U.S. patent application number 17/364618 was filed with the patent office on 2022-05-26 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, Youhei SATOU, Haruna SEO, Shin'ichi TAKEDA, Tatsushi WAKAYAMA.
Application Number | 20220162604 17/364618 |
Document ID | / |
Family ID | 1000006136024 |
Filed Date | 2022-05-26 |
United States Patent
Application |
20220162604 |
Kind Code |
A1 |
WAKAYAMA; Tatsushi ; et
al. |
May 26, 2022 |
ANTISENSE NUCLEIC ACIDS
Abstract
Provided is a drug that allows highly-efficient skipping of exon
51 in the human dystrophin gene. The present invention provides an
antisense oligomer which enables exon 51 in the human dystrophin
gene to be skipped.
Inventors: |
WAKAYAMA; Tatsushi;
(Ibaraki, JP) ; SEO; Haruna; (Tokyo, JP) ;
SATOU; Youhei; (Ibaraki, 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: |
1000006136024 |
Appl. No.: |
17/364618 |
Filed: |
June 30, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16437130 |
Jun 11, 2019 |
11053497 |
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17364618 |
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15902231 |
Feb 22, 2018 |
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16437130 |
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15122435 |
Aug 30, 2016 |
9988629 |
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PCT/JP2015/057180 |
Mar 11, 2015 |
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15902231 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/3525 20130101;
C12N 2310/3535 20130101; C12N 15/111 20130101; C12N 2310/315
20130101; C12N 2310/322 20130101; C12N 2310/11 20130101; C12N
2310/3521 20130101; C12N 2310/314 20130101; C12N 2310/3533
20130101; C12N 2310/3233 20130101; C12N 15/113 20130101; C12N
2310/321 20130101; C12N 2320/33 20130101; A61K 31/713 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 31/713 20060101 A61K031/713; C12N 15/11 20060101
C12N015/11 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 12, 2014 |
JP |
2014-048897 |
Claims
1-21. (canceled)
22. A morpholino oligomer that causes skipping of the 51st exon in
a human dystrophin pre-mRNA, consisting of the nucleobase sequence
of SEQ ID NO: 1 or SEQ ID NO: 2, or a pharmaceutically acceptable
salt or hydrate thereof.
23. The morpholino oligomer of claim 22, or a pharmaceutically
acceptable salt or hydrate thereof, wherein said morpholino
oligomer is a phosphorodiamidate morpholino oligomer (PMO).
24. The PMO of claim 23, or a pharmaceutically acceptable salt or
hydrate thereof, wherein each phosphorodiamidate morpholino monomer
of said PMO has the formula: ##STR00020## wherein each of R.sup.2
and R.sup.3 represents a methyl; and Base represent a
nucleobase.
25. The PMO of claim 24, or a pharmaceutically acceptable salt or
hydrate thereof, wherein the 5' end of said PMO is any one of
chemical formulae (1) to (3) below: ##STR00021##
26. The PMO of claim 24, or a pharmaceutically acceptable salt or
hydrate thereof, wherein the 5' end of said PMO is:
##STR00022##
27. A pharmaceutical composition for the treatment of muscular
dystrophy, comprising as an active ingredient the morpholino
oligomer of claim 22, or a pharmaceutically acceptable salt or
hydrate thereof.
28. The pharmaceutical composition according to claim 27,
comprising a pharmaceutically acceptable carrier.
29. A method for treatment of muscular dystrophy, which comprises
intravenously administering to a patient with muscular dystrophy
the morpholino oligomer of claim 22, or a pharmaceutically
acceptable salt or hydrate thereof.
30. The method for treatment of claim 29, wherein said patient with
muscular dystrophy is a patient with deletions of nucleotides
within exons 29-50, 50, 45-50, 48-50, 49-50, 52, 52-63, 13-50,
19-50, 43-50 or 47-50.
31. The method for treatment of claim 29, wherein said patient is a
human.
32. A solid-phase method of making the PMO of claim 26, comprising:
a) providing Compound 1: ##STR00023## wherein T represents trityl,
monomethoxytrityl, or dimethoxytrityl; and wherein B.sup.P is a
protected nucleobase; b) reacting said Compound 1 with an acid to
form Compound 2: ##STR00024## c) reacting said Compound 2 with a
morpholino monomer having the formula: ##STR00025## in the presence
of a base and a solvent; d) repeating steps b) and c) until
Compound 3 is complete; ##STR00026## e) reacting said Compound 3
with a deprotecting agent to form Compound 4: ##STR00027## and f)
reacting Compound 4 with an acid to form said PMO.
33. The method according to claim 32, wherein said acid used in
step b) is trifluoroacetic acid.
34. The method according to claim 32, wherein said base used in
step c) is N-ethylmorpholine, and said solvent used in step c) is
N,N-dimethylimidazolidone.
35. The method according to claim 32, wherein said deprotecting
agent in step e) is concentrated ammonia water used as a dilution
with a solvent or a mixture of solvents.
36. The method according to claim 32, wherein said acid used in
step 0 is selected from phosphoric acid and hydrochloric acid.
Description
TECHNICAL FIELD
[0001] The present invention relates to an antisense oligomer which
causes skipping of exon 51 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 of 11,058 bases, 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 13% of all DMD patients
may be treated by skipping the exon 51 (hereinafter referred to as
"exon Si"). In recent years, a plurality of research organizations
reported on the studies where exon 51 in the dystrophin gene was
targeted for exon skipping (Patent Documents 1 to 6; Non-Patent
Documents 5 to 6). However, a technique for skipping exon 51 with a
high efficiency has not yet been established.
