U.S. patent application number 16/229821 was filed with the patent office on 2019-04-25 for methods and means for efficient skipping of exon 45 in duchenne muscular dystrophy pre-mrna.
The applicant listed for this patent is Academisch Ziekenhuis Leiden, BioMarin Technologies B.V.. Invention is credited to Josephus Johannes De Kimpe, Gerard Johannes Platenburg, Adriana Marie Rus, Judith Christina Theodora Van Deutekom, Garrit-Jan Boudewijn Van Ommen.
Application Number | 20190119679 16/229821 |
Document ID | / |
Family ID | 39045623 |
Filed Date | 2019-04-25 |
United States Patent
Application |
20190119679 |
Kind Code |
A1 |
De Kimpe; Josephus Johannes ;
et al. |
April 25, 2019 |
METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE
MUSCULAR DYSTROPHY PRE-mRNA
Abstract
The. invention relates to a method for inducing or promoting
skipping of exon 45 of DMD pre-mRNA in a Duchenne Muscular
Dystrophy patient, preferably in an isolated (muscle) cell, the
method comprising providing said cell with an antisense molecule
that binds to a continuous stretch of at least 21 nucleotides
within said exon. The invention further relates to such antisense
molecule used in said method.
Inventors: |
De Kimpe; Josephus Johannes;
(Utrecht, NL) ; Rus; Adriana Marie; (Hoofddorp,
NL) ; Platenburg; Gerard Johannes; (Voorschoten,
NL) ; Van Deutekom; Judith Christina Theodora;
(Dordrecht, NL) ; Van Ommen; Garrit-Jan Boudewijn;
(Amsterdam, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BioMarin Technologies B.V.
Academisch Ziekenhuis Leiden |
Leiden
Leiden |
|
NL
NL |
|
|
Family ID: |
39045623 |
Appl. No.: |
16/229821 |
Filed: |
December 21, 2018 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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15390836 |
Dec 27, 2016 |
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16229821 |
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14542183 |
Nov 14, 2014 |
9528109 |
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15390836 |
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14200251 |
Mar 7, 2014 |
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14542183 |
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14134971 |
Dec 19, 2013 |
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14200251 |
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14097210 |
Dec 4, 2013 |
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14134971 |
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13094548 |
Apr 26, 2011 |
9926557 |
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14097210 |
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PCT/NL2009/050006 |
Jan 13, 2009 |
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13094548 |
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PCT/NL2008/050673 |
Oct 27, 2008 |
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PCT/NL2009/050006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/315 20130101;
C12N 2310/111 20130101; A61K 45/06 20130101; A61P 39/06 20180101;
C12N 2310/313 20130101; A61P 21/04 20180101; C12N 2310/3181
20130101; A61P 29/00 20180101; C12N 2310/3231 20130101; C12N
2310/31 20130101; A61K 31/56 20130101; A61K 38/1719 20130101; C12N
2310/346 20130101; A61K 31/56 20130101; A61P 21/00 20180101; C12N
15/113 20130101; C12N 2320/33 20130101; A61K 31/57 20130101; A61P
3/14 20180101; C12N 2310/314 20130101; A61K 48/00 20130101; C12N
2310/3233 20130101; A61P 43/00 20180101; C12N 2310/11 20130101;
C12N 2320/31 20130101; A61P 21/02 20180101; A61K 2300/00 20130101;
A61K 31/57 20130101; A61K 31/58 20130101; A61K 31/573 20130101;
A61K 31/7088 20130101; C12N 2310/321 20130101; A61K 2300/00
20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 31/57 20060101 A61K031/57; A61K 38/17 20060101
A61K038/17; A61K 45/06 20060101 A61K045/06; A61K 31/7088 20060101
A61K031/7088; A61K 31/56 20060101 A61K031/56; A61K 31/58 20060101
A61K031/58; A61K 31/573 20060101 A61K031/573 |
Claims
1-15. (canceled)
16. An antisense oligonucleotide of 25 nucleotides in length,
wherein the antisense oligonucleotide comprises at least 18
consecutive bases of a base sequence of the sequence
CUGUUGCCUCCGGUUCUGAAGGUG (SEQ ID NO: 115), in which uracil bases
are thymine bases, wherein the antisense oligonucleotide is a
morpholino phosphorodiamidate antisense oligonucleotide, and
wherein the antisense oligonucleotide induces exon 53 skipping of
human dystrophin pre-mRNA.
17. A pharmaceutical composition, comprising the oligonucleotide of
claim 16 and a pharmaceutically acceptable excipient.
18. A method for treating Duchenne Muscular Dystrophy (DMD) or
Becker Muscular Dystrophy (BMD), comprising administering to a
subject a therapeutically effective amount of the oligonucleotide
of claim 16.
Description
FIELD
[0001] The invention relates to the field of genetics, more
specifically human genetics. The invention in particular relates to
the modulation of splicing of the human Duchenne Muscular Dystrophy
pre-mRNA.
BACKGROUND OF THE INVENTION
[0002] Myopathies are disorders that result in functional
impairment of muscles. Muscular dystrophy (MD) refers to genetic
diseases that are characterized by progressive weakness and
degeneration of skeletal muscles. Duchenne muscular dystrophy (DMD)
and Becker muscular dystrophy (BMD) are the most common childhood
forms of muscular dystrophy. They are recessive disorders and
because the gene responsible for DMD and BMD resides on the
X-chromosome, mutations mainly affect males with an incidence of
about 1 in 3500 boys.
[0003] DMD and BMD are caused by genetic defects in the DMD gene
encoding dystrophin, a muscle protein that is required for
interactions between the cytoskeleton and the extracellular matrix
to maintain muscle fiber stability during contraction. DMD is a
severe, lethal neuromuscular disorder resulting in a dependency on
wheelchair support before the age of 12 and DMD patients often die
before the age of thirty due to respiratory- or heart failure. In
contrast, BMD patients often remain ambulatory until later in life,
and have near normal life expectancies. DMD mutations in the DMD
gene are mainly characterized by frame shifting insertions or
deletions or nonsense point mutations, resulting in the absence of
functional dystrophin. BMD mutations in general keep the reading
frame intact, allowing synthesis of a partly functional dystrophin.
During the last decade, specific modification of splicing in order
to restore the disrupted reading frame of the DMD transcript has
emerged as a promising therapy for Duchenne muscular dystrophy
(DMD) (van Ommen, van Deutekom, Aartsma-Rus, Curr Opin Mol Ther.
2008;10(2)140-9, Yokota, Duddy, Partidge, Acta Myol.
2007;26(3)179-84, van Deutekom et al., N Engl J Med.
2007;357(26)2677-86).
[0004] Using antisense oligonucleotides (AONs) interfering with
splicing signals the skipping of specific exons can be induced in
the DMD pre-mRNA, thus restoring the open reading frame and
converting the severe DMD into a milder BMD phenotype (van Deutekom
et al. Hum Mol Genet. 2001; 10: 1547-54; Aartsma-Rus et al., Hum
Mol Genet 2003; 12(8)907-14.). In vivo proof-of-concept was first
obtained in the mdx mouse model, which is dystrophin-deficient due
to a nonsense mutation in exon 23. Intramuscular and intravenous
injections of AONs targeting the mutated exon 23 restored
dystrophin expression for at least three months (Lu et al. Nat Med.
2003; 8: 1009-14; Lu et al., Proc Natl Acad Sci U S A.
2005;102(1)198-203). This was accompanied by restoration of
dystrophin-associated proteins at the fiber membrane as well as
functional improvement of the treated muscle. In vivo skipping of
human exons has also been achieved in the hDMD mouse model, which
contains a complete copy of the human DMD gene integrated in
chromosome 5 of the mouse (Bremmer-Bout et al. Molecular Therapy.
2004; 10: 232-40; 't Hoen et al. J Biol Chem. 2008; 283:
5899-907).
[0005] As the majority of DMD patients have deletions that cluster
in hotspot regions, the skipping of a small number of exons is
applicable to relatively large numbers of patients. The actual
applicability of exon skipping can be determined for deletions,
duplications and point mutations reported in DMD mutation databases
such as the Leiden DMD mutation database available at www.dmd.nl.
Therapeutic skipping of exon 45 of the DMD pre-mRNA would restore
the open reading frame of DMD patients having deletions including
but not limited to exons 12-44, 18-44, 44, 46, 46-47, 46-48, 46-49,
46-51, 46-53, 46-55, 46-59, 46-60 of the DMD pre-mRNA, occurring in
a total of 16% of all DMD patients with a deletion (Aartsma-Rus and
van Deutekom, 2007, Antisense Elements (Genetics) Research Focus,
2007 Nova Science Publishers, Inc). Furthermore, for some DMD
patients the simultaneous skipping of one of more exons in addition
to exon 45, such as exons 51 or 53 is required to restore the
correct reading frame. None-limiting examples include patients with
a deletion of exons 46-50 requiring the co-skipping of exons 45 and
51, or with a deletion of exons 46-52 requiring the co-skipping of
exons 45 and 53.
[0006] Recently, a first-in-man study was successfully completed
where an AON inducing the skipping of exon 51 was injected into a
small area of the tibialis anterior muscle of four DMD patients.
Novel dystrophin expression was observed in the majority of muscle
fibers in all four patients treated, and the AON was safe and well
tolerated (van Deutekom et al. N Engl J Med. 2007; 357:
2677-86).
[0007] Most AONs studied contain up to 20 nucleotides, and it has
been argued that this relatively short size improves the tissue
distribution and/or cell penetration of an AON. However, such short
AONs will result in a limited specificity due to an increased risk
for the presence of identical sequences elsewhere in the genome,
and a limited target binding or target affinity due to a low free
energy of the AON-target complex. Therefore the inventors decided
to design new and optionally improved oligonucleotides that would
not exhibit all of these drawbacks.
DESCRIPTION OF THE INVENTION
[0008] Method
[0009] In a first aspect, the invention provides a method for
inducing and/or promoting skipping of exon 45 of DMD pre-mRNA in a
patient, preferably in an isolated cell of said patient, the method
comprising providing said cell and/or said patient with a molecule
that binds to a continuous stretch of at least 21 nucleotides
within said exon.
[0010] Accordingly, a method is herewith provided for inducing
and/or promoting skipping of exon 45 of DMD pre-mRNA, preferably in
an isolated cell of a patient, the method comprising providing said
cell and/or said patient with a molecule that binds to a continuous
stretch of at least 21 nucleotides within said exon.
[0011] It is to be understood that said method encompasses an in
vitro, in vivo or ex vivo method.
[0012] As defined herein a DMD pre-mRNA preferably means the
pre-mRNA of a DMD gene of a DMD or BMD patient. The DMD gene or
protein corresponds to the dystrophin gene or protein.
[0013] A patient is preferably intended to mean a patient having
DMD or BMD as later defined herein or a patient susceptible to
develop DMD or BMD due to his or her genetic background.
[0014] Exon skipping refers to the induction in a cell of a mature
mRNA that does not contain a particular exon that is normally
present therein. Exon skipping is achieved by providing a cell
expressing the pre-mRNA of said mRNA with a molecule capable of
interfering with sequences such as, for example, the splice donor
or splice acceptor sequence that are both required for allowing the
enzymatic process of splicing, or a molecule that is capable of
interfering with an exon inclusion signal required for recognition
of a stretch of nucleotides as an exon to be included in the mRNA.
The term pre-mRNA refers to a non-processed or partly processed
precursor mRNA that is synthesized from a DNA template in the cell
nucleus by transcription.
[0015] Within the context of the invention inducing anchor
promoting skipping of an exon as indicated herein means that at
least 1%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or more of the
DMD mRNA in one or more (muscle) cells of a treated patient will
not contain said exon. This is preferably assessed by PCR as
described in the examples.
[0016] Preferably, a method of the invention by inducing or
promoting skipping of exon 45 of the DMD pre-mRNA in one or more
cells of a patient provides said patient with a functional
dystrophin protein and/or decreases the production of an aberrant
dystrophin protein in said patient. Therefore a preferred method is
a method, wherein a patient or a cell of said patient is provided
with a functional dystrophin protein and/or wherein the production
of an aberrant dystrophin protein in said patient or in a cell of
said patient is decreased
[0017] Decreasing the production of an aberrant dystrophin 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 dystrophin mRNA or protein. 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.
[0018] 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 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
dystrophin mRNA. 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 immuno fluorescence 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.
[0019] 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:l. In other words, 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
10%, 20%, 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 functional dystrophin is preferably
binding to actin and to the dystrophin-associated glycoprotein
complex (DGC) (Aartsma-Rus A et al, (2006), Entries in the leiden
Duchenne Muscular Dystrophy mutation database: an overview of
mutation types and paradoxical cases that confirm the reading-frame
rule, Muscle Nerve, 34: 135-144). 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 muscle
biopsy, as known to the skilled person.
[0020] Individuals or patients suffering from Duchenne muscular
dystrophy typically have a mutation in the DMD gene that prevent
synthesis of the complete dystrophin protein, i.e of a premature
stop prevents the synthesis of the C-terminus. In Becker muscular
dystrophy the DMD gene also comprises a mutation compared to the
wild type gene but the mutation does typically not induce a
premature stop and the C-terminus is typically synthesized. As a
result a functional dystrophin protein is synthesized that has at
least the same activity in kind as the wild type protein, not
although not necessarily the same amount of activity. 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
(Aartsma-Rus A et al, (2006), Entries in the leiden Duchenne
Muscular Dystrophy mutation database: an overview of mutation types
and paradoxical cases that confirm the reading-frame rule, Muscle
Nerve, 34: 135-144). Exon--skipping for the treatment of DMD is
typically directed to overcome a premature stop in the pre-mRNA by
skipping an exon in the rod-shaped domain to correct the reading
frame and allow synthesis of the 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 by
a method 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: typically
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 A et al, (2006), Entries in the
leiden Duchenne Muscular Dystrophy mutation database: an overview
of mutation types and paradoxical cases that confirm the
reading-frame rule, Muscle Nerve, 34: 135-144). The central
rod-shaped domain of wild type dystrophin comprises 24
spectrin-like repeats (Aartsma-Rus A et al, (2006), Entries in the
leiden Duchenne Muscular Dystrophy mutation database: an overview
of mutation types and paradoxical cases that confirm the
reading-frame rule, Muscle Nerve, 34: 135-144). 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.
[0021] A method of the invention may alleviate one or more
characteristics of a muscle cell from a DMD patient comprising
deletions including but not limited to exons 12-44, 18-44, 44, 46,
46-47, 46-48, 46-49, 46-51, 46-53, 46-55, 46.sup.-59, 46.sup.-60 of
the DMD pre-mRNA of said patient (Aartsma-Rus and van Deutekom,
2007, Antisense Elements (Genetics) Research Focus, 2007 Nova
Science Publishers, Inc) as well as from DMD patients requiring the
simultaneous skipping of one of more exons in addition to exon 45
including but not limited to patients with a deletion of exons
46-50 requiring the co-skipping of exons 45 and 51, or with a
deletion of exons 46-52 requiring the co-skipping of exons 45 and
53.
[0022] In a preferred method, one or more symptom(s) or
characteristic(s) of a myogenic cell or muscle cell from a DMD
patient is/are alleviated. Such symptoms or characteristics may be
assessed at the cellular, tissue level or on the patient self.
