U.S. patent application number 16/422241 was filed with the patent office on 2019-11-28 for compositions for treating muscular dystrophy.
The applicant listed for this patent is Sarepta Therapeutics, Inc.. Invention is credited to Edward M. KAYE.
Application Number | 20190359982 16/422241 |
Document ID | / |
Family ID | 50678303 |
Filed Date | 2019-11-28 |
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United States Patent
Application |
20190359982 |
Kind Code |
A1 |
KAYE; Edward M. |
November 28, 2019 |
COMPOSITIONS FOR TREATING MUSCULAR DYSTROPHY
Abstract
Improved compositions and methods for treating muscular
dystrophy by administering antisense molecules capable of binding
to a selected target site in the human dystrophin gene to induce
exon skipping are described.
Inventors: |
KAYE; Edward M.; (Cambridge,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sarepta Therapeutics, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
50678303 |
Appl. No.: |
16/422241 |
Filed: |
May 24, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15359152 |
Nov 22, 2016 |
10337003 |
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16422241 |
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14213629 |
Mar 14, 2014 |
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15359152 |
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61793463 |
Mar 15, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2320/30 20130101;
A61P 21/02 20180101; A61P 21/00 20180101; C12N 15/113 20130101;
A61K 31/7125 20130101; C12N 2310/33 20130101; A61P 1/00
20180101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 31/7125 20060101 A61K031/7125 |
Claims
1. A method of treating Duchenne muscular dystrophy or Becker
muscular dystrophy in a human subject comprising administering
about 30 mg/kg to about 50 mg/kg of a composition comprising an
antisense oligonucleotide of 20 to 50 nucleotides in length
comprising at least 10 consecutive nucleotides complementary to a
target region in an exon of the human dystrophin gene, wherein the
antisense oligonucleotide specifically hybridizes to the target
region inducing exon skipping, thereby treating Duchenne muscular
dystrophy or Becker muscular dystrophy in the subject.
2. The method of claim 1, wherein treatment increases the number of
dystrophin-positive fibers to at least 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90% or 95% of normal in the subject.
3. The method of claim 2, wherein the number of dystrophin-positive
fibers is increased to between 20-60% of normal in the human
subject.
4. The method of claim 2, wherein the number of dystrophin-positive
fibers is increased to between 30-50% of normal in the human
subject.
5. The method of claim 1, wherein about 30 mg/kg of the composition
is administered to the human subject.
6. The method of claim 1, wherein about 50 mg/kg of the composition
is administered to the human subject.
7. The method of claim 1, wherein treatment improves or maintains a
stable walking distance in a 6 minute walk test from a 20% deficit
in the subject relative to a healthy peer.
8. The method of claim 1, wherein the antisense oligonucleotide is
substantially uncharged.
9. The method of claim 1, wherein the antisense oligonucleotide
comprises morpholino subunits linked by phosphorus-containing
intersubunit linkages joining a morpholino nitrogen of one subunit
to a 5' exocyclic carbon of an adjacent subunit.
10. The method of claim 1, wherein the antisense oligonucleotide
comprises morpholino subunits linked by substantially uncharged
phosphorus-containing intersubunit linkages joining a morpholino
nitrogen of one subunit to a 5' exocyclic carbon of an adjacent
subunit.
11. The method of claim 1, wherein the antisense oligonucleotide
comprises morpholino subunits and phosphorodiamidate intersubunit
linkages.
12. The method of claim 1, wherein the antisense oligonucleotide is
chemically linked to one or more moieties or conjugates that
enhance the activity, cellular distribution, or cellular uptake of
the antisense oligonucleotide.
13. The method of claim 12, wherein the antisense oligonucleotide
is conjugated to an arginine-rich peptide.
14. The method of claim 1, wherein the antisense oligonucleotide is
30 to 50 nucleotides in length.
15. The method of claim 1, wherein the antisense oligonucleotide is
20 to 30 nucleotides in length.
16. The method of claim 1, wherein the exon in the human dystrophin
gene is selected from the group consisting of exon 51, exon 50,
exon 53, exon 45, exon 46, exon 44, exon 52, exon 55 and exon
8.
17. The method of claim 1, wherein the antisense oligonucleotide is
selected from the group consisting of SEQ ID NOS: 1-9.
18. The method of claim 1, wherein the antisense oligonucleotide is
SEQ ID NO: 1.
19. The method of claim 1, wherein the antisense oligonucleotide is
any one or a combination of the nucleotide sequences set forth in
Tables 3 and 4, wherein uracil bases in the antisense
oligonucleotide are optionally thymine bases.
20. The method of claim 1, wherein the composition further
comprises phosphate-buffered saline.
21. The method of claim 1, wherein the composition is administered
by systemic administration.
22. The method of claim 1, wherein the composition is administered
once weekly by infusion.
23. The method of claim 1, further comprising administering a
steroid to the human subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Patent
Application No. 15,359,152, filed Nov. 22, 2016, now pending, which
application is a continuation of U.S. patent application Ser. No.
14/213,629, filed Mar. 14, 2014, now abandoned, which claims
priority to U.S. Provisional Application No. 61/793,463, filed on
Mar. 15, 2013. The entire contents of the foregoing applications
are incorporated herein by reference.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The Sequence Listing associated with this application has
been submitted electronically in ASCII format, and is hereby
incorporated by reference into the specification in its entirety.
The name of the text file containing the sequence Listing is
4140_0030008_SL_ST25.txt. The text file is 190693 bytes, was
created on May 23, 2019 and is being submitted electronically via
EFS-Web.
FIELD OF THE INVENTION
[0003] The present invention relates to improved methods for
treating muscular dystrophy in a patient. It also provides
compositions suitable for facilitating exon skipping in the human
dystrophin gene.
BACKGROUND OF THE INVENTION
[0004] Antisense technologies are being developed using a range of
chemistries to affect gene expression at a variety of different
levels (transcription, splicing, stability, translation). Much of
that research has focused on the use of antisense compounds to
correct or compensate for abnormal or disease-associated genes in a
wide range of indications. Antisense molecules are able to inhibit
gene expression with specificity, and because of this, many
research efforts concerning oligonucleotides as modulators of gene
expression have focused on inhibiting the expression of targeted
genes or the function of cis-acting elements. The antisense
oligonucleotides are typically directed against RNA, either the
sense strand (e.g., mRNA), or minus-strand in the case of some
viral RNA targets. To achieve a desired effect of specific gene
down-regulation, the oligonucleotides generally either promote the
decay of the targeted mRNA, block translation of the mRNA or block
the function of cis-acting RNA elements, thereby effectively
preventing either de novo synthesis of the target protein or
replication of the viral RNA.
[0005] However, such techniques are not useful where the object is
to up-regulate production of the native protein or compensate for
mutations that induce premature termination of translation, such as
nonsense or frame-shifting mutations. In these cases, the defective
gene transcript should not be subjected to targeted degradation or
steric inhibition, so the antisense oligonucleotide chemistry
should not promote target mRNA decay or block translation.
[0006] In a variety of genetic diseases, the effects of mutations
on the eventual expression of a gene can be modulated through a
process of targeted exon skipping during the splicing process. The
splicing process is directed by complex multi-component machinery
that brings adjacent exon-intron junctions in pre-mRNA into close
proximity and performs cleavage of phosphodiester bonds at the ends
of the introns with their subsequent reformation between exons that
are to be spliced together. This complex and highly precise process
is mediated by sequence motifs in the pre-mRNA that are relatively
short, semi-conserved RNA segments to which various nuclear
splicing factors that are then involved in the splicing reactions
bind. By changing the way the splicing machinery reads or
recognizes the motifs involved in pre-mRNA processing, it is
possible to create differentially spliced mRNA molecules. It has
now been recognized that the majority of human genes are
alternatively spliced during normal gene expression, although the
mechanisms involved have not been identified. Bennett et al. (U.S.
Pat. No. 6,210,892) describe antisense modulation of wild-type
cellular mRNA processing using antisense oligonucleotide analogs
that do not induce RNAse H-mediated cleavage of the target RNA.
This finds utility in being able to generate alternatively spliced
mRNAs that lack specific exons (e.g., as described by (Sazani,
Kole, et al. 2007) for the generation of soluble TNF superfamily
receptors that lack exons encoding membrane spanning domains.
[0007] In cases where a normally functional protein is prematurely
terminated because of mutations therein, a means for restoring some
functional protein production through antisense technology has been
shown to be possible through intervention during the splicing
processes, and that if exons associated with disease-causing
mutations can be specifically deleted from some genes, a shortened
protein product can sometimes be produced that has similar
biological properties of the native protein or has sufficient
biological activity to ameliorate the disease caused by mutations
associated with the exon (see e.g., Sierakowska, Sambade et al.
1996; Wilton, Lloyd et al. 1999; van Deutekom, Bremmer-Bout et al.
2001; Lu, Mann et al. 2003; Aartsma-Rus, Janson et al. 2004). Kole
et al. (U.S. Pat. Nos. 5,627,274; 5,916,808; 5,976,879; and
5,665,593) disclose methods of combating aberrant splicing using
modified antisense oligonucleotide analogs that do not promote
decay of the targeted pre-mRNA. Bennett et al. (U.S. Pat. No.
6,210,892) describe antisense modulation of wild-type cellular mRNA
processing also using antisense oligonucleotide analogs that do not
induce RNAse H-mediated cleavage of the target RNA.
[0008] The process of targeted exon skipping is likely to be
particularly useful in long genes where there are many exons and
introns, where there is redundancy in the genetic constitution of
the exons or where a protein is able to function without one or
more particular exons. Efforts to redirect gene processing for the
treatment of genetic diseases associated with truncations caused by
mutations in various genes have focused on the use of antisense
oligonucleotides that either: (1) fully or partially overlap with
the elements involved in the splicing process; or (2) bind to the
pre-mRNA at a position sufficiently close to the element to disrupt
the binding and function of the splicing factors that would
normally mediate a particular splicing reaction which occurs at
that element.
[0009] Duchenne muscular dystrophy (DMD) is caused by a defect in
the expression of the protein dystrophin. The gene encoding the
protein contains 79 exons spread out over more than 2 million
nucleotides of DNA. Any exonic mutation that changes the reading
frame of the exon, or introduces a stop codon, or is characterized
by removal of an entire out of frame exon or exons, or duplications
of one or more exons, has the potential to disrupt production of
functional dystrophin, resulting in DMD.
[0010] Disease onset can be documented at birth with elevated
creatine kinase levels, and significant motor deficits may be
present in the first year of life. By the age of seven or eight,
most patients with DMD have an increasingly labored gait and are
losing the ability to rise from the floor and climb stairs; by ages
10 to 14, most are wheelchair-dependent. DMD is uniformly fatal;
affected individuals typically die of respiratory and/or cardiac
failure in their late teens or early 20s. The continuous
progression of DMD allows for therapeutic intervention at all
stages of the disease; however, treatment is currently limited to
glucocorticoids, which are associated with numerous side effects
including weight gain, behavioral changes, pubertal changes,
osteoporosis, Cushingoid facies, growth inhibition, and cataracts.
Consequently, developing better therapies to treat the underlying
cause of this disease is imperative.
[0011] A less severe form of muscular dystrophy, Becker muscular
dystrophy (BMD) has been found to arise where a mutation, typically
a deletion of one or more exons, results in a correct reading frame
along the entire dystrophin transcript, such that translation of
mRNA into protein is not prematurely terminated. If the joining of
the upstream and downstream exons in the processing of a mutated
dystrophin pre-mRNA maintains the correct reading frame of the
gene, the result is an mRNA coding for a protein with a short
internal deletion that retains some activity, resulting in a Becker
phenotype.
[0012] For many years it has been known that deletions of an exon
or exons which do not alter the reading frame of a dystrophin
protein would give rise to a BMD phenotype, whereas an exon
deletion that causes a frame-shift will give rise to DMD (Monaco,
Bertelson et al. 1988). In general, dystrophin mutations including
point mutations and exon deletions that change the reading frame
and thus interrupt proper protein translation result in DMD. It
should also be noted that some BMD and DMD patients have exon
deletions covering multiple exons.
[0013] Modulation of mutant dystrophin pre-mRNA splicing with
antisense oligoribonucleotides has been reported both in vitro and
in vivo (see e.g., Matsuo, Masumura et al. 1991; Takeshima, Nishio
et al. 1995; Pramono, Takeshima et al. 1996; Dunckley, Eperon et
al. 1997; Dunckley, Manoharan et al. 1998; Errington, Mann et al.
2003).
[0014] The first example of specific and reproducible exon skipping
in the mdx mouse model was reported by Wilton et al. (Wilton, Lloyd
et al. 1999). By directing an antisense molecule to the donor
splice site, consistent and efficient exon 23 skipping was induced
in the dystrophin mRNA within 6 hours of treatment of the cultured
cells. Wilton et al. also describe targeting the acceptor region of
the mouse dystrophin pre-mRNA with longer antisense
oligonucleotides. While the first antisense oligonucleotide
directed at the intron 23 donor splice site induced consistent exon
skipping in primary cultured myoblasts, this compound was found to
be much less efficient in immortalized cell cultures expressing
higher levels of dystrophin. However, with refined targeting and
antisense oligonucleotide design, the efficiency of specific exon
removal was increased by almost an order of magnitude (Mann,
Honeyman et al. 2002).
[0015] Recent studies have begun to address the challenge of
achieving sustained dystrophin expression accompanied by minimal
adverse effects in tissues affected by the absence of dystrophin.
Intramuscular injection of an antisense oligonucleotide targeted to
exon 51 (PRO051) into the tibialis anterior muscle in four patients
with DMD resulted in specific skipping of exon 51 without any
clinically apparent adverse effects (Mann, Honeyman et al. 2002;
van Deutekom, Janson et al. 2007). Studies looking at systemic
delivery of an antisense phosphorodiamidate morpholino oligomer
conjugated to a cell-penetrating peptide (PPMO) targeted to exon 23
in mdx mice produced high and sustained dystrophin protein
production in skeletal and cardiac muscles without detectable
toxicity (Jearawiriyapaisarn, Moulton et al. 2008; Wu, Moulton et
al. 2008; Yin, Moulton et al. 2008).
[0016] Recent clinical trials testing the safety and efficacy of
splice switching oligonucleotides (SSOs) for the treatment of DMD
are based on SSO technology to induce alternative splicing of
pre-mRNAs by steric blockade of the spliceosome (Cirak et al.,
2011; Goemans et al., 2011; Kinali et al., 2009; van Deutekom et
al., 2007). However, despite these successes, the pharmacological
options available for treating DMD are limited. Notably, an
antisense oligonucleotide (drisapersen), which utilizes a
negatively charged phosphorothioate backbone, has been associated
in clinical trials with proteinuria, increased urinary
.alpha.1-microglobulin, thrombocytopenia and injection site
reactions, such as erythema and inflammation.
[0017] Thus, there remains a need for improved compositions and
methods for treating muscular dystrophy, such as DMD and BMD in
patients.
SUMMARY OF THE INVENTION
[0018] The present invention is based, at least in part, on
compelling evidence of a therapeutic effect of an exon skipping
antisense oligonucleotide, eteplirsen, which represents a major
advance in the treatment of DMD by addressing the underlying cause
of the disease. The novel finding that treatment with an exon 51
skipping antisense oligonucleotide, eteplirsen, produced reliable
increases in novel dystrophin and stabilized walking ability (e.g.,
stabilization of ambulation), as measured by the 6 Minute Walk Test
(6MWT), underscores the potential alter the course of the disease.
Significantly, no drug-related adverse events were seen in 576
infusions administered over one year. When applied to other exons,
the use of exon skipping antisense oligonucleotides could treat an
estimated 70% to 80% of patients who have DMD due to a deletion in
the dystrophin gene.
[0019] Accordingly, the present invention relates to methods of
treating Duchenne muscular dystrophy (DMD) or Becker muscular
dystrophy (BMD) in patients by administering an effective amount of
a composition comprising an antisense oligonucleotide of 20 to 50
nucleotides in length comprising at least 10 consecutive
nucleotides complementary to a target region in an exon of the
human dystrophin gene to specifically hybridize to the target
region, induce exon skipping, and thereby treat the disease. In one
embodiment, an effective amount is at least 20 mg/kg for a period
of time sufficient to increase the number of dystrophin-positive
fibers in a subject to at least 20% of normal, and stabilize,
maintain, or improve walking distance from a 20% deficit, for
example in a 6 MWT, in the patient, relative to a healthy peer. In
another embodiment, an effective amount is at least 20 mg/kg to
about 30 mg/kg, about 25 mg/kg to about 30 mg/kg, or about 30 mg/kg
to about 50 mg/kg. In yet another embodiment, an effective amount
is about 30 mg/kg or about 50 mg/kg.