PRIOR ART DOCUMENT
Patent Document
[0009] Patent Document 1: International Publication WO 2004/048570
[0010] Patent Document 2: International Publication WO 2002/024906
[0011] Patent Document 3: International Publication WO 2010/048586
Patent Document 4: International Publication WO 2010/050801 [0012]
Patent Document 5: US 2010/0168212 [0013] Non-Patent Document 1:
Monaco A. P. et al., Genomics 1988; 2: p. 90-95 [0014] Non-Patent
Document 2: Matsuo M., Brain Dev 1996; 18: p. 167-172 Non-Patent
Document 3: Wilton S. D., e t al., Molecular Therapy 2007: 15: p.
1288-96 [0015] Non-Patent Document 4: Annemieke Aartsma-Rus et al.,
(2002) Neuromuscular Disorders 12: S71-S77 [0016] Non-Patent
Document 5: Aoki Y., et al., Molecular therapy 2010: 18: p.
1995-2005 Non-Patent Document 6: Nakano S., et al., Pediatr Int.
2011: 53: 524-429
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0017] Under the foregoing circumstances, antisense oligomers that
induce exon 51 skipping in the dystrophin gene with a high
efficiency and muscular dystrophy therapeutics comprising oligomers
thereof have been desired.
Means for Solving the Problem
[0018] As a result of detailed studies of the technical contents of
the above documents and the structure of the dystrophin gene, the
present inventors have found that exon 51 skipping can be induced
with a high efficiency by administering the antisense oligomer
having the nucleotide sequences represented by SEQ ID NO:1 and 2.
Based on this finding, the present inventors have accomplished the
present invention.
[0019] That is, the present invention is as follows.
[1] An antisense oligomer which is selected from a group consisting
of (a) to (d) below: [0020] (a) an antisense oligomer comprising a
nucleotide sequence of SEQ ID NO: 1 or 2; [0021] (b) an antisense
oligomer which consists of a nucleotide sequence having deletion,
substitution, insertion and/or addition of 1 to 5 nucleotides in
the nucleotide sequence of SEQ ID NO: 1 or 2, and has an activity
to cause skipping of the 51st exon in the human dystrophin gene;
[0022] (c) an antisense oligomer which has a nucleotide sequence
having at least 80% identity with a nucleotide sequence of SEQ ID
NO: 1 or 2 and has an activity to cause skipping of the 51st exon
in the human dystrophin gene; and [0023] (d) an antisense oligomer
that hybridizes under stringent conditions to an oligonucleotide
consisting of a nucleotide sequence complementary to the nucleotide
sequence of SEQ ID NO: 1 or 2 and has an activity to cause skipping
of the 51st exon in the human dystrophin gene. [2] An antisense
oligomer which is selected from a group consisting of (e) to (h)
below: [0024] (e) an antisense oligomer which consists of a
nucleotide sequence of SEQ ID NO: 1 or 2; [0025] (f) an antisense
oligomer which consists of a nucleotide sequence having deletion,
substitution, insertion and/or addition of 1 to 3 nucleotides in
the nucleotide sequence of SEQ ID NO: 1 or 2, and has an activity
to cause skipping of the 51st exon in the human dystrophin gene;
[0026] (g) an antisense oligomer which consists of a nucleotide
sequence having at least 80% identity with a nucleotide sequence of
SEQ ID NO: 1 or 2 and has an activity to cause skipping of the 51st
exon in the human dystrophin gene; and [0027] (h) an antisense
oligomer that hybridizes under high stringent conditions to an
oligonucleotide consisting of a nucleotide sequence complementary
to a nucleotide sequence of SEQ ID NO: 1 or 2 and has an activity
to cause skipping of the 51st exon in the human dystrophin gene.
[3] An antisense oligomer which is selected from a group consisting
of (i) and (j) below: [0028] (i) an antisense oligomer which
consists of a nucleotide sequence of SEQ ID NO: 1 or 2; and [0029]
(j) an antisense oligomer which has a nucleotide sequence having at
least 90% identity with a nucleotide sequence of SEQ ID NO: 1 or 2
and has an activity to cause skipping of the 51st exon in the human
dystrophin gene. [4] The antisense oligomer according to any one of
[1] to [3] above, which is an oligonucleotide. [5] The antisense
oligomer according to [4] above, wherein the sugar moiety and/or
the phosphate-binding region of at least one nucleotide
constituting the oligonucleotide is modified. [6] The antisense
oligomer according to [4] or [5] above, 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). [7] The antisense oligomer according
to any one of [4] to [6] above, 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. [8] The antisense
oligomer according to any one of [1] to [3] above, which is a
morpholino oligomer. [9] The antisense oligomer according to [8]
above, wherein the morpholine ring moiety, the phosphate-binding
region, 3'-end and/or 5'-end of at least one morpholino
constituting the morpholino oligomer is modified. [10] The
antisense oligomer according to [9] or [10] above, wherein the
phosphate-binding region of at least one morpholino constituting
the morpholino oligomer is any one selected from a
phosphorodiamidate bond, a phosphorothioate bond, a
phosphorodithioate bond, an alkylphosphonate bond, a
phosphoramidate bond and a boranophosphate bond. [11] The antisense
oligomer according to [10] above, which is a phosphorodiamidate
morpholino oligomer. [12] The antisense oligomer according to any
one of [9] to [11] above, wherein the 5' end is any one of chemical
formulae (1) to (3) below:
##STR00001##
[0029] [13] A pharmaceutical composition for the treatment of
muscular dystrophy, comprising as an active ingredient the
antisense oligomer according to any one of [1] to [12] above, or a
pharmaceutically acceptable salt or hydrate thereof. [14] The
pharmaceutical composition according to [13] above, comprising a
pharmaceutically acceptable carrier. [15] A method for treatment of
muscular dystrophy, which comprises administering to a patient with
muscular gystrophy the antisense oligomer according to any one of
[1] to [12] above or the pharmaceutical composition according to
[13] or [14] above. [16] The method for treatment according to [15]
above, wherein the patient with muscular dystrophy is a patient
with deletions of nucleotides within exons 29-50, 50, 45-50, 48-50,
49-50, 52, 52-63, 13-50, 19-50, 43-50 or 47-50. [17] The method for
treatment according to [15] or [16] above, wherein the patient is a
human. [18] The use of the antisense oligomer according to any one
of [1] to [12] above in manufacturing of the pharmaceutical
composition for the treatment of muscular gystrophy. [19] The
antisense oligomer according to any one of [1] to [12] above, for
use in the treatment of muscular dystrophy. [20] The antisense
oligomer according to [19] above, wherein the patient with muscular
dystrophy in the said treatment is a patient with deletions of
nucleotides within exons 29-50, 50, 45-50, 48-50, 49-50, 52, 52-63,
13-50, 19-50, 43-50 or 47-50. [21] The antisense oligomer according
to [19] or [20] above, wherein the patient is a human.
