NZ740562B2 - Antisense nucleic acid - Google Patents
Antisense nucleic acid Download PDFInfo
- Publication number
- NZ740562B2 NZ740562B2 NZ740562A NZ74056216A NZ740562B2 NZ 740562 B2 NZ740562 B2 NZ 740562B2 NZ 740562 A NZ740562 A NZ 740562A NZ 74056216 A NZ74056216 A NZ 74056216A NZ 740562 B2 NZ740562 B2 NZ 740562B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- nucleotide sequence
- oligomer
- pharmaceutically acceptable
- hydrate
- acceptable salt
- Prior art date
Links
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- 125000001971 neopentyl group Chemical group [H]C([*])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 230000001264 neutralization Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 150000002815 nickel Chemical class 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 150000003891 oxalate salts Chemical class 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 150000008103 phosphatidic acids Chemical class 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 150000004885 piperazines Chemical class 0.000 description 1
- 229920001601 polyetherimide Polymers 0.000 description 1
- 229920005990 polystyrene resin Polymers 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- 230000000750 progressive Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 201000004193 respiratory failure Diseases 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000003746 solid phase reaction Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000000087 stabilizing Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 150000003890 succinate salts Chemical class 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 125000000472 sulfonyl group Chemical group *S(*)(=O)=O 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 150000003892 tartrate salts Chemical class 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000001973 tert-pentyl group Chemical group [H]C([H])([H])C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- QEMXHQIAXOOASZ-UHFFFAOYSA-N tetramethylammonium Chemical class C[N+](C)(C)C QEMXHQIAXOOASZ-UHFFFAOYSA-N 0.000 description 1
- 150000003536 tetrazoles Chemical class 0.000 description 1
- 229950000329 thiouracil Drugs 0.000 description 1
- 125000005425 toluyl group Chemical group 0.000 description 1
- ITMCEJHCFYSIIV-UHFFFAOYSA-M triflate Chemical class [O-]S(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-M 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 125000003774 valeryl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
- 150000003751 zinc Chemical class 0.000 description 1
- ZGUNAGUHMKGQNY-UHFFFAOYSA-N α-phenylglycine Chemical compound OC(=O)C(N)C1=CC=CC=C1 ZGUNAGUHMKGQNY-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N2310/00—Structure or type of the nucleic acid
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- C12N2310/11—Antisense
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/314—Phosphoramidates
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- C12N2310/00—Structure or type of the nucleic acid
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- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
Abstract
The present invention provides an oligomer that enables skipping of exon 45 in the human dystrophin gene.
Description
SPECIFICATION
ANTISENSE NUCLEIC ACID
TECHNICAL FIELD
The present invention relates to an antisense oligomer which allows exon 45
skipping in the human dystrophin gene, and a pharmaceutical composition comprising
such an oligomer.
BACKGROUND ART
Duchenne muscular dystrophy (DMD) is an inherited progressive myopathy
with the highest incidence which occurs at a frequency of about one in 3,500 live male
births. In their infancy, DMD patients show almost the same motor function as in
normal humans, but they show signs of muscle weakness around the ages of 4 to 5 years.
Then, their muscle weakness progresses to the loss of ambulation until the age of about
12 years and eventually leads to death in their twenties due to heart failure or respiratory
failure. DMD is such a sever disease. Currently, there is no effective therapy for
DMD, and hence the development of a new therapeutic agent is strongly demanded.
DMD is known to be caused by mutations in the dystrophin gene. The
dystrophin gene is located on the X chromosome and is a huge gene consisting of 2.2
million DNA nucleotide pairs. This DNA is transcribed into precursor mRNA and
further spliced to remove introns, thereby resulting in mRNA consisting of 79 exons
joined together, which is 13,993 bases in length. This mRNA is translated into 3,685
amino acids to produce a dystrophin protein. The dystrophin protein is involved in
maintenance of the membrane stability of muscle cells and is required to make muscle
cells less prone to breakage. DMD patients have mutations in their dystrophin gene
and therefore show almost no expression of a functional dystrophin protein in their
muscle cells. For this reason, in the body of DMD patients, muscle cells can no longer
retain their structure and an abundance of calcium ions flows into the muscle cells. As
a result, a reaction similar to inflammation will occur to promote fibrosis, so that muscle
cells are difficult to regenerate.
Becker muscular dystrophy (BMD) is also caused by mutations in the
dystrophin gene. As its symptom, muscle weakness is observed, but is usually milder
and progresses slower than in DMD, so that BMD develops in adulthood in most cases.
Differences in clinical symptoms between DMD and BMD appear to arise from whether
mutations disrupt or maintain the amino acid reading frame during translation from
dystrophin mRNA into a dystrophin protein (Non-patent Document 1). Namely, DMD
patients show almost no expression of a functional dystrophin protein because of having
mutations responsible for shifting the amino acid reading frame, whereas in BMD
patients, mutations cause deletion of some exons but the amino acid reading frame is
maintained, so that a functional albeit incomplete dystrophin protein is produced.
As a therapy for DMD, the exon skipping therapy is promising. This therapy
involves modification of splicing to restore the amino acid reading frame in dystrophin
mRNA, thereby inducing the expression of a dystrophin protein with partially recovered
function (Non-patent Document 2). Amino acid sequence regions targeted by exon
skipping are deleted in this therapy. For this reason, a dystrophin protein expressed in
this therapy is shorter than the normal protein, but partially retains the function of
stabilizing muscle cells because the amino acid reading frame is maintained. It is
therefore expected that exon skipping allows DMD to present the same symptoms as
seen in BMD which is milder. The exon skipping therapy is now under clinical trial in
human DMD patients after animal experiments in mice and dogs.
Exon skipping can be induced by binding of antisense nucleic acids directed
against either or both of the 5’ and 3’ splice sites or against exon internal sequences.
An exon is included into mRNA only when its both splice sites are recognized by a
spliceosome complex. Thus, exon skipping can be induced when the splice sites are
targeted by antisense nucleic acids. Moreover, to induce exon recognition by the
splicing machinery, SR proteins rich in serine and arginine would be required to bind to
exon splicing enhancers (ESEs); and hence exon skipping can also be induced upon
targeting to ESEs.
DMD patients have different mutations in their dystrophin gene, and hence
various antisense nucleic acids are required depending on the position and type of gene
mutation. There are some reports of an antisense nucleic acid designed to induce exon
skipping of a single exon in the dystrophin gene by targeting a single continuous
sequence (Patent Documents 1 to 6, as well as Non-patent Documents 1 and 2). In
addition, there is a report showing that when two different antisense nucleic acids
directed against the same exon in the dystrophin gene are allowed to act in admixture
(double targeting), skipping activity may be enhanced as compared to when each
antisense nucleic acid is used alone (Patent Document 7).
However, there has been no report showing that connected single-stranded
antisense nucleic acids directed against two or more sites in the same exon (i.e.,
antisense nucleic acid of connected type) show skipping activity.
Prior Art Documents
Patent Documents
Patent Document 1: WO2004/048570
Patent Document 2: WO2009/139630
Patent Document 3: WO2010/048586
Patent Document 4: US2010/0168212
Patent Document 5: WO2011/057350
Patent Document 6: WO2006/000057
Patent Document 7: WO2007/135105
Non-patent Documents
Non-patent Document 1: Annemieke Aartsma-Rus et al., (2002) Neuromuscular
Disorders 12: S71-S77
Non-patent Document 2: Wilton S. D., et al., Molecular Therapy 2007: 15: p.
1288-96
SUMMARY OF THE INVENTION
Disclosed herein is a novel antisense oligomer of connected type which is
designed to induce exon skipping by targeting separate two nucleotide sequences in the
same exon of the dystrophin gene, and a therapeutic agent for muscular dystrophy
comprising such an oligomer.
As a result of detailed studies on the technical contents described in the above
documents and on the structure of the dystrophin gene, etc., the inventors of the present
invention have found that oligomers directed against two separate sites in exon 45 of the
human dystrophin gene are connected together and the resulting antisense oligomer can
induce skipping of this exon. The inventors of the present invention have completed the
present invention on the basis of this finding.
Namely, the present invention is as follows.
An antisense oligomer of 14 to 32 bases in length comprising connected two unit
oligomers selected from the group consisting of (a) to (e) shown below, or a
pharmaceutically acceptable salt or hydrate thereof, wherein the two unit oligomers are
not contiguous to each other or do not overlap with each other:
(a) a unit oligomer consisting of a nucleotide sequence complementary to a
nucleotide sequence consisting of contiguous 7 to 16 bases selected from a nucleotide
sequence located at positions -5 to 15 from the 5’-terminal end of exon 45 in the human
dystrophin gene;
(b) a unit oligomer consisting of a nucleotide sequence complementary to a
nucleotide sequence consisting of contiguous 7 to 16 bases selected from a nucleotide
sequence located at positions 48 to 70 from the 5’-terminal end of exon 45 in the human
dystrophin gene;
(c) a unit oligomer consisting of a nucleotide sequence complementary to a
nucleotide sequence consisting of contiguous 7 to 16 bases selected from a nucleotide
sequence located at positions 128 to 150 from the 5’-terminal end of exon 45 in the human
dystrophin gene;
(d) a unit oligomer consisting of a nucleotide sequence complementary to a
nucleotide sequence consisting of contiguous 7 to 16 bases selected from a nucleotide
sequence located at positions 15 to 40 from the 5’-terminal end of exon 45 in the human
dystrophin gene; and
(e) a unit oligomer consisting of a nucleotide sequence complementary to a
nucleotide sequence consisting of contiguous 7 to 16 bases selected from a nucleotide
sequence located at positions 110 to 125 from the 5’-terminal end of exon 45 in the human
dystrophin gene.
The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof
according to [1] above, wherein one of the two unit oligomers is (a).
The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof
according to [1] or [2] above, which consists of any one nucleotide sequence selected
from the group consisting of SEQ ID NOs: 7 to 12, 14 to 33, 40 to 52, 57, 64, 65 and 79
to 86.
The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof
according to any one of [1] to [3] above, which consists of any one nucleotide sequence
selected from the group consisting of SEQ ID NOs: 8, 10, 25, 30, 33, 79 and 80.
The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof
according to any one of [1] to [4] above, which is an oligonucleotide.
The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof
according to [5] above, wherein at least one nucleotide constituting the oligonucleotide is
modified at the sugar moiety and/or at the phosphate bond moiety.
The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof
according to [5] or [6] above, wherein the sugar moiety of at least one nucleotide
constituting the oligonucleotide is a ribose in which the -OH group at the 2’-position is
substituted with any group selected from the group consisting of OR, R, R’OR, SH, SR,
NH2, NHR, NR2, N3, CN, F, Cl, Br and I (wherein R represents alkyl or aryl, and R’
represents alkylene).
The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof
according to [6] or [7] above, wherein the phosphate bond moiety 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
phosphoroamidate bond and a boranophosphate bond.
The antisense oligomer according to any one of [1] to [4] above, which is a
morpholino oligomer, or pharmaceutically acceptable salt or hydrate thereof.
The antisense oligomer according to [9] above, which is a phosphorodiamidate
morpholino oligomer, or pharmaceutically acceptable salt or hydrate thereof.
The antisense oligomer according to [4] above, which is a phosphorodiamidate
morpholino oligomer, or pharmaceutically acceptable salt or hydrate thereof.
The antisense oligomer according to any one of [9] to [11] above, whose 5’-
terminal end is any one of the groups represented by chemical formulae (1) to (3) shown
below, or pharmaceutically acceptable salt or hydrate thereof.
[Formula 1]
A pharmaceutical composition for treatment of muscular dystrophy, which
comprises the antisense oligomer or pharmaceutically acceptable salt or hydrate thereof
according to any one of [1] to [12] above as an active ingredient.
The pharmaceutical composition according to [13] above, which further
comprises a pharmaceutically acceptable carrier.
A method for treatment of muscular dystrophy, which comprises the step of
administering a muscular dystrophy patient with the antisense oligomer or
pharmaceutically acceptable salt or hydrate thereof according to any one of [1] to [12]
above or with the pharmaceutical composition according to [13] or [14] above.
The method for treatment according to [15] above, wherein the muscular
dystrophy patient is a patient having a mutation to be targeted by exon 45 skipping in the
dystrophin gene.
The method for treatment according to [15] or [16] above, wherein the patient is
a human patient.
Use of the antisense oligomer or pharmaceutically acceptable salt or hydrate
thereof according to any one of [1] to [12] above in the manufacture of a pharmaceutical
composition for treatment of muscular dystrophy.
[18a] Use of the antisense oligomer or pharmaceutically acceptable salt or hydrate
thereof according to any one of [1] to [12] above in the manufacture of a medicament for
the treatment of muscular dystrophy in a patient.
The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof
according to any one of [1] to [12] above for use in the treatment of muscular dystrophy.
The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof
according to [19] above, wherein in the treatment, a muscular dystrophy patient has a
mutation to be targeted by exon 45 skipping in the dystrophin gene.
The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof
according to [19] or [20] above, wherein the patient is a human patient.
