6573.6
[0372] Found: 6572.4
Example 28
PMO. No. 32
[0373] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-(((2S,
6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-tritylmorphoi-
in-2-yl) methoxy)-4-oxobutanoic acid loaded onto aminopolystyrene
resin (REFERENCE EXAMPLE 3) was used as the starting material.
[0374] ESI-TOF-MS Clcd.: 6588.6
[0375] Found: 6588.3
Example 29
PMO. No. 33
[0376] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that
1,12-dioxo-1-(4-tritylpiperazin-1-yl)-2,5,8,11-tetraoxa-15-pentadecanoic
acid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 5) was
used as the starting material.
[0377] ESI-TOF-MS Clcd.: 7255.2
[0378] Found: 7254.7
TEST EXAMPLE 6
In Vitro Assay
[0379] Experiments were performed using the antisense oligomers of
2'-O-methoxy-phosphorothioates (2'-OMe-S-RNA) shown by SEQ ID NO:
82 to SEQ ID NO: 95 and SEQ ID NO: 109 to SEQ ID NO: 118. Various
antisense oligomers used for the assay were purchased from Japan
Bio Services. The sequences of various antisense oligomers are
given below.
TABLE-US-00019 TABLE 14 Antisense SEQ ID oligomer Nucleotide
sequence NO: H44_1-22 CUCAACAGAUCUGUCAAAUCGC 82 H44_6-27
CAUUUCUCAACAGAUCUGUCAA 105 H44_11-32 GCCGCCAUUUCUCAACAGAUCU 83
H44_16-37 AAAACGCCGCCAUUUCUCAACA 106 H44_2142
UAAUGAAAACGCCGCCAUUUCU 84 H44_26-47 UAUCAUAAUGAAAACGCCGCCA 85
H44_31-52 CUUUAUAUCAUAAUGAAAACGC 86 H44_36-57
AAUAUCUUUAUAUCAUAAUGAA 107 H44_41-62 GAUUAAAUAUCUUUAUAUCAUA 87
H44_51-72 GUUAGCCACUGAUUAAAUAUCU 88 H44_56-77
CUUCUGUUAGCCACUGAUUAAA 108 H44_61-82 UUCAGCUUCUGUUAGCCACUGA 89
H44_66-87 AACUGUUCAGCUUCUGUUAGCC 109 H44_71-92
UGAGAAACUGUUCAGCUUCUGU 90 H44_76-97 CUUUCUGAGAAACUGUUCAGCU 110
H44_81-102 UGUGUCUUUCUGAGAAACUGUU 91 H44_86-107
GAAUUUGUGUCUUUCUGAGAAA 111 H44_91-112 CUCAGGAAUUUGUGUCUUUCUG 112
H44_96-117 CAAUUCUCAGGAAUUUGUGUCU 113 H44_101-122
GUUCCCAAUUCUCAGGAAUUUG 92 H44_106-127 AGCAUGUUCCCAAUUCUCAGGA 114
H44_111-132 UAUUUAGCAUGUUCCCAAUUCU 93 H44_121-142
AUACCAUUUGUAUUUAGCAUGU 94 H44_62-81 UCAGCUUCUGUUAGCCACUG 95
[0380] Experiments were performed in accordance with the condition
and the procedure of exon 45 (TEST EXAMPLE 1).
Experimental Results
[0381] The results are shown in FIGS. 9 and 10. These experiments
revealed that, when the antisense oligomers were designed at the
11th to the 32nd, or the 26th to the 47th nucleotides from the 5'
end of exon 44 in the human dystrophin gene; exon 44 skipping of
these antisense oligomer could be caused with the same efficiency
with that of the antisense oligomer which is designed at the 62nd
to the 81st nucleotides from the 5' end of exon 44.
Test Example 7
In Vitro Assay
[0382] Experiments were performed in accordance with the condition
and the procedure of exon 45 (TEST EXAMPLE 2).
Experimental Results
[0383] The results are shown in FIGS. 11, 12 and 18. This
experiment revealed that in RD cells, the oligomers PMO No. 24 and
26 (H44_25-45 (OH) and H44_27-47 (OH)) of the present invention
caused exon 44 skipping with the same efficiency as the antisense
oligomer PMO No. 30 (H44_10-39(OH)) (FIG. 11, 12). The oligomer PMO
No. 26 of the present invention and the oligomer PMO No. 31
(H44_27-46(OH)), which is one base shorter than the oligomer PMO
No. 26, were found to cause exon 44 skipping with the same
efficiency (FIG. 18). Furthermore, the oligomer PMO No. 26 of the
present invention was found to cause exon 44 skipping with the same
efficiency as the oligomer PMO No. 33 (H44_27-47 (TEG)), which has
a different end structure from the oligomer PMO No. 26 (FIG.
18).
EXON 50
[0384] According to the descriptions in EXAMPLES 30 to 39 below,
various types of PMO shown by PMO Nos. 34-38 and 41-45 in TABLE 15
were synthesized. The PMO synthesized was dissolved in water for
injection (manufactured by Otsuka Pharmaceutical Factory, Inc.).
PMO Nos. 39 and 40 were purchased from Gene Tools, LLC.
TABLE-US-00020 TABLE 15 SEQ PMO ID No. Sequence name Note NO: 34
H50_103-123(OH) 5' end: group (3) 96 35 H50_104-124(OH) 5' end:
group (3) 97 36 H50_105-125(OH) 5' end: group (3) 98 37
H50_106-126(OH) 5' end: group (3) 99 38 H50_107-127(OH) 5' end:
group (3) 100 39 H50_90-114(GT) Sequence corresponding to 101 SEQ
ID NO: 287 in Patent Document 4, 5' end: group (2) 40
H50_103-127(GT) Sequence corresponding to 102 SEQ ID NO: 175 in
Patent Document 1, 5' end: group (2) 41 H50_107-126(OH) 5' end:
group (3) 119 42 H50_108-127(OH) 5' end: group (3) 120 43
H50_108-128(OH) 5' end: group (3) 121 44 H50_109-129(OH) 5' end:
group (3) 122 45 H50_107-127(TEG) 5' end: group (1) 100
Example 301
PMO. No. 34
[0385] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that
4-{[(2S,6R)-6-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1-yl)-4-tritylmorp-
holin-2-yl]methoxy}-4-oxobutanoic acid loaded onto aminopolystyrene
resin (REFERENCE EXAMPLE 3) was used as the starting material.