[0023] An alleviation of one or more symptoms or 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.
[0024] The improvement of muscle fiber function, integrity and/or
survival may also 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.
[0025] Creatine kinase may be detected in blood as described in
Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular
Disorders, 16: 591-602.2006). 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 DMD patient before treatment.
[0026] A detectable decrease of necrosis of muscle fibers is
preferably assessed in a muscle biopsy, more preferably as
described in Hodgetts et al (Hodgetts S., et al, (2006),
Neuromuscular Disorders, 16: 591-602.2006) 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 DMD patient before treatment.
[0027] A detectable increase of the homogeneity of the diameter of
muscle fibers is preferably assessed in a muscle biopsy
cross-section, more preferably as described in Hodgetts et al
(Hodgetts S., et al, (2006), Neuromuscular Disorders, 16:
591-602.2006). The increase is measured by comparison to the
homogeneity of the diameter of muscle fibers in a muscle biopsy
cross-section of a same DMD patient before treatment.
[0028] An alleviation of one or more symptoms or characteristics
may be assessed by any of the following assays on the patient self:
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 (Manzur A Y et al, (2008), Glucocorticoid
corticosteroids for Duchenne muscular dystrophy (review), Wiley
publishers, The Cochrane collaboration.) 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 has been
alleviated in an individual using a method of the invention.
Detectable improvement or prolongation is preferably a
statistically significant improvement or prolongation as described
in Hodgetts et al (Hodgetts S., et al, (2006), Neuromuscular
Disorders, 16: 591-602.2006). Alternatively, the alleviation of one
or more symptom(s) of Duchenne Muscular Dystrophy may be assessed
by measuring an improvement of a muscle fiber function, integrity
and/or survival as later defined herein.
[0029] A treatment in a method according to the invention may have
a duration of 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. The frequency of administration of an oligonucleotide,
composition, compound of the invention may depend on several
parameters such as the age of the patient, the type of mutation,
the number of molecules (dose), the formulation of said molecule.
The frequency may be ranged between at least once in a two weeks,
or three weeks or four weeks or five weeks or a longer time period.
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 and may be
administered directly in vivo, ex vivo or in vitro. An
oligonucleotide as used herein 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, 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, and may
be administered directly or indirectly in vivo, ex vivo or in
vitro. As Duchenne muscular dystrophy has 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.
[0030] A molecule or oligonucleotide or equivalent thereof can be
delivered as is to a cell. When administering said molecule,
oligonucleotide or equivalent thereof to an individual, it is
preferred that it 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 for a method of the invention is the use of
an excipient that will further enhance delivery of said molecule,
oligonucleotide or functional equivalent thereof as defined herein,
to a cell and into a cell, preferably a muscle cell. Preferred
excipient are defined in the section entitled "pharmaceutical
composition". In vitro, we obtained very good results using
polyethylenimine (PEI, ExGen500, MBI Fermentas) as shown in the
example.
[0031] In a preferred method of the invention, an additional
molecule is used which is able to induce and/or promote skipping of
a distinct exon of the DMD pre-mRNA of a patient. Preferably, the
second exon is selected from: exon 7, 44, 46, 51, 53, 59, 67 of the
dystrophin pre-mRNA of a patient. Molecules which can be used are
depicted in table 2. Preferred molecules comprise or consist of any
of the oligonucleotides as disclosed in table 2. Several
oligonucleotides may also be used in combination.This way,
inclusion of two or more exons of a DMD pre-mRNA in mRNA produced
from this pre-mRNA is prevented. This embodiment is further
referred to as double- or multi-exon skipping (Aartsma-Rus A,
Janson A A, Kaman W E, et al. Antisense-induced multiexon skipping
for Duchenne muscular dystrophy makes more sense. Am J Hum Genet
2004;74(1):83-92, Aartsma-Rus A, Kaman W E, Weij R, den Dunnen J T,
van Ommen G J, van Deutekom J C. Exploring the frontiers of
therapeutic exon skipping for Duchenne muscular dystrophy by double
targeting within one or multiple exons. Mol Ther 2006;14(3):401-7).
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-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.
[0032] 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.
[0033] In case, more than one compounds are used in a method of the
invention, said compounds can be administered to an individual in
any order. In one embodiment, said compounds are administered
simultaneously (meaning that said compounds are administered within
10 hours, preferably within one hour). This is however not
necessary. In another embodiment, said compounds are administered
sequentially.
[0034] Molecule
[0035] In a second aspect, there is provided a molecule for use in
a method as described in the previous section entitled "Method".
This molecule preferably comprises or consists of an
oligonucleotide, Said oligonucleotide is preferably an antisense
oligonucleotide (AON) or antisense oligoribonucleotide.
[0036] It was found by the present investigators that especially
exon 45 is specifically skipped at a high frequency using a
molecule that binds to a continuous stretch of at least 21
nucleotides within said exon. Although this effect can be
associated with a higher binding affinity of said molecule,
compared to a molecule that binds to a continuous stretch of less
than 21 nucleotides, there could be other intracellular parameters
involved that favor thermodynamic, kinetic, or structural
characteristics of the hybrid duplex. In a preferred embodiment, a
molecule that binds to a continuous stretch of at least 21, 25, 30,
35, 40, 45, 50 nucleotides within said exon is used.
[0037] In a preferred embodiment, a molecule or an oligonucleotide
of the invention which comprises a sequence that is complementary
to a part of exon 45 of DMD 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% and most preferably up to 100%. "A part of exon 45"
preferably means a stretch of at least 21 nucleotides. In a most
preferred embodiment, an oligonucleotide of the invention consists
of a sequence that is complementary to part of exon 45 dystrophin
pre-mRNA as defined herein. Alternatively, an oligonucleotide may
comprise a sequence that is complementary to part of exon 45
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 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. Several
types of flanking sequences may be used. Preferably, additional
flanking sequences are used to modify the binding of a protein to
said molecule or oligonucleotide, or to modify a thermodynamic
property of the oligonucleotide, more preferably to modify target
RNA binding affinity. In another preferred embodiment, additional
flanking sequences are complementary to sequences of the DMD
pre-mRNA which are not present in exon 45. Such flanking sequences
are preferably complementary to sequences comprising or consisting
of the splice site acceptor or donor consensus sequences of exon
45. In a preferred embodiment, such flanking sequences are
complementary to sequences comprising or consisting of sequences of
an intron of the DMD pre-mRNA which is adjacent to exon 45; i.e.
intron 44 or 45.
[0038] A continuous stretch of at least 21, 25, 30, 35, 40, 45, 50
nucleotides within exon 45 is preferably selected from the
sequence:
TABLE-US-00001 (SEQ ID NO 2)
5'-CCAGGAUGGCAUUGGGCAGCGGCAAACUGUUGUCAGA
ACAUUGAAUGCAACUGGGGAAGAAAUAAUUCAGCAAUC-3'.
[0039] It was found that a molecule that binds to a nucleotide
sequence comprising or consisting of a continuous stretch of at
least 21, 25, 30, 35, 40, 45, 50 nucleotides of SEQ ID NO. 2
results in highly efficient skipping of exon 45 in a cell provided
with this molecule. Molecules that bind to a nucleotide sequence
comprising a continuous stretch of less than 21 nucleotides of SEQ
ID NO:2 were found to induce exon skipping in a less efficient way
than the molecules of the invention. Therefore, in a preferred
embodiment, a method is provided wherein a molecule binds to a
continuous stretch of at least 21, 25, 30, 35 nucleotides within
SEQ ID NO:2. Contrary to what was generally thought, the inventors
surprisingly found that a higher specificity and efficiency of exon
skipping may be reached using an oligonucleotides having a length
of at least 21 nucleotides. None of the indicated sequences is
derived from conserved parts of splice-junction sites. Therefore,
said molecule is not likely to mediate differential splicing of
other exons from the DMD pre-mRNA or exons from other genes.
[0040] In one embodiment, a molecule of the invention capable of
interfering with the inclusion of exon 45 of the DMD pre-mRNA is a
compound molecule that binds to the specified sequence, or a
protein such as an RNA-binding protein or a non-natural zinc-finger
protein that has been modified to be able to bind to the indicated
nucleotide sequence on a RNA molecule. Methods for screening
compound molecules that bind specific nucleotide sequences are for
example disclosed in PCT/NL01/00697 and US patent 6875736, which
are herein enclosed by reference. Methods for designing RNA-binding
Zinc-finger proteins that bind specific nucleotide sequences are
disclosed by Friesen and Darby, Nature Structural Biology 5:
543-546 (1998) which is herein enclosed by reference.
[0041] In a further embodiment, a molecule of the invention capable
of interfering with the inclusion of exon 45 of the DMD pre-mRNA
comprises an antisense oligonucleotide that is complementary to and
can base-pair with the coding strand of the pre-mRNA of the DMD
gene. Said antisense oligonucleotide preferably contains a RNA
residue, a DNA residue, and/or a nucleotide analogue or equivalent,
as will be further detailed herein below.
[0042] A preferred molecule of the invention comprises a
nucleotide-based or nucleotide or an antisense oligonucleotide
sequence of between 21 and 50 nucleotides or bases, more preferred
between 21 and 40 nucleotides, more preferred between 21 and 30
nucleotides, such as 21 nucleotides, 22 nucleotides, 23
nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27
nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31
nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35
nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39
nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43
nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47
nucleotides, 48 nucleotides, 49 nucleotides or 50 nucleotides.
[0043] A most preferred molecule of the invention comprises a
nucleotide-based sequence of 25 nucleotides.
[0044] In a preferred embodiment, a molecule of the invention binds
to a continuous stretch of or is complementary to or is antisense
to at least a continuous stretch of at least 21 nucleotides within
the nucleotide sequence SEQ ID NO:2.
[0045] In a certain embodiment, the invention provides a molecule
comprising or consisting of an antisense nucleotide sequence
selected from the antisense nucleotide sequences as depicted in
Table 1, except SEQ ID NO:68. A molecule of the invention that is
antisense to the sequence of SEQ ID NO 2, which is present in exon
45 of the DMD gene preferably comprises or consists of the
antisense nucleotide sequence of SEQ ID NO 3; SEQ ID NO 4, SEQ ID
NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO
10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID
NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ
ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24,
SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO
29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID
NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ
ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43,
SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO
48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID
NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ
ID NO 58, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 61, SEQ ID NO 62,
SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 66 and/or SEQ
ID NO:67.
[0046] In a more preferred embodiment, the invention provides a
molecule comprising or consisting of the antisense nucleotide
sequence of SEQ ID NO 3; SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ
ID NO 7 and/or SEQ ID NO 8.
[0047] In a most preferred embodiment, the invention provides a
molecule comprising or consisting of the antisense nucleotide
sequence of SEQ ID NO 3. It was found that this molecule is very
efficient in modulating splicing of exon 45 of the DMD pre-mRNA in
a muscle cell.
[0048] A nucleotide sequence of a molecule of the invention may
contain a RNA residue, a DNA residue, a nucleotide analogue or
equivalent as will be further detailed herein below. In addition, a
molecule of the invention may encompass a functional equivalent of
a molecule of the invention as defined herein.
[0049] It is preferred that a molecule of the invention comprises a
or at least one residue that is modified to increase nuclease
resistance, and/or to increase the affinity of the antisense
nucleotide for the target sequence. Therefore, in a preferred
embodiment, an antisense nucleotide sequence comprises a or at
least one nucleotide analogue or equivalent, wherein a nucleotide
analogue or equivalent is defined as a residue having a modified
base, and/or a modified backbone, and/or a non-natural
internucleoside linkage, or a combination of these
modifications.
[0050] In a preferred embodiment, a nucleotide analogue or
equivalent comprises a modified backbone. Examples of such
backbones are provided by morpholino backbones, carbamate
backbones, siloxane backbones, sulfide, sulfoxide and sulfone
backbones, formacetyl and thioformacetyl backbones,
methyleneformacetyl backbones, riboacetyl backbones, alkene
containing backbones, sulfamate, sulfonate and sulfonamide
backbones, methyleneimino and methylenehydrazino backbones, and
amide backbones. Phosphorodiamidate morpholino oligomers are
modified backbone oligonucleotides that have previously been
investigated as antisense agents. Morpholino oligonucleotides have
an uncharged backbone in which the deoxyribose sugar of DNA is
replaced by a six membered ring and the phosphodiester linkage is
replaced by a phosphorodiamidate linkage. Morpholino
oligonucleotides are resistant to enzymatic degradation and appear
to function as antisense agents by arresting translation or
interfering with pre-mRNA splicing rather than by activating RNase
H. Morpholino oligonucleotides have been successfully delivered to
tissue culture cells by methods that physically disrupt the cell
membrane, and one study comparing several of these methods found
that scrape loading was the most efficient method of delivery;
however, because the morpholino backbone is uncharged, cationic
lipids are not effective mediators of morpholino oligonucleotide
uptake in cells. A recent report demonstrated triplex formation by
a morpholino oligonucleotide and, because of the non-ionic
backbone, these studies showed that the morpholino oligonucleotide
was capable of triplex formation in the absence of magnesium.
[0051] It is further preferred that the linkage between a residue
in a backbone does not include a phosphorus atom, such as a linkage
that is formed by short chain alkyl or cycloalkyl internucleoside
linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside
linkages, or one or more short chain heteroatomic or heterocyclic
internucleoside linkages.
[0052] A preferred nucleotide analogue or equivalent comprises a
Peptide Nucleic Acid (PNA), having a modified polyamide backbone
(Nielsen, et al. (1991) Science 254, 1497-1500). PNA-based
molecules are true mimics of DNA molecules in terms of base-pair
recognition. The backbone of the PNA is composed of
N-(2-aminoethyl)-glycine units linked by peptide bonds, wherein the
nucleobases are linked to the backbone by methylene carbonyl bonds.
An alternative backbone comprises a one-carbon extended pyrrolidine
PNA monomer (Govindaraju and Kumar (2005) Chem. Commun, 495-497).
Since the backbone of a PNA molecule contains no charged phosphate
groups, PNA-RNA hybrids are usually more stable than RNA-RNA or
RNA-DNA hybrids, respectively (Egholm et al (1993) Nature 365,
566-568).
[0053] A further preferred backbone comprises a morpholino
nucleotide analog or equivalent, in which the ribose or deoxyribose
sugar is replaced by a 6-membered morpholino ring. A most preferred
nucleotide analog or equivalent comprises a phosphorodiamidate
morpholino oligomer (PMO), in which the ribose or deoxyribose sugar
is replaced by a 6-membered morpholino ring, and the anionic
phosphodiester linkage between adjacent morpholino rings is
replaced by a non-ionic phosphorodiamidate linkage.
[0054] In yet a further embodiment, a nucleotide analogue or
equivalent of the invention comprises a substitution of at least
one of the non-bridging oxygens in the phosphodiester linkage. This
modification slightly destabilizes base-pairing but adds
significant resistance to nuclease degradation. A preferred
nucleotide analogue or equivalent comprises phosphorothioate,
chiral phosphorothioate, phosphorodithioate, phosphotriester,
aminoalkylphosphotriester, H-phosphonate, methyl and other alkyl
phosphonate including 3'-alkylene phosphonate, 5'-alkylene
phosphonate and chiral phosphonate, phosphinate, phosphoramidate
including 3'-amino phosphoramidate and aminoalkylphosphoramidate,
thionophosphoramidate, thionoalkylphosphonate,
thionoalkylphosphotriester, selenophosphate or boranophosphate.