EFFECTS OF THE INVENTION
The antisense oligomer of the present invention allows effective induction of
exon 45 skipping in the human dystrophin gene. In addition, the pharmaceutical
composition of the present invention, when administered, allows effective alleviation of
symptoms in Duchenne muscular dystrophy. Deleted exons in patients to be targeted
include exons 18-44, 44, 46, 46-47, 46-48, 46-49, 46-51, 46-53, etc.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 2 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 3 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 4 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 5 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 6 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 7 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 8 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 9 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 10 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 11 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 12 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 13 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 14 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 15 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 16 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 17 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 18 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 19 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 20 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 21 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 22 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 23 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 24 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
Figure 25 is a graph showing the efficiency of exon 45 skipping in the human
dystrophin gene in human rhabdomyosarcoma cells (RD cells).
DESCRIPTION OF EMBODIMENTS
The present invention will be described in more detail below. The following
embodiments are illustrated to describe the present invention, and it is not intended to
limit the present invention only to these embodiments. The present invention can be
implemented in various modes without departing from the spirit of the present invention.
It should be noted that all publications cited herein, including prior art
documents, patent gazettes and other patent documents, are incorporated herein by
reference. Moreover, this specification incorporates the contents disclosed in the
specification and drawings of Japanese Patent Application No. 2015-182145 (filed on
September 15, 2015), based on which the present application claims priority.
1. Antisense oligomer
The present invention provides an antisense oligomer which allows exon 45
skipping in the human dystrophin gene, or a pharmaceutically acceptable salt or hydrate
thereof (hereinafter collectively referred to as “the oligomer of the present invention”).
[Exon 45 in the human dystrophin gene]
In the context of the present invention, the term “gene” is intended to include
not only a genomic gene, but also cDNA, precursor mRNA, and mRNA. The gene is
preferably precursor mRNA, i.e., pre-mRNA.
In the human genome, the human dystrophin gene is located 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 in the human dystrophin gene constitute
only 14 kb and are distributed over 79 exons within the dystrophin gene (Roberts, RG.,
et al., Genomics, 16: 536-538 (1993)). Pre-mRNA transcribed from the human
dystrophin gene is spliced to generate mature mRNA of 14 kb. The nucleotide
sequence of the human wild-type dystrophin gene is known (GenBank Accession No.
NM_004006).
The nucleotide sequence of exon 45 in the human wild-type dystrophin gene is
shown in SEQ ID NO: 13. Moreover, in the nucleotide sequence (SEQ ID NO: 13) of
exon 45 in the human wild-type dystrophin gene, a sequence consisting of bases at
positions -5 to 15 counted from the 5’-terminal end is shown in SEQ ID NO: 3.
Likewise, a sequence consisting of bases at positions 48 to 70, a sequence consisting of
bases at positions 128 to 150, a sequence consisting of bases at positions 15 to 40 and a
sequence consisting of bases at positions 110 to 125 are shown in SEQ ID NOs: 4 to 6
and 143, respectively.
The oligomer of the present invention has now been prepared to cause exon 45
skipping in the human dystrophin gene with the aim of modifying a protein encoded by
the DMD dystrophin gene into a BMD dystrophin protein. Thus, exon 45 in the
dystrophin gene to be skipped by the oligomer of the present invention includes not
only wild-type, but also mutated forms.
More specifically, mutated exon 45 in the human dystrophin gene or a portion
thereof is a polynucleotide shown in (I) or (II) below:
(I) a polynucleotide hybridizable under stringent conditions with a polynucleotide
consisting of a nucleotide sequence complementary to any nucleotide sequence selected
from the group consisting of SEQ ID NO: 13, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID
NO: 5, SEQ ID NO: 6 and SEQ ID NO: 143; or
(II) a polynucleotide consisting of a nucleotide sequence sharing an identity of 90%
or more with any nucleotide sequence selected from the group consisting of SEQ ID
NO: 13, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID
NO: 143.
As used herein, the term “polynucleotide” is intended to mean DNA or RNA.
As used herein, the expression “polynucleotide hybridizable under stringent
conditions” is intended to mean, for example, a polynucleotide that can be obtained by
means of colony hybridization, plaque hybridization, Southern hybridization or other
hybridization techniques using, as a probe, the whole or a part of a polynucleotide
consisting of a nucleotide sequence complementary to any nucleotide sequence selected
from the group consisting of SEQ ID NO: 13, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID
NO: 5, SEQ ID NO: 6 and SEQ ID NO: 143. For hybridization, it is possible to use
techniques as described in, e.g., "Sambrook & Russell, Molecular Cloning: A
Laboratory Manual Vol. 3, Cold Spring Harbor, Laboratory Press 2001" and "Ausubel,
Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997."
As used herein, the expression “nucleotide sequence complementary” is not
limited only to a nucleotide sequence forming Watson-Crick pairs with a target
nucleotide sequence and also includes nucleotide sequences forming wobble base pairs
with a target nucleotide sequence. In this regard, a Watson-Crick pair is intended to
mean a base pair which forms hydrogen bonding between adenine and thymine,
between adenine and uracil or between guanine and cytosine, whereas a wobble base
pair is intended to mean a base pair which forms hydrogen bonding between guanine
and uracil, between inosine and uracil, between inosine and adenine or between inosine
and cytosine. Moreover, such a “nucleotide sequence complementary” does not
necessarily have 100% complementarity to a target nucleotide sequence and may
contain non-complementary bases (e.g., 1 to 3 bases, 1 or 2 bases, or a single base) to
the target nucleotide sequence.
As used herein, the term “stringent conditions” may be any of low stringent
conditions, moderately stringent conditions and high stringent conditions. “Low
stringent conditions” refer to, for example, conditions of 5 × SSC, 5 × Denhardt’s
solution, 0.5% SDS, 50% formamide at 32°C. Likewise, “moderately stringent
conditions” refer to, for example, conditions of 5 × SSC, 5 × Denhardt’s solution, 0.5%
SDS, 50% formamide at 42°C or conditions of 5 × SSC, 1% SDS, 50 mM Tris-HCl (pH
7.5), 50% formamide at 42°C. “High stringent conditions” refer to, for example,
conditions of 5 × SSC, 5 × Denhardt’s solution, 0.5% SDS, 50% formamide at 50°C or
conditions of 0.2 × SSC, 0.1% SDS at 65°C. Under these conditions, it can be
expected that a polynucleotide having a higher identity is more efficiently obtained at a
higher temperature. However, the stringency of hybridization would be affected by a
plurality of factors, including temperature, probe concentration, probe length, ionic
strength, reaction time, salt concentration and so on. Those skilled in the art would be
able to achieve the same stringency by selecting these factors as appropriate.
It should be noted that if a commercially available kit is used for hybridization,
an Alkphos Direct Labelling and Detection System (GE Healthcare) may be used for
this purpose, by way of example. In this case, hybridization may be accomplished in
accordance with the protocol included in the kit, i.e., after a membrane is incubated
overnight with a labeled probe and then washed with a primary washing buffer
containing 0.1% (w/v) SDS at 55°C, the hybridized polynucleotide can be detected.
Alternatively, if a commercially available reagent (e.g., PCR Labeling Mix (Roche
Diagnostics)) is used for digoxigenin (DIG) labeling of a probe during probe
preparation based on the whole or a part of a nucleotide sequence complementary to any
nucleotide sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID
NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 143 or selected
from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID
NO: 6 and SEQ ID NO: 143, a DIG Nucleic Acid Detection Kit (Roche Diagnostics)
may be used for detection of hybridization.
In addition to those listed above, other hybridizable polynucleotides include
polynucleotides sharing an identity of 90% or more, 91% or more, 92% or more, 93%
or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or
more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more,
99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more with a sequence
consisting of any polynucleotide selected from the group consisting of SEQ ID NO: 13,
SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 143, as
calculated by the homology search software BLAST using default parameters.
It should be noted that the identity of nucleotide sequences can be determined
by using the algorithm of Karlin and Altschul, BLAST (Basic Local Alignment Search
Tool) (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc Natl Acad Sci USA 90:
5873, 1993). Based on the algorithm of BLAST, programs called BLASTN and
BLASTX have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). If
BLASTN is used for nucleotide sequence analysis, parameters may be set to, for
example, score = 100 and wordlength = 12. If BLAST and Gapped BLAST programs
are used, default parameters in each program may be used.
In a certain embodiment, the oligomer of the present invention is an antisense
oligomer of 14 to 32 bases in length comprising connected two unit oligomers selected
from the group consisting of (a) to (e) shown below, or a pharmaceutically acceptable
salt or hydrate thereof:
(a) a unit oligomer consisting of a nucleotide sequence complementary to a
nucleotide sequence consisting of contiguous 7 to 16 bases selected from a nucleotide
sequence located at positions -5 to 15 from the 5’-terminal end of exon 45 in the human
dystrophin gene;
(b) a unit oligomer consisting of a nucleotide sequence complementary to a
nucleotide sequence consisting of contiguous 7 to 16 bases selected from a nucleotide
sequence located at positions 48 to 70 from the 5’-terminal end of exon 45 in the human
dystrophin gene;
(c) a unit oligomer consisting of a nucleotide sequence complementary to a
nucleotide sequence consisting of contiguous 7 to 16 bases selected from a nucleotide
sequence located at positions 128 to 150 from the 5’-terminal end of exon 45 in the
human dystrophin gene;
(d) a unit oligomer consisting of a nucleotide sequence complementary to a
nucleotide sequence consisting of contiguous 7 to 16 bases selected from a nucleotide
sequence located at positions 15 to 40 from the 5’-terminal end of exon 45 in the human
dystrophin gene; and
(e) a unit oligomer consisting of a nucleotide sequence complementary to a
nucleotide sequence consisting of contiguous 7 to 16 bases selected from a nucleotide
sequence located at positions 110 to 125 from the 5’-terminal end of exon 45 in the
human dystrophin gene.
The above unit oligomers (a) to (e) (hereinafter also simply referred to as
“units”) each have a size of 7 to 16 bases in length, preferably 8 to 16 bases in length,
more preferably 9 to 16 bases in length. The respective units may be of the same or
different size.
Moreover, when two unit oligomers are selected from the group consisting of
(a) to (e), these two unit oligomers may be a combination of the same units (i.e., (a) and
(a), (b) and (b), (c) and (c), (d) and (d), or (e) and (e)) or may be a combination of
different units, but preferably a combination of different units. For example, if (a) is
selected as one unit, the other unit is preferably any one of (b) to (e). Likewise, if (b)
is selected as one unit, the other unit is preferably (a), (c), (d) or (e), while if (c) is
selected as one unit, the other unit is preferably (a), (b), (d) or (e).
When two units are selected from (a) to (e), either of the selected two units may
be located at the 5’-terminal side. If (a) and (b) are selected, the unit (a) is preferably
connected to the 3’-terminal side. If (b) and (c) are selected, the unit (b) is preferably
connected to the 3’-terminal side. If (a) and (c) are selected, the unit (a) is preferably
connected to the 3’-terminal side. If (a) and (d) are selected, the unit (a) is preferably
connected to the 3’-terminal side. If (a) and (e) are selected, the unit (a) is preferably
connected to the 3’-terminal side.
As used here, the term “connected” is intended to mean that two units selected
from (a) to (e) are directly connected to each other. Namely, when two units are
connected, it means that the 3’-terminal end of the unit located at the 5’-terminal side
and the 5’-terminal end of the unit located at the 3’-terminal side form a phosphate bond
or any of the following groups:
[Formula 2]
1 1 1 2 3
(wherein X represents -OH, -CH R , -O-CH R , -S-CH R , -NR R or F;
2 2 2
R represents H or alkyl;
R and R , which may be the same or different, each represent H, alkyl,
cycloalkyl or aryl;
Y represents O, S, CH or NR ;
Y represents O, S or NR ; and
Z represents O or S).
The expression “allowing exon 45 skipping in the human dystrophin gene” is
intended to mean that upon binding the oligomer of the present invention to a site
corresponding to exon 45 in a transcript (e.g., pre-mRNA) of the human dystrophin
gene, the transcript is spliced to establish connection between a base corresponding to
the 3’-terminal end of exon 43 and a base corresponding to the 5’-terminal end of exon
46 in the case of DMD patients with deletion of exon 44, by way of example, to thereby
form mature mRNA free from codon frameshift.
The term “binding” is used here to mean that once the oligomer of the present
invention has been mixed with a transcript of the human dystrophin gene, both will be
hybridized with each other under physiological conditions to form a duplex. The
expression “under physiological conditions” is used here to mean conditions adjusted to
mimic in vivo pH, salt composition and temperature, as exemplified by conditions of
°C to 40°C, preferably 37°C, pH 5 to 8, preferably pH 7.4, and a sodium chloride
concentration of 150 mM.
To confirm whether or not exon 45 skipping was caused in the human
dystrophin gene, the oligomer of the present invention may be transfected into
dystrophin-expressing cells (e.g., human rhabdomyosarcoma cells) and a region around
exon 45 in mRNA of the human dystrophin gene may be amplified by RT-PCR from
the total RNA of the above dystrophin-expressing cells, followed by nested PCR or
sequencing analysis on the PCR amplification product. The efficiency of skipping
may be determined as follows: mRNA of the human dystrophin gene is collected from
test cells and the mRNA is measured for the polynucleotide level “A” in the band with
exon 45 skipping and the polynucleotide level “B” in the band without exon 45 skipping,
followed by calculation based on these measured values of “A” and “B” according to
the following equation.
Skipping efficiency (%) = A/(A + B) × 100
The oligomer of the present invention preferably causes exon 45 skipping with
an efficiency of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more,
60% or more, 70% or more, 80% or more, or 90% or more.