[0386] ESI-TOF-MS Clcd.: 6861.8
[0387] Found: 6861.8
Example 31
PMO. No. 35
[0388] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-{[(2S,
6R)-6-(6-benzamideprine-9-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutano-
ic acid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 4)
was used as the starting material.
[0389] ESI-TOF-MS Clcd.: 6885.8
[0390] Found: 6885.9
Example 32
PMO. No. 36
[0391] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that
4-[[(2S,6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purine-9-yl]-4-
-tritylmolphorin-2-yl]methoxy]-4-oxo-butanoic acid loaded onto
aminopolystyrene resin (REFERENCE EXAMPLE 2) was used as the
starting material.
[0392] ESI-TOF-MS Clcd.: 6925.9
[0393] Found: 6925.9
Example 33
PMO. No. 37
[0394] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-[[(2S,
6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purine-9-yl]-4-tritylm-
olphorin-2-yl]methoxy]-4-oxo-butanoic acid loaded onto
aminopolystyrene resin (REFERENCE EXAMPLE 2) was used as the
starting material.
[0395] ESI-TOF-MS Clcd.: 6950.9
[0396] Found: 6950.9
Example 34
PMO. No. 38
[0397] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-[[(2S,
6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purine-9-yl]-4-tritylm-
olphorin-2-yl]methoxy]-4-oxo-butanoic acid loaded onto
aminopolystyrene resin (REFERENCE EXAMPLE 2) was used as the
starting material.
[0398] ESI-TOF-MS Clcd.: 6990.9
[0399] Found: 6991.0
Example 351
PMO. No. 41.
[0400] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-[[(2S,
6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purine-9-yl]-4-tritylm-
olphorin-2-yl]methoxy]-4-oxo-butanoic acid loaded onto
aminopolystyrene resin (REFERENCE EXAMPLE 2) was used as the
starting material.
[0401] ESI-TOF-MS Clcd.: 6635.6
[0402] Found: 6635.0
Example 361
PMO. No. 42
[0403] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-[[(2S,
6R)-6-[6-(2-cyanoethoxy)-2-[(2-phenoxyacetyl)amino]purine-9-yl]-4-tritylm-
olphorin-2-yl]methoxy]-4-oxo-butanoic acid loaded onto
aminopolystyrene resin (REFERENCE EXAMPLE 2) was used as the
starting material.
[0404] ESI-TOF-MS Clcd.: 6635.6
[0405] Found: 6634.9
Example 371
PMO. No. 43
[0406] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that 4-(((2S,
6R)-6-(5-methyl-2,4-dioxo- 3,4-dihydropyrimidine 1(2H)-yl)
-4-tritylmorpholin-2-yl) methoxy)-4-oxobutanoic acid loaded onto
aminopolystyrene resin (REFERENCE EXAMPLE 3) was used as the
starting material.
[0407] ESI-TOF-MS Clcd.: 6965.9
[0408] Found: 6965.2
Example 38
PMO. No. 44
[0409] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that
4-{[(2S,6R)-6-(6-benzamidepurine-9-yl)-4-tritylmorpholin-2-yl]xnethoxy}-4-
-oxobutanoic acid loaded onto aminopolystyrene resin (REFERENCE
EXAMPLE 4) was used as the starting material.
[0410] ESI-TOF-MS Clcd.: 6949.9
[0411] Found: 6949.2
Example 39
PMO. No. 45
[0412] The title compound was produced in accordance with the
procedure of EXAMPLE 1, except that
1,12-dioxo-1-(4-tritylpiperazin-1-yl)-2,5,8,11-tetraoxa-15-pentadecanoic
acid loaded onto aminopolystyrene resin (REFERENCE EXAMPLE 5) was
used as the starting material.
[0413] ESI-TOF-MS Clcd.: 7342.2
[0414] Found: 7341.6
Test Example 8
In Vitro Assay
[0415] Experiments were performed in accordance with the condition
and the procedure of exon 45 (TEST EXAMPLE 2), except that the
RT-PCR was performed using the primers below in the concentrations
of 0.1, 0.3 or 1 .mu.M.
TABLE-US-00021 Forward primer: (SEQ ID NO: 103)
5'-AACAACCGGATGTGGAAGAG-3' Reverse primer: (SEQ ID NO: 104)
5'-TTGGAGATGGCAGTTTCCTT-3'
Experimental Results
[0416] The results are shown in FIGS. 13 and 19. These experiments
revealed that in RD cells the oligomers PMO No. 38 (H50_107-127
(OH)) of the present invention caused exon 50 skipping with a
higher efficiency than the antisense oligomer PMO No. 39 or 40
(H50_90-114 (GT), H50_103-127 (GT)). Also, the experiments revealed
that the oligomer PMO No. 38 caused exon 50 skipping with a higher
efficiency than the oligomer PMO No. 45 (H50_107-127 (TEG)), whose
end structure is different from that of the oligomer PMO No. 38
(FIG. 19).
Examination of Exon 44 Skipping
In Vitro Assay Using Human Fibroblasts
Test Example 9
[0417] The exon 44 skipping activity was determined using GM05112
cells (human DMD patient-derived fibroblasts with deletion of exon
45, Coriell Institute for Medical Research). As a growth medium,
there was used Dulbecco's Modified Eagle Medium: Nutrient Mixture
F-12 (DMEM/F-12) (Invitrogen Corp.) containing 10% FCS and 1%
Penicillin/Streptmycin (P/S) (Sigma-Aldrich, Inc.) and the cells
were cultured under conditions of 37.degree. C. and 5%
CO.sub.2.