[0055] A further preferred nucleotide analogue or equivalent of the
invention comprises one or more sugar moieties that are mono- or
disubstituted at the 2', 3' and/or 5' position such as a --OH; --F;
substituted or unsubstituted, linear or branched lower (C1-C10)
alkyl, alkenyl, alkynyl, alkaryl, allyl, aryl, or aralkyl, that may
be interrupted by one or more heteroatoms; O--, S--, or N-alkyl;
O--, S--, or N-alkenyl; O--, S-or N-alkynyl; O--, S--, or N-allyl;
O-alkyl-O-alkyl, -methoxy, -aminopropoxy; aminoxy, methoxyethoxy;
-dimethylaminooxyethoxy; and -dimethylaminoethoxyethoxy. The sugar
moiety can be a pyranose or derivative thereof, or a deoxypyranose
or derivative thereof, preferably a ribose or a derivative thereof,
or deoxyribose or derivative thereof. Such preferred derivatized
sugar moieties comprise Locked Nucleic Acid (LNA), in which the
2'-carbon atom is linked to the 3' or 4' carbon atom of the sugar
ring thereby forming a bicyclic sugar moiety. A preferred LNA
comprises 2'-O,4'-C-ethylene-bridged nucleic acid (Morita et al.
2001. Nucleic Acid Res Supplement No. 1: 241-242). These
substitutions render the nucleotide analogue or equivalent RNase H
and nuclease resistant and increase the affinity for the target
RNA.
[0056] It is understood by a skilled person that it is not
necessary for all positions in an antisense oligonucleotide to be
modified uniformly. In addition, more than one of the
aforementioned analogues or equivalents may be incorporated in a
single antisense oligonucleotide or even at a single position
within an antisense oligonucleotide. In certain embodiments, an
antisense oligonucleotide of the invention has at least two
different types of analogues or equivalents.
[0057] A preferred antisense oligonucleotide according to the
invention comprises a 2'-O -alkyl phosphorothioate antisense
oligonucleotide, such as 2'-O-methyl modified ribose (RNA),
2'-O-ethyl modified ribose, 2'-O-propyl modified ribose, and/or
substituted derivatives of these modifications such as halogenated
derivatives.
[0058] A most preferred antisense oligonucleotide according to the
invention comprises a 2'-O-methyl phosphorothioate ribose.
[0059] A functional equivalent of a molecule of the invention may
be defined as an oligonucleotide as defined herein wherein an
activity of said functional equivalent is retained to at least some
extent. Preferably, an activity of said functional equivalent is
inducing exon 45 skipping and providing a functional dystrophin
protein. Said activity of said functional equivalent is therefore
preferably assessed by detection of exon 45 skipping and
quantifying the amount of a functional dystrophin protein. A
functional dystrophin is herein preferably defined as being a
dystrophin able to bind actin and members of the DGC protein
complex. The assessment of said activity of an oligonucleotide is
preferably done by RT-PCR or by immunofluorescence or Western blot
analysis. 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. Throughout this application, when the word
oligonucleotide is used it may be replaced by a functional
equivalent thereof as defined herein.
[0060] It will also be understood by a skilled person that distinct
antisense oligonucleotides can be combined for efficiently skipping
of exon 45 of the human DMD pre-mRNA. In a preferred embodiment, a
combination of at least two antisense oligonucleotides are used in
a method of the invention, such as two distinct antisense
oligonucleotides, three distinct antisense oligonucleotides, four
distinct antisense oligonucleotides, or five distinct antisense
oligonucleotides or even more. It is also encompassed by the
present invention to combine several oligonucleotides or molecules
as depicted in table 1 except SEQ ID NO:68.
[0061] An antisense oligonucleotide can be linked to a moiety that
enhances uptake of the antisense oligonucleotide in cells,
preferably myogenic cells or muscle cells. Examples of such
moieties are cholesterols, carbohydrates, vitamins, biotin, lipids,
phospholipids, cell-penetrating peptides including but not limited
to antennapedia, TAT, transportan and positively charged amino
acids such as oligoarginine, poly-arginine, oligolysine or
polylysine, antigen-binding domains such as provided by an
antibody, a Fab fragment of an antibody, or a single chain antigen
binding domain such as a cameloid single domain antigen-binding
domain.
[0062] A preferred antisense oligonucleotide comprises a
peptide-linked PMO.
[0063] A preferred antisense oligonucleotide comprising one or more
nucleotide analogs or equivalents of the invention modulates
splicing in one or more muscle cells, including heart muscle cells,
upon systemic delivery. In this respect, systemic delivery of an
antisense oligonucleotide comprising a specific nucleotide analog
or equivalent might result in targeting a subset of muscle cells,
while an antisense oligonucleotide comprising a distinct nucleotide
analog or equivalent might result in targeting of a different
subset of muscle cells. Therefore, in one embodiment it is
preferred to use a combination of antisense oligonucleotides
comprising different nucleotide analogs or equivalents for
modulating skipping of exon 45 of the human DMD pre-mRNA.
[0064] A cell can be provided with a molecule capable of
interfering with essential sequences that result in highly
efficient skipping of exon 45 of the human DMD pre-mRNA by
plasmid-derived antisense oligonucleotide expression or viral
expression provided by viral-based vector. Such a viral-based
vector comprises an expression cassette that drives expression of
an antisense molecule as defined herein. Preferred virus-based
vectors include adenovirus- or adeno-associated virus-based
vectors. Expression is preferably driven by a polymerase III
promoter, such as a U1, a U6, or a U7 RNA promoter. A muscle or
myogenic cell can be provided with a plasmid for antisense
oligonucleotide expression by providing the plasmid in an aqueous
solution. Alternatively, a plasmid can be provided by transfection
using known transfection agentia such as, for example,
LipofectAMINETM 2000 (Invitrogen) or polyethyleneimine (PEI;
ExGen500 (MBI Fermentas)), or derivatives thereof.
[0065] One preferred antisense oligonucleotide expression system is
an adenovirus associated virus (AAV)-based vector. Single chain and
double chain AAV-based vectors have been developed that can be used
for prolonged expression of small antisense nucleotide sequences
for highly efficient skipping of exon 45 of the DMD pre-mRNA.
[0066] A preferred AAV-based vector comprises an expression
cassette that is driven by a polymerase III-promoter (Pol III). A
preferred Pol III promoter is, for example, a U1, a U6, or a U7 RNA
promoter.
[0067] The invention therefore also provides a viral-based vector,
comprising a Pol III-promoter driven expression cassette for
expression of one or more antisense sequences of the invention for
inducing skipping of exon 45 of the human DMD pre-mRNA.
Pharmaceutical Composition
[0068] If required, a molecule or a vector expressing an antisense
oligonucleotide of the invention can be incorporated into a
pharmaceutically active mixture or composition by adding a
pharmaceutically acceptable carrier.
[0069] Therefore, in a further aspect, the invention provides a
composition, preferably a pharmaceutical composition comprising a
molecule comprising an antisense oligonucleotide according to the
invention, and/or a viral-based vector expressing the antisense
sequence(s) according to the invention and a pharmaceutically
acceptable carrier.
[0070] A preferred pharmaceutical composition comprises a molecule
as defined herein and/or a vector as defined herein, and a
pharmaceutical acceptable carrier or excipient, optionally combined
with a molecule and/or a vector which is able to modulate skipping
of exon 7, 44, 46, 51, 53, 59, 67 of the DMD pre-mRNA.
[0071] Preferred excipients include excipients capable of forming
complexes, vesicles and/or liposomes that deliver such a molecule
as defined herein, preferably an oligonucleotide 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 and derivatives, or similar cationic polymers,
including polypropyleneimine or polyethylenimine copolymers (PECs)
and derivatives, synthetic amphiphils, lipofectin.TM., DOTAP and/or
viral capsid proteins that are capable of self assembly into
particles that can deliver such molecule, preferably an
oligonucleotide as defined herein to a cell, preferably a muscle
cell. Such excipients have been shown to efficiently deliver
(oligonucleotide such as antisense) nucleic acids to a wide variety
of cultured cells, including muscle cells. We obtained very good
results using polyethylenimine (PEI, ExGen500, MBI Fermentas) as
shown in the example. 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.
[0072] 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.
[0073] 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 a molecule or a compound as defined herein, preferably
an oligonucleotide across cell membranes into cells.
[0074] In addition to these common nanoparticle materials, the
cationic peptide protamine offers an alternative approach to
formulate a compound as defined herein, preferably an
oligonucleotide as 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 a compound as defined herein, preferably 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 a compound as defined herein, preferably an
oligonucleotide for use in the current invention to deliver said
compound for the treatment of Duchenne Muscular Dystrophy in
humans.
[0075] In addition, a compound as defined herein, preferably 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 a compound as defined herein,
preferably an oligonucleotide from vesicles, e.g. endosomes or
lysosomes.
[0076] Therefore, in a preferred embodiment, a compound as defined
herein, preferably an oligonucleotide are formulated in a
medicament which is provided with at least an excipient and/or a
targeting ligand for delivery and/or a delivery device of said
compound to a cell and/or enhancing its intracellular delivery.
Accordingly, the invention also encompasses a pharmaceutically
acceptable composition comprising a compound as defined herein,
preferably an oligonucleotide and further comprising at least one
excipient and/or a targeting ligand for delivery and/or a delivery
device of said compound to a cell and/or enhancing its
intracellular delivery.
[0077] It is to be understood that a molecule or compound or
oligonucleotide may not be formulated in one single composition or
preparation. Depending on their identity, the skilled person will
know which type of formulation is the most appropriate for each
compound.
[0078] In a preferred embodiment, an in vitro concentration of a
molecule or an oligonucleotide as defined herein, which is ranged
between 0.1 nM and 1 .mu.M is used. More preferably, the
concentration used is ranged between 0.3 to 400 nM, even more
preferably between 1 to 200 nM. 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. If several molecules or
oligonucleotides are used, these concentrations may refer to the
total concentration of oligonucleotides or the concentration of
each oligonucleotide added. The ranges of concentration of
oligonucleotide(s) as given above are preferred concentrations 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
of oligonucleotide(s) used may further vary and may need to be
optimised any further.
[0079] More preferably, a compound preferably an oligonucleotide
and an adjunct compound to be used in the invention to prevent,
treat DMD are synthetically produced and administered directly to a
cell, a tissue, an organ and/or patients in formulated form in a
pharmaceutically acceptable composition or preparation. The
delivery of a pharmaceutical composition to the subject is
preferably carried out by one or more parenteral injections, e.g.
intravenous and/or subcutaneous and/or intramuscular and/or
intrathecal and/or intraventricular administrations, preferably
injections, at one or at multiple sites in the human body.
Use
[0080] In yet a further aspect, the invention provides the use of
an antisense oligonucleotide or molecule according to the
invention, and/or a viral-based vector that expresses one or more
antisense sequences according to the invention and/or a
pharmaceutical composition, for inducing and/or promoting splicing
of the DMD pre-mRNA. The splicing is preferably modulated in a
human myogenic cell or a muscle cell in vitro. More preferred is
that splicing is modulated in human a myogenic cell or muscle cell
in vivo.
[0081] Accordingly, the invention further relates to the use of the
molecule as defined herein and/or the vector as defined herein
and/or or the pharmaceutical composition as defined herein for
inducing and/or promoting splicing of the DMD pre-mRNA or for the
preparation of a medicament for the treatment of a DMD patient.
[0082] 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 molecule
or a viral-based vector or a composition 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. 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".
[0083] Each embodiment as identified herein may be combined
together unless otherwise indicated. All patent and literature
references cited in the present specification are hereby
incorporated by reference in their entirety.
[0084] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
LEGENDS TO THE FIGURE
[0085] FIG. 1. In human control myotubes, a series of AONs (PS220
to PS225; SEQ ID NO: 3 to 8), all binding to a continuous stretch
of at least 21 nucleotides within a specific sequence of exon 45
(i.e. SEQ ID NO:2), were tested at two different concentrations
(200 and 500 nM). All six AONs were effective in inducing specific
exon 45 skipping, as confirmed by sequence analysis (not shown).
PS220 (SEQ ID NO:3) however, reproducibly induced highest levels of
exon 45 skipping (see FIG. 2). (NT: non-treated cells, M: size
marker).
[0086] FIG. 2. In human control myotubes, 25-mer PS220 (SEQ ID NO:
3) was tested at increasing concentration. Levels of exon 45
skipping of up to 75% (at 400 nM) were observed reproducibly, as
assessed by Agilent LabChip Analysis.
[0087] FIG. 3. In human control myotubes, the efficiencies of a
"short" 17-mer AON45-5 (SEQ ID NO:68) and its overlapping "long"
25-mer counterpart PS220 were directly compared at 200 nM and 500
nM. PS220 was markedly more efficient at both concentrations: 63%
when compared to 3% obtained with 45-5. (NT: non-treated cells, M:
size marker).
EXAMPLES
Examples 1 and 2
[0088] Materials and Methods
[0089] AON design was based on (partly) overlapping open secondary
structures of the target exon RNA as predicted by the m-fold
program (Zuker,M. (2003) Mfold web server for nucleic acid folding
and hybridization prediction. Nucleic Acids Res., 31, 3406-3415),
and on (partly) overlapping putative SR-protein binding sites as
predicted by numerous software programs such as ESEfinder
(Cartegni,L. et al.(2003) ESEfinder: A web resource to identify
exonic splicing enhancers. Nucleic Acids Res, 31, 3568-71; Smith,P.
J. et al. (2006) An increased specificity score matrix for the
prediction of SF2/ASF-specific exonic splicing enhancers.
Hum.Mol.Genet., 15, 2490-2508) that predicts binding sites for the
four most abundant SR proteins (SF2/ASF, SC35, SRp40 and SRp55).
AONs were synthesized by Prosensa Therapeutics B. V. (Leiden,
Netherlands), and contain 2'-O-methyl RNA and full-length
phosphorothioate (PS) backbones.
[0090] Tissue culturing, transfection and RT-PCR analysis Myotube
cultures derived from a healthy individual ("human control") were
obtained as described previously (Aartsma-Rus et al. Hum Mol Genet
2003; 12(8): 907-14). For the screening of AONs, myotube cultures
were transfected with 0 to 500 nM of each AON. The transfection
reagent polyethylenimine (PEI, ExGen500 MBI Fermentas) was used
according to manufacturer's instructions, with 2 .mu.l PEI per
.mu.g AON. Exon skipping efficiencies were determined by nested
RT-PCR analysis using primers in the exons flanking exon 45. PCR
fragments were isolated from agarose gels for sequence
verification. For quantification, the PCR products were analyzed
using the Agilent DNA 1000 LabChip Kit and the Agilent 2100
bioanalyzer (Agilent Technologies, USA).
[0091] Results
[0092] A series of AONs targeting sequences within SEQ ID NO:2
within exon 45 were designed and tested in normal myotube cultures,
by transfection and subsequent RT-PCR and sequence analysis of
isolated RNA. PS220 (SEQ ID NO: 3) reproducibly induced highest
levels of exon 45 skipping, when compared to PS221-PS225 (FIG. 1).