As to the calculation of skipping efficiency, reference may be made to
WO2012/029986.
The oligomer of the present invention may be exemplified by an
oligonucleotide, a morpholino oligomer or a peptide nucleic acid (PNA) oligomer, each
being 14 to 32 bases in length. The oligomer of the present invention is preferably 16
to 30 bases, 17 to 30 bases, 18 to 30 bases, 19 to 30 bases, 20 to 30 bases, 20 to 29 bases,
to 28 bases, 20 to 27 bases, 20 to 26 bases or 21 to 26 bases in length, and is
preferably a morpholino oligomer.
The above oligonucleotide (hereinafter referred to as “the oligonucleotide of
the present invention”) is an oligomer according to the present invention, whose
constituent unit is a nucleotide, and such a nucleotide may be any of a ribonucleotide, a
deoxyribonucleotide or a modified nucleotide.
A modified nucleotide refers to a ribonucleotide or deoxyribonucleotide whose
nucleobase, sugar moiety and phosphate bond moiety are all or partly modified.
Examples of a nucleobase include adenine, guanine, hypoxanthine, cytosine,
thymine, uracil, or modified bases thereof. Such modified bases may be exemplified
by pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosines (e.g., 5-
methylcytosine), 5-alkyluracils (e.g., 5-ethyluracil), 5-halouracils (e.g., 5-bromouracil),
6-azapyrimidine, 6-alkylpyrimidines (e.g., 6-methyluracil), 2-thiouracil, 4-thiouracil, 4-
acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5'-carboxymethylaminomethyl
thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-
methylhypoxanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-
methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl
thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-
methyloxyuracil, 5-methylthiouracil, 2-methylthio-N6-isopentenyladenine, uracil
oxyacetic acid, 2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine,
indole, imidazole, xanthine and so on, but are not limited thereto.
Modifications to the sugar moiety may be exemplified by modifications at the
2’-position of ribose and modifications at the other positions of sugar. Examples of
modifications at the 2’-position of ribose include modifications intended to replace
the -OH group at the 2’-position of ribose with OR, R, R’OR, SH, SR, NH , NHR, NR ,
N , CN, F, Cl, Br or I, wherein R represents alkyl or aryl, and R’ represents alkylene.
Examples of modifications at the other positions of sugar include replacement
of O with S at the 4’-position of ribose or deoxyribose, and bridging between 2’- and 4’-
positions of sugar, as exemplified by LNAs (locked nucleic acids) or ENAs (2’-O,4’-C-
ethylene-bridged nucleic acids), but are not limited thereto.
Modifications to the phosphate bond moiety may be exemplified by
modifications intended to replace the phosphodiester bond with a phosphorothioate
bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoroamidate bond
or a boranophosphate bond (Enya et al: Bioorganic & Medicinal Chemistry, 2008, 18,
9154-9160) (see, e.g., JP WO2006/129594 and JP WO2006/038608).
Alkyl is preferably a linear or branched alkyl containing 1 to 6 carbon atoms.
More specifically, examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl and isohexyl.
Such an alkyl may be substituted with 1 to 3 substituents including halogen, alkoxy,
cyano, nitro, etc.
Cycloalkyl is preferably a cycloalkyl containing 5 to 12 carbon atoms. More
specifically, examples include cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,
cyclodecyl and cyclododecyl.
Halogens include fluorine, chlorine, bromine and iodine.
Alkoxy may be a linear or branched alkoxy containing 1 to 6 carbon atoms, as
exemplified by methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-
butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy, isohexyloxy and so on.
Particularly preferred is an alkoxy containing 1 to 3 carbon atoms.
Aryl is preferably an aryl containing 6 to 10 carbon atoms. More specifically,
examples include phenyl, α-naphthyl and β-naphthyl. Particularly preferred is phenyl.
Such an aryl may be substituted with 1 to 3 substituents including alkyl, halogen,
alkoxy, cyano, nitro, etc.
Alkylene is preferably a linear or branched alkylene containing 1 to 6 carbon
atoms. More specifically, examples include methylene, ethylene, trimethylene,
tetramethylene, pentamethylene, hexamethylene, 2-(ethyl)trimethylene and 1-
(methyl)tetramethylene.
Acyl may be a linear or branched alkanoyl or an aroyl. Examples of such an
alkanoyl include formyl, acetyl, 2-methylacetyl, 2,2-dimethylacetyl, propionyl, butyryl,
isobutyryl, pentanoyl, 2,2-dimethylpropionyl, hexanoyl and so on. Examples of an
aroyl include benzoyl, toluoyl and naphthoyl. Such an aroyl may be substituted at any
substitutable position and may be substituted with alkyl(s).
The oligonucleotide of the present invention is preferably an oligomer
according to the present invention, whose constituent unit is a group represented by the
following general formula, in which the -OH group at the 2’-position of ribose is
substituted with methoxy and the phosphate bond moiety is a phosphorothioate bond:
[Formula 3]
(wherein Base represents a nucleobase).
The oligonucleotide of the present invention may be readily synthesized with
various automated synthesizers (e.g., AKTA oligopilot plus 10/100 (GE Healthcare)), or
alternatively, its synthesis may be entrusted to a third party (e.g., Promega or Takara),
etc.
The above morpholino oligomer is an oligomer according to the present
invention, whose constituent unit is a group represented by the following general
formula:
[Formula 4]
(wherein Base is the same as defined above; and
W represents a group shown by any of the following formulae:
[Formula 5]
1 1 1 2 3
(wherein X represents -CH R , -O-CH R , -S-CH R , -NR R or F;
2 2 2
R represents H or alkyl;
R and R , which may be the same or different, each represent H, alkyl,
cycloalkyl or aryl;
Y represents O, S, CH or NR ;
Y represents O, S or NR ; and
Z represents O or S)).
The morpholino oligomer is preferably an oligomer whose constituent unit is a
group represented by the following formula (i.e., a phosphorodiamidate morpholino
oligomer (hereinafter referred to as “PMO”)):
[Formula 6]
(wherein Base, R and R are the same as defined above).
For example, the morpholino oligomer may be prepared in accordance with
WO1991/009033 or WO2009/064471. In particular, PMO may be prepared in
accordance with the procedures described in WO2009/064471 or may be prepared in
accordance with the procedures described in WO2013/100190.
[Process for PMO preparation]
As one embodiment of PMO, a compound represented by the following general
formula (I) (hereinafter referred to as PMO (I)) may be given by way of example:
[Formula 7]
[wherein each Base, R and R are the same as defined above; and
n is any integer in the range of 1 to 99, preferably any integer in the range of 13
to 31].
PMO (I) may be prepared in accordance with known procedures, for example,
by conducting the operations shown in the following steps.
Compounds and reagents used in the following steps are not limited in any way
as long as they are commonly used for PMO preparation.
Moreover, all the following steps may be accomplished by the liquid phase
method or the solid phase method (repeating batch reactions or using a commercially
available solid phase automated synthesizer). When PMO is prepared by the solid
phase method, it is desirable to use an automated synthesizer in terms of simple
operation and accurate synthesis.
(1) Step A:
This is a step where a compound represented by the following general formula
(II) (hereinafter referred to as compound (II)) is treated with an acid to prepare a
compound represented by the following general formula (III) (hereinafter referred to as
compound (III)):
[Formula 8]
[wherein n, R and R are the same as defined above;
each B independently represents a nucleobase which may be protected;
T represents a trityl group, a monomethoxytrityl group or a dimethoxytrityl
group; and
L represents hydrogen, acyl or a group represented by the following general
formula (IV) (hereinafter referred to as group (IV))]:
[Formula 9]
“Nucleobases” possible for B may be exemplified by the same “nucleobases”
as listed for Base, provided that amino groups or hydroxyl groups in these nucleobases
for B may be protected.
Protecting groups for these amino groups are not limited in any way as long as
they are used as protecting groups for nucleic acids. More specifically, examples
include benzoyl, 4-methoxybenzoyl, acetyl, propionyl, butyryl, isobutyryl, phenylacetyl,
phenoxyacetyl, 4-tert-butylphenoxyacetyl, 4-isopropylphenoxyacetyl, and
(dimethylamino)methylene. Protecting groups for hydroxyl groups include, for
example, 2-cyanoethyl, 4-nitrophenethyl, phenylsulfonylethyl, methylsulfonylethyl,
trimethylsilylethyl, phenyl which may be substituted with 1 to 5 electron withdrawing
groups at any substitutable position(s), diphenylcarbamoyl, dimethylcarbamoyl,
diethylcarbamoyl, methylphenylcarbamoyl, 1-pyrrolidinylcarbamoyl,
morpholinocarbamoyl, 4-(tert-butylcarboxy)benzyl, 4-[(dimethylamino)carboxy]benzyl,
and 4-(phenylcarboxy)benzyl (see, e.g., WO2009/064471).
The “solid carrier” is not limited in any way as long as it is a carrier available
for use in the solid phase reaction of nucleic acids, but it is desirable to use, for example,
a carrier which (i) is sparingly soluble in reagents available for use in the synthesis of
morpholino nucleic acid derivatives (e.g., dichloromethane, acetonitrile, tetrazole, N-
methylimidazole, pyridine, acetic anhydride, lutidine, trifluoroacetic acid), (ii) is
chemically stable against the reagents available for use in the synthesis of morpholino
nucleic acid derivatives, (iii) can be chemically modified, (iv) can be loaded with
desired morpholino nucleic acid derivatives, (v) has strength sufficient to withstand high
pressure during processing, and (vi) has a certain range of particle size and distribution.
More specifically, examples include swellable polystyrene (e.g., aminomethyl
polystyrene resin crosslinked with 1% divinylbenzene (200 to 400 mesh) (2.4 to 3.0
mmol/g) (Tokyo Chemical Industry Co., Ltd., Japan), Aminomethylated Polystyrene
Resin HCl [divinylbenzene 1%, 100 to 200 mesh] (Peptide Institute, Inc., Japan)), non-
swellable polystyrene (e.g., Primer Support (GE Healthcare)), PEG chain-attached
polystyrenes (e.g., NH -PEG resin (Watanabe Chemical Industries, Ltd., Japan),
TentaGel resin), controlled pore glass (CPG) (e.g., CPG Inc.), oxalylated controlled
pore glass (see, e.g., Alul et al., Nucleic Acids Research, Vol. 19, 1527 (1991)),
TentaGel support-aminopolyethylene glycol-derivatized support (see, e.g., Wright et al.,
Tetrahedron Letters, Vol. 34, 3373 (1993)), and a Poros-polystyrene/divinylbenzene
copolymer.
As a “linker,” it is possible to use a known linker which is commonly used to
link a nucleic acid or a morpholino nucleic acid derivative, and examples include 3-
aminopropyl, succinyl, 2,2’-diethanol sulfonyl, and a long-chain alkylamino (LCAA).
This step may be accomplished by treating compound (II) with an acid.
Examples of an “acid” available for use in this step include trifluoroacetic acid,
dichloroacetic acid or trichloroacetic acid. The amount of an acid to be used is, for
example, reasonably in the range of 0.1 molar equivalents to 1000 molar equivalents,
preferably in the range of 1 molar equivalent to 100 molar equivalents, relative to 1
mole of compound (II).
Moreover, it is possible to use an organic amine together with the above acid.
Any organic amine may be used for this purpose, and examples include triethylamine.
The amount of an organic amine to be used is, for example, reasonably in the range of
0.01 molar equivalents to 10 molar equivalents, preferably in the range of 0.1 molar
equivalents to 2 molar equivalents, relative to 1 mole of the acid.
In a case where an acid and an organic amine are used as a salt or mixture in
this step, examples include a salt or mixture of trifluoroacetic acid and triethylamine,
more specifically a mixture containing 2 equivalents of trifluoroacetic acid and 1
equivalent of triethylamine.
An acid available for use in this step may be used by being diluted with an
appropriate solvent to give a concentration in the range of 0.1% to 30%. Any solvent
may be used for this purpose as long as it is inert to the reaction, and examples include
dichloromethane, acetonitrile, alcohols (e.g., ethanol, isopropanol, trifluoroethanol),
water, or mixtures thereof.
The reaction temperature in the above reaction is, for example, preferably in
the range of 10°C to 50 °C, more preferably in the range of 20 °C to 40°C, and even
more preferably in the range of 25°C to 35°C.
The reaction time will vary depending on the type of acid to be used and/or the
reaction temperature, but it is generally reasonably in the range of 0.1 minutes to 24
hours, and preferably in the range of 1 minute to 5 hours.
Moreover, after completion of this step, a base may optionally be added to
neutralize the acid remaining in the system. Any “base” may be used for this purpose
and examples include diisopropylethylamine. Such a base may be used by being
diluted with an appropriate solvent to give a concentration in the range of 0.1% (v/v) to
% (v/v).
Any solvent may be used in this step as long as it is inert to the reaction, and
examples include dichloromethane, acetonitrile, alcohols (e.g., ethanol, isopropanol,
trifluoroethanol), water, or mixtures thereof. The reaction temperature is, for example,
preferably in the range of 10 °C to 50°C, more preferably in the range of 20°C to 40°C,
and even more preferably in the range of 25°C to 35°C.