Effects of the Invention
[0030] The antisense oligomer of the present invention can induce
skipping of exon 51 in the human dystrophin gene with a high
efficiency. Also, the symptoms of Duchenne muscular dystrophy can
be effectively alleviated by administering the pharmaceutical
composition of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 shows the efficiency of exon 51 skipping in the human
dystrophin gene in human rhabdomyosarcoma cell line (RD cells).
MODE FOR CARRYING OUT THE INVENTION
[0032] 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.
1. Antisense Oligomer
[0033] The present invention provides the antisense oligomer
(hereinafter referred to as the "antisense oligomer of the present
invention") which causes skipping of exon 51 in the human
dystrophin gene with a high efficiency.
[Exon 51 in Human Dystrophin Gene]
[0034] 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.
[0035] In the human genome, the human dystrophin gene locates at
locus Xp21.2. The human dystrophin gene has a size of 2.2 million
nucleotide pairs 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).
[0036] The nucleotide sequence of exon 51 in the human wild-type
dystrophin gene is represented by SEQ ID NO: 3.
[0037] [Antisense Oligomer]
[0038] The antisense oligomer of the present invention is designed
to cause skipping of exon 51 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 51 in the
dystrophin gene that is the target of exon skipping by the
antisense oligomer includes both wild and mutant types.
[0039] The antisense oligomer of the present invention is
specifically the antisense oligomer which is selected from a group
consisting of (a) to (d) below.
(a) an antisense oligomer comprising a nucleotide sequence of SEQ
ID NO: 1 or 2; (b) an antisense oligomer which consists of a
nucleotide sequence having deletion, substitution, insertion and/or
addition of 1 to 5 nucleotides in the nucleotide sequence of SEQ ID
NO: 1 or 2, and has an activity to cause skipping of the 51st exon
in the human dystrophin gene; (c) an antisense oligomer which has a
nucleotide sequence having at least 80% identity with a nucleotide
sequence of SEQ ID NO: 1 or 2 and has an activity to cause skipping
of exon 51 in the human dystrophin gene; and (d) an antisense
oligomer that hybridizes under stringent conditions to an
oligonucleotide consisting of a nucleotide sequence complementary
to the nucleotide sequence of SEQ ID NO: 1 or 2 and has an activity
to cause skipping of exon 51 in the human dystrophin gene.
[0040] The antisense oligomers of (b) to (d) are mutants of the
antisense oligomer of (a) in particular and are intended to
correspond to mutations of the dystrophin gene of the patients,
e.g. polymorphism.
[0041] As another embodiment, the antisense oligomer of the present
invention is specifically the antisense oligomer which is selected
from a group consisting of (k) to (n) below.
(k) An antisense oligomer comprising the nucleotide sequence shown
by any one of the SEQ ID NOS: 6 to 33; (l) An antisense oligomer
which consists of a nucleotide sequence having deletion,
substitution, insertion and/or addition of 1 to 5 nucleotides in
the nucleotide sequence shown by any one of the SEQ ID NOS: 6 to
33, and has an activity to cause skipping of exon 51 in the human
dystrophin gene; (m) An antisense oligomer which has a nucleotide
sequence having at least 80% identity with a nucleotide sequence of
any one of the SEQ ID NOS: 6 to 33 and has an activity to cause
skipping of exon 51 in the human dystrophin gene; and (n) An
antisense oligomer that hybridizes under stringent conditions to an
oligonucleotide consisting of a nucleotide sequence complementary
to the nucleotide sequence shown by any one of the SEQ ID NOS: 6 to
33 and has an activity to cause skipping of exon 51 in the human
dystrophin gene.
[0042] The antisense oligomers of (1) to (n) are mutants of the
antisense oligomer of (k) in particular and are intended to
correspond to mutations, of the dystrophin gene of the patients,
e.g. polymorphism.
[0043] Also, the antisense oligomer of the present invention is the
antisense oligomer which is selected from a group consisting of (o)
to (r) below.