[0020] In another aspect, an effective amount is at least 20 mg/kg,
about 25 mg/kg, about 30 mg/kg, or about 30 mg/kg to about 50
mg/kg, for at least 24 weeks, at least 36 weeks, or at least 48
weeks, to thereby increase the number of dystrophin-positive fibers
in a subject to at least 20%, about 30%, about 40%, about 50%,
about 60%, about 70%, about 80%, about 90%, about 95% of normal,
and stabilize or improve walking distance from a 20% deficit, for
example in a 6 MWT, in the patient relative to a healthy peer. In
one embodiment, treatment increases the number of
dystrophin-positive fibers to 20-60%, or 30-50% of normal in the
patient. In some embodiments, treatment is by systemic
administration, such as once weekly by infusion. In other
embodiments, treatment includes administering another therapeutic
agent, such as a steroid to the subject.
[0021] In another aspect, the present invention provides a method
of treating DMD or BMD in a patient by administering about 30 mg/kg
to about 50 mg/kg of a composition comprising an antisense
oligonucleotide of 20 to 50 nucleotides in length comprising at
least 10 consecutive nucleotides complementary to a target region
in an exon of the human dystrophin gene, wherein the antisense
oligonucleotide specifically hybridizes to the target region
inducing exon skipping, thereby treating the subject. In one
embodiment, the antisense oligonucleotide is substantially
uncharged. In another embodiment, the antisense oligonucleotide
comprises morpholino subunits linked by phosphorus-containing
intersubunit linkages joining a morpholino nitrogen of one subunit
to a 5' exocyclic carbon of an adjacent subunit. In yet another
embodiment, the antisense oligonucleotide comprises morpholino
subunits linked by substantially uncharged phosphorus-containing
intersubunit linkages joining a morpholino nitrogen of one subunit
to a 5' exocyclic carbon of an adjacent subunit. In other aspects,
the antisense oligonucleotide comprises morpholino subunits and
phosphorodiamidate intersubunit linkages.
[0022] In some embodiments, the antisense oligonucleotide is 20 to
50, 30 to 50, or 20 to 30 nucleotides in length comprising at least
10, 12, 15, 17, or 20 consecutive nucleotides complementary to a
target region in an exon of the human dystrophin gene selected from
the group consisting of exon 51, exon 50, exon 53, exon 45, exon
46, exon 44, exon 52, exon 55 and exon 8. In one embodiment, the
antisense is 20 to 50, 30 to 50, or 20 to 30 nucleotides in length
and includes at least 20 consecutive nucleotides eteplirsen (SEQ ID
NO: 1). In another embodiment, the antisense oligonucleotide is 20
to 50, 30 to 50, or 20 to 30 nucleotides in length and includes at
least 10, 12, 15, 17, or 20 consecutive nucleotides of the
antisense oligonucleotide set forth as SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:
8, or SEQ ID NO: 9. In yet another embodiment, the antisense
oligonucleotide is 20 to 50, 30 to 50, or 20 to 30 nucleotides in
length and includes at least 10, 12, 15, 17, or 20 consecutive
nucleotides of a nucleotide sequences set forth in Tables 3 and 4,
wherein uracil bases in the antisense oligonucleotide are
optionally thymine bases.
[0023] In one embodiment, the composition includes eteplirsen (SEQ
ID NO: 1), and, optionally, a pharmaceutically acceptable carrier.
In another embodiment, the composition includes an antisense
oligonucleotide selected from the group consisting of SEQ ID NOS:
1-9, such as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ
ID NO: 9. In yet another embodiment, the antisense oligonucleotide
is any one or a combination of the nucleotide sequences set forth
in Tables 3 and 4, wherein uracil bases in the antisense
oligonucleotide are optionally thymine bases. In some aspects, the
antisense oligonucleotide is chemically linked to one or more
moieties or conjugates that enhance the activity, cellular
distribution, or cellular uptake of the antisense oligonucleotide,
such as an arginine-rich peptide.
[0024] In another aspect, the present invention provides a method
of treating DMD or BMD in a patient by administering at least 20
mg/kg of a composition comprising eteplirsen (SEQ ID NO: 1) for a
period of time sufficient to increase the number of
dystrophin-positive fibers in a subject to at least about 20% of
normal, and stabilize or improve walking distance from a 20%
deficit, for example in a 6 MWT, in the patient, relative to a
healthy peer. In another embodiment, an effective amount is at
least 20 mg/kg to about 30 mg/kg, about 25 mg/kg to about 30 mg/kg,
or about 30 mg/kg to about 50 mg/kg of a composition comprising
eteplirsen (SEQ ID NO: 1), and, optionally, a pharmaceutically
acceptable carrier, such as phosphate-buffered saline.
[0025] In another aspect, an effective amount of a composition
comprising eteplirsen (SEQ ID NO: 1) is at least 20 mg/kg, about 25
mg/kg, about 30 mg/kg, or about 30 mg/kg to about 50 mg/kg, for at
least 24 weeks, at least 36 weeks, or at least 48 weeks, to thereby
increase the number of dystrophin-positive fibers in a subject to
at least about 20%, about 30%, about 40%, about 50%, about 60%,
about 70%, about 80%, about 90%, about 95% of normal, and stabilize
or improve walking distance from a 20% deficit, for example in a 6
MWT, in the patient relative to a healthy peer. In some
embodiments, treatment with antisense oligonucleotides of the
present invention slows or reduces the loss of ambulation that
would be expected without treatment. In some embodiments, treatment
with the antisense oligonucleotides of the present invention
stabilizes, maintains, or increases a stable walking distance in a
patient. For example, treatment may increase the stable walking
distance in the patient from baseline to greater than 3, 5, 6, 7,
8, 9, 10, 15, 20, 25, 30 or 50 meters (including all integers in
between).
BRIEF DESCRIPTION OF THE FIGURES
[0026] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0027] FIG. 1A shows an exemplary morpholino oligomer structure
with a phosphorodiamidate linkage.
[0028] FIG. 1B shows a conjugate of an arginine-rich peptide and an
antisense oligomer, in accordance with an embodiment of the
invention.
[0029] FIG. 1C shows a conjugate as in FIG. 1B, wherein the
backbone linkages contain one or more positively charged
groups.
[0030] FIGS. 1D-G show the repeating subunit segment of exemplary
morpholino oligonucleotides, designated D through G.
[0031] FIG. 2 is a schematic representation of the study design for
treating DMD patients. Twelve DMD patients were randomized to one
of three cohorts in the double-blind, placebo-controlled study,
201: Cohort 1, eteplirsen 30 mg/kg/wk; Cohort 2, eteplirsen 50
mg/kg/wk; and Cohort 3, placebo/delayed eteplirsen. At week 25,
placebo-treated patients in Cohort 3 switched to open-label
treatment with 30 or 50 mg/kg/week eteplirsen. Patients were
maintained on their same dose of eteplirsen under the open-label
extension study, 202. Muscle Biopsies. Patients underwent biceps
biopsies at baseline and deltoid biopsies at week 48 for analysis
of dystrophin. Additional biceps biopsies were obtained at week 12
(from patients in Cohort 2 and two patients in Cohort 3) or week 24
(from patients in Cohort 1 and two patients in Cohort 3). Efficacy
Evaluations. The 6MWT was used as a functional outcome measure and
was performed pre-treatment and every 12 weeks post treatment
through week 48.
[0032] FIG. 3 depicts dystrophin-positive muscle fibers after 12,
24, and 48 weeks of eteplirsen. Panels A and B show the mean
absolute change from baseline in the percentage of
dystrophin-positive fibers at weeks 12, 24, and week 48 by
treatment group. In Panel A: P-value is for comparison between
eteplirsen and placebo using the pooled results from weeks 12 and
24, and is based on an analysis of covariance model for ranked data
with treatment as a fixed effect and baseline value and time since
DMD diagnosis as covariates. Mean changes shown are based on
descriptive statistics. .sup..dagger.P-value is from a paired
t-test comparing the week 48 value to baseline. Results from the
placebo-treated patients biopsied at weeks 12 and 24 are pooled.
.sctn. Placebo/delayed eteplirsen patients began receiving
eteplirsen at week 25 and had received a total of 24 doses at week
48. Abbreviations: BL=baseline; NA=not applicable; ND=not done;
NS=not significant; SE=standard error.
[0033] FIG. 4 shows the effects of eteplirsen on the
dystrophin-associated glycoprotein complex. (A) Representative
examples of time-dependent increases in dystrophin-positive fibers
in relation to treatment for all participating study patients. (B)
nNOS .mu. staining in muscle from DMD (a) and normal (c) control
patients (not in study), and from patient 6 at baseline (b) and
week 48 (d), demonstrates restoration of nNOS.mu. binding with
eteplirsen. .beta.-sarcoglycan (e) and .gamma.-sarcoglycan (0
staining in patient 6 at week 48 demonstrate restoration of the
sarcoglycan complex with eteplirsen. (C) RT-PCR shows skipped
product (289 bp) post-treatment in the muscle of patient 12.
[0034] FIG. 5 graphically depicts the functional efficacy of
eteplirsen. The dark purple line shows the change from baseline in
distance walked on the 6MWT over time for the 6 evaluable patients
who received eteplirsen from the start of 201 (two boys were unable
to at or beyond week 24 were excluded from this analysis). The gray
line shows change from baseline in distance walked on the 6MWT for
the 4 patients who received placebo for the first 24 weeks and
eteplirsen for the last 24 weeks.
DETAILED DESCRIPTION
[0035] Embodiments of the present invention relate to improved
methods for treating muscular dystrophy, such as DMD and BMD, by
administering antisense compounds that are specifically designed to
induce exon skipping in the human dystrophin gene. Dystrophin plays
a vital role in muscle function, and various muscle-related
diseases are characterized by mutated forms of this gene. Hence, in
certain embodiments, the improved methods described herein may be
used for inducing exon skipping in mutated forms of the human
dystrophin gene, such as the mutated dystrophin genes found in DMD
and BMD.
[0036] Due to aberrant mRNA splicing events caused by mutations,
these mutated human dystrophin genes either express defective
dystrophin protein or express no measurable dystrophin at all, a
condition that leads to various forms of muscular dystrophy. To
remedy this condition, the antisense compounds of the present
invention hybridize to selected regions of a pre-processed RNA of a
mutated human dystrophin gene, induce exon skipping and
differential splicing in that otherwise aberrantly spliced
dystrophin mRNA, and thereby allow muscle cells to produce an mRNA
transcript that encodes a functional dystrophin protein. In certain
embodiments, the resulting dystrophin protein is not necessarily
the "wild-type" form of dystrophin, but is rather a truncated, yet
functional or semi-functional, form of dystrophin.
[0037] By increasing the levels of functional dystrophin protein in
muscle cells, these and related embodiments are useful in the
prophylaxis and treatment of muscular dystrophy, especially those
forms of muscular dystrophy, such as DMD and BMD, that are
characterized by the expression of defective dystrophin proteins
due to aberrant mRNA splicing. The methods described herein further
provide improved treatment options for patients with muscular
dystrophy and offer significant and practical advantages over
alternate methods of treating relevant forms of muscular dystrophy.
For example, in some embodiments, the improved methods relate to
the administration of an antisense compound for inducing exon
skipping in the human dystrophin gene at a higher dose and/or for a
longer duration than prior approaches.
[0038] Thus, the invention relates to improved methods for treating
muscular dystrophy such as DMD and BMD, by inducing exon skipping
in a patient. In some embodiments, exon skipping is induced by
administering an effective amount of a composition which includes a
charge-neutral, phosphorodiamidate morpholino oligomer (PMO), such
as eteplirsen, which selectively binds to a target sequence in an
exon of dystrophin pre-mRNA. In some embodiments, the invention
relates to methods of treating DMD or BMD in which an effective
amount of a composition e.g., at least 20 mg/kg, about 25 mg/kg,
about 30 mg/kg or about 30 mg/kg to about 50 mg/kg, which includes
an antisense as described herein, such as eteplirsen, over a period
of time sufficient to treat the disease.
[0039] Some embodiments of the present invention relate to the use
of eteplirsen as a disease-modifying therapy for treating DMD. In a
clinical study described in the Examples, no drug-related adverse
events were seen in 576 infusions of eteplirsen administered over
one year, nor were there any infusion-associated reactions, cardiac
abnormalities, or any other organ system involvement. There was
also no evidence of glomerular or renal tubular dysfunction in
eteplirsen-treated patients over the course of the study (e.g., no
proteinuria, elevations in serum cytstatin C, or urine cystatin C
or KIM-1). Without being bound by theory, the safety profile of
eteplirsen and other exon-skipping antisense oligonucleotides
described herein may be attributed to its unique chemical
composition, which is characterized by nucleotides bound to
morpholine rings linked through charge-neutral phosphorodiamidate
moieties.
[0040] In DMD patients treated with eteplirsen for one year, the
mean percentage of dystrophin-positive fibers was increased to 47%
of normal, relative to baseline. The magnitude of the increase was
dependent upon treatment duration. Significant increases in
dystrophin levels were observed in the 24-week biopsies taken from
patients in Cohort 1 (30 mg/kg) and in the 48-week biopsies from
patients in Cohort 3 (who started eteplirsen at week 25).
[0041] Eteplirsen's clinical benefit mirrored its ability to induce
exon skipping and restore functional dystrophin production.
Clinical effect was assessed with the 6MWT, a measure of endurance
and muscular capacity that goes beyond the assessment of strength
in individual muscle groups. Patients who received 30 or 50 mg/kg
eteplirsen from the beginning maintained a stable walking distance
over 48 weeks, consistent with eteplirsen-induced increases in
novel dystrophin expression between weeks 12 and 24. In contrast,
patients in the placebo/delayed eteplirsen cohort lost 70 meters by
week 36, but appeared to stabilize by week 48 (24 weeks after
initiating eteplirsen). This is the same timeframe in which a
clinical impact was seen in patients who received 30 or 50 mg/kg
eteplirsen once a week from the start of the study.
[0042] The present invention is based, at least in part, on the
evidence of a therapeutic effect of eteplirsen, which represents a
major advance in the treatment of DMD by addressing the underlying
cause of the disease. Accordingly, the invention relates to methods
of treating DMD or BMD in patients by administering an effective
amount of a composition which includes an antisense
oligonucleotide, such as eteplirsen, which is complementary to a
target region in an exon of the human dystrophin gene to
specifically hybridize to the target region, induce exon skipping,
and treat the disease. In one embodiment, treatment is by
administering one or more antisense oligonucleotides of the present
invention (e.g., a nucleotide sequence shown in Tables 3 and 4),
optionally as part of a pharmaceutical formulation or dosage form,
to a subject in need thereof. Treatment includes inducing
exon-skipping in a subject by administering an effective amount of
one or more antisense oligonucleotides, in which the exon is any
one or more of exons 1-79 from the dystrophin gene. Preferably, the
exon is exon 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 or
8 from the human dystrophin gene.
[0043] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which the invention belongs.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, preferred methods and materials are described.
For the purposes of the present invention, the following terms are
defined below.
I. DEFINITIONS
[0044] By "about" is meant a quantity, level, value, number,
frequency, percentage, dimension, size, amount, weight or length
that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3,
2 or 1% to a reference quantity, level, value, number, frequency,
percentage, dimension, size, amount, weight or length.
[0045] The terms "complementary" and "complementarity" refer to
polynucleotides (i.e., a sequence of nucleotides) related by
base-pairing rules. For example, the sequence "T-G-A (5'-3')," is
complementary to the sequence "T-C-A (5'-3')." Complementarity may
be "partial," in which only some of the nucleic acids' bases are
matched according to base pairing rules. Or, there may be
"complete" or "total" complementarity between the nucleic acids.