The reaction time will vary depending on the type of base to be used and/or the
reaction temperature, but it is generally reasonably in the range of 0.1 minutes to 24
hours, and preferably in the range of 1 minute to 5 hours.
It should be noted that compound (II) in which n = 1 and L is group (IV), i.e., a
compound represented by the following general formula (IIa) (hereinafter referred to as
compound (IIa)) may be prepared in accordance with the following procedures:
[Formula 10]
[wherein B , T, Linker and Solid carrier are the same as defined above].
Step 1:
This is a step where a compound represented by the following general formula
(V) is treated with an acylating agent to prepare a compound represented by the
following general formula (VI) (hereinafter referred to as compound (VI)):
[Formula 11]
[wherein B , T and Linker are the same as defined above; and
R represents a hydroxyl group, halogen or amino].
This step may be accomplished starting from compound (V) by any known
reaction for linker introduction.
In particular, a compound represented by the following general formula (VIa)
may be prepared by any process known as esterification reaction with the use of
compound (V) and succinic anhydride:
[Formula 12]
[wherein B and T are the same as defined above].
Step 2:
This is a step where compound (VI) is reacted with a solid carrier by being
treated with a condensing agent or the like to prepare compound (IIa):
[Formula 13]
[wherein B , R , T, Linker and Solid carrier are the same as defined above].
This step may be accomplished by any process known as condensation reaction
with the use of compound (VI) and a solid carrier.
Compound (II) in which n = 2 to 99 and L is group (IV), i.e., a compound
represented by the following general formula (IIa2) may be prepared starting from
compound (IIa) by repeating desired times Steps A and B of the process for PMO
preparation disclosed herein:
[Formula 14]
P 2 3
[wherein B , R , R , T, Linker and Solid carrier are the same as defined above; and
n’ represents 1 to 98].
Likewise, compound (II) in which n = 1 and L is hydrogen, i.e., a compound
represented by the following general formula (IIb) may be prepared, for example, by the
procedures described in WO1991/009033:
[Formula 15]
[wherein B and T are the same as defined above].
Compound (II) in which n = 2 to 99 and L is hydrogen, i.e., a compound
represented by the following general formula (IIb2) may be prepared starting from
compound (IIb) by repeating desired times Steps A and B of the process for PMO
preparation disclosed herein:
[Formula 16]
P 2 3
[wherein B , n’, R , R and T are the same as defined above].
Likewise, compound (II) in which n = 1 and L is acyl, i.e., a compound
represented by the following general formula (IIc) may be prepared from compound
(IIb) by any process known as acylation reaction:
[Formula 17]
[wherein B and T are the same as defined above; and
R represents acyl].
Compound (II) in which n = 2 to 99 and L is acyl, i.e., a compound represented
by the following general formula (IIc2) may be prepared starting from compound (IIc)
by repeating desired times Steps A and B of the process for PMO preparation disclosed
herein:
[Formula 18]
P 2 3 5
[wherein B , n’, R , R , R and T are the same as defined above].
(2) Step B:
This is a step where compound (III) is treated with a morpholino monomer
compound in the presence of a base to prepare a compound represented by the following
general formula (VII) (hereinafter referred to as compound (VII)):
[Formula 19]
P 2 3
[wherein each B , L, n, R , R and T are the same as defined above].
This step may be accomplished by treating compound (III) with a morpholino
monomer compound in the presence of a base.
Such a morpholino monomer compound may be exemplified by a compound
represented by the following general formula (VIII):
[Formula 20]
P 2 3
[wherein B , R , R and T are the same as defined above].
Examples of a “base” available for use in this step include
diisopropylethylamine, triethylamine or N-ethylmorpholine. The amount of a base to
be used is, for example, reasonably in the range of 1 molar equivalent to 1000 molar
equivalents, preferably in the range of 10 molar equivalents to 100 molar equivalents,
relative to 1 mole of compound (III).
Such a morpholino monomer compound and a base available for use in this
step may be used by being diluted with an appropriate solvent to give a concentration of
0.1% to 30%. Any solvent may be used for this purpose as long as it is inert to the
reaction, and examples include N,N-dimethylimidazolidinone, N-methylpiperidone,
DMF, dichloromethane, acetonitrile, tetrahydrofuran, or mixtures thereof.
The reaction temperature is, for example, preferably in the range of 0°C to
100 °C, and more preferably in the range of 10°C to 50°C.
The reaction time will vary depending on the type of base to be used and/or the
reaction temperature, but it is generally reasonably in the range of 1 minute to 48 hours,
and preferably in the range of 30 minutes to 24 hours.
Moreover, after completion of this step, an acylating agent may optionally be
added. Examples of an “acylating agent” include acetic anhydride, acetyl chloride and
phenoxyacetic anhydride. Such an acylating agent may be used by being diluted with
an appropriate solvent to give a concentration in the range of 0.1% to 30%, by way of
example. Any solvent may be used for this purpose as long as it is inert to the reaction,
and examples include dichloromethane, acetonitrile, alcohols (e.g., ethanol, isopropanol,
trifluoroethanol), water, or mixtures thereof.
If necessary, it is possible to use a base (e.g., pyridine, lutidine, collidine,
triethylamine, diisopropylethylamine, N-ethylmorpholine) together with an acylating
agent. The amount of an acylating agent to be used is preferably in the range of 0.1
molar equivalents to 10000 molar equivalents, and more preferably in the range of 1
molar equivalent to 1000 molar equivalents. The amount of a base to be used is, for
example, reasonably in the range of 0.1 molar equivalents to 100 molar equivalents,
preferably in the range of 1 molar equivalent to 10 molar equivalents, relative to 1 mole
of an acylating agent.
The reaction temperature in this reaction is preferably in the range of 10 °C to
50 °C, more preferably in the range of 10 °C to 50°C, even more preferably in the range
of 20 °C to 40°C, and still even more preferably in the range of 25°C to 35°C. The
reaction time will vary, e.g., depending on the type of acylating agent to be used and/or
the reaction temperature, but it is generally reasonably in the range of 0.1 minutes to 24
hours, and preferably in the range of 1 minute to 5 hours.
(3) Step C:
This is a step where a deprotecting agent is used to remove the protecting
groups from compound (VII) prepared in Step B, thereby preparing a compound
represented by general formula (IX):
[Formula 21]
P 2 3
[wherein Base, B , L, n, R , R and T are the same as defined above].
This step may be accomplished by treating compound (VII) with a deprotecting
agent.
Examples of a “deprotecting agent” include concentrated aqueous ammonia
and methylamine. Such a “deprotecting agent” available for use in this step may be
used by being diluted with water, methanol, ethanol, isopropyl alcohol, acetonitrile,
tetrahydrofuran, DMF, N,N-dimethylimidazolidinone, N-methylpiperidone, or a mixed
solvent thereof. Among them, preferred is ethanol. The amount of a deprotecting
agent to be used is, for example, reasonably in the range of 1 molar equivalent to
100000 molar equivalents, preferably in the range of 10 molar equivalents to 1000
molar equivalents, relative to 1 mole of compound (VII), by way of example.
The reaction temperature is, for example, reasonably in the range of 15 °C to
75°C, preferably in the range of 40°C to 70°C, and more preferably in the range of 50°C
to 60°C. The reaction time for deprotection will vary depending on the type of
compound (VII) and/or the reaction temperature, etc., but it is reasonably in the range of
minutes to 30 hours, preferably in the range of 30 minutes to 24 hours, and more
preferably in the range of 5 hours to 20 hours.
(4) Step D:
This is a step where compound (IX) prepared in Step C is treated with an acid
to prepare PMO (I):
[Formula 22]
[wherein Base, n, R , R and T are the same as defined above].
This step may be accomplished by adding an acid to compound (IX).
Examples of an “acid” available for use in this step include trichloroacetic acid,
dichloroacetic acid, acetic acid, phosphoric acid and hydrochloric acid, etc. As to the
amount of an acid to be used, it is reasonable to use the acid in an amount to give a
solution pH, for example, in the range of 0.1 to 4.0, more preferably in the range of 1.0
to 3.0. Any solvent may be used in this step as long as it is inert to the reaction, and
examples include acetonitrile, water, or mixed solvents thereof.
The reaction temperature is preferably in the range of 10 °C to 50°C, more
preferably in the range of 20°C to 40°C, and even more preferably in the range of 25 °C
to 35°C. The reaction time for deprotection will vary depending on the type of
compound (IX) and/or the reaction temperature, etc., but it is reasonably in the range of
0.1 minutes to 5 hours, preferably in the range of 1 minute to 1 hour, and more
preferably in the range of 1 minute to 30 minutes.
PMO (I) may be obtained from the reaction mixture obtained in this step by
commonly used separation and purification means including extraction, concentration,
neutralization, filtration, centrifugation, recrystallization, C to C reversed-phase
8 18
column chromatography, cation exchange column chromatography, anion exchange
column chromatography, gel filtration column chromatography, high performance liquid
chromatography, dialysis, ultrafiltration and other means, which may be used either
alone or in combination, whereby desired PMO (I) can be isolated and purified (see, e.g.,
WO1991/09033).
In the case of using reversed-phase chromatography for purification of PMO (I),
a mixed solution of 20 mM triethylamine/acetate buffer and acetonitrile may be used as
an elution solvent, by way of example.
Likewise, in the case of using ion exchange chromatography for purification of
PMO (I), a mixed solution of 1 M aqueous sodium chloride and 10 mM aqueous sodium
hydroxide may be used, by way of example.
The above peptide nucleic acid oligomer is an oligomer according to the
present invention, whose constituent unit is a group represented by the following
general formula:
[Formula 23]
(wherein Base is the same as defined above).
Peptide nucleic acids may be prepared, for example, in accordance with the
documents listed below.
1) P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, Science, 254, 1497 (1991)
2) M. Egholm, O. Buchardt, P. E. Nielsen, R. H. Berg, Jacs., 114, 1895 (1992)
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)
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)
) T. Koch, H. F. Hansen, P. Andersen, T. Larsen, H. G. Batz, K. Otteson, H. Orum,
J. Pept. Res., 49, 80 (1997)
Moreover, the oligomer of the present invention may be configured such that
its 5’-terminal end is any one of the groups represented by chemical formulae (1) to (3)
shown below, with (3) -OH being preferred.
[Formula 24]
The groups represented by the above formulae (1), (2) and (3) are hereinafter
referred to as “group (1),” “group (2)” and “ group (3),” respectively.
2. Pharmaceutical composition
The oligomer of the present invention allows exon 45 skipping in the
dystrophin gene. It is therefore expected that the symptoms of muscular dystrophy can
be alleviated when a pharmaceutical composition comprising the oligomer of the
present invention is administered to DMD patients having a mutation targeted by exon
45 skipping (i.e., a mutation is converted to in-flame by exon 45 skipping) in their
dystrophin gene. Moreover, because of its short chain length, the oligomer of the
present invention is advantageous in that its preparation steps are simple and further in
that its preparation costs can be reduced.
Thus, in another embodiment, the present invention provides a pharmaceutical
composition for treatment of muscular dystrophy, which comprises the oligomer of the
present invention, a pharmaceutically acceptable salt or hydrate thereof as an active
ingredient (hereinafter referred to as “the composition of the present invention”).
Examples of a pharmaceutically acceptable salt of the oligomer of the present
invention contained in the composition of the present invention include alkali metal
salts (e.g., sodium salt, potassium salt, lithium salt); alkaline earth metal salts (e.g.,
calcium salt, magnesium salt); metal salts (e.g., aluminum salt, iron salt, zinc salt,
copper salt, nickel salt, cobalt salt); ammonium salt; organic amine salts (e.g., t-
octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine
alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt,
diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-
dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-
benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt,
tris(hydroxymethyl)aminomethane salt); halogenated hydroacid salts (e.g.,
hydrofluoride salt, hydrochloride salt, hydrobromide salt, hydroiodide salt); inorganic
acid salts (i.e., nitrate salt, perchlorate salt, sulfate salt, phosphate salt); lower
alkanesulfonic acid salts (e.g., methanesulfonate salt, trifluoromethanesulfonate salt,
ethanesulfonate salt); arylsulfonic acid salts (e.g., benzenesulfonate salt, p-
toluenesulfonate salt); organic acid salts (e.g., acetate salt, malate salt, fumarate salt,
succinate salt, citrate salt, tartrate salt, oxalate salt, maleate salt); amino acid salts (e.g.,
glycine salt, lysine salt, arginine salt, ornithine salt, glutamate salt, aspartate salt), etc.
These salts may be prepared in any known manner. Alternatively, the oligomer of the
present invention contained in the composition of the present invention may be in the
form of a hydrate thereof.
The composition of the present invention may be administered in any
pharmaceutically acceptable mode, which may be selected as appropriate for the
intended therapeutic method. However, in terms of easy delivery to muscle tissue,
preferred are intravenous administration, intraarterial administration, intramuscular
administration, subcutaneous administration, oral administration, interstitial
administration, percutaneous administration and so on. Moreover, the composition of
the present invention may be in any dosage form, and examples include various types of
injections, oral formulations, drops, inhalants, ointments, lotions, etc.
In a case where the oligomer of the present invention is administered to
muscular dystrophy patients, the composition of the present invention preferably
comprises a carrier which promotes the delivery of the oligomer to muscle tissue.