(o) An antisense oligomer which consists of the nucleotide sequence
shown by any one of the SEQ ID NOS: 6 to 33; (p) An antisense
oligomer which consists of a nucleotide sequence having deletion,
substitution, insertion and/or addition of 1 to 3 nucleotides in
the nucleotide sequence shown by any one of the SEQ ID NOS: 6 to
33, and has an activity to cause skipping of exon 51 in the human
dystrophin gene; (q) An antisense oligomer which has a nucleotide
sequence having at least 80% identity with a nucleotide sequence of
any one of the SEQ ID NOS: 6 to 33 and has an activity to cause
skipping of exon 51 in the human dystrophin gene; and (r) An
antisense oligomer that hybridizes under high stringent conditions
to an oligonucleotide consisting of a nucleotide sequence
complementary to the nucleotide sequence shown by any one of the
SEQ ID NOS: 6 to 33 and has an activity to cause skipping of exon
51 in the human dystrophin gene.
[0044] Furthermore, the antisense oligomer of the present invention
is the antisense oligomer which is selected from a group consisting
of (i) and (j) below:
(i) an antisense oligomer consisting of a nucleotide sequence of
any one of the SEQ ID NOS: 6 to 33; or (j) an antisense oligomer
which consists of a nucleotide sequence having at least 90%
identity with a nucleotide sequence of any one of the SEQ ID NOS: 6
to 33 and has an activity to cause skipping of exon 51 in the human
dystrophin gene.
[0045] As used herein, the term "antisense oligomer that hybridizes
under stringent conditions" refers to, for example, an antisense
oligomer obtained by colony hybridization, plaque hybridization,
Southern hybridization or the like, using as a probe all or part of
an oligonucleotide consisting of a nucleotide sequence
complementary to the nucleotide sequence of, e.g., SEQ ID NO: 1.
The hybridization method which may be used includes methods
described in, for example, "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.
[0046] 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, (1) 5.times.SSC,
5.times.Denhardt's solution, 0.5% SDS, 50% formamide at 50.degree.
C., (2) 0.2.times.SSC, 0.1% SDS at 60.degree. C., (3)
0.2.times.SSC, 0.1% SDS at 62.degree. C., (4) 0.2.times.SSC, 0.1%
SDS at 65.degree. C., or (5) 0.1.times.SSC, 0.1% SDS at 65.degree.
C., but is not limited thereto. Under these conditions, antisense
oligomer 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.
[0047] 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 antisense
oligomer. 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: 3, hybridization can be detected with a DIG Nucleic Acid
Detection Kit (Roche Diagnostics).
[0048] In addition to the antisense oligomer described above, other
antisense oligomer that can be hybridized include antisense
oligomers 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, and 99.9% or higher
identity with the nucleotide sequence of SEQ ID NO: 1 or 2, as
calculated by homology search software such as FASTA and BLAST
using the default parameters.
[0049] The identity between nucleotide sequences may be determined
using FASTA (Science 227 (4693): 1435-1441, (1985)) or 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, blastx, tblastn
and tblastx 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.
[0050] The term "cause skipping of the exon 51 in the human
dystrophin gene" is intended to mean that by binding of the
antisense oligomer of the present invention to the site
corresponding to exon 51 of the transcript (e.g., pre-mRNA) of the
human dystrophin gene, for example, the nucleotide sequence
corresponding to the 5' end of exon 53 is spliced at the nucleotide
sequence corresponding to the 3' end of exon 50 in DMD patients
with deletion of exon 52 when the transcript undergoes splicing,
thus resulting in formation of mature mRNA which is free of codon
frame shift.
[0051] Herein, the term "binding" described above is intended to
mean that when the antisense 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
nucleic acid. 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.
[0052] Whether the skipping of exon 51 in the human dystrophin gene
is caused or not can be confirmed by introducing the antisense
oligomer of the present invention into a dystrophin expression cell
(e.g., human rhabdomyosarcoma cells), amplifying the region
surrounding exon 51 of mRNA of the human dystrophin gene from the
total RNA of the dystrophin expression cell by RT-PCR and
performing nested PCR or sequence analysis on the PCR amplified
product.
[0053] 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 51 is
skipped and the polynucleotide level "B" of the band where exon 51
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
[0054] Preferably, the antisense oligomer of the present invention
cause skipping of exon 51 with the efficiency of 10% or higher, 20%
or higher, 30% or higher, 40% or higher, 50% or higher, 60% or
higher, 70% or higher, 80% or higher, and 90% or higher. For
calculation of the efficiency of skipping, International
Publication WO2012/029986 may be referred.
[0055] The antisense oligomer of the present invention includes,
for example, an oligonucleotide, morpholino oligomer or peptide
nucleic acid (PNA), having a length of 16 to 35 nucleotides. The
length is preferably from 19 to 32, from 20 to 31, 21 or 30
nucleotides and morpholino oligomers are preferred.
[0056] The oligonucleotide described above (hereinafter referred to
as "the oligonucleotide of the present invention") is the antisense
oligomer of the present invention composed of nucleotides as
constituent units. Such nucleotides may be any of ribonucleotides,
deoxyribonucleotides and modified nucleotides.
[0057] 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.
[0058] In the present invention, the nucleobase includes, for
example, adenine, guanine, hypoxanthine, cytosine, thymine, uracil,
and modified bases thereof. Examples of such modified bases
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-alkylpyrimidines
(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.
[0059] 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.
[0060] 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'O,4'C-ethylene-bridged nucleic acids), but is not limited
thereto.
[0061] 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).
[0062] In this invention, 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, tertbutyl, 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.
