U.S. patent application number 14/678517 was filed with the patent office on 2015-07-23 for oligonucleotide comprising an inosine for treating dmd.
The applicant listed for this patent is Prosensa Technologies B.V.. Invention is credited to Josephus Johannes de Kimpe, Gerard Johannes Platenburg, Judith Christina Theodora van Deutekom.
Application Number | 20150203849 14/678517 |
Document ID | / |
Family ID | 40940166 |
Filed Date | 2015-07-23 |
United States Patent
Application |
20150203849 |
Kind Code |
A1 |
van Deutekom; Judith Christina
Theodora ; et al. |
July 23, 2015 |
Oligonucleotide comprising an inosine for treating DMD
Abstract
The invention provides an oligonucleotide comprising an inosine,
and/or a nucleotide containing a base able to form a wobble base
pair or a functional equivalent thereof, wherein the
oligonucleotide, or a functional equivalent thereof, comprises a
sequence which is complementary to at least part of a dystrophin
pre-m RNA exon or at least part of a non-exon region of a
dystrophin pre-m RNA said part being a contiguous stretch
comprising at least 8 nucleotides. The invention further provides
the use of said oligonucleotide for preventing or treating DMD or
BMD.
Inventors: |
van Deutekom; Judith Christina
Theodora; (Dordrecht, NL) ; de Kimpe; Josephus
Johannes; (Utrecht, NL) ; Platenburg; Gerard
Johannes; (Voorschoten, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Prosensa Technologies B.V. |
Leiden |
|
NL |
|
|
Family ID: |
40940166 |
Appl. No.: |
14/678517 |
Filed: |
April 3, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13266110 |
Oct 24, 2011 |
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PCT/NL2010/050230 |
Apr 26, 2010 |
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14678517 |
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61172506 |
Apr 24, 2009 |
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Current U.S.
Class: |
514/44A ;
435/375; 536/24.5 |
Current CPC
Class: |
C12N 2310/336 20130101;
A61K 48/00 20130101; C12N 2310/315 20130101; C12N 2320/33 20130101;
A61P 29/00 20180101; C12N 2310/3181 20130101; C12N 2310/3231
20130101; C12N 2310/331 20130101; C12N 2310/333 20130101; C12N
2310/321 20130101; C12N 2310/11 20130101; C12N 15/113 20130101;
A61P 21/00 20180101; C12N 15/111 20130101; C12N 2310/321 20130101;
C12N 2310/3521 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 24, 2009 |
EP |
09158731.1 |
Claims
1. An isolated antisense oligonucleotide 8 to 50 nucleotides in
length comprising a base sequence which is complementary to a
consecutive part comprising 8 nucleotides of an exon of a
dystrophin pre-mRNA exon, wherein said oligonucleotide comprises at
least one inosine base and is functional to induce skipping of said
exon.
2. The oligonucleotide according to claim 1, wherein said
consecutive part comprises 13 to 50 nucleotides.
3. The oligonucleotide according to claim 2, wherein said
consecutive part comprises 14 to 25 nucleotides.
4. The oligonucleotide according to claim 1, wherein said exon is
selected from the group consisting of 51, 45, 53, 44, 46, 52, 50,
43 and 55.
5. The oligonucleotide according to claim 1, wherein said
oligonucleotide comprises RNA bases.
6. The oligonucleotide of claim 1, wherein said oligonucleotide
comprises a modified base, and/or a modified sugar moiety and/or a
modified internucleoside linkage.
7. The oligonucleotide according to claim 6, wherein said
oligonucleotide comprises a modified backbone.
8. The oligonucleotide according to claim 7, wherein said modified
backbone is selected from the group consisting of a morpholino
backbone, a carbamate backbone, a siloxane backbone, a sulfide
backbone, a sulfoxide backbone, a sulfone backbone, a formacetyl
backbone, a thioformacetyl backbone, a methyleneformacetyl
backbone, a riboacetyl backbone, an alkene containing backbone, a
sulfamate backbone, a sulfonate backbone, a sulfonamide backbone, a
methyleneimino backbone, a methylenehydrazino backbone and an amide
backbone.
9. The oligonucleotide according to claim 6, wherein said
oligonucleotide comprises a phosphorodiamidate morpholino oligomer
(PMO), peptide nucleic acid (PNA), and/or locked nucleic acid
(LNA).
10. The oligonucleotide according to claim 6, wherein said
oligonucleotide comprises a phosphorothioate internucleoside
linkage and a 2'-O-methyl substituted ribose moiety.
11. The oligonucleotide according to claim 6, wherein said modified
internucleoside linkage is a phosphorothioate moiety, said modified
sugar moiety is a 2'-O-methyl substituted ribose and wherein said
oligonucleotide comprises the sequence
5'-UUUGCCICUGCCCAAUGCCAUCCUG-3' (SEQ ID NO:557).
12. The oligonucleotide according to claim 1, wherein said
oligonucleotide comprises the base sequence
5'-UUUGCCICUGCCCAAUGCCAUCCUG-3' (SEQ ID NO:557).
13. The isolated antisense oligonucleotide of claim 12, wherein
said oligonucleotide comprises a modified base, and/or a modified
sugar moiety and/or a modified internucleoside linkage.
14. An isolated antisense oligonucleotide comprising or consisting
of a base or a nucleotide sequence selected from the group
consisting of: SEQ ID NO: 2-473, 539-556 and 558-576, wherein said
oligonucleotide comprises at least one inosine base.
15. The oligonucleotide of claim 14, wherein said oligonucleotide
comprises RNA bases.
16. The oligonucleotide of claim 14, wherein said oligonucleotide
comprises a modified base, and/or a modified sugar moiety and/or a
modified internucleoside linkage.
17. An isolated antisense oligonucleotide, comprising a base
sequence consisting of the base sequence
5'-UUUGCCICUGCCCAAUGCCAUCCUG-3' (SEQ ID NO:557), wherein the
oligonucleotide consists of phosphorothioate internucleoside
linkages and 2'-O-methyl substituted ribose moieties.
18. An isolated antisense oligonucleotide of 13 to 50 nucleotides
in length, said oligonucleotide comprising a sequence which is
fully complementary to a consecutive part comprising 8 nucleotides
of exon 45 of the human dystrophin pre-mRNA, wherein said
consecutive part is fully complementary to a portion of the base
sequence 5'-UUUGCCICUGCCCAAUGCCAUCCUG-3' (SEQ ID NO: 557).
19. The isolated oligonucleotide of claim 18, said consecutive part
comprising 13 nucleotides of exon 45.
20. The isolated oligonucleotide of claim 18, said consecutive part
comprising 25 nucleotides of exon 45.
21. The oligonucleotide of claim 18, wherein said oligonucleotide
comprises a modified base, and/or a modified sugar moiety, and/or a
modified internucleoside linkage.
22. The oligonucleotide claim 21, wherein said modified sugar
moiety is mono- or di-substituted at the 2', 3' and/or 5'
position.
23. The oligonucleotide of claim 21, wherein said modified
internucleoside linkage is a phosphorothioate moiety and said
modified sugar moiety is a 2'-O-substituted ribose.
24. The oligonucleotide of claim 23, wherein said modified sugar
moiety is a 2'-O-methyl ribose.
25. The oligonucleotide of claim 24, wherein each sugar moiety is a
2'-O-methyl ribose and each internucleoside linkage is a
phosphorothioate.
26. The oligonucleotide of claim 18, wherein said oligonucleotide
comprises RNA bases.
27. A method for inducing skipping of an exon of human dystrophin
pre-mRNA in a muscle cell, the method comprising contacting said
cell with an oligonucleotide of claim 1 for a time and under
conditions which permit exon skipping.
28. A method for inducing skipping of an exon of human dystrophin
pre-mRNA in a human subject, the method comprising administering an
oligonucleotide of claim 1 to said subject in an amount and for a
time which is effective to induce exon skipping.
29. A method for alleviating one or more symptom(s) of Duchenne
Muscular Dystrophy or Becker Muscular Dystrophy in an individual,
the method comprising administering to said individual an
oligonucleotide of claim 1, wherein said oligonucleotide induces
skipping of an exon of a dystrophin pre-mRNA.
30. A method for inducing and/or promoting skipping of exon 43, 44,
45, 46, 50, 51, 52 or 53 of the dystrophin pre-mRNA in a patient,
the method comprising administering an oligonucleotide of claim 4
to said patient.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the fields of molecular biology and
medicine.
BACKGROUND OF THE INVENTION
[0002] A muscle disorder is a disease that usually has a
significant impact on the life of an individual. A muscle disorder
can have either a genetic cause or a non-genetic cause. An
important group of muscle diseases with a genetic cause are Becker
Muscular Dystrophy (BMD) and Duchenne Muscular Dystrophy (DMD).
These disorders are caused by defects in a gene for a muscle
protein.
[0003] Becker Muscular Dystrophy and Duchenne Muscular Dystrophy
are genetic muscular dystrophies with a relatively high incidence.
In both Duchenne and Becker muscular dystrophy the muscle protein
dystrophin is affected. In Duchenne dystrophin is absent, whereas
in Becker some dystrophin is present but its production is most
often not sufficient and/or the dystrophin present is abnormally
formed. Both diseases are associated with recessive X-linked
inheritance. DMD results from a frameshift mutation in the DMD
gene. The frameshift in the DMD gene's transcript (mRNA) results in
the production of a truncated non-functional dystrophin protein,
resulting in progressive muscle wasting and weakness. BMD occurs as
a mutation does not cause a frame-shift in the DMD transcript
(mRNA). As in BMD some partly to largely functional dystrophin is
present in contrast to DMD where dystrophin is absent, BMD has
generally less severe symptoms then DMD. The onset of DMD is
earlier than BMD. DMD usually manifests itself in early childhood,
BMD in the teens or in early adulthood. The progression of BMD is
slower and less predictable than DMD. Patients with BMD can survive
into mid to late adulthood. Patients with DMD rarely survive beyond
their thirties.
[0004] Dystrophin plays an important structural role in the muscle
fiber, connecting the extracellular matrix and the cytoskeleton.
The N-terminal region binds actin, whereas the C-terminal end is
part of the dystrophin glycoprotein complex (DGC), which spans the
sarcolemma. In the absence of dystrophin, mechanical stress leads
to sarcolemmal ruptures, causing an uncontrolled influx of calcium
into the muscle fiber interior, thereby triggering
calcium-activated proteases and fiber necrosis.
[0005] For most genetic muscular dystrophies no clinically
applicable and effective therapies are currently available. Exon
skipping techniques are nowadays explored in order to combat
genetic muscular dystrophies. Promising results have recently been
reported by us and others on a genetic therapy aimed at restoring
the reading frame of the dystrophin pre-mRNA in cells from the mdx
mouse, the GRMD dog (reference 59) and DMD patients.sup.1-11. By
the targeted skipping of a specific exon, a DMD phenotype (lacking
dystrophin) is converted into a milder BMD phenotype (partly to
largely functional dystrophin). The skipping of an exon is
preferably induced by the binding of antisense oligoribonucleotides
(AONs) targeting either one or both of the splice sites, or
exon-internal sequences. Since an exon will only be included in the
mRNA when both the splice sites are recognised by the spliceosome
complex, splice sites have been considered obvious targets for
AONs. More preferably, one or more AONs are used which are specific
for at least part of one or more exonic sequences involved in
correct splicing of the exon. Using exon-internal AONs specific for
an exon 46 sequence, we were previously able to modulate the
splicing pattern in cultured myotubes from two different DMD
patients with an exon 45 deletion.sup.11. Following AON treatment,
exon 46 was skipped, which resulted in a restored reading frame and
the induction of dystrophin synthesis in at least 75% of the cells.
We have recently shown that exon skipping can also efficiently be
induced in human control and patient muscle cells for 39 different
DMD exons using exon-internal AONs.sup.1, 2, 11-15.
[0006] Hence, exon skipping techniques applied on the dystrophin
gene result in the generation of at least partially
functional--albeit shorter--dystrophin protein in DMD patients.
Since DMD is caused by a dysfunctional dystrophin protein, it would
be expected that the symptoms of DMD are sufficiently alleviated
once a DMD patient has been provided with functional dystrophin
protein. However, the present invention provides the insight that,
even though exon skipping techniques are capable of inducing
dystrophin synthesis, the oligonucleotide used for exon skipping
technique can be improved any further by incorporating an inosine
and/or a nucleotide containing a base able to form a wobble base
pair in said oligonucleotide.
DESCRIPTION OF THE INVENTION
Oligonucleotide
[0007] In a first aspect, there is provided an oligonucleotide
comprising an inosine and/or a nucleotide containing a base able to
form a wobble base pair or a functional equivalent thereof, wherein
the oligonucleotide, or a functional equivalent thereof, comprises
a sequence which is complementary to at least part of a dystrophin
pre-mRNA exon or at least part of a non-exon region of a dystrophin
pre-mRNA said part being a contiguous stretch comprising at least 8
nucleotides.
[0008] The use of an inosine and/or a nucleotide containing a base
able to form a wobble base pair in an oligonucleotide of the
invention is very attractive as explained below. Inosine for
example is a known modified base which can pair with three bases:
uracil, adenine, and cytosine. Inosine is a nucleoside that is
formed when hypoxanthine is attached to a ribose ring (also known
as a ribofuranose) via a .beta.-N9-glycosidic bond. Inosine is
commonly found in tRNAs and is essential for proper translation of
the genetic code in wobble base pairs. A wobble base pair is a G-U
and I-U/I-A/I-C pair fundamental in RNA secondary structure. Its
thermodynamic stability is comparable to that of the Watson-Crick
base pair. Wobble base pairs are critical for the proper
translation of the genetic code. The genetic code makes up for
disparities in the number of amino acids (20) for triplet codons
(64), by using modified base pairs in the first base of the
anti-codon. Similarly, when designing primers for polymerase chain
reaction, inosine is useful in that it will indiscriminately pair
with adenine, thymine, or cytosine. A first advantage of using such
a base allows one to design a primer that spans a single nucleotide
polymorphism (SNP), without worry that the polymorphism will
disrupt the primer's annealing efficiency. Therefore in the
invention, the use of such a base allows to design an
oligonucleotide that may be used for an individual having a SNP
within the dystrophin pre-mRNA stretch which is targeted by an
oligonucleotide of the invention. A second advantage of using an
inosine and/or a base able to form a wobble base pair in an
oligonucleotide of the invention is when said oligonucleotide would
normally contain a CpG if one would have designed it as being
complementary to at least part of a dystrophin pre-mRNA exon or at
least part of a non-exon region of a dystrophin pre-mRNA said part
being a contiguous stretch comprising at least 8 nucleotides. The
presence of a CpG in an oligonucleotide is usually associated with
an increased immunogenicity of said oligonucleotide (reference 60).
This increased immunogenicity is undesired since it may induce the
breakdown of muscle fibers. Replacing one, two or more CpG by the
corresponding inosine and/or a base able to form a wobble base pair
in said oligonucleotide is expected to provide an oligonucleotide
with a decreased and/or acceptable level of immunogenicity.
Immunogenicity may be assessed in an animal model by assessing the
presence of CD4.sup.+ and/or CD8.sup.+ cells and/or inflammatory
mononucleocyte infiltration in muscle biopsy of said animal.
Immunogenicity may also be assessed in blood of an animal or of a
human being treated with an oligonucleotide of the invention by
detecting the presence of a neutralizing antibody and/or an
antibody recognizing said oligonucleotide using a standard
immunoassay known to the skilled person.
[0009] An increase in immunogenicity preferably corresponds to a
detectable increase of at least one of these cell types by
comparison to the amount of each cell type in a corresponding
muscle biopsy of an animal before treatment or treated with a
corresponding oligonucleotide having at least one inosine and/or a
base able to form a wobble base pair. Alternatively, an increase in
immunogenicity may be assessed by detecting the presence or an
increasing amount of a neutralizing antibody or an antibody
recognizing said oligonucleotide using a standard immunoassay.
[0010] A decrease in immunogenicity preferably corresponds to a
detectable decrease of at least one of these cell types by
comparison to the amount of corresponding cell type in a
corresponding muscle biopsy of an animal before treatment or
treated with a corresponding oligonucleotide having no inosine
and/or a base able to form a wobble base pair. Alternatively a
decrease in immunogenicity may be assessed by the absence of or a
decreasing amount of said compound and/or neutralizing antibodies
using a standard immunoassay.
[0011] A third advantage of using an inosine and/or a base able to
form a wobble base pair in an oligonucleotide of the invention is
to avoid or decrease a potential multimerisation or aggregation of
oligonucleotides. It is for example known that an oligonucleotide
comprising a G-quartet motif has the tendency to form a quadruplex,
a multimer or aggregate formed by the Hoogsteen base-pairing of
four single-stranded oligonucleotides (reference 61), which is of
course not desired: as a result the efficiency of the
oligonucleotide is expected to be decreased. Multimerisation or
aggregation is preferably assessed by standard polyacrylamide
non-denaturing gel electrophoresis techniques known to the skilled
person. In a preferred embodiment, less than 20% or 15%, 10%, 7%,
5% or less of a total amount of an oligonucleotide of the invention
has the capacity to multimerise or aggregate assessed using the
assay mentioned above.
[0012] A fourth advantage of using an inosine and/or a base able to
form a wobble base pair in an oligonucleotide of the invention is
thus also to avoid quadruplex structures which have been associated
with antithrombotic activity (reference 62) as well as with the
binding to, and inhibition of, the macrophage scavenger receptor
(reference 63.).
[0013] A fifth advantage of using an inosine and/or a base able to
form a wobble base pair in an oligonucleotide of the invention is
to allow to design an oligonucleotide with improved RNA binding
kinetics and/or thermodynamic properties. The RNA binding kinetics
and/or thermodynamic properties are at least in part determined by
the melting temperature of an oligonucleotide (Tm; calculated with
the oligonucleotide properties calculator
(http://www.unc.edu/.about.cail/biotool/oligo/index.html) for
single stranded RNA using the basic Tm and the nearest neighbour
model), and/or the free energy of the AON-target exon complex
(using RNA structure version 4.5). If a Tm is too high, the
oligonucleotide is expected to be less specific. An acceptable Tm
and free energy depend on the sequence of the oligonucleotide.
Therefore, it is difficult to give preferred ranges for each of
these parameters. An acceptable Tm may be ranged between 35 and
65.degree. C. and an acceptable free energy may be ranged between
15 and 45 kcal/mol.
[0014] The skilled person may therefore first choose an
oligonucleotide as a potential therapeutic compound. In a second
step, he may use the invention to further optimise said
oligonucleotide by decreasing its immunogenicity and/or avoiding
aggregation and/or quadruplex formation and/or by optimizing its Tm
and/or free energy of the AON-target complex. He may try to
introduce at least one inosine and/or a base able to form a wobble
base pair in said oligonucleotide at a suitable position and assess
how the immunogenicity and/or aggregation and/or quadruplex
formation and/or Tm and/or free energy of the AON-target complex
have been altered by the presence of said inosine and/or a base
able to form a wobble base pair. If the alteration does not provide
the desired alteration or decrease of immunogenicity and/or
aggregation and/or quadruplex formation and/or its Tm and/or free
energy of the AON-target complex he may choose to introduce a
further inosine and/or a base able to form a wobble base pair in
said oligonucleotide and/or to introduce a given inosine and/or a
base able to form a wobble base pair at a distinct suitable
position within said oligonucleotide.
[0015] An oligonucleotide comprising an inosine and/or a base able
to form a wobble base pair may be defined as an oligonucleotide
wherein at least one nucleotide has been substituted with an
inosine and/or a base able to form a wobble base pair. The skilled
person knows how to test whether a nucleotide contains a base able
to form a wobble base pair. Since for example inosine can form a
base pair with uracil, adenine, and/or cytosine, it means that at
least one nucleotide able to form a base pair with uracil, adenine
and/or cytosine has been substituted with inosine. However, in
order to safeguard specificity, the inosine containing
oligonucleotide preferably comprises the substitution of at least
one, two, three, four nucleotide(s) able to form a base pair with
uracil or adenine or cytosine as long as an acceptable level of a
functional activity of said oligonucleotide is retained as defined
later herein.
[0016] An oligonucleotide comprising an inosine and/or a base able
to form a wobble base pair is preferably an olignucleotide, which
is still able to exhibit an acceptable level of a functional
activity of a corresponding oligonucleotide not comprising an
inosine and/or a base able to form a wobble base pair. A functional
activity of said oligonucleotide is preferably to provide an
individual with a functional dystrophin protein and/or mRNA and/or
at least in part decreasing the production of an aberrant
dystrophin protein and/or mRNA. Each of these features are later
defined herein. An acceptable level of such a functional activity
is preferably at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the
functional activity of the corresponding oligonucleotide which does
not comprise an inosine and/or a base able to form a wobble base
pair. Such functional activity may be as measured in a muscular
tissue or in a muscular cell of an individual or in vitro in a cell
by comparison to the functional activity of a corresponding
oligonucleotides not comprising an inosine and/or a base able to
form a wobble base pair. The assessment of the functionality may be
carried out at the mRNA level, preferably using RT-PCR. The
assessment of the functionality may be carried out at the protein
level, preferably using western blot analysis or immunofluorescence
analysis of cross-sections.
[0017] Within the context of the invention, an inosine and/or a
base able to form a wobble base pair as present in an
oligonucleotide is/are present in a part of said oligonucleotide
which is complementary to at least part of a dystrophin pre-mRNA
exon or at least part of a non-exon region of a dystrophin pre-mRNA
said part being a contiguous stretch comprising at least 8
nucleotides. Therefore, in a preferred embodiment, an
oligonucleotide comprising an inosine and/or a nucleotide
containing a base able to form a wobble base pair or a functional
equivalent thereof, wherein the oligonucleotide, or a functional
equivalent thereof, comprises a sequence which is complementary to
at least part of a dystrophin pre-mRNA exon or at least part of a
non-exon region of a dystrophin pre-mRNA said part being a
contiguous stretch comprising at least 8 nucleotides and wherein
said inosine and/or a nucleotide containing a base able is/are
present within the oligonucleotide sequence which is complementary
to at least part of a dystrophin pre-mRNA as defined in previous
sentence.
[0018] However, as later defined herein such inosine and/or a base
able to form a wobble base pair may also be present in a linking
moiety present in an oligonucleotide of the invention. Preferred
linking moieties are later defined herein.
[0019] In a preferred embodiment, such oligonucleotide is
preferably a medicament. More preferably, said medicament is for
preventing or treating Duchenne Muscular Dystrophy or Becker
Muscular Dystrophy in an individual or a patient. As defined herein
a DMD pre-mRNA preferably means the pre-mRNA of a DMD gene of a DMD
or BMD patient. A patient is preferably intended to mean a patient
having DMD or BMD or a patient susceptible to develop DMD or BMD
due to his or her genetic background. In the case of a DMD patient,
an oligonucleotide used will preferably correct at least one of the
DMD mutations as present in the DMD gene of said patient and
therefore will preferably create a dystrophin that will look like a
BMD dystrophin: said dystrophin will preferably be a functional
dystrophin as later defined herein.
[0020] In the case of a BMD patient, an oligonucleotide as used
will preferably correct at least one of the BMD mutations as
present in the DMD gene of said patient and therefore will
preferably create a, or more of a, dystrophin, which will be more
functional than the dystrophin which was originally present in said
BMD patient. Even more preferably, said medicament provides an
individual with a functional or more (of a) functional dystrophin
protein and/or mRNA and/or at least in part decreases the
production of an aberrant dystrophin protein and/or mRNA.
Preferably, a method of the invention by inducing and/or promoting
skipping of at least one exon of the DMD pre-mRNA as identified
herein in one or more cells, preferably muscle cells of a patient,
provides said patient with an increased production of a more (of a)
functional dystrophin protein and/or mRNA and/or decreases the
production of an aberrant or less functional dystrophin protein
and/or mRNA in said patient. Providing a patient with a more
functional dystrophin protein and/or mRNA and/or decreasing the
production of an aberrant dystrophin protein and/or mRNA in said
patient is typically applied in a DMD patient. Increasing the
production of a more functional or functional dystrophin and/or
mRNA is typically applied in a BMD patient.
[0021] Therefore a preferred method is a method, wherein a patient
or one or more cells of said patient is provided with an increased
production of a more functional or functional dystrophin protein
and/or mRNA and/or wherein the production of an aberrant dystrophin
protein and/or mRNA in said patient is decreased, wherein the level
of said aberrant or more functional dystrophin protein and/or mRNA
is assessed by comparison to the level of said dystrophin and/or
mRNA in said patient at the onset of the method.
[0022] As defined herein, a functional dystrophin is preferably a
wild type dystrophin corresponding to a protein having the amino
acid sequence as identified in SEQ ID NO: 1. A functional
dystrophin is preferably a dystrophin, which has an actin binding
domain in its N terminal part (first 240 amino acids at the N
terminus), a cystein-rich domain (amino acid 3361 till 3685) and a
C terminal domain (last 325 amino acids at the C terminus) each of
these domains being present in a wild type dystrophin as known to
the skilled person. The amino acids indicated herein correspond to
amino acids of the wild type dystrophin being represented by SEQ ID
NO: 1. In another embodiment, a functional dystrophin is a
dystrophin, which exhibits at least to some extent an activity of a
wild type dystrophin. "At least to some extent" preferably means at
least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of a
corresponding activity of a wild type functional dystrophin. In
this context, an activity of a wild type dystrophin is preferably
binding to actin and to the dystrophin-associated glycoprotein
complex (DGC).sup.56. Binding of dystrophin to actin and to the DGC
complex may be visualized by either co-immunoprecipitation using
total protein extracts or immunofluorescence analysis of
cross-sections, from a biopsy of a muscle suspected to be
dystrophic, as known to the skilled person.
[0023] Individuals suffering from Duchenne muscular dystrophy
typically have a mutation in the gene encoding dystrophin that
prevents synthesis of the complete protein, i.e a premature stop
prevents the synthesis of the C-terminus of the protein. In Becker
muscular dystrophy the dystrophin gene also comprises a mutation
compared to the wild type but the mutation does typically not
include a premature stop and the C-terminus of the protein is
typically synthesized. As a result a functional dystrophin protein
is synthesized that has at least the same activity in kind as a
wild type protein, although not necessarily the same amount of
activity. In a preferred embodiment, a functional dystrophin
protein means an in frame dystrophin gene. The genome of a BMD
individual typically encodes a dystrophin protein comprising the N
terminal part (first 240 amino acids at the N terminus), a
cystein-rich domain (amino acid 3361 till 3685) and a C terminal
domain (last 325 amino acids at the C terminus) but its central rod
shaped domain may be shorter than the one of a wild type
dystrophin.sup.56. Exon-skipping for the treatment of DMD is
preferably but not exclusively directed to overcome a premature
stop in the pre-mRNA by skipping an exon in the rod-domain shaped
domain to correct the reading frame and allow synthesis of
remainder of the dystrophin protein including the C-terminus,
albeit that the protein is somewhat smaller as a result of a
smaller rod domain. In a preferred embodiment, an individual having
DMD and being treated using an oligonucleotide as defined herein
will be provided a dystrophin, which exhibits at least to some
extent an activity of a wild type dystrophin. More preferably, if
said individual is a Duchenne patient or is suspected to be a
Duchenne patient, a functional dystrophin is a dystrophin of an
individual having BMD: preferably said dystrophin is able to
interact with both actin and the DGC, but its central rod shaped
domain may be shorter than the one of a wild type dystrophin
(Aartsma-Rus et al (2006, ref 56). The central rod domain of wild
type dystrophin comprises 24 spectrin-like repeats.sup.56. For
example, a central rod shaped domain of a dystrophin as provided
herein may comprise 5 to 23, 10 to 22 or 12 to 18 spectrin-like
repeats as long as it can bind to actin and to DGC. Decreasing the
production of an aberrant dystrophin in said patient or in a cell
of said patient may be assessed at the mRNA level and preferably
means that 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or
less of the initial amount of aberrant dystrophin mRNA, is still
detectable by RT PCR. An aberrant dystrophin mRNA or protein is
also referred to herein as a non-functional or less to
non-functional or semi-functional dystrophin mRNA or protein. A
non-functional pre-mRNA dystrophin is preferably leads to an out of
frame dystrophin protein, which means that no dystrophin protein
will be produced and/or detected. A non functional dystrophin
protein is preferably a dystrophin protein which is not able to
bind actin and/or members of the DGC protein complex. A
non-functional dystrophin protein or dystrophin mRNA does typically
not have, or does not encode a dystrophin protein with an intact
C-terminus of the protein.
[0024] Increasing the production of a functional dystrophin in said
patient or in a cell of said patient may be assessed at the mRNA
level (by RT-PCR analysis) and preferably means that a detectable
amount of a functional or in frame dystrophin mRNA is detectable by
RT PCR. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90% or more of the detectable dystrophin mRNA is a
functional or in frame dystrophin mRNA.
[0025] Increasing the production of a functional dystrophin in said
patient or in a cell of said patient may be assessed at the protein
level (by immunofluorescence and western blot analyses) and
preferably means that a detectable amount of a functional
dystrophin protein is detectable by immunofluorescence or western
blot analysis. In another embodiment, 1%, 5%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90% or more of the detectable dystrophin
protein is a functional dystrophin protein.
[0026] An increase or a decrease is preferably assessed in a
muscular tissue or in a muscular cell of an individual or a patient
by comparison to the amount present in said individual or patient
before treatment with said molecule or composition of the
invention. Alternatively, the comparison can be made with a
muscular tissue or cell of said individual or patient, which has
not yet been treated with said oligonucleotide or composition in
case the treatment is local.
[0027] In a preferred method, one or more symptom(s) from a DMD or
a BMD patient is/are alleviated and/or one or more
characteristic(s) of a muscle cell or tissue from a DMD or a BMD
patient is/are alleviated using a molecule or a composition of the
invention. Such symptoms may be assessed on the patient self. Such
characteristics may be assessed at the cellular, tissue level of a
given patient. An alleviation of one or more characteristics may be
assessed by any of the following assays on a myogenic cell or
muscle cell from a patient: reduced calcium uptake by muscle cells,
decreased collagen synthesis, altered morphology, altered lipid
biosynthesis, decreased oxidative stress, and/or improved muscle
fiber function, integrity, and/or survival. These parameters are
usually assessed using immunofluorescence and/or histochemical
analyses of cross sections of muscle biopsies.
[0028] Alleviating one or more symptom(s) of Duchenne Muscular
Dystrophy or Becker Muscular Dystrophy in an individual using a
molecule or a composition of the invention may be assessed by any
of the following assays: prolongation of time to loss of walking,
improvement of muscle strength, improvement of the ability to lift
weight, improvement of the time taken to rise from the floor,
improvement in the nine-meter walking time, improvement in the time
taken for four-stairs climbing, improvement of the leg function
grade, improvement of the pulmonary function, improvement of
cardiac function, improvement of the quality of life. Each of these
assays is known to the skilled person. As an example, the
publication of Manzur at al (2008, ref 58) gives an extensive
explanation of each of these assays. For each of these assays, as
soon as a detectable improvement or prolongation of a parameter
measured in an assay has been found, it will preferably mean that
one or more symptoms of Duchenne Muscular Dystrophy or Becker
Muscular Dystrophy has been alleviated in an individual using a
molecule or composition of the invention. Detectable improvement or
prolongation is preferably a statistically significant improvement
or prolongation as described in Hodgetts et al (2006, ref 57).
Alternatively, the alleviation of one or more symptom(s) of
Duchenne Muscular Dystrophy or Becker Muscular Dystrophy may be
assessed by measuring an improvement of a muscle fiber function,
integrity and/or survival as later defined herein.