The degree of complementarity between nucleic acid strands has
significant effects on the efficiency and strength of hybridization
between nucleic acid strands. While perfect complementarity is
often desired, some embodiments can include one or more but
preferably 6, 5, 4, 3, 2, or 1 mismatches with respect to the
target RNA. Variations at any location within the oligomer are
included. In certain embodiments, variations in sequence near the
termini of an oligomer are generally preferable to variations in
the interior, and if present are typically within about 6, 5, 4, 3,
2, or 1 nucleotides of the 5' and/or 3' terminus.
[0046] The terms "cell penetrating peptide" and "CPP" are used
interchangeably and refer to cationic cell penetrating peptides,
also called transport peptides, carrier peptides, or peptide
transduction domains. The peptides, as shown herein, have the
capability of inducing cell penetration within 100% of cells of a
given cell culture population and allow macromolecular
translocation within multiple tissues in vivo upon systemic
administration. A preferred CPP embodiment is an arginine-rich
peptide as described further below.
[0047] The terms "antisense oligomer" and "antisense compound" and
"antisense oligonucleotide" are used interchangeably and refer to a
sequence of cyclic subunits, each bearing a base-pairing moiety,
linked by intersubunit linkages that allow the base-pairing
moieties to hybridize to a target sequence in a nucleic acid
(typically an RNA) by Watson-Crick base pairing, to form a nucleic
acid:oligomer heteroduplex within the target sequence. The cyclic
subunits are based on ribose or another pentose sugar or, in a
preferred embodiment, a morpholino group (see description of
morpholino oligomers below). The oligomer may have exact or near
sequence complementarity to the target sequence; variations in
sequence near the termini of an oligomer are generally preferable
to variations in the interior.
[0048] Such an antisense oligomer can be designed to block or
inhibit translation of mRNA or to inhibit natural pre-mRNA splice
processing, and may be said to be "directed to" or "targeted
against" a target sequence with which it hybridizes. The target
sequence is typically a region including an AUG start codon of an
mRNA, a Translation Suppressing Oligomer, or splice site of a
pre-processed mRNA, a Splice Suppressing Oligomer (SSO). The target
sequence for a splice site may include an mRNA sequence having its
5' end 1 to about 25 base pairs downstream of a normal splice
acceptor junction in a preprocessed mRNA. A preferred target
sequence is any region of a preprocessed mRNA that includes a
splice site or is contained entirely within an exon coding sequence
or spans a splice acceptor or donor site. An oligomer is more
generally said to be "targeted against" a biologically relevant
target, such as a protein, virus, or bacteria, when it is targeted
against the nucleic acid of the target in the manner described
above.
[0049] The terms "morpholino oligomer" or "PMO" (phosphoramidate-
or phosphorodiamidate morpholino oligomer) refer to an
oligonucleotide analog composed of morpholino subunit structures,
where (i) the structures are linked together by
phosphorus-containing linkages, one to three atoms long, preferably
two atoms long, and preferably uncharged or cationic, joining the
morpholino nitrogen of one subunit to a 5' exocyclic carbon of an
adjacent subunit, and (ii) each morpholino ring bears a purine or
pyrimidine base-pairing moiety effective to bind, by base specific
hydrogen bonding, to a base in a polynucleotide. See, for example,
the structure in FIG. 1A, which shows a preferred
phosphorodiamidate linkage type. Variations can be made to this
linkage as long as they do not interfere with binding or activity.
For example, the oxygen attached to phosphorus may be substituted
with sulfur (thiophosphorodiamidate). The 5' oxygen may be
substituted with amino or lower alkyl substituted amino. The
pendant nitrogen attached to phosphorus may be unsubstituted,
monosubstituted, or disubstituted with (optionally substituted)
lower alkyl. The purine or pyrimidine base pairing moiety is
typically adenine, cytosine, guanine, uracil, thymine or inosine.
The synthesis, structures, and binding characteristics of
morpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685,
5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, 5,506,337,
8,076,476, 8,299,206 and 7,943,762 (cationic linkages), all of
which are incorporated herein by reference. Modified intersubunit
linkages and terminal groups are detailed in PCT application
US2011/038459 and publication WO/2011/150408 which are incorporated
herein by reference in their entirety.
[0050] An "amino acid subunit" or "amino acid residue" can refer to
an .alpha.-amino acid residue (--CO--CHR--NH--) or a .beta.- or
other amino acid residue (e.g. --CO--(CH.sub.2).sub.nCHR--NH--),
where R is a side chain (which may include hydrogen) and n is 1 to
6, preferably 1 to 4.
[0051] The term "naturally occurring amino acid" refers to an amino
acid present in proteins found in nature. The term "non-natural
amino acids" refers to those amino acids not present in proteins
found in nature, examples include beta-alanine (.beta.-Ala),
6-aminohexanoic acid (Ahx) and 6-aminopentanoic acid.
[0052] An "exon" refers to a defined section of nucleic acid that
encodes for a protein, or a nucleic acid sequence that is
represented in the mature form of an RNA molecule after either
portions of a pre-processed (or precursor) RNA have been removed by
splicing. The mature RNA molecule can be a messenger RNA (mRNA) or
a functional form of a non-coding RNA, such as rRNA or tRNA. The
human dystrophin gene has about 79 exons.
[0053] An "intron" refers to a nucleic acid region (within a gene)
that is not translated into a protein. An intron is a non-coding
section that is transcribed into a precursor mRNA (pre-mRNA), and
subsequently removed by splicing during formation of the mature
RNA.
[0054] An "effective amount" or "therapeutically effective amount"
refers to an amount of therapeutic compound, such as an antisense
oligonucleotide, administered to a human subject, either as a
single dose or as part of a series of doses, which is effective to
produce a desired therapeutic effect. For an antisense
oligonucleotide, this effect is typically brought about by
inhibiting translation or natural splice-processing of a selected
target sequence. In some embodiments, an effective amount is at
least 20 mg/kg of a composition including an antisense
oligonucleotide for a period of time to treat the subject. In one
embodiment, an effective amount is at least 20 mg/kg of a
composition including an antisense oligonucleotide to increase the
number of dystrophin-positive fibers in a subject to at least 20%
of normal. In another embodiment, an effective amount is at least
20 mg/kg of a composition including an antisense oligonucleotide to
stabilize, maintain, or improve walking distance from a 20%
deficit, for example in a 6 MWT, in a patient, relative to a
healthy peer. In another embodiment, an effective amount is at
least 20 mg/kg to about 30 mg/kg, about 25 mg/kg to about 30 mg/kg,
or about 30 mg/kg to about 50 mg/kg. In yet another embodiment, an
effective amount is about 30 mg/kg or about 50 mg/kg. In another
aspect, an effective amount is at least 20 mg/kg, about 25 mg/kg,
about 30 mg/kg, or about 30 mg/kg to about 50 mg/kg, for at least
24 weeks, at least 36 weeks, or at least 48 weeks, to thereby
increase the number of dystrophin-positive fibers in a subject to
at least 20%, about 30%, about 40%, about 50%, about 60%, about
70%, about 80%, about 90%, about 95% of normal, and stabilize or
improve walking distance from a 20% deficit, for example in a 6
MWT, in the patient relative to a healthy peer. In one embodiment,
treatment increases the number of dystrophin-positive fibers to
20-60%, or 30-50% of normal in the patient.
[0055] "Exon skipping" refers generally to the process by which an
entire exon, or a portion thereof, is removed from a given
pre-processed RNA, and is thereby excluded from being present in
the mature RNA, such as the mature mRNA that is translated into a
protein. Hence, the portion of the protein that is otherwise
encoded by the skipped exon is not present in the expressed form of
the protein, typically creating an altered, though still
functional, form of the protein. In certain embodiments, the exon
being skipped is an aberrant exon from the human dystrophin gene,
which may contain a mutation or other alteration in its sequence
that otherwise causes aberrant splicing. In certain embodiments,
the exon being skipped is any one or more of exons 1-79 of the
human dystrophin gene, such as 3-8, 10-16, 19-40, 42-47, and 50-55,
though exons 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 and
8 of the human dystrophin gene are preferred.
[0056] "Dystrophin" is a rod-shaped cytoplasmic protein, and a
vital part of the protein complex that connects the cytoskeleton of
a muscle fiber to the surrounding extracellular matrix through the
cell membrane. Dystrophin contains multiple functional domains. For
instance, dystrophin contains an actin binding domain at about
amino acids 14-240 and a central rod domain at about amino acids
253-3040. This large central domain is formed by 24 spectrin-like
triple-helical elements of about 109 amino acids, which have
homology to alpha-actinin and spectrin. The repeats are typically
interrupted by four proline-rich non-repeat segments, also referred
to as hinge regions. Repeats 15 and 16 are separated by an 18 amino
acid stretch that appears to provide a major site for proteolytic
cleavage of dystrophin. The sequence identity between most repeats
ranges from 10-25%. One repeat contains three alpha-helices: 1, 2
and 3. Alpha-helices 1 and 3 are each formed by 7 helix turns,
probably interacting as a coiled-coil through a hydrophobic
interface. Alpha-helix 2 has a more complex structure and is formed
by segments of four and three helix turns, separated by a Glycine
or Proline residue. Each repeat is encoded by two exons, typically
interrupted by an intron between amino acids 47 and 48 in the first
part of alpha-helix 2. The other intron is found at different
positions in the repeat, usually scattered over helix-3. Dystrophin
also contains a cysteine-rich domain at about amino acids
3080-3360), including a cysteine-rich segment (i.e., 15 Cysteines
in 280 amino acids) showing homology to the C-terminal domain of
the slime mold (Dictyostelium discoideum) alpha-actinin. The
carboxy-terminal domain is at about amino acids 3361-3685.
[0057] The amino-terminus of dystrophin binds to F-actin and the
carboxy-terminus binds to the dystrophin-associated protein complex
(DAPC) at the sarcolemma. The DAPC includes the dystroglycans,
sarcoglycans, integrins and caveolin, and mutations in any of these
components cause autosomally inherited muscular dystrophies. The
DAPC is destabilized when dystrophin is absent, which results in
diminished levels of the member proteins, and in turn leads to
progressive fibre damage and membrane leakage. In various forms of
muscular dystrophy, such as Duchenne's muscular dystrophy (DMD) and
Becker's muscular dystrophy (BMD), muscle cells produce an altered
and functionally defective form of dystrophin, or no dystrophin at
all, mainly due to mutations in the gene sequence that lead to
incorrect splicing. The predominant expression of the defective
dystrophin protein, or the complete lack of dystrophin or a
dystrophin-like protein, leads to rapid progression of muscle
degeneration, as noted above. In this regard, a "defective"
dystrophin protein may be characterized by the forms of dystrophin
that are produced in certain subjects with DMD or BMD, as known in
the art, or by the absence of detectable dystrophin.
[0058] As used herein, the terms "function" and "functional" and
the like refer to a biological, enzymatic, or therapeutic
function.
[0059] A "functional" dystrophin protein refers generally to a
dystrophin protein having sufficient biological activity to reduce
the progressive degradation of muscle tissue that is otherwise
characteristic of muscular dystrophy, typically as compared to the
altered or "defective" form of dystrophin protein that is present
in certain subjects with DMD or BMD. In certain embodiments, a
functional dystrophin protein may have about 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, or 100% (including all integers in
between) of the in vitro or in vivo biological activity of
wild-type dystrophin, as measured according to routine techniques
in the art. As one example, dystrophin-related activity in muscle
cultures in vitro can be measured according to myotube size,
myofibril organization (or disorganization), contractile activity,
and spontaneous clustering of acetylcholine receptors (see, e.g.,
Brown et al., Journal of Cell Science. 112:209-216, 1999). Animal
models are also valuable resources for studying the pathogenesis of
disease, and provide a means to test dystrophin-related activity.
Two of the most widely used animal models for DMD research are the
mdx mouse and the golden retriever muscular dystrophy (GRMD) dog,
both of which are dystrophin negative (see, e.g., Collins &
Morgan, Int J Exp Pathol 84: 165-172, 2003). These and other animal
models can be used to measure the functional activity of various
dystrophin proteins. Included are truncated forms of dystrophin,
such as those forms that are produced by certain of the
exon-skipping antisense compounds of the present invention.
[0060] The term "restoration" of dystrophin synthesis or production
refers generally to the production of a dystrophin protein
including truncated forms of dystrophin in a patient with muscular
dystrophy following treatment with an antisense oligonucleotide as
described herein. In some embodiments, treatment results in an
increase in novel dystrophin production in a patient by 1%, 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% (including all
integers in between). In some embodiments, treatment increases the
number of dystrophin-positive fibers to at least 20%, about 30%,
about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or
about 95% to 100% of normal in the subject. In other embodiments,
treatment increases the number of dystrophin-positive fibers to
about 20% to about 60%, or about 30% to about 50% of normal in the
subject. The percent of dystrophin-positive fibers in a patient
following treatment can be determined by a muscle biopsy using
known techniques. For example, a muscle biopsy may be taken from a
suitable muscle, such as the biceps brachii muscle in a
patient.
[0061] Analysis of the percentage of positive dystrophin fibers may
be performed pre-treatment and/or post-treatment or at time points
throughout the course of treatment. In some embodiments, a
post-treatment biopsy is taken from the contralateral muscle from
the pre-treatment biopsy. Pre- and post-treatment dystrophin
expression studies may be performed using any suitable assay for
dystrophin. In one embodiment, immunohistochemical detection is
performed on tissue sections from the muscle biopsy using an
antibody that is a marker for dystrophin, such as a monoclonal or a
polyclonal antibody. For example, the MANDYS106 antibody can be
used which is a highly sensitive marker for dystrophin. Any
suitable secondary antibody may be used.
[0062] In some embodiments, the percent dystrophin-positive fibers
are calculated by dividing the number of positive fibers by the
total fibers counted. Normal muscle samples have 100%
dystrophin-positive fibers. Therefore, the percent
dystrophin-positive fibers can be expressed as a percentage of
normal. To control for the presence of trace levels of dystrophin
in the pretreatment muscle as well as revertant fibers a baseline
can be set using sections of pre-treatment muscles from each
patient when counting dystrophin-positive fibers in post-treatment
muscles. This may be used as a threshold for counting
dystrophin-positive fibers in sections of post-treatment muscle in
that patient. In other embodiments, antibody-stained tissue
sections can also be used for dystrophin quantification using
Bioquant image analysis software (Bioquant Image Analysis
Corporation, Nashville, Tenn.). The total dystrophin fluorescence
signal intensity can be reported as a percentage of normal. In
addition, Western blot analysis with monoclonal or polyclonal
anti-dystrophin antibodies can be used to determine the percentage
of dystrophin positive fibers. For example, the anti-dystrophin
antibody NCL-Dys1 from Novacastra may be used. The percentage of
dystrophin-positive fibers can also be analyzed by determining the
expression of the components of the sarcoglycan complex
(.beta.,.gamma.) and/or neuronal NOS.
[0063] By "isolated" is meant material that is substantially or
essentially free from components that normally accompany it in its
native state. For example, an "isolated polynucleotide," as used
herein, may refer to a polynucleotide that has been purified or
removed from the sequences that flank it in a naturally-occurring
state, e.g., a DNA fragment that has been removed from the
sequences that are normally adjacent to the fragment.
[0064] As used herein, "sufficient length" refers to an antisense
oligonucleotide that is complementary to at least 8, more typically
8-30, contiguous nucleobases in a target dystrophin pre-mRNA. In
some embodiments, an antisense of sufficient length includes at
least 8, 9, 10, 11, 12, 13, 14, or 15 contiguous nucleobases in the
target dystrophin pre-mRNA. In other embodiments an antisense of
sufficient length includes at least 16, 17, 18, 19, 20, 21, 22, 23,
24, or 25 contiguous nucleobases in the target dystrophin pre-mRNA.
An antisense oligonucleotide of sufficient length has at least a
minimal number of nucleotides to be capable of specifically
hybridizing to any one or more of exons 1-79 of the dystrophin
gene. Preferably, the antisense oligonucleotide of the invention
has a minimal number of nucleotides to be capable of specifically
hybridizing to any one or more of exons 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56 or 8 of the human dystrophin gene.
Preferably an oligonucleotide of sufficient length is from about 10
to about 50 nucleotides in length, including oligonucleotides of
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40 or more
nucleotides. In one embodiment, an oligonucleotide of sufficient
length is from 10 to about 30 nucleotides in length. In another
embodiment, an oligonucleotide of sufficient length is from 15 to
about 25 nucleotides in length. In yet another embodiment, an
oligonucleotide of sufficient length is from 20 to 30, or 20 to 50,
nucleotides in length. In yet another embodiment, an
oligonucleotide of sufficient length is from 25 to 28 nucleotides
in length.