[0418] The cells were cultured in T225 flask and the 2.5 mL of
retrovirus (ZsGreen1 coexpression) expressing human derived MyoD
(SEQ ID NO: 38) and a final concentration of 8 .mu.g/mL of
polybrene (Sigma-Aldrich, Inc.) were added to 35 mL of the growth
medium. After incubation at 32.degree. C. for 2 days, the medium
was exchanged to a fresh growth medium and incubation was further
continued at 37.degree. C. for 3 days. ZsGreen1-positive
MyoD-transformed fibroblasts were collected by BD FACSAria Cell
Sorter (BD Bioscience) and plated at 9.times.10.sup.4 cells/well
into a collagen-coated 24-well plate. The next day, the medium was
replaced by a differentiation medium (DMEM/F-12 containing 2%
equine serum (Invitrogen Corp.), 1% P/S and ITS Liquid Media
Supplement (Sigma, Inc.)). The medium was exchanged every 2 to 3
days and incubation was continued to differentiate into
myotubes.
[0419] On the 7th day after the medium was changed to the
differentiation medium, the medium was replaced by a
differentiation medium containing 6 .mu.M at a final concentration
of Endo-Porter (Gene Tools), and 1, 3, 10 .mu.M of the oligomers
PMO No. 26 and 31 were added thereto at a final concentration.
After the cells were incubated for 7 days, the cells were collected
to extract total RNA using RNeasy Mini Kit (QIAGEN). RT-PCR was
performed with 50 ng of the extracted total RNA using a QIAGEN
OneStep RT-PCR Kit. A reaction solution was prepared in accordance
with the protocol attached to the kit. An iCycler (manufactured by
Bio-Rad) was used as a thermal cycler. The RT-PCR program used is
as follows.
[0420] 50.degree. C., 30 mins: reverse transcription
[0421] 95.degree. C., 15 mins: thermal denaturation
[0422] [94.degree. C., 1 mins; 60.degree. C., 1 mins; 72.degree.
C., 1 mins].times.35 cycles: PCR amplification
[0423] 72.degree. C., 7 mins: final extension reaction
[0424] The nucleotide sequences of the forward primer and reverse
primer used for RT-PCR are given below.
TABLE-US-00022 Forward primer: (SEQ ID NO: 36)
5'-GCTCAGGTCGGATTGACATT-3' Reverse primer: (SEQ ID NO: 37)
5'-GGGCAACTCTTCCACCAGTA-3'
[0425] The reaction product of RT-PCR above was separated by 2%
agarose gel electrophoresis and gel images were captured with an
image analyzer ImageQuant LAS 4000 mini (manufactured by FUJI
Film). Using the attached soft, the polynucleotide level "A" of the
band with exon 44 skipping and the polynucleotide level "B" of the
band without exon 44 skipping were measured. Based on these
measurement values of "A", and "B", the skipping efficiency was
determined by the following equation:
Skipping efficiency (%)=A/(A+B).times.100
Experimental Results
[0426] The result is shown in FIG. 20. These experiments revealed
that in GM05112 cells the oligomers PMO No. 26 and 31 of the
present invention caused exon 44 skipping with a high
efficiency.
Test Example 10
[0427] A MyoD-transformed fibroblasts were prepared using GM05112
cells in accordance with the procedure of TEST EXAMPLE 9, and the
cells were differentiated into myotubes. Subsequently, the
differentiation medium was replaced by a differentiation medium
containing 6 .mu.M at a final concentration of Endo-Porter (Gene
Tools), and the oligomers PMO Nos. 26 and 31 were added to the
cells at a final concentration of 10 .mu.M on the 6th day after the
medium was changed to the differentiation medium. After incubation
for 14 days, the cells were collected by a scraper using a cell
lysis buffer RIPA buffer (manufactured by Pierce) containing a
protease inhibitor cocktail Complete Mini (manufactured by Roche).
The cell lysate were extracted from the cells by disrupting the
cells by a ultrasonic crusher Bioruptor UCD-250 (Tosho Denki) and
collecting the supernatant after centrifugation. The protein
concentrations were quantified using a Pierce BCA protein assay kit
(Pierce). The absorbance of 544 nm of wavelength was detected using
a plate reader Thermo Appliskan Type2001 (Thermo Electron).
[0428] The 3 .mu.g of cell lysates were electrophoresed in
acrylamide gel NuPAGE Novex Tris-Acetate Gel 3-8% (manufactured by
Invitrogen) and transferred onto a Immobilon-P membrane
(manufactured by Millipore) using a semi-dry blotter. The
transferred membrane was washed with PBS (PBST) containing 0.1%
Tween20 and blocked with PBST containing 5% Amersham ECL Prime
Blocking agent (GE Healthcare) in the refrigerator overnight. After
the membrane was washed with PBST, the membrane was incubated in a
solution of anti-dystrophin antibody (manufactured by NCL-Dys1,
Novocastra) 50-fold diluted with Can Get Signal1 (manufactured by
TOYOBO) at room temperature for 1 hour. After washing with PBST,
the membrane was incubated in a solution of peroxidase-conjugated
goat-antimouse IgG antibody (170-6516, Bio-Rad) 2,500-fold diluted
with Can Get Signal1 (manufactured by TOYOBO) at room temperature
for 10 minutes. After washing with PBST, the membrane was stained
with ECL Plus Western Blotting Detection System (GE Healthcare).
The chemiluminescence of the dystrophin protein deleted exon 44-45
was detected by lumino image analyzer ImageQuant LAS 4000 mini
(FUJI Film).
Experimental Results
[0429] The results of Western blotting are shown in FIG. 21. In
FIG. 21, the arrowhead represents a band of dystrophin protein of
which the expression was confirmed. This experiment reveals that
the oligomers PMO No. 26 and 31 of the present invention induced
expression of dystrophin proteins in GM05112 cells.
Study of Exon 50 Skipping
In Vitro Assay Using Human Fibroblasts
Test Example 11
[0430] MyoD-transformed fibroblasts were prepared using GM05112
cells to differentiate into myotubes in accordance with the
procedure of TEST EXAMPLE 9.
[0431] Subsequently, the differentiation medium was replaced by a
differentiation medium containing 6 .mu.M of Endo-Porter (Gene
Tools), and a oligomer PMO No. 38 was added thereto at a final
concentration of 0.1, 0.3, 1, 3, 10 .mu.M on the 12th day after the
medium was changed to the differentiation medium. After incubation
for 2 days, the cells were collected. Total RNA was extracted from
the cells, RT-PCR was performed and the skipping efficiency was
determined in accordance with the procedure of TEST EXAMPLE 9,
except that the nucleotide sequences of the forward primer and
reverse primer given below were used for RT-PCR.