[0029] An oligonucleotide as used herein preferably comprises an
antisense oligonucleotide or antisense oligoribonucleotide. In a
preferred embodiment an exon skipping technique is applied. Exon
skipping interferes with the natural splicing processes occurring
within a eukaryotic cell. In higher eukaryotes the genetic
information for proteins in the DNA of the cell is encoded in exons
which are separated from each other by intronic sequences. These
introns are in some cases very long. The transcription machinery of
eukaryotes generates a pre-mRNA which contains both exons and
introns, while the splicing machinery, often already during the
production of the pre-mRNA, generates the actual coding region for
the protein by splicing together the exons present in the
pre-mRNA.
[0030] Exon-skipping results in mature mRNA that lacks at least one
skipped exon. Thus, when said exon codes for amino acids, exon
skipping leads to the expression of an altered product. Technology
for exon-skipping is currently directed towards the use of
antisense oligonucleotides (AONs). Much of this work is done in the
mdx mouse model for Duchenne muscular dystrophy. The mdx mouse
carries a nonsense mutation in exon 23. Despite the mdx mutation,
which should preclude the synthesis of a functional dystrophin
protein, rare, naturally occurring dystrophin positive fibers have
been observed in mdx muscle tissue. These dystrophin-positive
fibers are thought to have arisen from an apparently naturally
occurring exon-skipping mechanism, either due to somatic mutations
or through alternative splicing. AONs directed to, respectively,
the 3' and/or 5' splice sites of introns 22 and 23 in dystrophin
pre-mRNA, have been shown to interfere with factors normally
involved in removal of intron 23 so that also exon 23 was removed
from the mRNA.sup.3, 5, 6, 39, 40.
[0031] By the targeted skipping of a specific exon, a DMD phenotype
is converted into a milder BMD phenotype. The skipping of an exon
is preferably induced by the binding of AONs targeting either one
or both of the splice sites, or exon-internal sequences. An
oligonucleotide directed toward an exon internal sequence typically
exhibits no overlap with non-exon sequences. It preferably does not
overlap with the splice sites at least not insofar, as these are
present in the intron. An oligonucleotide directed toward an exon
internal sequence preferably does not contain a sequence
complementary to an adjacent intron. Further provided is thus an
oligonucleotide according to the invention, wherein said
oligonucleotide, or a functional equivalent thereof, is for
inhibiting inclusion of an exon of a dystrophin pre-mRNA into mRNA
produced from splicing of said pre-mRNA. An exon skipping technique
is preferably applied such that the absence of an exon from mRNA
produced from dystrophin pre-mRNA generates a coding region for a
more functional--albeit shorter--dystrophin protein. In this
context, inhibiting inclusion of an exon preferably means that the
detection of the original, aberrant dystrophin mRNA and/or protein
is decreased as earlier defined herein.
[0032] Since an exon of a dystrophin pre-mRNA will only be included
into the resulting mRNA when both the splice sites are recognised
by the spliceosome complex, splice sites have been obvious targets
for AONs. One embodiment therefore provides an oligonucleotide, or
a functional equivalent thereof, comprising a sequence which is
complementary to a non-exon region of a dystrophin pre mRNA. In one
embodiment an AON is used which is solely complementary to a
non-exon region of a dystrophin pre mRNA. This is however not
necessary: it is also possible to use an AON which comprises an
intron-specific sequence as well as exon-specific sequence. Such
AON comprises a sequence which is complementary to a non-exon
region of a dystrophin pre mRNA, as well as a sequence which is
complementary to an exon region of a dystrophin pre mRNA. Of
course, an AON is not necessarily complementary to the entire
sequence of a dystrophin exon or intron. AONs, which are
complementary to a part of such exon or intron are preferred. An
AON is preferably complementary to at least part of a dystrophin
exon and/or intron, said part having at least 8, 10, 13, 15, 20
nucleotides.
[0033] Splicing of a dystrophin pre-mRNA occurs via two sequential
transesterification reactions. First, the 2'OH of a specific
branch-point nucleotide within the intron that is defined during
spliceosome assembly performs a nucleophilic attack on the first
nucleotide of the intron at the 5' splice site forming the lariat
intermediate. Second, the 3'OH of the released 5' exon then
performs a nucleophilic attack at the last nucleotide of the intron
at the 3' splice site thus joining the exons and releasing the
intron lariat. The branch point and splice sites of an intron are
thus involved in a splicing event. Hence, an oligonucleotide
comprising a sequence, which is complementary to such branch point
and/or splice site is preferably used for exon skipping. Further
provided is therefore an oligonucleotide, or a functional
equivalent thereof, which comprises a sequence which is
complementary to a splice site and/or branch point of a dystrophin
pre mRNA.
[0034] Since splice sites contain consensus sequences, the use of
an oligonucleotide or a functional equivalent thereof (herein also
called an AON) comprising a sequence which is complementary of a
splice site involves the risk of promiscuous hybridization.
Hybridization of AONs to other splice sites than the sites of the
exon to be skipped could easily interfere with the accuracy of the
splicing process. To overcome these and other potential problems
related to the use of AONs which are complementary to an intron
sequence, one preferred embodiment provides an oligonucleotide, or
a functional equivalent thereof, comprising a sequence which is
complementary to a dystrophin pre-mRNA exon. Preferably, said AON
is capable of specifically inhibiting an exon inclusion signal of
at least one exon in said dystrophin pre-mRNA. Interfering with an
exon inclusion signal (EIS) has the advantage that such elements
are located within the exon. By providing an AON for the interior
of the exon to be skipped, it is possible to interfere with the
exon inclusion signal thereby effectively masking the exon from the
splicing apparatus. The failure of the splicing apparatus to
recognize the exon to be skipped thus leads to exclusion of the
exon from the final mRNA. This embodiment does not interfere
directly with the enzymatic process of the splicing machinery (the
joining of the exons). It is thought that this allows the method to
be more specific and/or reliable. It is thought that an EIS is a
particular structure of an exon that allows splice acceptor and
donor to assume a particular spatial conformation. In this concept,
it is the particular spatial conformation that enables the splicing
machinery to recognize the exon. However, the invention is
certainly not limited to this model. In a preferred embodiment, use
is made of an oligonucleotide, which is capable of binding to an
exon and is capable of inhibiting an EIS. An AON may specifically
contact said exon at any point and still be able to specifically
inhibit said EIS.
[0035] Within the context of the invention, a functional equivalent
of an oligonucleotide preferably means an oligonucleotide as
defined herein wherein one or more nucleotides have been
substituted and wherein an activity of said functional equivalent
is retained to at least some extent.
[0036] Preferably, an activity of said functional equivalent is
providing a functional dystrophin protein. Said activity of said
functional equivalent is therefore preferably assessed by
quantifying the amount of a functional dystrophin protein or by
quantifying the amount of a functional dystrophin mRNA. A
functional dystrophin protein (or a functional dystrophin mRNA) is
herein preferably defined as being a dystrophin protein (or a
dystrophin protein encoded by said mRNA) able to bind actin and
members of the DGC protein. The assessment of said activity of an
oligonucleotide is preferably done by RT-PCR (m-RNA) or by
immunofluorescence or Western blot analyses (protein). Said
activity is preferably retained to at least some extent when it
represents at least 50%, or at least 60%, or at least 70% or at
least 80% or at least 90% or at least 95% or more of corresponding
activity of said oligonucleotide the functional equivalent derives
from. Such activity may be measured in a muscular tissue or in a
muscular cell of an individual or in vitro in a cell by comparison
to an activity of a corresponding oligonucleotide of said
oligonucleotide the functional equivalent derives from. Throughout
this application, when the word oligonucleotide is used it may be
replaced by a functional equivalent thereof as defined herein.
[0037] Hence, an oligonucleotide, or a functional equivalent
thereof, comprising or consisting of a sequence which is
complementary to a dystrophin pre-mRNA exon provides good DMD
therapeutic results. In one preferred embodiment an
oligonucleotide, or a functional equivalent thereof, is used which
comprises or consists of a sequence which is complementary to at
least part of either dystrophin pre-mRNA exons 2 to 75 said part
having or comprising at least 13 nucleotides. However, said part
may also have at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50
nucleotides. A part of dystrophin pre-mRNA to which an
oligonucleotide is complementary may also be called a contiguous
stretch of dystrophin pre-mRNA.
[0038] Most preferably an AON is used which comprises or consists
of a sequence which is complementary to at least part of dystrophin
pre-mRNA exon 51, 45, 53, 44, 46, 52, 50, 43, 6, 7, 8, 55, 2, 11,
17, 19, 21, 57, 59, 62, 63, 65, 66, 69, and/or 75 said part having
or comprising at least 13 nucleotides. However, said part may also
have at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides. More
preferred oligonucleotides are represented by a sequence that
comprises or consists of each of the following sequences SEQ ID NO:
2 to SEQ ID NO:539 wherein at least one inosine and/or a base able
to form a wobble base pair is present in said sequence. Preferably,
an inosine has been introduced in one of these sequences to replace
a guanosine, adenine, or a uracil. Accordingly, an even more
preferred oligonucleotide as used herein is represented by a
sequence that comprises or consists of SEQ ID NO:2 to SEQ ID NO:486
or SEQ ID NO:539, even more preferably SEQ ID NO:2 to NO 237 or SEQ
ID NO:539, most preferably SEQ ID NO:76 wherein at least one
inosine and/or a base able to form a wobble base pair is present in
said sequence. Preferably, an inosine has been introduced in one of
these sequences to replace a guanosine, adenine, or a uracil.
[0039] Accordingly, in another preferred embodiment, an
oligonucleotide as used herein is represented by a sequence that
comprises or consists of SEQ ID NO:540 to SEQ ID NO:576. More
preferably, an oligonucleotide as used herein is represented by a
sequence that comprises or consists of SEQ ID NO:557.
[0040] Said exons are listed in decreasing order of patient
population applicability. Hence, the use of an AON comprising a
sequence, which is complementary to at least part of dystrophin
pre-mRNA exon 51 is suitable for use in a larger part of the DMD
patient population as compared to an AON comprising a sequence
which is complementary to dystrophin pre-mRNA exon 44, et
cetera.
[0041] In a preferred embodiment, an oligonucleotide of the
invention, which comprises a sequence that is complementary to part
of dystrophin pre-mRNA is such that the complementary part is at
least 50% of the length of the oligonucleotide of the invention,
more preferably at least 60%, even more preferably at least 70%,
even more preferably at least 80%, even more preferably at least
90% or even more preferably at least 95%, or even more preferably
98% or even more preferably at least 99%, or even more preferably
100%. In a most preferred embodiment, the oligonucleotide of the
invention consists of a sequence that is complementary to part of
dystrophin pre-mRNA as defined herein. As an example, an
oligonucleotide may comprise a sequence that is complementary to
part of dystrophin pre-mRNA as defined herein and additional
flanking sequences. In a more preferred embodiment, the length of
said complementary part of said oligonucleotide is of at least 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50 nucleotides. Preferably, additional
flanking sequences are used to modify the binding of a protein to
the oligonucleotide, or to modify a thermodynamic property of the
oligonucleotide, more preferably to modify target RNA binding
affinity.
[0042] One preferred embodiment provides an oligonucleotide, or a
functional equivalent thereof which comprises:
[0043] a sequence which is complementary to a region of a
dystrophin pre-mRNA exon that is hybridized to another part of a
dystrophin pre-mRNA exon (closed structure), and
[0044] a sequence which is complementary to a region of a
dystrophin pre-mRNA exon that is not hybridized in said dystrophin
pre-mRNA (open structure).
[0045] For this embodiment, reference is made to WO 2004/083432,
which is incorporated by reference in its entirety. RNA molecules
exhibit strong secondary structures, mostly due to base pairing of
complementary or partly complementary stretches within the same
RNA. It has long since been thought that structures in the RNA play
a role in the function of the RNA. Without being bound by theory,
it is believed that the secondary structure of the RNA of an exon
plays a role in structuring the splicing process. The structure of
an exon is one parameter which is believed to direct its inclusion
into the mRNA. However, other parameters may also play a role
therein. Herein this signalling function is referred to as an exon
inclusion signal. A complementary oligonucleotide of this
embodiment is capable of interfering with the structure of the exon
and thereby capable of interfering with the exon inclusion signal
of the exon. It has been found that many complementary
oligonucleotides indeed comprise this capacity, some more efficient
than others. Oligonucleotides of this preferred embodiment, i.e.
those with the said overlap directed towards open and closed
structures in the native exon RNA, are a selection from all
possible oligonucleotides. The selection encompasses
oligonucleotides that can efficiently interfere with an exon
inclusion signal. Without being bound by theory it is thought that
the overlap with an open structure improves the invasion efficiency
of the oligonucleotide and prevents the binding of splicing factors
(i.e. increases the efficiency with which the oligonucleotide can
enter the structure), whereas the overlap with the closed structure
subsequently increases the efficiency of interfering with the
secondary structure of the RNA of the exon, and thereby interfere
with the exon inclusion signal. It is found that the length of the
partial complementarity to both the closed and the open structure
is not extremely restricted. We have observed high efficiencies
with oligonucleotides with variable lengths of complementarity in
either structure. The term complementarity is used herein to refer
to a stretch of nucleic acids that can hybridise to another stretch
of nucleic acids under physiological conditions. It is thus not
absolutely required that all the bases in the region of
complementarity are capable of pairing with bases in the opposing
strand. For instance, when designing the oligonucleotide one may
want to incorporate for instance a residue that does not base pair
with the base on the complementary strand. Mismatches may, to some
extent, be allowed, if under the circumstances in the cell, the
stretch of nucleotides is sufficiently capable of hybridising to
the complementary part. In this context, "sufficiently" preferably
means that using a gel mobility shift assay as described in example
1 of EP 1 619 249, binding of an oligonucleotide is detectable.
Optionally, said oligonucleotide may further be tested by
transfection into muscle cells of patients. Skipping of the
targeted exon may be assessed by RT-PCR (as described in EP 1 619
249). The complementary regions are preferably designed such that,
when combined, they are specific for the exon in the pre-mRNA. Such
specificity may be created with various lengths of complementary
regions as this depends on the actual sequences in other (pre-)mRNA
in the system. The risk that also one or more other pre-mRNA will
be able to hybridise to the oligonucleotide decreases with
increasing size of the oligonucleotide. It is clear that
oligonucleotides comprising mismatches in the region of
complementarity but that retain the capacity to hybridise to the
targeted region(s) in the pre-mRNA, can be used in the present
invention. However, preferably at least the complementary parts do
not comprise such mismatches as these typically have a higher
efficiency and a higher specificity, than oligonucleotides having
such mismatches in one or more complementary regions. It is
thought, that higher hybridisation strengths, (i.e. increasing
number of interactions with the opposing strand) are favourable in
increasing the efficiency of the process of interfering with the
splicing machinery of the system. Preferably, the complementarity
is between 90 and 100%. In general this allows for approximately 1
or 2 mismatch(es) in an oligonucleotide of around 20
nucleotides
[0046] The secondary structure is best analysed in the context of
the pre-mRNA wherein the exon resides. Such structure may be
analysed in the actual RNA. However, it is currently possible to
predict the secondary structure of an RNA molecule (at lowest
energy costs) quite well using structure-modelling programs. A
non-limiting example of a suitable program is RNA mfold version 3.1
server.sup.41. A person skilled in the art will be able to predict,
with suitable reproducibility, a likely structure of the exon,
given the nucleotide sequence. Best predictions are obtained when
providing such modelling programs with both the exon and flanking
intron sequences. It is typically not necessary to model the
structure of the entire pre-mRNA.
[0047] The open and closed structure to which the oligonucleotide
is directed, are preferably adjacent to one another. It is thought,
that in this way the annealing of the oligonucleotide to the open
structure induces opening of the closed structure whereupon
annealing progresses into this closed structure. Through this
action the previously closed structure assumes a different
conformation. The different conformation results in the disruption
of the exon inclusion signal. However, when potential (cryptic)
splice acceptor and/or donor sequences are present within the
targeted exon, occasionally a new exon inclusion signal is
generated defining a different (neo) exon, i.e. with a different 5'
end, a different 3' end, or both. This type of activity is within
the scope of the present invention as the targeted exon is excluded
from the mRNA. The presence of a new exon, containing part of the
targeted exon, in the mRNA does not alter the fact that the
targeted exon, as such, is excluded. The inclusion of a neo-exon
can be seen as a side effect, which occurs only occasionally. There
are two possibilities when exon skipping is used to restore (part
of) an open reading frame of dystrophin that is disrupted as a
result of a mutation. One is that the neo-exon is functional in the
restoration of the reading frame, whereas in the other case the
reading frame is not restored. When selecting oligonucleotides for
restoring dystrophin reading frames by means of exon-skipping it is
of course clear that under these conditions only those
oligonucleotides are selected that indeed result in exon-skipping
that restores the dystrophin open reading frame, with or without a
neo-exon.
[0048] Further provided is an oligonucleotide, or a functional
equivalent thereof, comprising a sequence that is complementary to
a binding site for a serine-arginine (SR) protein in RNA of an exon
of a dystrophin pre-mRNA. In WO 2006/112705 we have disclosed the
presence of a correlation between the effectively of an
exon-internal antisense oligonucleotide (AON) in inducing exon
skipping and the presence of a (for example by ESE finder)
predicted SR binding site in the target pre-mRNA site of said
AON.
[0049] Therefore, in one embodiment an oligonucleotide is generated
comprising determining a (putative) binding site for an SR
(Ser-Arg) protein in RNA of a dystrophin exon and producing an
oligonucleotide that is complementary to said RNA and that at least
partly overlaps said (putative) binding site. The term "at least
partly overlaps" is defined herein as to comprise an overlap of
only a single nucleotide of an SR binding site as well as multiple
nucleotides of said binding site as well as a complete overlap of
said binding site. This embodiment preferably further comprises
determining from a secondary structure of said RNA, a region that
is hybridised to another part of said RNA (closed structure) and a
region that is not hybridised in said structure (open structure),
and subsequently generating an oligonucleotide that at least partly
overlaps said (putative) binding site and that overlaps at least
part of said closed structure and overlaps at least part of said
open structure. In this way we increase the chance of obtaining an
oligonucleotide that is capable of interfering with the exon
inclusion from the pre-mRNA into mRNA. It is possible that a first
selected SR-binding region does not have the requested open-closed
structure in which case another (second) SR protein binding site is
selected which is then subsequently tested for the presence of an
open-closed structure. This process is continued until a sequence
is identified which contains an SR protein binding site as well as
a(n) (partly overlapping) open-closed structure. This sequence is
then used to design an oligonucleotide which is complementary to
said sequence.
[0050] Such a method, for generating an oligonucleotide, is also
performed by reversing the described order, i.e. first generating
an oligonucleotide comprising determining, from a secondary
structure of RNA from a dystrophin exon, a region that assumes a
structure that is hybridised to another part of said RNA (closed
structure) and a region that is not hybridised in said structure
(open structure), and subsequently generating an oligonucleotide,
of which at least a part of said oligonucleotide is complementary
to said closed structure and of which at least another part of said
oligonucleotide is complementary to said open structure. This is
then followed by determining whether an SR protein binding site at
least overlaps with said open/closed structure. In this way the
method of WO 2004/083432 is improved. In yet another embodiment the
selections are performed simultaneously.
[0051] Without wishing to be bound by any theory it is currently
thought that use of an oligonucleotide directed to an SR protein
binding site results in (at least partly) impairing the binding of
an SR protein to the binding site of an SR protein which results in
disrupted or impaired splicing.
[0052] Preferably, an open/closed structure and an SR protein
binding site partly overlap and even more preferred an open/closed
structure completely overlaps an SR protein binding site or an SR
protein binding site completely overlaps an open/closed structure.
This allows for an improved disruption of exon inclusion.
[0053] Besides consensus splice sites sequences, many (if not all)
exons contain splicing regulatory sequences such as exonic splicing
enhancer (ESE) sequences to facilitate the recognition of genuine
splice sites by the spliceosome.sup.42, 43. A subgroup of splicing
factors, called the SR proteins, can bind to these ESEs and recruit
other splicing factors, such as U1 and U2AF to (weakly defined)
splice sites. The binding sites of the four most abundant SR
proteins (SF2/ASF, SC35, SRp40 and SRp55) have been analyzed in
detail and these results are implemented in ESE finder, a web
source that predicts potential binding sites for these SR
proteins.sup.42, 43. There is a correlation between the
effectiveness of an AON and the presence/absence of an SF2/ASF,
SC35 and SRp40 binding site. In a preferred embodiment, the
invention thus provides a combination as described above, wherein
said SR protein is SF2/ASF or SC35 or SRp40.
[0054] In one embodiment an oligonucleotide, or a functional
equivalent thereof is capable of specifically binding a regulatory
RNA sequence which is required for the correct splicing of a
dystrophin exon in a transcript. Several cis-acting RNA sequences
are required for the correct splicing of exons in a transcript. In
particular, supplementary elements such as intronic or exonic
splicing enhancers (ISEs and ESEs) or silencers (ISSs and ESEs) are
identified to regulate specific and efficient splicing of
constitutive and alternative exons. Using sequence-specific
antisense oligonucleotides (AONs) that bind to the elements, their
regulatory function is disturbed so that the exon is skipped, as
shown for DMD. Hence, in one preferred embodiment an
oligonucleotide or functional equivalent thereof is used which is
complementary to an intronic splicing enhancer (ISE), an exonic
splicing enhancer (ESE), an intronic splicing silencer (ISS) and/or
an exonic splicing silencer (ESS). As already described herein
before, a dystrophin exon is in one preferred embodiment skipped by
an agent capable of specifically inhibiting an exon inclusion
signal of said exon, so that said exon is not recognized by the
splicing machinery as a part that needs to be included in the mRNA.
As a result, a mRNA without said exon is formed.
[0055] An AON used herein is preferably complementary to a
consecutive part or a contiguous stretch of between 8 and 50
nucleotides of dystrophin exon RNA or dystrophin intron RNA. In one
embodiment an AON used herein is complementary to a consecutive
part or a contiguous stretch of between 14 and 50 nucleotides of a
dystrophin exon RNA or dystrophin intron RNA. Preferably, said AON
is complementary to a consecutive part or contiguous stretch of
between 14 and 25 nucleotides of said exon RNA. More preferably, an
AON is used which comprises a sequence which is complementary to a
consecutive part or a contiguous stretch of between 20 and 25
nucleotides of a dystrophin exon RNA or a dystrophin intron
RNA.
[0056] Different types of nucleic acid may be used to generate an
oligonucleotide. Preferably, said oligonucleotide comprises RNA, as
RNA/RNA hybrids are very stable. Since one of the aims of the exon
skipping technique is to direct splicing in subjects it is
preferred that the oligonucleotide RNA comprises a modification
providing the RNA with an additional property, for instance
resistance to endonucleases, exonucleases, and RNaseH, additional
hybridisation strength, increased stability (for instance in a
bodily fluid), increased or decreased flexibility, reduced
toxicity, increased intracellular transport, tissue-specificity,
etc. Preferably, said modification comprises a
2'-O-methyl-phosphorothioate oligoribonucleotide modification.
Preferably, said modification comprises a
2'-O-methyl-phosphorothioate oligodeoxyribonucleotide modification.
One embodiment thus provides an oligonucleotide is used which
comprises RNA which contains a modification, preferably a
2'-O-methyl modified ribose (RNA) or deoxyribose (DNA)
modification.
[0057] In one embodiment the invention provides a hybrid
oligonucleotide comprising an oligonucleotide comprising a
2'-O-methyl-phosphorothioate oligo(deoxy)ribonucleotide
modification and locked nucleic acid. This particular
oligonucleotide comprises better sequence specificity compared to
an equivalent consisting of locked nucleic acid, and comprises
improved effectively when compared with an oligonucleotide
consisting of 2'-O-methyl-phosphorothioate
oligo(deoxy)ribonucleotide modification.
[0058] With the advent of nucleic acid mimicking technology it has
become possible to generate molecules that have a similar,
preferably the same hybridisation characteristics in kind not
necessarily in amount as nucleic acid itself. Such functional
equivalents are of course also suitable for use in the invention.
Preferred examples of functional equivalents of an oligonucleotide
are peptide nucleic acid and/or locked nucleic acid. Most
preferably, a morpholino phosphorodiamidate is used. Suitable but
non-limiting examples of equivalents of oligonucleotides of the
invention can be found in.sup.44-50. Hybrids between one or more of
the equivalents among each other and/or together with nucleic acid
are of course also suitable. In a preferred embodiment locked
nucleic acid is used as a functional equivalent of an
oligonucleotide, as locked nucleic acid displays a higher target
affinity and reduced toxicity and therefore shows a higher
efficiency of exon skipping.
[0059] In one embodiment an oligonucleotide, or a functional
equivalent thereof, which is capable of inhibiting inclusion of a
dystrophin exon into dystrophin mRNA is combined with at least one
other oligonucleotide, or functional equivalent thereof, that is
capable of inhibiting inclusion of another dystrophin exon into
dystrophin mRNA. This way, inclusion of two or more exons of a
dystrophin pre-mRNA in mRNA produced from this pre-mRNA is
prevented. This embodiment is further referred to as double- or
multi-exon skipping.sup.2, 15. In most cases double-exon skipping
results in the exclusion of only the two targeted exons from the
dystrophin pre-mRNA. However, in other cases it was found that the
targeted exons and the entire region in between said exons in said
pre-mRNA were not present in the produced mRNA even when other
exons (intervening exons) were present in such region. This
multi-exon skipping was notably so for the combination of
oligonucleotides derived from the DMD gene, wherein one
oligonucleotide for exon 45 and one oligonucleotide for exon 51 was
added to a cell transcribing the DMD gene. Such a set-up resulted
in mRNA being produced that did not contain exons 45 to 51.
Apparently, the structure of the pre-mRNA in the presence of the
mentioned oligonucleotides was such that the splicing machinery was
stimulated to connect exons 44 and 52 to each other. Other
preferred examples of multi-exon skipping are:
[0060] the use of an oligonucleotide targeting exon 17, and a
second one exon 48 which may result in the skipping of said both
exons or of the entire region between exon 17 and exon 48.
[0061] the use of an oligonucleotide targeting exon 17, and a
second one exon 51 which may result in the skipping of said both
exons or of the entire region between exon 17 and exon 51.
[0062] the use of an oligonucleotide targeting exon 42, and a
second one exon 55 which may result in the skipping of said both
exons or of the entire region between exon 42 and exon 55.
[0063] the use of an oligonucleotide targeting exon 43, and a
second one exon 51 which may result in the skipping of said both
exons or of the entire region between exon 43 and exon 51.
[0064] the use of an oligonucleotide targeting exon 43, and a
second one exon 55 which may result in the skipping of said both
exons or of the entire region between exon 43 and exon 55.
[0065] the use of an oligonucleotide targeting exon 45, and a
second one exon 55 which may result in the skipping of said both
exons or of the entire region between exon 45 and exon 55.
[0066] the use of an oligonucleotide targeting exon 45, and a
second one exon 59 which may result in the skipping of said both
exons or of the entire region between exon 45 and exon 59.
[0067] the use of an oligonucleotide targeting exon 48, and a
second one exon 59 which may result in the skipping of said both
exons or of the entire region between exon 48 and exon 59.
[0068] the use of an oligonucleotide targeting exon 50, and a
second one exon 51 which may result in the skipping of said both
exons.
[0069] the use of an oligonucleotide targeting exon 51, and a
second one exon 52 which may result in the skipping of said both
exons.
[0070] Further provided is therefore an oligonucleotide which
comprises at least 8, preferably between 16 to 80, consecutive
nucleotides that are complementary to a first exon of a dystrophin
pre-mRNA and wherein a nucleotide sequence is used which comprises
at least 8, preferably between 16 to 80, consecutive nucleotides
that are complementary to a second exon of said dystrophin
pre-mRNA. Said first and said second exon may be the same.
[0071] In one preferred embodiment said first and said second exon
are separated in said dystrophin pre-mRNA by at least one exon to
which said oligonucleotide is not complementary. Alternatively,
said first and said second exon are adjacent.
[0072] It is possible to specifically promote the skipping of also
the intervening exons by providing a linkage between the two
complementary oligonucleotides. Hence, in one embodiment stretches
of nucleotides complementary to at least two dystrophin exons are
separated by a linking moiety. The at least two stretches of
nucleotides are thus linked in this embodiment so as to form a
single molecule. Further provided is therefore an oligonucleotide,
or functional equivalent thereof which is complementary to at least
two exons in a dystrophin pre-mRNA, said oligonucleotide or
functional equivalent comprising at least two parts wherein a first
part comprises an oligonucleotide having at least 8, preferably
between 16 to 80, consecutive nucleotides that are complementary to
a first of said at least two exons and wherein a second part
comprises an oligonucleotide having at least 8, preferably between
16 to 80, consecutive nucleotides that are complementary to a
second exon in said dystrophin pre-mRNA. The linkage may be through
any means, but is preferably accomplished through a nucleotide
linkage. In the latter case, the number of nucleotides that do not
contain an overlap between one or the other complementary exon can
be zero, but is preferably between 4 to 40 nucleotides. The linking
moiety can be any type of moiety capable of linking
oligonucleotides. Preferably, said linking moiety comprises at
least 4 uracil nucleotides. Currently, many different compounds are
available that mimic hybridisation characteristics of
oligonucleotides. Such a compound, called herein a functional
equivalent of an oligonucleotide, is also suitable for the present
invention if such equivalent comprises similar hybridisation
characteristics in kind not necessarily in amount. Suitable
functional equivalents are mentioned earlier in this description.
As mentioned, oligonucleotides of the invention do not have to
consist of only oligonucleotides that contribute to hybridisation
to the targeted exon. There may be additional material and/or
nucleotides added.
[0073] The DMD gene is a large gene, with many different exons.
Considering that the gene is located on the X-chromosome, it is
mostly boys that are affected, although girls can also be affected
by the disease, as they may receive a bad copy of the gene from
both parents, or are suffering from a particularly biased
inactivation of the functional allele due to a particularly biased
X chromosome inactivation in their muscle cells. The protein is
encoded by a plurality of exons (79) over a range of at least 2.4
Mb. Defects may occur in any part of the DMD gene. Skipping of a
particular exon or particular exons can, very often, result in a
restructured mRNA that encodes a shorter than normal but at least
partially functional dystrophin protein. A practical problem in the
development of a medicament based on exon-skipping technology is
the plurality of mutations that may result in a deficiency in
functional dystrophin protein in the cell. Despite the fact that
already multiple different mutations can be corrected for by the
skipping of a single exon, this plurality of mutations, requires
the generation of a series of different pharmaceuticals as for
different mutations different exons need to be skipped. An
advantage of an oligonucleotide or of a composition comprising at
least two distinct oligonucleotide as later defined herein capable
of inducing skipping of two or more exons, is that more than one
exon can be skipped with a single pharmaceutical. This property is
not only practically very useful in that only a limited number of
pharmaceuticals need to be generated for treating many different
DMD or particular, severe BMD mutations. Another option now open to
the person skilled in the art is to select particularly functional
restructured dystrophin proteins and produce compounds capable of
generating these preferred dystrophin proteins. Such preferred end
results are further referred to as mild phenotype dystrophins.
[0074] Dose ranges of oligonucleotide according to the invention
are preferably designed on the basis of rising dose studies in
clinical trials (in vivo use) for which rigorous protocol
requirements exist. A molecule or an oligonucleotide as defined
herein may be used at a dose which is ranged between 0.1 and 20
mg/kg, preferably 0.5 and 10 mg/kg.