[0065] By "enhance" or "enhancing," or "increase" or "increasing,"
or "stimulate" or "stimulating," refers generally to the ability of
one or antisense compounds or compositions to produce or cause a
greater physiological response (i.e., downstream effects) in a cell
or a subject, as compared to the response caused by either no
antisense compound or a control compound. A measurable
physiological response may include increased expression of a
functional form of a dystrophin protein, or increased
dystrophin-related biological activity in muscle tissue, among
other responses apparent from the understanding in the art and the
description herein. Increased muscle function can also be measured,
including increases or improvements in muscle function by about 1%,
2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,
17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, or 100%. The percentage of muscle
fibers that express a functional dystrophin can also be measured,
including increased dystrophin expression in about 1%, 2%, %, 15%,
16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of muscle fibers. For
instance, it has been shown that around 40% of muscle function
improvement can occur if 25-30% of fibers express dystrophin (see,
e.g., DelloRusso et al, Proc Natl Acad Sci USA 99: 12979-12984,
2002). An "increased" or "enhanced" amount is typically a
"statistically significant" amount, and may include an increase
that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or
more times (e.g., 500, 1000 times) (including all integers and
decimal points in between and above 1), e.g., 1.5, 1.6, 1.7, 1.8,
etc.) the amount produced by no antisense compound (the absence of
an agent) or a control compound.
[0066] The term "reduce" or "inhibit" may relate generally to the
ability of one or more antisense compounds of the invention to
"decrease" a relevant physiological or cellular response, such as a
symptom of a disease or condition described herein, as measured
according to routine techniques in the diagnostic art. Relevant
physiological or cellular responses (in vivo or in vitro) will be
apparent to persons skilled in the art, and may include reductions
in the symptoms or pathology of muscular dystrophy, or reductions
in the expression of defective forms of dystrophin, such as the
altered forms of dystrophin that are expressed in individuals with
DMD or BMD. A "decrease" in a response may be statistically
significant as compared to the response produced by no antisense
compound or a control composition, and may include a 1%, 2%, 3%,
4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,
18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, or 100% decrease, including all integers
in between.
[0067] Also included are vector delivery systems that are capable
of expressing the oligomeric, dystrophin-targeting sequences of the
present invention, such as vectors that express a polynucleotide
sequence comprising any one or more of the sequences shown in
Tables 3 and 4, and variants thereof, as described herein. By
"vector" or "nucleic acid construct" is meant a polynucleotide
molecule, preferably a DNA molecule derived, for example, from a
plasmid, bacteriophage, yeast or virus, into which a polynucleotide
can be inserted or cloned. A vector preferably contains one or more
unique restriction sites and can be capable of autonomous
replication in a defined host cell including a target cell or
tissue or a progenitor cell or tissue thereof, or be integrated
with the genome of the defined host such that the cloned sequence
is reproducible. Accordingly, the vector can be an autonomously
replicating vector, i.e., a vector that exists as an
extra-chromosomal entity, the replication of which is independent
of chromosomal replication, e.g., a linear or closed circular
plasmid, an extra-chromosomal element, a mini-chromosome, or an
artificial chromosome. The vector can contain any means for
assuring self-replication. Alternatively, the vector can be one
which, when introduced into the host cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated.
[0068] "Treatment" of an individual (e.g. a mammal, such as a
human) or a cell is any type of intervention used in an attempt to
alter the natural course of the individual or cell. Treatment
includes, but is not limited to, administration of a pharmaceutical
composition, and may be performed either prophylactically or
subsequent to the initiation of a pathologic event or contact with
an etiologic agent. Treatment includes any desirable effect on the
symptoms or pathology of a disease or condition associated with the
dystrophin protein, as in certain forms of muscular dystrophy, and
may include, for example, minimal changes or improvements in one or
more measurable markers of the disease or condition being treated.
Also included are "prophylactic" treatments, which can be directed
to reducing the rate of progression of the disease or condition
being treated, delaying the onset of that disease or condition, or
reducing the severity of its onset. "Treatment" or "prophylaxis"
does not necessarily indicate complete eradication, cure, or
prevention of the disease or condition, or associated symptoms
thereof.
[0069] In one embodiment, treatment with an antisense
oligonucleotide of the invention increases novel dystrophin
production and slows or reduces the loss of ambulation that would
be expected without treatment. For example, treatment may
stabilize, maintain, improve or increase walking ability (e.g.,
stabilization of ambulation) in the subject. In some embodiments,
treatment maintains or increases a stable walking distance in a
patient, as measured by, for example, the 6 Minute Walk Test
(6MWT), described by McDonald, et al. (Muscle Nerve, 2010;
42:966-74, herein incorporated by reference). A change in the 6
Minute Walk Distance (6MWD) may be expressed as an absolute value,
a percentage change or a change in the %-predicted value. In some
embodiments, treatment maintains or improves a stable walking
distance in a 6MWT from a 20% deficit in the subject relative to a
healthy peer. The performance of a DMD patient in the 6MWT relative
to the typical performance of a healthy peer can be determined by
calculating a %-predicted value. For example, the %-predicted 6MWD
may be calculated using the following equation for males:
196.72+(39.81.times.age)-(1.36.times.age.sup.2)+(132.28.times.heig-
ht in meters). For females, the %-predicted 6MWD may be calculated
using the following equation:
188.61+(51.50.times.age)-(1.86.times.age.sup.2)+(86.10.times.height
in meters) (Henricson et al. PLoS Curr., 2012, version 2, herein
incorporated by reference). In some embodiments, treatment with an
antisense oligonucleotide increases the stable walking distance in
the patient from baseline to greater than 3, 5, 6, 7, 8, 9, 10, 15,
20, 25, 30 or 50 meters (including all integers in between).
[0070] Loss of muscle function in patients with DMD may occur
against the background of normal childhood growth and development.
Indeed, younger children with DMD may show an increase in distance
walked during 6MWT over the course of about 1 year despite
progressive muscular impairment. In some embodiments, the 6MWD from
patients with DMD is compared to typically developing control
subjects and to existing normative data from age and sex matched
subjects. In some embodiments, normal growth and development can be
accounted for using an age and height based equation fitted to
normative data. Such an equation can be used to convert 6MWD to a
percent-predicted (%-predicted) value in subjects with DMD. In
certain embodiments, analysis of %-predicted 6MWD data represents a
method to account for normal growth and development, and may show
that gains in function at early ages (e.g., less than or equal to
age 7) represent stable rather than improving abilities in patients
with DMD (Henricson et al. PLoS Curr., 2012, version 2, herein
incorporated by reference).
[0071] A "subject," as used herein, includes any animal that
exhibits a symptom, or is at risk for exhibiting a symptom, which
can be treated with an antisense compound of the invention, such as
a subject that has or is at risk for having DMD or BMD, or any of
the symptoms associated with these conditions (e.g., muscle fibre
loss). Suitable subjects (patients) include laboratory animals
(such as mouse, rat, rabbit, or guinea pig), farm animals, and
domestic animals or pets (such as a cat or dog). Non-human primates
and, preferably, human patients, are included.
[0072] "Alkyl" or "alkylene" both refer to a saturated straight or
branched chain hydrocarbon radical containing from 1 to 18 carbons.
Examples include without limitation methyl, ethyl, propyl,
iso-propyl, butyl, iso-butyl, tert-butyl, n-pentyl and n-hexyl. The
term "lower alkyl" refers to an alkyl group, as defined herein,
containing between 1 and 8 carbons.
[0073] "Alkenyl" refers to an unsaturated straight or branched
chain hydrocarbon radical containing from 2 to 18 carbons and
comprising at least one carbon to carbon double bond. Examples
include without limitation ethenyl, propenyl, iso-propenyl,
butenyl, iso-butenyl, tert-butenyl, n-pentenyl and n-hexenyl. The
term "lower alkenyl" refers to an alkenyl group, as defined herein,
containing between 2 and 8 carbons.
[0074] "Alkynyl" refers to an unsaturated straight or branched
chain hydrocarbon radical containing from 2 to 18 carbons
comprising at least one carbon to carbon triple bond. Examples
include without limitation ethynyl, propynyl, iso-propynyl,
butynyl, iso-butynyl, tert-butynyl, pentynyl and hexynyl. The term
"lower alkynyl" refers to an alkynyl group, as defined herein,
containing between 2 and 8 carbons.
[0075] "Cycloalkyl" refers to a mono- or poly-cyclic alkyl radical.
Examples include without limitation cyclobutyl, cyclopentyl,
cyclohexyl, cycloheptyl and cyclooctyl.
[0076] "Aryl" refers to a cyclic aromatic hydrocarbon moiety
containing from to 18 carbons having one or more closed ring(s).
Examples include without limitation phenyl, benzyl, naphthyl,
anthracenyl, phenanthracenyl and biphenyl.
[0077] "Aralkyl" refers to a radical of the formula RaRb where Ra
is an alkylene chain as defined above and Rb is one or more aryl
radicals as defined above, for example, benzyl, diphenylmethyl and
the like.
[0078] "Thioalkoxy" refers to a radical of the formula --SRc where
Rc is an alkyl radical as defined herein. The term "lower
thioalkoxy" refers to an alkoxy group, as defined herein,
containing between 1 and 8 carbons.
[0079] "Alkoxy" refers to a radical of the formula --ORda where Rd
is an alkyl radical as defined herein. The term "lower alkoxy"
refers to an alkoxy group, as defined herein, containing between 1
and 8 carbons. Examples of alkoxy groups include, without
limitation, methoxy and ethoxy.
[0080] "Alkoxyalkyl" refers to an alkyl group substituted with an
alkoxy group.
[0081] "Carbonyl" refers to the C(.dbd.O)-- radical.
[0082] "Guanidynyl" refers to the H.sub.2N(C.dbd.NH.sub.2)--NH--
radical.
[0083] "Amidinyl" refers to the H.sub.2N(C.dbd.NH.sub.2)CH--
radical.
[0084] "Amino" refers to the NH.sub.2 radical.
[0085] "Alkylamino" refers to a radical of the formula --NHRd or
--NRdRd where each Rd is, independently, an alkyl radical as
defined herein. The term "lower alkylamino" refers to an alkylamino
group, as defined herein, containing between 1 and 8 carbons.
[0086] "Heterocycle" means a 5- to 7-membered monocyclic, or 7- to
10-membered bicyclic, heterocyclic ring which is either saturated,
unsaturated, or aromatic, and which contains from 1 to 4
heteroatoms independently selected from nitrogen, oxygen and
sulfur, and wherein the nitrogen and sulfur heteroatoms may be
optionally oxidized, and the nitrogen heteroatom may be optionally
quaternized, including bicyclic rings in which any of the above
heterocycles are fused to a benzene ring. The heterocycle may be
attached via any heteroatom or carbon atom. Heterocycles include
heteroaryls as defined below. Thus, in addition to the heteroaryls
listed below, heterocycles also include morpholinyl,
pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizinyl,
hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,
tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothiophenyl,
tetrahydrothiopyranyl, tetrahydropyrimidinyl,
tetrahydrothiopyranyl, and the like.
[0087] "Heteroaryl" means an aromatic heterocycle ring of 5- to 10
members and having at least one heteroatom selected from nitrogen,
oxygen and sulfur, and containing at least 1 carbon atom, including
both mono- and bicyclic ring systems. Representative heteroaryls
are pyridyl, furyl, benzofuranyl, thiophenyl, benzothiophenyl,
quinolinyl, pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl,
benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl,
isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,
cinnolinyl, phthalazinyl, and quinazolinyl.
[0088] The terms "optionally substituted alkyl", "optionally
substituted alkenyl", "optionally substituted alkoxy", "optionally
substituted thioalkoxy", "optionally substituted alkyl amino",
"optionally substituted lower alkyl", "optionally substituted lower
alkenyl", "optionally substituted lower alkoxy", "optionally
substituted lower thioalkoxy", "optionally substituted lower alkyl
amino" and "optionally substituted heterocyclyl" mean that, when
substituted, at least one hydrogen atom is replaced with a
substituent. In the case of an oxo substituent (.dbd.O) two
hydrogen atoms are replaced. In this regard, substituents include:
deuterium, optionally substituted alkyl, optionally substituted
alkenyl, optionally substituted alkynyl, optionally substituted
aryl, optionally substituted heterocycle, optionally substituted
cycloalkyl, oxo, halogen, --CN, --ORx, NRxRy, NRxC(.dbd.O)Ry,
NRxSO2Ry, --NRxC(.dbd.O)NRxRy, C(.dbd.O)Rx, C(.dbd.O)ORx,
C(.dbd.O)NRxRy, --SOmRx and --SOmNRxRy, wherein m is 0, 1 or 2, Rx
and Ry are the same or different and independently hydrogen,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted aryl,
optionally substituted heterocycle or optionally substituted
cycloalkyl and each of said optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted aryl, optionally substituted heterocycle and
optionally substituted cycloalkyl substituents may be further
substituted with one or more of oxo, halogen, --CN, --ORx, NRxRy,
NRxC(.dbd.O)Ry, NRxSO2Ry, --NRxC(.dbd.O)NRxRy, C(.dbd.O)Rx,
C(.dbd.O)ORx, C(.dbd.O)NRxRy, --SOmRx and --SOmNRxRy.
[0089] An antisense molecule nomenclature system was proposed and
published to distinguish between the different antisense molecules
(see Mann et al., (2002) J Gen Med 4, 644-654). This nomenclature
became especially relevant when testing several slightly different
antisense molecules, all directed at the same target region, as
shown below:
H#A/D(x:y).
[0090] The first letter designates the species (e.g. H: human, M:
murine, C: canine). "#" designates target dystrophin exon number.
"A/D" indicates acceptor or donor splice site at the beginning and
end of the exon, respectively. (x y) represents the annealing
coordinates where "-" or "+" indicate intronic or exonic sequences
respectively. For example, A(-6+18) would indicate the last 6 bases
of the intron preceding the target exon and the first 18 bases of
the target exon. The closest splice site would be the acceptor so
these coordinates would be preceded with an "A". Describing
annealing coordinates at the donor splice site could be D(+2-18)
where the last 2 exonic bases and the first 18 intronic bases
correspond to the annealing site of the antisense molecule.
Entirely exonic annealing coordinates that would be represented by
A(+65+85), that is the site between the 65th and 85th nucleotide
from the start of that exon.
II. CONSTRUCTING THE ANTISENSE OLIGONUCLEOTIDE
[0091] Exemplary embodiments of the invention relate to morpholino
oligonucleotides having phosphorus-containing backbone linkages are
illustrated in FIGS. 1A-1C. Preferred is a
phosphorodiamidate-linked morpholino oligonucleotide such as shown
in FIG. 1C, which is modified, in accordance with one aspect of the
present invention, to contain positively charged groups at
preferably 10%-50% of its backbone linkages. Morpholino
oligonucleotides with uncharged backbone linkages, including
antisense oligonucleotides, are detailed, for example, in
(Summerton and Weller 1997) and in co-owned U.S. Pat. Nos.
5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185, 444,
5,521,063, 5,506,337, 8,076,476, 8,299,206 and 7,943,762 all of
which are expressly incorporated by reference herein.
[0092] Important properties of the morpholino-based subunits
include: 1) the ability to be linked in a oligomeric form by
stable, uncharged or positively charged backbone linkages; 2) the
ability to support a nucleotide base (e.g. adenine, cytosine,
guanine, thymidine, uracil and inosine) such that the polymer
formed can hybridize with a complementary-base target nucleic acid,
including target RNA, Tm values above about 45.degree. C. in
relatively short oligonucleotides (e.g., 10-15 bases); 3) the
ability of the oligonucleotide to be actively or passively
transported into mammalian cells; and 4) the ability of the
antisense oligonucleotide:RNA heteroduplex to resist RNAse and
RNase H degradation, respectively.