High levels of exon 45 skipping of up to 75% were already obtained
at 400 nM PS220 (FIG. 2). In a direct comparison, PS220 (a 25-mer)
was reproducibly more efficient in inducing exon 45 skipping than
its shorter 17-mer counterpart AON 45-5 (SEQ ID NO: 68; previously
published as h45AON5 (Aartsma-Rus et al. Am J Hum Genet 2004;74:
83-92)), at both AON concentrations of 200 nM and 500 nM and with
63% versus 3% respectively at 500 nM (FIG. 3). This result is
probably due to the fact that the extended length of PS220, in fact
completely overlapping AON 45-5, increases the free energy of the
AON-target complex such that the efficiency of inducing exon 45
skipping is also increased.
TABLE-US-00002 TABLE 1 AONs in exon 45 SEQ ID NO 3
UUUGCCGCUGCCCAAUGCCAUCCUG SEQ ID NO 36 GUUGCAUUCAAUGUUCUGACAACAG
(PS220) SEQ ID NO 4 AUUCAAUGUUCUGACAACAGUUUGC SEQ ID NO 37
UUGCAUUCAAUGUUCUGACAACAGU (PS221) SEQ ID NO 5
CCAGUUGCAUUCAAUGUUCUGACAA SEQ ID NO 38 UGCAUUCAAUGUUCUGACAACAGUU
(PS222) SEQ ID NO 6 CAGUUGCAUUCAAUGUUCUGAC SEQ ID NO 39
GCAUUCAAUGUUCUGACAACAGUUU (PS223) SEQ ID NO 7 AGUUGCAUUCAAUGUUCUGA
SEQ ID NO 40 CAUUCAAUGUUCUGACAACAGUUUG (PS224) SEQ ID NO 8
GAUUGCUGAAUUAUUUCUUCC SEQ ID NO 41 AUUCAAUGUUCUGACAACAGUUUGC
(PS225) SEQ ID NO 9 GAUUGCUGAAUUAUUUCUUCCCCAG SEQ ID NO 42
UCAAUGUUCUGACAACAGUUUGCCG SEQ ID NO 10 AUUGCUGAAUUAUUUCUUCCCCAGU
SEQ ID NO 43 CAAUGUUCUGACAACAGUUUGCCGC SEQ ID NO 11
UUGCUGAAUUAUUUCUUCCCCAGUU SEQ ID NO 44 AAUGUUCUGACAACAGUUUGCCGCU
SEQ ID NO 12 UGCUGAAUUAUUUCUUCCCCAGUUG SEQ ID NO 45
AUGUUCUGACAACAGUUUGCCGCUG SEQ ID NO 13 GCUGAAUUAUUUCUUCCCCAGUUGC
SEQ ID NO 46 UGUUCUGACAACAGUUUGCCGCUGC SEQ ID NO 14
CUGAAUUAUUUCUUCCCCAGUUGCA SEQ ID NO 47 GUUCUGACAACAGUUUGCCGCUGCC
SEQ ID NO 15 UGAAUUAUUUCUUCCCCAGUUGCAU SEQ ID NO 48
UUCUGACAACAGUUUGCCGCUGCCC SEQ ID NO 16 GAAUUAUUUCUUCCCCAGUUGCAUU
SEQ ID NO 49 UCUGACAACAGUUUGCCGCUGCCCA SEQ ID NO 17
AAUUAUUUCUUCCCCAGUUGCAUUC SEQ ID NO 50 CUGACAACAGUUUGCCGCUGCCCAA
SEQ ID NO 18 AUUAUUUCUUCCCCAGUUGCAUUCA SEQ ID NO 51
UGACAACAGUUUGCCGCUGCCCAAU SEQ ID NO 19 UUAUUUCUUCCCCAGUUGCAUUCAA
SEQ ID NO 52 GACAACAGUUUGCCGCUGCCCAAUG SEQ ID NO 20
UAUUUCUUCCCCAGUUGCAUUCAAU SEQ ID NO 53 ACAACAGUUUGCCGCUGCCCAAUGC
SEQ ID NO 21 AUUUCUUCCCCAGUUGCAUUCAAUG SEQ ID NO 54
CAACAGUUUGCCGCUGCCCAAUGCC SEQ ID NO 22 UUUCUUCCCCAGUUGCAUUCAAUGU
SEQ ID NO 55 AACAGUUUGCCGCUGCCCAAUGCCA SEQ ID NO 23
UUCUUCCCCAGUUGCAUUCAAUGUU SEQ ID NO 56 ACAGUUUGCCGCUGCCCAAUGCCAU
SEQ ID NO 24 UCUUCCCCAGUUGCAUUCAAUGUUC SEQ ID NO 57
CAGUUUGCCGCUGCCCAAUGCCAUC SEQ ID NO 25 CUUCCCCAGUUGCAUUCAAUGUUCU
SEQ ID NO 58 AGUUUGCCGCUGCCCAAUGCCAUCC SEQ ID NO 26
UUCCCCAGUUGCAUUCAAUGUUCUG SEQ ID NO 59 GUUUGCCGCUGCCCAAUGCCAUCCU
SEQ ID NO 27 UCCCCAGUUGCAUUCAAUGUUCUGA SEQ ID NO 60
UUUGCCGCUGCCCAAUGCCAUCCUG SEQ ID NO 28 CCCCAGUUGCAUUCAAUGUUCUGAC
SEQ ID NO 61 UUGCCGCUGCCCAAUGCCAUCCUGG SEQ ID NO 29
CCCAGUUGCAUUCAAUGUUCUGACA SEQ ID NO 62 UGCCGCUGCCCAAUGCCAUCCUGGA
SEQ ID NO 30 CCAGUUGCAUUCAAUGUUCUGACAA SEQ ID NO 63
GCCGCUGCCCAAUGCCAUCCUGGAG SEQ ID NO 31 CAGUUGCAUUCAAUGUUCUGACAAC
SEQ ID NO 64 CCGCUGCCCAAUGCCAUCCUGGAGU SEQ ID NO 32
AGUUGCAUUCAAUGUUCUGACAACA SEQ ID NO 65 CGCUGCCCAAUGCCAUCCUGGAGUU
SEQ ID NO 33 UCC UGU AGA AUA CUG GCA UC SEQ ID NO 66 UGU UUU UGA
GGA UUG CUG AA SEQ ID NO 34 UGC AGA CCU CCU GCC ACC GCA SEQ ID NO
67 UGUUCUGACAACAGUUUGCCGCUGCCCAAU GAU UCA GCCAUCCUGG SEQ ID NO 35
UUGCAGACCUCCUGCCACCGCAGAUUC SEQ ID NO 68 GCCCAAUGCCAUCCUGG AGGCUUC
(45-5)
TABLE-US-00003 TABLE 2 AONs in exons 51, 53, 7, 44, 46, 59, and 67
DMD Gene Exon 51 SEQ ID NO 69 AGAGCAGGUACCUCCAACAUCAAGG SEQ ID NO
91 UCAAGGAAGAUGGCAUUUCUAGUUU SEQ ID NO 70 GAGCAGGUACCUCCAACAUCAAGGA
SEQ ID NO 92 UCAAGGAAGAUGGCAUUUCU SEQ ID NO 71
AGCAGGUACCUCCAACAUCAAGGAA SEQ ID NO 93 CAAGGAAGAUGGCAUUUCUAGUUUG
SEQ ID NO 72 GCAGGUACCUCCAACAUCAAGGAAG SEQ ID NO 94
AAGGAAGAUGGCAUUUCUAGUUUGG SEQ ID NO 73 CAGGUACCUCCAACAUCAAGGAAGA
SEQ ID NO 95 AGGAAGAUGGCAUUUCUAGUUUGGA SEQ ID NO 74
AGGUACCUCCAACAUCAAGGAAGAU SEQ ID NO 96 GGAAGAUGGCAUUUCUAGUUUGGAG
SEQ ID NO 75 GGUACCUCCAACAUCAAGGAAGAUG SEQ ID NO 97
GAAGAUGGCAUUUCUAGUUUGGAGA SEQ ID NO 76 GUACCUCCAACAUCAAGGAAGAUGG
SEQ ID NO 98 AAGAUGGCAUUUCUAGUUUGGAGAU SEQ ID NO 77
UACCUCCAACAUCAAGGAAGAUGGC SEQ ID NO 99 AGAUGGCAUUUCUAGUUUGGAGAUG
SEQ ID NO 78 ACCUCCAACAUCAAGGAAGAUGGCA SEQ ID NO 100
GAUGGCAUUUCUAGUUUGGAGAUGG SEQ ID NO 79 CCUCCAACAUCAAGGAAGAUGGCAU
SEQ ID NO 101 AUGGCAUUUCUAGUUUGGAGAUGGC SEQ ID NO 80
CUCCAACAUCAAGGAAGAUGGCAUU SEQ ID NO 102 UGGCAUUUCUAGUUUGGAGAUGGCA
SEQ ID NO 81 CUCCAACAUCAAGGAAGAUGGCAUUUCUAG SEQ ID NO 103
GGCAUUUCUAGUUUGGAGAUGGCAG SEQ ID NO 82 UCCAACAUCAAGGAAGAUGGCAUUU
SEQ ID NO 104 GCAUUUCUAGUUUGGAGAUGGCAGU SEQ ID NO 83
CCAACAUCAAGGAAGAUGGCAUUUC SEQ ID NO 105 CAUUUCUAGUUUGGAGAUGGCAGUU
SEQ ID NO 84 CAACAUCAAGGAAGAUGGCAUUUCU SEQ ID NO 106
AUUUCUAGUUUGGAGAUGGCAGUUU SEQ ID NO 85 AACAUCAAGGAAGAUGGCAUUUCUA
SEQ ID NO 107 UUUCUAGUUUGGAGAUGGCAGUUUC SEQ ID NO 86
ACAUCAAGGAAGAUGGCAUUUCUAG SEQ ID NO 108 UUCUAGUUUGGAGAUGGCAGUUUCC
SEQ ID NO 87 ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG SEQ ID NO 88
ACAUCAAGGAAGAUGGCAUUUCUAG SEQ ID NO 89 CAUCAAGGAAGAUGGCAUUUCUAGU
SEQ ID NO 90 AUCAAGGAAGAUGGCAUUUCUAGUU DMD Gene Exon 53 SEQ ID NO
109 CCAUUGUGUUGAAUCCUUUAACAUU SEQ ID NO 116
CAUUCAACUGUUGCCUCCGGUUCUGAAGGUG SEQ ID NO 110
CCAUUGUGUUGAAUCCUUUAAC SEQ ID NO 117 CUGAAGGUGUUCUUGUACUUCAUCC SEQ
ID NO 111 AUUGUGUUGAAUCCUUUAAC SEQ ID NO 118
UGUAUAGGGACCCUCCUUCCAUGACUC SEQ ID NO 112 CCUGUCCUAAGACCUGCUCA SEQ
ID NO 119 AUCCCACUGAUUCUGAAUUC SEQ ID NO 113
CUUUUGGAUUGCAUCUACUGUAUAG SEQ ID NO 120 UUGGCUCUGGCCUGUCCUAAGA SEQ
ID NO 114 CAUUCAACUGUUGCCUCCGGUUCUG SEQ ID NO 121
AAGACCUGCUCAGCUUCUUCCUUAGCUUCCAGCCA SEQ ID NO 115
CUGUUGCCUCCGGUUCUGAAGGUG DMD Gene Exon 7 SEQ ID NO 122
UGCAUGUUCCAGUCGUUGUGUGG SEQ ID NO 124 AUUUACCAACCUUCAGGAUCGAGUA SEQ
ID NO 123 CACUAUUCCAGUCAAAUAGGUCUGG SEQ ID NO 125
GGCCUAAAACACAUACACAUA DMD Gene Exon 44 SEQ ID NO 126
UCAGCUUCUGUUAGCCACUG SEQ ID NO 151 AGCUUCUGUUAGCCACUGAUUAAA SEQ ID
NO 127 UUCAGCUUCUGUUAGCCACU SEQ ID NO 152 CAGCUUCUGUUAGCCACUGAUUAAA
SEQ ID NO 128 UUCAGCUUCUGUUAGCCACUG SEQ ID NO 153
AGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 129 UCAGCUUCUGUUAGCCACUGA SEQ ID
NO 154 AGCUUCUGUUAGCCACUGAU SEQ ID NO 130 UUCAGCUUCUGUUAGCCACUGA
SEQ ID NO 155 GCUUCUGUUAGCCACUGAUU SEQ ID NO 131
UCAGCUUCUGUUAGCCACUGA SEQ ID NO 156 AGCUUCUGUUAGCCACUGAUU SEQ ID NO
132 UUCAGCUUCUGUUAGCCACUGA SEQ ID NO 157 GCUUCUGUUAGCCACUGAUUA SEQ
ID NO 133 UCAGCUUCUGUUAGCCACUGAU SEQ ID NO 158
AGCUUCUGUUAGCCACUGAUUA SEQ ID NO 134 UUCAGCUUCUGUUAGCCACUGAU SEQ ID
NO 159 GCUUCUGUUAGCCACUGAUUAA SEQ ID NO 135 UCAGCUUCUGUUAGCCACUGAUU
SEQ ID NO 160 AGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 136
UUCAGCUUCUGUUAGCCACUGAUU SEQ ID NO 161 GCUUCUGUUAGCCACUGAUUAAA SEQ
ID NO 137 UCAGCUUCUGUUAGCCACUGAUUA SEQ ID NO 162
AGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 138 UUCAGCUUCUGUUAGCCACUGAUA SEQ
ID NO 163 GCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 139
UCAGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 164 CCAUUUGUAUUUAGCAUGUUCCC SEQ
ID NO 140 UUCAGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 165
AGAUACCAUUUGUAUUUAGC SEQ ID NO 141 UCAGCUUCUGUUAGCCACUGAUUAAA SEQ
ID NO 166 GCCAUUUCUCAACAGAUCU SEQ ID NO 142
UUCAGCUUCUGUUAGCCACUGAUUAAA SEQ ID NO 167 GCCAUUUCUCAACAGAUCUGUCA
SEQ ID NO 143 CAGCUUCUGUUAGCCACUG SEQ ID NO 168
AUUCUCAGGAAUUUGUGUCUUUC SEQ ID NO 144 CAGCUUCUGUUAGCCACUGAU SEQ ID
NO 169 UCUCAGGAAUUUGUGUCUUUC SEQ ID NO 145 AGCUUCUGUUAGCCACUGAUU
SEQ ID NO 170 GUUCAGCUUCUGUUAGCC SEQ ID NO 146
CAGCUUCUGUUAGCCACUGAUU SEQ ID NO 171 CUGAUUAAAUAUCUUUAUAU C SEQ ID
NO 147 AGCUUCUGUUAGCCACUGAUUA SEQ ID NO 172 GCCGCCAUUUCUCAACAG SEQ
ID NO 148 CAGCUUCUGUUAGCCACUGAUUA SEQ ID NO 173 GUAUUUAGCAUGUUCCCA
SEQ ID NO 149 AGCUUCUGUUAGCCACUGAUUAA SEQ ID NO 174
CAGGAAUUUGUGUCUUUC SEQ ID NO 150 CAGCUUCUGUUAGCCACUGAUUAA DMD Gene
Exon 46 SEQ ID NO 175 GCUUUUCUUUUAGUUGCUGCUCUUU SEQ ID NO 203
AGGUUCAAGUGGGAUACUAGCAAUG SEQ ID NO 176 CUUUUCUUUUAGUUGCUGCUCUUUU
SEQ ID NO 204 GGUUCAAGUGGGAUACUAGCAAUGU SEQ ID NO 177
UUUUCUUUUAGUUGCUGCUCUUUUC SEQ ID NO 205 GUUCAAGUGGGAUACUAGCAAUGUU
SEQ ID NO 178 UUUCUUUUAGUUGCUGCUCUUUUCC SEQ ID NO 206
UUCAAGUGGGAUACUAGCAAUGUUA SEQ ID NO 179 UUCUUUUAGUUGCUGCUCUUUUCCA
SEQ ID NO 207 UCAAGUGGGAUACUAGCAAUGUUAU SEQ ID NO 180
UCUUUUAGUUGCUGCUCUUUUCCAG SEQ ID NO 208 CAAGUGGGAUACUAGCAAUGUUAUC
SEQ ID NO 181 CUUUUAGUUGCUGCUCUUUUCCAGG SEQ ID NO 209
AAGUGGGAUACUAGCAAUGUUAUCU SEQ ID NO 182 UUUUAGUUGCUGCUCUUUUCCAGGU
SEQ ID NO 210 AGUGGGAUACUAGCAAUGUUAUCUG SEQ ID NO 183
UUUAGUUGCUGCUCUUUUCCAGGUU SEQ ID NO 211 GUGGGAUACUAGCAAUGUUAUCUGC
SEQ ID NO 184 UUAGUUGCUGCUCUUUUCCAGGUUC SEQ ID NO 212
UGGGAUACUAGCAAUGUUAUCUGCU SEQ ID NO 185 UAGUUGCUGCUCUUUUCCAGGUUCA
SEQ ID NO 213 GGGAUACUAGCAAUGUUAUCUGCUU SEQ ID NO 186
AGUUGCUGCUCUUUUCCAGGUUCAA SEQ ID NO 214 GGAUACUAGCAAUGUUAUCUGCUUC
SEQ ID NO 187 GUUGCUGCUCUUUUCCAGGUUCAAG SEQ ID NO 215
GAUACUAGCAAUGUUAUCUGCUUCC SEQ ID NO 188 UUGCUGCUCUUUUCCAGGUUCAAGU
SEQ ID NO 216 AUACUAGCAAUGUUAUCUGCUUCCU SEQ ID NO 189
UGCUGCUCUUUUCCAGGUUCAAGUG SEQ ID NO 217 UACUAGCAAUGUUAUCUGCUUCCUC
SEQ ID NO 190 GCUGCUCUUUUCCAGGUUCAAGUGG SEQ ID NO 218
ACUAGCAAUGUUAUCUGCUUCCUCC SEQ ID NO 191 CUGCUCUUUUCCAGGUUCAAGUGGG
SEQ ID NO 219 CUAGCAAUGUUAUCUGCUUCCUCCA SEQ ID NO 192
UGCUCUUUUCCAGGUUCAAGUGGGA SEQ ID NO 220 UAGCAAUGUUAUCUGCUUCCUCCAA