[0075] In a preferred embodiment, a concentration of an
oligonucleotide as defined herein, which is ranged between 0.1 nM
and 1 .mu.M is used. Preferably, this range is for in vitro use in
a cellular model such as muscular cells or muscular tissue. More
preferably, the concentration used is ranged between 0.3 to 400 nM,
even more preferably between 1 to 200 nM. If several
oligonucleotides are used, this concentration or dose may refer to
the total concentration or dose of oligonucleotides or the
concentration or dose of each oligonucleotide added.
[0076] The ranges of concentration or dose of oligonucleotide(s) as
given above are preferred concentrations or doses for in vitro or
ex vivo uses. The skilled person will understand that depending on
the oligonucleotide(s) used, the target cell to be treated, the
gene target and its expression levels, the medium used and the
transfection and incubation conditions, the concentration or dose
of oligonucleotide(s) used may further vary and may need to be
optimised any further.
[0077] An oligonucleotide as defined herein for use according to
the invention may be suitable for administration to a cell, tissue
and/or an organ in vivo of individuals affected by or at risk of
developing DMD or BMD, and may be administered in vivo, ex vivo or
in vitro. Said oligonucleotide may be directly or indirectly
administrated to a cell, tissue and/or an organ in vivo of an
individual affected by or at risk of developing DMD or BMD, and may
be administered directly or indirectly in vivo, ex vivo or in
vitro. As Duchenne and Becker muscular dystrophy have a pronounced
phenotype in muscle cells, it is preferred that said cells are
muscle cells, it is further preferred that said tissue is a
muscular tissue and/or it is further preferred that said organ
comprises or consists of a muscular tissue. A preferred organ is
the heart. Preferably, said cells comprise a gene encoding a mutant
dystrophin protein. Preferably, said cells are cells of an
individual suffering from DMD or BMD.
[0078] An oligonucleotide of the invention may be indirectly
administrated using suitable means known in the art. An
oligonucleotide may for example be provided to an individual or a
cell, tissue or organ of said individual in the form of an
expression vector wherein the expression vector encodes a
transcript comprising said oligonucleotide. The expression vector
is preferably introduced into a cell, tissue, organ or individual
via a gene delivery vehicle. In a preferred embodiment, there is
provided a viral-based expression vector comprising an expression
cassette or a transcription cassette that drives expression or
transcription of a molecule as identified herein. A preferred
delivery vehicle is a viral vector such as an adeno-associated
virus vector (AAV), or a retroviral vector such as a lentivirus
vector.sup.4, 51, 52 and the like. Also, plasmids, artificial
chromosomes, plasmids suitable for targeted homologous
recombination and integration in the human genome of cells may be
suitably applied for delivery of an oligonucleotide as defined
herein. Preferred for the current invention are those vectors
wherein transcription is driven from PolIII promoters, and/or
wherein transcripts are in the form fusions with U1 or U7
transcripts, which yield good results for delivering small
transcripts. It is within the skill of the artisan to design
suitable transcripts. Preferred are PolIII driven transcripts.
Preferably, in the form of a fusion transcript with an U1 or U7
transcript.sup.4, 51, 52. Such fusions may be generated as
described.sup.53, 54. The oligonucleotide may be delivered as is.
However, the oligonucleotide may also be encoded by the viral
vector. Typically, this is in the form of an RNA transcript that
comprises the sequence of the oligonucleotide in a part of the
transcript.
[0079] Improvements in means for providing an individual or a cell,
tissue, organ of said individual with an oligonucleotide and/or an
equivalent thereof, are anticipated considering the progress that
has already thus far been achieved. Such future improvements may of
course be incorporated to achieve the mentioned effect on
restructuring of mRNA using a method of the invention. An
oligonucleotide and/or an equivalent thereof can be delivered as is
to an individual, a cell, tissue or organ of said individual. When
administering an oligonucleotide and/or an equivalent thereof, it
is preferred that an oligonucleotide and/or an equivalent thereof
is dissolved in a solution that is compatible with the delivery
method. For intravenous, subcutaneous, intramuscular, intrathecal
and/or intraventricular administration it is preferred that the
solution is a physiological salt solution. Particularly preferred
in the invention is the use of an excipient that will aid in
delivery of each of the constituents as defined herein to a cell
and/or into a cell, preferably a muscle cell. Preferred are
excipients capable of forming complexes, nanoparticles, micelles,
vesicles and/or liposomes that deliver each constituent as defined
herein, complexed or trapped in a vesicle or liposome through a
cell membrane. Many of these excipients are known in the art.
Suitable excipients comprise polyethylenimine (PEI), or similar
cationic polymers, including polypropyleneimine or polyethylenimine
copolymers (PECs) and derivatives,
synthetic amphiphils (SAINT-18), Lipofectin.TM., DOTAP and/or viral
capsid proteins that are capable of self assembly into particles
that can deliver each constitutent as defined herein to a cell,
preferably a muscle cell. Such excipients have been shown to
efficiently deliver an oligonucleotide such as antisense nucleic
acids to a wide variety of cultured cells, including muscle cells.
Their high transfection potential is combined with an excepted low
to moderate toxicity in terms of overall cell survival. The ease of
structural modification can be used to allow further modifications
and the analysis of their further (in vivo) nucleic acid transfer
characteristics and toxicity.
[0080] Lipofectin represents an example of a liposomal transfection
agent. It consists of two lipid components, a cationic lipid
N-[1-(2,3 dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral
lipid dioleoylphosphatidylethanolamine (DOPE). The neutral
component mediates the intracellular release. Another group of
delivery systems are polymeric nanoparticles.
[0081] Polycations such like diethylaminoethylaminoethyl
(DEAE)-dextran, which are well known as DNA transfection reagent
can be combined with butylcyanoacrylate (PBCA) and
hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that
can deliver each constituent as defined herein, preferably an
oligonucleotide across cell membranes into cells.
[0082] In addition to these common nanoparticle materials, the
cationic peptide protamine offers an alternative approach to
formulate an oligonucleotide with colloids. This colloidal
nanoparticle system can form so called proticles, which can be
prepared by a simple self-assembly process to package and mediate
intracellular release of an oligonucleotide. The skilled person may
select and adapt any of the above or other commercially available
alternative excipients and delivery systems to package and deliver
an oligonucleotide for use in the current invention to deliver it
for the treatment of Duchenne Muscular Dystrophy or Becker Muscular
Dystrophy in humans.
[0083] In addition, an oligonucleotide could be covalently or
non-covalently linked to a targeting ligand specifically designed
to facilitate the uptake in to the cell, cytoplasm and/or its
nucleus. Such ligand could comprise (i) a compound (including but
not limited to peptide(-like) structures) recognising cell, tissue
or organ specific elements facilitating cellular uptake and/or (ii)
a chemical compound able to facilitate the uptake in to cells
and/or the intracellular release of an oligonucleotide from
vesicles, e.g. endosomes or lysosomes.
[0084] Therefore, in a preferred embodiment, an oligonucleotide is
formulated in a composition or a medicament or a composition, which
is provided with at least an excipient and/or a targeting ligand
for delivery and/or a delivery device thereof to a cell and/or
enhancing its intracellular delivery. Accordingly, the invention
also encompasses a pharmaceutically acceptable composition
comprising an oligonucleotide and further comprising at least one
excipient and/or a targeting ligand for delivery and/or a delivery
device of said oligonucleotide to a cell and/or enhancing its
intracellular delivery. It is to be understood that if a
composition comprises an additional constituent such as an adjunct
compound as later defined herein, each constituent of the
composition may not be formulated in one single combination or
composition or preparation. Depending on their identity, the
skilled person will know which type of formulation is the most
appropriate for each constituent as defined herein. In a preferred
embodiment, the invention provides a composition or a preparation
which is in the form of a kit of parts comprising an
oligonucleotide and a further adjunct compound as later defined
herein.
[0085] A preferred oligonucleotide is for preventing or treating
Duchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy
(BMD) in an individual. An individual, which may be treated using
an oligonucleotide of the invention may already have been diagnosed
as having a DMD or a BMD. Alternatively, an individual which may be
treated using an oligonucleotide of the invention may not have yet
been diagnosed as having a DMD or a BMD but may be an individual
having an increased risk of developing a DMD or a BMD in the future
given his or her genetic background. A preferred individual is a
human being.
Composition
[0086] In a further aspect, there is provided a composition
comprising an oligonucleotide as defined herein. Preferably, said
composition comprises at least two distinct oligonucleotide as
defined herein. More preferably, these two distinct
oligonucleotides are designed to skip distinct two or more exons as
earlier defined herein for multi-exon skipping.
[0087] In a preferred embodiment, said composition being preferably
a pharmaceutical composition said pharmaceutical composition
comprising a pharmaceutically acceptable carrier, adjuvant, diluent
and/or excipient. Such a pharmaceutical composition may comprise
any pharmaceutically acceptable carrier, filler, preservative,
adjuvant, solubilizer, diluent and/or excipient is also provided.
Such pharmaceutically acceptable carrier, filler, preservative,
adjuvant, solubilizer, diluent and/or excipient may for instance be
found in Remington: The Science and Practice of Pharmacy, 20th
Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000.
Each feature of said composition has earlier been defined
herein.
[0088] If several oligonucleotides are used, concentration or dose
already defined herein may refer to the total concentration or dose
of all oligonucleotides used or the concentration or dose of each
oligonucleotide used or added. Therefore in one embodiment, there
is provided a composition wherein each or the total amount of
oligonucleotide used is dosed in an amount ranged between 0.5 mg/kg
and 10 mg/kg.
[0089] A preferred composition additionally comprises: [0090] a) an
adjunct compound for reducing inflammation, preferably for reducing
muscle tissue inflammation, and/or [0091] b) an adjunct compound
for improving muscle fiber function, integrity and/or survival
and/or [0092] c) a compound exhibiting readthrough activity.
[0093] It has surprisingly been found that the skipping frequency
of a dystrophin exon from a pre-MRNA comprising said exon, when
using an oligonucleotide directed toward the exon or to one or both
splice sites of said exon, is enhanced if cells expressing said
pre-mRNA are also provided with an adjunct compound for reducing
inflammation, preferably for reducing muscle tissue inflammation,
and/or an adjunct compound for improving muscle fiber function,
integrity and/or survival. The enhanced skipping frequency also
increases the level of functional dystrophin protein produced in a
muscle cell of a DMD or BMD individual.
[0094] According to the present invention, even when a dystrophin
protein deficiency has been restored in a DMD patient by
administering an oligonucleotide of the invention, the presence of
tissue inflammation and damaged muscle cells still continues to
contribute to the symptoms of DMD. Hence, even though the cause of
DMD--i.e. a dysfunctional dystrophin protein--is alleviated,
treatment of DMD is still further improved by additionally using an
adjunct therapy according to the present invention. Furthermore,
the present invention provides the insight that a reduction of
inflammation does not result in significant reduction of AON uptake
by muscle cells. This is surprising because, in general,
inflammation enhances the trafficking of cells, blood and other
compounds. As a result, AON uptake/delivery is also enhanced during
inflammation. Hence, before the present invention it would be
expected that an adjunct therapy counteracting inflammation
involves the risk of negatively influencing AON therapy. This,
however, appears not to be the case.
[0095] An adjunct compound for reducing inflammation comprises any
therapy which is capable of at least in part reducing inflammation,
preferably inflammation caused by damaged muscle cells. Said
adjunct compound is most preferably capable of reducing muscle
tissue inflammation. Inflammation is preferably assessed by
detecting an increase in the number of infiltrating immune cells
such as neutrophils and/or mast cells and/or dendritic cells and/or
lymphocytes in muscle tissue suspected to be dystrophic. This
assessment is preferably carried out in cross-sections of a
biopsy.sup.57 of muscle tissue suspected to be dystrophic after
having specifically stained immune cells as identified above. The
quantification is preferably carried out under the microscope.
Reducing inflammation is therefore preferably assessed by detecting
a decrease in the number of immune cells in a cross-section of
muscle tissue suspected to be dystrophic. Detecting a decrease
preferably means that the number of at least one sort of immune
cells as identified above is decreased of at least 1%, 2%, 3%, 5%,
7%, 10%, 12%, 15%, 17%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or
more compared to the number of a corresponding immune cell in a
same individual before treatment. Most preferably, no infiltrating
immune cells are detected in cross-sections of said biopsy.
[0096] An adjunct compound for improving muscle fiber function,
integrity and/or survival comprises any therapy, which is capable
of measurably enhancing muscle fiber function, integrity and/or
survival as compared to an otherwise similar situation wherein said
adjunct compound is not present. The improvement of muscle fiber
function, integrity and/or survival may be assessed using at least
one of the following assays: a detectable decrease of creatine
kinase in blood, a detectable decrease of necrosis of muscle fibers
in a biopsy cross-section of a muscle suspected to be dystrophic,
and/or a detectable increase of the homogeneity of the diameter of
muscle fibers in a biopsy cross-section of a muscle suspected to be
dystrophic. Each of these assays is known to the skilled
person.
[0097] Creatine kinase may be detected in blood as described in 57.
A detectable decrease in creatine kinase may mean a decrease of 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the
concentration of creatine kinase in a same individual before
treatment.
[0098] A detectable decrease of necrosis of muscle fibers is
preferably assessed in a muscle biopsy, more preferably as
described in 57 using biopsy cross-sections. A detectable decrease
of necrosis may be a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90% or more of the area wherein necrosis has been
identified using biopsy cross-sections. The decrease is measured by
comparison to the necrosis as assessed in a same individual before
treatment.
[0099] A detectable increase of the homogeneity of the diameter of
a muscle fiber is preferably assessed in a muscle biopsy
cross-section, more preferably as described in 57.
[0100] In one embodiment, an adjunct compound for increasing
turnover of damaged muscle cells is used. An adjunct compound for
increasing turnover of damaged muscle cells comprises any therapy,
which is capable of at least in part inducing and/or increasing
turnover of damaged muscle cells. Damaged muscle cells are muscle
cells, which have significantly less clinically measurable
functionality than a healthy, intact muscle cell. In the absence of
dystrophin, mechanical stress leads to sarcolemmal ruptures,
causing an uncontrolled influx of calcium into the muscle fiber
interior, thereby triggering calcium-activated proteases and fiber
necrosis, resulting in damaged muscle cells. Increasing turnover of
damaged muscle cells means that damaged muscle cells are more
quickly broken down and/or removed as compared to a situation
wherein turnover of damaged muscle cells is not increased. Turnover
of damaged muscle cells is preferably assessed in a muscle biopsy,
more preferably as described in 57 using a cross-section of a
biopsy. A detectable increase of turnover may be an increase of 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the area
wherein turnover has been identified using a biopsy cross-section.
The increase is measured by comparison to the turnover as assessed
in a same individual before treatment.
[0101] Without wishing to be bound to theory, it is believed that
increasing turnover of muscle cells is preferred because this
reduces inflammatory responses.
[0102] According to the present invention, a composition of the
invention further comprising an adjunct therapy for reducing
inflammation, preferably for reducing muscle tissue inflammation in
an individual, is particularly suitable for use as a medicament.
Such composition is even better capable of alleviating one or more
symptom(s) of Duchenne Muscular Dystrophy or Becker Muscular
Dystrophy as compared to a combination not comprising said adjunct
compound. This embodiment also enhances the skipping frequency of a
dystrophin exon from a pre-mRNA comprising said exon, when using an
oligonucleotide directed toward the exon or to one or both splice
sites of said exon. The enhanced skipping frequency also increases
the level of functional dystrophin protein produced in a muscle
cell of a DMD or BMD individual.
[0103] Further provided is therefore a composition further
comprising an adjunct compound for reducing inflammation,
preferably for reducing muscle tissue inflammation in said
individual, for use as a medicament, preferably for treating or
preventing counteracting DMD. In one embodiment, said composition
is used in order to alleviate one or more symptom(s) of a severe
form of BMD wherein a very short dystrophin protein or altered or
truncated dystrophin mRNA or protein is formed which is not
sufficiently functional.
[0104] Preferred adjunct compound for reducing inflammation include
a steroid, a TNF.alpha. inhibitor, a source of mIGF-1 and/or an
antioxidant. However, any other compound able to reduce
inflammation as defined herein is also encompassed within the
present invention. Each of these compounds is later on extensively
presented. Each of the compounds extensively presented may be used
separately or in combination with each other and/or in combination
with one or more of the adjunct compounds used for improving muscle
fiber function, integrity and/or survival.
[0105] Furthermore, a composition comprising an adjunct therapy for
improving muscle fiber function, integrity and/or survival in an
individual is particularly suitable for use as a medicament,
preferably for treating or preventing DMD. Such composition is even
better capable of alleviating one or more symptom(s) of Duchenne
Muscular Dystrophy as compared to a composition not comprising said
adjunct compound.
[0106] Preferred adjunct compounds for improving muscle fiber
function, integrity and/or survival include an ion channel
inhibitor, a protease inhibitor, L-arginine and/or an angiotensin
II type I receptor blocker. However, any other compound able to
improving muscle fiber function, integrity and/or survival as
defined herein is also encompassed within the present invention.
Each of these compounds is later on extensively presented. Each of
the compounds extensively presented may be used separately or in
combination with each other and/or in combination with one or more
of the adjunct compounds used for reducing inflammation.
[0107] In a particularly preferred embodiment, a composition
further comprises a steroid. Such composition results in
significant alleviation of DMD symptoms. This embodiment also
enhances the skipping frequency of a dystrophin exon from a
pre-mRNA comprising said exon, when using an oligonucleotide
directed toward the exon or to one or both splice sites of said
exon. The enhanced skipping frequency also increases the level of
functional dystrophin protein produced in a muscle cell of a DMD or
BMD individual.
[0108] In one embodiment, said composition is used in order to
alleviate one or more symptom(s) of a severe form of BMD wherein a
very short dystrophin protein is formed which is not sufficiently
functional.
[0109] A steroid is a terpenoid lipid characterized by a carbon
skeleton with four fused rings, generally arranged in a 6-6-6-5
fashion. Steroids vary by the functional groups attached to these
rings and the oxidation state of the rings. Steroids include
hormones and drugs, which are usually used to relieve swelling and
inflammation, such as for instance prednisone, dexamethasone and
vitamin D.
[0110] According to the present invention, supplemental effects of
adjunct steroid therapy in DMD patients include reduction of tissue
inflammation, suppression of cytotoxic cells, and improved calcium
homeostasis. Most positive results are obtained in younger boys.
Preferably, the steroid is a corticosteroid, more preferably, a
glucocorticosteroid. Preferably, prednisone steroids such as
prednisone, prednizolone or deflazacort are used in a combination
according to the invention.sup.21. Dose ranges of steroid or of a
glucocorticosteroid to be used in the therapeutic applications as
described herein are designed on the basis of rising dose studies
in clinical trials for which rigorous protocol requirements exist.
The usual doses are 0.5-1.0 mg/kg/day, preferably 0.75 mg/kg/day
for prednisone and prednisolone, and 0.4-1.4 mg/kg/day, preferably
0.9 mg/kg/day for deflazacort.
[0111] In one embodiment, a steroid is administered to said
individual prior to administering a composition as earlier defined
herein. In this embodiment, it is preferred that said steroid is
administered at least one day, more preferred at least one week,
more preferred at least two weeks, more preferred at least three
weeks prior to administering said composition.
[0112] In another preferred embodiment, a combination further
comprises a tumour necrosis factor-alpha (TNF.alpha.) inhibitor.
Tumour necrosis factor-alpha (TNF.alpha.) is a pro-inflammatory
cytokine that stimulates the inflammatory response. Pharmacological
blockade of TNF.alpha. activity with the neutralising antibody
infliximab (Remicade) is highly effective clinically at reducing
symptoms of inflammatory diseases. In mdx mice, both infliximab and
etanercept delay and reduce the necrosis of dystrophic
muscle.sup.24, 25, with additional physiological benefits on muscle
strength, chloride channel function and reduced CK levels being
demonstrated in chronically treated exercised adult mdx
mice.sup.26. Such highly specific anti-inflammatory drugs designed
for use in other clinical conditions, are attractive alternatives
to the use of steroids for DMD. In one embodiment, the use of a
TNF.alpha. inhibitor is limited to periods of intensive muscle
growth in boys when muscle damage and deterioration are especially
pronounced.
[0113] A composition further comprising a TNF.alpha. inhibitor for
use as a medicament is also provided. In one embodiment, said
composition is used in order to alleviate one or more symptom(s) of
a severe form of BMD wherein a very short dystrophin protein is
formed which is not sufficiently functional. A preferred TNF.alpha.
inhibitor is a dimeric fusion protein consisting of the
extracellular ligand-binding domain of the human p75 receptor of
TNF.alpha. linked to the Fc portion of human IgG1. A more preferred
TNF.alpha. inhibitor is etanercept (Amgen, America).sup.26. The
usual doses of etanercept is about 0.2 mg/kg, preferably about 0.5
mg/kg twice a week. The administration is preferably
subcutaneous.
[0114] In another preferred embodiment, a composition of the
invention further comprises a source of mIGF-1. As defined herein,
a source of IGF-1 preferably encompasses mIGF-1 itself, a compound
able of enhancing mIGF-1 expression and/or activity. Enhancing is
herein synonymous with increasing. Expression of mIGF-1 is
synonymous with amount of mIGF-1. mIGF-1 promotes regeneration of
muscles through increase in satellite cell activity, and reduces
inflammation and fibrosis.sup.27. Local injury of muscle results in
increased mIGF-1 expression. In transgenic mice with extra IGF-1
genes, muscle hypertrophy and enlarged muscle fibers are
observed.sup.27. Similarly, transgenic mdx mice show reduced muscle
fiber degeneration.sup.28. Upregulation of the mIGF-1 gene and/or
administration of extra amounts of mIGF-1 protein or a functional
equivalent thereof (especially the mIGF-1 Ea isoform [as described
in 27, human homolog IGF-1 isoform 4: SEQ ID NO: 577]) thus
promotes the effect of other, preferably genetic, therapies for
DMD, including antisense-induced exon skipping. The additional
mIGF-1 levels in the above mentioned transgenic mice do not induce
cardiac problems nor promote cancer, and have no pathological side
effects. As stated before, the amount of mIGF-1 is for instance
increased by enhancing expression of the mIGF-1 gene and/or by
administration of mIGF-1 protein and/or a functional equivalent
thereof (especially the mIGF-1 Ea isoform [as described in 27,
human homolog IGF-1 isoform 4: SEQ ID NO: 577]). A composition of
the invention further preferably comprises mIGF-1, a compound
capable of enhancing mIGF-1 expression and/or an mIGF-1 activity,
for use as a medicament is also provided. Said medicament is
preferably for alleviating one or more symptom(s) of DMD. In one
embodiment, such composition is used in order to alleviate one or
more symptom(s) of a severe form of BMD wherein a very short
dystrophin protein is formed which is not sufficiently
functional.
[0115] Within the context of the invention, an increased amount or
activity of mIGF-1 may be reached by increasing the gene expression
level of an IGF-1 gene, by increasing the amount of a corresponding
IGF-1 protein and/or by increasing an activity of an IGF1-protein.
A preferred mIGF-1 protein has been earlier defined herein. An
increase of an activity of said protein is herein understood to
mean any detectable change in a biological activity exerted by said
protein or in the steady state level of said protein as compared to
said activity or steady-state in a individual who has not been
treated. Increased amount or activity of mIGF-1 is preferably
assessed by detection of increased expression of muscle hypertrophy
biomarker GATA-2 (as described in 27).
[0116] Gene expression level is preferably assessed using classical
molecular biology techniques such as (real time) PCR, arrays or
Northern analysis. A steady state level of a protein is determined
directly by quantifying the amount of a protein. Quantifying a
protein amount may be carried out by any known technique such as
Western blotting or immunoassay using an antibody raised against a
protein. The skilled person will understand that alternatively or
in combination with the quantification of a gene expression level
and/or a corresponding protein, the quantification of a substrate
of a corresponding protein or of any compound known to be
associated with a function or activity of a corresponding protein
or the quantification of said function or activity of a
corresponding protein using a specific assay may be used to assess
the alteration of an activity or steady state level of a
protein.
[0117] In the invention, an activity or steady-state level of a
said protein may be altered at the level of the protein itself,
e.g. by providing a protein to a cell from an exogenous source.
[0118] Preferably, an increase or an up-regulation of the
expression level of a said gene means an increase of at least 5% of
the expression level of said gene using arrays. More preferably, an
increase of the expression level of said gene means an increase of
at least 10%, even more preferably at least 20%, at least 30%, at
least 40%, at least 50%, at least 70%, at least 90%, at least 150%
or more. In another preferred embodiment, an increase of the
expression level of said protein means an increase of at least 5%
of the expression level of said protein using Western blotting
and/or using ELISA or a suitable assay. More preferably, an
increase of the expression level of a protein means an increase of
at least 10%, even more preferably at least 20%, at least 30%, at
least 40%, at least 50%, at least 70%, at least 90%, at least 150%
or more.
[0119] In another preferred embodiment, an increase of a
polypeptide activity means an increase of at least 5% of a
polypeptide activity using a suitable assay. More preferably, an
increase of a polypeptide activity means an increase of at least
10%, even more preferably at least 20%, at least 30%, at least 40%,
at least 50%, at least 70%, at least 90%, at least 150% or more.
The increase is preferably assessed by comparison to corresponding
activity in the individual before treatment.
[0120] A preferred way of providing a source of mIGF1 is to
introduce a transgene encoding mIGF1, preferably an mIGF-1 Ea
isoform (as described in 27, human homolog IGF-1 isoform 4: SEQ ID
NO: 577), more preferably in an AAV vector as later defined herein.
Such source of mIGF1 is specifically expressed in muscle tissue as
described in mice in 27.
[0121] In another preferred embodiment, a composition further
comprises an antioxidant. Oxidative stress is an important factor
in the progression of DMD and promotes chronic inflammation and
fibrosis.sup.29. The most prevalent products of oxidative stress,
the peroxidized lipids, are increased by an average of 35% in
Duchenne boys. Increased levels of the enzymes superoxide dismutase
and catalase reduce the excessive amount of free radicals causing
these effects. In fact, a dietary supplement Protandim.RTM.
(LifeVantage) was clinically tested and found to increase levels of
superoxide dismutase (up to 30%) and catalase (up to 54%), which
indeed significantly inhibited the peroxidation of lipids in 29
healthy persons.sup.30. Such effective management of oxidative
stress thus preserves muscle quality and so promotes the positive
effect of DMD therapy. Idebenone is another potent antioxidant with
a chemical structure derived from natural coenzyme Q10. It protects
mitochondria where adenosine triphosphate, ATP, is generated by
oxidative phosphorylation. The absence of dystrophin in DMD
negatively affects this process in the heart, and probably also in
skeletal muscle. Idebenone was recently applied in clinical trials
in the US and Europe demonstrating efficacy on neurological aspects
of Friedreich's Ataxia.sup.31. A phase-IIa double-blind,
placebo-controlled randomized clinical trial with Idebenone has
recently been started in Belgium, including 21 Duchenne boys at 8
to 16 years of age. The primary objective of this study is to
determine the effect of Idebenone on heart muscle function. In
addition, several different tests will be performed to detect the
possible functional benefit on muscle strength in the patients.
When effective, Idebenone is a preferred adjunct compound for use
in a combination according to the present invention in order to
enhance the therapeutic effect of DMD therapy, especially in the
heart. A composition further comprising an antioxidant for use as a
medicament is also provided. Said medicament is preferably for
alleviating one or more symptom(s) of DMD. In one embodiment, said
composition is used in order to alleviate one or more symptom(s) of
a severe form of BMD wherein a very short dystrophin protein is
formed which is not sufficiently functional. Depending on the
identity of the antioxidant, the skilled person will know which
quantities are preferably used. An antioxidant may include
bacoside, silymarin, curcumin and/or a polyphenol. Preferably, a
polyphenol is or comprises epigallocatechin-3-gallate (EGCG).
Preferably, an antioxidant is a mixture of antioxidants as the
dietary supplement Protandim.RTM. (LifeVantage). A daily capsule of
675 mg of Protandim.RTM. comprises 150 mg of B. monniera (45%
bacosides), 225 mg of S. marianum (70-80% silymarin), 150 mg of W.
somnifera powder, 75 mg green tea (98% polyphenols wherein 45%
EGCG) and 75 mg turmeric (95% curcumin).
[0122] In another preferred embodiment, a composition further
comprises an ion channel inhibitor. The presence of damaged muscle
membranes in DMD disturbs the passage of calcium ions into the
myofibers, and the consequently disrupted calcium homeostasis
activates many enzymes, e.g. proteases, that cause additional
damage and muscle necrosis. Ion channels that directly contribute
to the pathological accumulation of calcium in dystrophic muscle
are potential targets for adjunct compounds to treat DMD. There is
evidence that some drugs, such as pentoxifylline, block
exercise-sensitive calcium channels.sup.32 and antibiotics that
block stretch activated channels reduce myofibre necrosis in mdx
mice and CK levels in DMD boys.sup.33. A composition further
comprising an ion channel inhibitor for use as a medicament is also
provided. Said medicament is preferably for alleviating one or more
symptom(s) of DMD. In one embodiment, said composition is used in
order to alleviate one or more symptom(s) of a severe form of BMD
wherein a very short dystrophin protein is formed which is not
sufficiently functional.
[0123] Preferably, an ion channel inhibitor of the class of
xanthines is used. More preferably, said xanthines are derivatives
of methylxanthines, and most preferably, said methylxanthine
derivates are chosen from the group consisting of pentoxifylline,
furafylline, lisofylline, propentofylline, pentifylline,
theophylline, torbafylline, albifylline, enprofylline and
derivatives thereof. Most preferred is the use of pentoxifylline.
Ion channel inhibitors of the class of xanthines enhance the
skipping frequency of a dystrophin exon from a pre-mRNA comprising
said exon, when using an oligonucleotide directed toward the exon
or to one or both splice sites of said exon. The enhanced skipping
frequency also increases the level of functional dystrophin protein
produced in a muscle cell of a DMD or BMD individual.
[0124] Depending on the identity of the ion channel inhibitor, the
skilled person will know which quantities are preferably used.
Suitable dosages of pentoxifylline are between 1 mg/kg/day to 100
mg/kg/day, preferred dosages are between 10 mg/kg/day to 50
mg/kg/day. Typical dosages used in humans are 20 mg/kg/day.
[0125] In one embodiment, an ion channel inhibitor is administered
to said individual prior to administering a composition comprising
an oligonucleotide. In this embodiment, it is preferred that said
ion channel inhibitor is administered at least one day, more
preferred at least one week, more preferred at least two weeks,
more preferred at least three weeks prior to administering a
composition comprising an oligonucleotide.
[0126] In another preferred embodiment, a composition further
comprises a protease inhibitor. Calpains are calcium-activated
proteases that are increased in dystrophic muscle and account for
myofiber degeneration. Calpain inhibitors such as calpastatin,
leupeptin.sup.34, calpeptin, calpain inhibitor III, or PD150606 are
therefore applied to reduce the degeneration process. A new
compound, BN 82270 (Ipsen) that has dual action as both a calpain
inhibitor and an antioxidant increased muscle strength, decreased
serum CK and reduced fibrosis of the mdx diaphragm, indicating a
therapeutic effect with this new compound.sup.35. Another compound
of Leupeptin/Carnitine (Myodur) has recently been proposed for
clinical trials in DMD patients.