[0063] In this invention, the cycloalkyl is preferably a cycloalkyl
having 5 to 12 carbon atoms. Specific examples include cyclopentyl,
cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl and
cyclododecyl.
[0064] In this invention, the halogen includes fluorine, chlorine,
bromine and iodine.
[0065] 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, npentyloxy,
isopentyloxy, n-hexyloxy, isohexyloxy, etc. Among others, an alkoxy
having 1 to 3 carbon atoms is preferred.
[0066] In this invention, the aryl is preferably an aryl having 6
to 10 carbon atoms. Specific examples include phenyl,
.alpha.-naphthyl and 6-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.
[0067] In this invention, 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.
[0068] In this invention, 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).
[0069] Preferably, the oligonucleotide of the present invention is
the antisense 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.
[0070] 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., Takara Co., or Japan Bio Service Co.),
etc.
[0071] The morpholino oligomer of the present invention is the
antisense 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;
[0072] R.sup.1 represents H or an alkyl;
[0073] R.sup.2 and R.sup.3, which may be the same or different,
each represents H, an alkyl, a cycloalkyl or an aryl;
[0074] Y.sub.1 represents O, S, CH.sub.2 or NR.sup.1;
[0075] Y.sub.2 represents O, S or NR.sup.1;
[0076] Z represents O or S.
[0077] 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.
[0078] The morpholino oligomer of the present invention comprises
one having fully or partly modified nucleobases, morpholine ring
moieties, phosphate-binding regions, 3'-end and/or 5'-end which
constitute the morpholino oligomer.
[0079] A modification of the phosphate-binding region includes, for
example, a modification of replacing with phosphorodiamidate bond,
phosphorothioate bond, phosphorodithioate bond, alkylphosphonate
bond, phosphoramidate bond and boranophosphate bond (Enya et al:
Bioorganic & Medicinal Chemistry, 2008, 18, 9154-9160)(cf.,
eg., Japan Domestic Re-Publications of PCT Application Nos.
2006/129594 and 2006/038608).
[0080] The morpholino oligomer may be produced in accordance with,
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]
[0081] An embodiment of PMO is, for example, the compound
represented by general formula (I) below (hereinafter PMO (I)).
##STR00006##
wherein Base, R.sup.2 and R.sup.3 have the same significance as
defined above; and,
[0082] n is a given integer of 1 to 99, preferably a given integer
of 24 to 34, 27 to 31 or 28 to 30, preferably 29.
[0083] PMO (I) can be produced in accordance with a known method,
for example, can be produced by performing the procedures in the
following steps.
[0084] The compounds and reagents used in the steps below are not
particularly limited so long as they are commonly used to prepare
PMO.
[0085] 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:
[0086] 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 (IV) below
(hereinafter referred to as group (IV)).
##STR00008##
[0087] 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. 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).
[0088] 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, N-methylimidazole,
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% divinilbenzene
crosslinked (200-400 mesh) (2.4-3.0 mmol/g) (manufactured by Tokyo
Chemical Industry), Aminomethylated Polystyrene Resin HCl
[divinylbenzene 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.,
e.g., Alul et al., Nucleic Acids Research, Vol. 19, 1527 (1991)),
TentaGel support-aminopolyethylene glycol-derivatized support
(e.g., Wright et al., cf., Tetrahedron Letters, Vol. 34, 3373
(1993)), and a copolymer of Poros-polystyrene/divinylbenzene.
[0089] 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).
[0090] This step can be performed by reacting Compound (II) with an
acid.
[0091] 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).
[0092] 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.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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).
[0098] 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.
[0099] 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.
[0100] 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:
[0101] 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, carboxyl group
or amino.
[0102] This step can be carried out by known procedures for
introducing linkers, using Compound (V) as the starting
material.
[0103] 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:
[0104] 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.
[0105] This step can be performed using Compound (VI) and a solid
carrier in accordance with a process known as condensation
reaction.
[0106] In Compound (II), the compound represented by general
formula (IIa2) below wherein n is 2 to 99 (preferably a given
integer of 25 to 35, 28 to 32, or 29 to 31, preferably 30) 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
(in a specific embodiment, n' is 1 to 34, 1 to 33, 1 to 32, 1 to
31, 1 to 30, 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to 25, 1 to
24).
(2) Step B
[0107] 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)):
##STR00014##
wherein B.sup.P, L, n, R.sup.2, R.sup.3 and T have the same
significance as defined above.
[0108] This step can be performed by reacting Compound (III) with
the morpholino monomer compound in the presence of a base.
[0109] The morpholino monomer compound includes, for example,
compounds represented by general formula (VIII) below:
##STR00015##
wherein B.sup.P, R.sup.2, R.sup.3 and T have the same significance
as defined above.
[0110] 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
(III), preferably, 10 mol equivalents to 100 mol equivalents based
on 1 mol of Compound (III).
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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,
tetrahydrofuran an alcohol(s) (ethanol, isopropanol,
trifluoroethanol, etc.), water, or a mixture thereof.
[0115] 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.
[0116] 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:
[0117] 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).
##STR00016##
wherein Base, B.sup.P, L, n, R.sup.2, R.sup.3 and T have the same
significance as defined above.
[0118] This step can be performed by reacting Compound (VII) with a
deprotecting agent.
[0119] 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 them, ethanol is preferred. The amount of the
deprotecting agent used is appropriately in a range of, 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).
[0120] 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:
[0121] PMO (I) is produced by reacting Compound (IX) produced in
step C with an acid:
##STR00017##
[0122] wherein Base, n, R.sup.2, R.sup.3 and T have the same
significance as defined above.