TABLE-US-00023 Forward primer: (SEQ ID NO: 103)
5'-AACAACCGGATGTGGAAGAG-3' Reverse primer: (SEQ ID NO: 104)
5'-TTGGAGATGGCAGTTTCCTT-3'
Experimental Results
[0432] The result of RT-PCR is shown in FIG. 22 and the result of
skipping efficiency is shown in FIG. 23. These experiments revealed
that in GM05112 cells the oligomer PMO No. 38 of the present
invention caused exon 50 skipping with a high efficiency and the
value of EC.sub.50 was 1.3 .mu.M.
TEST EXAMPLE 12
[0433] Experiments for skipping were performed in accordance with
the condition and the procedure of TEST EXAMPLE 11, except that
11-0627 cells (human DMD patient derived fibroblasts with
duplication of exons 8-9, National Center of Neurology and
Psychiatry neuromuscular disorder research resource repository)
were used and the oligomer PMO No. 38 was added at a final
concentration of 0.1, 1, 10 .mu.M.
Experimental Results
[0434] The result of RT-PCR is shown in FIG. 26 and the skipping
efficiency is shown in FIG. 27. These experiments revealed that in
11-0627 cells the oligomer PMO No. 38 of the present invention
caused exon 50 skipping with a high efficiency.
Test Example 13
[0435] pLVX-MyoD-ZsGreen1 Lentivirus Preparation
[0436] pLVZ-puro (8120 bp, Clontech) was linearized by deleting
1164 bp nucleotides which is located on from XhoI site in the
multicloning site (at 2816) to the site (at 3890) adjacent to the
3' end of Puromycin resistant gene coding region to prepare a
linearized vector. Subsequently, the nucleotide sequences (2272 bp)
which encodes human MyoD gene, IRES sequence, ZsGreen1 gene was
integrated into the linearized vector in turn and then the
lentivirus expression vector pLVX-MyoD-ZsGreen1 (9210 bp) was
prepared.
[0437] Lenti-X 293T cells were plated onto 10 cm collagen coated
dish in accordance with the protocol attached to Lenti-X HTX
Packaging System (Clontech). Lentivirus expression vector and
packaging vector were transfected into fibroblasts three days
before infection. After four hours, the medium was exchanged and
the cells were incubated for three days without exchanging medium.
On the clay of infection, the culture supernatant was collected as
a virus solution (about 9 mL for 10 cm dish). The culture
supernatant was filtrated by cellstrainer (40 .mu.m) and then
centrifuged by 500.times.g, 10 min. This supernatant was
concentrated in accordance with the protocol attached to Lenti-X
Concentrator (Clontech) and then dissolved in DMEM/F12 medium to
ten times the concentration of the collected culture supernatant.
This solution was used as a virus solution.
Virus Infection into Fibroblasts
[0438] GM04364 cells (human DMD patient derived fibroblasts deleted
exons 51-55, Conicll Institute for Medical Research) were plated on
a collagen-coated 24-well plate by 3.times.10.sup.4/well by the day
of infection. On the day of infection, 400 .mu.L of the
differentiation medium, 100 .mu.L of the virus solution, and 8
.mu.g/mL of polybrene at a final concentration per well were added.
The day after infection, the medium containing virus was exchanged
into 500 ti L of the differentiation medium. The differentiation
medium was exchanged every 2 or 3 days and the cells were incubated
for 12 days to induce differentiation into myotubes.
[0439] On the 12th day after the medium was exchanged into the
differentiation medium, the medium was replaced by a
differentiation medium containing 6 .mu.M Endo-Porter (Gene Tools)
at a final concentration, and 0.1, 0.3, 1, 3, 10 .mu.M of the
oligomer PMO No. 38 was added thereto at a final concentration.
After incubation for 2 days, the cells were collected. The skipping
efficiency was determined in accordance with the procedure of TEST
EXAMPLE 11, except that the nucleotide sequences of the forward
primer and reverse primer given below were used for RT-PCR.
TABLE-US-00024 Forward primer: (SEQ ID NO: 103)
5'-AACAACCGGATGTGGAAGAG-3' Reverse primer: (SEQ ID NO: 70)
5'-CTGCCGGCTTAATTCATCAT-3'
Experimental Results
[0440] The result of RT-PCR is shown in FIG. 28 and the skipping
efficiency is shown in FIG. 29. These experiments revealed that in
GM04364 cells the oligomer PMO No. 38 of the present invention
caused exon 50 skipping with a high efficiency.
Study of Exon 55 Skipping
In Vitro Assay Using Human Fibroblasts
Test Example 141
[0441] Experiments were performed in accordance with the condition
and the procedure of TEST EXAMPLE 11, except that the oligomers PMO
No. 14 and 21 were used and the RT-PCR was performed using the
primers below.
TABLE-US-00025 Forward primer: (SEQ ID NO: 69)
5'-CATGGAAGGAGGGTCCCTAT-3' Reverse primer: (SEQ ID NO: 70)
5'-CTGCCGGCTTAATTCATCAT-3'
Experimental Results
[0442] The result of RT-PCR is shown in FIG. 24 and the skipping
efficiency is shown in FIG. 25. These experiments revealed that in
GM05112 cells the oligomers PMO No. 14 and 21 of the present
invention caused exon 55 skipping with a high efficiency and the
value of EC.sub.50 was 3.5 .mu.M and 7.5 .mu.M, respectively.
Test Example 15
[0443] Experiments were performed in accordance with the condition
and the procedure of TEST EXAMPLE 13, except that the 04-035 cells
(human DMD patient derived cells with single deletion of exon 54,
National Center of Neurology and Psychiatry neuromuscular disorder
research resource repository) were used and 1, 3, 10 .mu.M of the
the oligomers PMO No. 14 and 21 at a final concentration were added
and the RT-PCR was performed using the primers below.