[0127] MG132 is another proteasomal inhibitor that has shown to
reduce muscle membrane damage, and to ameliorate the
histopathological signs of muscular dystrophy.sup.36. MG-132
(CBZ-leucyl-leucyl-leucinal) is a cell-permeable, proteasomal
inhibitor (Ki=4 nM), which inhibits NFkappaB activation by
preventing IkappaB degradation (IC50=3 .mu.M). In addition, it is a
peptide aldehyde that inhibits ubiquitin-mediated proteolysis by
binding to and inactivating 20S and 26S proteasomes. MG-132 has
shown to inhibit the proteasomal degradation of
dystrophin-associated proteins in the dystrophic mdx mouse
model.sup.36. This compound is thus also suitable for use as an
adjunct pharmacological compound for DMD. A composition further
comprising a protease inhibitor for use as a medicament is also
provided. Said medicament is preferably for alleviating one or more
symptom(s) of DMD. In one embodiment, said combination is used in
order to alleviate one or more symptom(s) of a severe form of BMD
wherein a very short dystrophin protein is formed which is not
sufficiently functional. Depending on the identity of the protease
inhibitor, the skilled person will know which quantities are
preferably used.
[0128] In another preferred embodiment, a composition further
comprises L-arginine. Dystrophin-deficiency is associated with the
loss of the DGC-complex at the fiber membranes, including neuronal
nitric oxide synthase (nNOS). Expression of a nNOS transgene in mdx
mice greatly reduced muscle membrane damage. Similarly,
administration of L-arginine (the substrate for nitric oxide
synthase) increased NO production and upregulated utrophin
expression in mdx mice. Six weeks of L-arginine treatment improved
muscle pathology and decreased serum CK in mdx mice.sup.37. The use
of L-arginine as a further constituent in a composition of the
invention has not been disclosed.
[0129] A composition further comprising L-arginine for use as a
medicament is also provided. Said medicament is preferably for
alleviating one or more symptom(s) of DMD. In one embodiment, said
composition is used in order to alleviate one or more symptom(s) of
a severe form of BMD wherein a very short dystrophin protein is
formed which is not sufficiently functional.
[0130] In another preferred embodiment, a composition further
comprises angiotensin II type 1 receptor blocker Losartan, which
normalizes muscle architecture, repair and function, as shown in
the dystrophin-deficient mdx mouse model.sup.23. A composition
further comprising angiotensin II type 1 receptor blocker Losartan
for use as a medicament is also provided. Said medicament is
preferably for alleviating one or more symptom(s) of DMD. In one
embodiment, said composition is used in order to alleviate one or
more symptom(s) of a severe form of BMD wherein a very short
dystrophin protein is formed which is not sufficiently functional.
Depending on the identity of the angiotensin II type 1 receptor
blocker, the skilled person will know which quantities are
preferably used.
[0131] In another preferred embodiment, a composition further
comprises an angiotensin-converting enzyme (ACE) inhibitor,
preferably perindopril. ACE inhibitors are capable of lowering
blood pressure. Early initiation of treatment with perindopril is
associated with a lower mortality in DMD patients.sup.22. A
composition further comprising an ACE inhibitor, preferably
perindopril for use as a medicament is also provided. Said
medicament is preferably for alleviating one or more symptom(s) of
DMD. In one embodiment, said composition is used in order to
alleviate one or more symptom(s) of a severe form of BMD wherein a
very short dystrophin protein is formed which is not sufficiently
functional. The usual doses of an ACE inhibitor, preferably
perindopril are about 2 to 4 mg/day.sup.22. In a more preferred
embodiment, an ACE inhibitor is combined with at least one of the
previously identified adjunct compounds.
[0132] In another preferred embodiment, a composition further
comprises a compound exhibiting a readthrough activity. A compound
exhibiting a readthrough activity may be any compound, which is
able to suppress a stop codon. For 20% of DMD patients, the
mutation in the dystrophin gene is comprising a point mutation, of
which 13% is a nonsense mutation. A compound exhibiting a
readthrough activity or which is able to suppress a stop codon is a
compound which is able to provide an increased amount of a
functional dystrophin mRNA or protein and/or a decreased amount of
an aberrant or truncated dystrophin mRNA or protein. Increased
preferably means increased of at least 1%, 2%, 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 100% or more. Decreased preferably means decreased of at
least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more. An increase
or a decrease of said protein is preferably assessed in a muscular
tissue or in a muscular cell of an individual by comparison to the
amount present in said individual before treatment with said
compound exhibiting a readthrough activity. Alternatively, the
comparison can be made with a muscular tissue or cell of said
individual, which has not yet been treated with said compound in
case the treatment is local. The assessment of an amount at the
protein level is preferably carried out using western blot
analysis.
[0133] Preferred compounds exhibiting a readthrough activity
comprise or consist of aminoglycosides, including, but not limited
to, geneticin (G418), paromomycin, gentamycin and/or
3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid), and
derivatives thereof (references 64, 65). A more preferred compound
exhibiting a readthrough activity comprises or consists of
PTC124.TM., and/or a functional equivalent thereof. PTC124.TM. is a
registered trademark of PTC Therapeutics, Inc. South Plainfield,
N.J. 3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) also
known as PTC124.TM. (references 16, 17) belongs to a new class of
small molecules that mimics at lower concentrations the readthrough
activity of gentamicin (reference 55). A functional equivalent of
3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) or of
gentamicin is a compound which is able to exhibit a readthrough
activity as earlier defined herein. Most preferably, a compound
exhibiting a readthrough activity comprises or consists of
gentamycin and/or
3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) also known
as PTC124.TM.. A composition further comprising a compound
exhibiting a readthrough activity, preferably comprising or
consisting of gentamycin and/or
3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) for use as
a medicament is also provided. Said medicament is preferably for
alleviating one or more symptom(s) of DMD. In one embodiment, said
composition is used in order to alleviate one or more symptom(s) of
a severe form of BMD wherein a very short dystrophin protein is
formed which is not sufficiently functional. The usual doses of a
compound exhibiting a readthrough activity, preferably
3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)benzoic acid) or of
gentamicin are ranged between 3 mg/kg/day to 200 mg/kg/day,
preferred dosages are between 10 mg/kg to 50 mg/kg per day or twice
a day.
[0134] In a more preferred embodiment, a compound exhibiting a
readthrough activity is combined with at least one of the
previously identified adjunct compounds.
[0135] In another preferred embodiment, a composition further
comprises a compound, which is capable of enhancing exon skipping
and/or inhibiting spliceosome assembly and/or splicing. Small
chemical compounds, such as for instance specific indole
derivatives, have been shown to selectively inhibit spliceosome
assembly and splicing.sup.38, for instance by interfering with the
binding of serine- and arginine-rich (SR) proteins to their cognate
splicing enhancers (ISEs or ESEs) and/or by interfering with the
binding of splicing repressors to silencer sequences (ESSs or
ISSs). These compounds are therefore suitable for applying as
adjunct compounds that enhance exon skipping. A composition further
comprising a compound for enhancing exon skipping and/or inhibiting
spliceosome assembly and/or splicing for use as a medicament is
also provided. Said medicament is preferably for alleviating one or
more symptom(s) of DMD. In one embodiment, said composition is used
in order to alleviate one or more symptom(s) of a severe form of
BMD wherein a very short dystrophin protein is formed which is not
sufficiently functional. Depending on the identity of the compound,
which is capable of enhancing exon skipping and/or inhibiting
spliceosome assembly and/or splicing, the skilled person will know
which quantities are preferably used. In a more preferred
embodiment, a compound for enhancing exon skipping and/or
inhibiting spliceosome assembly and/or splicing is combined with a
ACE inhibitor and/or with any adjunct compounds as identified
earlier herein.
[0136] The invention thus provides a composition further comprising
an adjunct compound, wherein said adjunct compound comprises a
steroid, an ACE inhibitor (preferably perindopril), angiotensin II
type 1 receptor blocker Losartan, a tumour necrosis factor-alpha
(TNF.alpha.) inhibitor, a source of mIGF-1, preferably mIGF-1, a
compound for enhancing mIGF-1 expression, a compound for enhancing
mIGF-1 activity, an antioxidant, an ion channel inhibitor, a
protease inhibitor, L-arginine, a compound exhibiting a readthrough
activity and/or inhibiting spliceosome assembly and/or
splicing.
[0137] In one embodiment an individual is further provided with a
functional dystrophin protein using a vector, preferably a viral
vector, comprising a micro-mini-dystrophin gene. Most preferably, a
recombinant adeno-associated viral (rAAV) vector is used. AAV is a
single-stranded DNA parvovirus that is non-pathogenic and shows a
helper-dependent life cycle. In contrast to other viruses
(adenovirus, retrovirus, and herpes simplex virus), rAAV vectors
have demonstrated to be very efficient in transducing mature
skeletal muscle. Application of rAAV in classical DMD "gene
addition" studies has been hindered by its restricted packaging
limits (<5 kb). Therefore, rAAV is preferably applied for the
efficient delivery of a much smaller micro- or mini-dystrophin
gene. Administration of such micro- or mini-dystrophin gene results
in the presence of an at least partially functional dystrophin
protein. Reference is made to.sup.18-20.
[0138] Each constituent of a composition can be administered to an
individual in any order. In one embodiment, each constituent is
administered simultaneously (meaning that each constituent is
administered within 10 hours, preferably within one hour). This is
however not necessary. In one embodiment at least one adjunct
compound is administered to an individual in need thereof before
administration of an oligonucleotide. Alternatively, an
oligonucleotide is administered to an individual in need thereof
before administration of at least one adjunct compound.
Use
[0139] In a further aspect, there is provided the use of a
oligonucleotide or of a composition as defined herein for the
manufacture of a medicament for preventing or treating Duchenne
Muscular Dystrophy or Becker Muscular Dystrophy in an individual.
Each feature of said use has earlier been defined herein.
[0140] A treatment in a use or in a method according to the
invention is at least one week, at least one month, at least
several months, at least one year, at least 2, 3, 4, 5, 6 years or
more. Each molecule or oligonucleotide or equivalent thereof as
defined herein for use according to the invention may be suitable
for direct administration to a cell, tissue and/or an organ in vivo
of individuals affected by or at risk of developing DMD or BMD, and
may be administered directly in vivo, ex vivo or in vitro. The
frequency of administration of an oligonucleotide, composition,
compound or adjunct compound of the invention may depend on several
parameters such as the age of the patient, the mutation of the
patient, the number of molecules (i.e. dose), the formulation of
said molecule. The frequency may be ranged between at least once in
two weeks, or three weeks or four weeks or five weeks or a longer
time period.
Method
[0141] In a further aspect, there is provided a method for
alleviating one or more symptom(s) of Duchenne Muscular Dystrophy
or Becker Muscular Dystrophy in an individual or alleviate one or
more characteristic(s) of a myogenic or muscle cell of said
individual, the method comprising administering to said individual
an oligonucleotide or a composition as defined herein.
[0142] There is further provided a method for enhancing, inducing
or promoting skipping of an exon from a dystrophin pre-mRNA in a
cell expressing said pre-mRNA in an individual suffering from
Duchenne Muscular Dystrophy or Becker Muscular Dystrophy, the
method comprising administering to said individual an
oligonucleotide or a composition as defined herein. Further
provided is a method for increasing the production of a functional
dystrophin protein and/or decreasing the production of an aberrant
dystrophin protein in a cell, said cell comprising a pre-mRNA of a
dystrophin gene encoding an aberrant dystrophin protein, the method
comprising providing said cell with an oligonucleotide or
composition of the invention and allowing translation of mRNA
produced from splicing of said pre-mRNA. In one embodiment, said
method is performed in vitro, for instance using a cell culture.
Preferably, said method is in vivo.
[0143] In this context, increasing the production of a functional
dystrophin protein has been earlier defined herein.
[0144] Unless otherwise indicated each embodiment as described
herein may be combined with another embodiment as described
herein.
[0145] In this document and in its claims, the verb "to comprise"
and its conjugations is used in its non-limiting sense to mean that
items following the word are included, but items not specifically
mentioned are not excluded. In addition the verb "to consist" may
be replaced by "to consist essentially of" meaning that a compound
or adjunct compound as defined herein may comprise additional
component(s) than the ones specifically identified, said additional
component(s) not altering the unique characteristic of the
invention.
[0146] In addition, reference to an element by the indefinite
article "a" or "an" does not exclude the possibility that more than
one of the element is present, unless the context clearly requires
that there be one and only one of the elements. The indefinite
article "a" or "an" thus usually means "at least one".
[0147] The word "approximately" or "about" when used in association
with a numerical value (approximately 10, about 10) preferably
means that the value may be the given value of 10 more or less 1%
of the value.
[0148] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety. Each embodiment as identified herein may be combined
together unless otherwise indicated.
[0149] The invention is further explained in the following
examples. These examples do not limit the scope of the invention,
but merely serve to clarify the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0150] FIG. 1. In human control myotubes, PS220 and PS305 both
targeting an identical sequence within exon 45, were directly
compared for relative skipping efficiencies. PS220 reproducibly
induced highest levels of exon 45 skipping (up to 73%), whereas
with PS305 maximum exon 45 skipping levels of up to 46% were
obtained. No exon 45 skipping was observed in non-treated cells.
(M: DNA size marker; NT: non-treated cells)
[0151] FIG. 2. Graph showing relative exon 45 skipping levels of
inosine-containing AONs as assessed by RT-PCR analysis. In human
control myotubes, a series of new AONs, all targeting exon 45 and
containing one inosine for guanosine substitution were tested for
relative exon 45 skipping efficiencies when compared with PS220 and
PS305 (see FIG. 1). All new inosine-containing AONs were effective,
albeit at variable levels (between 4% and 25%). PS220 induced
highest levels of exon 45 skipping (up to 72%), whereas with PS305
maximum exon 45 skipping levels of up to 63% were obtained. No exon
45 skipping was observed in non-treated cells. (M: DNA size marker;
NT: non-treated cells).
EXAMPLES
Example 1
Materials and Methods
[0152] AON design was based on (partly) overlapping open secondary
structures of the target exon RNA as predicted by the m-fold
program, on (partly) overlapping putative SR-protein binding sites
as predicted by the ESE-finder software. AONs were synthesized by
Prosensa Therapeutics B.V. (Leiden, Netherlands), and contain
2'-O-methyl RNA and full-length phosphorothioate (PS)
backbones.
Tissue Culturing, Transfection and RT-PCR Analysis
[0153] Myotube cultures derived from a healthy individual ("human
control") (examples 1, 3, and 4; exon 43, 50, 52 skipping) or a DMD
patient carrying an exon 45 deletion (example 2; exon 46 skipping)
were processed as described previously (Aartsma-Rus et al.,
Neuromuscul. Disord. 2002; 12: S71-77 and Hum Mol Genet 2003;
12(8): 907-14). For the screening of AONs, myotube cultures were
transfected with 200 nM for each AON (PS220 and PS305).
Transfection reagent UNIFectylin (Prosensa Therapeutics BV,
Netherlands) was used, with 2 .mu.l UNIFectylin per .mu.g AON. Exon
skipping efficiencies were determined by nested RT-PCR analysis
using primers in the exons flanking the targeted exon 45. PCR
fragments were isolated from agarose gels for sequence
verification. For quantification, the PCR products were analyzed
using the DNA 1000 LabChip Kit on the Agilent 2100 bioanalyzer
(Agilent Technologies, USA).
Results
[0154] DMD exon 45 skipping.
[0155] Two AONs, PS220 (SEQ ID NO: 76;
5'-UUUGCCGCUGCCCAAUGCCAUCCUG-3') and PS305 (SEQ ID NO: 557;
5'-UUUGCCICUGCCCAAUGCCAUCCUG-3') both targeting an identical
sequence within exon 45, were directly compared for relative
skipping efficiencies in healthy control myotube cultures.
Subsequent RT-PCR and sequence analysis of isolated RNA
demonstrated that both AONs were indeed capable of inducing exon 45
skipping. PS220, consisting a GCCGC stretch, reproducibly induced
highest levels of exon 45 skipping (up to 73%), as shown in FIG. 1.
However, PS305, which is identical to PS220 but containing an
inosine for a G substitution at position 4 within that stretch is
also effective and leading to exon 45 skipping levels of up to 46%.
No exon 45 skipping was observed in non-treated cells (NT).
Example 2
Materials and Methods
[0156] AON design was based on (partly) overlapping open secondary
structures of the target exon 45 RNA as predicted by the m-fold
program, on (partly) overlapping putative SR-protein binding sites
as predicted by the ESE-finder software. AONs were synthesized by
Prosensa Therapeutics B.V. (Leiden, Netherlands), and contain
2'-O-methyl RNA, full-length phosphorothioate (PS) backbones and
one inosine for guanosine substitution.
Tissue Culturing, Transfection and RT-PCR Analysis
[0157] Myotube cultures derived from a healthy individual ("human
control") were processed as described previously (Aartsma-Rus et
al., Neuromuscul. Disord. 2002; 12: S71-77 and Hum Mol Genet 2003;
12(8): 907-14). For the screening of AONs, myotube cultures were
transfected with 200 nM for each AON. Transfection reagent
UNIFectylin (Prosensa Therapeutics BV, Netherlands) was used, with
2 .mu.l UNIFectylin per .mu.g AON. Exon skipping efficiencies were
determined by nested RT-PCR analysis using primers in the exons
flanking the targeted exon 45. PCR fragments were isolated from
agarose gels for sequence verification. For quantification, the PCR
products were analyzed using the DNA 1000 LabChip Kit on the
Agilent 2100 bioanalyzer (Agilent Technologies, USA).
Results
[0158] DMD exon 45 skipping.
[0159] An additional series of AONs targeting exon 45 and
containing one inosine-substitution were tested in healthy control
myotube cultures for exon 45 skipping efficiencies, and directly
compared to PS220 (without inosine; SEQ ID NO: 76)) and PS305
(identical sequence as PS220 but with inosine substitution; SEQ ID
NO: 557). Subsequent RT-PCR and sequence analysis of isolated RNA
demonstrated that all new AONs (PS309 to PS316) were capable of
inducing exon 45 skipping between 4% (PS311) and 25% (PS310) as
shown in FIG. 2. When compared to PS220 and PS305, PS220 induced
highest levels of exon 45 skipping (up to 72%). Of the new
inosine-containing AONs PS305 was most effective, showing exon 45
skipping levels of up to 63%. No exon 45 skipping was observed in
non-treated cells (NT).
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TABLE-US-00001 [0225] Sequence listing DMD gene amino acid sequence
SEQ ID NO 1:
MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGL
TGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIIL
HWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLALNALIH
SHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYIT
SLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSP
KPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEV
LSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGYMMDLTAHQGRVGNILQLGSKLIGT
GKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELNDWLT
KTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQVRVNSLTHMVVVVDESSG
DHATAALEEQLKVLGDRWANICRWTEDRWVLLQDILLKWQRLTEEQCLFSAWLSEKE
DAVNKIHTTGFKDQNEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVT
QKTEAWLDNFARCWDNLVQKLEKSTAQISQAVTTTQPSLTQTTVMETVTTVTTREQILV
KHAQEELPPPPPQKKRQITVDSEIRKRLDVDITELHSWITRSEAVLQSPEFAIFRKEGNF
SDLKEKVNAIEREKAEKFRKLQDASRSAQALVEQMVNEGVNADSIKQASEQLNSRWIE
FCQLLSERLNWLEYQNNHAFYNQLQQLEQMTTTAENWLKIQPTTPSEPTAIKSQLKIC
KDEVNRLSGLQPQIERLKIQSIALKEKGQGPMFLDADFVAFTNHFKQVFSDVQAREKEL
QTIFDTLPPMRYQETMSAIRTWVQQSETKLSIPQLSVTDYEIMEQRLGELQALQSSLQE
QQSGLYYLSTTVKEMSKKAPSEISRKYQSEFEEIEGRWKKLSSQLVEHCQKLEEQMNK
LRKIQNHIQTLKKWMAEVDVFLKEEWPALGDSEILKKQLKQCRLLVSDIQTIQPSLNSV
NEGGQKIKNEAEPEFASRLETELKELNTQWDHMCQQVYARKEALKGGLEKTVSLQKD
LSEMHEWMTQAEEEYLERDFEYKTPDELQKAVEEMKRAKEEAQQKEAKVKLLTESV
NSVIAQAPPVAQEALKKELETLTTNYQWLCTRLNGKCKTLEEVWACWHELLSYLEKAN
KWLNEVEFKLKTTENIPGGAEEISEVLDSLENLMRHSEDNPNQIRILAQTLTDGGVMD
ELINEELETFNSRWRELHEEAVRRQKLLEQSIQSAQETEKSLHLIQESLTFIDKQLAAYI
ADKVDAAQMPQEAQKIQSDLTSHEISLEEMKKHNQGKEAAQRVLSQIDVAQKKLQDVS
MKFRLFQKPANFEQRLQESKMILDEVKMHLPALETKSVEQEVVQSQLNHCVNLYKSLS
EVKSEVEMVIKTGRQIVQKKQTENPKELDERVTALKLHYNELGAKVTERKQQLEKCLK
LSRKMRKEMNVLTEWLAATDMELTKRSAVEGMPSNLDSEVAWGKATQKEIEKQKVH
LKSITEVGEALKTVLGKKETLVEDKLSLLNSNWIAVTSRAEEWLNLLLEYQKHMETFD
QNVDHITKWITQADTLLDESEKKKPQQKEDVLKRLKAELNDIRPKVDSTRDQAANLMA
NRGDHCRKLVEPQISELNHRFAAISHRIKTGKASIPLKELEQFNSDIQKLLEPLEAEIQQ
GVNLKEEDFNKDMNEDNEGTVKELLQRGDNLQQRITDERKREEIKIKQQLLQTKHNA
LKDLRSQRRKKALEISHQWYQYKRQADDLLKCLDDIEKKLASLPEPRDERKIKEIDREL
QKKKEELNAVRRQAEGLSEDGAAMAVEPTQIQLSKRWREIESKFAQFRRLNFAQIHTV
REETMMVMTEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQE
ESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKD
RQGRFDRSVEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGI
GQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQKN
ILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPLRQGILKQL
NETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEKK
LEDLEEQLNHLLLWLSPIRNQLEIYNQPNQEGPFDVQETEIAVQAKQPDVEEILSKGQH
LYKEKPATQPVKRKLEDLSSEWKAVNRLLQELRAKQPDLAPGLTTIGASPTQTVTLVTQ
PVVTKETAISKLEMPSSLMLEVPALADFNRAWTELTDWLSLLDQVIKSQRVMVGDLEDI
NEMIIKQKATMQDLEQRRPQLEELITAAQNLKNKTSNQEARTIITDRIERIQNQWDEVQ
EHLQNRRQQLNEMLKDSTQWLEAKEEAEQVLGQARAKLESWKEGPYTVDAIQKKITE
TKQLAKDLRQWQTNVDVANDLALKLLRDYSADDTRKVHMITENINASWRSIHKRVSER
EAALEETHRLLQQFPLDLEKFLAWLTEAETTANVLQDATRKERLLEDSKGVKELMKQ
WQDLQGEIEAHTDVYHNLDENSQKILRSLEGSDDAVLLQRRLDNMNFKWSELRKKSL
NIRSHLEASSDQWKRLHLSLQELLVWLQLKDDELSRQAPIGGDFPAVQKQNDVHRAFK
RELKTKEPVIMSTLETVRIFLTEQPLEGLEKLYQEPRELPPEERAQNVTRLLRKQAEEV
NTEWEKLNLHSADWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLID
SLQDHLEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQ
VAVEDRVRQLHEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHP
KMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQ
PMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTG
IISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSV
RSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPI
IGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNK
FRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRI
EHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILIS
LESEERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSPRD
AELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQPQAEAKVNGTTVSS
PSTSLQRSDSSQPMLLRVVGSQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRG
RNTPGKPMREDTM DMD Gene Exon 51 SEQ ID GUACCUCCAACAUCAAGGAAGAUGG SEQ
ID GAGAUGGCAGUUUCCUUAGUAACCA NO 2 NO 39 SEQ ID
UACCUCCAACAUCAAGGAAGAUGGC SEQ ID AGAUGGCAGUUUCCUUAGUAACCAC NO 3 NO
40 SEQ ID ACCUCCAACAUCAAGGAAGAUGGCA SEQ ID
GAUGGCAGUUUCCUUAGUAACCACA NO 4 NO 41 SEQ ID
CCUCCAACAUCAAGGAAGAUGGCAU SEQ ID AUGGCAGUUUCCUUAGUAACCACAG NO 5 NO
42 SEQ ID CUCCAACAUCAAGGAAGAUGGCAUU SEQ ID
UGGCAGUUUCCUUAGUAACCACAGG NO 6 NO 43 SEQ ID
UCCAACAUCAAGGAAGAUGGCAUUU SEQ ID GGCAGUUUCCUUAGUAACCACAGGU NO 7 NO
44 SEQ ID CCAACAUCAAGGAAGAUGGCAUUUC SEQ ID
GCAGUUUCCUUAGUAACCACAGGUU NO 8 NO 45 SEQ ID
CAACAUCAAGGAAGAUGGCAUUUCU SEQ ID CAGUUUCCUUAGUAACCACAGGUUG NO 9 NO
46 SEQ ID AACAUCAAGGAAGAUGGCAUUUCUA SEQ ID
AGUUUCCUUAGUAACCACAGGUUGU NO 10 NO 47 SEQ ID
ACAUCAAGGAAGAUGGCAUUUCUAG SEQ ID GUUUCCUUAGUAACCACAGGUUGUG NO 11 NO
48 SEQ ID CAUCAAGGAAGAUGGCAUUUCUAGU SEQ ID
UUUCCUUAGUAACCACAGGUUGUGU NO 12 NO 49 SEQ ID
AUCAAGGAAGAUGGCAUUUCUAGUU SEQ ID UUCCUUAGUAACCACAGGUUGUGUC NO 13 NO
50 SEQ ID UCAAGGAAGAUGGCAUUUCUAGUUU SEQ ID
UCCUUAGUAACCACAGGUUGUGUCA NO 14 NO 51 SEQ ID
CAAGGAAGAUGGCAUUUCUAGUUUG SEQ ID CCUUAGUAACCACAGGUUGUGUCAC NO 15 NO
52 SEQ ID AAGGAAGAUGGCAUUUCUAGUUUGG SEQ ID
CUUAGUAACCACAGGUUGUGUCACC NO 16 NO 53 SEQ ID
AGGAAGAUGGCAUUUCUAGUUUGGA SEQ ID UUAGUAACCACAGGUUGUGUCACCA NO 17 NO
54 SEQ ID GGAAGAUGGCAUUUCUAGUUUGGAG SEQ ID
UAGUAACCACAGGUUGUGUCACCAG NO 18 NO 55 SEQ ID
GAAGAUGGCAUUUCUAGUUUGGAGA SEQ ID AGUAACCACAGGUUGUGUCACCAGA NO 19 NO
56 SEQ ID AAGAUGGCAUUUCUAGUUUGGAGAU SEQ ID
GUAACCACAGGUUGUGUCACCAGAG NO 20 NO 57 SEQ ID
AGAUGGCAUUUCUAGUUUGGAGAUG SEQ ID UAACCACAGGUUGUGUCACCAGAGU NO 21 NO
58 SEQ ID GAUGGCAUUUCUAGUUUGGAGAUGG SEQ ID
AACCACAGGUUGUGUCACCAGAGUA NO 22 NO 59 SEQ ID
AUGGCAUUUCUAGUUUGGAGAUGGC SEQ ID ACCACAGGUUGUGUCACCAGAGUAA NO 23 NO
60 SEQ ID UGGCAUUUCUAGUUUGGAGAUGGCA SEQ ID
CCACAGGUUGUGUCACCAGAGUAAC NO 24 NO 61 SEQ ID
GGCAUUUCUAGUUUGGAGAUGGCAG SEQ ID CACAGGUUGUGUCACCAGAGUAACA NO 25 NO
62 SEQ ID GCAUUUCUAGUUUGGAGAUGGCAGU SEQ ID
ACAGGUUGUGUCACCAGAGUAACAG NO 26 NO 63 SEQ ID
CAUUUCUAGUUUGGAGAUGGCAGUU SEQ ID CAGGUUGUGUCACCAGAGUAACAGU NO 27 NO
64 SEQ ID AUUUCUAGUUUGGAGAUGGCAGUUU SEQ ID
AGGUUGUGUCACCAGAGUAACAGUC NO 28 NO 65 SEQ ID
UUUCUAGUUUGGAGAUGGCAGUUUC SEQ ID GGUUGUGUCACCAGAGUAACAGUCU NO 29 NO
66 SEQ ID UUCUAGUUUGGAGAUGGCAGUUUCC SEQ ID
GUUGUGUCACCAGAGUAACAGUCUG NO 30 NO 67 SEQ ID
UCUAGUUUGGAGAUGGCAGUUUCCU SEQ ID UUGUGUCACCAGAGUAACAGUCUGA NO 31 NO
68 SEQ ID CUAGUUUGGAGAUGGCAGUUUCCUU SEQ ID
UGUGUCACCAGAGUAACAGUCUGAG NO 32 NO 69 SEQ ID
UAGUUUGGAGAUGGCAGUUUCCUUA SEQ ID GUGUCACCAGAGUAACAGUCUGAGU NO 33 NO
70 SEQ ID AGUUUGGAGAUGGCAGUUUCCUUAG SEQ ID
UGUCACCAGAGUAACAGUCUGAGUA NO 34 NO 71 SEQ ID
GUUUGGAGAUGGCAGUUUCCUUAGU SEQ ID GUCACCAGAGUAACAGUCUGAGUAG NO 35 NO
72 SEQ ID UUUGGAGAUGGCAGUUUCCUUAGUA SEQ ID
UCACCAGAGUAACAGUCUGAGUAGG NO 36 NO 73 SEQ ID
UUGGAGAUGGCAGUUUCCUUAGUAA SEQ ID CACCAGAGUAACAGUCUGAGUAGGA NO 37 NO
74 SEQ ID UGGAGAUGGCAGUUUCCUUAGUAAC SEQ ID
ACCAGAGUAACAGUCUGAGUAGGAG NO 38 NO 75 SEQ ID UCAAGGAAGAUGGCAUUUCU
SEQ ID UCAAGGAAGAUGGCAUIUCU NO 539 NO 548 SEQ ID
UCAAIGAAGAUGGCAUUUCU SEQ ID UCAAGGAAGAUGGCAUUICU NO 540 NO 549 SEQ
ID UCAAGIAAGAUGGCAUUUCU SEQ ID UCAAGGAAGAUGGCAUUUCI NO 541 NO 550
SEQ ID UCAAGGAAIAUGGCAUUUCU SEQ ID UCIAGGAAGAUGGCAUUUCU NO 542 NO
551 SEQ ID UCAAGGAAGAUIGCAUUUCU SEQ ID UCAIGGAAGAUGGCAUUUCU NO 543
NO 552 SEQ ID UCAAGGAAGAUGICAUUUCU SEQ ID UCAAGGIAGAUGGCAUUUCU NO
544 NO 553 SEQ ID ICAAGGAAGAUGGCAUUUCU SEQ ID UCAAGGAIGAUGGCAUUUCU
NO 545 NO 554 SEQ ID UCAAGGAAGAIGGCAUUUCU SEQ ID
UCAAGGAAGIUGGCAUUUCU NO 546 NO 555 SEQ ID UCAAGGAAGAUGGCAIUUCU SEQ
ID UCAAGGAAGAUGGCIUUUCU NO 547 NO 556 DMD Gene Exon 45 SEQ ID
UUUGCCGCUGCCCAAUGCCAUCCUG SEQ ID GUUGCAUUCAAUGUUCUGACAACAG NO 76 NO
109 PS220 SEQ ID AUUCAAUGUUCUGACAACAGUUUGC SEQ ID
UUGCAUUCAAUGUUCUGACAACAGU NO 77 NO 110 SEQ ID
CCAGUUGCAUUCAAUGUUCUGACAA SEQ ID UGCAUUCAAUGUUCUGACAACAGUU NO 78 NO
111 SEQ ID CAGUUGCAUUCAAUGUUCUGAC SEQ ID GCAUUCAAUGUUCUGACAACAGUUU
NO 79 NO 112 SEQ ID AGUUGCAUUCAAUGUUCUGA SEQ ID
CAUUCAAUGUUCUGACAACAGUUUG NO 80 NO 113 SEQ ID GAUUGCUGAAUUAUUUCUUCC
SEQ ID AUUCAAUGUUCUGACAACAGUUUGC NO 81 NO 114 SEQ ID
GAUUGCUGAAUUAUUUCUUCCCCAG SEQ ID UCAAUGUUCUGACAACAGUUUGCCG NO 82 NO
115 SEQ ID AUUGCUGAAUUAUUUCUUCCCCAGU SEQ ID
CAAUGUUCUGACAACAGUUUGCCGC NO 83 NO 116 SEQ ID
UUGCUGAAUUAUUUCUUCCCCAGUU SEQ ID AAUGUUCUGACAACAGUUUGCCGCU NO 84 NO
117 SEQ ID UGCUGAAUUAUUUCUUCCCCAGUUG SEQ ID
AUGUUCUGACAACAGUUUGCCGCUG NO 85 NO 118 SEQ ID
GCUGAAUUAUUUCUUCCCCAGUUGC SEQ ID UGUUCUGACAACAGUUUGCCGCUGC NO 86 NO
119 SEQ ID CUGAAUUAUUUCUUCCCCAGUUGCA SEQ ID
GUUCUGACAACAGUUUGCCGCUGCC NO 87 NO 120 SEQ ID
UGAAUUAUUUCUUCCCCAGUUGCAU SEQ ID UUCUGACAACAGUUUGCCGCUGCCC NO 88 NO
121
SEQ ID GAAUUAUUUCUUCCCCAGUUGCAUU SEQ ID UCUGACAACAGUUUGCCGCUGCCCA
NO 89 NO 122 SEQ ID AAUUAUUUCUUCCCCAGUUGCAUUC SEQ ID
CUGACAACAGUUUGCCGCUGCCCAA NO 90 NO 123 SEQ ID
AUUAUUUCUUCCCCAGUUGCAUUCA SEQ ID UGACAACAGUUUGCCGCUGCCCAAU NO 91 NO
124 SEQ ID UUAUUUCUUCCCCAGUUGCAUUCAA SEQ ID
GACAACAGUUUGCCGCUGCCCAAUG NO 92 NO 125 SEQ ID
UAUUUCUUCCCCAGUUGCAUUCAAU SEQ ID ACAACAGUUUGCCGCUGCCCAAUGC NO 93 NO
126 SEQ ID AUUUCUUCCCCAGUUGCAUUCAAUG SEQ ID
CAACAGUUUGCCGCUGCCCAAUGCC NO 94 NO 127 SEQ ID
UUUCUUCCCCAGUUGCAUUCAAUGU SEQ ID AACAGUUUGCCGCUGCCCAAUGCCA NO 95 NO
128 SEQ ID UUCUUCCCCAGUUGCAUUCAAUGUU SEQ ID
ACAGUUUGCCGCUGCCCAAUGCCAU NO 96 NO 129 SEQ ID
UCUUCCCCAGUUGCAUUCAAUGUUC SEQ ID CAGUUUGCCGCUGCCCAAUGCCAUC NO 97 NO
130 SEQ ID CUUCCCCAGUUGCAUUCAAUGUUCU SEQ ID
AGUUUGCCGCUGCCCAAUGCCAUCC NO 98 NO 131 SEQ ID
UUCCCCAGUUGCAUUCAAUGUUCUG SEQ ID GUUUGCCGCUGCCCAAUGCCAUCCU NO 99 NO
132 SEQ ID UCCCCAGUUGCAUUCAAUGUUCUGA SEQ ID
UUUGCCGCUGCCCAAUGCCAUCCUG NO 100 NO 133 SEQ ID
CCCCAGUUGCAUUCAAUGUUCUGAC SEQ ID UUGCCGCUGCCCAAUGCCAUCCUGG NO 101
NO 134 SEQ ID CCCAGUUGCAUUCAAUGUUCUGACA SEQ ID
UGCCGCUGCCCAAUGCCAUCCUGGA NO 102 NO 135 SEQ ID
CCAGUUGCAUUCAAUGUUCUGACAA SEQ ID GCCGCUGCCCAAUGCCAUCCUGGAG NO 103
NO 136 SEQ ID CAGUUGCAUUCAAUGUUCUGACAAC SEQ ID
CCGCUGCCCAAUGCCAUCCUGGAGU NO 104 NO 137 SEQ ID
AGUUGCAUUCAAUGUUCUGACAACA SEQ ID CGCUGCCCAAUGCCAUCCUGGAGUU NO 105
NO 138 SEQ ID UCC UGU AGA AUA CUG GCA UC SEQ ID
UGUUUUUGAGGAUUGCUGAA NO 106 NO 139 SEQ ID UGCAGACCUCCUGCCACCGCAGAUU
SEQ ID UGUUCUGACAACAGUUUGCCGCUGCC NO 107 CA NO 140 CAAUGCCAUCCUGG
SEQ ID UUGCAGACCUCCUGCCACCGCAGAU SEQ ID NO 108 UCAGGCUUC NO 557
UUUGCCICUGCCCAAUGCCAUCCUG PS305 SEQ ID UUUGCCGCUICCCAAUGCCAUCCUG
SEQ ID UUUGCCGCUGCCCAIUGCCAUCCUG NO 558 NO 566 SEQ ID
UUUGCCGCUGCCCAAUICCAUCCUG SEQ ID UUUGCCGCUGCCCAAUGCCIUCCUG NO 559
NO 567 SEQ ID UUUICCGCUGCCCAAUGCCAUCCUG SEQ ID
UUUICCICUGCCCAAUGCCAUCCUG NO 560 NO 568 SEQ ID
UUUGCCGCUGCCCAAUGCCAUCCUI SEQ ID UUUGCCGCUGCCCAAIGCCAUCCUG NO 561
NO 569 SEQ ID IUUGCCGCUGCCCAAUGCCAUCCUG SEQ ID
UUUGCCGCUGCCCAAUGCCAICCUG NO 562 NO 570 SEQ ID
UIUGCCGCUGCCCAAUGCCAUCCUG SEQ ID UUUGCCGCUGCCCAAUGCCAUCCIG NO 563
NO 571 SEQ ID UUIGCCGCUGCCCAAUGCCAUCCUG SEQ ID
UUUGCCGCUGCCCIAUGCCAUCCUG NO 564 NO 572 SEQ ID
UUUGCCGCIGCCCAAUGCCAUCCUG NO 565 DMD Gene Exon 53 SEQ ID
CUCUGGCCUGUCCUAAGACCUGCUC SEQ ID CAGCUUCUUCCUUAGCUUCCAGCCA NO 141
NO 165 SEQ ID UCUGGCCUGUCCUAAGACCUGCUCA SEQ ID
AGCUUCUUCCUUAGCUUCCAGCCAU NO 142 NO 166 SEQ ID
CUGGCCUGUCCUAAGACCUGCUCAG SEQ ID GCUUCUUCCUUAGCUUCCAGCCAUU NO 143
NO 167 SEQ ID UGGCCUGUCCUAAGACCUGCUCAGC SEQ ID
CUUCUUCCUUAGCUUCCAGCCAUUG NO 144 NO 168 SEQ ID
GGCCUGUCCUAAGACCUGCUCAGCU SEQ ID UUCUUCCUUAGCUUCCAGCCAUUGU NO 145
NO 169 SEQ ID GCCUGUCCUAAGACCUGCUCAGCUU SEQ ID
UCUUCCUUAGCUUCCAGCCAUUGUG NO 146 NO 170 SEQ ID
CCUGUCCUAAGACCUGCUCAGCUUC SEQ ID CUUCCUUAGCUUCCAGCCAUUGUGU NO 147
NO 171 SEQ ID CUGUCCUAAGACCUGCUCAGCUUCU SEQ ID
UUCCUUAGCUUCCAGCCAUUGUGUU NO 148 NO 172 SEQ ID
UGUCCUAAGACCUGCUCAGCUUCUU SEQ ID UCCUUAGCUUCCAGCCAUUGUGUUG NO 149
NO 173 SEQ ID GUCCUAAGACCUGCUCAGCUUCUUC SEQ ID
CCUUAGCUUCCAGCCAUUGUGUUGA NO 150 NO 174 SEQ ID
UCCUAAGACCUGCUCAGCUUCUUCC SEQ ID CUUAGCUUCCAGCCAUUGUGUUGAA NO 151
NO 175 SEQ ID CCUAAGACCUGCUCAGCUUCUUCCU SEQ ID
UUAGCUUCCAGCCAUUGUGUUGAAU NO 152 NO 176 SEQ ID
CUAAGACCUGCUCAGCUUCUUCCUU SEQ ID UAGCUUCCAGCCAUUGUGUUGAAUC NO 153
NO 177 SEQ ID UAAGACCUGCUCAGCUUCUUCCUUA SEQ ID
AGCUUCCAGCCAUUGUGUUGAAUCC NO 154 NO 178 SEQ ID
AAGACCUGCUCAGCUUCUUCCUUAG SEQ ID GCUUCCAGCCAUUGUGUUGAAUCCU NO 155
NO 179 SEQ ID AGACCUGCUCAGCUUCUUCCUUAGC SEQ ID
CUUCCAGCCAUUGUGUUGAAUCCUU NO 156 NO 180 SEQ ID
GACCUGCUCAGCUUCUUCCUUAGCU SEQ ID UUCCAGCCAUUGUGUUGAAUCCUUU NO 157
NO 181 SEQ ID ACCUGCUCAGCUUCUUCCUUAGCUU SEQ ID
UCCAGCCAUUGUGUUGAAUCCUUUA NO 158 NO 182 SEQ ID
CCUGCUCAGCUUCUUCCUUAGCUUC SEQ ID CCAGCCAUUGUGUUGAAUCCUUUAA NO 159
NO 183 SEQ ID CUGCUCAGCUUCUUCCUUAGCUUCC SEQ ID
CAGCCAUUGUGUUGAAUCCUUUAAC NO 160 NO 184 SEQ ID
UGCUCAGCUUCUUCCUUAGCUUCCA SEQ ID AGCCAUUGUGUUGAAUCCUUUAACA NO 161
NO 185 SEQ ID GCUCAGCUUCUUCCUUAGCUUCCAG SEQ ID
GCCAUUGUGUUGAAUCCUUUAACAU NO 162 NO 186 SEQ ID
CUCAGCUUCUUCCUUAGCUUCCAGC SEQ ID CCAUUGUGUUGAAUCCUUUAACAUU NO 163
NO 187 SEQ ID UCAGCUUCUUCCUUAGCUUCCAGCC SEQ ID
CAUUGUGUUGAAUCCUUUAACAUUU NO 164 NO 188 DMD Gene Exon 44 SEQ ID
UCAGCUUCUGUUAGCCACUG SEQ ID AGCUUCUGUUAGCCACUGAUUAAA NO 189 NO 214
SEQ ID UUCAGCUUCUGUUAGCCACU SEQ ID CAGCUUCUGUUAGCCACUGAUUAAA NO 190
NO 215 SEQ ID UUCAGCUUCUGUUAGCCACUG SEQ ID AGCUUCUGUUAGCCACUGAUUAAA
NO 191 NO 216 SEQ ID UCAGCUUCUGUUAGCCACUGA SEQ ID
AGCUUCUGUUAGCCACUGAU NO 192 NO 217 SEQ ID UUCAGCUUCUGUUAGCCACUGA
SEQ ID GCUUCUGUUAGCCACUGAUU NO 193 NO 218 SEQ ID
UCAGCUUCUGUUAGCCACUGA SEQ ID AGCUUCUGUUAGCCACUGAUU NO 194 NO 219
SEQ ID UUCAGCUUCUGUUAGCCACUGA SEQ ID GCUUCUGUUAGCCACUGAUUA NO 195
NO 220 SEQ ID UCAGCUUCUGUUAGCCACUGAU SEQ ID AGCUUCUGUUAGCCACUGAUUA
NO 196 NO 221 SEQ ID UUCAGCUUCUGUUAGCCACUGAU SEQ ID
GCUUCUGUUAGCCACUGAUUAA NO 197 NO 222 SEQ ID UCAGCUUCUGUUAGCCACUGAUU
SEQ ID AGCUUCUGUUAGCCACUGAUUAA NO 198 NO 223 SEQ ID
UUCAGCUUCUGUUAGCCACUGAUU SEQ ID GCUUCUGUUAGCCACUGAUUAAA NO 199 NO
224 SEQ ID UCAGCUUCUGUUAGCCACUGAUUA SEQ ID AGCUUCUGUUAGCCACUGAUUAAA
NO 200 NO 225 SEQ ID UUCAGCUUCUGUUAGCCACUGAUA SEQ ID
GCUUCUGUUAGCCACUGAUUAAA NO 201 NO 226 SEQ ID
UCAGCUUCUGUUAGCCACUGAUUAA SEQ ID CCAUUUGUAUUUAGCAUGUUCCC NO 202 NO
227 SEQ ID UUCAGCUUCUGUUAGCCACUGAUUAA SEQ ID AGAUACCAUUUGUAUUUAGC
NO 203 NO 228 SEQ ID UCAGCUUCUGUUAGCCACUGAUUAAA SEQ ID
GCCAUUUCUCAACAGAUCU NO 204 NO 229 SEQ ID
UUCAGCUUCUGUUAGCCACUGAUUAAA SEQ ID GCCAUUUCUCAACAGAUCUGUCA NO 205
NO 230 SEQ ID CAGCUUCUGUUAGCCACUG SEQ ID AUUCUCAGGAAUUUGUGUCUUUC NO
206 NO 231 SEQ ID CAGCUUCUGUUAGCCACUGAU SEQ ID
UCUCAGGAAUUUGUGUCUUUC NO 207 NO 232 SEQ ID AGCUUCUGUUAGCCACUGAUU
SEQ ID GUUCAGCUUCUGUUAGCC NO 208 NO 233 SEQ ID
CAGCUUCUGUUAGCCACUGAUU SEQ ID CUGAUUAAAUAUCUUUAUAU C NO 209 NO 234
SEQ ID AGCUUCUGUUAGCCACUGAUUA SEQ ID GCCGCCAUUUCUCAACAG NO 210 NO
235 SEQ ID CAGCUUCUGUUAGCCACUGAUUA SEQ ID GUAUUUAGCAUGUUCCCA NO 211
NO 236 SEQ ID AGCUUCUGUUAGCCACUGAUUAA SEQ ID CAGGAAUUUGUGUCUUUC NO
212 NO 237 SEQ ID CAGCUUCUGUUAGCCACUGAUUAA SEQ ID
UCAICUUCUGUUAGCCACUG NO 213 NO 575 SEQ ID UCAGCUUCUIUUAGCCACUG SEQ
ID UCAGCUUCUGUUAGCCACUI NO 573 NO 576 SEQ ID UCAGCUUCUGUUAICCACUG
NO 574 DMD Gene Exon 46 SEQ ID GCUUUUCUUUUAGUUGCUGCUCUUU SEQ ID
CCAGGUUCAAGUGGGAUACUAGCAA NO 238 NO 265 SEQ ID
CUUUUCUUUUAGUUGCUGCUCUUUU SEQ ID CAGGUUCAAGUGGGAUACUAGCAAU NO 239
NO 266 SEQ ID UUUUCUUUUAGUUGCUGCUCUUUUC SEQ ID
AGGUUCAAGUGGGAUACUAGCAAUG NO 240 NO 267 SEQ ID
UUUCUUUUAGUUGCUGCUCUUUUCC SEQ ID GGUUCAAGUGGGAUACUAGCAAUGU NO 241
NO 268
SEQ ID UUCUUUUAGUUGCUGCUCUUUUCCA SEQ ID GUUCAAGUGGGAUACUAGCAAUGUU
NO 242 NO 269 SEQ ID UCUUUUAGUUGCUGCUCUUUUCCAG SEQ ID
UUCAAGUGGGAUACUAGCAAUGUUA NO 243 NO 270 SEQ ID
CUUUUAGUUGCUGCUCUUUUCCAGG SEQ ID UCAAGUGGGAUACUAGCAAUGUUAU NO 244
NO 271 SEQ ID UUUUAGUUGCUGCUCUUUUCCAGGU SEQ ID
CAAGUGGGAUACUAGCAAUGUUAUC NO 245 NO 272 SEQ ID
UUUAGUUGCUGCUCUUUUCCAGGUU SEQ ID AAGUGGGAUACUAGCAAUGUUAUCU NO 246
NO 273 SEQ ID UUAGUUGCUGCUCUUUUCCAGGUUC SEQ ID
AGUGGGAUACUAGCAAUGUUAUCUG NO 247 NO 274 SEQ ID
UAGUUGCUGCUCUUUUCCAGGUUCA SEQ ID GUGGGAUACUAGCAAUGUUAUCUGC NO 248
NO 275 SEQ ID AGUUGCUGCUCUUUUCCAGGUUCAA SEQ ID
UGGGAUACUAGCAAUGUUAUCUGCU NO 249 NO 276 SEQ ID
GUUGCUGCUCUUUUCCAGGUUCAAG SEQ ID GGGAUACUAGCAAUGUUAUCUGCUU NO 250
NO 277 SEQ ID UUGCUGCUCUUUUCCAGGUUCAAGU SEQ ID
GGAUACUAGCAAUGUUAUCUGCUUC NO 251 NO 278 SEQ ID
UGCUGCUCUUUUCCAGGUUCAAGUG SEQ ID GAUACUAGCAAUGUUAUCUGCUUCC NO 252
NO 279 SEQ ID GCUGCUCUUUUCCAGGUUCAAGUGG SEQ ID
AUACUAGCAAUGUUAUCUGCUUCCU NO 253 NO 280 SEQ ID
CUGCUCUUUUCCAGGUUCAAGUGGG SEQ ID UACUAGCAAUGUUAUCUGCUUCCUC NO 254
NO 281 SEQ ID UGCUCUUUUCCAGGUUCAAGUGGGA SEQ ID
ACUAGCAAUGUUAUCUGCUUCCUCC NO 255 NO 282 SEQ ID
GCUCUUUUCCAGGUUCAAGUGGGAC SEQ ID CUAGCAAUGUUAUCUGCUUCCUCCA NO 256
NO 283 SEQ ID CUCUUUUCCAGGUUCAAGUGGGAUA SEQ ID
UAGCAAUGUUAUCUGCUUCCUCCAA NO 257 NO 284 SEQ ID
UCUUUUCCAGGUUCAAGUGGGAUAC SEQ ID AGCAAUGUUAUCUGCUUCCUCCAAC NO 258
NO 285 SEQ ID UCUUUUCCAGGUUCAAGUGG SEQ ID GCAAUGUUAUCUGCUUCCUCCAACC
NO 259 NO 286 SEQ ID CUUUUCCAGGUUCAAGUGGGAUACU SEQ ID
CAAUGUUAUCUGCUUCCUCCAACCA NO 260 NO 287 SEQ ID
UUUUCCAGGUUCAAGUGGGAUACUA SEQ ID AAUGUUAUCUGCUUCCUCCAACCAU NO 261
NO 288 SEQ ID UUUCCAGGUUCAAGUGGGAUACUAG SEQ ID
AUGUUAUCUGCUUCCUCCAACCAUA NO 262 NO 289 SEQ ID
UUCCAGGUUCAAGUGGGAUACUAGC SEQ ID UGUUAUCUGCUUCCUCCAACCAUAA NO 263
NO 290 SEQ ID UCCAGGUUCAAGUGGGAUACUAGCA NO 264 DMD Gene Exon 52 SEQ
ID AGCCUCUUGAUUGCUGGUCUUGUUU SEQ ID UUGGGCAGCGGUAAUGAGUUCUUCC NO
291 NO 326 SEQ ID GCCUCUUGAUUGCUGGUCUUGUUUU SEQ ID
UGGGCAGCGGUAAUGAGUUCUUCCA NO 292 NO 327 SEQ ID
CCUCUUGAUUGCUGGUCUUGUUUUU SEQ ID GGGCAGCGGUAAUGAGUUCUUCCAA NO 293
NO 328 SEQ ID CCUCUUGAUUGCUGGUCUUG SEQ ID GGCAGCGGUAAUGAGUUCUUCCAAC
NO 294 NO 329 SEQ ID CUCUUGAUUGCUGGUCUUGUUUUUC SEQ ID
GCAGCGGUAAUGAGUUCUUCCAACU NO 295 NO 330 SEQ ID
UCUUGAUUGCUGGUCUUGUUUUUCA SEQ ID CAGCGGUAAUGAGUUCUUCCAACUG NO 296
NO 331 SEQ ID CUUGAUUGCUGGUCUUGUUUUUCAA SEQ ID
AGCGGUAAUGAGUUCUUCCAACUGG NO 297 NO 332 SEQ ID
UUGAUUGCUGGUCUUGUUUUUCAAA SEQ ID GCGGUAAUGAGUUCUUCCAACUGGG NO 298
NO 333 SEQ ID UGAUUGCUGGUCUUGUUUUUCAAAU SEQ ID
CGGUAAUGAGUUCUUCCAACUGGGG NO 299 NO 334 SEQ ID
GAUUGCUGGUCUUGUUUUUCAAAUU SEQ ID GGUAAUGAGUUCUUCCAACUGGGGA NO 300
NO 335 SEQ ID GAUUGCUGGUCUUGUUUUUC SEQ ID GGUAAUGAGUUCUUCCAACUGG NO
301 NO 336 SEQ ID AUUGCUGGUCUUGUUUUUCAAAUUU SEQ ID
GUAAUGAGUUCUUCCAACUGGGGAC NO 302 NO 337 SEQ ID
UUGCUGGUCUUGUUUUUCAAAUUUU SEQ ID UAAUGAGUUCUUCCAACUGGGGACG NO 303
NO 338 SEQ ID UGCUGGUCUUGUUUUUCAAAUUUUG SEQ ID
AAUGAGUUCUUCCAACUGGGGACGC NO 304 NO 339 SEQ ID
GCUGGUCUUGUUUUUCAAAUUUUGG SEQ ID AUGAGUUCUUCCAACUGGGGACGCC NO 305
NO 340 SEQ ID CUGGUCUUGUUUUUCAAAUUUUGGG SEQ ID
UGAGUUCUUCCAACUGGGGACGCCU NO 306 NO 341 SEQ ID
UGGUCUUGUUUUUCAAAUUUUGGGC SEQ ID GAGUUCUUCCAACUGGGGACGCCUC NO 307
NO 342 SEQ ID GGUCUUGUUUUUCAAAUUUUGGGCA SEQ ID
AGUUCUUCCAACUGGGGACGCCUCU NO 308 NO 343 SEQ ID
GUCUUGUUUUUCAAAUUUUGGGCAG SEQ ID GUUCUUCCAACUGGGGACGCCUCUG NO 309
NO 344 SEQ ID UCUUGUUUUUCAAAUUUUGGGCAGC SEQ ID
UUCUUCCAACUGGGGACGCCUCUGU NO 310 NO 345 SEQ ID
CUUGUUUUUCAAAUUUUGGGCAGCG SEQ ID UCUUCCAACUGGGGACGCCUCUGUU NO 311
NO 346 SEQ ID UUGUUUUUCAAAUUUUGGGCAGCGG SEQ ID
CUUCCAACUGGGGACGCCUCUGUUC NO 312 NO 347 SEQ ID
UGUUUUUCAAAUUUUGGGCAGCGGU SEQ ID UUCCAACUGGGGACGCCUCUGUUCC NO 313
NO 348 SEQ ID GUUUUUCAAAUUUUGGGCAGCGGUA SEQ ID
UCCAACUGGGGACGCCUCUGUUCCA NO 314 NO 349 SEQ ID
UUUUUCAAAUUUUGGGCAGCGGUAA SEQ ID CCAACUGGGGACGCCUCUGUUCCAA NO 315
NO 350 SEQ ID UUUUCAAAUUUUGGGCAGCGGUAAU SEQ ID
CAACUGGGGACGCCUCUGUUCCAAA NO 316 NO 351 SEQ ID
UUUCAAAUUUUGGGCAGCGGUAAUG SEQ ID AACUGGGGACGCCUCUGUUCCAAAU NO 317
NO 352 SEQ ID UUCAAAUUUUGGGCAGCGGUAAUGA SEQ ID
ACUGGGGACGCCUCUGUUCCAAAUC NO 318 NO 353 SEQ ID
UCAAAUUUUGGGCAGCGGUAAUGAG SEQ ID CUGGGGACGCCUCUGUUCCAAAUCC NO 319
NO 354 SEQ ID CAAAUUUUGGGCAGCGGUAAUGAGU SEQ ID
UGGGGACGCCUCUGUUCCAAAUCCU NO 320 NO 355 SEQ ID
AAAUUUUGGGCAGCGGUAAUGAGUU SEQ ID GGGGACGCCUCUGUUCCAAAUCCUG NO 321
NO 356 SEQ ID AAUUUUGGGCAGCGGUAAUGAGUUC SEQ ID
GGGACGCCUCUGUUCCAAAUCCUGC NO 322 NO 357 SEQ ID
AUUUUGGGCAGCGGUAAUGAGUUCU SEQ ID GGACGCCUCUGUUCCAAAUCCUGCA NO 323
NO 358 SEQ ID UUUUGGGCAGCGGUAAUGAGUUCUU SEQ ID
GACGCCUCUGUUCCAAAUCCUGCAU NO 324 NO 359 SEQ ID
UUUGGGCAGCGGUAAUGAGUUCUUC NO 325 DMD Gene Exon 50 SEQ ID
CCAAUAGUGGUCAGUCCAGGAGCUA SEQ ID CUAGGUCAGGCUGCUUUGCCCUCAG NO 360
NO 386 SEQ ID CAAUAGUGGUCAGUCCAGGAGCUAG SEQ ID
UAGGUCAGGCUGCUUUGCCCUCAGC NO 361 NO 387 SEQ ID
AAUAGUGGUCAGUCCAGGAGCUAGG SEQ ID AGGUCAGGCUGCUUUGCCCUCAGCU NO 362
NO 388 SEQ ID AUAGUGGUCAGUCCAGGAGCUAGGU SEQ ID
GGUCAGGCUGCUUUGCCCUCAGCUC NO 363 NO 389 SEQ ID
AUAGUGGUCAGUCCAGGAGCU SEQ ID GUCAGGCUGCUUUGCCCUCAGCUCU NO 364 NO
390 SEQ ID UAGUGGUCAGUCCAGGAGCUAGGUC SEQ ID
UCAGGCUGCUUUGCCCUCAGCUCUU NO 365 NO 391 SEQ ID
AGUGGUCAGUCCAGGAGCUAGGUCA SEQ ID CAGGCUGCUUUGCCCUCAGCUCUUG NO 366
NO 392 SEQ ID GUGGUCAGUCCAGGAGCUAGGUCAG SEQ ID
AGGCUGCUUUGCCCUCAGCUCUUGA NO 367 NO 393 SEQ ID
UGGUCAGUCCAGGAGCUAGGUCAGG SEQ ID GGCUGCUUUGCCCUCAGCUCUUGAA NO 368
NO 394 SEQ ID GGUCAGUCCAGGAGCUAGGUCAGGC SEQ ID
GCUGCUUUGCCCUCAGCUCUUGAAG NO 369 NO 395 SEQ ID
GUCAGUCCAGGAGCUAGGUCAGGCU SEQ ID CUGCUUUGCCCUCAGCUCUUGAAGU NO 370
NO 396 SEQ ID UCAGUCCAGGAGCUAGGUCAGGCUG SEQ ID
UGCUUUGCCCUCAGCUCUUGAAGUA NO 371 NO 397 SEQ ID
CAGUCCAGGAGCUAGGUCAGGCUGC SEQ ID GCUUUGCCCUCAGCUCUUGAAGUAA NO 372
NO 398 SEQ ID AGUCCAGGAGCUAGGUCAGGCUGCU SEQ ID
CUUUGCCCUCAGCUCUUGAAGUAAA NO 373 NO 399 SEQ ID
GUCCAGGAGCUAGGUCAGGCUGCUU SEQ ID UUUGCCCUCAGCUCUUGAAGUAAAC NO 374
NO 400 SEQ ID UCCAGGAGCUAGGUCAGGCUGCUUU SEQ ID
UUGCCCUCAGCUCUUGAAGUAAACG NO 375 NO 401 SEQ ID
CCAGGAGCUAGGUCAGGCUGCUUUG SEQ ID UGCCCUCAGCUCUUGAAGUAAACGG NO 376
NO 402 SEQ ID CAGGAGCUAGGUCAGGCUGCUUUGC SEQ ID
GCCCUCAGCUCUUGAAGUAAACGGU NO 377 NO 403 SEQ ID
AGGAGCUAGGUCAGGCUGCUUUGCC SEQ ID CCCUCAGCUCUUGAAGUAAACGGUU NO 378