[0123] This step can be performed by adding an acid to Compound
(IX).
[0124] 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.
[0125] 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 deprotection
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.
[0126] 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).
[0127] 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.
[0128] 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.
[0129] A peptide nucleic acid is the oligomer of the present
invention having a group represented by the following general
formula as the constituent unit:
##STR00018##
wherein Base has the same significance as defined above.
[0130] Peptide nucleic acids can be prepared by referring to, e.g.,
the following literatures. [0131] 1) P. E. Nielsen, M. Egholm, R.
H. Berg, O. Buchardt, Science, 254, 1497 (1991) [0132] 2) M.
Egholm, O. Buchardt, P. E. Nielsen, R. H. Berg, Jacs., 114, 1895
(1992) [0133] 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) [0134]
4) L. Christensen, R. Fitzpatrick, B. Gildea, K. H. Petersen, H. F.
Hansen, T. Koch, M. Egholm, O. Buchardt, P. E. Nielsen, J. Coull,
R. H. Berg, J. Pept. Sci., 1, 175 (1995) [0135] 5) T. Koch, H. F.
Hansen, P. Andersen, T. Larsen, H. G. Batz, K. Otteson, H. Orum, J.
Pept. Res., 49, 80 (1997)
[0136] 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.
##STR00019##
[0137] 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
[0138] The oligomer of the present invention causes exon 51
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 efficience by
administering the pharmaceutical composition comprising the
oligomer of the present invention to DMD patients, who has mutation
converting to in-frame by Exon 51 skipping, for example, patients
with deletion of exon 29-50, patients with deletion of exon 50,
patients with deletion of exon 45-50, patients with deletion of
exon 48-50, patients with deletion of exon 49-50, patients with
deletion of exon 52, patients with deletion of exon 52-63, patients
with deletion of exon 13-50, patients with deletion of exon 19-50,
patients with deletion of exon 43-50, or patients with deletion of
exon 47-50. 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.
[0139] 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").
[0140] Also, the present invention provides a method for treatment
of muscular dystrophy, which comprises administering to a patient
of DMD the oligomer of the present invention.
[0141] In the said method for treatment, the oligomer of the
present invention can be administered as the pharmaceutical
composition for the treatment of muscular dystrophy.
[0142] Furthermore, the present invention provides the use of the
oligomer of the present invention in manufacturing of the
pharmaceutical composition for treating muscular dystrophy and the
oligomer of the present invention applied for the treatment of
muscular dystrophy.
[0143] 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.
[0144] 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.
[0145] In administration of the oligomer of the present invention
to patients with muscular dystrophy, the composition of the present
invention may contain 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 Mirus) 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. For
further details, U.S. Pat. Nos. 4,235,871, 4,737,323, WO96/14057,
"New RRC, Liposomes: A practical approach, IRL Press, Oxford (1990)
pages 33-104", etc. can be referred,
[0146] 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 100 nM to 10
.mu.M. A weight ratio of the oligomer of the present invention
contained in the composition of the present invention and the
carrier (carrier/antisense 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 0.1 to 10.
[0147] 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.
[0148] 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.
[0149] 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 about 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.
[0150] 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.
[0151] 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 antisense 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.
[0152] 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 100 nM to 10 .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
0.1 to 10. 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.
[0153] 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.
EXAMPLES
Reference Example 1
4-{[(2S,
6R)-6-(4-Benzamido-2-oxopyrimidin-1-yl)-4-tritylmorpholin-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)-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic Acid
[0154] 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 0.5M 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
[0155] After 4.0 g of
4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-y-
l]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.
[0156] 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 nmol/g.
Conditions of UV Measurement
[0157] Apparatus: U-2910 (Hitachi, Ltd.)
[0158] Solvent: methanesulfonic acid
[0159] Wavelength: 265 nm
[0160] .epsilon. value: 45000
[0161] According to the descriptions in EXAMPLES 1, 2 and REFERENCE
EXAMPLE 1 below, PMO shown by PMO Nos. 1-3 in TABLE 1 were
synthesized. The synthesized PMO was dissolved in water for
injection (manufactured by Otsuka Pharmaceutical Factory,
Inc.).
TABLE-US-00001 TABLE 1 PMO No. Note SEQ ID NO: 1 5' end: group (3)
1 2 5' end: group (3) 2 3 Sequence corresponding to SEQ ID 4 NO;
588 in Patent Document 3, 5' end: group (3)
Example 1
PMO No. 1
[0162] 0.2 g 4-{[(2S, 6R)-6-(4-benzamide-2-oxopyrimidin-1
(2H)-yl)-4-tritylmorpholin-2-yl]methoxy} 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 (see the Table 2
below).
TABLE-US-00002 TABLE 2 Step Reagent Volume (mL) Time (min) 1
deblocking solution 18-32 1.8-3.2 2 neutralizing and washing
solution 30 1.5 3 coupling solution B 5 0.5 4 coupling solution A
1.3 0.25 5 coupling reaction by the reagents 120-300 added in the
steps 3 and 4 6 acetonitrile 20 1.0 7 capping solution 9 2.0 8
acetonitrile 30 2.0
[0163] The deblocking solution used was dichloromethane solution
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.
[0164] 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
shown in Table 3 below.
TABLE-US-00003 TABLE 3 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 CV: column
volume
[0165] 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).
[0166] The resulting aqueous solution containing the objective
product was purified by an anionic exchange resin column. The
conditions used are as shown in Table 4 below.