TABLE-US-00026 Forward primer: (SEQ ID NO: 69)
5'-CATGGAAGGAGGGTCCCTAT-3' Reverse primer: (SEQ ID NO: 70)
5'-CTGCCGGCTTAATTCATCAT-3'
Experimental Results
[0444] The result of RT-PCR is shown in FIG. 30 and the skipping
efficiency is shown in FIG. 31. These experiments revealed that in
human DMD patient derived cells with single deletion of exon 54,
the oligomers PMO No. 14 and 21 of the present invention caused
exon 55 skipping with a high efficiency.
INDUSTRIAL APPLICABILITY
[0445] Experimental results in TEST EXAMPLES demonstrate that the
oligomers of the present invention caused exon skipping with a
markedly high efficiency in both RD cells and DMD patients derived
cells.
[0446] Therefore, the oligomers of the present invention are
extremely useful for the treatment of DMD.
Sequence Listing Free Text
[0447] SEQ ID NO: 9: synthetic nucleic acid
[0448] SEQ ID NO: 10: synthetic nucleic acid
[0449] SEQ ID NO: 11: synthetic nucleic acid
[0450] SEQ ID NO: 12: synthetic nucleic acid
[0451] SEQ ID NO: 13: synthetic nucleic acid
[0452] SEQ ID NO: 14: synthetic nucleic acid
[0453] SEQ ID NO: 15: synthetic nucleic acid
[0454] SEQ ID NO: 16: synthetic nucleic acid
[0455] SEQ ID NO: 17: synthetic nucleic acid
[0456] SEQ ID NO: 18: synthetic nucleic acid
[0457] SEQ ID NO: 19: synthetic nucleic acid
[0458] SEQ ID NO: 20: synthetic nucleic acid
[0459] SEQ ID NO: 21: synthetic nucleic acid
[0460] SEQ ID NO: 22: synthetic nucleic acid
[0461] SEQ ID NO: 23: synthetic nucleic acid
[0462] SEQ ID NO: 24: synthetic nucleic acid
[0463] SEQ ID NO: 25: synthetic nucleic acid
[0464] SEQ ID NO: 26: synthetic nucleic acid
[0465] SEQ ID NO: 27: synthetic nucleic acid
[0466] SEQ ID NO: 28: synthetic nucleic acid
[0467] SEQ ID NO: 29: synthetic nucleic acid
[0468] SEQ ID NO: 30: synthetic nucleic acid
[0469] SEQ ID NO: 31: synthetic nucleic acid
[0470] SEQ ID NO: 32: synthetic nucleic acid
[0471] SEQ ID NO: 33: synthetic nucleic acid
[0472] SEQ ID NO: 34: synthetic nucleic acid
[0473] SEQ ID NO: 35: synthetic nucleic acid
[0474] SEQ ID NO: 36: synthetic nucleic acid
[0475] SEQ ID NO: 37: synthetic nucleic acid
[0476] SEQ ID NO: 38: synthetic nucleic acid
[0477] SEQ ID NO: 39: synthetic nucleic acid
[0478] SEQ ID NO: 40: synthetic nucleic acid
[0479] SEQ ID NO: 41: synthetic nucleic acid
[0480] SEQ ID NO: 42: synthetic nucleic acid
[0481] SEQ ID NO: 43: synthetic nucleic acid
[0482] SEQ ID NO: 45: synthetic nucleic acid
[0483] SEQ ID NO: 46: synthetic nucleic acid
[0484] SEQ ID NO: 47: synthetic nucleic acid
[0485] SEQ ID NO: 48: synthetic nucleic acid
[0486] SEQ ID NO: 49: synthetic nucleic acid
[0487] SEQ ID NO: 50: synthetic nucleic acid
[0488] SEQ ID NO: 51: synthetic nucleic acid
[0489] SEQ ID NO: 52: synthetic nucleic acid
[0490] SEQ ID NO: 53: synthetic nucleic acid
[0491] SEQ ID NO: 54: synthetic nucleic acid
[0492] SEQ ID NO: 55: synthetic nucleic acid
[0493] SEQ ID NO: 56: synthetic nucleic acid
[0494] SEQ ID NO: 57: synthetic nucleic acid
[0495] SEQ ID NO: 58: synthetic nucleic acid
[0496] SEQ ID NO: 59: synthetic nucleic acid
[0497] SEQ ID NO: 60: synthetic nucleic acid
[0498] SEQ ID NO: 61: synthetic nucleic acid
[0499] SEQ ID NO; 62: synthetic nucleic acid
[0500] SEQ ID NO: 63: synthetic nucleic acid
[0501] SEQ ID NO: 64: synthetic nucleic acid
[0502] SEQ ID NO: 65: synthetic nucleic acid
[0503] SEQ ID NO: 66: synthetic nucleic acid
[0504] SEQ ID NO: 67: synthetic nucleic acid
[0505] SEQ ID NO: 68: synthetic nucleic acid
[0506] SEQ ID NO: 69: synthetic nucleic acid
[0507] SEQ ID NO: 70: synthetic nucleic acid
[0508] SEQ ID NO: 71: synthetic nucleic acid
[0509] SEQ ID NO: 72: synthetic nucleic acid
[0510] SEQ ID NO: 73: synthetic nucleic acid
[0511] SEQ ID NO: 74: synthetic nucleic acid
[0512] SEQ ID NO: 75: synthetic nucleic acid
[0513] SEQ ID NO: 76: synthetic nucleic acid
[0514] SEQ ID NO: 77: synthetic nucleic acid
[0515] SEQ ID NO: 78: synthetic nucleic acid
[0516] SEQ ID NO: 79: synthetic nucleic acid
[0517] SEQ ID NO: 80: synthetic nucleic acid
[0518] SEQ ID NO: 81: synthetic nucleic acid
[0519] SEQ ID NO: 82: synthetic nucleic acid
[0520] SEQ ID NO: 83: synthetic nucleic acid