SEQ ID NO 193 GCUCUUUUCCAGGUUCAAGUGGGAC SEQ ID NO 221
AGCAAUGUUAUCUGCUUCCUCCAAC SEQ ID NO 194 CUCUUUUCCAGGUUCAAGUGGGAUA
SEQ ID NO 222 GCAAUGUUAUCUGCUUCCUCCAACC SEQ ID NO 195
UCUUUUCCAGGUUCAAGUGGGAUAC SEQ ID NO 223 CAAUGUUAUCUGCUUCCUCCAACCA
SEQ ID NO 196 CUUUUCCAGGUUCAAGUGGGAUACU SEQ ID NO 224
AAUGUUAUCUGCUUCCUCCAACCAU SEQ ID NO 197 UUUUCCAGGUUCAAGUGGGAUACUA
SEQ ID NO 225 AUGUUAUCUGCUUCCUCCAACCAUA SEQ ID NO 198
UUUCCAGGUUCAAGUGGGAUACUAG SEQ ID NO 226 UGUUAUCUGCUUCCUCCAACCAUAA
SEQ ID NO 199 UUCCAGGUUCAAGUGGGAUACUAGC SEQ ID NO 227
GUUAUCUGCUUCCUCCAACCAUAAA SEQ ID NO 200 UCCAGGUUCAAGUGGGAUACUAGCA
SEQ ID NO 228 GCUGCUCUUUUCCAGGUUC SEQ ID NO 201
CCAGGUUCAAGUGGGAUACUAGCAA SEQ ID NO 229 UCUUUUCCAGGUUCAAGUGG SEQ ID
NO 202 CAGGUUCAAGUGGGAUACUAGCAAU SEQ ID NO 230 AGGUUCAAGUGGGAUACUA
DMD Gene Exon 59 SEQ ID NO 231 CAAUUUUUCCCACUCAGUAUU SEQ ID NO 233
UCCUCAGGAGGCAGCUCUAAAU SEQ ID NO 232 UUGAAGUUCCUGGAGUCUU DMD Gene
Exon 67 SEQ ID NO 234 GCGCUGGUCACAAAAUCCUGUUGAAC SEQ ID NO 236
GGUGAAUAACUUACAAAUUUGGAAGC SEQ ID NO 235
CACUUGCUUGAAAAGGUCUACAAAGGA
Sequence CWU 1
1
23613685PRThomo sapiens 1Met Leu Trp Trp Glu Glu Val Glu Asp Cys
Tyr Glu Arg Glu Asp Val1 5 10 15Gln Lys Lys Thr Phe Thr Lys Trp Val
Asn Ala Gln Phe Ser Lys Phe 20 25 30Gly Lys Gln His Ile Glu Asn Leu
Phe Ser Asp Leu Gln Asp Gly Arg 35 40 45Arg Leu Leu Asp Leu Leu Glu
Gly Leu Thr Gly Gln Lys Leu Pro Lys 50 55 60Glu Lys Gly Ser Thr Arg
Val His Ala Leu Asn Asn Val Asn Lys Ala65 70 75 80Leu Arg Val Leu
Gln Asn Asn Asn Val Asp Leu Val Asn Ile Gly Ser 85 90 95Thr Asp Ile
Val Asp Gly Asn His Lys Leu Thr Leu Gly Leu Ile Trp 100 105 110Asn
Ile Ile Leu His Trp Gln Val Lys Asn Val Met Lys Asn Ile Met 115 120
125Ala Gly Leu Gln Gln Thr Asn Ser Glu Lys Ile Leu Leu Ser Trp Val
130 135 140Arg Gln Ser Thr Arg Asn Tyr Pro Gln Val Asn Val Ile Asn
Phe Thr145 150 155 160Thr Ser Trp Ser Asp Gly Leu Ala Leu Asn Ala
Leu Ile His Ser His 165 170 175Arg Pro Asp Leu Phe Asp Trp Asn Ser
Val Val Cys Gln Gln Ser Ala 180 185 190Thr Gln Arg Leu Glu His Ala
Phe Asn Ile Ala Arg Tyr Gln Leu Gly 195 200 205Ile Glu Lys Leu Leu
Asp Pro Glu Asp Val Asp Thr Thr Tyr Pro Asp 210 215 220Lys Lys Ser
Ile Leu Met Tyr Ile Thr Ser Leu Phe Gln Val Leu Pro225 230 235
240Gln Gln Val Ser Ile Glu Ala Ile Gln Glu Val Glu Met Leu Pro Arg
245 250 255Pro Pro Lys Val Thr Lys Glu Glu His Phe Gln Leu His His
Gln Met 260 265 270His Tyr Ser Gln Gln Ile Thr Val Ser Leu Ala Gln
Gly Tyr Glu Arg 275 280 285Thr Ser Ser Pro Lys Pro Arg Phe Lys Ser
Tyr Ala Tyr Thr Gln Ala 290 295 300Ala Tyr Val Thr Thr Ser Asp Pro
Thr Arg Ser Pro Phe Pro Ser Gln305 310 315 320His Leu Glu Ala Pro
Glu Asp Lys Ser Phe Gly Ser Ser Leu Met Glu 325 330 335Ser Glu Val
Asn Leu Asp Arg Tyr Gln Thr Ala Leu Glu Glu Val Leu 340 345 350Ser
Trp Leu Leu Ser Ala Glu Asp Thr Leu Gln Ala Gln Gly Glu Ile 355 360
365Ser Asn Asp Val Glu Val Val Lys Asp Gln Phe His Thr His Glu Gly
370 375 380Tyr Met Met Asp Leu Thr Ala His Gln Gly Arg Val Gly Asn
Ile Leu385 390 395 400Gln Leu Gly Ser Lys Leu Ile Gly Thr Gly Lys
Leu Ser Glu Asp Glu 405 410 415Glu Thr Glu Val Gln Glu Gln Met Asn
Leu Leu Asn Ser Arg Trp Glu 420 425 430Cys Leu Arg Val Ala Ser Met
Glu Lys Gln Ser Asn Leu His Arg Val 435 440 445Leu Met Asp Leu Gln
Asn Gln Lys Leu Lys Glu Leu Asn Asp Trp Leu 450 455 460Thr Lys Thr
Glu Glu Arg Thr Arg Lys Met Glu Glu Glu Pro Leu Gly465 470 475
480Pro Asp Leu Glu Asp Leu Lys Arg Gln Val Gln Gln His Lys Val Leu
485 490 495Gln Glu Asp Leu Glu Gln Glu Gln Val Arg Val Asn Ser Leu
Thr His 500 505 510Met Val Val Val Val Asp Glu Ser Ser Gly Asp His
Ala Thr Ala Ala 515 520 525Leu Glu Glu Gln Leu Lys Val Leu Gly Asp
Arg Trp Ala Asn Ile Cys 530 535 540Arg Trp Thr Glu Asp Arg Trp Val
Leu Leu Gln Asp Ile Leu Leu Lys545 550 555 560Trp Gln Arg Leu Thr
Glu Glu Gln Cys Leu Phe Ser Ala Trp Leu Ser 565 570 575Glu Lys Glu
Asp Ala Val Asn Lys Ile His Thr Thr Gly Phe Lys Asp 580 585 590Gln
Asn Glu Met Leu Ser Ser Leu Gln Lys Leu Ala Val Leu Lys Ala 595 600
605Asp Leu Glu Lys Lys Lys Gln Ser Met Gly Lys Leu Tyr Ser Leu Lys
610 615 620Gln Asp Leu Leu Ser Thr Leu Lys Asn Lys Ser Val Thr Gln
Lys Thr625 630 635 640Glu Ala Trp Leu Asp Asn Phe Ala Arg Cys Trp
Asp Asn Leu Val Gln 645 650 655Lys Leu Glu Lys Ser Thr Ala Gln Ile
Ser Gln Ala Val Thr Thr Thr 660 665 670Gln Pro Ser Leu Thr Gln Thr
Thr Val Met Glu Thr Val Thr Thr Val 675 680 685Thr Thr Arg Glu Gln
Ile Leu Val Lys His Ala Gln Glu Glu Leu Pro 690 695 700Pro Pro Pro
Pro Gln Lys Lys Arg Gln Ile Thr Val Asp Ser Glu Ile705 710 715
720Arg Lys Arg Leu Asp Val Asp Ile Thr Glu Leu His Ser Trp Ile Thr
725 730 735Arg Ser Glu Ala Val Leu Gln Ser Pro Glu Phe Ala Ile Phe
Arg Lys 740 745 750Glu Gly Asn Phe Ser Asp Leu Lys Glu Lys Val Asn
Ala Ile Glu Arg 755 760 765Glu Lys Ala Glu Lys Phe Arg Lys Leu Gln
Asp Ala Ser Arg Ser Ala 770 775 780Gln Ala Leu Val Glu Gln Met Val
Asn Glu Gly Val Asn Ala Asp Ser785 790 795 800Ile Lys Gln Ala Ser
Glu Gln Leu Asn Ser Arg Trp Ile Glu Phe Cys 805 810 815Gln Leu Leu
Ser Glu Arg Leu Asn Trp Leu Glu Tyr Gln Asn Asn Ile 820 825 830Ile
Ala Phe Tyr Asn Gln Leu Gln Gln Leu Glu Gln Met Thr Thr Thr 835 840
845Ala Glu Asn Trp Leu Lys Ile Gln Pro Thr Thr Pro Ser Glu Pro Thr
850 855 860Ala Ile Lys Ser Gln Leu Lys Ile Cys Lys Asp Glu Val Asn
Arg Leu865 870 875 880Ser Gly Leu Gln Pro Gln Ile Glu Arg Leu Lys
Ile Gln Ser Ile Ala 885 890 895Leu Lys Glu Lys Gly Gln Gly Pro Met
Phe Leu Asp Ala Asp Phe Val 900 905 910Ala Phe Thr Asn His Phe Lys
Gln Val Phe Ser Asp Val Gln Ala Arg 915 920 925Glu Lys Glu Leu Gln
Thr Ile Phe Asp Thr Leu Pro Pro Met Arg Tyr 930 935 940Gln Glu Thr
Met Ser Ala Ile Arg Thr Trp Val Gln Gln Ser Glu Thr945 950 955
960Lys Leu Ser Ile Pro Gln Leu Ser Val Thr Asp Tyr Glu Ile Met Glu
965 970 975Gln Arg Leu Gly Glu Leu Gln Ala Leu Gln Ser Ser Leu Gln
Glu Gln 980 985 990Gln Ser Gly Leu Tyr Tyr Leu Ser Thr Thr Val Lys
Glu Met Ser Lys 995 1000 1005Lys Ala Pro Ser Glu Ile Ser Arg Lys
Tyr Gln Ser Glu Phe Glu 1010 1015 1020Glu Ile Glu Gly Arg Trp Lys
Lys Leu Ser Ser Gln Leu Val Glu 1025 1030 1035His Cys Gln Lys Leu
Glu Glu Gln Met Asn Lys Leu Arg Lys Ile 1040 1045 1050Gln Asn His
Ile Gln Thr Leu Lys Lys Trp Met Ala Glu Val Asp 1055 1060 1065Val
Phe Leu Lys Glu Glu Trp Pro Ala Leu Gly Asp Ser Glu Ile 1070 1075
1080Leu Lys Lys Gln Leu Lys Gln Cys Arg Leu Leu Val Ser Asp Ile
1085 1090 1095Gln Thr Ile Gln Pro Ser Leu Asn Ser Val Asn Glu Gly
Gly Gln 1100 1105 1110Lys Ile Lys Asn Glu Ala Glu Pro Glu Phe Ala
Ser Arg Leu Glu 1115 1120 1125Thr Glu Leu Lys Glu Leu Asn Thr Gln
Trp Asp His Met Cys Gln 1130 1135 1140Gln Val Tyr Ala Arg Lys Glu
Ala Leu Lys Gly Gly Leu Glu Lys 1145 1150 1155Thr Val Ser Leu Gln
Lys Asp Leu Ser Glu Met His Glu Trp Met 1160 1165 1170Thr Gln Ala
Glu Glu Glu Tyr Leu Glu Arg Asp Phe Glu Tyr Lys 1175 1180 1185Thr
Pro Asp Glu Leu Gln Lys Ala Val Glu Glu Met Lys Arg Ala 1190 1195
1200Lys Glu Glu Ala Gln Gln Lys Glu Ala Lys Val Lys Leu Leu Thr
1205 1210 1215Glu Ser Val Asn Ser Val Ile Ala Gln Ala Pro Pro Val
Ala Gln 1220 1225 1230Glu Ala Leu Lys Lys Glu Leu Glu Thr Leu Thr
Thr Asn Tyr Gln 1235 1240 1245Trp Leu Cys Thr Arg Leu Asn Gly Lys
Cys Lys Thr Leu Glu Glu 1250 1255 1260Val Trp Ala Cys Trp His Glu
Leu Leu Ser Tyr Leu Glu Lys Ala 1265 1270 1275Asn Lys Trp Leu Asn
Glu Val Glu Phe Lys Leu Lys Thr Thr Glu 1280 1285 1290Asn Ile Pro
Gly Gly Ala Glu Glu Ile Ser Glu Val Leu Asp Ser 1295 1300 1305Leu
Glu Asn Leu Met Arg His Ser Glu Asp Asn Pro Asn Gln Ile 1310 1315
1320Arg Ile Leu Ala Gln Thr Leu Thr Asp Gly Gly Val Met Asp Glu
1325 1330 1335Leu Ile Asn Glu Glu Leu Glu Thr Phe Asn Ser Arg Trp
Arg Glu 1340 1345 1350Leu His Glu Glu Ala Val Arg Arg Gln Lys Leu
Leu Glu Gln Ser 1355 1360 1365Ile Gln Ser Ala Gln Glu Thr Glu Lys
Ser Leu His Leu Ile Gln 1370 1375 1380Glu Ser Leu Thr Phe Ile Asp
Lys Gln Leu Ala Ala Tyr Ile Ala 1385 1390 1395Asp Lys Val Asp Ala
Ala Gln Met Pro Gln Glu Ala Gln Lys Ile 1400 1405 1410Gln Ser Asp
Leu Thr Ser His Glu Ile Ser Leu Glu Glu Met Lys 1415 1420 1425Lys
His Asn Gln Gly Lys Glu Ala Ala Gln Arg Val Leu Ser Gln 1430 1435
1440Ile Asp Val Ala Gln Lys Lys Leu Gln Asp Val Ser Met Lys Phe
1445 1450 1455Arg Leu Phe Gln Lys Pro Ala Asn Phe Glu Gln Arg Leu
Gln Glu 1460 1465 1470Ser Lys Met Ile Leu Asp Glu Val Lys Met His
Leu Pro Ala Leu 1475 1480 1485Glu Thr Lys Ser Val Glu Gln Glu Val
Val Gln Ser Gln Leu Asn 1490 1495 1500His Cys Val Asn Leu Tyr Lys
Ser Leu Ser Glu Val Lys Ser Glu 1505 1510 1515Val Glu Met Val Ile
Lys Thr Gly Arg Gln Ile Val Gln Lys Lys 1520 1525 1530Gln Thr Glu
Asn Pro Lys Glu Leu Asp Glu Arg Val Thr Ala Leu 1535 1540 1545Lys
Leu His Tyr Asn Glu Leu Gly Ala Lys Val Thr Glu Arg Lys 1550 1555
1560Gln Gln Leu Glu Lys Cys Leu Lys Leu Ser Arg Lys Met Arg Lys
1565 1570 1575Glu Met Asn Val Leu Thr Glu Trp Leu Ala Ala Thr Asp
Met Glu 1580 1585 1590Leu Thr Lys Arg Ser Ala Val Glu Gly Met Pro
Ser Asn Leu Asp 1595 1600 1605Ser Glu Val Ala Trp Gly Lys Ala Thr
Gln Lys Glu Ile Glu Lys 1610 1615 1620Gln Lys Val His Leu Lys Ser
Ile Thr Glu Val Gly Glu Ala Leu 1625 1630 1635Lys Thr Val Leu Gly
Lys Lys Glu Thr Leu Val Glu Asp Lys Leu 1640 1645 1650Ser Leu Leu
Asn Ser Asn Trp Ile Ala Val Thr Ser Arg Ala Glu 1655 1660 1665Glu