Such a carrier is not limited in any way as long as it is pharmaceutically acceptable, and
examples include cationic carriers (e.g., cationic liposomes, cationic polymers) or viral
envelope-based carriers. Examples of cationic liposomes include liposomes formed
from 2-O-(2-diethylaminoethyl)carbamoyl-1,3-O-dioleoyl glycerol and a phospholipid
as essential constituent members (hereinafter referred to as “liposome A”),
Oligofectamine (Invitrogen), Lipofectin (Invitrogen), Lipofectamine (Invitrogen),
Lipofectamine 2000 (Invitrogen), DMRIE-C (Invitrogen), GeneSilencer (Gene
Therapy Systems), TransMessenger (QIAGEN), TransIT TKO (Mirus) and
Nucleofector II (Lonza). Among them, preferred is liposome A. Examples of
cationic polymers include JetSI (Qbiogene) and Jet-PEI (polyethyleneimine,
Qbiogene). Examples of viral envelope-based carriers include GenomeOne (HVJ-E
liposomes, Ishihara Sangyo Kaisha, Ltd., Japan). Alternatively, it is also possible to
use the pharmaceutical device shown in Japanese Patent No. 2924179 or the cationic
carriers shown in JP WO2006/129594 and JP WO2008/096690.
The concentration of the oligomer of the present invention contained in the
composition of the present invention will vary, e.g., depending on the type of carrier,
but it is reasonably in the range of 0.1 nM to 100 µM, preferably in the range of 1 nM to
µM, and more preferably in the range of 10 nM to 1 µM. Likewise, the weight
ratio of the carrier to the oligomer of the present invention contained in the composition
of the present invention (i.e., the carrier/oligomer ratio) will vary, e.g., depending on the
properties of the oligomer and the type of the carrier, but it is reasonably in the range of
0.1 to 100, preferably in the range of 1 to 50, and more preferably in the range of 10 to
The composition of the present invention may optionally comprise a
pharmaceutically acceptable additive, in addition to the oligomer of the present
invention and the carrier described above. Examples of such an additive include an
emulsifier aid (e.g., a fatty acid containing 6 to 22 carbon atoms or a pharmaceutically
acceptable salt thereof, albumin, dextran), a stabilizing agent (e.g., cholesterol,
phosphatidic acid), an isotonizing agent (e.g., sodium chloride, glucose, maltose, lactose,
sucrose, trehalose), and a pH adjuster (e.g., hydrochloric acid, sulfuric acid, phosphoric
acid, acetic acid, sodium hydroxide, potassium hydroxide, triethanolamine). These
additives may be used either alone or in combination. The content of the additive(s) in
the composition of the present invention is reasonably 90% by weight or less, preferably
70% by weight or less, and more preferably 50% by weight or less.
The composition of the present invention may be prepared by adding the
oligomer of the present invention to a dispersion of a carrier, followed by adequate
stirring. An additive(s) may be added at any appropriate stage, either before or after
adding the oligomer of the present invention. Any aqueous solvent may be used for
adding the oligomer of the present invention as long as it is pharmaceutically acceptable,
and examples include injectable water, injectable distilled water, electrolytic solutions
(e.g., physiological saline), and sugar solutions (e.g., glucose solution, maltose solution).
Moreover, in this case, conditions including pH and temperature may be selected as
appropriate by those skilled in the art.
The composition of the present invention may be formulated into a solution or
a lyophilized formulation thereof, by way of example. Such a lyophilized formulation
may be prepared in a standard manner by freeze-drying the composition of the present
invention in a solution form. For example, the composition of the present invention in
a solution form may be sterilized as appropriate and then dispensed in given amounts
into vial bottles, followed by preliminary freezing under conditions of about -40°C to -
°C for about 2 hours, primary drying at about 0°C to 10°C under reduced pressure
and then secondary drying at about 15°C to 25°C under reduced pressure. Moreover,
in most cases, the vials may be purged with a nitrogen gas and then capped, thereby
giving a lyophilized formulation of the composition of the present invention.
Such a lyophilized formulation of the composition of the present invention may
generally be used after being reconstituted by addition of any appropriate solution (i.e.,
a reconstituting solution). Examples of such a reconstituting solution include
injectable water, physiological saline, and other commonly used infusion solutions.
The volume of such a reconstituting solution will vary, e.g., depending on the intended
use and is not limited in any way, but it is reasonably 0.5- to 2-fold greater than the
solution volume before freeze-drying, or 500 mL or less.
The dose for administration of the composition of the present invention is
desirably adjusted in consideration of the type of the oligomer of the present invention
contained therein, the intended dosage form, the condition of a patient such as age and
body weight, the route of administration, and the nature and severity of a disease.
However, the daily dose for adults is generally in the range of 0.1 mg to 10 g/human,
preferably in the range of 1 mg to 1 g/human, calculated as the amount of the oligomer
of the present invention. This numerical range may vary depending on the type of
disease to be targeted, the mode of administration, and/or the type of target molecule.
Thus, a dose lower than this range may be sufficient in some cases, or conversely, a
dose higher than this range should be required in some cases. Moreover, the
composition of the present invention may be administered once to several times a day or
at intervals of one to several days.
In another embodiment, the composition of the present invention may be a
pharmaceutical composition comprising a vector capable of expressing the
oligonucleotide of the present invention and a carrier as described above. Such an
expression vector may be capable of expressing a plurality of oligonucleotides
according to the present invention. Such a composition may optionally comprise a
pharmaceutically acceptable additive, as in the case of the composition of the present
invention comprising the oligomer of the present invention. The concentration of the
expression vector contained in this composition will vary, e.g., depending on the type of
carrier, but it is reasonably in the range of 0.1 nM to 100 µM, preferably in the range of
1 nM to 10 µM, and more preferably in the range of 10 nM to 1 µM. The weight ratio
of the carrier to the expression vector contained in this composition (i.e., the
carrier/expression vector ratio) will vary, e.g., depending on the properties of the
expression vector and the type of the carrier, but it is reasonably in the range of 0.1 to
100, preferably in the range of 1 to 50, and more preferably in the range of 10 to 20.
Moreover, the content of the carrier contained in this composition is the same as in the
case of the composition of the present invention comprising the oligomer of the present
invention, and procedures for preparation are also the same as in the case of the
composition of the present invention.
The present invention will be further described in more detail below by way of
the following illustrative examples and test examples, although the present invention is
not limited thereto.
EXAMPLES
[Reference Example 1]
4-{[(2S,6R)(4-Benzamidooxopyrimidinyl)tritylmorpholinyl]methoxy}
oxobutanoic acid loaded on aminopolystyrene resin
Step 1: Preparation of 4-{[(2S,6R)(4-benzamidooxopyrimidin-1(2H)-yl)-
4-tritylmorpholinyl]methoxy}oxobutanoic acid
Under an argon atmosphere, N-{1-[(2R,6S)(hydroxymethyl)
tritylmorpholinyl]oxo-1,2-dihydropyrimidinyl}benzamide (3.44 g) and 4-
dimethylaminopyridine (4-DMAP) (1.1 g) were suspended in dichloromethane (50 mL),
and succinic anhydride (0.90 g) was then added thereto, followed by stirring at room
temperature for 3 hours. The reaction solution was mixed with methanol (10 mL) and
concentrated under reduced pressure. The residue was extracted with ethyl acetate and
0.5 M aqueous potassium dihydrogen phosphate. The resulting organic layer was
washed sequentially with 0.5 M aqueous potassium dihydrogen phosphate, water and
saturated aqueous sodium chloride. The resulting organic layer was dried over sodium
sulfate and concentrated under reduced pressure to obtain 4.0 g of the desired product.
Step 2: Preparation of 4-{[(2S,6R)(4-benzamidooxopyrimidinyl)
tritylmorpholinyl]methoxy}oxobutanoic acid loaded on aminopolystyrene resin
4-{[(2S,6R)(4-Benzamidooxopyrimidin-1(2H)-yl)tritylmorpholin
yl]methoxy}oxobutanoic acid (4.0 g) was dissolved in pyridine (dehydrated) (200
mL), followed by addition of 4-DMAP (0.73 g) and 1-ethyl(3-dimethylaminopropyl)-
carbodiimide hydrochloride (11.5 g). Then, aminopolystyrene resin Primer support
200 amino (GE Healthcare Japan, 1797) (25.0 g) and triethylamine (8.5 mL)
were added to this mixture, followed by shaking at room temperature for 4 days. After
the reaction, the resin was collected by filtration. The resulting resin was washed
sequentially with pyridine, methanol and dichloromethane, and then dried under
reduced pressure. To the resulting resin, tetrahydrofuran (dehydrated) (200 mL), acetic
anhydride (15 mL) and 2,6-lutidine (15 mL) were added, followed by shaking at room
temperature for 2 hours. The resin was collected by filtration, washed sequentially
with pyridine, methanol and dichloromethane, and then dried under reduced pressure to
obtain 26.7 g of the desired product.
To determine the loading amount of the desired product, the molar amount of
trityl per gram of the resin was measured in a known manner as UV absorbance at 409
nm. The loading amount on the resin was found to be 129.2 µmol/g.
Conditions for UV measurement
Instrument: U-2910 (Hitachi, Ltd., Japan)
Solvent: methanesulfonic acid
Wavelength: 409 nm
ε value: 45000
[Reference Example 2]
4-{[(2S,6R)(5-Methyl-2,4-dioxopyrimidinyl)tritylmorpholinyl]methoxy}
oxobutanoic acid loaded on aminopolystyrene resin
The same procedures as shown in Reference Example 1 were repeated to
prepare the titled compound, except that N-{1-[(2R,6S)(hydroxymethyl)
tritylmorpholinyl]oxo-1,2-dihydropyrimidinyl}benzamide used in Step 1 of
Reference Example 1 was replaced in this step with 1-[(2R,6S)(hydroxymethyl)
tritylmorpholinyl]methylpyrimidine-2,4(1H,3H)-dione.
To determine the loading amount of the desired product, the molar amount of
trityl per gram of the resin was measured in a known manner as UV absorbance at 409
nm. The loading amount on the resin was found to be 164.0 µmol/g.
[Reference Example 3]
4-{[(2S,6R)(6-Benzamidopurinyl)tritylmorpholinyl]methoxy}
oxobutanoic acid loaded on aminopolystyrene resin
The same procedures as shown in Reference Example 1 were repeated to
prepare the titled compound, except that N-{1-[(2R,6S)(hydroxymethyl)
tritylmorpholinyl]oxo-1,2-dihydropyrimidinyl}benzamide used in Step 1 of
Reference Example 1 was replaced in this step with N-{9-[(2R,6S)(hydroxymethyl)-
4-tritylmorpholinyl]purinyl}benzamide.
To determine the loading amount of the desired product, the molar amount of
trityl per gram of the resin was measured in a known manner as UV absorbance at 409
nm. The loading amount on the resin was found to be 185.7 µmol/g.
[Reference Example 4]
4-{{(2S,6R){6-(2-Cyanoethoxy)[(2-phenoxyacetyl)amino]purinyl}
tritylmorpholinyl}methoxy}oxobutanoic acid loaded on aminopolystyrene resin
The same procedures as shown in Reference Example 1 were repeated to
prepare the titled compound, except that N-{1-[(2R,6S)(hydroxymethyl)
tritylmorpholinyl]oxo-1,2-dihydropyrimidinyl}benzamide used in Step 1 of
Reference Example 1 was replaced in this step with N-{6-(2-cyanoethoxy)[(2R,6S)-
6-(hydroxymethyl)tritylmorpholinyl]purinyl}phenoxyacetamide.
To determine the loading amount of the desired product, the molar amount of
trityl per gram of the resin was measured in a known manner as UV absorbance at 409
nm. The loading amount on the resin was found to be 164.8 µmol/g.
In accordance with the descriptions in Example 1 shown below, PMOs having
the nucleotide sequences of PMO Nos. 1 to 81 indicated in Table 1 were synthesized
(wherein R and R are each methyl, and the 5’-terminal end is group (3)). The thus
synthesized PMOs were each dissolved in water for injection (Otsuka Pharmaceutical
Factory, Inc., Japan).