TABLE-US-00004 TABLE4 Column Source 15Q (GE Healthcare, .phi.10
.times. 108 mm, 1 CV = 8.5 mL) Flow rate 8.5 mL/min Column room
temperature 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
[0167] 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 in Table 5
below.
TABLE-US-00005 TABLE 5 Column XBridge 5 .mu.m C8 (Waters, .phi.10
.times. 50 mm, 1 CV = 4 ml) Flow rate 4 mL/min Column 60.degree. C.
temperature Solution A water Solution B CH.sub.3CN Gradient (B)
conc. 0.fwdarw.50%/20 CV
[0168] 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 the objective compound as a white cotton-like solid.
ESI-TOF-MS Clcd.: 10021.46
[0169] Found: 10021.91
Example 2
PMO No. 2
[0170] The title compound was produced in accordance with the
procedure of EXAMPLE 1.
ESI-TOF-MS Clcd.: 9916.71
[0171] Found: 9916.43
Comparative Example 1
PMO No. 3
[0172] The title compound was produced in accordance with the
procedure of EXAMPLE 1.
ESI-TOF-MS Clcd.: 9949.46
[0173] Found: 9949.41
Test Example 1
In Vitro Assay
[0174] Experiments were performed using the antisense oligomers PMO
Nos. 1 and 2 of the present invention and the antisense oligomer
PMO No. 3. The sequences of various antisense oligomers are given
in Table 6 below.
TABLE-US-00006 TABLE 6 SEQ PMO ID Nucleotide sequence No. NO:
CGGTAAGTTCTGTCCTCAAGGAAGATGGCA 1 1 CTCATACCTTCTGCTTCAAGGAAGATGGCA 2
2 CTCCAACATCAAGGAAGATGGCATTTCTAG 3 4
[0175] Using an Amaxa Cell Line Nucleofector Kit L on Nucleofector
II (Lonza), 0.3, 1, 3, 10 .mu.M of the oligomers PMO Nos. 1 and 2
of the present invention and the antisense oligomer PMO No. 3 were
transfected with 3.5.times.10.sup.5 of RD cells (human
rhabdomyosarcoma cell line). The Program T-030 was used.
[0176] After transfection, the cells were cultured for three 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% CO2. The cells were washed with PBS (manufactured by Nissui,
hereinafter the same) and 500 .mu.l of ISOGEN II (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).
[0177] One-Step RT-PCR was performed with 400 ng of the extracted
total RNA using a Qiagen One Step 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.
[0178] 50.degree. C., 30 mins: reverse transcription reaction
[0179] 95.degree. C., 15 mins: thermal denaturation
[0180] [94.degree. C., 30 seconds; 60.degree. C., 30 seconds;
72.degree. C., 60 seconds].times.35 cycles: PCR amplification
72.degree. C., 10 mins: final extension
[0181] The nucleotide sequences of the forward primer and reverse
primer used for RT-PCR are given below.
TABLE-US-00007 (SEQ ID NO: 5) Forward primer: 5'-
CTGAGTGGAAGGCGGTAAAC-3' (SEQ ID NO: 6) Reverse primer: 5'-
GAAGTTTCAGGGCCAAGTCA-3'
[0182] The reaction product, 1 .mu.L of the RT-PCR above was
analyzed using a Bioanalyzer (manufactured by Agilent Technologies,
Inc.). The polynucleotide level "A" of the band with exon 51
skipping and the polynucleotide level "B" of the band without exon
51 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
[0183] The results are shown in FIG. 1. This experiment revealed
that, the antisense oligomers of the present invention could cuase
exon 51 skipping with a markedly higher efficiency than the
antisense oligomer PMO No. 3.
Example 3
PMO No. 4-6
[0184] The title compound was produced in accordance with the
procedure of EXAMPLE 1. The sequences of various antisense
oligomers are given below.
TABLE-US-00008 TABLE 7 SEQ PMO ESI-TOF-MS ID No. Sequence MW Found
Note NO 4 AACATCAAGGAAGAT 7007.96 7007.97 5' end: 7 GGCATT group
(3) 5 TCCAACATCAAGGAA 6968.97 6968.42 5' end: 8 GATGGC group (3) 6
ACCTCCAACATCAAG 6912.91 6912.85 5' end: 9 GAAGAT group (3)
Example 4
[0185] 2'-O-methoxy-phosphorothioates Shown by SEQ ID NOS: 9 to
33
[0186] Various antisense oligomers of the title were produced by
outsourcing to Japan Bio Service Co. The sequence of various
antisense oligomers are given in Table 8.