Trp Leu Asn Leu Leu Leu Glu Tyr Gln Lys His Met Glu Thr 1670 1675
1680Phe Asp Gln Asn Val Asp His Ile Thr Lys Trp Ile Ile Gln Ala
1685 1690 1695Asp Thr Leu Leu Asp Glu Ser Glu Lys Lys Lys Pro Gln
Gln Lys 1700 1705 1710Glu Asp Val Leu Lys Arg Leu Lys Ala Glu Leu
Asn Asp Ile Arg 1715 1720 1725Pro Lys Val Asp Ser Thr Arg Asp Gln
Ala Ala Asn Leu Met Ala 1730 1735 1740Asn Arg Gly Asp His Cys Arg
Lys Leu Val Glu Pro Gln Ile Ser 1745 1750 1755Glu Leu Asn His Arg
Phe Ala Ala Ile Ser His Arg Ile Lys Thr 1760 1765 1770Gly Lys Ala
Ser Ile Pro Leu Lys Glu Leu Glu Gln Phe Asn Ser 1775 1780 1785Asp
Ile Gln Lys Leu Leu Glu Pro Leu Glu Ala Glu Ile Gln Gln 1790 1795
1800Gly Val Asn Leu Lys Glu Glu Asp Phe Asn Lys Asp Met Asn Glu
1805 1810 1815Asp Asn Glu Gly Thr Val Lys Glu Leu Leu Gln Arg Gly
Asp Asn 1820 1825 1830Leu Gln Gln Arg Ile Thr Asp Glu Arg Lys Arg
Glu Glu Ile Lys 1835 1840 1845Ile Lys Gln Gln Leu Leu Gln Thr Lys
His Asn Ala Leu Lys Asp 1850 1855 1860Leu Arg Ser Gln Arg Arg Lys
Lys Ala Leu Glu Ile Ser His Gln 1865 1870 1875Trp Tyr Gln Tyr Lys
Arg Gln Ala Asp Asp Leu Leu Lys Cys Leu 1880 1885 1890Asp Asp Ile
Glu Lys Lys Leu Ala Ser Leu Pro Glu Pro Arg Asp 1895 1900 1905Glu
Arg Lys Ile Lys Glu Ile Asp Arg Glu Leu Gln Lys Lys Lys 1910 1915
1920Glu Glu Leu Asn Ala Val Arg Arg Gln Ala Glu Gly Leu Ser Glu
1925 1930 1935Asp Gly Ala Ala Met Ala Val Glu Pro Thr Gln Ile Gln
Leu Ser 1940 1945 1950Lys Arg Trp Arg Glu Ile Glu Ser Lys Phe Ala
Gln Phe Arg Arg 1955 1960 1965Leu Asn Phe Ala Gln Ile His Thr Val
Arg Glu Glu Thr Met Met 1970 1975 1980Val Met Thr Glu Asp Met Pro
Leu Glu Ile Ser Tyr Val Pro Ser 1985 1990 1995Thr Tyr Leu Thr Glu
Ile Thr His Val Ser Gln Ala Leu Leu Glu 2000 2005 2010Val Glu Gln
Leu Leu Asn Ala Pro Asp Leu Cys Ala Lys Asp Phe 2015 2020 2025Glu
Asp Leu Phe Lys Gln Glu Glu Ser Leu Lys Asn Ile Lys Asp 2030 2035
2040Ser Leu Gln Gln Ser Ser Gly Arg Ile Asp Ile Ile His Ser Lys
2045 2050 2055Lys Thr Ala Ala Leu Gln Ser Ala Thr Pro Val Glu Arg
Val Lys 2060 2065 2070Leu Gln Glu Ala Leu Ser Gln Leu Asp Phe Gln
Trp Glu Lys Val 2075 2080 2085Asn Lys Met Tyr Lys Asp Arg Gln Gly
Arg Phe Asp Arg Ser Val 2090 2095 2100Glu Lys Trp Arg Arg Phe His
Tyr Asp Ile Lys Ile Phe Asn Gln 2105 2110 2115Trp Leu Thr Glu Ala
Glu Gln Phe Leu Arg Lys Thr Gln Ile Pro 2120 2125 2130Glu Asn Trp
Glu His Ala Lys Tyr Lys Trp Tyr Leu Lys Glu Leu 2135 2140 2145Gln
Asp Gly Ile Gly Gln Arg Gln Thr Val Val Arg Thr Leu Asn 2150 2155
2160Ala Thr Gly Glu Glu Ile Ile Gln Gln Ser Ser Lys Thr Asp Ala
2165 2170 2175Ser Ile Leu Gln Glu Lys Leu Gly Ser Leu Asn Leu Arg
Trp Gln 2180 2185 2190Glu Val Cys Lys Gln Leu Ser Asp Arg Lys Lys
Arg Leu Glu Glu 2195 2200 2205Gln Lys Asn Ile Leu Ser Glu Phe Gln
Arg Asp Leu Asn Glu Phe 2210 2215 2220Val Leu Trp Leu Glu Glu Ala
Asp Asn Ile Ala Ser Ile Pro Leu 2225 2230 2235Glu Pro Gly Lys Glu
Gln Gln Leu Lys Glu Lys Leu Glu Gln Val 2240 2245 2250Lys Leu Leu
Val Glu Glu Leu Pro Leu Arg Gln Gly Ile Leu Lys 2255 2260 2265Gln
Leu Asn Glu Thr Gly Gly Pro Val Leu Val Ser Ala Pro Ile 2270 2275
2280Ser Pro Glu Glu Gln Asp Lys Leu Glu Asn Lys Leu Lys Gln Thr
2285 2290 2295Asn Leu Gln Trp Ile Lys Val Ser Arg Ala Leu Pro Glu
Lys Gln 2300 2305 2310Gly Glu Ile Glu Ala Gln Ile Lys Asp Leu Gly
Gln Leu Glu Lys 2315 2320 2325Lys Leu Glu Asp Leu Glu Glu Gln Leu
Asn His Leu Leu Leu Trp 2330 2335 2340Leu Ser Pro Ile Arg Asn Gln
Leu Glu Ile Tyr Asn Gln Pro Asn 2345 2350 2355Gln Glu Gly Pro Phe
Asp Val Gln Glu Thr Glu Ile Ala Val Gln 2360 2365 2370Ala Lys Gln
Pro Asp Val Glu Glu Ile Leu Ser Lys Gly Gln His 2375 2380 2385Leu
Tyr Lys Glu Lys Pro Ala Thr Gln Pro Val Lys Arg Lys Leu 2390 2395
2400Glu Asp Leu Ser Ser Glu Trp Lys Ala Val Asn Arg Leu Leu Gln
2405 2410 2415Glu Leu Arg Ala Lys Gln Pro Asp Leu Ala Pro Gly Leu
Thr Thr 2420 2425 2430Ile Gly Ala Ser Pro Thr Gln Thr Val Thr Leu
Val Thr Gln Pro 2435 2440 2445Val
Val Thr Lys Glu Thr Ala Ile Ser Lys Leu Glu Met Pro Ser 2450 2455
2460Ser Leu Met Leu Glu Val Pro Ala Leu Ala Asp Phe Asn Arg Ala
2465 2470 2475Trp Thr Glu Leu Thr Asp Trp Leu Ser Leu Leu Asp Gln
Val Ile 2480 2485 2490Lys Ser Gln Arg Val Met Val Gly Asp Leu Glu
Asp Ile Asn Glu 2495 2500 2505Met Ile Ile Lys Gln Lys Ala Thr Met
Gln Asp Leu Glu Gln Arg 2510 2515 2520Arg Pro Gln Leu Glu Glu Leu
Ile Thr Ala Ala Gln Asn Leu Lys 2525 2530 2535Asn Lys Thr Ser Asn
Gln Glu Ala Arg Thr Ile Ile Thr Asp Arg 2540 2545 2550Ile Glu Arg
Ile Gln Asn Gln Trp Asp Glu Val Gln Glu His Leu 2555 2560 2565Gln
Asn Arg Arg Gln Gln Leu Asn Glu Met Leu Lys Asp Ser Thr 2570 2575
2580Gln Trp Leu Glu Ala Lys Glu Glu Ala Glu Gln Val Leu Gly Gln
2585 2590 2595Ala Arg Ala Lys Leu Glu Ser Trp Lys Glu Gly Pro Tyr
Thr Val 2600 2605 2610Asp Ala Ile Gln Lys Lys Ile Thr Glu Thr Lys
Gln Leu Ala Lys 2615 2620 2625Asp Leu Arg Gln Trp Gln Thr Asn Val
Asp Val Ala Asn Asp Leu 2630 2635 2640Ala Leu Lys Leu Leu Arg Asp
Tyr Ser Ala Asp Asp Thr Arg Lys 2645 2650 2655Val His Met Ile Thr
Glu Asn Ile Asn Ala Ser Trp Arg Ser Ile 2660 2665 2670His Lys Arg
Val Ser Glu Arg Glu Ala Ala Leu Glu Glu Thr His 2675 2680 2685Arg
Leu Leu Gln Gln Phe Pro Leu Asp Leu Glu Lys Phe Leu Ala 2690 2695
2700Trp Leu Thr Glu Ala Glu Thr Thr Ala Asn Val Leu Gln Asp Ala
2705 2710 2715Thr Arg Lys Glu Arg Leu Leu Glu Asp Ser Lys Gly Val
Lys Glu 2720 2725 2730Leu Met Lys Gln Trp Gln Asp Leu Gln Gly Glu
Ile Glu Ala His 2735 2740 2745Thr Asp Val Tyr His Asn Leu Asp Glu
Asn Ser Gln Lys Ile Leu 2750 2755 2760Arg Ser Leu Glu Gly Ser Asp
Asp Ala Val Leu Leu Gln Arg Arg 2765 2770 2775Leu Asp Asn Met Asn
Phe Lys Trp Ser Glu Leu Arg Lys Lys Ser 2780 2785 2790Leu Asn Ile
Arg Ser His Leu Glu Ala Ser Ser Asp Gln Trp Lys 2795 2800 2805Arg
Leu His Leu Ser Leu Gln Glu Leu Leu Val Trp Leu Gln Leu 2810 2815
2820Lys Asp Asp Glu Leu Ser Arg Gln Ala Pro Ile Gly Gly Asp Phe
2825 2830 2835Pro Ala Val Gln Lys Gln Asn Asp Val His Arg Ala Phe
Lys Arg 2840 2845 2850Glu Leu Lys Thr Lys Glu Pro Val Ile Met Ser
Thr Leu Glu Thr 2855 2860 2865Val Arg Ile Phe Leu Thr Glu Gln Pro
Leu Glu Gly Leu Glu Lys 2870 2875 2880Leu Tyr Gln Glu Pro Arg Glu
Leu Pro Pro Glu Glu Arg Ala Gln 2885 2890 2895Asn Val Thr Arg Leu
Leu Arg Lys Gln Ala Glu Glu Val Asn Thr 2900 2905 2910Glu Trp Glu
Lys Leu Asn Leu His Ser Ala Asp Trp Gln Arg Lys 2915 2920 2925Ile
Asp Glu Thr Leu Glu Arg Leu Gln Glu Leu Gln Glu Ala Thr 2930 2935
2940Asp Glu Leu Asp Leu Lys Leu Arg Gln Ala Glu Val Ile Lys Gly
2945 2950 2955Ser Trp Gln Pro Val Gly Asp Leu Leu Ile Asp Ser Leu
Gln Asp 2960 2965 2970His Leu Glu Lys Val Lys Ala Leu Arg Gly Glu
Ile Ala Pro Leu 2975 2980 2985Lys Glu Asn Val Ser His Val Asn Asp
Leu Ala Arg Gln Leu Thr 2990 2995 3000Thr Leu Gly Ile Gln Leu Ser
Pro Tyr Asn Leu Ser Thr Leu Glu 3005 3010 3015Asp Leu Asn Thr Arg
Trp Lys Leu Leu Gln Val Ala Val Glu Asp 3020 3025 3030Arg Val Arg
Gln Leu His Glu Ala His Arg Asp Phe Gly Pro Ala 3035 3040 3045Ser
Gln His Phe Leu Ser Thr Ser Val Gln Gly Pro Trp Glu Arg 3050 3055
3060Ala Ile Ser Pro Asn Lys Val Pro Tyr Tyr Ile Asn His Glu Thr
3065 3070 3075Gln Thr Thr Cys Trp Asp His Pro Lys Met Thr Glu Leu
Tyr Gln 3080 3085 3090Ser Leu Ala Asp Leu Asn Asn Val Arg Phe Ser
Ala Tyr Arg Thr 3095 3100 3105Ala Met Lys Leu Arg Arg Leu Gln Lys
Ala Leu Cys Leu Asp Leu 3110 3115 3120Leu Ser Leu Ser Ala Ala Cys
Asp Ala Leu Asp Gln His Asn Leu 3125 3130 3135Lys Gln Asn Asp Gln
Pro Met Asp Ile Leu Gln Ile Ile Asn Cys 3140 3145 3150Leu Thr Thr
Ile Tyr Asp Arg Leu Glu Gln Glu His Asn Asn Leu 3155 3160 3165Val
Asn Val Pro Leu Cys Val Asp Met Cys Leu Asn Trp Leu Leu 3170 3175
3180Asn Val Tyr Asp Thr Gly Arg Thr Gly Arg Ile Arg Val Leu Ser
3185 3190 3195Phe Lys Thr Gly Ile Ile Ser Leu Cys Lys Ala His Leu
Glu Asp 3200 3205 3210Lys Tyr Arg Tyr Leu Phe Lys Gln Val Ala Ser
Ser Thr Gly Phe 3215 3220 3225Cys Asp Gln Arg Arg Leu Gly Leu Leu
Leu His Asp Ser Ile Gln 3230 3235 3240Ile Pro Arg Gln Leu Gly Glu
Val Ala Ser Phe Gly Gly Ser Asn 3245 3250 3255Ile Glu Pro Ser Val
Arg Ser Cys Phe Gln Phe Ala Asn Asn Lys 3260 3265 3270Pro Glu Ile
Glu Ala Ala Leu Phe Leu Asp Trp Met Arg Leu Glu 3275 3280 3285Pro
Gln Ser Met Val Trp Leu Pro Val Leu His Arg Val Ala Ala 3290 3295
3300Ala Glu Thr Ala Lys His Gln Ala Lys Cys Asn Ile Cys Lys Glu
3305 3310 3315Cys Pro Ile Ile Gly Phe Arg Tyr Arg Ser Leu Lys His
Phe Asn 3320 3325 3330Tyr Asp Ile Cys Gln Ser Cys Phe Phe Ser Gly
Arg Val Ala Lys 3335 3340 3345Gly His Lys Met His Tyr Pro Met Val
Glu Tyr Cys Thr Pro Thr 3350 3355 3360Thr Ser Gly Glu Asp Val Arg
Asp Phe Ala Lys Val Leu Lys Asn 3365 3370 3375Lys Phe Arg Thr Lys
Arg Tyr Phe Ala Lys His Pro Arg Met Gly 3380 3385 3390Tyr Leu Pro
Val Gln Thr Val Leu Glu Gly Asp Asn Met Glu Thr 3395 3400 3405Pro
Val Thr Leu Ile Asn Phe Trp Pro Val Asp Ser Ala Pro Ala 3410 3415
3420Ser Ser Pro Gln Leu Ser His Asp Asp Thr His Ser Arg Ile Glu
3425 3430 3435His Tyr Ala Ser Arg Leu Ala Glu Met Glu Asn Ser Asn
Gly Ser 3440 3445 3450Tyr Leu Asn Asp Ser Ile Ser Pro Asn Glu Ser
Ile Asp Asp Glu 3455 3460 3465His Leu Leu Ile Gln His Tyr Cys Gln
Ser Leu Asn Gln Asp Ser 3470 3475 3480Pro Leu Ser Gln Pro Arg Ser
Pro Ala Gln Ile Leu Ile Ser Leu 3485 3490 3495Glu Ser Glu Glu Arg
Gly Glu Leu Glu Arg Ile Leu Ala Asp Leu 3500 3505 3510Glu Glu Glu
Asn Arg Asn Leu Gln Ala Glu Tyr Asp Arg Leu Lys 3515 3520 3525Gln