[Table 1-1]
PMO SEQ ID
Nucleotide sequence Sequence name
No. NO:
1 TTGCCGCTGCCCACATCCTGGAGTTC H45_1-13_18-30 14
2 GTTTGCCGCTGCCTCCTGGAGTTCCT H45_11_20-32 7
3 GCCGCTGCCCACATCCTGGAGTTCCT H45_13_18-28 15
4 CCGCTGCCCAATGTCCTGGAGTTCCT H45_11_15-27 16
TTGCCGCTGCCCATCCTGGAGTTCCT H45_11_18-30 17
6 TTTGCCGCTGCCATCCTGGAGTTCCT H45_13_21-31 18
7 TTTGCCGCTGCCTCCTGGAGTTCC H45_11_20-31 19
8 TGCCGCTGCCCGCCATCCTGGAGTTC H45_1-15_19-29 20
9 GTTTGCCGCTGCCCTGGAGTTCCT H45_10_21-32 8
CAGTTTGCCGCTGCCCATCCTGGAGTTCCT H45_13_20-34 9
11 CAGTTTGCCGCTGCCCTGGAGTTCCT H45_8_19-34 10
12 GTTTGCCGCTGCCATCCTGGAGTTC H45_1-12_20-32 21
13 CAGTTTGCCGCTGCTGGAGTTCCT H45_8_21-34 22
14 ACAGTTTGCCGCTCTGGAGTTCCT H45_9_23-35 23
CAGTTTGCCGCTGCCGGAGTTCCT H45_7_20-34 24
16 GTTTGCCGCTGCCCTGGAGTTCC H45_8_19-32 25
17 CAGTTTGCCGCTGCCGGAGTTCCTG H45_7_20-34 26
18 CCGCTGCCCAATGTGGAGTTCCTGT H45_8_15-27 27
19 CAGTTTGCCGCTGCCCTGGAGTTC H45_1-8_19-34 28
CCGCTGCCCAATCTGGAGTTCCT H45_9_16-27 29
21 CAGTTTGCCGCTGCCCTGGAGTTCC H45_8_19-34 30
22 TTGCCGCTGCCCACTGGAGTTCCT H45_9_18-30 31
23 TTGCCGCTGCCCACTGGAGTTCCTGT H45_9_18-30 32
24 ACAGTTTGCCGCCTGGAGTTCC H45_10_25-35 33
GTTTGCCGCTGC H45_21-32 34
26 CCTGGAGTTCCT H45_10 35
27 TGGAGTTCCT H45_8 36
28 CAGTTTGCCGCTGCCC H45_19-34 37
29 TCTTCCCCAGTTGCCATCCTGGAGTT H45_2-14_53-65 38
AGACCTCCTGCCACCATCCTGGAGTT H45_2-14_136-148 39
31 TTCTTCCCCAGTTGCGCCATCCTGGAGTTC H45_1-15_52-66 11
32 CAGACCTCCTGCCACGCCATCCTGGAGTTC H45_1-15_135-149 12
33 GACCTCCTGCCACCATCCTGGAGTTC H45_1-14_136-147 40
[Table 1-2]
34 TCCCCAGTTGCGCCATCCTGGAGTTC H45_1-15_52-62 41
GACCTCCTGCCGCCATCCTGGAGTTC H45_1-15_137-147 42
36 CTTCCCCAGTTGCCATCCTGGAGTTC H45_1-14_53-64 43
37 TTCCCCAGTTGCACATCCTGGAGTTC H45_1-13_51-63 44
38 CCTCCTGCCACCGCATCCTGGAGTTC H45_1-13_133-145 45
39 ACCTCCTGCCACCCATCCTGGAGTTC H45_1-13_134-146 46
40 TTTCTTCCCCAGTCATCCTGGAGTTC H45_1-13_55-67 47
41 GCAGACCTCCTGCCATCCTGGAGTTC H45_1-13_138-150 48
42 TTCTTCCCCAGTTGCCATCCTGGAGTTC H45_1-13_52-66 49
43 CCCCAGTTGCATCTGGAGTTCCT H45_9_50-61 50
44 TTCTTCCCCAGTTGCCCTGGAGTTCC H45_10_52-66 51
45 CTTCCCCAGTTGCCATCCTGGAGTTCCT H45_13_52-64 52
46 CAGACCTCCTGCCACTCCTGGAGTTC H45_1-11_135-149 53
47 TGCAGACCTCCTGCCTCCTGGAGTTC H45_1-11_137-151 54
48 CTGTTTGCAGACCCATCCTGGAGTTC H45_1-13_144-156 55
49 TTTGCAGACCTCCTGGAGTTCCTGTA H45_8_141-153 56
50 CCTGCCACCGCAGATGCCATCCTGGAGTTC H45_1-15_128-142 57
51 ACCTCCTGCCACCGCTTGCCGCTGCCCAAT H45_16-30_132-146 58
52 TCCTGTAGAATACCATCCTGGAGTTC H45_1-13_98-110 59
53 CTCCTGCCACCGCTGGCATCTGTTTT H45_85-97_132-144 60
54 ACCTCCTGCCACCGCTCTTCCCCAGTTGCA H45_51-65_132-146 61
55 TGGCATCTGTTTTCATCCTGGAGTTC H45_1-13_85-97 62
56 TTATTTCTTCCCCAGTTCCTGTAAGA H45_5_58-70 63
57 GCTTCCCAATGCCATCCTGGAGTTCC H45_15_114-123 64
58 GGCTTCCCAATGCCATCCTGGAGTTC H45_1-15_114-124 65
59 TTTCTGTCTGACAGCTCCTGCCACCGCAGA H45_129-143_156-170 66
60 TCCTGCCACCGCAGAGAGGATTGCTGAATT H45_69-83_129-143 67
61 TCCTGCCACCGCAGACTGGCATCTGTTTTT H45_84-98_129-143 68
62 TCCTGCCACCGCAGATTTTCCTGTAGAATA H45_99-113_129-143 69
63 GCCATCCTGGAGTTC H45_1-15 70
64 TTCTTCCCCAGTTGC H45_52-66 71
65 CAGACCTCCTGCCAC H45_135-149 72
66 TCCTGGAGTTCCT H45_11 73
67 GTTTGCCGCTGCC H45_20-32 74
68 CTCCTGCCACCGCGCCGCTGCCCAAT H45_16-28_132-144 75
[Table 1-3]
69 ATTCAGGCTTCCCTTCCCCAGTTGCA H45_51-63_117-129 76
70 TGGAGTTCC H45_8 77
71 TGGAGTTC H45_1-8 78
72 CAGTTTGCCGCCTGGAGTTCC H45_10_25-34 79
73 ACAGTTTGCCGCTGGAGTTCCT H45_9_25-35 80
74 GTTTGCCGCTGCCTGGAGTTCC H45_8_20-32 81
75 AACAGTTTGCCCCTGGAGTTCC H45_10_26-36 82
76 CAGTTTGCCGCCTGGAGTTC H45_1-10_25-34 83
77 CAGTTTGCCGCTCCTGGAGTTC H45_1-11_24-34 84
78 AGTTTGCCGCTCCTGGAGTTC H45_1-11_24-33 85
79 ACAGTTTGCCGCTGGAGTTCC H45_9_25-35 86
80 TGCCGCTGCCCATCCTGGAGTTCC H45_11_18-29 87
81 CTGCCACCGCAGCCGCTGCCCAATGC H45_14-27_130-141 88
82 CCTGGAGTTCC H45_10 144
83 CAGTTTGCCG H45_25-34 145
84 ACAGTTTGCCG H45_25-35 146
[Example 1]
4-{[(2S,6R)(4-Benzamidooxopyrimidin-1(2H)-yl)tritylmorpholin
yl]methoxy}oxobutanoic acid loaded on aminopolystyrene resin (Reference Example
1) or 4-{[(2S,6R)(5-methyl-2,4-dioxopyrimidinyl)tritylmorpholin
yl]methoxy}oxobutanoic acid loaded on aminopolystyrene resin (Reference Example
2) or 4-{[(2S,6R)(6-benzamidopurinyl)tritylmorpholinyl]methoxy}
oxobutanoic acid loaded on aminopolystyrene resin (Reference Example 3) or 4-
{{(2S,6R){6-(2-cyanoethoxy)[(2-phenoxyacetyl)amino]purinyl}
tritylmorpholinyl}methoxy}oxobutanoic acid loaded on aminopolystyrene resin
(Reference Example 4), each corresponding to the 5’-terminal base, was filled in an
amount of 0.2 g into a column equipped with a filter to initiate the following synthesis
cycles using a nucleic acid synthesizer (AKTA Oligopilot 10 plus). To give the
nucleotide sequence of each compound indicated in Table 1, a desired morpholino
monomer compound was added in each coupling cycle (see Table 2 below).
[Table 2]
Volume Time
Step Reagent
(mL) (min)
1 Deblocking solution 18 to 32 1.8 to 3.2
2 Neutralizing/washing solution 30 1.5
3 Coupling solution B 5 0.5
4 Coupling solution A 1.3 0.25
Coupling reaction with the reagents
120 to 300
charged in Steps 3 and 4
6 Acetonitrile 20 1.0
7 Capping solution 9 2.0
8 Acetonitrile 30 2.0
(Note) Only in the case of 3’-terminal acetylation, Steps 1, 2, 7 and 8
were repeated again after the final cycle.
It should be noted that the deblocking solution used was a dichloromethane
solution containing 3% (w/v) trifluoroacetic acid. The neutralizing/washing solution
used was prepared by dissolving N,N-diisopropylethylamine at 10% (v/v) and
tetrahydrofuran at 5% (v/v) in a dichloromethane solution containing 35% (v/v)
acetonitrile. The coupling solution A used was prepared by dissolving a morpholino
monomer compound at 0.10 M in tetrahydrofuran. The coupling solution B used was
prepared by dissolving N,N-diisopropylethylamine at 20% (v/v) and tetrahydrofuran at
% (v/v) in acetonitrile. The capping solution used was prepared by dissolving acetic
anhydride at 20% (v/v) and 2,6-lutidine at 30% (v/v) in acetonitrile.
The aminopolystyrene resin loaded with PMO synthesized as above was
collected from the reaction vessel and dried at room temperature for 2 hours or longer
under reduced pressure. The dried PMO loaded on the aminopolystyrene resin was
charged into a reaction vessel and 5 mL of 28% aqueous ammonia-ethanol (1/4) was
added thereto, followed by stirring at 55°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 mixed solvent containing 20 mM acetic acid-triethylamine
buffer (TEAA buffer) and acetonitrile (4/1), and then filtered through a membrane filter.
The resulting filtrate was purified by reversed-phase HPLC. The conditions used are
as indicated in Table 3 below.
[Table 3]
Column
XBridge 5 µm C18 (Waters, φ19 × 50 mm, 1 CV = 14 mL)
Flow rate 10 mL/minute
Column temperature room temperature
Solution A 20 mM TEAA buffer
Solution B CH CN
Gradient
(B) conc. 10% → 70%/15 CV
CV: column volume
The fractions were each analyzed to collect the desired product, which was
then concentrated under reduced pressure. The concentrated residue was diluted with
2 M aqueous phosphoric acid (0.5 mL) and stirred for 15 minutes. Further, the residue
was made alkaline with 2 M aqueous sodium hydroxide (2 mL) and filtered through a
membrane filter (0.45 µm).
The resulting aqueous solution containing the desired product was purified
through an anion exchange resin column. The conditions used are as indicated in
Table 4 below.
[Table 4]
Column
Source 15Q (GE Healthcare, φ10 × 108 mm, 1 CV = 8.5 mL)
Flow rate 8.5 mL/min
Column temperature room temperature
Solution A 10 mM aqueous sodium hydroxide
Solution B 10 mM aqueous sodium hydroxide,
1 M aqueous sodium chloride
Gradient
(B) conc. 1% → 50%/40 CV
The fractions were each analyzed (by HPLC) to obtain the desired product as
an aqueous solution. The resulting aqueous solution was neutralized with 0.1 M
phosphate buffer (pH 6.0) and then desalted by reversed-phase HPLC under the
conditions indicated in Table 5 below.
[Table 5]
Column
XBridge 5 µm C8 (Waters, φ10 × 50 mm, 1 CV = 4 mL)
Flow rate 4 mL/minute
Column temperature
60°C
Solution A water
Solution B CH CN
Gradient
(B) conc. 0% → 50%/20 CV
The desired product was collected and concentrated under reduced pressure.
The resulting residue was dissolved in water and freeze-dried to obtain the desired
compound as a white flocculent solid. The calculated and measured values of ESI-
TOF-MS are shown in Table 6.