[0093] Exemplary backbone structures for antisense oligonucleotides
of the claimed subject matter include the morpholino subunit types
shown in FIGS. 1D-G, each linked by an uncharged or positively
charged, phosphorus-containing subunit linkage. FIG. 1D shows a
phosphorus-containing linkage which forms the five atom
repeating-unit backbone, where the morpholino rings are linked by a
1-atom phosphoamide linkage. FIG. 1E shows a linkage which produces
a 6-atom repeating-unit backbone. In this structure, the atom Y
linking the 5' morpholino carbon to the phosphorus group may be
sulfur, nitrogen, carbon or, preferably, oxygen. The X moiety
pendant from the phosphorus may be fluorine, an alkyl or
substituted alkyl, an alkoxy or substituted alkoxy, a thioalkoxy or
substituted thioalkoxy, or unsubstituted, monosubstituted, or
disubstituted nitrogen, including cyclic structures, such as
morpholines or piperidines. Alkyl, alkoxy and thioalkoxy preferably
include 1-6 carbon atoms. The Z moieties are sulfur or oxygen, and
are preferably oxygen.
[0094] The linkages shown in FIGS. 1F and 1G are designed for
7-atom unit-length backbones. In structure 1F, the X moiety is as
in Structure 1E, and the Y moiety may be methylene, sulfur, or,
preferably, oxygen. In Structure 1G, the X and Y moieties are as in
Structure 1E. Particularly preferred morpholino oligonucleotides
include those composed of morpholino subunit structures of the form
shown in FIG. 1E, where X.dbd.NH.sub.2, N(CH.sub.3).sub.2, or
1-piperazine or other charged group, Y.dbd.O, and Z.dbd.O.
[0095] A substantially uncharged oligonucleotide may be modified,
in accordance with an aspect of the invention, to include charged
linkages, e.g., up to about 1 per every 2-5 uncharged linkages,
such as about 4-5 per every 10 uncharged linkages. In certain
embodiments, optimal improvement in antisense activity may be seen
when about 25% of the backbone linkages are cationic. In certain
embodiments, enhancement may be seen with a small number e.g.,
10-20% cationic linkages, or where the number of cationic linkages
are in the range 50-80%, such as about 60%.
[0096] Oligomers having any number of cationic linkages are
provided, including fully cationic-linked oligomers. Preferably,
however, the oligomers are partially charged, having, for example,
10%-80%. In preferred embodiments, about 10% to 60%, and preferably
20% to 50% of the linkages are cationic.
[0097] In one embodiment, the cationic linkages are interspersed
along the backbone. The partially charged oligomers preferably
contain at least two consecutive uncharged linkages; that is, the
oligomer preferably does not have a strictly alternating pattern
along its entire length.
[0098] Also considered are oligomers having blocks of cationic
linkages and blocks of uncharged linkages; for example, a central
block of uncharged linkages may be flanked by blocks of cationic
linkages, or vice versa. In one embodiment, the oligomer has
approximately equal-length 5', 3' and center regions, and the
percentage of cationic linkages in the center region is greater
than about 50%, preferably greater than about 70%.
[0099] In certain embodiments, the antisense compounds can be
prepared by stepwise solid-phase synthesis, employing methods
detailed in the references cited above, and below with respect to
the synthesis of oligonucleotides having a mixture or uncharged and
cationic backbone linkages. In some cases, it may be desirable to
add additional chemical moieties to the antisense compound, e.g.,
to enhance pharmacokinetics or to facilitate capture or detection
of the compound. Such a moiety may be covalently attached,
according to standard synthetic methods. For example, addition of a
polyethylene glycol moiety or other hydrophilic polymer, e.g., one
having 1-100 monomeric subunits, may be useful in enhancing
solubility.
[0100] A reporter moiety, such as fluorescein or a radiolabeled
group, may be attached for purposes of detection. Alternatively,
the reporter label attached to the oligomer may be a ligand, such
as an antigen or biotin, capable of binding a labeled antibody or
streptavidin. In selecting a moiety for attachment or modification
of an antisense compound, it is generally of course desirable to
select chemical compounds of groups that are biocompatible and
likely to be tolerated by a subject without undesirable side
effects.
[0101] Oligomers for use in antisense applications generally range
in length from about 10 to about 50 subunits, more preferably about
10 to 30 subunits, and typically 15-25 bases. For example, an
oligomer of the invention having 19-20 subunits, a useful length
for an antisense compound, may ideally have two to ten, e.g., four
to eight, cationic linkages, and the remainder uncharged linkages.
An oligomer having 14-15 subunits may ideally have two to seven,
e.g., 3, 4, or 5, cationic linkages and the remainder uncharged
linkages. In a preferred embodiment, the oligomers have 25 to 28
subunits.
[0102] Each morpholino ring structure supports a base pairing
moiety, to form a sequence of base pairing moieties which is
typically designed to hybridize to a selected antisense target in a
cell or in a subject being treated. The base pairing moiety may be
a purine or pyrimidine found in native DNA or RNA (e.g., A, G, C, T
or U) or an analog, such as hypoxanthine (the base component of the
nucleoside inosine) or 5-methyl cytosine.
[0103] As noted above, certain embodiments are directed to
oligomers comprising novel intersubunit linkages, including PMO-X
oligomers and those having modified terminal groups. In some
embodiments, these oligomers have higher affinity for DNA and RNA
than do the corresponding unmodified oligomers and demonstrate
improved cell delivery, potency, and/or tissue distribution
properties compared to oligomers having other intersubunit
linkages. The structural features and properties of the various
linkage types and oligomers are described in more detail in the
following discussion. The synthesis of these and related oligomers
is described in co-owned U.S. application Ser. No. 13/118,298,
which is incorporated by reference in its entirety.
[0104] In certain embodiments, the invention provides for an
oligonucleotide having a sequence complementary to the target
sequence which is associated with a human disease, and comprises a
sequence of nucleotides having a formula:
##STR00001##
[0105] wherein Nu is a nucleobase;
[0106] R.sub.1 has the formula
##STR00002##
[0107] q is 0, 1, or 2;
[0108] R.sub.2 is selected from the group consisting of hydrogen,
C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 aralkyl, and a formamidinyl
group, and
[0109] R.sub.3 is selected from the group consisting of hydrogen,
C.sub.1-C.sub.10 acyl, C.sub.1-C.sub.10 aminoacyl, acyl moiety of a
natural or unnatural alpha or beta amino acid, C.sub.1-C.sub.10
aralkyl, and C.sub.1-C.sub.10 alkyl, or
[0110] R.sub.2 and R.sub.3 are joined to form a 5-7 membered ring
where the ring may be optionally substituted with a substituent
selected from the group consisting of C.sub.1-C.sub.10 alkyl,
phenyl, halogen, and C.sub.1-C.sub.10 aralkyl;
[0111] R.sub.4 is selected from the group consisting of an electron
pair, hydrogen, a C.sub.1-C.sub.6 alkyl and C.sub.1-C.sub.6
aralkyl;
[0112] Rx is selected from the group consisting of sarcosinamide,
hydroxyl, a nucleotide, a cell penetrating peptide moiety, and
piperazinyl;
[0113] Ry is selected from the group consisting of hydrogen, a
C.sub.1-C.sub.6 alkyl, a nucleotide a cell penetrating peptide
moiety, an amino acid, a formamidinyl group, and C.sub.1-C.sub.6
acyl; and,
[0114] Rz is selected from the group consisting of an electron
pair, hydrogen, a C.sub.1-C.sub.6 alkyl, and C.sub.1-C.sub.6 acyl
pharmaceutically acceptable salts thereof.
[0115] Nu may be selected from the group consisting of adenine,
guanine, thymine, uracil, cytosine, and hypoxanthine. More
preferably Nu is thymine or uracil.
[0116] In preferred embodiments, the invention provides an
oligonucleotide having a sequence of nucleotides having a
formula:
##STR00003##
[0117] wherein Nu is a nucleobase;
[0118] R.sub.1 is selected from the group consisting of R.sub.1'
and R.sub.1'' wherein R.sub.1' is dimethyl-amino and R.sub.1'' has
the formula
##STR00004##
[0119] wherein at least one R.sub.1 is R.sub.1'';
[0120] q is 0, 1, or 2; with the proviso that at least one of
R.sub.1 is a piperidinyl moiety;
[0121] R.sub.2 is selected from the group consisting of hydrogen,
C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.5 aralkyl, and a formamidinyl
group, and
[0122] R.sub.3 is selected from the group consisting of hydrogen,
C.sub.1-C.sub.10 acyl, C.sub.1-C.sub.10 aminoacyl, acyl moiety of a
natural or unnatural alpha or beta amino acid, C.sub.1-C.sub.10
aralkyl, and C.sub.1-C.sub.10 alkyl, or
[0123] R.sub.2 and R.sub.3 are joined to form a 5-7 membered ring
where the ring may be optionally substituted with a substituent
selected from the group consisting of C.sub.1-C.sub.10 alkyl,
phenyl, halogen, and C.sub.1-C.sub.10 aralkyl;
[0124] R.sub.4 is selected from the group consisting of an electron
pair, hydrogen, a C.sub.1-C.sub.6 alkyl and aralkyl;
[0125] Rx is selected from the group consisting of sarcosinamide,
hydroxyl, a nucleotide, a cell penetrating peptide moiety, and
piperazinyl;
[0126] Ry is selected from the group consisting of hydrogen, a
C.sub.1-C.sub.6 alkyl, a nucleotide a cell penetrating peptide
moiety, an amino acid, a formamidinyl group, and C.sub.1-C.sub.6
acyl; and,
[0127] Rz is selected from the group consisting of an electron
pair, hydrogen, a C.sub.1-C.sub.6 alkyl, and C.sub.1-C.sub.6 acyl
pharmaceutically acceptable salts thereof.
[0128] Nu may be selected from the group consisting of adenine,
guanine, thymine, uracil, cytosine, and hypoxanthine. More
preferably Nu is thymine or uracil.
[0129] About 90-50% of the R.sub.1 groups are dimethylamino (i.e.
R.sub.1'). More, preferably, 90-50% of the R.sub.1 groups are
dimethylamino. Most, preferably about 66% of the R.sub.1 groups are
dimethylamino.
[0130] R.sub.1 may be selected from the group consisting of
##STR00005##
[0131] Preferably, at least one nucleotide of the oligonucleotide
has the formula:
##STR00006##
[0132] wherein Rx, Ry, Rz, and Nu are as stated above. Most
preferably, Nu is thymine or uracil.
[0133] Although thymine (T) is the preferred base pairing moiety
(Nu or Pi) containing the chemical modifications described above,
any base subunit known to a person of skill in the art can be used
as the base pairing moiety.
[0134] The oligonucleotide and the DNA or RNA are complementary to
each other when a sufficient number of corresponding positions in
each molecule are occupied by nucleotides which can hydrogen bond
with each other. Thus, "specifically hybridizable" and
"complementary" are terms which are used to indicate a sufficient
degree of complementarity or precise pairing such that stable and
specific binding occurs between the oligonucleotide and the DNA or
RNA target. It is understood in the art that the sequence of an
antisense molecule need not be 100% complementary to that of its
target sequence to be specifically hybridizable. An antisense
molecule is specifically hybridizable when binding of the compound
to the target DNA or RNA molecule interferes with the normal
function of the target DNA or RNA to cause a loss of utility, and
there is a sufficient degree of complementarity to avoid
non-specific binding of the antisense compound to non-target
sequences under conditions in which specific binding is desired,
i.e., under physiological conditions in the case of in vivo assays
or therapeutic treatment, and in the case of in vitro assays, under
conditions in which the assays are performed.
[0135] While the above method may be used to select antisense
molecules capable of deleting any exon from within a protein that
is capable of being shortened without affecting its biological
function, the exon deletion should not lead to a reading frame
shift in the shortened transcribed mRNA. Thus, if in a linear
sequence of three exons the end of the first exon encodes two of
three nucleotides in a codon and the next exon is deleted then the
third exon in the linear sequence must start with a single
nucleotide that is capable of completing the nucleotide triplet for
a codon. If the third exon does not commence with a single
nucleotide there will be a reading frame shift that would lead to
the generation of truncated or a non-functional protein.
[0136] It will be appreciated that the codon arrangements at the
end of exons in structural proteins may not always break at the end
of a codon, consequently there may be a need to delete more than
one exon from the pre-mRNA to ensure in-frame reading of the mRNA.
In such circumstances, a plurality of antisense oligonucleotides
may need to be selected by the method of the invention wherein each
is directed to a different region responsible for inducing splicing
in the exons that are to be deleted.
[0137] The length of an antisense molecule may vary so long as it
is capable of binding selectively to the intended location within
the pre-mRNA molecule. The length of such sequences can be
determined in accordance with selection procedures described
herein. Generally, the antisense molecule will be from about 10
nucleotides in length up to about 50 nucleotides in length. It will
be appreciated however that any length of nucleotides within this
range may be used in the method. Preferably, the length of the
antisense molecule is between 10-30 nucleotides in length.
[0138] The most common method for producing antisense molecules is
the methylation of the 2' hydroxyribose position and the
incorporation of a phosphorothioate backbone produces molecules
that superficially resemble RNA but that are much more resistant to
nuclease degradation.
[0139] To avoid degradation of pre-mRNA during duplex formation
with the antisense molecules, the antisense molecules used in the
method may be adapted to minimize or prevent cleavage by endogenous
RNase H. This property is highly preferred as the treatment of the
RNA with the unmethylated oligonucleotides either intracellularly
or in crude extracts that contain RNase H leads to degradation of
the pre-mRNA: antisense oligonucleotide duplexes. Any form of
modified antisense molecules that is capable of by-passing or not
inducing such degradation may be used in the present method. An
example of antisense molecules which when duplexed with RNA are not
cleaved by cellular RNase H is 2'-O-methyl derivatives.
2'-O-methyl-oligoribonucleotides are very stable in a cellular
environment and in animal tissues, and their duplexes with RNA have
higher Tm values than their ribo- or deoxyribo-counterparts.
[0140] Antisense molecules that do not activate RNase H can be made
in accordance with known techniques (see, e.g., U.S. Pat. No.
5,149,797). Such antisense molecules, which may be
deoxyribonucleotide or ribonucleotide sequences, simply contain any
structural modification which sterically hinders or prevents
binding of RNase H to a duplex molecule containing the
oligonucleotide as one member thereof, which structural
modification does not substantially hinder or disrupt duplex
formation. Because the portions of the oligonucleotide involved in
duplex formation are substantially different from those portions
involved in RNase H binding thereto, numerous antisense molecules
that do not activate RNase H are available. For example, such
antisense molecules may be oligonucleotides wherein at least one,
or all, of the inter-nucleotide bridging phosphate residues are
modified phosphates, such as methyl phosphonates, methyl
phosphorothioates, phosphoromorpholidates, phosphoropiperazidates
and phosphoramidates. For example, every other one of the
internucleotide bridging phosphate residues may be modified as
described. In another non-limiting example, such antisense
molecules are molecules wherein at least one, or all, of the
nucleotides contain a 2' lower alkyl moiety (e.g., C.sub.1-C.sub.4,
linear or branched, saturated or unsaturated alkyl, such as methyl,
ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). For
example, every other one of the nucleotides may be modified as
described.
[0141] While antisense oligonucleotides are a preferred form of the
antisense molecules, the present invention comprehends other
oligomeric antisense molecules, including but not limited to
oligonucleotide mimetics such as are described below.
[0142] Specific examples of preferred antisense compounds useful in
this invention include oligonucleotides containing modified
backbones or non-natural inter-nucleoside linkages. As defined in
this specification, oligonucleotides having modified backbones
include those that retain a phosphorus atom in the backbone and
those that do not have a phosphorus atom in the backbone. For the
purposes of this specification, and as sometimes referenced in the
art, modified oligonucleotides that do not have a phosphorus atom
in their inter-nucleoside backbone can also be considered to be
oligonucleosides.
[0143] In other preferred oligonucleotide mimetics, both the sugar
and the inter-nucleoside linkage, i.e., the backbone, of the
nucleotide units are replaced with novel groups. The base units are
maintained for hybridization with an appropriate nucleic acid
target compound. One such oligomeric compound, an oligonucleotide
mimetic that has been shown to have excellent hybridization
properties, is referred to as a peptide nucleic acid (PNA). In PNA
compounds, the sugar-backbone of an oligonucleotide is replaced
with an amide containing backbone, in particular an
aminoethylglycine backbone. The nucleo-bases are retained and are
bound directly or indirectly to aza nitrogen atoms of the amide
portion of the backbone.