NO 404 SEQ ID GGAGCUAGGUCAGGCUGCUUUGCCC SEQ ID CCUCAGCUCUUGAAGUAAAC
NO 379 NO 405 SEQ ID GAGCUAGGUCAGGCUGCUUUGCCCU SEQ ID
CCUCAGCUCUUGAAGUAAACG NO 380 NO 406 SEQ ID
AGCUAGGUCAGGCUGCUUUGCCCUC SEQ ID CUCAGCUCUUGAAGUAAACG NO 381 NO 407
SEQ ID GCUAGGUCAGGCUGCUUUGCCCUCA SEQ ID CCUCAGCUCUUGAAGUAAACGGUUU
NO 382 NO 408 SEQ ID CUCAGCUCUUGAAGUAAACGGUUUA SEQ ID
UCAGCUCUUGAAGUAAACGGUUUAC NO 383 NO 409 SEQ ID
CAGCUCUUGAAGUAAACGGUUUACC SEQ ID AGCUCUUGAAGUAAACGGUUUACCG NO 384
NO 410
SEQ ID GCUCUUGAAGUAAACGGUUUACCGC SEQ ID CUCUUGAAGUAAACGGUUUACCGCC
NO 385 NO 411 DMD Gene Exon 43 SEQ ID CCACAGGCGUUGCACUUUGCAAUGC SEQ
ID UCUUCUUGCUAUGAAUAAUGUCAAU NO 412 NO 443 SEQ ID
CACAGGCGUUGCACUUUGCAAUGCU SEQ ID CUUCUUGCUAUGAAUAAUGUCAAUC NO 413
NO 444 SEQ ID ACAGGCGUUGCACUUUGCAAUGCUG SEQ ID
UUCUUGCUAUGAAUAAUGUCAAUCC NO 414 NO 445 SEQ ID
CAGGCGUUGCACUUUGCAAUGCUGC SEQ ID UCUUGCUAUGAAUAAUGUCAAUCCG NO 415
NO 446 SEQ ID AGGCGUUGCACUUUGCAAUGCUGCU SEQ ID
CUUGCUAUGAAUAAUGUCAAUCCGA NO 416 NO 447 SEQ ID
GGCGUUGCACUUUGCAAUGCUGCUG SEQ ID UUGCUAUGAAUAAUGUCAAUCCGAC NO 417
NO 448 SEQ ID GCGUUGCACUUUGCAAUGCUGCUGU SEQ ID
UGCUAUGAAUAAUGUCAAUCCGACC NO 418 NO 449 SEQ ID
CGUUGCACUUUGCAAUGCUGCUGUC SEQ ID GCUAUGAAUAAUGUCAAUCCGACCU NO 419
NO 450 SEQ ID CGUUGCACUUUGCAAUGCUGCUG SEQ ID
CUAUGAAUAAUGUCAAUCCGACCUG NO 420 NO 451 SEQ ID
GUUGCACUUUGCAAUGCUGCUGUCU SEQ ID UAUGAAUAAUGUCAAUCCGACCUGA NO 421
NO 452 SEQ ID UUGCACUUUGCAAUGCUGCUGUCUU SEQ ID
AUGAAUAAUGUCAAUCCGACCUGAG NO 422 NO 453 SEQ ID
UGCACUUUGCAAUGCUGCUGUCUUC SEQ ID UGAAUAAUGUCAAUCCGACCUGAGC NO 423
NO 454 SEQ ID GCACUUUGCAAUGCUGCUGUCUUCU SEQ ID
GAAUAAUGUCAAUCCGACCUGAGCU NO 424 NO 455 SEQ ID
CACUUUGCAAUGCUGCUGUCUUCUU SEQ ID AAUAAUGUCAAUCCGACCUGAGCUU NO 425
NO 456 SEQ ID ACUUUGCAAUGCUGCUGUCUUCUUG SEQ ID
AUAAUGUCAAUCCGACCUGAGCUUU NO 426 NO 457 SEQ ID
CUUUGCAAUGCUGCUGUCUUCUUGC SEQ ID UAAUGUCAAUCCGACCUGAGCUUUG NO 427
NO 458 SEQ ID UUUGCAAUGCUGCUGUCUUCUUGCU SEQ ID
AAUGUCAAUCCGACCUGAGCUUUGU NO 428 NO 459 SEQ ID
UUGCAAUGCUGCUGUCUUCUUGCUA SEQ ID AUGUCAAUCCGACCUGAGCUUUGUU NO 429
NO 460 SEQ ID UGCAAUGCUGCUGUCUUCUUGCUAU SEQ ID
UGUCAAUCCGACCUGAGCUUUGUUG NO 430 NO 461 SEQ ID
GCAAUGCUGCUGUCUUCUUGCUAUG SEQ ID GUCAAUCCGACCUGAGCUUUGUUGU NO 431
NO 462 SEQ ID CAAUGCUGCUGUCUUCUUGCUAUGA SEQ ID
UCAAUCCGACCUGAGCUUUGUUGUA NO 432 NO 463 SEQ ID
AAUGCUGCUGUCUUCUUGCUAUGAA SEQ ID CAAUCCGACCUGAGCUUUGUUGUAG NO 433
NO 464 SEQ ID AUGCUGCUGUCUUCUUGCUAUGAAU SEQ ID
AAUCCGACCUGAGCUUUGUUGUAGA NO 434 NO 465 SEQ ID
UGCUGCUGUCUUCUUGCUAUGAAUA SEQ ID AUCCGACCUGAGCUUUGUUGUAGAC NO 435
NO 466 SEQ ID GCUGCUGUCUUCUUGCUAUGAAUAA SEQ ID
UCCGACCUGAGCUUUGUUGUAGACU NO 436 NO 467 SEQ ID
CUGCUGUCUUCUUGCUAUGAAUAAU SEQ ID CCGACCUGAGCUUUGUUGUAGACUA NO 437
NO 468 SEQ ID UGCUGUCUUCUUGCUAUGAAUAAUG SEQ ID CGACCUGAGCUUUGUUGUAG
NO 438 NO 469 SEQ ID GCUGUCUUCUUGCUAUGAAUAAUGU SEQ ID
CGACCUGAGCUUUGUUGUAGACUAU NO 439 NO 470 SEQ ID
CUGUCUUCUUGCUAUGAAUAAUGUC SEQ ID GACCUGAGCUUUGUUGUAGACUAUC NO 440
NO 471 SEQ ID UGUCUUCUUGCUAUGAAUAAUGUCA SEQ ID
ACCUGAGCUUUGUUGUAGACUAUCA NO 441 NO 472 SEQ ID
GUCUUCUUGCUAUGAAUAAUGUCAA SEQ ID CCUGA GCUUU GUUGU AGACU AUC NO 442
NO 473 DMD Gene Exon 6 SEQ ID CAUUUUUGACCUACAUGUGG SEQ ID
AUUUUUGACCUACAUGGGAAA G NO 474 NO 479 SEQ ID UUUGACCUACAUGUGGAAAG
SEQ ID UACGAGUUGAUUGUCGGACCCAG NO 475 NO 480 SEQ ID
UACAUUUUUGACCUACAUGUGGAAAG SEQ ID GUGGUCUCCUUACCUAUGACUGUGG NO 476
NO 481 SEQ ID GGUCUCCUUACCUAUGA SEQ ID UGUCUCAGUAAUCUUCUUACCUAU NO
477 NO 482 SEQ ID UCUUACCUAUGACUAUGGAUGAGA NO 478 DMD Gene Exon 7
SEQ ID UGCAUGUUCCAGUCGUUGUGUGG SEQ ID AUUUACCAACCUUCAGGAUCGAGUA NO
483 NO 485 SEQ ID CACUAUUCCAGUCAAAUAGGUCUGG SEQ ID
GGCCUAAAACACAUACACAUA NO 484 NO 486 DMD Gene Exon 8 SEQ ID
GAUAGGUGGUAUCAACAUCUGUAA SEQ ID UGUUGUUGUUUAUGCUCAUU NO 487 NO 490
SEQ ID GAUAGGUGGUAUCAACAUCUG SEQ ID GUACAUUAAGAUGGACUUC NO 488 NO
491 SEQ ID CUUCCUGGAUGGCUUGAAU NO 489 DMD Gene Exon 55 SEQ ID
CUGUUGCAGUAAUCUAUGAG SEQ ID UGCCAUUGUUUCAUCAGCUCUUU NO 492 NO 495
SEQ ID UGCAGUAAUCUAUGAGUUUC SEQ ID UCCUGUAGGACAUUGGCAGU NO 493 NO
496 SEQ ID GAGUCUUCUAGGAGCCUU SEQ ID CUUGGAGUCUUCUAGGAGCC NO 494 NO
497 DMD Gene Exon 2 SEQ ID CCAUUUUGUGAAUGUUUUCUUUUG SEQ ID
GAAAAUUGUGCAUUUACCCAUUUU NO 498 AACAUC NO 500 SEQ ID
CCCAUUUUGUGAAUGUUUUCUUUU SEQ ID UUGUGCAUUUACCCAUUUUGUG NO 499 NO
501 DMD Gene Exon 11 SEQ ID CCCUGAGGCAUUCCCAUCUUGAAU SEQ ID
CUUGAAUUUAGGAGAUUCAUCUG NO 502 NO 504 SEQ ID AGGACUUACUUGCUUUGUUU
SEQ ID CAUCUUCUGAUAAUUUUCCUGUU NO 503 NO 505 DMD Gene Exon 17 SEQ
ID CCAUUACAGUUGUCUGUGUU SEQ ID UAAUCUGCCUCUUCUUUUGG NO 506 NO 508
SEQ ID UGACAGCCUGUGAAAUCUGUGAG NO 507 DMD Gene Exon 19 SEQ ID
CAGCAGUAGUUGUCAUCUGC SEQ ID GCCUGAGCUGAUCUGCUGGCAUCUUG NO 509 NO
511 CAGUU SEQ ID GCCUGAGCUGAUCUGCUGGCAUCUU SEQ ID UCUGCUGGCAUCUUGC
NO 510 GC NO 512 DMD Gene Exon 21 SEQ ID GCCGGUUGACUUCAUCCUGUGC SEQ
ID CUGCAUCCAGGAACAUGGGUCC NO 513 NO 516 SEQ ID
GUCUGCAUCCAGGAACAUGGGUC SEQ ID GUUGAAGAUCUGAUAGCCGGUUGA NO 514 NO
517 SEQ ID UACUUACUGUCUGUAGCUCUUUCU NO 515 DMD Gene Exon 57 SEQ ID
UAGGUGCCUGCCGGCUU SEQ ID CUGAACUGCUGGAAAGUCGCC NO 518 NO 520 SEQ ID
UUCAGCUGUAGCCACACC SEQ ID CUGGCUUCCAAAUGGGACCUGAAAAA NO 519 NO 521
GAAC DMD Gene Exon 59 SEQ ID CAAUUUUUCCCACUCAGUAUU SEQ ID
UCCUCAGGAGGCAGCUCUAAAU NO 522 NO 524 SEQ ID UUGAAGUUCCUGGAGUCUU NO
523 DMD Gene Exon 62 SEQ ID UGGCUCUCUCCCAGGG SEQ ID
GGGCACUUUGUUUGGCG NO 525 NO 527 SEQ ID GAGAUGGCUCUCUCCCAGGGACCCU NO
526 GG DMD Gene Exon 63 SEQ ID GGUCCCAGCAAGUUGUUUG SEQ ID
GUAGAGCUCUGUCAUUUUGGG NO 528 NO 530 SEQ ID
UGGGAUGGUCCCAGCAAGUUGUUUG NO 529 DMD Gene Exon 65 SEQ ID
GCUCAAGAGAUCCACUGCAAAAAAC SEQ ID UCUGCAGGAUAUCCAUGGGCUGGUC NO 531
NO 533 SEQ ID GCCAUACGUACGUAUCAUAAACAUU NO 532 C DMD Gene Exon 66
SEQ ID GAUCCUCCCUGUUCGUCCCCUAUUA NO 534 UG DMD Gene Exon 69 SEQ ID
UGCUUUAGACUCCUGUACCUGAUA NO 535 DMD Gene Exon 75 SEQ ID
GGCGGCCUUUGUGUUGAC SEQ ID CCUUUAUGUUCGUGCUGCU NO 536 NO 538 SEQ ID
GGACAGGCCUUUAUGUUCGUGCUGC NO 537 Human IGF-1 Isoform 4 amino acid
sequence SEQ ID NO 577:
MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAGPETLCGAELV
DALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA
RSVRAQRHTDMPKTQKEVHLKNASRGSAGNKNYRM
Sequence CWU 1
1
57713685PRTHomo sapiens 1Met Leu Trp Trp Glu Glu Val Glu Asp Cys
Tyr Glu Arg Glu Asp Val 1 5 10 15 Gln Lys Lys Thr Phe Thr Lys Trp
Val Asn Ala Gln Phe Ser Lys Phe 20 25 30 Gly Lys Gln His Ile Glu
Asn Leu Phe Ser Asp Leu Gln Asp Gly Arg 35 40 45 Arg Leu Leu Asp
Leu Leu Glu Gly Leu Thr Gly Gln Lys Leu Pro Lys 50 55 60 Glu Lys
Gly Ser Thr Arg Val His Ala Leu Asn Asn Val Asn Lys Ala 65 70 75 80
Leu Arg Val Leu Gln Asn Asn Asn Val Asp Leu Val Asn Ile Gly Ser 85
90 95 Thr Asp Ile Val Asp Gly Asn His Lys Leu Thr Leu Gly Leu Ile
Trp 100 105 110 Asn Ile Ile Leu His Trp Gln Val Lys Asn Val Met Lys
Asn Ile Met 115 120 125 Ala Gly Leu Gln Gln Thr Asn Ser Glu Lys Ile
Leu Leu Ser Trp Val 130 135 140 Arg Gln Ser Thr Arg Asn Tyr Pro Gln
Val Asn Val Ile Asn Phe Thr 145 150 155 160 Thr Ser Trp Ser Asp Gly
Leu Ala Leu Asn Ala Leu Ile His Ser His 165 170 175 Arg Pro Asp Leu
Phe Asp Trp Asn Ser Val Val Cys Gln Gln Ser Ala 180 185 190 Thr Gln
Arg Leu Glu His Ala Phe Asn Ile Ala Arg Tyr Gln Leu Gly 195 200 205
Ile Glu Lys Leu Leu Asp Pro Glu Asp Val Asp Thr Thr Tyr Pro Asp 210
215 220 Lys Lys Ser Ile Leu Met Tyr Ile Thr Ser Leu Phe Gln Val Leu
Pro 225 230 235 240 Gln Gln Val Ser Ile Glu Ala Ile Gln Glu Val Glu
Met Leu Pro Arg 245 250 255 Pro Pro Lys Val Thr Lys Glu Glu His Phe
Gln Leu His His Gln Met 260 265 270 His Tyr Ser Gln Gln Ile Thr Val
Ser Leu Ala Gln Gly Tyr Glu Arg 275 280 285 Thr Ser Ser Pro Lys Pro
Arg Phe Lys Ser Tyr Ala Tyr Thr Gln Ala 290 295 300 Ala Tyr Val Thr
Thr Ser Asp Pro Thr Arg Ser Pro Phe Pro Ser Gln 305 310 315 320 His
Leu Glu Ala Pro Glu Asp Lys Ser Phe Gly Ser Ser Leu Met Glu 325 330
335 Ser Glu Val Asn Leu Asp Arg Tyr Gln Thr Ala Leu Glu Glu Val Leu
340 345 350 Ser Trp Leu Leu Ser Ala Glu Asp Thr Leu Gln Ala Gln Gly
Glu Ile 355 360 365 Ser Asn Asp Val Glu Val Val Lys Asp Gln Phe His
Thr His Glu Gly 370 375 380 Tyr Met Met Asp Leu Thr Ala His Gln Gly
Arg Val Gly Asn Ile Leu 385 390 395 400 Gln Leu Gly Ser Lys Leu Ile
Gly Thr Gly Lys Leu Ser Glu Asp Glu 405 410 415 Glu Thr Glu Val Gln
Glu Gln Met Asn Leu Leu Asn Ser Arg Trp Glu 420 425 430 Cys Leu Arg
Val Ala Ser Met Glu Lys Gln Ser Asn Leu His Arg Val 435 440 445 Leu
Met Asp Leu Gln Asn Gln Lys Leu Lys Glu Leu Asn Asp Trp Leu 450 455
460 Thr Lys Thr Glu Glu Arg Thr Arg Lys Met Glu Glu Glu Pro Leu Gly
465 470 475 480 Pro Asp Leu Glu Asp Leu Lys Arg Gln Val Gln Gln His
Lys Val Leu 485 490 495 Gln Glu Asp Leu Glu Gln Glu Gln Val Arg Val
Asn Ser Leu Thr His 500 505 510 Met Val Val Val Val Asp Glu Ser Ser
Gly Asp His Ala Thr Ala Ala 515 520 525 Leu Glu Glu Gln Leu Lys Val
Leu Gly Asp Arg Trp Ala Asn Ile Cys 530 535 540 Arg Trp Thr Glu Asp
Arg Trp Val Leu Leu Gln Asp Ile Leu Leu Lys 545 550 555 560 Trp Gln
Arg Leu Thr Glu Glu Gln Cys Leu Phe Ser Ala Trp Leu Ser 565 570 575
Glu Lys Glu Asp Ala Val Asn Lys Ile His Thr Thr Gly Phe Lys Asp 580
585 590 Gln Asn Glu Met Leu Ser Ser Leu Gln Lys Leu Ala Val Leu Lys
Ala 595 600 605 Asp Leu Glu Lys Lys Lys Gln Ser Met Gly Lys Leu Tyr
Ser Leu Lys 610 615 620 Gln Asp Leu Leu Ser Thr Leu Lys Asn Lys Ser
Val Thr Gln Lys Thr 625 630 635 640 Glu Ala Trp Leu Asp Asn Phe Ala
Arg Cys Trp Asp Asn Leu Val Gln 645 650 655 Lys Leu Glu Lys Ser Thr
Ala Gln Ile Ser Gln Ala Val Thr Thr Thr 660 665 670 Gln Pro Ser Leu
Thr Gln Thr Thr Val Met Glu Thr Val Thr Thr Val 675 680 685 Thr Thr
Arg Glu Gln Ile Leu Val Lys His Ala Gln Glu Glu Leu Pro 690 695 700
Pro Pro Pro Pro Gln Lys Lys Arg Gln Ile Thr Val Asp Ser Glu Ile 705
710 715 720 Arg Lys Arg Leu Asp Val Asp Ile Thr Glu Leu His Ser Trp
Ile Thr 725 730 735 Arg Ser Glu Ala Val Leu Gln Ser Pro Glu Phe Ala
Ile Phe Arg Lys 740 745 750 Glu Gly Asn Phe Ser Asp Leu Lys Glu Lys
Val Asn Ala Ile Glu Arg 755 760 765 Glu Lys Ala Glu Lys Phe Arg Lys
Leu Gln Asp Ala Ser Arg Ser Ala 770 775 780 Gln Ala Leu Val Glu Gln
Met Val Asn Glu Gly Val Asn Ala Asp Ser 785 790 795 800 Ile Lys Gln
Ala Ser Glu Gln Leu Asn Ser Arg Trp Ile Glu Phe Cys 805 810 815 Gln
Leu Leu Ser Glu Arg Leu Asn Trp Leu Glu Tyr Gln Asn Asn Ile 820 825
830 Ile Ala Phe Tyr Asn Gln Leu Gln Gln Leu Glu Gln Met Thr Thr Thr
835 840 845 Ala Glu Asn Trp Leu Lys Ile Gln Pro Thr Thr Pro Ser Glu
Pro Thr 850 855 860 Ala Ile Lys Ser Gln Leu Lys Ile Cys Lys Asp Glu
Val Asn Arg Leu 865 870 875 880 Ser Gly Leu Gln Pro Gln Ile Glu Arg
Leu Lys Ile Gln Ser Ile Ala 885 890 895 Leu Lys Glu Lys Gly Gln Gly
Pro Met Phe Leu Asp Ala Asp Phe Val 900 905 910 Ala Phe Thr Asn His
Phe Lys Gln Val Phe Ser Asp Val Gln Ala Arg 915 920 925 Glu Lys Glu
Leu Gln Thr Ile Phe Asp Thr Leu Pro Pro Met Arg Tyr 930 935 940 Gln
Glu Thr Met Ser Ala Ile Arg Thr Trp Val Gln Gln Ser Glu Thr 945 950
955 960 Lys Leu Ser Ile Pro Gln Leu Ser Val Thr Asp Tyr Glu Ile Met
Glu 965 970 975 Gln Arg Leu Gly Glu Leu Gln Ala Leu Gln Ser Ser Leu
Gln Glu Gln 980 985 990 Gln Ser Gly Leu Tyr Tyr Leu Ser Thr Thr Val
Lys Glu Met Ser Lys 995 1000 1005 Lys Ala Pro Ser Glu Ile Ser Arg
Lys Tyr Gln Ser Glu Phe Glu 1010 1015 1020 Glu Ile Glu Gly Arg Trp
Lys Lys Leu Ser Ser Gln Leu Val Glu 1025 1030 1035 His Cys Gln Lys
Leu Glu Glu Gln Met Asn Lys Leu Arg Lys Ile 1040 1045 1050 Gln Asn
His Ile Gln Thr Leu Lys Lys Trp Met Ala Glu Val Asp 1055 1060 1065
Val Phe Leu Lys Glu Glu Trp Pro Ala Leu Gly Asp Ser Glu Ile 1070
1075 1080 Leu Lys Lys Gln Leu Lys Gln Cys Arg Leu Leu Val Ser Asp
Ile 1085 1090 1095 Gln Thr Ile Gln Pro Ser Leu Asn Ser Val Asn Glu
Gly Gly Gln 1100 1105 1110 Lys Ile Lys Asn Glu Ala Glu Pro Glu Phe
Ala Ser Arg Leu Glu 1115 1120 1125 Thr Glu Leu Lys Glu Leu Asn Thr
Gln Trp Asp His Met Cys Gln 1130 1135 1140 Gln Val Tyr Ala Arg Lys
Glu Ala Leu Lys Gly Gly Leu Glu Lys 1145 1150 1155 Thr Val Ser Leu
Gln Lys Asp Leu Ser Glu Met His Glu Trp Met 1160 1165 1170 Thr Gln
Ala Glu Glu Glu Tyr Leu Glu Arg Asp Phe Glu Tyr Lys 1175 1180 1185
Thr Pro Asp Glu Leu Gln Lys Ala Val Glu Glu Met Lys Arg Ala 1190
1195 1200 Lys Glu Glu Ala Gln Gln Lys Glu Ala Lys Val Lys Leu Leu
Thr 1205 1210 1215 Glu Ser Val Asn Ser Val Ile Ala Gln Ala Pro Pro
Val Ala Gln 1220 1225 1230 Glu Ala Leu Lys Lys Glu Leu Glu Thr Leu
Thr Thr Asn Tyr Gln 1235 1240 1245 Trp Leu Cys Thr Arg Leu Asn Gly
Lys Cys Lys Thr Leu Glu Glu 1250 1255 1260 Val Trp Ala Cys Trp His
Glu Leu Leu Ser Tyr Leu Glu Lys Ala 1265 1270 1275 Asn Lys Trp Leu
Asn Glu Val Glu Phe Lys Leu Lys Thr Thr Glu 1280 1285 1290 Asn Ile
Pro Gly Gly Ala Glu Glu Ile Ser Glu Val Leu Asp Ser 1295 1300 1305
Leu Glu Asn Leu Met Arg His Ser Glu Asp Asn Pro Asn Gln Ile 1310
1315 1320 Arg Ile Leu Ala Gln Thr Leu Thr Asp Gly Gly Val Met Asp
Glu 1325 1330 1335 Leu Ile Asn Glu Glu Leu Glu Thr Phe Asn Ser Arg
Trp Arg Glu 1340 1345 1350 Leu His Glu Glu Ala Val Arg Arg Gln Lys
Leu Leu Glu Gln Ser 1355 1360 1365 Ile Gln Ser Ala Gln Glu Thr Glu
Lys Ser Leu His Leu Ile Gln 1370 1375 1380 Glu Ser Leu Thr Phe Ile
Asp Lys Gln Leu Ala Ala Tyr Ile Ala 1385 1390 1395 Asp Lys Val Asp
Ala Ala Gln Met Pro Gln Glu Ala Gln Lys Ile 1400 1405 1410 Gln Ser
Asp Leu Thr Ser His Glu Ile Ser Leu Glu Glu Met Lys 1415 1420 1425
Lys His Asn Gln Gly Lys Glu Ala Ala Gln Arg Val Leu Ser Gln 1430
1435 1440 Ile Asp Val Ala Gln Lys Lys Leu Gln Asp Val Ser Met Lys
Phe 1445 1450 1455 Arg Leu Phe Gln Lys Pro Ala Asn Phe Glu Gln Arg
Leu Gln Glu 1460 1465 1470 Ser Lys Met Ile Leu Asp Glu Val Lys Met
His Leu Pro Ala Leu 1475 1480 1485 Glu Thr Lys Ser Val Glu Gln Glu
Val Val Gln Ser Gln Leu Asn 1490 1495 1500 His Cys Val Asn Leu Tyr
Lys Ser Leu Ser Glu Val Lys Ser Glu 1505 1510 1515 Val Glu Met Val
Ile Lys Thr Gly Arg Gln Ile Val Gln Lys Lys 1520 1525 1530 Gln Thr
Glu Asn Pro Lys Glu Leu Asp Glu Arg Val Thr Ala Leu 1535 1540 1545
Lys Leu His Tyr Asn Glu Leu Gly Ala Lys Val Thr Glu Arg Lys 1550
1555 1560 Gln Gln Leu Glu Lys Cys Leu Lys Leu Ser Arg Lys Met Arg
Lys 1565 1570 1575 Glu Met Asn Val Leu Thr Glu Trp Leu Ala Ala Thr
Asp Met Glu 1580 1585 1590 Leu Thr Lys Arg Ser Ala Val Glu Gly Met
Pro Ser Asn Leu Asp 1595 1600 1605 Ser Glu Val Ala Trp Gly Lys Ala
Thr Gln Lys Glu Ile Glu Lys 1610 1615 1620 Gln Lys Val His Leu Lys
Ser Ile Thr Glu Val Gly Glu Ala Leu 1625 1630 1635 Lys Thr Val Leu
Gly Lys Lys Glu Thr Leu Val Glu Asp Lys Leu 1640 1645 1650 Ser Leu
Leu Asn Ser Asn Trp Ile Ala Val Thr Ser Arg Ala Glu 1655 1660 1665
Glu Trp Leu Asn Leu Leu Leu Glu Tyr Gln Lys His Met Glu Thr 1670
1675 1680 Phe Asp Gln Asn Val Asp His Ile Thr Lys Trp Ile Ile Gln
Ala 1685 1690 1695 Asp Thr Leu Leu Asp Glu Ser Glu Lys Lys Lys Pro
Gln Gln Lys 1700 1705 1710 Glu Asp Val Leu Lys Arg Leu Lys Ala Glu
Leu Asn Asp Ile Arg 1715 1720 1725 Pro Lys Val Asp Ser Thr Arg Asp
Gln Ala Ala Asn Leu Met Ala 1730 1735 1740 Asn Arg Gly Asp His Cys
Arg Lys Leu Val Glu Pro Gln Ile Ser 1745 1750 1755 Glu Leu Asn His
Arg Phe Ala Ala Ile Ser His Arg Ile Lys Thr 1760 1765 1770 Gly Lys
Ala Ser Ile Pro Leu Lys Glu Leu Glu Gln Phe Asn Ser 1775 1780 1785
Asp Ile Gln Lys Leu Leu Glu Pro Leu Glu Ala Glu Ile Gln Gln 1790
1795 1800 Gly Val Asn Leu Lys Glu Glu Asp Phe Asn Lys Asp Met Asn
Glu 1805 1810 1815 Asp Asn Glu Gly Thr Val Lys Glu Leu Leu Gln Arg
Gly Asp Asn 1820 1825 1830 Leu Gln Gln Arg Ile Thr Asp Glu Arg Lys
Arg Glu Glu Ile Lys 1835 1840 1845 Ile Lys Gln Gln Leu Leu Gln Thr
Lys His Asn Ala Leu Lys Asp 1850 1855 1860 Leu Arg Ser Gln Arg Arg
Lys Lys Ala Leu Glu Ile Ser His Gln 1865 1870 1875 Trp Tyr Gln Tyr
Lys Arg Gln Ala Asp Asp Leu Leu Lys Cys Leu 1880 1885 1890 Asp Asp
Ile Glu Lys Lys Leu Ala Ser Leu Pro Glu Pro Arg Asp 1895 1900 1905
Glu Arg Lys Ile Lys Glu Ile Asp Arg Glu Leu Gln Lys Lys Lys 1910
1915 1920 Glu Glu Leu Asn Ala Val Arg Arg Gln Ala Glu Gly Leu Ser
Glu 1925 1930 1935 Asp Gly Ala Ala Met Ala Val Glu Pro Thr Gln Ile
Gln Leu Ser 1940 1945 1950 Lys Arg Trp Arg Glu Ile Glu Ser Lys Phe
Ala Gln Phe Arg Arg 1955 1960 1965 Leu Asn Phe Ala Gln Ile His Thr
Val Arg Glu Glu Thr Met Met 1970 1975 1980 Val Met Thr Glu Asp Met
Pro Leu Glu Ile Ser Tyr Val Pro Ser 1985 1990 1995 Thr Tyr Leu Thr
Glu Ile Thr His Val Ser Gln Ala Leu Leu Glu 2000 2005 2010 Val Glu
Gln Leu Leu Asn Ala Pro Asp Leu Cys Ala Lys Asp Phe 2015 2020 2025
Glu Asp Leu Phe Lys Gln Glu Glu Ser Leu Lys Asn Ile Lys Asp 2030
2035 2040 Ser Leu Gln Gln Ser Ser Gly Arg Ile Asp Ile Ile His Ser
Lys 2045 2050 2055 Lys Thr Ala Ala Leu Gln Ser Ala Thr Pro Val Glu
Arg Val Lys 2060 2065 2070 Leu Gln Glu Ala Leu Ser Gln Leu Asp Phe
Gln Trp Glu Lys Val 2075 2080 2085 Asn Lys Met Tyr Lys Asp Arg Gln
Gly Arg Phe Asp Arg Ser Val 2090 2095 2100 Glu Lys Trp Arg Arg Phe
His Tyr Asp Ile Lys Ile Phe Asn Gln 2105 2110 2115 Trp Leu Thr Glu
Ala Glu Gln Phe Leu Arg Lys Thr Gln Ile Pro 2120 2125 2130 Glu Asn
Trp Glu His Ala Lys Tyr Lys Trp Tyr Leu Lys Glu Leu 2135 2140 2145
Gln Asp Gly Ile Gly Gln Arg Gln Thr Val Val Arg Thr Leu Asn 2150
2155 2160 Ala Thr Gly Glu Glu Ile Ile Gln Gln Ser Ser Lys Thr Asp
Ala 2165 2170 2175 Ser Ile Leu Gln Glu Lys Leu Gly Ser Leu Asn Leu
Arg Trp Gln 2180 2185 2190 Glu Val Cys Lys Gln Leu Ser Asp Arg Lys
Lys Arg Leu Glu Glu 2195 2200 2205 Gln Lys Asn Ile Leu Ser Glu Phe
Gln Arg Asp Leu Asn Glu Phe 2210 2215 2220 Val Leu Trp Leu Glu Glu
Ala Asp Asn Ile Ala Ser Ile Pro Leu 2225 2230 2235 Glu Pro Gly Lys
Glu Gln Gln
Leu Lys Glu Lys Leu Glu Gln Val 2240 2245 2250 Lys Leu Leu Val Glu
Glu Leu Pro Leu Arg Gln Gly Ile Leu Lys 2255 2260 2265 Gln Leu Asn
Glu Thr Gly Gly Pro Val Leu Val Ser Ala Pro Ile 2270 2275 2280 Ser
Pro Glu Glu Gln Asp Lys Leu Glu Asn Lys Leu Lys Gln Thr 2285 2290
2295 Asn Leu Gln Trp Ile Lys Val Ser Arg Ala Leu Pro Glu Lys Gln
2300 2305 2310 Gly Glu Ile Glu Ala Gln Ile Lys Asp Leu Gly Gln Leu
Glu Lys 2315 2320 2325 Lys Leu Glu Asp Leu Glu Glu Gln Leu Asn His
Leu Leu Leu Trp 2330 2335 2340 Leu Ser Pro Ile Arg Asn Gln Leu Glu
Ile Tyr Asn Gln Pro Asn 2345 2350 2355 Gln Glu Gly Pro Phe Asp Val
Gln Glu Thr Glu Ile Ala Val Gln 2360 2365 2370 Ala Lys Gln Pro Asp
Val Glu Glu Ile Leu Ser Lys Gly Gln His 2375 2380 2385 Leu Tyr Lys
Glu Lys Pro Ala Thr Gln Pro Val Lys Arg Lys Leu 2390 2395 2400 Glu
Asp Leu Ser Ser Glu Trp Lys Ala Val Asn Arg Leu Leu Gln 2405 2410
2415 Glu Leu Arg Ala Lys Gln Pro Asp Leu Ala Pro Gly Leu Thr Thr
2420 2425 2430 Ile Gly Ala Ser Pro Thr Gln Thr Val Thr Leu Val Thr
Gln Pro 2435 2440 2445 Val Val Thr Lys Glu Thr Ala Ile Ser Lys Leu
Glu Met Pro Ser 2450 2455 2460 Ser Leu Met Leu Glu Val Pro Ala Leu
Ala Asp Phe Asn Arg Ala 2465 2470 2475 Trp Thr Glu Leu Thr Asp Trp
Leu Ser Leu Leu Asp Gln Val Ile 2480 2485 2490 Lys Ser Gln Arg Val
Met Val Gly Asp Leu Glu Asp Ile Asn Glu 2495 2500 2505 Met Ile Ile
Lys Gln Lys Ala Thr Met Gln Asp Leu Glu Gln Arg 2510 2515 2520 Arg
Pro Gln Leu Glu Glu Leu Ile Thr Ala Ala Gln Asn Leu Lys 2525 2530
2535 Asn Lys Thr Ser Asn Gln Glu Ala Arg Thr Ile Ile Thr Asp Arg
2540 2545 2550 Ile Glu Arg Ile Gln Asn Gln Trp Asp Glu Val Gln Glu
His Leu 2555 2560 2565 Gln Asn Arg Arg Gln Gln Leu Asn Glu Met Leu
Lys Asp Ser Thr 2570 2575 2580 Gln Trp Leu Glu Ala Lys Glu Glu Ala
Glu Gln Val Leu Gly Gln 2585 2590 2595 Ala Arg Ala Lys Leu Glu Ser
Trp Lys Glu Gly Pro Tyr Thr Val 2600 2605 2610 Asp Ala Ile Gln Lys
Lys Ile Thr Glu Thr Lys Gln Leu Ala Lys 2615 2620 2625 Asp Leu Arg
Gln Trp Gln Thr Asn Val Asp Val Ala Asn Asp Leu 2630 2635 2640 Ala
Leu Lys Leu Leu Arg Asp Tyr Ser Ala Asp Asp Thr Arg Lys 2645 2650
2655 Val His Met Ile Thr Glu Asn Ile Asn Ala Ser Trp Arg Ser Ile
2660 2665 2670 His Lys Arg Val Ser Glu Arg Glu Ala Ala Leu Glu Glu
Thr His 2675 2680 2685 Arg Leu Leu Gln Gln Phe Pro Leu Asp Leu Glu
Lys Phe Leu Ala 2690 2695 2700 Trp Leu Thr Glu Ala Glu Thr Thr Ala
Asn Val Leu Gln Asp Ala 2705 2710 2715 Thr Arg Lys Glu Arg Leu Leu
Glu Asp Ser Lys Gly Val Lys Glu 2720 2725 2730 Leu Met Lys Gln Trp
Gln Asp Leu Gln Gly Glu Ile Glu Ala His 2735 2740 2745 Thr Asp Val
Tyr His Asn Leu Asp Glu Asn Ser Gln Lys Ile Leu 2750 2755 2760 Arg
Ser Leu Glu Gly Ser Asp Asp Ala Val Leu Leu Gln Arg Arg 2765 2770
2775 Leu Asp Asn Met Asn Phe Lys Trp Ser Glu Leu Arg Lys Lys Ser
2780 2785 2790 Leu Asn Ile Arg Ser His Leu Glu Ala Ser Ser Asp Gln
Trp Lys 2795 2800 2805 Arg Leu His Leu Ser Leu Gln Glu Leu Leu Val
Trp Leu Gln Leu 2810 2815 2820 Lys Asp Asp Glu Leu Ser Arg Gln Ala
Pro Ile Gly Gly Asp Phe 2825 2830 2835 Pro Ala Val Gln Lys Gln Asn
Asp Val His Arg Ala Phe Lys Arg 2840 2845 2850 Glu Leu Lys Thr Lys
Glu Pro Val Ile Met Ser Thr Leu Glu Thr 2855 2860 2865 Val Arg Ile
Phe Leu Thr Glu Gln Pro Leu Glu Gly Leu Glu Lys 2870 2875 2880 Leu
Tyr Gln Glu Pro Arg Glu Leu Pro Pro Glu Glu Arg Ala Gln 2885 2890
2895 Asn Val Thr Arg Leu Leu Arg Lys Gln Ala Glu Glu Val Asn Thr
2900 2905 2910 Glu Trp Glu Lys Leu Asn Leu His Ser Ala Asp Trp Gln
Arg Lys 2915 2920 2925 Ile Asp Glu Thr Leu Glu Arg Leu Gln Glu Leu
Gln Glu Ala Thr 2930 2935 2940 Asp Glu Leu Asp Leu Lys Leu Arg Gln
Ala Glu Val Ile Lys Gly 2945 2950 2955 Ser Trp Gln Pro Val Gly Asp
Leu Leu Ile Asp Ser Leu Gln Asp 2960 2965 2970 His Leu Glu Lys Val
Lys Ala Leu Arg Gly Glu Ile Ala Pro Leu 2975 2980 2985 Lys Glu Asn
Val Ser His Val Asn Asp Leu Ala Arg Gln Leu Thr 2990 2995 3000 Thr
Leu Gly Ile Gln Leu Ser Pro Tyr Asn Leu Ser Thr Leu Glu 3005 3010
3015 Asp Leu Asn Thr Arg Trp Lys Leu Leu Gln Val Ala Val Glu Asp