[Table 6-1]
Calculated Measured
PMO No. Nucleotide sequence
value value
1 TTGCCGCTGCCCACATCCTGGAGTTC 8520.95 8520.65
2 GTTTGCCGCTGCCTCCTGGAGTTCCT 8542.94 8542.57
3 GCCGCTGCCCACATCCTGGAGTTCCT 8505.95 8506.57
4 CCGCTGCCCAATGTCCTGGAGTTCCT 8520.95 8521.37
TTGCCGCTGCCCATCCTGGAGTTCCT 8511.94 8511.70
6 TTTGCCGCTGCCATCCTGGAGTTCCT 8526.94 8527.07
7 TTTGCCGCTGCCTCCTGGAGTTCC 7857.71 7857.32
8 TGCCGCTGCCCGCCATCCTGGAGTTC 8521.95 8521.98
9 GTTTGCCGCTGCCCTGGAGTTCCT 7897.72 7897.71
CAGTTTGCCGCTGCCCATCCTGGAGTTCCT 9851.40 9851.60
11 CAGTTTGCCGCTGCCCTGGAGTTCCT 8551.95 8551.80
12 GTTTGCCGCTGCCATCCTGGAGTTC 8236.84 8236.69
13 CAGTTTGCCGCTGCTGGAGTTCCT 7921.73 7921.91
14 ACAGTTTGCCGCTCTGGAGTTCCT 7905.73 7905.53
CAGTTTGCCGCTGCCGGAGTTCCT 7906.73 7906.65
16 GTTTGCCGCTGCCCTGGAGTTCC 7567.61 7567.35
17 CAGTTTGCCGCTGCCGGAGTTCCTG 8261.85 8261.67
18 CCGCTGCCCAATGTGGAGTTCCTGT 8245.85 8245.68
19 CAGTTTGCCGCTGCCCTGGAGTTC 7906.73 7906.70
CCGCTGCCCAATCTGGAGTTCCT 7520.61 7520.60
21 CAGTTTGCCGCTGCCCTGGAGTTCC 8221.84 8221.48
22 TTGCCGCTGCCCACTGGAGTTCCT 7866.72 7866.77
23 TTGCCGCTGCCCACTGGAGTTCCTGT 8551.95 8552.23
24 ACAGTTTGCCGCCTGGAGTTCC 7245.51 7245.48
GTTTGCCGCTGC 3912.36 3912.16
26 CCTGGAGTTCCT 3896.36 3896.12
27 TGGAGTTCCT 3266.14 3265.99
28 CAGTTTGCCGCTGCCC 5196.81 5196.30
29 TCTTCCCCAGTTGCCATCCTGGAGTT 8510.93 8511.8
AGACCTCCTGCCACCATCCTGGAGTT 8513.95 8513.72
31 TTCTTCCCCAGTTGCGCCATCCTGGAGTTC 9826.39 9826.15
32 CAGACCTCCTGCCACGCCATCCTGGAGTTC 9814.41 9813.82
33 GACCTCCTGCCACCATCCTGGAGTTC 8489.95 8490.01
[Table 6-2]
34 TCCCCAGTTGCGCCATCCTGGAGTTC 8520.95 8520.97
GACCTCCTGCCGCCATCCTGGAGTTC 8505.95 8506.48
36 CTTCCCCAGTTGCCATCCTGGAGTTC 8495.94 8495.43
37 TTCCCCAGTTGCACATCCTGGAGTTC 8519.95 8520.35
38 CCTCCTGCCACCGCATCCTGGAGTTC 8465.94 8466.23
39 ACCTCCTGCCACCCATCCTGGAGTTC 8449.94 8449.88
40 TTTCTTCCCCAGTCATCCTGGAGTTC 8485.93 8486.01
41 GCAGACCTCCTGCCATCCTGGAGTTC 8529.96 8529.54
42 TTCTTCCCCAGTTGCCATCCTGGAGTTC 9156.16 9156.62
43 CCCCAGTTGCATCTGGAGTTCCT 7535.61 7535.92
44 TTCTTCCCCAGTTGCCCTGGAGTTCC 8486.93 8486.27
45 CTTCCCCAGTTGCCATCCTGGAGTTCCT 9141.16 9141.18
46 CAGACCTCCTGCCACTCCTGGAGTTC 8489.95 8489.65
47 TGCAGACCTCCTGCCTCCTGGAGTTC 8520.95 8520.58
48 CTGTTTGCAGACCCATCCTGGAGTTC 8559.96 8560.66
49 TTTGCAGACCTCCTGGAGTTCCTGTA 8574.96 8574.85
50 CCTGCCACCGCAGATGCCATCCTGGAGTTC 9854.42 9854.07
51 ACCTCCTGCCACCGCTTGCCGCTGCCCAAT 9750.39 9750.67
52 TCCTGTAGAATACCATCCTGGAGTTC 8567.97 8567.11
53 CTCCTGCCACCGCTGGCATCTGTTTT 8486.93 8486.39
54 ACCTCCTGCCACCGCTCTTCCCCAGTTGCA 9725.38 9725.57
55 TGGCATCTGTTTTCATCCTGGAGTTC 8580.95 8580.81
56 TTATTTCTTCCCCAGTTCCTGTAAGA 8508.94 8508.7
57 GCTTCCCAATGCCATCCTGGAGTTCC 8504.95 8504.88
58 GGCTTCCCAATGCCATCCTGGAGTTC 8544.96 8544.72
59 TTTCTGTCTGACAGCTCCTGCCACCGCAGA 9844.41 9844.1
60 TCCTGCCACCGCAGAGAGGATTGCTGAATT 9957.45 9957.8
61 TCCTGCCACCGCAGACTGGCATCTGTTTTT 9850.4 9850.45
62 TCCTGCCACCGCAGATTTTCCTGTAGAATA 9867.42 9867.85
63 GCCATCCTGGAGTTC 4905.71 4905.02
64 TTCTTCCCCAGTTGC 4831.68 4831.14
65 CAGACCTCCTGCCAC 4819.7 4819.64
66 TCCTGGAGTTCCT 4226.47 4226.03
67 GTTTGCCGCTGCC 4227.47 4227.48
68 CTCCTGCCACCGCGCCGCTGCCCAAT 8435.93 8436.58
[Table 6-3]
69 ATTCAGGCTTCCCTTCCCCAGTTGCA 8479.93 8479.03
70 TGGAGTTCC 2936.03 2936.07
71 TGGAGTTC 2620.92 2620.97
72 CAGTTTGCCGCCTGGAGTTCC 6906.39 6906.44
73 ACAGTTTGCCGCTGGAGTTCCT 7260.51 7260.67
74 GTTTGCCGCTGCCTGGAGTTCC 7252.5 7252.48
75 AACAGTTTGCCCCTGGAGTTCC 7229.51 7229.07
76 CAGTTTGCCGCCTGGAGTTC 6591.28 6591.07
77 CAGTTTGCCGCTCCTGGAGTTC 7236.5 7236.76
78 AGTTTGCCGCTCCTGGAGTTC 6921.39 6921.06
79 ACAGTTTGCCGCTGGAGTTCC 6930.4 6930.42
80 TGCCGCTGCCCATCCTGGAGTTCC 7851.72 7852.1
81 CTGCCACCGCAGCCGCTGCCCAATGC 8484.96 8484.68
82 CCTGGAGTTCC 3566.25 3566.51
83 CAGTTTGCCG 3251.14 3251.19
84 ACAGTTTGCCG 3590.26 3590.04
[Test Example 1]
In vitro assay
Into 3.5 × 10 RD cells (human rhabdomyosarcoma cell line), the antisense
oligomers shown in Table 1 were each transfected at 1 to 10 µM through Nucleofector
II (Lonza) using an Amaxa Cell Line Nucleofector Kit L. The program used was T-
030.
After transfection, the cells were cultured for three nights at 37°C under 5%
CO conditions in 2 mL of Eagle’s minimal essential medium (EMEM) (SIGMA; the
same applies hereinafter) containing 10% fetal bovine serum (FBS) (Invitrogen).
After the cells were washed once with PBS (Nissui Pharmaceutical Co., Ltd.,
Japan; the same applies hereinafter), 350 µL of Buffer RLT (QIAGEN) containing 1%
2-mercaptoethanol (Nacalai Tesque, Inc., Japan) was added to the cells, and the cells
were lysed by being allowed to stand at room temperature for a few minutes. The cell
lysate was collected into a QIAshredder homogenizer (QIAGEN) and centrifuged at
,000 rpm for 2 minutes to prepare a homogenate. The total RNA was extracted in
accordance with the protocol attached to an RNeasy Mini Kit (QIAGEN). The
concentration of the extracted total RNA was measured with a NanoDrop ND-1000
spectrophotometer (LMS Co., Ltd., Japan).
One-Step RT-PCR was performed on 400 ng of the extracted total RNA using
a QIAGEN OneStep RT-PCR Kit (QIAGEN). A reaction solution was prepared in
accordance with the protocol attached to the kit. The thermal cycler used was PTC-
100 (MJ Research) or TaKaRa PCR Thermal Cycler Dice Touch (Takara Bio Inc.,
Japan). The RT-PCR program used is as shown below.
50°C for 30 minutes: reverse transcription reaction
95°C for 15 minutes: polymerase activation, reverse transcriptase inactivation,
cDNA thermal denaturation
[94°C for 30 seconds; 60°C for 30 seconds; 72 °C for 1 minute] × 35 cycles:
PCR amplification
72°C for 10 minutes: final elongation reaction
The nucleotide sequences of the forward and reverse primers used for RT-PCR
are as shown below.
Forward primer: 5’-GCTCAGGTCGGATTGACATT-3’ (SEQ ID NO: 1)
Reverse primer: 5’-GGGCAACTCTTCCACCAGTA-3’ (SEQ ID NO: 2)
The above PCR reaction product (1 µL) was analyzed using a Bioanalyzer
(Agilent) and a MultiNA system (Shimadzu Corporation, Japan).
The polynucleotide level “A” in the band with exon 45 skipping and the
polynucleotide level “B” in the band without exon 45 skipping were measured. Based
on these measured values of “A” and “B,” the skipping efficiency was determined
according to the following equation.
Skipping efficiency (%) = A/(A + B) × 100
Experimental results
The results obtained are shown in Figures 1 to 5, 8, 10, 11 and 16 to 24. This
experiment indicated that the oligomer of the present invention effectively caused exon
45 skipping.
[Test Example 2]
In vitro assay
The same procedures as shown in Test Example 1 were repeated to conduct
this experiment, except that 3.5 × 10 RD cells (human rhabdomyosarcoma cell line)
were transfected with the oligomer of the present invention alone (PMO No. 11 or PMO
No. 9) or with either of the two unit oligomers constituting the oligomer of the present
invention or with a mixture thereof at a concentration of 3 µM through Nucleofector II
(Lonza) using an Amaxa Cell Line Nucleofector Kit L. The program used was T-030.
Combinations of the sequences transfected are as shown below.
[Table 7]
Combinations of the sequences transfected
Sequence combination
Transfection concentration ( µM)
PMO No. 11
3 µM
(PMO No. 27 and PMO No. 28 connected together)
PMO No. 27
3 µM
PMO No. 28
3 µM
PMO No. 27 and PMO No. 28 3 µM each
PMO No. 9
3 µM
(PMO No. 25 and PMO No. 26 connected together)
PMO No. 25
3 µM
PMO No. 26
3 µM
PMO No. 25 and PMO No. 26
3 µM each
PMO No. 72
3 µM
(PMO No. 82 and PMO No. 83 connected together)
PMO No. 82
3 µM
PMO No. 83
3 µM
PMO No. 82 and PMO No. 83
3 µM each
Experimental results
The results obtained are shown in Figures 6 and 25. This experiment
indicated that the oligomer of the present invention, i.e., PMO No. 11 (SEQ ID NO: 10),
PMO No. 9 (SEQ ID NO: 8) or PMO No. 72 (SEQ ID NO: 79), each being consisting
of connected two antisense oligomers targeting different sites in exon 45, caused exon
45 skipping with higher efficiency when compared to the respective antisense oligomers
constituting each oligomer (i.e., PMO No. 27 (SEQ ID NO: 36), PMO No. 28 (SEQ ID
NO: 37), PMO No. 25 (SEQ ID NO: 34), PMO No. 26 (SEQ ID NO: 35), PMO No. 82
(SEQ ID NO: 144) or PMO No. 83 (SEQ ID NO: 145)) or a mixture thereof (i.e., PMO
No. 27 and PMO No. 28, PMO No. 25 and PMO No. 26, or PMO No. 82 and PMO No.
83).
[Test Example 3]
In vitro assay
This experiment was conducted by using the antisense oligomers in 2’-O-
methoxy-phosphorothioate form (2’-OMe-S-RNA) shown in SEQ ID NOs: 89 to 141,
11 and 12. These various antisense oligomers used for assay were purchased from
Japan Bio Services Co., Ltd. The sequences of these various antisense oligomers are
shown below.