[0144] Modified oligonucleotides may also contain one or more
substituted sugar moieties. Oligonucleotides may also include
nucleobase (often referred to in the art simply as "base")
modifications or substitutions. Certain nucleo-bases are
particularly useful for increasing the binding affinity of the
oligomeric compounds of the invention. These include 5-substituted
pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted
purines, including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. 5-methylcytosine substitutions have been shown
to increase nucleic acid duplex stability by 0.6-1.2.degree. C. and
are presently preferred base substitutions, even more particularly
when combined with 2'-O-methoxyethyl sugar modifications.
[0145] Another modification of the oligonucleotides of the
invention involves chemically linking to the oligonucleotide one or
more moieties or conjugates that enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. Such
moieties include but are not limited to lipid moieties such as a
cholesterol moiety, cholic acid, a thioether, e.g.,
hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,
dodecandiol or undecyl residues, a phospholipid, e.g.,
di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a
polyethylene glycol chain, or adamantane acetic acid, a palmityl
moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol
moiety.
[0146] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within an oligonucleotide.
The present invention also includes antisense compounds that are
chimeric compounds. "Chimeric" antisense compounds or "chimeras,"
in the context of this invention, are antisense molecules,
particularly oligonucleotides, which contain two or more chemically
distinct regions, each made up of at least one monomer unit, i.e.,
a nucleotide in the case of an oligonucleotide compound. These
oligonucleotides typically contain at least one region wherein the
oligonucleotide is modified so as to confer upon the increased
resistance to nuclease degradation, increased cellular uptake, and
an additional region for increased binding affinity for the target
nucleic acid.
III. PEPTIDE TRANSPORTERS
[0147] The antisense compounds of the invention may include an
oligonucleotide moiety conjugated to a CPP, preferably an
arginine-rich peptide transport moiety effective to enhance
transport of the compound into cells. The transport moiety is
preferably attached to a terminus of the oligomer, as shown, for
example, in FIGS. 1B and 1C. The peptides have the capability of
inducing cell penetration within 30%, 40%, 50%, 60%, 70%, 80%, 90%
or 100% of cells of a given cell culture population, including all
integers in between, and allow macromolecular translocation within
multiple tissues in vivo upon systemic administration. In one
embodiment, the cell-penetrating peptide may be an arginine-rich
peptide transporter. In another embodiment, the cell-penetrating
peptide may be Penetratin or the Tat peptide. These peptides are
well known in the art and are disclosed, for example, in US
Publication No. 2010-0016215 A1, incorporated by reference in its
entirety. A particularly preferred approach to conjugation of
peptides to antisense oligonucleotides can be found in PCT
publication WO2012/150960, which is incorporated by reference in
its entirety. A preferred embodiment of a peptide conjugated
oligonucleotide of the present invention utilizes glycine as the
linker between the CPP and the antisense oligonucleotide. For
example, a preferred peptide conjugated PMO consists of
R.sub.6-G-PMO.
[0148] The transport moieties as described above have been shown to
greatly enhance cell entry of attached oligomers, relative to
uptake of the oligomer in the absence of the attached transport
moiety. Uptake is preferably enhanced at least ten fold, and more
preferably twenty fold, relative to the unconjugated compound.
[0149] The use of arginine-rich peptide transporters (i.e.,
cell-penetrating peptides) are particularly useful in practicing
the present invention. Certain peptide transporters have been shown
to be highly effective at delivery of antisense compounds into
primary cells including muscle cells (Marshall, Oda et al. 2007;
Jearawiriyapaisarn, Moulton et al. 2008; Wu, Moulton et al. 2008).
Furthermore, compared to other known peptide transporters such as
Penetratin and the Tat peptide, the peptide transporters described
herein, when conjugated to an antisense PMO, demonstrate an
enhanced ability to alter splicing of several gene transcripts
(Marshall, Oda et al. 2007).
[0150] Exemplary peptide transporters, excluding linkers are given
below in Table 1.
TABLE-US-00001 TABLE 1 Exemplary peptide transporters SEQ NAME
(DESIGNATION) SEQUENCE ID NO.sup.A rTAT RRRQRRKKR 876 Tat RKKRRQRRR
877 R.sub.9F.sub.2 RRRRRRRRRFF 878 R.sub.5F.sub.2R.sub.4
RRRRRFFRRRR 879 R.sub.4 RRRR 880 R.sub.5 RRRRR 881 R.sub.6 RRRRRR
882 R.sub.7 RRRRRRR 883 R.sub.8 RRRRRRRR 884 R.sub.9 RRRRRRRRR 885
(RX).sub.8 RXRXRXRXRXRXRXRX 886 (RAhxR).sub.4; (P007)
RAhxRRAhxRRAhxRRAhxR 887 (RAhxR).sub.5; RAhxRRAhxRRAhxRRAhxRRAhxR
888 (CP04057) (RAhxRRBR).sub.2; RAhxRRBRRAhxRRBR 889 (CP06062)
(RAR).sub.4F.sub.2 RARRARRARRARFF 890 (RGR).sub.4F.sub.2
RGRRGRRGRRGRFF 891 .sup.ASequences assigned to SEQ ID NOs do not
include the linkage portion (e.g., C, G, P, Ahx, B, AhxB where Ahx
and B refer to 6-aminohexanoic acid and beta-alanine,
respectively).
IV. FORMULATIONS AND TREATMENT
[0151] In certain embodiments, the present invention provides
formulations or compositions suitable for the therapeutic delivery
of antisense oligomers, as described herein. Hence, in certain
embodiments, the present invention provides pharmaceutically
acceptable compositions that comprise a therapeutically-effective
amount of one or more of the oligomers described herein, formulated
together with one or more pharmaceutically acceptable carriers
(additives) and/or diluents. While it is possible for an oligomer
of the present invention to be administered alone, it is preferable
to administer the compound as a pharmaceutical formulation
(composition).
[0152] The compositions of the present invention may be
administered alone or in combination with another therapeutic. The
additional therapeutic may be administered prior, concurrently or
subsequently to the administration of the composition of the
present invention. For example, the compositions may be
administered in combination with a steroid and/or an antibiotic.
The steroid may be a glucocorticoid or prednisone. Other agents
which can be administered include an antagonist of the ryanodine
receptor, such as dantrolene, which has been shown to enhance
antisense-mediated exon skipping in patient cells and a mouse model
of DMD (G. Kendall et al. Sci Tranl Med 4 164ra160 (2012),
incorporated herein by reference).
[0153] Methods for the delivery of nucleic acid molecules are
described, for example, in Akhtar et al., 1992, Trends Cell Bio.,
2:139; and Delivery Strategies for Antisense Oligonucleotide
Therapeutics, ed. Akhtar; Sullivan et al., PCT WO 94/02595. These
and other protocols can be utilized for the delivery of virtually
any nucleic acid molecule, including the isolated oligomers of the
present invention.
[0154] As detailed below, the pharmaceutical compositions of the
present invention may be specially formulated for administration in
solid or liquid form, including those adapted for the following:
(1) oral administration, for example, drenches (aqueous or
non-aqueous solutions or suspensions), tablets, e.g., those
targeted for buccal, sublingual, and systemic absorption, boluses,
powders, granules, pastes for application to the tongue; (2)
parenteral administration, for example, by subcutaneous,
intramuscular, intravenous or epidural injection as, for example, a
sterile solution or suspension, or sustained-release formulation;
(3) topical application, for example, as a cream, ointment, or a
controlled-release patch or spray applied to the skin; (4)
intravaginally or intrarectally, for example, as a pessary, cream
or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8)
nasally.
[0155] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0156] The phrase "pharmaceutically-acceptable carrier" as used
herein means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc
stearate, or steric acid), or solvent encapsulating material,
involved in carrying or transporting the subject compound from one
organ, or portion of the body, to another organ, or portion of the
body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
injurious to the patient.
[0157] Some examples of materials that can serve as
pharmaceutically-acceptable carriers include, without limitation:
(1) sugars, such as lactose, glucose and sucrose; (2) starches,
such as corn starch and potato starch; (3) cellulose, and its
derivatives, such as sodium carboxymethyl cellulose, ethyl
cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt;
(6) gelatin; (7) talc; (8) excipients, such as cocoa butter and
suppository waxes; (9) oils, such as peanut oil, cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil;
(10) glycols, such as propylene glycol; (11) polyols, such as
glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,
such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering
agents, such as magnesium hydroxide and aluminum hydroxide; (15)
alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)
Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions;
(21) polyesters, polycarbonates and/or polyanhydrides; and (22)
other non-toxic compatible substances employed in pharmaceutical
formulations.
[0158] Additional non-limiting examples of agents suitable for
formulation with the antisense oligomers of the instant invention
include: PEG conjugated nucleic acids, phospholipid conjugated
nucleic acids, nucleic acids containing lipophilic moieties,
phosphorothioates, P-glycoprotein inhibitors (such as Pluronic P85)
which can enhance entry of drugs into various tissues;
biodegradable polymers, such as poly (DL-lactide-coglycolide)
microspheres for sustained release delivery after implantation
(Emerich, D F et al., 1999, Cell Transplant, 8, 47-58) Alkermes,
Inc. Cambridge, Mass.; and loaded nanoparticles, such as those made
of polybutylcyanoacrylate, which can deliver drugs across the blood
brain barrier and can alter neuronal uptake mechanisms (Prog
Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).
[0159] The invention also features the use of the composition
comprising surface-modified liposomes containing poly (ethylene
glycol) lipids (PEG-modified, branched and unbranched or
combinations thereof, or long-circulating liposomes or stealth
liposomes). Oligomers of the invention can also comprise covalently
attached PEG molecules of various molecular weights. These
formulations offer a method for increasing the accumulation of
drugs in target tissues. This class of drug carriers resists
opsonization and elimination by the mononuclear phagocytic system
(MPS or RES), thereby enabling longer blood circulation times and
enhanced tissue exposure for the encapsulated drug (Lasic et al.
Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull.
1995, 43, 1005-1011). Such liposomes have been shown to accumulate
selectively in tumors, presumably by extravasation and capture in
the neovascularized target tissues (Lasic et al., Science 1995,
267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238,
86-90). The long-circulating liposomes enhance the pharmacokinetics
and pharmacodynamics of DNA and RNA, particularly compared to
conventional cationic liposomes which are known to accumulate in
tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42,
24864-24870; Choi et al., International PCT Publication No. WO
96/10391; Ansell et al., International PCT Publication No. WO
96/10390; Holland et al., International PCT Publication No. WO
96/10392). Long-circulating liposomes are also likely to protect
drugs from nuclease degradation to a greater extent compared to
cationic liposomes, based on their ability to avoid accumulation in
metabolically aggressive MPS tissues such as the liver and
spleen.
[0160] In a further embodiment, the present invention includes
oligomer compositions prepared for delivery as described in U.S.
Pat. Nos. 6,692,911, 7,163,695 and 7,070,807. In this regard, in
one embodiment, the present invention provides an oligomer of the
present invention in a composition comprising copolymers of lysine
and histidine (HK) (as described in U.S. Pat. Nos. 7,163,695,
7,070,807, and 6,692,911) either alone or in combination with PEG
(e.g., branched or unbranched PEG or a mixture of both), in
combination with PEG and a targeting moiety or any of the foregoing
in combination with a crosslinking agent. In certain embodiments,
the present invention provides antisense oligomers in compositions
comprising gluconic-acid-modified polyhistidine or
gluconylated-polyhistidine/transferrin-polylysine. One skilled in
the art will also recognize that amino acids with properties
similar to His and Lys may be substituted within the
composition.
[0161] Certain embodiments of the oligomers described herein may
contain a basic functional group, such as amino or alkylamino, and
are, thus, capable of forming pharmaceutically-acceptable salts
with pharmaceutically-acceptable acids. The term
"pharmaceutically-acceptable salts" in this respect, refers to the
relatively non-toxic, inorganic and organic acid addition salts of
compounds of the present invention. These salts can be prepared in
situ in the administration vehicle or the dosage form manufacturing
process, or by separately reacting a purified compound of the
invention in its free base form with a suitable organic or
inorganic acid, and isolating the salt thus formed during
subsequent purification. Representative salts include the
hydrobromide, hydrochloride, sulfate, bisulfate, phosphate,
nitrate, acetate, valerate, oleate, palmitate, stearate, laurate,
benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,
succinate, tartrate, napthylate, mesylate, glucoheptonate,
lactobionate, and laurylsulphonate salts and the like. (See, e.g.,
Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci.
66:1-19).
[0162] The pharmaceutically acceptable salts of the subject
oligomers include the conventional nontoxic salts or quaternary
ammonium salts of the compounds, e.g., from non-toxic organic or
inorganic acids. For example, such conventional nontoxic salts
include those derived from inorganic acids such as hydrochloride,
hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like;
and the salts prepared from organic acids such as acetic,
propionic, succinic, glycolic, stearic, lactic, malic, tartaric,
citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic,
glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic,
fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic,
oxalic, isothionic, and the like.
[0163] In certain embodiments, the oligomers of the present
invention may contain one or more acidic functional groups and,
thus, are capable of forming pharmaceutically-acceptable salts with
pharmaceutically-acceptable bases. The term
"pharmaceutically-acceptable salts" in these instances refers to
the relatively non-toxic, inorganic and organic base addition salts
of compounds of the present invention. These salts can likewise be
prepared in situ in the administration vehicle or the dosage form
manufacturing process, or by separately reacting the purified
compound in its free acid form with a suitable base, such as the
hydroxide, carbonate or bicarbonate of a
pharmaceutically-acceptable metal cation, with ammonia, or with a
pharmaceutically-acceptable organic primary, secondary or tertiary
amine. Representative alkali or alkaline earth salts include the
lithium, sodium, potassium, calcium, magnesium, and aluminum salts
and the like. Representative organic amines useful for the
formation of base addition salts include ethylamine, diethylamine,
ethylenediamine, ethanolamine, diethanolamine, piperazine and the
like. (See, e.g., Berge et al., supra).
[0164] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0165] Examples of pharmaceutically-acceptable antioxidants
include: (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0166] Formulations of the present invention include those suitable
for oral, nasal, topical (including buccal and sublingual), rectal,
vaginal and/or parenteral administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any methods well known in the art of pharmacy. The amount of
active ingredient that can be combined with a carrier material to
produce a single dosage form will vary depending upon the host
being treated, the particular mode of administration. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form will generally be that amount of the
compound which produces a therapeutic effect. Generally, out of one
hundred percent, this amount will range from about 0.1 percent to
about ninety-nine percent of active ingredient, preferably from
about 5 percent to about 70 percent, most preferably from about 10
percent to about 30 percent.
[0167] In certain embodiments, a formulation of the present
invention comprises an excipient selected from cyclodextrins,
celluloses, liposomes, micelle forming agents, e.g., bile acids,
and polymeric carriers, e.g., polyesters and polyanhydrides; and an
oligomer of the present invention. In certain embodiments, an
aforementioned formulation renders orally bioavailable an oligomer
of the present invention.
[0168] Methods of preparing these formulations or compositions
include the step of bringing into association an oligomer of the
present invention with the carrier and, optionally, one or more
accessory ingredients. In general, the formulations are prepared by
uniformly and intimately bringing into association a compound of
the present invention with liquid carriers, or finely divided solid
carriers, or both, and then, if necessary, shaping the product.
[0169] Formulations of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, lozenges (using a flavored basis, usually sucrose and
acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of a compound of the
present invention as an active ingredient. An oligomer of the
present invention may also be administered as a bolus, electuary or
paste.
[0170] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules, trouches and the like), the active ingredient may be
mixed with one or more pharmaceutically-acceptable carriers, such
as sodium citrate or dicalcium phosphate, and/or any of the
following: (1) fillers or extenders, such as starches, lactose,
sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such
as, for example, carboxymethylcellulose, alginates, gelatin,
polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such
as glycerol; (4) disintegrating agents, such as agar-agar, calcium
carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate; (5) solution retarding agents,
such as paraffin; (6) absorption accelerators, such as quaternary
ammonium compounds and surfactants, such as poloxamer and sodium
lauryl sulfate; (7) wetting agents, such as, for example, cetyl
alcohol, glycerol monostearate, and non-ionic surfactants; (8)
absorbents, such as kaolin and bentonite clay; (9) lubricants, such
as talc, calcium stearate, magnesium stearate, solid polyethylene
glycols, sodium lauryl sulfate, zinc stearate, sodium stearate,
stearic acid, and mixtures thereof; (10) coloring agents; and (11)
controlled release agents such as crospovidone or ethyl cellulose.