3020 3025 3030 Arg Val Arg Gln Leu His Glu Ala His Arg Asp Phe Gly
Pro Ala 3035 3040 3045 Ser Gln His Phe Leu Ser Thr Ser Val Gln Gly
Pro Trp Glu Arg 3050 3055 3060 Ala Ile Ser Pro Asn Lys Val Pro Tyr
Tyr Ile Asn His Glu Thr 3065 3070 3075 Gln Thr Thr Cys Trp Asp His
Pro Lys Met Thr Glu Leu Tyr Gln 3080 3085 3090 Ser Leu Ala Asp Leu
Asn Asn Val Arg Phe Ser Ala Tyr Arg Thr 3095 3100 3105 Ala Met Lys
Leu Arg Arg Leu Gln Lys Ala Leu Cys Leu Asp Leu 3110 3115 3120 Leu
Ser Leu Ser Ala Ala Cys Asp Ala Leu Asp Gln His Asn Leu 3125 3130
3135 Lys Gln Asn Asp Gln Pro Met Asp Ile Leu Gln Ile Ile Asn Cys
3140 3145 3150 Leu Thr Thr Ile Tyr Asp Arg Leu Glu Gln Glu His Asn
Asn Leu 3155 3160 3165 Val Asn Val Pro Leu Cys Val Asp Met Cys Leu
Asn Trp Leu Leu 3170 3175 3180 Asn Val Tyr Asp Thr Gly Arg Thr Gly
Arg Ile Arg Val Leu Ser 3185 3190 3195 Phe Lys Thr Gly Ile Ile Ser
Leu Cys Lys Ala His Leu Glu Asp 3200 3205 3210 Lys Tyr Arg Tyr Leu
Phe Lys Gln Val Ala Ser Ser Thr Gly Phe 3215 3220 3225 Cys Asp Gln
Arg Arg Leu Gly Leu Leu Leu His Asp Ser Ile Gln 3230 3235 3240 Ile
Pro Arg Gln Leu Gly Glu Val Ala Ser Phe Gly Gly Ser Asn 3245 3250
3255 Ile Glu Pro Ser Val Arg Ser Cys Phe Gln Phe Ala Asn Asn Lys
3260 3265 3270 Pro Glu Ile Glu Ala Ala Leu Phe Leu Asp Trp Met Arg
Leu Glu 3275 3280 3285 Pro Gln Ser Met Val Trp Leu Pro Val Leu His
Arg Val Ala Ala 3290 3295 3300 Ala Glu Thr Ala Lys His Gln Ala Lys
Cys Asn Ile Cys Lys Glu 3305 3310 3315 Cys Pro Ile Ile Gly Phe Arg
Tyr Arg Ser Leu Lys His Phe Asn 3320 3325 3330 Tyr Asp Ile Cys Gln
Ser Cys Phe Phe Ser Gly Arg Val Ala Lys 3335 3340 3345 Gly His Lys
Met His Tyr Pro Met Val Glu Tyr Cys Thr Pro Thr 3350 3355 3360 Thr
Ser Gly Glu Asp Val Arg Asp Phe Ala Lys Val Leu Lys Asn 3365 3370
3375 Lys Phe Arg Thr Lys Arg Tyr Phe Ala Lys His Pro Arg Met Gly
3380 3385 3390 Tyr Leu Pro Val Gln Thr Val Leu Glu Gly Asp Asn Met
Glu Thr 3395 3400 3405 Pro Val Thr Leu Ile Asn Phe Trp Pro Val Asp
Ser Ala Pro Ala 3410 3415 3420 Ser Ser Pro Gln Leu Ser His Asp Asp
Thr His Ser Arg Ile Glu 3425 3430 3435 His Tyr Ala Ser Arg Leu Ala
Glu Met Glu Asn Ser Asn Gly Ser 3440 3445 3450 Tyr Leu Asn Asp Ser
Ile Ser Pro Asn Glu Ser Ile Asp Asp Glu 3455 3460 3465 His Leu Leu
Ile Gln His Tyr Cys Gln Ser Leu Asn Gln Asp Ser 3470 3475 3480 Pro
Leu Ser Gln Pro Arg Ser Pro Ala Gln Ile Leu Ile Ser Leu 3485 3490
3495 Glu Ser Glu Glu Arg Gly Glu Leu Glu Arg Ile Leu Ala Asp Leu
3500 3505 3510 Glu Glu Glu Asn Arg Asn Leu Gln Ala Glu Tyr Asp Arg
Leu Lys 3515 3520 3525 Gln Gln His Glu His Lys Gly Leu Ser Pro Leu
Pro Ser Pro Pro 3530 3535 3540 Glu Met Met Pro Thr Ser Pro Gln Ser
Pro Arg Asp Ala Glu Leu 3545 3550 3555 Ile Ala Glu Ala Lys Leu Leu
Arg Gln His Lys Gly Arg Leu Glu 3560 3565 3570 Ala Arg Met Gln Ile
Leu Glu Asp His Asn Lys Gln Leu Glu Ser 3575 3580 3585 Gln Leu His
Arg Leu Arg Gln Leu Leu Glu Gln Pro Gln Ala Glu 3590 3595 3600 Ala
Lys Val Asn Gly Thr Thr Val Ser Ser Pro Ser Thr Ser Leu 3605 3610
3615 Gln Arg Ser Asp Ser Ser Gln Pro Met Leu Leu Arg Val Val Gly
3620 3625 3630 Ser Gln Thr Ser Asp Ser Met Gly Glu Glu Asp Leu Leu
Ser Pro 3635 3640 3645 Pro Gln Asp Thr Ser Thr Gly Leu Glu Glu Val
Met Glu Gln Leu 3650 3655 3660 Asn Asn Ser Phe Pro Ser Ser Arg Gly
Arg Asn Thr Pro Gly Lys 3665 3670 3675 Pro Met Arg Glu Asp Thr Met
3680 3685 225RNAArtificialoligonucleotide 2guaccuccaa caucaaggaa
gaugg 25325RNAArtificialoligonucleotide 3uaccuccaac aucaaggaag
auggc 25425RNAArtificialoligonucleotide 4accuccaaca ucaaggaaga
uggca 25525RNAArtificialoligonucleotide 5ccuccaacau caaggaagau
ggcau 25625RNAArtificialoligonucleotide 6cuccaacauc aaggaagaug
gcauu 25725RNAArtificialoligonucleotide 7uccaacauca aggaagaugg
cauuu 25825RNAArtificialoligonucleotide 8ccaacaucaa ggaagauggc
auuuc 25925RNAArtificialoligonucleotide 9caacaucaag gaagauggca
uuucu 251025RNAArtificialoligonucleotide 10aacaucaagg aagauggcau
uucua 251125RNAArtificialoligonucleotide 11acaucaagga agauggcauu
ucuag 251225RNAArtificialoligonucleotide 12caucaaggaa gauggcauuu
cuagu 251325RNAArtificialoligonucleotide 13aucaaggaag auggcauuuc
uaguu 251425RNAArtificialoligonucleotide 14ucaaggaaga uggcauuucu
aguuu 251525RNAArtificialoligonucleotide 15caaggaagau ggcauuucua
guuug 251625RNAArtificialoligonucleotide 16aaggaagaug gcauuucuag
uuugg 251725RNAArtificialoligonucleotide 17aggaagaugg cauuucuagu
uugga 251825RNAArtificialoligonucleotide 18ggaagauggc auuucuaguu
uggag 251925RNAArtificialoligonucleotide 19gaagauggca uuucuaguuu
ggaga 252025RNAArtificialoligonucleotide 20aagauggcau uucuaguuug
gagau 252125RNAArtificialoligonucleotide 21agauggcauu ucuaguuugg
agaug 252225RNAArtificialoligonucleotide 22gauggcauuu cuaguuugga
gaugg 252325RNAArtificialoligonucleotide 23auggcauuuc uaguuuggag
auggc 252425RNAArtificialoligonucleotide 24uggcauuucu aguuuggaga
uggca 252525RNAArtificialoligonucleotide 25ggcauuucua guuuggagau
ggcag 252625RNAArtificialoligonucleotide 26gcauuucuag uuuggagaug
gcagu 252725RNAArtificialoligonucleotide 27cauuucuagu uuggagaugg
caguu 252825RNAArtificialoligonucleotide 28auuucuaguu uggagauggc
aguuu 252925RNAArtificialoligonucleotide 29uuucuaguuu ggagauggca
guuuc 253025RNAArtificialoligonucleotide 30uucuaguuug gagauggcag
uuucc 253125RNAArtificialoligonucleotide 31ucuaguuugg agauggcagu
uuccu 253225RNAArtificialoligonucleotide 32cuaguuugga gauggcaguu
uccuu 253325RNAArtificialoligonucleotide 33uaguuuggag auggcaguuu
ccuua 253425RNAArtificialoligonucleotide 34aguuuggaga uggcaguuuc
cuuag 253525RNAArtificialoligonucleotide 35guuuggagau ggcaguuucc
uuagu 253625RNAArtificialoligonucleotide 36uuuggagaug gcaguuuccu
uagua 253725RNAArtificialoligonucleotide 37uuggagaugg caguuuccuu
aguaa 253825RNAArtificialoligonucleotide 38uggagauggc aguuuccuua
guaac 253925RNAArtificialoligonucleotide 39gagauggcag uuuccuuagu
aacca 254025RNAArtificialoligonucleotide 40agauggcagu uuccuuagua
accac 254125RNAArtificialoligonucleotide 41gauggcaguu uccuuaguaa
ccaca 254225RNAArtificialoligonucleotide 42auggcaguuu ccuuaguaac
cacag 254325RNAArtificialoligonucleotide 43uggcaguuuc cuuaguaacc
acagg 254425RNAArtificialoligonucleotide 44ggcaguuucc uuaguaacca
caggu 254525RNAArtificialoligonucleotide 45gcaguuuccu uaguaaccac
agguu 254625RNAArtificialoligonucleotide 46caguuuccuu aguaaccaca
gguug 254725RNAArtificialoligonucleotide 47aguuuccuua guaaccacag
guugu 254825RNAArtificialoligonucleotide 48guuuccuuag uaaccacagg
uugug 254925RNAArtificialoligonucleotide 49uuuccuuagu aaccacaggu
ugugu 255025RNAArtificialoligonucleotide 50uuccuuagua accacagguu
guguc 255125RNAArtificialoligonucleotide 51uccuuaguaa ccacagguug
uguca 255225RNAArtificialoligonucleotide 52ccuuaguaac cacagguugu
gucac 255325RNAArtificialoligonucleotide 53cuuaguaacc acagguugug
ucacc 255425RNAArtificialoligonucleotide 54uuaguaacca cagguugugu
cacca 255525RNAArtificialoligonucleotide 55uaguaaccac agguuguguc
accag 255625RNAArtificialoligonucleotide 56aguaaccaca gguuguguca
ccaga 255725RNAArtificialoligonucleotide 57guaaccacag guugugucac
cagag 255825RNAArtificialoligonucleotide 58uaaccacagg uugugucacc
agagu 255925RNAArtificialoligonucleotide
59aaccacaggu ugugucacca gagua 256025RNAArtificialoligonucleotide
60accacagguu gugucaccag aguaa 256125RNAArtificialoligonucleotide
61ccacagguug ugucaccaga guaac 256225RNAArtificialoligonucleotide
62cacagguugu gucaccagag uaaca 256325RNAArtificialoligonucleotide
63acagguugug ucaccagagu aacag 256425RNAArtificialoligonucleotide
64cagguugugu caccagagua acagu 256525RNAArtificialoligonucleotide
65agguuguguc accagaguaa caguc 256625RNAArtificialoligonucleotide
66gguuguguca ccagaguaac agucu 256725RNAArtificialoligonucleotide
67guugugucac cagaguaaca gucug 256825RNAArtificialoligonucleotide
68uugugucacc agaguaacag ucuga 256925RNAArtificialoligonucleotide
69ugugucacca gaguaacagu cugag 257025RNAArtificialoligonucleotide
70gugucaccag aguaacaguc ugagu 257125RNAArtificialoligonucleotide
71ugucaccaga guaacagucu gagua 257225RNAArtificialoligonucleotide
72gucaccagag uaacagucug aguag 257325RNAArtificialoligonucleotide
73ucaccagagu aacagucuga guagg 257425RNAArtificialoligonucleotide
74caccagagua acagucugag uagga 257525RNAArtificialoligonucleotide
75accagaguaa cagucugagu aggag 257625RNAArtificialoligonucleotide
76uuugccgcug cccaaugcca uccug 257725RNAArtificialoligonucleotide
77auucaauguu cugacaacag uuugc 257825RNAArtificialoligonucleotide
78ccaguugcau ucaauguucu gacaa 257922RNAArtificialoligonucleotide
79caguugcauu caauguucug ac 228020RNAArtificialoligonucleotide
80aguugcauuc aauguucuga 208121RNAArtificialoligonucleotide
81gauugcugaa uuauuucuuc c 218225RNAArtificialoligonucleotide
82gauugcugaa uuauuucuuc cccag 258325RNAArtificialoligonucleotide
83auugcugaau uauuucuucc ccagu 258425RNAArtificialoligonucleotide
84uugcugaauu auuucuuccc caguu 258525RNAArtificialoligonucleotide
85ugcugaauua uuucuucccc aguug 258625RNAArtificialoligonucleotide
86gcugaauuau uucuucccca guugc 258725RNAArtificialoligonucleotide
87cugaauuauu ucuuccccag uugca 258825RNAArtificialoligonucleotide
88ugaauuauuu cuuccccagu ugcau 258925RNAArtificialoligonucleotide
89gaauuauuuc uuccccaguu gcauu 259025RNAArtificialoligonucleotide
90aauuauuucu uccccaguug cauuc 259125RNAArtificialoligonucleotide
91auuauuucuu ccccaguugc auuca 259225RNAArtificialoligonucleotide
92uuauuucuuc cccaguugca uucaa 259325RNAArtificialoligonucleotide
93uauuucuucc ccaguugcau ucaau 259425RNAArtificialoligonucleotide
94auuucuuccc caguugcauu caaug 259525RNAArtificialoligonucleotide
95uuucuucccc aguugcauuc aaugu 259625RNAArtificialoligonucleotide
96uucuucccca guugcauuca auguu 259725RNAArtificialoligonucleotide
97ucuuccccag uugcauucaa uguuc 259825RNAArtificialoligonucleotide
98cuuccccagu ugcauucaau guucu 259925RNAArtificialoligonucleotide
99uuccccaguu gcauucaaug uucug 2510025RNAArtificialoligonucleotide
100uccccaguug cauucaaugu ucuga 2510125RNAArtificialoligonucleotide
101ccccaguugc auucaauguu cugac 2510225RNAArtificialoligonucleotide
102cccaguugca uucaauguuc ugaca 2510325RNAArtificialoligonucleotide
103ccaguugcau ucaauguucu gacaa 2510425RNAArtificialoligonucleotide
104caguugcauu caauguucug acaac 2510525RNAArtificialoligonucleotide
105aguugcauuc aauguucuga caaca 2510620RNAArtificialoligonucleotide
106uccuguagaa uacuggcauc 2010727RNAArtificialoligonucleotide
107ugcagaccuc cugccaccgc agauuca
2710834RNAArtificialoligonucleotide 108uugcagaccu ccugccaccg
cagauucagg cuuc 3410925RNAArtificialoligonucleotide 109guugcauuca
auguucugac aacag 2511025RNAArtificialoligonucleotide 110uugcauucaa
uguucugaca acagu 2511125RNAArtificialoligonucleotide 111ugcauucaau
guucugacaa caguu 2511225RNAArtificialoligonucleotide 112gcauucaaug
uucugacaac aguuu 2511325RNAArtificialoligonucleotide 113cauucaaugu
ucugacaaca guuug 2511425RNAArtificialoligonucleotide 114auucaauguu
cugacaacag uuugc 2511525RNAArtificialoligonucleotide 115ucaauguucu
gacaacaguu ugccg 2511625RNAArtificialoligonucleotide 116caauguucug
acaacaguuu gccgc 2511725RNAArtificialoligonucleotide 117aauguucuga
caacaguuug ccgcu 2511825RNAArtificialoligonucleotide 118auguucugac
aacaguuugc cgcug 2511925RNAArtificialoligonucleotide 119uguucugaca
acaguuugcc gcugc 2512025RNAArtificialoligonucleotide 120guucugacaa
caguuugccg cugcc 2512125RNAArtificialoligonucleotide 121uucugacaac
aguuugccgc ugccc 2512225RNAArtificialoligonucleotide 122ucugacaaca
guuugccgcu gccca 2512325RNAArtificialoligonucleotide 123cugacaacag
uuugccgcug cccaa 2512425RNAArtificialoligonucleotide 124ugacaacagu
uugccgcugc ccaau 2512525RNAArtificialoligonucleotide 125gacaacaguu
ugccgcugcc caaug 2512625RNAArtificialoligonucleotide 126acaacaguuu
gccgcugccc aaugc 2512725RNAArtificialoligonucleotide 127caacaguuug
ccgcugccca augcc 2512825RNAArtificialoligonucleotide 128aacaguuugc
cgcugcccaa ugcca 2512925RNAArtificialoligonucleotide 129acaguuugcc
gcugcccaau gccau 2513025RNAArtificialoligonucleotide 130caguuugccg
cugcccaaug ccauc 2513125RNAArtificialoligonucleotide 131aguuugccgc
ugcccaaugc caucc 2513225RNAArtificialoligonucleotide 132guuugccgcu
gcccaaugcc auccu 2513325RNAArtificialoligonucleotide 133uuugccgcug
cccaaugcca uccug 2513425RNAArtificialoligonucleotide 134uugccgcugc
ccaaugccau ccugg 2513525RNAArtificialoligonucleotide 135ugccgcugcc
caaugccauc cugga 2513625RNAArtificialoligonucleotide 136gccgcugccc
aaugccaucc uggag 2513725RNAArtificialoligonucleotide 137ccgcugccca
augccauccu ggagu 2513825RNAArtificialoligonucleotide 138cgcugcccaa
ugccauccug gaguu 2513920RNAArtificialoligonucleotide 139uguuuuugag
gauugcugaa 2014040RNAArtificialoligonucleotide 140uguucugaca
acaguuugcc gcugcccaau gccauccugg
4014125RNAArtificialoligonucleotide 141cucuggccug uccuaagacc ugcuc
2514225RNAArtificialoligonucleotide 142ucuggccugu ccuaagaccu gcuca
2514325RNAArtificialoligonucleotide 143cuggccuguc cuaagaccug cucag
2514425RNAArtificialoligonucleotide 144uggccugucc uaagaccugc ucagc
2514525RNAArtificialoligonucleotide 145ggccuguccu aagaccugcu cagcu
2514625RNAArtificialoligonucleotide 146gccuguccua agaccugcuc agcuu
2514725RNAArtificialoligonucleotide 147ccuguccuaa gaccugcuca gcuuc
2514825RNAArtificialoligonucleotide 148cuguccuaag accugcucag cuucu
2514925RNAArtificialoligonucleotide 149uguccuaaga ccugcucagc uucuu
2515025RNAArtificialoligonucleotide 150guccuaagac cugcucagcu ucuuc
2515125RNAArtificialoligonucleotide 151uccuaagacc ugcucagcuu cuucc
2515225RNAArtificialoligonucleotide 152ccuaagaccu gcucagcuuc uuccu
2515325RNAArtificialoligonucleotide 153cuaagaccug cucagcuucu uccuu
2515425RNAArtificialoligonucleotide 154uaagaccugc ucagcuucuu ccuua
2515525RNAArtificialoligonucleotide 155aagaccugcu cagcuucuuc cuuag
2515625RNAArtificialoligonucleotide 156agaccugcuc agcuucuucc uuagc
2515725RNAArtificialoligonucleotide 157gaccugcuca gcuucuuccu uagcu
2515825RNAArtificialoligonucleotide 158accugcucag cuucuuccuu agcuu
2515925RNAArtificialoligonucleotide 159ccugcucagc uucuuccuua gcuuc
2516025RNAArtificialoligonucleotide 160cugcucagcu ucuuccuuag cuucc
2516125RNAArtificialoligonucleotide 161ugcucagcuu cuuccuuagc uucca
2516225RNAArtificialoligonucleotide 162gcucagcuuc uuccuuagcu uccag
2516325RNAArtificialoligonucleotide 163cucagcuucu uccuuagcuu ccagc
2516425RNAArtificialoligonucleotide 164ucagcuucuu ccuuagcuuc cagcc
2516525RNAArtificialoligonucleotide 165cagcuucuuc cuuagcuucc agcca
2516625RNAArtificialoligonucleotide 166agcuucuucc uuagcuucca gccau
2516725RNAArtificialoligonucleotide 167gcuucuuccu uagcuuccag ccauu
2516825RNAArtificialoligonucleotide 168cuucuuccuu agcuuccagc cauug
2516925RNAArtificialoligonucleotide 169uucuuccuua gcuuccagcc auugu
2517025RNAArtificialoligonucleotide 170ucuuccuuag cuuccagcca uugug
2517125RNAArtificialoligonucleotide 171cuuccuuagc uuccagccau ugugu
2517225RNAArtificialoligonucleotide 172uuccuuagcu uccagccauu guguu
2517325RNAArtificialoligonucleotide 173uccuuagcuu ccagccauug uguug
2517425RNAArtificialoligonucleotide 174ccuuagcuuc cagccauugu guuga
2517525RNAArtificialoligonucleotide 175cuuagcuucc agccauugug uugaa
2517625RNAArtificialoligonucleotide 176uuagcuucca gccauugugu ugaau
2517725RNAArtificialoligonucleotide 177uagcuuccag ccauuguguu gaauc
2517825RNAArtificialoligonucleotide 178agcuuccagc cauuguguug aaucc
2517925RNAArtificialoligonucleotide 179gcuuccagcc auuguguuga auccu
2518025RNAArtificialoligonucleotide 180cuuccagcca uuguguugaa uccuu
2518125RNAArtificialoligonucleotide 181uuccagccau uguguugaau ccuuu
2518225RNAArtificialoligonucleotide 182uccagccauu guguugaauc cuuua
2518325RNAArtificialoligonucleotide 183ccagccauug uguugaaucc uuuaa
2518425RNAArtificialoligonucleotide 184cagccauugu guugaauccu uuaac
2518525RNAArtificialoligonucleotide 185agccauugug uugaauccuu uaaca
2518625RNAArtificialoligonucleotide 186gccauugugu ugaauccuuu aacau
2518725RNAArtificialoligonucleotide 187ccauuguguu gaauccuuua acauu
2518825RNAArtificialoligonucleotide 188cauuguguug aauccuuuaa cauuu
2518920RNAArtificialoligonucleotide 189ucagcuucug uuagccacug
2019020RNAArtificialoligonucleotide 190uucagcuucu guuagccacu
2019121RNAArtificialoligonucleotide 191uucagcuucu guuagccacu g
2119221RNAArtificialoligonucleotide 192ucagcuucug uuagccacug a
2119322RNAArtificialoligonucleotide 193uucagcuucu guuagccacu ga
2219421RNAArtificialoligonucleotide 194ucagcuucug uuagccacug a
2119522RNAArtificialoligonucleotide 195uucagcuucu guuagccacu ga
2219622RNAArtificialoligonucleotide 196ucagcuucug uuagccacug au
2219723RNAArtificialoligonucleotide 197uucagcuucu guuagccacu gau
2319823RNAArtificialoligonucleotide 198ucagcuucug uuagccacug auu
2319924RNAArtificialoligonucleotide 199uucagcuucu guuagccacu gauu
2420024RNAArtificialoligonucleotide 200ucagcuucug uuagccacug auua
2420124RNAArtificialoligonucleotide 201uucagcuucu guuagccacu gaua
2420225RNAArtificialoligonucleotide 202ucagcuucug uuagccacug auuaa
2520326RNAArtificialoligonucleotide 203uucagcuucu guuagccacu gauuaa
2620426RNAArtificialoligonucleotide 204ucagcuucug uuagccacug auuaaa
2620527RNAArtificialoligonucleotide 205uucagcuucu guuagccacu
gauuaaa 2720619RNAArtificialoligonucleotide 206cagcuucugu uagccacug
1920721RNAArtificialoligonucleotide 207cagcuucugu uagccacuga u
2120821RNAArtificialoligonucleotide 208agcuucuguu agccacugau u
2120922RNAArtificialoligonucleotide 209cagcuucugu uagccacuga uu
2221022RNAArtificialoligonucleotide 210agcuucuguu agccacugau ua
2221123RNAArtificialoligonucleotide 211cagcuucugu uagccacuga uua
2321223RNAArtificialoligonucleotide 212agcuucuguu agccacugau uaa
2321324RNAArtificialoligonucleotide 213cagcuucugu uagccacuga uuaa
2421424RNAArtificialoligonucleotide 214agcuucuguu agccacugau uaaa
2421525RNAArtificialoligonucleotide 215cagcuucugu uagccacuga uuaaa
2521624RNAArtificialoligonucleotide 216agcuucuguu agccacugau uaaa
2421720RNAArtificialoligonucleotide 217agcuucuguu agccacugau
2021820RNAArtificialoligonucleotide 218gcuucuguua gccacugauu
2021921RNAArtificialoligonucleotide 219agcuucuguu agccacugau u
2122021RNAArtificialoligonucleotide 220gcuucuguua gccacugauu a
2122122RNAArtificialoligonucleotide 221agcuucuguu agccacugau ua
2222222RNAArtificialoligonucleotide 222gcuucuguua gccacugauu aa
2222323RNAArtificialoligonucleotide 223agcuucuguu agccacugau uaa
2322423RNAArtificialoligonucleotide 224gcuucuguua gccacugauu aaa
2322524RNAArtificialoligonucleotide 225agcuucuguu agccacugau uaaa
2422623RNAArtificialoligonucleotide 226gcuucuguua gccacugauu
aaa
2322723RNAArtificialoligonucleotide 227ccauuuguau uuagcauguu ccc
2322820RNAArtificialoligonucleotide 228agauaccauu uguauuuagc
2022919RNAArtificialoligonucleotide 229gccauuucuc aacagaucu
1923023RNAArtificialoligonucleotide 230gccauuucuc aacagaucug uca
2323123RNAArtificialoligonucleotide 231auucucagga auuugugucu uuc
2323221RNAArtificialoligonucleotide 232ucucaggaau uugugucuuu c
2123318RNAArtificialoligonucleotide 233guucagcuuc uguuagcc
1823421RNAArtificialoligonucleotide 234cugauuaaau aucuuuauau c
2123518RNAArtificialoligonucleotide 235gccgccauuu cucaacag
1823618RNAArtificialoligonucleotide 236guauuuagca uguuccca
1823718RNAArtificialoligonucleotide 237caggaauuug ugucuuuc
1823825RNAArtificialoligonucleotide 238gcuuuucuuu uaguugcugc ucuuu
2523925RNAArtificialoligonucleotide 239cuuuucuuuu aguugcugcu cuuuu
2524025RNAArtificialoligonucleotide 240uuuucuuuua guugcugcuc uuuuc
2524125RNAArtificialoligonucleotide 241uuucuuuuag uugcugcucu uuucc
2524225RNAArtificialoligonucleotide 242uucuuuuagu ugcugcucuu uucca
2524325RNAArtificialoligonucleotide 243ucuuuuaguu gcugcucuuu uccag
2524425RNAArtificialoligonucleotide 244cuuuuaguug cugcucuuuu ccagg
2524525RNAArtificialoligonucleotide 245uuuuaguugc ugcucuuuuc caggu
2524625RNAArtificialoligonucleotide 246uuuaguugcu gcucuuuucc agguu
2524725RNAArtificialoligonucleotide 247uuaguugcug cucuuuucca gguuc
2524825RNAArtificialoligonucleotide 248uaguugcugc ucuuuuccag guuca
2524925RNAArtificialoligonucleotide 249aguugcugcu cuuuuccagg uucaa
2525025RNAArtificialoligonucleotide 250guugcugcuc uuuuccaggu ucaag
2525125RNAArtificialoligonucleotide 251uugcugcucu uuuccagguu caagu
2525225RNAArtificialoligonucleotide 252ugcugcucuu uuccagguuc aagug
2525325RNAArtificialoligonucleotide 253gcugcucuuu uccagguuca agugg
2525425RNAArtificialoligonucleotide 254cugcucuuuu ccagguucaa guggg
2525525RNAArtificialoligonucleotide 255ugcucuuuuc cagguucaag uggga
2525625RNAArtificialoligonucleotide 256gcucuuuucc agguucaagu gggac
2525725RNAArtificialoligonucleotide 257cucuuuucca gguucaagug ggaua
2525825RNAArtificialoligonucleotide 258ucuuuuccag guucaagugg gauac
2525920RNAArtificialoligonucleotide 259ucuuuuccag guucaagugg
2026025RNAArtificialoligonucleotide 260cuuuuccagg uucaaguggg auacu
2526125RNAArtificialoligonucleotide 261uuuuccaggu ucaaguggga uacua
2526225RNAArtificialoligonucleotide 262uuuccagguu caagugggau acuag
2526325RNAArtificialoligonucleotide 263uuccagguuc aagugggaua cuagc
2526425RNAArtificialoligonucleotide 264uccagguuca agugggauac uagca
2526525RNAArtificialoligonucleotide 265ccagguucaa gugggauacu agcaa
2526625RNAArtificialoligonucleotide 266cagguucaag ugggauacua gcaau
2526725RNAArtificialoligonucleotide 267agguucaagu gggauacuag caaug
2526825RNAArtificialoligonucleotide 268gguucaagug ggauacuagc aaugu
2526925RNAArtificialoligonucleotide 269guucaagugg gauacuagca auguu