[0521] SEQ ID NO: 84: synthetic nucleic acid
[0522] SEQ ID NO: 85: synthetic nucleic acid
[0523] SEQ ID NO: 86: synthetic nucleic acid
[0524] SEQ ID NO: 87: synthetic nucleic acid
[0525] SEQ ID NO: 88: synthetic nucleic acid
[0526] SEQ ID NO: 89: synthetic nucleic acid
[0527] SEQ ID NO: 90: synthetic nucleic acid
[0528] SEQ ID NO: 91: synthetic nucleic acid
[0529] SEQ ID NO: 92: synthetic nucleic acid
[0530] SEQ ID NO: 93: synthetic nucleic acid
[0531] SEQ ID NO: 94: synthetic nucleic acid
[0532] SEQ ID NO: 95: synthetic nucleic acid
[0533] SEQ ID NO: 96: synthetic nucleic acid
[0534] SEQ ID NO: 97: synthetic nucleic acid
[0535] SEQ ID NO: 98: synthetic nucleic acid
[0536] SEQ ID NO: 99: synthetic nucleic acid
[0537] SEQ ID NO: 100: synthetic nucleic acid
[0538] SEQ ID NO: 101: synthetic nucleic acid
[0539] SEQ ID NO: 102: synthetic nucleic acid
[0540] SEQ ID NO: 103: synthetic nucleic acid
[0541] SEQ ID NO: 104: synthetic nucleic acid
[0542] SEQ ID NO: 105: synthetic nucleic acid
[0543] SEQ ID NO: 106: synthetic nucleic acid
[0544] SEQ ID NO: 107: synthetic nucleic acid
[0545] SEQ ID NO: 108: synthetic nucleic acid
[0546] SEQ ID NO: 109: synthetic nucleic acid
[0547] SEQ ID NO: 110: synthetic nucleic acid
[0548] SEQ ID NO: 111: synthetic nucleic acid
[0549] SEQ ID NO: 112: synthetic nucleic acid
[0550] SEQ ID NO: 113: synthetic nucleic acid
[0551] SEQ ID NO: 114: synthetic nucleic acid
[0552] SEQ ID NO: 115: synthetic nucleic acid
[0553] SEQ ID NO: 116: synthetic nucleic acid
[0554] SEQ ID NO: 117: synthetic nucleic acid
[0555] SEQ ID NO: 118: synthetic nucleic acid
[0556] SEQ ID NO: 119: synthetic nucleic acid
[0557] SEQ ID NO: 120: synthetic nucleic acid
[0558] SEQ ID NO: 121: synthetic nucleic acid
[0559] SEQ ID NO: 122: synthetic nucleic acid
SEQUENCE LISTING
Sequence CWU 1
1
1221192DNAHomo sapiens 1agggtgagtg agcgagaggc tgctttggaa gaaactcata
gattactgca acagttcccc 60ctggacctgg aaaagtttct tgcctggctt acagaagctg
aaacaactgc caatgtccta 120caggatgcta cccgtaagga aaggctccta
gaagactcca agggagtaaa agagctgatg 180aaacaatggc aa 1922179DNAHomo
sapiens 2caggaactcc aggatggcat tgggcagcgg caaactgttg tcagaacatt
gaatgcaact 60ggggaagaaa taattcagca atcctcaaaa acagatgcca gtattctaca
ggaaaaattg 120ggaagcctga atctgcggtg gcaggaggtc tgcaaacagc
tgtcagacag aaaaaagag 1793129DNAHomo sapiens 3aggaagttag aagatctgag
ctctgagtgg aaggcggtaa accgtttact tcaagagctg 60agggcaaagc agcctgacct
agctcctgga ctgaccacta ttggagcctg taagtatact 120ggatcccat
1294148DNAHomo sapiens 4gcgatttgac agatctgttg agaaatggcg gcgttttcat
tatgatataa agatatttaa 60tcagtggcta acagaagctg aacagtttct cagaaagaca
caaattcctg agaattggga 120acatgctaaa tacaaatggt atcttaag
1485192DNAHomo sapiens 5ttgccattgt ttcatcagct cttttactcc cttggagtct
tctaggagcc tttccttacg 60ggtagcatcc tgtaggacat tggcagttgt ttcagcttct
gtaagccagg caagaaactt 120ttccaggtcc agggggaact gttgcagtaa
tctatgagtt tcttccaaag cagcctctcg 180ctcactcacc ct 1926179DNAHomo
sapiens 6ctcttttttc tgtctgacag ctgtttgcag acctcctgcc accgcagatt
caggcttccc 60aatttttcct gtagaatact ggcatctgtt tttgaggatt gctgaattat
ttcttcccca 120gttgcattca atgttctgac aacagtttgc cgctgcccaa
tgccatcctg gagttcctg 1797129DNAHomo sapiens 7atgggatcca gtatacttac
aggctccaat agtggtcagt ccaggagcta ggtcaggctg 60ctttgccctc agctcttgaa
gtaaacggtt taccgccttc cactcagagc tcagatcttc 120taacttcct
1298148DNAHomo sapiens 8cttaagatac catttgtatt tagcatgttc ccaattctca
ggaatttgtg tctttctgag 60aaactgttca gcttctgtta gccactgatt aaatatcttt
atatcataat gaaaacgccg 120ccatttctca acagatctgt caaatcgc
148921DNAArtificialSynthetic Nucleic Acid 9caatgccatc ctggagttcc t
211021DNAArtificialSynthetic Nucleic Acid 10ccaatgccat cctggagttc c
211121DNAArtificialSynthetic Nucleic Acid 11cccaatgcca tcctggagtt c
211221DNAArtificialSynthetic Nucleic Acid 12gcccaatgcc atcctggagt t
211321DNAArtificialSynthetic Nucleic Acid 13tgcccaatgc catcctggag t
211425DNAArtificialSynthetic Nucleic Acid 14cccaatgcca tcctggagtt
cctgt 251530DNAArtificialSynthetic Nucleic Acid 15cagtttgccg
ctgcccaatg ccatcctgga 301620DNAArtificialSynthetic Nucleic Acid
16ccaatgccat cctggagttc 201720DNAArtificialSynthetic Nucleic Acid
17cccaatgcca tcctggagtt 201821DNAArtificialSynthetic Nucleic Acid