In the case of capsules, tablets and pills, the pharmaceutical
compositions may also comprise buffering agents. Solid compositions
of a similar type may also be employed as fillers in soft and
hard-shelled gelatin capsules using such excipients as lactose or
milk sugars, as well as high molecular weight polyethylene glycols
and the like.
[0171] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (e.g., gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid
diluent.
[0172] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be formulated for rapid release, e.g.,
freeze-dried. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions which
can be dissolved in sterile water, or some other sterile injectable
medium immediately before use. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain portion of the gastrointestinal tract, optionally, in
a delayed manner. Examples of embedding compositions which can be
used include polymeric substances and waxes. The active ingredient
can also be in micro-encapsulated form, if appropriate, with one or
more of the above-described excipients.
[0173] Liquid dosage forms for oral administration of the compounds
of the invention include pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert diluents commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof.
[0174] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0175] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0176] Formulations for rectal or vaginal administration may be
presented as a suppository, which may be prepared by mixing one or
more compounds of the invention with one or more suitable
nonirritating excipients or carriers comprising, for example, cocoa
butter, polyethylene glycol, a suppository wax or a salicylate, and
which is solid at room temperature, but liquid at body temperature
and, therefore, will melt in the rectum or vaginal cavity and
release the active compound.
[0177] Formulations or dosage forms for the topical or transdermal
administration of an oligomer as provided herein include powders,
sprays, ointments, pastes, creams, lotions, gels, solutions,
patches and inhalants. The active oligomers may be mixed under
sterile conditions with a pharmaceutically-acceptable carrier, and
with any preservatives, buffers, or propellants which may be
required. The ointments, pastes, creams and gels may contain, in
addition to an active compound of this invention, excipients, such
as animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0178] Powders and sprays can contain, in addition to an oligomer
of the present invention, excipients such as lactose, talc, silicic
acid, aluminum hydroxide, calcium silicates and polyamide powder,
or mixtures of these substances. Sprays can additionally contain
customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0179] Transdermal patches have the added advantage of providing
controlled delivery of an oligomer of the present invention to the
body. Such dosage forms can be made by dissolving or dispersing the
oligomer in the proper medium. Absorption enhancers can also be
used to increase the flux of the agent across the skin. The rate of
such flux can be controlled by either providing a rate controlling
membrane or dispersing the agent in a polymer matrix or gel, among
other methods known in the art.
[0180] Pharmaceutical compositions suitable for parenteral
administration may comprise one or more oligomers of the invention
in combination with one or more pharmaceutically-acceptable sterile
isotonic aqueous or nonaqueous solutions, dispersions, suspensions
or emulsions, or sterile powders which may be reconstituted into
sterile injectable solutions or dispersions just prior to use,
which may contain sugars, alcohols, antioxidants, buffers,
bacteriostats, solutes which render the formulation isotonic with
the blood of the intended recipient or suspending or thickening
agents. Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0181] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms upon the subject
oligomers may be ensured by the inclusion of various antibacterial
and antifungal agents, for example, paraben, chlorobutanol, phenol
sorbic acid, and the like. It may also be desirable to include
isotonic agents, such as sugars, sodium chloride, and the like into
the compositions. In addition, prolonged absorption of the
injectable pharmaceutical form may be brought about by the
inclusion of agents which delay absorption such as aluminum
monostearate and gelatin.
[0182] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility, among other methods known in the art. The
rate of absorption of the drug then depends upon its rate of
dissolution which, in turn, may depend upon crystal size and
crystalline form. Alternatively, delayed absorption of a
parenterally-administered drug form is accomplished by dissolving
or suspending the drug in an oil vehicle.
[0183] Injectable depot forms may be made by forming microencapsule
matrices of the subject oligomers in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of oligomer to
polymer, and the nature of the particular polymer employed, the
rate of oligomer release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations may also prepared
by entrapping the drug in liposomes or microemulsions that are
compatible with body tissues.
[0184] When the oligomers of the present invention are administered
as pharmaceuticals, to humans and animals, they can be given per se
or as a pharmaceutical composition containing, for example, 0.1 to
99% (more preferably, 10 to 30%) of active ingredient in
combination with a pharmaceutically acceptable carrier.
[0185] As noted above, the formulations or preparations of the
present invention may be given orally, parenterally, systemically,
topically, rectally or intramuscular administartion. They are
typically given in forms suitable for each administration route.
For example, they are administered in tablets or capsule form, by
injection, inhalation, eye lotion, ointment, suppository, etc.
administration by injection, infusion or inhalation; topical by
lotion or ointment; and rectal by suppositories.
[0186] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
[0187] The phrases "systemic administration," "administered
systemically," "peripheral administration" and "administered
peripherally" as used herein mean the administration of a compound,
drug or other material other than directly into the central nervous
system, such that it enters the patient's system and, thus, is
subject to metabolism and other like processes, for example,
subcutaneous administration.
[0188] Regardless of the route of administration selected, the
oligomers of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, may be formulated into
pharmaceutically-acceptable dosage forms by conventional methods
known to those of skill in the art. Actual dosage levels of the
active ingredients in the pharmaceutical compositions of this
invention may be varied so as to obtain an amount of the active
ingredient which is effective to achieve the desired therapeutic
response for a particular patient, composition, and mode of
administration, without being unacceptably toxic to the
patient.
[0189] The selected dosage level will depend upon a variety of
factors including the activity of the particular oligomer of the
present invention employed, or the ester, salt or amide thereof,
the route of administration, the time of administration, the rate
of excretion or metabolism of the particular oligomer being
employed, the rate and extent of absorption, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular oligomer employed, the age, sex,
weight, condition, general health and prior medical history of the
patient being treated, and like factors well known in the medical
arts.
[0190] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the compounds of the invention
employed in the pharmaceutical composition at levels lower than
that required in order to achieve the desired therapeutic effect
and gradually increase the dosage until the desired effect is
achieved. In general, a suitable daily dose of a compound of the
invention will be that amount of the compound which is the lowest
dose effective to produce a therapeutic effect. Such an effective
dose will generally depend upon the factors described above.
Generally, oral, intravenous, intracerebroventricular,
intramuscular and subcutaneous doses of the compounds of this
invention for a patient, when used for the indicated effects, will
range from about 0.0001 to about 100 mg per kilogram of body weight
per day.
[0191] Preferred doses of the oligomers of the present invention
(e.g., phosphorodiamidate morpholino oligomers; eteplirsen) are
administered generally from about 20-100 mg/kg. In some cases,
doses of greater than 100 mg/kg may be necessary. For i.v.
administration, preferred doses are from about 0.5 mg to 100 mg/kg.
In some embodiments, the oligomers are administered at doses of
about 20 mg/kg, 21 mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg,
29 mg/kg, 30 mg/kg, 31 mg/kg, 32 mg/kg, 33 mg/kg, 34 mg/kg, 35
mg/kg, 36 mg/kg, 37 mg/kg, 38 mg/kg, 39 mg/kg, 40 mg/kg, 41 mg/kg,
42 mg/kg, 43 mg/kg, 44 mg/kg, 45 mg/kg, 46 mg/kg, 47 mg/kg, 48
mg/kg, 49 mg/kg 50 mg/kg, 51 mg/kg, 52 mg/kg, 53 mg/kg, 54 mg/kg,
55 mg/kg, 56 mg/kg, 57 mg/kg, 58 mg/kg, 59 mg/kg, 60 mg/kg, 65
mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg,
100 mg/kg, including all integers in between. In a preferred
embodiment, the oligomer is administered at 30 mg/kg. In another
preferred embodiment, the oligomer is administered at 50 mg/kg.
[0192] If desired, the effective daily dose of the active compound
may be administered as two, three, four, five, six or more
sub-doses administered separately at appropriate intervals
throughout the day, optionally, in unit dosage forms. In certain
situations, dosing is one administration per day. In certain
embodiments, dosing is one or more administration per every 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or every 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12 months, as needed, to maintain the desired expression of
a functional dystrophin protein.
[0193] In some embodiments, the oligomers of the present invention
(e.g., phosphorodiamidate morpholino oligomers; eteplirsen) are
administered, generally at regular intervals (e.g., daily, weekly,
biweekly, monthly, bimonthly). The oligomers may be administered at
regular intervals, e.g., daily; once every two days; once every
three days; once every 3 to 7 days; once every 3 to 10 days; once
every 7 to 10 days; once every week; once every two weeks; once
monthly. For example, the oligomers may be administered once weekly
by intravenous infusion. The oligomers may be administered
intermittently over a longer period of time, e.g., for several
weeks, months or years. For example, the oligomers may be
administered once every one, two, three, four, five, six, seven,
eight, nine, ten, eleven or twelve months. In addition, the
oligomers may be administered once every one, two, three, four or
five years. Administration may be followed by, or concurrent with,
administration of an antibiotic, steroid or other therapeutic
agent. The treatment regimen may be adjusted (dose, frequency,
route, etc.) as indicated, based on the results of immunoassays,
other biochemical tests and physiological examination of the
subject under treatment.
[0194] Nucleic acid molecules can be administered to cells by a
variety of methods known to those familiar to the art, including,
but not restricted to, encapsulation in liposomes, by
iontophoresis, or by incorporation into other vehicles, such as
hydrogels, cyclodextrins, biodegradable nanocapsules, and
bioadhesive microspheres, as described herein and known in the art.
In certain embodiments, microemulsification technology may be
utilized to improve bioavailability of lipophilic (water insoluble)
pharmaceutical agents. Examples include Trimetrine (Dordunoo, S.
K., et al., Drug Development and Industrial Pharmacy, 17(12),
1685-1713, 1991 and REV 5901 (Sheen, P. C., et al., J Pharm Sci
80(7), 712-714, 1991). Among other benefits, microemulsification
provides enhanced bioavailability by preferentially directing
absorption to the lymphatic system instead of the circulatory
system, which thereby bypasses the liver, and prevents destruction
of the compounds in the hepatobiliary circulation.
[0195] In one aspect of invention, the formulations contain
micelles formed from an oligomer as provided herein and at least
one amphiphilic carrier, in which the micelles have an average
diameter of less than about 100 nm. More preferred embodiments
provide micelles having an average diameter less than about 50 nm,
and even more preferred embodiments provide micelles having an
average diameter less than about 30 nm, or even less than about 20
nm.
[0196] While all suitable amphiphilic carriers are contemplated,
the presently preferred carriers are generally those that have
Generally-Recognized-as-Safe (GRAS) status, and that can both
solubilize the compound of the present invention and microemulsify
it at a later stage when the solution comes into a contact with a
complex water phase (such as one found in human gastro-intestinal
tract). Usually, amphiphilic ingredients that satisfy these
requirements have HLB (hydrophilic to lipophilic balance) values of
2-20, and their structures contain straight chain aliphatic
radicals in the range of C-6 to C-20. Examples are
polyethylene-glycolized fatty glycerides and polyethylene
glycols.
[0197] Examples of amphiphilic carriers include saturated and
monounsaturated polyethyleneglycolyzed fatty acid glycerides, such
as those obtained from fully or partially hydrogenated various
vegetable oils. Such oils may advantageously consist of tri-, di-,
and mono-fatty acid glycerides and di- and mono-polyethyleneglycol
esters of the corresponding fatty acids, with a particularly
preferred fatty acid composition including capric acid 4-10, capric
acid 3-9, lauric acid 40-50, myristic acid 14-24, palmitic acid
4-14 and stearic acid 5-15%. Another useful class of amphiphilic
carriers includes partially esterified sorbitan and/or sorbitol,
with saturated or mono-unsaturated fatty acids (SPAN-series) or
corresponding ethoxylated analogs (TWEEN-series).
[0198] Commercially available amphiphilic carriers may be
particularly useful, including Gelucire-series, Labrafil, Labrasol,
or Lauroglycol (all manufactured and distributed by Gattefosse
Corporation, Saint Priest, France), PEG-mono-oleate, PEG-di-oleate,
PEG-mono-laurate and di-laurate, Lecithin, Polysorbate 80, etc
(produced and distributed by a number of companies in USA and
worldwide).
[0199] In certain embodiments, the delivery may occur by use of
liposomes, nanocapsules, microparticles, microspheres, lipid
particles, vesicles, and the like, for the introduction of the
compositions of the present invention into suitable host cells. In
particular, the compositions of the present invention may be
formulated for delivery either encapsulated in a lipid particle, a
liposome, a vesicle, a nanosphere, a nanoparticle or the like. The
formulation and use of such delivery vehicles can be carried out
using known and conventional techniques.
[0200] Hydrophilic polymers suitable for use in the present
invention are those which are readily water-soluble, can be
covalently attached to a vesicle-forming lipid, and which are
tolerated in vivo without toxic effects (i.e., are biocompatible).
Suitable polymers include polyethylene glycol (PEG), polylactic
(also termed polylactide), polyglycolic acid (also termed
polyglycolide), a polylactic-polyglycolic acid copolymer, and
polyvinyl alcohol. In certain embodiments, polymers have a
molecular weight of from about 100 or 120 daltons up to about 5,000
or 10,000 daltons, or from about 300 daltons to about 5,000
daltons. In other embodiments, the polymer is polyethyleneglycol
having a molecular weight of from about 100 to about 5,000 daltons,
or having a molecular weight of from about 300 to about 5,000
daltons. In certain embodiments, the polymer is polyethyleneglycol
of 750 daltons (PEG(750)). Polymers may also be defined by the
number of monomers therein; a preferred embodiment of the present
invention utilizes polymers of at least about three monomers, such
PEG polymers consisting of three monomers (approximately 150
daltons).
[0201] Other hydrophilic polymers which may be suitable for use in
the present invention include polyvinylpyrrolidone,
polymethoxazoline, polyethyloxazoline, polyhydroxypropyl
methacrylamide, polymethacrylamide, polydimethylacrylamide, and
derivatized celluloses such as hydroxymethylcellulose or
hydroxyethylcellulose.
[0202] In certain embodiments, a formulation of the present
invention comprises a biocompatible polymer selected from the group
consisting of polyamides, polycarbonates, polyalkylenes, polymers
of acrylic and methacrylic esters, polyvinyl polymers,
polyglycolides, polysiloxanes, polyurethanes and co-polymers
thereof, celluloses, polypropylene, polyethylenes, polystyrene,
polymers of lactic acid and glycolic acid, polyanhydrides,
poly(ortho)esters, poly(butic acid), poly(valeric acid),
poly(lactide-co-caprolactone), polysaccharides, proteins,
polyhyaluronic acids, polycyanoacrylates, and blends, mixtures, or
copolymers thereof.
[0203] Cyclodextrins are cyclic oligosaccharides, consisting of 6,
7 or 8 glucose units, designated by the Greek letter .alpha.,
.beta., or .gamma., respectively. The glucose units are linked by
.alpha.-1,4-glucosidic bonds. As a consequence of the chair
conformation of the sugar units, all secondary hydroxyl groups (at
C-2, C-3) are located on one side of the ring, while all the
primary hydroxyl groups at C-6 are situated on the other side. As a
result, the external faces are hydrophilic, making the
cyclodextrins water-soluble. In contrast, the cavities of the
cyclodextrins are hydrophobic, since they are lined by the hydrogen
of atoms C-3 and C-5, and by ether-like oxygens. These matrices
allow complexation with a variety of relatively hydrophobic
compounds, including, for instance, steroid compounds such as
17.alpha.-estradiol (see, e.g., van Uden et al. Plant Cell Tiss.
Org. Cult. 38:1-3-113 (1994)). The complexation takes place by Van
der Waals interactions and by hydrogen bond formation. For a
general review of the chemistry of cyclodextrins, see, Wenz, Agnew.
Chem. Int. Ed. Engl., 33:803-822 (1994).
[0204] The physico-chemical properties of the cyclodextrin
derivatives depend strongly on the kind and the degree of
substitution. For example, their solubility in water ranges from
insoluble (e.g., triacetyl-beta-cyclodextrin) to 147% soluble (w/v)
(G-2-beta-cyclodextrin). In addition, they are soluble in many
organic solvents. The properties of the cyclodextrins enable the
control over solubility of various formulation components by
increasing or decreasing their solubility.