[Table 8-1]
Sequence name Nucleotide sequence SEQ ID NO:
H45_1-15_48-62 UCCCCAGUUGCAUUCGCCAUCCUGGAGUUC 89
H45_1-15_56-70 UUAUUUCUUCCCCAGGCCAUCCUGGAGUUC 90
H45_1-15_131-145 CCUCCUGCCACCGCAGCCAUCCUGGAGUUC 91
H45_13_131-145 CCUCCUGCCACCGCACAUCCUGGAGUUCCU 92
H45_13_135-149 CAGACCUCCUGCCACCAUCCUGGAGUUCCU 93
H45_13_48-62 UCCCCAGUUGCAUUCCAUCCUGGAGUUCCU 94
H45_13_52-66 UUCUUCCCCAGUUGCCAUCCUGGAGUUCCU 95
H45_13_56-70 UUAUUUCUUCCCCAGCAUCCUGGAGUUCCU 96
H45_13_18-32 GUUUGCCGCUGCCCACAUCCUGGAGUUCCU 97
H45_13_139-153 UUUGCAGACCUCCUGCAUCCUGGAGUUCCU 98
H45_1-17_135-147 GACCUCCUGCCACAUGCCAUCCUGGAGUUC 99
H45_1-17_52-64 CUUCCCCAGUUGCAUGCCAUCCUGGAGUUC 100
H45_1-15_139-153 UUUGCAGACCUCCUGGCCAUCCUGGAGUUC 101
H45_13_99-113 UUUUCCUGUAGAAUACAUCCUGGAGUUCCU 102
H45_53-67_132-146 ACCUCCUGCCACCGCUUUCUUCCCCAGUUG 103
H45_16-30_99-113 UUUUCCUGUAGAAUAUUGCCGCUGCCCAAU 104
H45_1-15_153-167 CUGUCUGACAGCUGUGCCAUCCUGGAGUUC 105
H45_1-15_67-81 GGAUUGCUGAAUUAUGCCAUCCUGGAGUUC 106
H45_1-15_99-113 UUUUCCUGUAGAAUAGCCAUCCUGGAGUUC 107
H45_1-13_46-58 CAGUUGCAUUCAACAUCCUGGAGUUC 108
H45_1-13_54-66 UUCUUCCCCAGUUCAUCCUGGAGUUC 109
H45_1-13_62-74 UGAAUUAUUUCUUCAUCCUGGAGUUC 110
H45_6-18_46-58 CAGUUGCAUUCAAAAUGCCAUCCUGG 111
H45_6-18_54-66 UUCUUCCCCAGUUAAUGCCAUCCUGG 112
H45_6-18_62-74 UGAAUUAUUUCUUAAUGCCAUCCUGG 113
H45_1-13_121-133 GCAGAUUCAGGCUCAUCCUGGAGUUC 114
H45_1-13_129-141 CUGCCACCGCAGACAUCCUGGAGUUC 115
H45_1-13_137-149 CAGACCUCCUGCCCAUCCUGGAGUUC 116
H45_6-18_121-133 GCAGAUUCAGGCUAAUGCCAUCCUGG 117
H45_6-18_129-141 CUGCCACCGCAGAAAUGCCAUCCUGG 118
H45_6-18_137-149 CAGACCUCCUGCCAAUGCCAUCCUGG 142
H45_16-28_116-128 UUCAGGCUUCCCAGCCGCUGCCCAAU 119
H45_16-28_124-136 ACCGCAGAUUCAGGCCGCUGCCCAAU 120
[Table 8-2]
H45_16-28_132-144 CUCCUGCCACCGCGCCGCUGCCCAAU 121
H45_26-38_116-128 UUCAGGCUUCCCAACAACAGUUUGCC 122
H45_26-38_124-136 ACCGCAGAUUCAGACAACAGUUUGCC 123
H45_26-38_132-144 CUCCUGCCACCGCACAACAGUUUGCC 124
H45_51-63_110-122 CUUCCCAAUUUUUUUCCCCAGUUGCA 125
H45_51-63_117-129 AUUCAGGCUUCCCUUCCCCAGUUGCA 126
H45_51-63_124-136 ACCGCAGAUUCAGUUCCCCAGUUGCA 127
H45_60-72_110-122 CUUCCCAAUUUUUAAUUAUUUCUUCC 128
H45_60-72_117-129 AUUCAGGCUUCCCAAUUAUUUCUUCC 129
H45_60-72_124-136 ACCGCAGAUUCAGAAUUAUUUCUUCC 130
H45_68-80_110-122 CUUCCCAAUUUUUGAUUGCUGAAUUA 131
H45_68-80_117-129 AUUCAGGCUUCCCGAUUGCUGAAUUA 132
H45_68-80_124-136 ACCGCAGAUUCAGGAUUGCUGAAUUA 133
H45_5_52-66 UUCUUCCCCAGUUGCAGUUCCUGUAAGAUA 134
H45_5_135-149 CAGACCUCCUGCCACAGUUCCUGUAAGAUA 135
H45_69-83_95-109 CCUGUAGAAUACUGGGAGGAUUGCUGAAUU 136
H45_16-30_84-98 CUGGCAUCUGUUUUUUUGCCGCUGCCCAAU 137
H45_16-30_53-67 UUUCUUCCCCAGUUGUUGCCGCUGCCCAAU 138
H45_1-15_84-98 CUGGCAUCUGUUUUUGCCAUCCUGGAGUUC 139
H45_84-98_132-146 ACCUCCUGCCACCGCCUGGCAUCUGUUUUU 140
H45_53-67_99-113 UUUUCCUGUAGAAUAUUUCUUCCCCAGUUG 141
H45_1-15_52-66 UUCUUCCCCAGUUGCGCCAUCCUGGAGUUC 11
H45_1-15_135-149 CAGACCUCCUGCCACGCCAUCCUGGAGUUC 12
In 24-well plates, 5 × 10 RD cells (human rhabdomyosarcoma cell line) were
seeded per well and cultured overnight at 37°C under 5% CO conditions in 0.5 mL of
Eagle’s minimal essential medium (EMEM) (SIGMA; the same applies hereinafter)
containing 10% fetal calf serum (FCS) (Invitrogen). The above various antisense
oligomers for exon 45 skipping (Japan Bio Services Co., Ltd., Japan) (1 µM or 300 nM)
were formed into conjugates with Lipofectamine 2000 (Invitrogen), and each conjugate
was added to the RD cells, which had been replaced in 0.45 mL fresh medium, in a
volume of 50 µL per well to give a final concentration of 100 nM or 30 nM.
After addition, the cells were cultured overnight. After the cells were washed
once with PBS (Nissui Pharmaceutical Co., Ltd., Japan; the same applies hereinafter),
350 µL of Buffer RLT (QIAGEN) containing 1% 2-mercaptoethanol (Nacalai Tesque,
Inc., Japan) was added to the cells, and the cells were lysed by being allowed to stand at
room temperature for a few minutes. The cell lysate was collected into a QIAshredder
homogenizer (QIAGEN) and centrifuged at 15,000 rpm for 2 minutes to prepare a
homogenate. The total RNA was extracted in accordance with the protocol attached to
an RNeasy Mini Kit (QIAGEN). The concentration of the extracted total RNA was
measured with a NanoDrop ND-1000 spectrophotometer (LMS Co., Ltd., Japan).
One-Step RT-PCR was performed on 400 ng of the extracted total RNA using
a QIAGEN OneStep RT-PCR Kit (QIAGEN). A reaction solution was prepared in
accordance with the protocol attached to the kit. The thermal cycler used was PTC-
100 (MJ Research) or TaKaRa PCR Thermal Cycler Dice Touch (Takara Bio Inc.,
Japan). The RT-PCR program used is as shown below.
50 °C for 30 minutes: reverse transcription reaction
95 °C for 15 minutes: polymerase activation, reverse transcriptase inactivation,
cDNA thermal denaturation
[94 °C for 30 seconds; 60 °C for 30 seconds; 72 °C for 1 minute] × 35 cycles:
PCR amplification
72 °C for 10 minutes: final elongation reaction
The nucleotide sequences of the forward and reverse primers used for RT-PCR
are as shown below.
Forward primer: 5’-GCTCAGGTCGGATTGACATT-3’ (SEQ ID NO: 1)
Reverse primer: 5’-GGGCAACTCTTCCACCAGTA-3’ (SEQ ID NO: 2)
The above PCR reaction product (1 µL) was analyzed by a Bioanalyzer
(Agilent) and a MultiNA system (Shimadzu Corporation, Japan).
The polynucleotide level “A” in the band with exon 45 skipping and the
polynucleotide level “B” in the band without exon 45 skipping were measured. Based
on these measured values of “A” and “B,” the skipping efficiency was determined
according to the following equation.
Skipping efficiency (%) = A/(A + B) × 100
Experimental results
The results obtained are shown in Figures 7 and 12 to 15. This experiment
indicated that the antisense oligomer of the present invention effectively caused exon 45
skipping.
[Test Example 4]
In vitro assay
The same procedures as shown in Test Example 1 were repeated to conduct
this experiment, except that 3.5 × 10 RD cells (human rhabdomyosarcoma cell line)
were transfected with the oligomer of the present invention alone (PMO No. 2, PMO
No. 31 or PMO No. 32) or with either of the two unit oligomers constituting the
oligomer of the present invention at a concentration of 3 µM or 10 µM through
Nucleofector II (Lonza) using an Amaxa Cell Line Nucleofector Kit L. The program
used was T-030. Combinations of the sequences transfected are as shown below.
[Table 9]
Sequence Transfection concentration
PMO No. 2 3 µM or 10 µM
(PMO No. 66 and PMO No. 67 connected together)
PMO No. 66
3 µM or 10 µM
PMO No. 67
3 µM or 10 µM
PMO No. 31
3 µM or 10 µM
(PMO No. 63 and PMO No. 64 connected together)
PMO No. 63 3 µM or 10 µM
PMO No. 64
3 µM or 10 µM
PMO No. 32
3 µM or 10 µM
(PMO No. 63 and PMO No. 65 connected together)
PMO No. 65
3 µM or 10 µM
Experimental results
The results obtained are shown in Figure 9. This experiment indicated that
the oligomer of the present invention, i.e., PMO No. 2 (SEQ ID NO: 7), PMO No. 31
(SEQ ID NO: 11) and PMO No. 32 (SEQ ID NO: 12), each being consisting of
connected two antisense nucleic acids targeting different sites in exon 45, caused exon
45 skipping with higher efficiency when compared to the respective antisense nucleic
acids constituting each oligomer (i.e., PMO No. 66, PMO No. 63, PMO No. 64 or PMO
No. 65).
INDUSTRIAL APPLICABILITY
As can be seen from the experimental results shown in the test examples, the
oligomer of the present invention consisting of short oligomers connected together was
found to cause exon 45 skipping in RD cells. Thus, the oligomer of the present
invention is very useful in the treatment of DMD.
Claims (19)
1. An antisense oligomer of 14 to 32 bases in length comprising connected two unit oligomers selected from the group consisting of (a) to (e) shown below, or a pharmaceutically acceptable salt or hydrate thereof, wherein the two unit oligomers are not contiguous to each other or do not overlap with each other: (a) a unit oligomer consisting of a nucleotide sequence complementary to a nucleotide sequence consisting of contiguous 7 to 16 bases selected from a nucleotide sequence located at positions -5 to 15 from the 5’-terminal end of exon 45 in the human dystrophin gene; (b) a unit oligomer consisting of a nucleotide sequence complementary to a nucleotide sequence consisting of contiguous 7 to 16 bases selected from a nucleotide sequence located at positions 48 to 70 from the 5’-terminal end of exon 45 in the human dystrophin gene; (c) a unit oligomer consisting of a nucleotide sequence complementary to a nucleotide sequence consisting of contiguous 7 to 16 bases selected from a nucleotide sequence located at positions 128 to 150 from the 5’-terminal end of exon 45 in the human dystrophin gene; (d) a unit oligomer consisting of a nucleotide sequence complementary to a nucleotide sequence consisting of contiguous 7 to 16 bases selected from a nucleotide sequence located at positions 15 to 40 from the 5’-terminal end of exon 45 in the human dystrophin gene; and (e) a unit oligomer consisting of a nucleotide sequence complementary to a nucleotide sequence consisting of contiguous 7 to 16 bases selected from a nucleotide sequence located at positions 110 to 125 from the 5’-terminal end of exon 45 in the human dystrophin gene.
2. The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof according to claim 1, wherein one of the two unit oligomers is (a).
3. The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof according to claim 1 or 2, which consists of any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 7 to 12, 14 to 33, 40 to 52, 57, 64, 65 and 79 to 86.
4. The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof according to any one of claims 1 to 3, which consists of any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 8, 10, 25, 30, 33, 79 and 80.
5. The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof according to any one of claims 1 to 4, which is an oligonucleotide.
6. The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof according to claim 5, wherein at least one nucleotide constituting the oligonucleotide is modified at the sugar moiety and/or at the phosphate bond moiety.
7. The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof according to claim 5 or 6, wherein the sugar moiety of at least one nucleotide constituting the oligonucleotide is a ribose in which the -OH group at the 2’-position is substituted with any group selected from the group consisting of OR, R, R’OR, SH, SR, NH , NHR, NR , N , CN, 2 2 3 F, Cl, Br and I (wherein R represents alkyl or aryl, and R’ represents alkylene).
8. The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof according to claim 6 or 7, wherein the phosphate bond moiety 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 phosphoroamidate bond and a boranophosphate bond.
9. The antisense oligomer according to any one of claims 1 to 4, which is a morpholino oligomer, or pharmaceutically acceptable salt or hydrate thereof.
10. The antisense oligomer according to claim 9, which is a phosphorodiamidate morpholino oligomer, or pharmaceutically acceptable salt or hydrate thereof.
11. The antisense oligomer according to claim 4, which is a phosphorodiamidate morpholino oligomer or pharmaceutically acceptable salt or hydrate thereof.
12. The antisense oligomer according to any one of claims 9 to 11, whose 5’-terminal end is any one of the groups represented by chemical formulae (1) to (3) shown below, or pharmaceutically acceptable salt or hydrate thereof. [Formula 25]
13. A pharmaceutical composition for treatment of muscular dystrophy, which comprises the antisense oligomer or pharmaceutically acceptable salt or hydrate thereof according to any one of claims 1 to 12 as an active ingredient.
14. The pharmaceutical composition according to claim 13, which further comprises a pharmaceutically acceptable carrier.
15. Use of the antisense oligomer or pharmaceutically acceptable salt or hydrate thereof according to any one of claims 1 to 12 in the manufacture of a medicament for the treatment of muscular dystrophy in a patient.
16. The use according to claim 15, wherein the muscular dystrophy patient is a patient having a mutation to be targeted by exon 45 skipping in the dystrophin gene.
17. The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof according to any one of claims 1 to 12, for use in the treatment of muscular dystrophy in a patient.
18. The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof according to claim 17, wherein the muscular dystrophy patient has a mutation to be targeted by exon 45 skipping in the dystrophin gene.
19. The antisense oligomer or pharmaceutically acceptable salt or hydrate thereof according to claim 17 or 18, wherein the patient is a human patient.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2015182145 | 2015-09-15 | ||
JP2015-182145 | 2015-09-15 | ||
PCT/JP2016/077305 WO2017047707A1 (en) | 2015-09-15 | 2016-09-15 | Antisense nucleic acid |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ740562A NZ740562A (en) | 2021-11-26 |
NZ740562B2 true NZ740562B2 (en) | 2022-03-01 |
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