2527025RNAArtificialoligonucleotide 270uucaaguggg auacuagcaa uguua
2527125RNAArtificialoligonucleotide 271ucaaguggga uacuagcaau guuau
2527225RNAArtificialoligonucleotide 272caagugggau acuagcaaug uuauc
2527325RNAArtificialoligonucleotide 273aagugggaua cuagcaaugu uaucu
2527425RNAArtificialoligonucleotide 274agugggauac uagcaauguu aucug
2527525RNAArtificialoligonucleotide 275gugggauacu agcaauguua ucugc
2527625RNAArtificialoligonucleotide 276ugggauacua gcaauguuau cugcu
2527725RNAArtificialoligonucleotide 277gggauacuag caauguuauc ugcuu
2527825RNAArtificialoligonucleotide 278ggauacuagc aauguuaucu gcuuc
2527925RNAArtificialoligonucleotide 279gauacuagca auguuaucug cuucc
2528025RNAArtificialoligonucleotide 280auacuagcaa uguuaucugc uuccu
2528125RNAArtificialoligonucleotide 281uacuagcaau guuaucugcu uccuc
2528225RNAArtificialoligonucleotide 282acuagcaaug uuaucugcuu ccucc
2528325RNAArtificialoligonucleotide 283cuagcaaugu uaucugcuuc cucca
2528425RNAArtificialoligonucleotide 284uagcaauguu aucugcuucc uccaa
2528525RNAArtificialoligonucleotide 285agcaauguua ucugcuuccu ccaac
2528625RNAArtificialoligonucleotide 286gcaauguuau cugcuuccuc caacc
2528725RNAArtificialoligonucleotide 287caauguuauc ugcuuccucc aacca
2528825RNAArtificialoligonucleotide 288aauguuaucu gcuuccucca accau
2528925RNAArtificialoligonucleotide 289auguuaucug cuuccuccaa ccaua
2529025RNAArtificialoligonucleotide 290uguuaucugc uuccuccaac cauaa
2529125RNAArtificialoligonucleotide 291agccucuuga uugcuggucu uguuu
2529225RNAArtificialoligonucleotide 292gccucuugau ugcuggucuu guuuu
2529325RNAArtificialoligonucleotide 293ccucuugauu gcuggucuug uuuuu
2529420RNAArtificialoligonucleotide 294ccucuugauu gcuggucuug
2029525RNAArtificialoligonucleotide 295cucuugauug cuggucuugu uuuuc
2529625RNAArtificialoligonucleotide 296ucuugauugc uggucuuguu uuuca
2529725RNAArtificialoligonucleotide 297cuugauugcu ggucuuguuu uucaa
2529825RNAArtificialoligonucleotide 298uugauugcug gucuuguuuu ucaaa
2529925RNAArtificialoligonucleotide 299ugauugcugg ucuuguuuuu caaau
2530025RNAArtificialoligonucleotide 300gauugcuggu cuuguuuuuc aaauu
2530120RNAArtificialoligonucleotide 301gauugcuggu cuuguuuuuc
2030225RNAArtificialoligonucleotide 302auugcugguc uuguuuuuca aauuu
2530325RNAArtificialoligonucleotide 303uugcuggucu uguuuuucaa auuuu
2530425RNAArtificialoligonucleotide 304ugcuggucuu guuuuucaaa uuuug
2530525RNAArtificialoligonucleotide 305gcuggucuug uuuuucaaau uuugg
2530625RNAArtificialoligonucleotide 306cuggucuugu uuuucaaauu uuggg
2530725RNAArtificialoligonucleotide 307uggucuuguu uuucaaauuu ugggc
2530825RNAArtificialoligonucleotide 308ggucuuguuu uucaaauuuu gggca
2530925RNAArtificialoligonucleotide 309gucuuguuuu ucaaauuuug ggcag
2531025RNAArtificialoligonucleotide 310ucuuguuuuu caaauuuugg gcagc
2531125RNAArtificialoligonucleotide 311cuuguuuuuc aaauuuuggg cagcg
2531225RNAArtificialoligonucleotide 312uuguuuuuca aauuuugggc agcgg
2531325RNAArtificialoligonucleotide 313uguuuuucaa auuuugggca gcggu
2531425RNAArtificialoligonucleotide 314guuuuucaaa uuuugggcag cggua
2531525RNAArtificialoligonucleotide 315uuuuucaaau uuugggcagc gguaa
2531625RNAArtificialoligonucleotide 316uuuucaaauu uugggcagcg guaau
2531725RNAArtificialoligonucleotide 317uuucaaauuu ugggcagcgg uaaug
2531825RNAArtificialoligonucleotide 318uucaaauuuu gggcagcggu aauga
2531925RNAArtificialoligonucleotide 319ucaaauuuug ggcagcggua augag
2532025RNAArtificialoligonucleotide 320caaauuuugg gcagcgguaa ugagu
2532125RNAArtificialoligonucleotide 321aaauuuuggg cagcgguaau gaguu
2532225RNAArtificialoligonucleotide 322aauuuugggc agcgguaaug aguuc
2532325RNAArtificialoligonucleotide 323auuuugggca gcgguaauga guucu
2532425RNAArtificialoligonucleotide 324uuuugggcag cgguaaugag uucuu
2532525RNAArtificialoligonucleotide 325uuugggcagc gguaaugagu ucuuc
2532625RNAArtificialoligonucleotide 326uugggcagcg guaaugaguu cuucc
2532725RNAArtificialoligonucleotide 327ugggcagcgg uaaugaguuc uucca
2532825RNAArtificialoligonucleotide 328gggcagcggu aaugaguucu uccaa
2532925RNAArtificialoligonucleotide 329ggcagcggua augaguucuu ccaac
2533025RNAArtificialoligonucleotide 330gcagcgguaa ugaguucuuc caacu
2533125RNAArtificialoligonucleotide 331cagcgguaau gaguucuucc aacug
2533225RNAArtificialoligonucleotide 332agcgguaaug aguucuucca acugg
2533325RNAArtificialoligonucleotide 333gcgguaauga guucuuccaa cuggg
2533425RNAArtificialoligonucleotide 334cgguaaugag uucuuccaac ugggg
2533525RNAArtificialoligonucleotide 335gguaaugagu ucuuccaacu gggga
2533622RNAArtificialoligonucleotide 336gguaaugagu ucuuccaacu gg
2233725RNAArtificialoligonucleotide 337guaaugaguu cuuccaacug gggac
2533825RNAArtificialoligonucleotide 338uaaugaguuc uuccaacugg ggacg
2533925RNAArtificialoligonucleotide 339aaugaguucu uccaacuggg gacgc
2534025RNAArtificialoligonucleotide 340augaguucuu ccaacugggg acgcc
2534125RNAArtificialoligonucleotide 341ugaguucuuc caacugggga cgccu
2534225RNAArtificialoligonucleotide 342gaguucuucc aacuggggac gccuc
2534325RNAArtificialoligonucleotide 343aguucuucca acuggggacg ccucu
2534425RNAArtificialoligonucleotide 344guucuuccaa cuggggacgc cucug
2534525RNAArtificialoligonucleotide 345uucuuccaac uggggacgcc ucugu
2534625RNAArtificialoligonucleotide 346ucuuccaacu ggggacgccu cuguu
2534725RNAArtificialoligonucleotide 347cuuccaacug gggacgccuc uguuc
2534825RNAArtificialoligonucleotide 348uuccaacugg ggacgccucu guucc
2534925RNAArtificialoligonucleotide 349uccaacuggg gacgccucug uucca
2535025RNAArtificialoligonucleotide 350ccaacugggg acgccucugu uccaa
2535125RNAArtificialoligonucleotide 351caacugggga cgccucuguu ccaaa
2535225RNAArtificialoligonucleotide 352aacuggggac gccucuguuc caaau
2535325RNAArtificialoligonucleotide 353acuggggacg ccucuguucc aaauc
2535425RNAArtificialoligonucleotide 354cuggggacgc cucuguucca aaucc
2535525RNAArtificialoligonucleotide 355uggggacgcc ucuguuccaa auccu
2535625RNAArtificialoligonucleotide 356ggggacgccu cuguuccaaa uccug
2535725RNAArtificialoligonucleotide 357gggacgccuc uguuccaaau ccugc
2535825RNAArtificialoligonucleotide 358ggacgccucu guuccaaauc cugca
2535925RNAArtificialoligonucleotide 359gacgccucug uuccaaaucc ugcau
2536025RNAArtificialoligonucleotide 360ccaauagugg ucaguccagg agcua
2536125RNAArtificialoligonucleotide 361caauaguggu caguccagga gcuag
2536225RNAArtificialoligonucleotide 362aauagugguc aguccaggag cuagg
2536325RNAArtificialoligonucleotide 363auagugguca guccaggagc uaggu
2536421RNAArtificialoligonucleotide 364auagugguca guccaggagc u
2136525RNAArtificialoligonucleotide 365uaguggucag uccaggagcu agguc
2536625RNAArtificialoligonucleotide 366aguggucagu ccaggagcua gguca
2536725RNAArtificialoligonucleotide 367guggucaguc caggagcuag gucag
2536825RNAArtificialoligonucleotide 368uggucagucc aggagcuagg ucagg
2536925RNAArtificialoligonucleotide 369ggucagucca ggagcuaggu caggc
2537025RNAArtificialoligonucleotide 370gucaguccag gagcuagguc aggcu
2537125RNAArtificialoligonucleotide 371ucaguccagg agcuagguca ggcug
2537225RNAArtificialoligonucleotide 372caguccagga gcuaggucag gcugc
2537325RNAArtificialoligonucleotide 373aguccaggag cuaggucagg cugcu
2537425RNAArtificialoligonucleotide 374guccaggagc uaggucaggc ugcuu
2537525RNAArtificialoligonucleotide 375uccaggagcu aggucaggcu gcuuu
2537625RNAArtificialoligonucleotide 376ccaggagcua ggucaggcug cuuug
2537725RNAArtificialoligonucleotide 377caggagcuag gucaggcugc uuugc
2537825RNAArtificialoligonucleotide 378aggagcuagg ucaggcugcu uugcc
2537925RNAArtificialoligonucleotide 379ggagcuaggu caggcugcuu ugccc
2538025RNAArtificialoligonucleotide 380gagcuagguc aggcugcuuu gcccu
2538125RNAArtificialoligonucleotide 381agcuagguca ggcugcuuug cccuc
2538225RNAArtificialoligonucleotide 382gcuaggucag gcugcuuugc ccuca
2538325RNAArtificialoligonucleotide 383cucagcucuu gaaguaaacg guuua
2538425RNAArtificialoligonucleotide 384cagcucuuga aguaaacggu uuacc
2538525RNAArtificialoligonucleotide 385gcucuugaag uaaacgguuu accgc
2538625RNAArtificialoligonucleotide 386cuaggucagg cugcuuugcc cucag
2538725RNAArtificialoligonucleotide 387uaggucaggc ugcuuugccc ucagc
2538825RNAArtificialoligonucleotide 388aggucaggcu gcuuugcccu cagcu
2538925RNAArtificialoligonucleotide 389ggucaggcug cuuugcccuc agcuc
2539025RNAArtificialoligonucleotide 390gucaggcugc uuugcccuca gcucu
2539125RNAArtificialoligonucleotide 391ucaggcugcu uugcccucag cucuu
2539225RNAArtificialoligonucleotide 392caggcugcuu ugcccucagc ucuug
2539325RNAArtificialoligonucleotide 393aggcugcuuu gcccucagcu
cuuga
2539425RNAArtificialoligonucleotide 394ggcugcuuug cccucagcuc uugaa
2539525RNAArtificialoligonucleotide 395gcugcuuugc ccucagcucu ugaag
2539625RNAArtificialoligonucleotide 396cugcuuugcc cucagcucuu gaagu
2539725RNAArtificialoligonucleotide 397ugcuuugccc ucagcucuug aagua
2539825RNAArtificialoligonucleotide 398gcuuugcccu cagcucuuga aguaa
2539925RNAArtificialoligonucleotide 399cuuugcccuc agcucuugaa guaaa
2540025RNAArtificialoligonucleotide 400uuugcccuca gcucuugaag uaaac
2540125RNAArtificialoligonucleotide 401uugcccucag cucuugaagu aaacg
2540225RNAArtificialoligonucleotide 402ugcccucagc ucuugaagua aacgg
2540325RNAArtificialoligonucleotide 403gcccucagcu cuugaaguaa acggu
2540425RNAArtificialoligonucleotide 404cccucagcuc uugaaguaaa cgguu
2540520RNAArtificialoligonucleotide 405ccucagcucu ugaaguaaac
2040621RNAArtificialoligonucleotide 406ccucagcucu ugaaguaaac g
2140720RNAArtificialoligonucleotide 407cucagcucuu gaaguaaacg
2040825RNAArtificialoligonucleotide 408ccucagcucu ugaaguaaac gguuu
2540925RNAArtificialoligonucleotide 409ucagcucuug aaguaaacgg uuuac
2541025RNAArtificialoligonucleotide 410agcucuugaa guaaacgguu uaccg
2541125RNAArtificialoligonucleotide 411cucuugaagu aaacgguuua ccgcc
2541225RNAArtificialoligonucleotide 412ccacaggcgu ugcacuuugc aaugc
2541325RNAArtificialoligonucleotide 413cacaggcguu gcacuuugca augcu
2541425RNAArtificialoligonucleotide 414acaggcguug cacuuugcaa ugcug
2541525RNAArtificialoligonucleotide 415caggcguugc acuuugcaau gcugc
2541625RNAArtificialoligonucleotide 416aggcguugca cuuugcaaug cugcu
2541725RNAArtificialoligonucleotide 417ggcguugcac uuugcaaugc ugcug
2541825RNAArtificialoligonucleotide 418gcguugcacu uugcaaugcu gcugu
2541925RNAArtificialoligonucleotide 419cguugcacuu ugcaaugcug cuguc
2542023RNAArtificialoligonucleotide 420cguugcacuu ugcaaugcug cug
2342125RNAArtificialoligonucleotide 421guugcacuuu gcaaugcugc ugucu
2542225RNAArtificialoligonucleotide 422uugcacuuug caaugcugcu gucuu
2542325RNAArtificialoligonucleotide 423ugcacuuugc aaugcugcug ucuuc
2542425RNAArtificialoligonucleotide 424gcacuuugca augcugcugu cuucu
2542525RNAArtificialoligonucleotide 425cacuuugcaa ugcugcuguc uucuu
2542625RNAArtificialoligonucleotide 426acuuugcaau gcugcugucu ucuug
2542725RNAArtificialoligonucleotide 427cuuugcaaug cugcugucuu cuugc
2542825RNAArtificialoligonucleotide 428uuugcaaugc ugcugucuuc uugcu
2542925RNAArtificialoligonucleotide 429uugcaaugcu gcugucuucu ugcua
2543025RNAArtificialoligonucleotide 430ugcaaugcug cugucuucuu gcuau
2543125RNAArtificialoligonucleotide 431gcaaugcugc ugucuucuug cuaug
2543225RNAArtificialoligonucleotide 432caaugcugcu gucuucuugc uauga
2543325RNAArtificialoligonucleotide 433aaugcugcug ucuucuugcu augaa
2543425RNAArtificialoligonucleotide 434augcugcugu cuucuugcua ugaau
2543525RNAArtificialoligonucleotide 435ugcugcuguc uucuugcuau gaaua
2543625RNAArtificialoligonucleotide 436gcugcugucu ucuugcuaug aauaa
2543725RNAArtificialoligonucleotide 437cugcugucuu cuugcuauga auaau
2543825RNAArtificialoligonucleotide 438ugcugucuuc uugcuaugaa uaaug
2543925RNAArtificialoligonucleotide 439gcugucuucu ugcuaugaau aaugu
2544025RNAArtificialoligonucleotide 440cugucuucuu gcuaugaaua auguc
2544125RNAArtificialoligonucleotide 441ugucuucuug cuaugaauaa uguca
2544225RNAArtificialoligonucleotide 442gucuucuugc uaugaauaau gucaa
2544325RNAArtificialoligonucleotide 443ucuucuugcu augaauaaug ucaau
2544425RNAArtificialoligonucleotide 444cuucuugcua ugaauaaugu caauc
2544525RNAArtificialoligonucleotide 445uucuugcuau gaauaauguc aaucc
2544625RNAArtificialoligonucleotide 446ucuugcuaug aauaauguca auccg
2544725RNAArtificialoligonucleotide 447cuugcuauga auaaugucaa uccga
2544825RNAArtificialoligonucleotide 448uugcuaugaa uaaugucaau ccgac
2544925RNAArtificialoligonucleotide 449ugcuaugaau aaugucaauc cgacc
2545025RNAArtificialoligonucleotide 450gcuaugaaua augucaaucc gaccu
2545125RNAArtificialoligonucleotide 451cuaugaauaa ugucaauccg accug
2545225RNAArtificialoligonucleotide 452uaugaauaau gucaauccga ccuga
2545325RNAArtificialoligonucleotide 453augaauaaug ucaauccgac cugag
2545425RNAArtificialoligonucleotide 454ugaauaaugu caauccgacc ugagc
2545525RNAArtificialoligonucleotide 455gaauaauguc aauccgaccu gagcu
2545625RNAArtificialoligonucleotide 456aauaauguca auccgaccug agcuu
2545725RNAArtificialoligonucleotide 457auaaugucaa uccgaccuga gcuuu
2545825RNAArtificialoligonucleotide 458uaaugucaau ccgaccugag cuuug
2545925RNAArtificialoligonucleotide 459aaugucaauc cgaccugagc uuugu
2546025RNAArtificialoligonucleotide 460augucaaucc gaccugagcu uuguu
2546125RNAArtificialoligonucleotide 461ugucaauccg accugagcuu uguug
2546225RNAArtificialoligonucleotide 462gucaauccga ccugagcuuu guugu
2546325RNAArtificialoligonucleotide 463ucaauccgac cugagcuuug uugua
2546425RNAArtificialoligonucleotide 464caauccgacc ugagcuuugu uguag
2546525RNAArtificialoligonucleotide 465aauccgaccu gagcuuuguu guaga
2546625RNAArtificialoligonucleotide 466auccgaccug agcuuuguug uagac
2546725RNAArtificialoligonucleotide 467uccgaccuga gcuuuguugu agacu
2546825RNAArtificialoligonucleotide 468ccgaccugag cuuuguugua gacua
2546920RNAArtificialoligonucleotide 469cgaccugagc uuuguuguag
2047025RNAArtificialoligonucleotide 470cgaccugagc uuuguuguag acuau
2547125RNAArtificialoligonucleotide 471gaccugagcu uuguuguaga cuauc
2547225RNAArtificialoligonucleotide 472accugagcuu uguuguagac uauca
2547323RNAArtificialoligonucleotide 473ccugagcuuu guuguagacu auc
2347420RNAArtificialoligonucleotide 474cauuuuugac cuacaugugg
2047520RNAArtificialoligonucleotide 475uuugaccuac auguggaaag
2047626RNAArtificialoligonucleotide 476uacauuuuug accuacaugu ggaaag
2647717RNAArtificialoligonucleotide 477ggucuccuua ccuauga
1747824RNAArtificialoligonucleotide 478ucuuaccuau gacuauggau gaga
2447922RNAArtificialoligonucleotide 479auuuuugacc uacaugggaa ag
2248023RNAArtificialoligonucleotide 480uacgaguuga uugucggacc cag
2348125RNAArtificialoligonucleotide 481guggucuccu uaccuaugac ugugg
2548224RNAArtificialoligonucleotide 482ugucucagua aucuucuuac cuau
2448323RNAArtificialoligonucleotide 483ugcauguucc agucguugug ugg
2348425RNAArtificialoligonucleotide 484cacuauucca gucaaauagg ucugg
2548525RNAArtificialoligonucleotide 485auuuaccaac cuucaggauc gagua
2548621RNAArtificialoligonucleotide 486ggccuaaaac acauacacau a
2148724RNAArtificialoligonucleotide 487gauagguggu aucaacaucu guaa
2448821RNAArtificialoligonucleotide 488gauagguggu aucaacaucu g
2148919RNAArtificialoligonucleotide 489cuuccuggau ggcuugaau
1949020RNAArtificialoligonucleotide 490uguuguuguu uaugcucauu
2049119RNAArtificialoligonucleotide 491guacauuaag auggacuuc
1949220RNAArtificialoligonucleotide 492cuguugcagu aaucuaugag
2049320RNAArtificialoligonucleotide 493ugcaguaauc uaugaguuuc
2049418RNAArtificialoligonucleotide 494gagucuucua ggagccuu
1849523RNAArtificialoligonucleotide 495ugccauuguu ucaucagcuc uuu
2349620RNAArtificialoligonucleotide 496uccuguagga cauuggcagu
2049720RNAArtificialoligonucleotide 497cuuggagucu ucuaggagcc
2049830RNAArtificialoligonucleotide 498ccauuuugug aauguuuucu
uuugaacauc 3049924RNAArtificialoligonucleotide 499cccauuuugu
gaauguuuuc uuuu 2450024RNAArtificialoligonucleotide 500gaaaauugug
cauuuaccca uuuu 2450122RNAArtificialoligonucleotide 501uugugcauuu
acccauuuug ug 2250224RNAArtificialoligonucleotide 502cccugaggca
uucccaucuu gaau 2450320RNAArtificialoligonucleotide 503aggacuuacu
ugcuuuguuu 2050423RNAArtificialoligonucleotide 504cuugaauuua
ggagauucau cug 2350523RNAArtificialoligonucleotide 505caucuucuga
uaauuuuccu guu 2350620RNAArtificialoligonucleotide 506ccauuacagu
ugucuguguu 2050723RNAArtificialoligonucleotide 507ugacagccug
ugaaaucugu gag 2350820RNAArtificialoligonucleotide 508uaaucugccu
cuucuuuugg 2050920RNAArtificialoligonucleotide 509cagcaguagu
ugucaucugc 2051027RNAArtificialoligonucleotide 510gccugagcug
aucugcuggc aucuugc 2751131RNAArtificialoligonucleotide
511gccugagcug aucugcuggc aucuugcagu u
3151216RNAArtificialoligonucleotide 512ucugcuggca ucuugc
1651322RNAArtificialoligonucleotide 513gccgguugac uucauccugu gc
2251423RNAArtificialoligonucleotide 514gucugcaucc aggaacaugg guc
2351524RNAArtificialoligonucleotide 515uacuuacugu cuguagcucu uucu
2451622RNAArtificialoligonucleotide 516cugcauccag gaacaugggu cc
2251724RNAArtificialoligonucleotide 517guugaagauc ugauagccgg uuga
2451817RNAArtificialoligonucleotide 518uaggugccug ccggcuu
1751918RNAArtificialoligonucleotide 519uucagcugua gccacacc
1852021RNAArtificialoligonucleotide 520cugaacugcu ggaaagucgc c
2152130RNAArtificialoligonucleotide 521cuggcuucca aaugggaccu
gaaaaagaac 3052221RNAArtificialoligonucleotide 522caauuuuucc
cacucaguau u 2152319RNAArtificialoligonucleotide 523uugaaguucc
uggagucuu 1952422RNAArtificialoligonucleotide 524uccucaggag
gcagcucuaa au 2252516RNAArtificialoligonucleotide 525uggcucucuc
ccaggg 1652627RNAArtificialoligonucleotide 526gagauggcuc ucucccaggg
acccugg 2752717RNAArtificialoligonucleotide 527gggcacuuug uuuggcg
1752819RNAArtificialoligonucleotide 528ggucccagca aguuguuug
1952925RNAArtificialoligonucleotide 529ugggaugguc ccagcaaguu guuug
2553021RNAArtificialoligonucleotide 530guagagcucu gucauuuugg g
2153125RNAArtificialoligonucleotide 531gcucaagaga uccacugcaa aaaac
2553226RNAArtificialoligonucleotide 532gccauacgua cguaucauaa acauuc
2653325RNAArtificialoligonucleotide 533ucugcaggau auccaugggc ugguc
2553427RNAArtificialoligonucleotide 534gauccucccu guucgucccc
uauuaug 2753524RNAArtificialoligonucleotide 535ugcuuuagac
uccuguaccu gaua 2453618RNAArtificialoligonucleotide 536ggcggccuuu
guguugac 1853725RNAArtificialoligonucleotide 537ggacaggccu
uuauguucgu gcugc 2553819RNAArtificialoligonucleotide 538ccuuuauguu
cgugcugcu 1953920RNAArtificialoligonucleotide 539ucaaggaaga
uggcauuucu 2054020RNAArtificialoligonucleotide 540ucaangaaga
uggcauuucu 2054120RNAArtificialoligonucleotide 541ucaagnaaga
uggcauuucu 2054220RNAArtificialoligonucleotide 542ucaaggaana
uggcauuucu 2054320RNAArtificialoligonucleotide 543ucaaggaaga
ungcauuucu 2054420RNAArtificialoligonucleotide 544ucaaggaaga
ugncauuucu 2054520RNAArtificialoligonucleotide 545ncaaggaaga
uggcauuucu 2054620RNAArtificialoligonucleotide 546ucaaggaaga
nggcauuucu 2054720RNAArtificialoligonucleotide 547ucaaggaaga
uggcanuucu 2054820RNAArtificialoligonucleotide 548ucaaggaaga
uggcaunucu 2054920RNAArtificialoligonucleotide 549ucaaggaaga
uggcauuncu 2055020RNAArtificialoligonucleotide 550ucaaggaaga
uggcauuucn 2055120RNAArtificialoligonucleotide 551ucnaggaaga
uggcauuucu 2055220RNAArtificialoligonucleotide 552ucanggaaga
uggcauuucu 2055320RNAArtificialoligonucleotide 553ucaaggnaga
uggcauuucu 2055420RNAArtificialoligonucleotide 554ucaagganga
uggcauuucu 2055520RNAArtificialoligonucleotide 555ucaaggaagn
uggcauuucu 2055620RNAArtificialoligonucleotide 556ucaaggaaga
uggcnuuucu 2055725RNAArtificialoligonucleotide 557uuugccncug
cccaaugcca uccug 2555825RNAArtificialoligonucleotide 558uuugccgcun
cccaaugcca uccug 2555925RNAArtificialoligonucleotide 559uuugccgcug
cccaauncca uccug 2556025RNAArtificialoligonucleotide 560uuunccgcug
cccaaugcca uccug 2556125RNAArtificialoligonucleotide
561uuugccgcug cccaaugcca uccun 2556225RNAArtificialoligonucleotide
562nuugccgcug cccaaugcca uccug 2556325RNAArtificialoligonucleotide
563unugccgcug cccaaugcca uccug 2556425RNAArtificialoligonucleotide
564uungccgcug cccaaugcca uccug 2556525RNAArtificialoligonucleotide
565uuugccgcng cccaaugcca uccug 2556625RNAArtificialoligonucleotide
566uuugccgcug cccanugcca uccug 2556725RNAArtificialoligonucleotide
567uuugccgcug cccaaugccn uccug 2556825RNAArtificialoligonucleotide
568uuunccncug cccaaugcca uccug 2556925RNAArtificialoligonucleotide
569uuugccgcug cccaangcca uccug 2557025RNAArtificialoligonucleotide
570uuugccgcug cccaaugcca nccug 2557125RNAArtificialoligonucleotide
571uuugccgcug cccaaugcca uccng 2557225RNAArtificialoligonucleotide
572uuugccgcug cccnaugcca uccug 2557320RNAArtificialoligonucleotide
573ucagcuucun uuagccacug 2057420RNAArtificialoligonucleotide
574ucagcuucug uuanccacug 2057520RNAArtificialoligonucleotide
575ucancuucug uuagccacug 2057620RNAArtificialoligonucleotide
576ucagcuucug uuagccacun 20577153PRTHomo sapiens 577Met Gly Lys Ile
Ser Ser Leu Pro Thr Gln Leu Phe Lys Cys Cys Phe 1 5 10 15 Cys Asp
Phe Leu Lys Val Lys Met His Thr Met Ser Ser Ser His Leu 20 25 30
Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala 35
40 45 Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln
Phe 50 55 60 Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr
Gly Tyr Gly 65 70 75 80 Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile
Val Asp Glu Cys Cys 85 90 95 Phe Arg Ser Cys Asp Leu Arg Arg Leu
Glu Met Tyr Cys Ala Pro Leu 100 105 110 Lys Pro Ala Lys Ser Ala Arg
Ser Val Arg Ala Gln Arg His Thr Asp 115 120 125 Met Pro Lys Thr Gln
Lys Glu Val His Leu Lys Asn Ala Ser Arg Gly 130 135 140 Ser Ala Gly
Asn Lys Asn Tyr Arg Met 145 150
* * * * *
References