18cccaatgcca tcctggagtt c 211925DNAArtificialSynthetic Nucleic Acid
19gcugcccaau gccauccugg aguuc 252025DNAArtificialSynthetic Nucleic
Acid 20uugccgcugc ccaaugccau ccugg 252125DNAArtificialSynthetic
Nucleic Acid 21acaguuugcc gcugcccaau gccau
252225DNAArtificialSynthetic Nucleic Acid 22ugacaacagu uugccgcugc
ccaau 252325DNAArtificialSynthetic Nucleic Acid 23uguucugaca
acaguuugcc gcugc 252425DNAArtificialSynthetic Nucleic Acid
24uucaauguuc ugacaacagu uugcc 252525DNAArtificialSynthetic Nucleic
Acid 25uugcauucaa uguucugaca acagu 252625DNAArtificialSynthetic
Nucleic Acid 26cccaguugca uucaauguuc ugaca
252725DNAArtificialSynthetic Nucleic Acid 27ucuuccccag uugcauucaa
uguuc 252825DNAArtificialSynthetic Nucleic Acid 28uuauuucuuc
cccaguugca uucaa 252925DNAArtificialSynthetic Nucleic Acid
29cugaauuauu ucuuccccag uugca 253025DNAArtificialSynthetic Nucleic
Acid 30gauugcugaa uuauuucuuc cccag 253125DNAArtificialSynthetic
Nucleic Acid 31uugaggauug cugaauuauu ucuuc
253225DNAArtificialSynthetic Nucleic Acid 32uguuuuugag gauugcugaa
uuauu 253325DNAArtificialSynthetic Nucleic Acid 33gcaucuguuu
uugaggauug cugaa 253425DNAArtificialSynthetic Nucleic Acid
34uacuggcauc uguuuuugag gauug 253525DNAArtificialSynthetic Nucleic
Acid 35uuugccgcug cccaaugcca uccug 253620DNAArtificialSynthetic
Nucleic Acid 36gctcaggtcg gattgacatt 203720DNAArtificialSynthetic
Nucleic Acid 37gggcaactct tccaccagta 2038963DNAHomo sapiens
38atggagctac tgtcgccacc gctccgcgac gtagacctga cggcccccga cggctctctc
60tgctcctttg ccacaacgga cgacttctat gacgacccgt gtttcgactc cccggacctg
120cgcttcttcg aagacctgga cccgcgcctg atgcacgtgg gcgcgctcct
gaaacccgaa 180gagcactcgc acttccccgc ggcggtgcac ccggccccgg
gcgcacgtga ggacgagcat 240gtgcgcgcgc ccagcgggca ccaccaggcg
ggccgctgcc tactgtgggc ctgcaaggcg 300tgcaagcgca agaccaccaa
cgccgaccgc cgcaaggccg ccaccatgcg cgagcggcgc 360cgcctgagca
aagtaaatga ggcctttgag acactcaagc gctgcacgtc gagcaatcca
420aaccagcggt tgcccaaggt ggagatcctg cgcaacgcca tccgctatat
cgagggcctg 480caggctctgc tgcgcgacca ggacgccgcg ccccctggcg
ccgcagccgc cttctatgcg 540ccgggcccgc tgcccccggg ccgcggcggc
gagcactaca gcggcgactc cgacgcgtcc 600agcccgcgct ccaactgctc
cgacggcatg atggactaca gcggcccccc gagcggcgcc 660cggcggcgga
actgctacga aggcgcctac tacaacgagg cgcccagcga acccaggccc
720gggaagagtg cggcggtgtc gagcctagac tgcctgtcca gcatcgtgga
gcgcatctcc 780accgagagcc ctgcggcgcc cgccctcctg ctggcggacg
tgccttctga gtcgcctccg 840cgcaggcaag aggctgccgc ccccagcgag
ggagagagca gcggcgaccc cacccagtca 900ccggacgccg ccccgcagtg
ccctgcgggt gcgaacccca acccgatata ccaggtgctc 960tga
9633920DNAArtificialSynthetic Nucleic Acid 39cctgagaatt gggaacatgc
204020DNAArtificialSynthetic Nucleic Acid 40ttgctgctct tttccaggtt
204121DNAArtificialSynthetic Nucleic Acid 41agcagcctct cgctcactca c
214221DNAArtificialSynthetic Nucleic Acid 42ttccaaagca gcctctcgct c
214321DNAArtificialSynthetic Nucleic Acid 43ttcttccaaa gcagcctctc g
214421DNAArtificialSynthetic Nucleic Acid 44agtttcttcc aaagcagcct c
214521DNAArtificialSynthetic Nucleic Acid 45tttcttccaa agcagcctct c
214621DNAArtificialSynthetic Nucleic Acid 46gtttcttcca aagcagcctc t
214721DNAArtificialSynthetic Nucleic Acid 47gagtttcttc caaagcagcc t
214821DNAArtificialSynthetic Nucleic Acid 48tgagtttctt ccaaagcagc c
214921DNAArtificialSynthetic Nucleic Acid 49gcagccucuc gcucacucac c
215021DNAArtificialSynthetic Nucleic Acid 50ccaaagcagc cucucgcuca c
215121DNAArtificialSynthetic Nucleic Acid 51uucuuccaaa gcagccucuc g
215221DNAArtificialSynthetic Nucleic Acid 52aucuaugagu uucuuccaaa g
215321DNAArtificialSynthetic Nucleic Acid 53uguugcagua aucuaugagu u
215421DNAArtificialSynthetic Nucleic Acid 54cagggggaac uguugcagua a
215521DNAArtificialSynthetic Nucleic Acid 55uuuccagguc cagggggaac u
215621DNAArtificialSynthetic Nucleic Acid 56gcaagaaacu uuuccagguc c
215721DNAArtificialSynthetic Nucleic Acid 57uguaagccag gcaagaaacu u
215821DNAArtificialSynthetic Nucleic Acid 58uuucagcuuc uguaagccag g
215921DNAArtificialSynthetic Nucleic Acid 59uuggcaguug uuucagcuuc u
216021DNAArtificialSynthetic Nucleic Acid 60cuguaggaca uuggcaguug u
216121DNAArtificialSynthetic Nucleic Acid 61ggguagcauc cuguaggaca u
216221DNAArtificialSynthetic Nucleic Acid 62cuuuccuuac ggguagcauc c