Gln His Glu His Lys Gly Leu Ser Pro Leu Pro Ser Pro Pro 3530 3535
3540Glu Met Met Pro Thr Ser Pro Gln Ser Pro Arg Asp Ala Glu Leu
3545 3550 3555Ile Ala Glu Ala Lys Leu Leu Arg Gln His Lys Gly Arg
Leu Glu 3560 3565 3570Ala Arg Met Gln Ile Leu Glu Asp His Asn Lys
Gln Leu Glu Ser 3575 3580 3585Gln Leu His Arg Leu Arg Gln Leu Leu
Glu Gln Pro Gln Ala Glu 3590 3595 3600Ala Lys Val Asn Gly Thr Thr
Val Ser Ser Pro Ser Thr Ser Leu 3605 3610 3615Gln Arg Ser Asp Ser
Ser Gln Pro Met Leu Leu Arg Val Val Gly 3620 3625 3630Ser Gln Thr
Ser Asp Ser Met Gly Glu Glu Asp Leu Leu Ser Pro 3635 3640 3645Pro
Gln Asp Thr Ser Thr Gly Leu Glu Glu Val Met Glu Gln Leu 3650 3655
3660Asn Asn Ser Phe Pro Ser Ser Arg Gly Arg Asn Thr Pro Gly Lys
3665 3670 3675Pro Met Arg Glu Asp Thr Met 3680 3685275RNAArtificial
Sequenceexon 2ccaggauggc auugggcagc ggcaaacugu ugucagaaca
uugaaugcaa cuggggaaga 60aauaauucag caauc 75325RNAArtificial
Sequenceoligonucleotide 3uuugccgcug cccaaugcca uccug
25425RNAArtificial Sequenceoligonucleotide 4auucaauguu cugacaacag
uuugc 25525RNAArtificial Sequenceoligonucleotide 5ccaguugcau
ucaauguucu gacaa 25622RNAArtificial Sequenceoligonucleotide
6caguugcauu caauguucug ac 22720RNAArtificial
Sequenceoligonucleotide 7aguugcauuc aauguucuga 20821RNAArtificial
Sequenceoligonucleotide 8gauugcugaa uuauuucuuc c 21925RNAArtificial
Sequenceoligonucleotide 9gauugcugaa uuauuucuuc cccag
251025RNAArtificial Sequenceoligonucleotide 10auugcugaau uauuucuucc
ccagu 251125RNAArtificial Sequenceoligonucleotide 11uugcugaauu
auuucuuccc caguu 251225RNAArtificial Sequenceoligonucleotide
12ugcugaauua uuucuucccc aguug 251325RNAArtificial
Sequenceoligonucleotide 13gcugaauuau uucuucccca guugc
251425RNAArtificial Sequenceoligonucleotide 14cugaauuauu ucuuccccag
uugca 251525RNAArtificial Sequenceoligonucleotide 15ugaauuauuu
cuuccccagu ugcau 251625RNAArtificial Sequenceoligonucleotide
16gaauuauuuc uuccccaguu gcauu 251725RNAArtificial
Sequenceoligonucleotide 17aauuauuucu uccccaguug cauuc
251825RNAArtificial Sequenceoligonucleotide 18auuauuucuu ccccaguugc
auuca 251925RNAArtificial Sequenceoligonucleotide 19uuauuucuuc
cccaguugca uucaa 252025RNAArtificial Sequenceoligonucleotide
20uauuucuucc ccaguugcau ucaau 252125RNAArtificial
Sequenceoligonucleotide 21auuucuuccc caguugcauu caaug
252225RNAArtificial Sequenceoligonucleotide 22uuucuucccc aguugcauuc
aaugu 252325RNAArtificial Sequenceoligonucleotide 23uucuucccca
guugcauuca auguu 252425RNAArtificial Sequenceoligonucleotide
24ucuuccccag uugcauucaa uguuc 252525RNAArtificial
Sequenceoligonucleotide 25cuuccccagu ugcauucaau guucu
252625RNAArtificial Sequenceoligonucleotide 26uuccccaguu gcauucaaug
uucug 252725RNAArtificial Sequenceoligonucleotide 27uccccaguug
cauucaaugu ucuga 252825RNAArtificial Sequenceoligonucleotide
28ccccaguugc auucaauguu cugac 252925RNAArtificial
Sequenceoligonucleotide 29cccaguugca uucaauguuc ugaca
253025RNAArtificial Sequenceoligonucleotide 30ccaguugcau ucaauguucu
gacaa 253125RNAArtificial Sequenceoligonucleotide 31caguugcauu
caauguucug acaac 253225RNAArtificial Sequenceoligonucleotide
32aguugcauuc aauguucuga caaca 253320RNAArtificial
Sequenceoligonucleotide 33uccuguagaa uacuggcauc 203427RNAArtificial
Sequenceoligonucleotide 34ugcagaccuc cugccaccgc agauuca
273534RNAArtificial Sequenceoligonucleotide 35uugcagaccu ccugccaccg
cagauucagg cuuc 343625RNAArtificial Sequenceoligonucleotide
36guugcauuca auguucugac aacag 253725RNAArtificial
Sequenceoligonucleotide 37uugcauucaa uguucugaca acagu
253825RNAArtificial Sequenceoligonucleotide 38ugcauucaau guucugacaa
caguu 253925RNAArtificial Sequenceoligonucleotide 39gcauucaaug
uucugacaac aguuu 254025RNAArtificial Sequenceoligonucleotide
40cauucaaugu ucugacaaca guuug 254125RNAArtificial
Sequenceoligonucleotide 41auucaauguu cugacaacag uuugc
254225RNAArtificial Sequenceoligonucleotide 42ucaauguucu gacaacaguu
ugccg 254325RNAArtificial Sequenceoligonucleotide 43caauguucug
acaacaguuu gccgc 254425RNAArtificial Sequenceoligonucleotide
44aauguucuga caacaguuug ccgcu 254525RNAArtificial
Sequenceoligonucleotide 45auguucugac aacaguuugc cgcug
254625RNAArtificial Sequenceoligonucleotide 46uguucugaca acaguuugcc
gcugc 254725RNAArtificial Sequenceoligonucleotide 47guucugacaa
caguuugccg cugcc 254825RNAArtificial Sequenceoligonucleotide
48uucugacaac aguuugccgc ugccc 254925RNAArtificial
Sequenceoligonucleotide 49ucugacaaca guuugccgcu gccca
255025RNAArtificial Sequenceoligonucleotide 50cugacaacag uuugccgcug
cccaa 255125RNAArtificial Sequenceoligonucleotide 51ugacaacagu
uugccgcugc ccaau 255225RNAArtificial Sequenceoligonucleotide
52gacaacaguu ugccgcugcc caaug 255325RNAArtificial
Sequenceoligonucleotide 53acaacaguuu gccgcugccc aaugc
255425RNAArtificial Sequenceoligonucleotide 54caacaguuug ccgcugccca
augcc 255525RNAArtificial Sequenceoligonucleotide 55aacaguuugc
cgcugcccaa ugcca 255625RNAArtificial Sequenceoligonucleotide
56acaguuugcc gcugcccaau gccau 255725RNAArtificial
Sequenceoligonucleotide 57caguuugccg cugcccaaug ccauc
255825RNAArtificial Sequenceoligonucleotide 58aguuugccgc ugcccaaugc
caucc 255925RNAArtificial Sequenceoligonucleotide 59guuugccgcu
gcccaaugcc auccu 256025RNAArtificial Sequenceoligonucleotide
60uuugccgcug cccaaugcca uccug 256125RNAArtificial
Sequenceoligonucleotide 61uugccgcugc ccaaugccau ccugg
256225RNAArtificial Sequenceoligonucleotide 62ugccgcugcc caaugccauc
cugga 256325RNAArtificial Sequenceoligonucleotide 63gccgcugccc
aaugccaucc uggag 256425RNAArtificial Sequenceoligonucleotide
64ccgcugccca augccauccu ggagu 256525RNAArtificial
Sequenceoligonucleotide 65cgcugcccaa ugccauccug gaguu
256620RNAArtificial Sequenceoligoncleotide 66uguuuuugag gauugcugaa
206740RNAArtificial Sequenceoligonucleotide 67uguucugaca acaguuugcc
gcugcccaau gccauccugg 406817RNAArtificial Sequenceoligonucleotide
68gcccaaugcc auccugg 176925RNAArtificial Sequenceoligonucleotide
69agagcaggua ccuccaacau caagg 257025RNAArtificial
Sequenceoligonucleotide 70gagcagguac cuccaacauc aagga
257125RNAArtificial Sequenceoligonucleotide 71agcagguacc uccaacauca
aggaa 257225RNAArtificial Sequenceoligonucleotide 72gcagguaccu
ccaacaucaa ggaag 257325RNAArtificial Sequenceoligonucleotide
73cagguaccuc caacaucaag gaaga 257425RNAArtificial
Sequenceoligonucleotide 74agguaccucc aacaucaagg aagau
257525RNAArtificial Sequenceoligonucleotide 75gguaccucca acaucaagga
agaug
257625RNAArtificial Sequenceoligonucleotide 76guaccuccaa caucaaggaa
gaugg 257725RNAArtificial Sequenceoligonucleotide 77uaccuccaac
aucaaggaag auggc 257825RNAArtificial Sequenceoligonucleotide
78accuccaaca ucaaggaaga uggca 257925RNAArtificial
Sequenceoligonucleotide 79ccuccaacau caaggaagau ggcau
258025RNAArtificial Sequenceoligonucleotide 80cuccaacauc aaggaagaug
gcauu 258130RNAArtificial Sequenceoligonucleotide 81cuccaacauc
aaggaagaug gcauuucuag 308225RNAArtificial Sequenceoligonucleotide
82uccaacauca aggaagaugg cauuu 258325RNAArtificial
Sequenceoligonucleotide 83ccaacaucaa ggaagauggc auuuc
258425RNAArtificial Sequenceoligonucleotide 84caacaucaag gaagauggca
uuucu 258525RNAArtificial Sequenceoligonucleotide 85aacaucaagg
aagauggcau uucua 258625RNAArtificial Sequenceoligonucleotide
86acaucaagga agauggcauu ucuag 258730RNAArtificial
Sequenceoligonucleotide 87acaucaagga agauggcauu ucuaguuugg
308825RNAArtificial Sequenceoligonucleotide 88acaucaagga agauggcauu
ucuag 258925RNAArtificial Sequenceoligonucleotide 89caucaaggaa
gauggcauuu cuagu 259025RNAArtificial Sequenceoligonucleotide
90aucaaggaag auggcauuuc uaguu 259125RNAArtificial
Sequenceoligonucleotide 91ucaaggaaga uggcauuucu aguuu
259220RNAArtificial Sequenceoligonucleotide 92ucaaggaaga uggcauuucu
209325RNAArtificial Sequenceoligonucleotide 93caaggaagau ggcauuucua
guuug 259425RNAArtificial Sequenceoligonucleotide 94aaggaagaug
gcauuucuag uuugg 259525RNAArtificial Sequenceoligonucleotide
95aggaagaugg cauuucuagu uugga 259625RNAArtificial
Sequenceoligonucleotide 96ggaagauggc auuucuaguu uggag
259725RNAArtificial Sequenceoligonucleotide 97gaagauggca uuucuaguuu
ggaga 259825RNAArtificial Sequenceoligonucleotide 98aagauggcau
uucuaguuug gagau 259925RNAArtificial Sequenceoligonucleotide
99agauggcauu ucuaguuugg agaug 2510025RNAArtificial
Sequenceoligonucleotide 100gauggcauuu cuaguuugga gaugg
2510125RNAArtificial Sequenceoligonucleotide 101auggcauuuc
uaguuuggag auggc 2510225RNAArtificial Sequenceoligonucleotide
102uggcauuucu aguuuggaga uggca 2510325RNAArtificial
Sequenceoligonucleotide 103ggcauuucua guuuggagau ggcag
2510425RNAArtificial Sequenceoligonucleotide 104gcauuucuag
uuuggagaug gcagu 2510525RNAArtificial Sequenceoligonucleotide
105cauuucuagu uuggagaugg caguu 2510625RNAArtificial
Sequenceoligonucleotide 106auuucuaguu uggagauggc aguuu
2510725RNAArtificial Sequenceoligonucleotide 107uuucuaguuu
ggagauggca guuuc 2510825RNAArtificial Sequenceoligonucleotide
108uucuaguuug gagauggcag uuucc 2510925RNAArtificial
Sequenceoligonucleotide 109ccauuguguu gaauccuuua acauu
2511022RNAArtificial Sequenceoligonucleotide 110ccauuguguu
gaauccuuua ac 2211120RNAArtificial Sequenceoligonucleotide
111auuguguuga auccuuuaac 2011220RNAArtificial
Sequenceoligonucleotide 112ccuguccuaa gaccugcuca
2011325RNAArtificial Sequenceoligonucleotide 113cuuuuggauu
gcaucuacug uauag 2511425RNAArtificial Sequenceoligonucleotide