TABLE-US-00009 TABLE 8 SEQ ID ESI-TOF-MS NO: Sequence MW Found 10
GAGUAACAGUCUGAGUAGGAG 7453 7452.876 11 UGUGUCACCAGAGUAACAGUC 7310
7313.793 12 AACCACAGGUUGUGUCACCAG 7309 7311.199 13
UUUCCUUAGUAACCACAGGUU 7209 7211.436 14 GAGAUGGCAGUUUCCUUAGUA 7328
7331.388 15 UUCUAGUUUGGAGAUGGCAGU 7345 7347.440 16
AAGAUGGCAUUUCUAGUUUGG 7329 7329.982 17 AACAUCAAGGAAGAUGGCAUU 7381
7381.059 18 AGGUACCUCCAACAUCAAGGA 7316 7318.395 19
CUGCCAGAGCAGGUACCUCCA 7284 7286.932 20 CGGUUGAAAUCUGCCAGAGCA 7349
7351.895 21 UGUCCAAGCCCGGUUGAAAUC 7286 7286.00 22
CGGUAAGUUCUGUCCAAGCCC 7262 7262.929 23 GAAAGCCAGUCGGUAAGUUCU 7350
7351.869 24 AUCAAGCAGAGAAAGCCAGUC 7379 7378.383 25
UUAUAACUUGAUCAAGCAGAG 7319 7320.149 26 CUCUGUGAUUUUAUAACUUGA 7211
7212.295 27 CACCAUCACCCUCUGUGAUUU 7144 7145.555 28
CAAGGUCACCCACCAUCACCC 7187 7187.709 29 UUGAUAUCCUCAAGGUCACCC 7207
7210.071 30 GAUCAUCUCGUUGAUAUCCUC 7185 71882.39 31
UCUGCUUGAUGAUCAUCUCGU 7202 7203.926 32 GGCAUUUCUAGUUUGGAGAUG 7346
7346.562 33 CAAGGAAGAUGGCAUUUCUAG 7375 7375.678 34
CCUCCAACAUCAAGGAAGAUG 7317 7318.343
INDUSTRIAL APPLICABILITY
[0187] Experimental results in TEST EXAMPLES demonstrate that the
oligomers of the present invention caused exon 51 skipping with a
markedly high efficiency in RD cells. Therefore, the oligomers of
the present invention are extremely useful for the treatment of
DMD.
Sequence CWU 1
1
34130DNAArtificial SequenceSynthetic Nucleic Acid 1cggtaagttc
tgtcctcaag gaagatggca 30230DNAArtificial SequenceSynthetic Nucleic
Acid 2ctcatacctt ctgcttcaag gaagatggca 303233DNAHomo sapiens
3ctcctactca gactgttact ctggtgacac aacctgtggt tactaaggaa actgccatct
60ccaaactaga aatgccatct tccttgatgt tggaggtacc tgctctggca gatttcaacc
120gggcttggac agaacttacc gactggcttt ctctgcttga tcaagttata
aaatcacaga 180gggtgatggt gggtgacctt gaggatatca acgagatgat
catcaagcag aag 233430DNAArtificial SequenceSynthetic Nucleic Acid
4ctccaacatc aaggaagatg gcatttctag 30520DNAArtificial
SequenceSynthetic Nucleic Acid 5ctgagtggaa ggcggtaaac
20620DNAArtificial SequenceSynthetic Nucleic Acid 6gaagtttcag
ggccaagtca 20721DNAArtificial SequenceSynthetic Nucleic Acid
7aacatcaagg aagatggcat t 21821DNAArtificial SequenceSynthetic
Nucleic Acid 8tccaacatca aggaagatgg c 21921DNAArtificial
SequenceSynthetic Nucleic Acid 9acctccaaca tcaaggaaga t
211021DNAArtificial SequenceSynthetic Nucleic Acid 10gaguaacagu
cugaguagga g 211121DNAArtificial SequenceSynthetic Nucleic Acid
11ugugucacca gaguaacagu c 211221DNAArtificial SequenceSynthetic
Nucleic Acid 12aaccacaggu ugugucacca g 211321DNAArtificial
SequenceSynthetic Nucleic Acid 13uuuccuuagu aaccacaggu u
211421DNAArtificial SequenceSynthetic Nucleic Acid 14gagauggcag
uuuccuuagu a 211521DNAArtificial SequenceSynthetic Nucleic Acid
15uucuaguuug gagauggcag u 211621DNAArtificial SequenceSynthetic
Nucleic Acid 16aagauggcau uucuaguuug g 211721DNAArtificial
SequenceSynthetic Nucleic Acid 17aacaucaagg aagauggcau u
211821DNAArtificial SequenceSynthetic Nucleic Acid 18agguaccucc
aacaucaagg a 211921DNAArtificial SequenceSynthetic Nucleic Acid
19cugccagagc agguaccucc a 212021DNAArtificial SequenceSynthetic
Nucleic Acid 20cgguugaaau cugccagagc a 212121DNAArtificial
SequenceSynthetic Nucleic Acid 21uguccaagcc cgguugaaau c
212221DNAArtificial SequenceSynthetic Nucleic Acid 22cgguaaguuc
uguccaagcc c 212321DNAArtificial SequenceSynthetic Nucleic Acid
23gaaagccagu cgguaaguuc u 212421DNAArtificial SequenceSynthetic
Nucleic Acid 24aucaagcaga gaaagccagu c 212521DNAArtificial
SequenceSynthetic Nucleic Acid 25uuauaacuug aucaagcaga g
212621DNAArtificial SequenceSynthetic Nucleic Acid 26cucugugauu
uuauaacuug a 212721DNAArtificial SequenceSynthetic Nucleic Acid
27caccaucacc cucugugauu u 212821DNAArtificial SequenceSynthetic
Nucleic Acid 28caaggucacc caccaucacc c 212921DNAArtificial
SequenceSynthetic Nucleic Acid 29uugauauccu caaggucacc c
213021DNAArtificial SequenceSynthetic Nucleic Acid 30gaucaucucg
uugauauccu c 213121DNAArtificial SequenceSynthetic Nucleic Acid
31ucugcuugau gaucaucucg u 213221DNAArtificial SequenceSynthetic
Nucleic Acid 32ggcauuucua guuuggagau g 213321DNAArtificial
SequenceSynthetic Nucleic Acid 33caaggaagau ggcauuucua g
213421DNAArtificial SequenceSynthetic Nucleic Acid 34ccuccaacau
caaggaagau g 21
* * * * *