216321DNAArtificialSynthetic Nucleic Acid 63uucuaggagc cuuuccuuac g
216421DNAArtificialSynthetic Nucleic Acid 64ccuuggaguc uucuaggagc c
216521DNAArtificialSynthetic Nucleic Acid 65ucuuuuacuc ccuuggaguc u
216621DNAArtificialSynthetic Nucleic Acid 66uuucaucagc ucuuuuacuc c
216720DNAArtificialSynthetic Nucleic Acid 67uugccauugu uucaucagcu
206820DNAArtificialSynthetic Nucleic Acid 68uccuguagga cauuggcagu
206920DNAArtificialSynthetic Nucleic Acid 69catggaagga gggtccctat
207020DNAArtificialSynthetic Nucleic Acid 70ctgccggctt aattcatcat
207121DNAArtificialSynthetic Nucleic Acid 71ataatgaaaa cgccgccatt t
217221DNAArtificialSynthetic Nucleic Acid 72tcataatgaa aacgccgcca t
217321DNAArtificialSynthetic Nucleic Acid 73atcataatga aaacgccgcc a
217421DNAArtificialSynthetic Nucleic Acid 74tatcataatg aaaacgccgc c
217521DNAArtificialSynthetic Nucleic Acid 75atatcataat gaaaacgccg c
217621DNAArtificialSynthetic Nucleic Acid 76tatatcataa tgaaaacgcc g
217721DNAArtificialSynthetic Nucleic Acid 77ttatatcata atgaaaacgc c
217830DNAArtificialSynthetic Nucleic Acid 78tgaaaacgcc gccatttctc
aacagatctg 307920DNAArtificialSynthetic Nucleic Acid 79atcataatga
aaacgccgcc 208020DNAArtificialSynthetic Nucleic Acid 80tatcataatg
aaaacgccgc 208121DNAArtificialSynthetic Nucleic Acid 81tatcataatg
aaaacgccgc c 218222DNAArtificialSynthetic Nucleic Acid 82cucaacagau
cugucaaauc gc 228322DNAArtificialSynthetic Nucleic Acid
83gccgccauuu cucaacagau cu 228422DNAArtificialSynthetic Nucleic
Acid 84uaaugaaaac gccgccauuu cu 228522DNAArtificialSynthetic
Nucleic Acid 85uaucauaaug aaaacgccgc ca
228622DNAArtificialSynthetic Nucleic Acid 86cuuuauauca uaaugaaaac
gc 228722DNAArtificialSynthetic Nucleic Acid 87gauuaaauau
cuuuauauca ua 228822DNAArtificialSynthetic Nucleic Acid
88guuagccacu gauuaaauau cu 228922DNAArtificialSynthetic Nucleic
Acid 89uucagcuucu guuagccacu ga 229022DNAArtificialSynthetic
Nucleic Acid 90ugagaaacug uucagcuucu gu
229122DNAArtificialSynthetic Nucleic Acid 91ugugucuuuc ugagaaacug
uu 229222DNAArtificialSynthetic Nucleic Acid 92guucccaauu
cucaggaauu ug 229322DNAArtificialSynthetic Nucleic Acid
93uauuuagcau guucccaauu cu 229422DNAArtificialSynthetic Nucleic
Acid 94auaccauuug uauuuagcau gu 229520DNAArtificialSynthetic
Nucleic Acid 95ucagcuucug uuagccacug 209621DNAArtificialSynthetic
Nucleic Acid 96tccagtatac ttacaggctc c 219721DNAArtificialSynthetic
Nucleic Acid 97atccagtata cttacaggct c 219821DNAArtificialSynthetic
Nucleic Acid 98gatccagtat acttacaggc t 219921DNAArtificialSynthetic
Nucleic Acid 99ggatccagta tacttacagg c
2110021DNAArtificialSynthetic Nucleic Acid 100gggatccagt atacttacag
g 2110125DNAArtificialSynthetic Nucleic Acid 101cttacaggct
ccaatagtgg tcagt 2510225DNAArtificialSynthetic Nucleic Acid
102gggatccagt atacttacag gctcc 2510320DNAArtificialSynthetic
Nucleic Acid 103aacaaccgga tgtggaagag 2010420DNAArtificialSynthetic
Nucleic Acid 104ttggagatgg cagtttcctt 2010522DNAArtificialSynthetic
Nucleic Acid 105cauuucucaa cagaucuguc aa
2210622DNAArtificialSynthetic Nucleic Acid 106aaaacgccgc cauuucucaa
ca 2210722DNAArtificialSynthetic Nucleic Acid 107aauaucuuua
uaucauaaug aa 2210822DNAArtificialSynthetic Nucleic Acid
108cuucuguuag ccacugauua aa 2210922DNAArtificialSynthetic Nucleic
Acid 109aacuguucag cuucuguuag cc 2211022DNAArtificialSynthetic
Nucleic Acid 110cuuucugaga aacuguucag cu
2211122DNAArtificialSynthetic Nucleic Acid 111gaauuugugu cuuucugaga
aa 2211222DNAArtificialSynthetic Nucleic Acid 112cucaggaauu
ugugucuuuc ug 2211322DNAArtificialSynthetic Nucleic Acid
113caauucucag gaauuugugu cu 2211422DNAArtificialSynthetic Nucleic
Acid 114agcauguucc caauucucag ga 2211518DNAArtificialSynthetic
Nucleic Acid 115gagtcttcta ggagcctt 1811620DNAArtificialSynthetic
Nucleic Acid 116gtttcttcca aagcagcctc 2011720DNAArtificialSynthetic
Nucleic Acid 117agtttcttcc aaagcagcct 2011821DNAArtificialSynthetic
Nucleic Acid 118agtttcttcc aaagcagcct c
2111920DNAArtificialSynthetic Nucleic Acid 119ggatccagta tacttacagg
2012020DNAArtificialSynthetic Nucleic Acid 120gggatccagt atacttacag
2012121DNAArtificialSynthetic Nucleic Acid 121tgggatccag tatacttaca
g 2112221DNAArtificialSynthetic Nucleic Acid 122atgggatcca
gtatacttac a 21
* * * * *