114cauucaacug uugccuccgg uucug 2511524RNAArtificial
Sequenceoligonucleotide 115cuguugccuc cgguucugaa ggug
2411631RNAArtificial Sequenceoligonucleotide 116cauucaacug
uugccuccgg uucugaaggu g 3111725RNAArtificial
Sequenceoligonucleotide 117cugaaggugu ucuuguacuu caucc
2511827RNAArtificial Sequenceoligonucleotide 118uguauaggga
cccuccuucc augacuc 2711920RNAArtificial Sequenceoligonucleotide
119aucccacuga uucugaauuc 2012022RNAArtificial
Sequenceoligonucleotide 120uuggcucugg ccuguccuaa ga
2212135RNAArtificial Sequenceoligonucleotide 121aagaccugcu
cagcuucuuc cuuagcuucc agcca 3512223RNAArtificial
Sequenceoligonucleotide 122ugcauguucc agucguugug ugg
2312325RNAArtificial Sequenceoligonucleotide 123cacuauucca
gucaaauagg ucugg 2512425RNAArtificial Sequenceoligonucleotide
124auuuaccaac cuucaggauc gagua 2512521RNAArtificial
Sequenceoligonucleotide 125ggccuaaaac acauacacau a
2112620RNAArtificial Sequenceoligonucleotide 126ucagcuucug
uuagccacug 2012720RNAArtificial Sequenceoligonucleotide
127uucagcuucu guuagccacu 2012821RNAArtificial
Sequenceoligonucleotide 128uucagcuucu guuagccacu g
2112921RNAArtificial Sequenceoligonucleotide 129ucagcuucug
uuagccacug a 2113022RNAArtificial Sequenceoligonucleotide
130uucagcuucu guuagccacu ga 2213121RNAArtificial
Sequenceoligonucleotide 131ucagcuucug uuagccacug a
2113222RNAArtificial Sequenceoligonucleotide 132uucagcuucu
guuagccacu ga 2213322RNAArtificial Sequenceoligonucleotide
133ucagcuucug uuagccacug au 2213423RNAArtificial
Sequenceoligonucleotide 134uucagcuucu guuagccacu gau
2313523RNAArtificial Sequenceoligonucleotide 135ucagcuucug
uuagccacug auu 2313624RNAArtificial Sequenceoligonucleotide
136uucagcuucu guuagccacu gauu 2413724RNAArtificial
Sequenceoligonucleotide 137ucagcuucug uuagccacug auua
2413824RNAArtificial Sequenceoligonucleotide 138uucagcuucu
guuagccacu gaua 2413925RNAArtificial Sequenceoligonucleotide
139ucagcuucug uuagccacug auuaa 2514026RNAArtificial
Sequenceoligonucleotide 140uucagcuucu guuagccacu gauuaa
2614126RNAArtificial Sequenceoligonucleotide 141ucagcuucug
uuagccacug auuaaa 2614227RNAArtificial Sequenceoligonucleotide
142uucagcuucu guuagccacu gauuaaa 2714319RNAArtificial
Sequenceoligonucleotide 143cagcuucugu uagccacug
1914421RNAArtificial Sequenceoligonucleotide 144cagcuucugu
uagccacuga u 2114521RNAArtificial Sequenceoligonucleotide
145agcuucuguu agccacugau u 2114622RNAArtificial
Sequenceoligonucleotide 146cagcuucugu uagccacuga uu
2214722RNAArtificial Sequenceoligonucleotide 147agcuucuguu
agccacugau ua 2214823RNAArtificial Sequenceoligonculeotide
148cagcuucugu uagccacuga uua 2314923RNAArtificial
Sequenceoligonucleotide 149agcuucuguu agccacugau uaa
2315024RNAArtificial Sequenceoligonucleotide 150cagcuucugu
uagccacuga uuaa 2415124RNAArtificial Sequenceoligonucleotide
151agcuucuguu agccacugau uaaa 2415225RNAArtificial
Sequenceoligonucleotide 152cagcuucugu uagccacuga uuaaa
2515324RNAArtificial Sequenceoligonucleotide 153agcuucuguu
agccacugau uaaa 2415420RNAArtificial Sequenceoligonucleotide
154agcuucuguu agccacugau 2015520RNAArtificial
Sequenceoligonucleotide 155gcuucuguua gccacugauu
2015621RNAArtificial Sequenceoligonucleotide 156agcuucuguu
agccacugau u 2115721RNAArtificial Sequenceoligonucleotide
157gcuucuguua gccacugauu a 2115822RNAArtificial
Sequenceoligonculeotide 158agcuucuguu agccacugau ua
2215922RNAArtificial Sequenceoligonucleotide 159gcuucuguua
gccacugauu aa 2216023RNAArtificial Sequenceoligonucleotide
160agcuucuguu agccacugau uaa 2316123RNAArtificial
Sequenceoligonucleotide 161gcuucuguua gccacugauu aaa
2316224RNAArtificial Sequenceoligonucleotide 162agcuucuguu
agccacugau uaaa 2416323RNAArtificial Sequenceoligonucleotide
163gcuucuguua gccacugauu aaa 2316423RNAArtificial
Sequenceoligonucleotide 164ccauuuguau uuagcauguu ccc
2316520RNAArtificial Sequenceoligonucleotide 165agauaccauu
uguauuuagc 2016619RNAArtificial Sequenceoligonucleotide
166gccauuucuc aacagaucu 1916723RNAArtificial
Sequenceoligonucleotide 167gccauuucuc aacagaucug uca
2316823RNAArtificial Sequenceoligonucleotide 168auucucagga
auuugugucu uuc 2316921RNAArtificial Sequenceoligonucleotide
169ucucaggaau uugugucuuu c 2117018RNAArtificial
Sequenceoligonucleotide 170guucagcuuc uguuagcc 1817121RNAArtificial
Sequenceoligonucleotide 171cugauuaaau aucuuuauau c
2117218RNAArtificial Sequenceoligonucleotide 172gccgccauuu cucaacag
1817318RNAArtificial Sequenceoligonucleotide 173guauuuagca uguuccca
1817418RNAArtificial Sequenceoligonucleotide 174caggaauuug ugucuuuc
1817525RNAArtificial Sequenceoligonucleotide 175gcuuuucuuu
uaguugcugc ucuuu 2517625RNAArtificial Sequenceoligonucleotide
176cuuuucuuuu aguugcugcu cuuuu 2517725RNAArtificial
Sequenceoligonucleotide 177uuuucuuuua guugcugcuc uuuuc
2517825RNAArtificial Sequenceoligonucleotide 178uuucuuuuag
uugcugcucu uuucc 2517925RNAArtificial Sequenceoligonucleotide
179uucuuuuagu ugcugcucuu uucca 2518025RNAArtificial
Sequenceoligonucleotide 180ucuuuuaguu gcugcucuuu uccag
2518125RNAArtificial Sequenceoligonucleotide 181cuuuuaguug
cugcucuuuu ccagg 2518225RNAArtificial Sequenceoligonucleotide
182uuuuaguugc ugcucuuuuc caggu 2518325RNAArtificial
Sequenceoligonucleotide 183uuuaguugcu gcucuuuucc agguu
2518425RNAArtificial Sequenceoligonucleotide 184uuaguugcug
cucuuuucca gguuc 2518525RNAArtificial Sequenceoligonucleotide
185uaguugcugc ucuuuuccag guuca 2518625RNAArtificial
Sequenceoligonucleotide 186aguugcugcu cuuuuccagg uucaa
2518725RNAArtificial Sequenceoligonucleotide 187guugcugcuc
uuuuccaggu ucaag 2518825RNAArtificial Sequenceoligonucleotide
188uugcugcucu uuuccagguu caagu 2518925RNAArtificial
Sequenceoligonucleotide 189ugcugcucuu uuccagguuc aagug
2519025RNAArtificial Sequenceoligonucleotide 190gcugcucuuu
uccagguuca agugg 2519125RNAArtificial Sequenceoligonucleotide
191cugcucuuuu ccagguucaa guggg 2519225RNAArtificial
Sequenceoligonucleotide 192ugcucuuuuc cagguucaag uggga
2519325RNAArtificial Sequenceoligonucleotide 193gcucuuuucc
agguucaagu gggac 2519425RNAArtificial Sequenceoligonucleotide
194cucuuuucca gguucaagug ggaua 2519525RNAArtificial
Sequenceoligonucleotide 195ucuuuuccag guucaagugg gauac
2519625RNAArtificial Sequenceoligonucleotide 196cuuuuccagg
uucaaguggg auacu 2519725RNAArtificial Sequenceoligonucleotide
197uuuuccaggu ucaaguggga uacua 2519825RNAArtificial
Sequenceoligonucleotide 198uuuccagguu caagugggau acuag
2519925RNAArtificial Sequenceoligonucleotide 199uuccagguuc
aagugggaua cuagc 2520025RNAArtificial Sequenceoligonucleotide
200uccagguuca agugggauac uagca 2520125RNAArtificial
Sequenceoligonucleotide 201ccagguucaa gugggauacu agcaa
2520225RNAArtificial Sequenceoligonucleotide 202cagguucaag
ugggauacua gcaau 2520325RNAArtificial Sequenceoligonucleotide
203agguucaagu gggauacuag caaug 2520425RNAArtificial
Sequenceoligonucleotide 204gguucaagug ggauacuagc aaugu
2520525RNAArtificial Sequenceoligonucleotide 205guucaagugg
gauacuagca auguu 2520625RNAArtificial Sequenceoligonucleotide
206uucaaguggg auacuagcaa uguua 2520725RNAArtificial
Sequenceoligonucleotide 207ucaaguggga uacuagcaau guuau
2520825RNAArtificial Sequenceoligonucleotide 208caagugggau
acuagcaaug uuauc 2520925RNAArtificial Sequenceoligonucleotide
209aagugggaua cuagcaaugu uaucu 2521025RNAArtificial
Sequenceoligonucleotide 210agugggauac uagcaauguu aucug
2521125RNAArtificial Sequenceoligonucleotide 211gugggauacu
agcaauguua ucugc 2521225RNAArtificial Sequenceoligonucleotide
212ugggauacua gcaauguuau cugcu 2521325RNAArtificial
Sequenceoligonucleotide 213gggauacuag caauguuauc ugcuu
2521425RNAArtificial Sequenceoligonucleotide 214ggauacuagc
aauguuaucu gcuuc 2521525RNAArtificial Sequenceoligonucleotide
215gauacuagca auguuaucug cuucc 2521625RNAArtificial
Sequenceoligonucleotide 216auacuagcaa uguuaucugc uuccu
2521725RNAArtificial Sequenceoligonucleotide 217uacuagcaau
guuaucugcu uccuc 2521825RNAArtificial Sequenceoligonucleotide
218acuagcaaug uuaucugcuu ccucc 2521925RNAArtificial
Sequenceoligonucleotide 219cuagcaaugu uaucugcuuc cucca
2522025RNAArtificial Sequenceoligonucleotide 220uagcaauguu
aucugcuucc uccaa 2522125RNAArtificial Sequenceoligonucleotide
221agcaauguua ucugcuuccu ccaac 2522225RNAArtificial
Sequenceoligonucleotide 222gcaauguuau cugcuuccuc caacc
2522325RNAArtificial Sequenceoligonucleotide 223caauguuauc
ugcuuccucc aacca 2522425RNAArtificial Sequenceoligonucleotide
224aauguuaucu gcuuccucca accau 2522525RNAArtificial
Sequenceoligonucleotide 225auguuaucug cuuccuccaa ccaua
2522625RNAArtificial
Sequenceoligonucleotide 226uguuaucugc uuccuccaac cauaa
2522725RNAArtificial Sequenceoligonucleotide 227guuaucugcu
uccuccaacc auaaa 2522819RNAArtificial Sequenceoligonucleotide
228gcugcucuuu uccagguuc 1922920RNAArtificial
Sequenceoligonucleotide 229ucuuuuccag guucaagugg
2023019RNAArtificial Sequenceoligonucleotide 230agguucaagu
gggauacua 1923121RNAArtificial Sequenceoligonucleotide
231caauuuuucc cacucaguau u 2123219RNAArtificial
Sequenceoligonucleotide 232uugaaguucc uggagucuu
1923322RNAArtificial Sequenceoligonucleotide 233uccucaggag
gcagcucuaa au 2223426RNAArtificial Sequenceoligonucleotide
234gcgcugguca caaaauccug uugaac 2623527RNAArtificial
Sequenceoligonucleotide 235cacuugcuug aaaaggucua caaagga
2723626RNAArtificial Sequenceoligonucleotide 236ggugaauaac
uuacaaauuu ggaagc 26
* * * * *
References