[0205] Numerous cyclodextrins and methods for their preparation
have been described. For example, Parmeter (I), et al. (U.S. Pat.
No. 3,453,259) and Gramera, et al. (U.S. Pat. No. 3,459,731)
described electroneutral cyclodextrins. Other derivatives include
cyclodextrins with cationic properties [Parmeter (II), U.S. Pat.
No. 3,453,257], insoluble crosslinked cyclodextrins (Solms, U.S.
Pat. No. 3,420,788), and cyclodextrins with anionic properties
[Parmeter (III), U.S. Pat. No. 3,426,011]. Among the cyclodextrin
derivatives with anionic properties, carboxylic acids, phosphorous
acids, phosphinous acids, phosphonic acids, phosphoric acids,
thiophosphonic acids, thiosulphinic acids, and sulfonic acids have
been appended to the parent cyclodextrin [see, Parmeter (III),
supra]. Furthermore, sulfoalkyl ether cyclodextrin derivatives have
been described by Stella, et al. (U.S. Pat. No. 5,134,127).
[0206] Liposomes consist of at least one lipid bilayer membrane
enclosing an aqueous internal compartment. Liposomes may be
characterized by membrane type and by size. Small unilamellar
vesicles (SUVs) have a single membrane and typically range between
0.02 and 0.05 .mu.m in diameter; large unilamellar vesicles (LUVS)
are typically larger than 0.05 .mu.m. Oligolamellar large vesicles
and multilamellar vesicles have multiple, usually concentric,
membrane layers and are typically larger than 0.1 .mu.m. Liposomes
with several nonconcentric membranes, i.e., several smaller
vesicles contained within a larger vesicle, are termed
multivesicular vesicles.
[0207] One aspect of the present invention relates to formulations
comprising liposomes containing an oligomer of the present
invention, where the liposome membrane is formulated to provide a
liposome with increased carrying capacity. Alternatively or in
addition, the compound of the present invention may be contained
within, or adsorbed onto, the liposome bilayer of the liposome. An
oligomer of the present invention may be aggregated with a lipid
surfactant and carried within the liposome's internal space; in
these cases, the liposome membrane is formulated to resist the
disruptive effects of the active agent-surfactant aggregate.
[0208] According to one embodiment of the present invention, the
lipid bilayer of a liposome contains lipids derivatized with
polyethylene glycol (PEG), such that the PEG chains extend from the
inner surface of the lipid bilayer into the interior space
encapsulated by the liposome, and extend from the exterior of the
lipid bilayer into the surrounding environment.
[0209] Active agents contained within liposomes of the present
invention are in solubilized form. Aggregates of surfactant and
active agent (such as emulsions or micelles containing the active
agent of interest) may be entrapped within the interior space of
liposomes according to the present invention. A surfactant acts to
disperse and solubilize the active agent, and may be selected from
any suitable aliphatic, cycloaliphatic or aromatic surfactant,
including but not limited to biocompatible lysophosphatidylcholines
(LPGs) of varying chain lengths (for example, from about C14 to
about C20). Polymer-derivatized lipids such as PEG-lipids may also
be utilized for micelle formation as they will act to inhibit
micelle/membrane fusion, and as the addition of a polymer to
surfactant molecules decreases the CMC of the surfactant and aids
in micelle formation. Preferred are surfactants with CMOs in the
micromolar range; higher CMC surfactants may be utilized to prepare
micelles entrapped within liposomes of the present invention.
[0210] Liposomes according to the present invention may be prepared
by any of a variety of techniques that are known in the art. See,
e.g., U.S. Pat. No. 4,235,871; Published PCT applications WO
96/14057; New RRC, Liposomes: A practical approach, IRL Press,
Oxford (1990), pages 33-104; Lasic DD, Liposomes from physics to
applications, Elsevier Science Publishers BV, Amsterdam, 1993. For
example, liposomes of the present invention may be prepared by
diffusing a lipid derivatized with a hydrophilic polymer into
preformed liposomes, such as by exposing preformed liposomes to
micelles composed of lipid-grafted polymers, at lipid
concentrations corresponding to the final mole percent of
derivatized lipid which is desired in the liposome. Liposomes
containing a hydrophilic polymer can also be formed by
homogenization, lipid-field hydration, or extrusion techniques, as
are known in the art.
[0211] In another exemplary formulation procedure, the active agent
is first dispersed by sonication in a lysophosphatidylcholine or
other low CMC surfactant (including polymer grafted lipids) that
readily solubilizes hydrophobic molecules. The resulting micellar
suspension of active agent is then used to rehydrate a dried lipid
sample that contains a suitable mole percent of polymer-grafted
lipid, or cholesterol. The lipid and active agent suspension is
then formed into liposomes using extrusion techniques as are known
in the art, and the resulting liposomes separated from the
unencapsulated solution by standard column separation.
[0212] In one aspect of the present invention, the liposomes are
prepared to have substantially homogeneous sizes in a selected size
range. One effective sizing method involves extruding an aqueous
suspension of the liposomes through a series of polycarbonate
membranes having a selected uniform pore size; the pore size of the
membrane will correspond roughly with the largest sizes of
liposomes produced by extrusion through that membrane. See e.g.,
U.S. Pat. No. 4,737,323 (Apr. 12, 1988). In certain embodiments,
reagents such as DharmaFECT.RTM. and Lipofectamine.RTM. may be
utilized to introduce polynucleotides or proteins into cells.
[0213] The release characteristics of a formulation of the present
invention depend on the encapsulating material, the concentration
of encapsulated drug, and the presence of release modifiers. For
example, release can be manipulated to be pH dependent, for
example, using a pH sensitive coating that releases only at a low
pH, as in the stomach, or a higher pH, as in the intestine. An
enteric coating can be used to prevent release from occurring until
after passage through the stomach. Multiple coatings or mixtures of
cyanamide encapsulated in different materials can be used to obtain
an initial release in the stomach, followed by later release in the
intestine. Release can also be manipulated by inclusion of salts or
pore forming agents, which can increase water uptake or release of
drug by diffusion from the capsule. Excipients which modify the
solubility of the drug can also be used to control the release
rate. Agents which enhance degradation of the matrix or release
from the matrix can also be incorporated. They can be added to the
drug, added as a separate phase (i.e., as particulates), or can be
co-dissolved in the polymer phase depending on the compound. In
most cases the amount should be between 0.1 and thirty percent (w/w
polymer). Types of degradation enhancers include inorganic salts
such as ammonium sulfate and ammonium chloride, organic acids such
as citric acid, benzoic acid, and ascorbic acid, inorganic bases
such as sodium carbonate, potassium carbonate, calcium carbonate,
zinc carbonate, and zinc hydroxide, and organic bases such as
protamine sulfate, spermine, choline, ethanolamine, diethanolamine,
and triethanolamine and surfactants such as Tween.RTM. and
Pluronic.RTM.. Pore forming agents which add microstructure to the
matrices (i.e., water soluble compounds such as inorganic salts and
sugars) are added as particulates. The range is typically between
one and thirty percent (w/w polymer).
[0214] Uptake can also be manipulated by altering residence time of
the particles in the gut. This can be achieved, for example, by
coating the particle with, or selecting as the encapsulating
material, a mucosal adhesive polymer. Examples include most
polymers with free carboxyl groups, such as chitosan, celluloses,
and especially polyacrylates (as used herein, polyacrylates refers
to polymers including acrylate groups and modified acrylate groups
such as cyanoacrylates and methacrylates).
[0215] An oligomer may be formulated to be contained within, or,
adapted to release by a surgical or medical device or implant. In
certain aspects, an implant may be coated or otherwise treated with
an oligomer. For example, hydrogels, or other polymers, such as
biocompatible and/or biodegradable polymers, may be used to coat an
implant with the compositions of the present invention (i.e., the
composition may be adapted for use with a medical device by using a
hydrogel or other polymer). Polymers and copolymers for coating
medical devices with an agent are well-known in the art. Examples
of implants include, but are not limited to, stents, drug-eluting
stents, sutures, prosthesis, vascular catheters, dialysis
catheters, vascular grafts, prosthetic heart valves, cardiac
pacemakers, implantable cardioverter defibrillators, IV needles,
devices for bone setting and formation, such as pins, screws,
plates, and other devices, and artificial tissue matrices for wound
healing.
[0216] In addition to the methods provided herein, the oligomers
for use according to the invention may be formulated for
administration in any convenient way for use in human or veterinary
medicine, by analogy with other pharmaceuticals. The antisense
oligomers and their corresponding formulations may be administered
alone or in combination with other therapeutic strategies in the
treatment of muscular dystrophy, such as myoblast transplantation,
stem cell therapies, administration of aminoglycoside antibiotics,
proteasome inhibitors, and up-regulation therapies (e.g.,
upregulation of utrophin, an autosomal paralogue of
dystrophin).
[0217] The routes of administration described are intended only as
a guide since a skilled practitioner will be able to determine
readily the optimum route of administration and any dosage for any
particular animal and condition. Multiple approaches for
introducing functional new genetic material into cells, both in
vitro and in vivo have been attempted (Friedmann (1989) Science,
244:1275-1280). These approaches include integration of the gene to
be expressed into modified retroviruses (Friedmann (1989) supra;
Rosenberg (1991) Cancer Research 51(18), suppl.: 5074S-5079S);
integration into non-retrovirus vectors (e.g., adeno-associated
viral vectors) (Rosenfeld, et al. (1992) Cell, 68:143-155;
Rosenfeld, et al. (1991) Science, 252:431-434); or delivery of a
transgene linked to a heterologous promoter-enhancer element via
liposomes (Friedmann (1989), supra; Brigham, et al. (1989) Am. J.
Med. Sci., 298:278-281; Nabel, et al. (1990) Science,
249:1285-1288; Hazinski, et al. (1991) Am. J. Resp. Cell Molec.
Biol., 4:206-209; and Wang and Huang (1987) Proc. Natl. Acad. Sci.
(USA), 84:7851-7855); coupled to ligand-specific, cation-based
transport systems (Wu and Wu (1988) J. Biol. Chem.,
263:14621-14624) or the use of naked DNA, expression vectors (Nabel
et al. (1990), supra); Wolff et al. (1990) Science, 247:1465-1468).
Direct injection of transgenes into tissue produces only localized
expression (Rosenfeld (1992) supra); Rosenfeld et al. (1991) supra;
Brigham et al. (1989) supra; Nabel (1990) supra; and Hazinski et
al. (1991) supra). The Brigham et al. group (Am. J. Med. Sci.
(1989) 298:278-281 and Clinical Research (1991) 39 (abstract)) have
reported in vivo transfection only of lungs of mice following
either intravenous or intratracheal administration of a DNA
liposome complex. An example of a review article of human gene
therapy procedures is: Anderson, Science (1992) 256:808-813.
V. KITS
[0218] The invention also provides kits for treatment of a patient
with a genetic disease which kit comprises at least an antisense
molecule (e.g., one or more antisense oligonucleotides capable of
specifically hybridizing to any one or more of exons 1-79 of the
dystrophin gene; for example, Exon 51 as set forth in Tables 3 and
4 herein), packaged in a suitable container, together with
instructions for its use. The kits may also contain peripheral
reagents such as buffers, stabilizers, etc. Those of ordinary skill
in the field should appreciate that applications of the above
method has wide application for identifying antisense molecules
suitable for use in the treatment of many other diseases.
VII. EXAMPLES
Materials and Methods
Patients
[0219] Eligible patients were between 7 and 13 years of age
(inclusive), with out-of-frame deletions of the DMD gene that could
be corrected by skipping exon 51. Patients were confirmed to have
stable cardiac and pulmonary function and a stable dose of
glucocorticoids for at least 24 weeks prior to enrollment. Only
patients who could walk between 200 and 400 meters (.+-.10%) on the
6-Minute Walk Test (6MWT) at baseline were enrolled.
Study Design
[0220] This one-year trial was conducted in two phases: (1)
treatment was double-blind through week 24 and (2) open-label
thereafter. Primary endpoints were change in percent dystrophin
fibers and ambulation as measured by the 6-Minute Walk Test
(6MWT).
[0221] Study 201 was a single-site, randomized, double-blind,
placebo-controlled, multiple-dose efficacy, safety and tolerability
trial of eteplirsen. Twelve patients with DMD were randomized to
one of three groups: eteplirsen 30 mg/kg/week (Cohort 1);
eteplirsen 50 mg/kg/week (Cohort 2); or placebo/delayed eteplirsen
(Cohort 3). All patients received weekly intravenous eteplirsen or
placebo/delayed eteplirsen dosing. Placebo-treated patients crossed
over to weekly eteplirsen 30 (n=2) or 50 mg/kg (n=2) at week 25.
Efficacy and safety were assessed at scheduled visits, and an
independent Data Safety Monitoring Board ensured the welfare of all
patients. All patients had bicep biopsies at baseline. Follow-up
biopsies were performed in the opposite arm (biceps) at week 12 for
the 50 mg/kg group and two placebo-treated patients and at week 24
for the 30 mg/kg group and two placebo-treated patients.
[0222] Patients continued weekly dosing with 30 or 50 mg/kg
eteplirsen under Study 202, a long-term, open-label extension
study. All efficacy assessments continued to be performed during
Study 202, including a third biopsy (in the left deltoid muscle) in
all patients at week 48. Monitoring of adverse events continued
throughout the study. A schematic of the study design is shown in
FIG. 1.
Study Drug
[0223] Eteplirsen [sequence 5'-CTCCAACATCAAGGAAGATGGCATTTCTAG-3']
(SEQ ID NO:1) was supplied by Sarepta Therapeutics, Inc. in
single-use vials of phosphate-buffered saline (100 mg/ml).
Eteplirsen was reconstituted with 150 ml normal saline and infused
over 60 minutes. Placebo, administered during the first 24 weeks of
Study 201, was supplied as identical vials of phosphate-buffered
saline and was administered in the same manner as eteplirsen.
Safety and Tolerability Monitoring
[0224] Safety was assessed by evaluation of adverse events, vital
signs, physical examinations, electrocardiograms, echocardiograms,
and clinical laboratory testing. In addition, kidney function was
monitored via regular assessments of serum cystatin C and urine
cystatin C and KIM-1.
Pharmacokinetic and Immune Assessments
[0225] Pharmacokinetic parameters of eteplirsen were established
from plasma and urine taken after the twelfth dose using a
validated and sensitive anion exchange high-performance liquid
chromatography with fluorescence detection bioanalytical method.
Single samples for analysis of plasma concentrations were taken at
weeks 24, 25, and 36. Immune response to novel dystrophin protein
was measured every six weeks through week 24 with ELISPOT following
methods previously published.
Biochemical Efficacy Assessments
[0226] Pre- and post-treatment dystrophin expression studies were
based on MANDYS106 [a gift from Glen Morris, MDA Monoclonal
Antibody Library], a highly sensitive marker for dystrophin used in
prior studies of eteplirsen and other exon skipping candidates.
Three 10 .mu.m frozen sections, separated by at least 200 .mu.m,
were stained with MANDYS106, followed by a secondary antibody
(Alexa Fluor 594 goat antimouse antibody). Percent
dystrophin-positive fibers were calculated by dividing the number
of positive fibers by the total fibers counted. As normal muscle
samples have 100% dystrophin-positive fibers, percent
dystrophin-positive fibers is expressed as a percentage of normal.
The same antibody-stained sections were used for dystrophin
quantification using Bioquant image analysis software. The total
dystrophin fluorescence signal intensity was reported as a
percentage of normal.
[0227] Supportive measurements included expression of the
components of the sarcoglycan complex (.beta.,.gamma.), neuronal
NOS, and Western blot (with the anti-dystrophin antibody NCL-Dys1
from Novacastra). RT-PCR analysis, for confirmation of exon
skipping, was performed on 400 ng of total RNA using
dystrophin-specific reverse primers as previously described.
Clinical Efficacy Assessments
[0228] The 6MWT was administered using the protocol established for
patients with DMD by McDonald, et al. (Muscle Nerve, 2010;
42:966-74, herein incorporated by reference). Exploratory
functional outcomes included the North Star Ambulatory Assessment,
quantitative muscle testing, the 9-Hole Peg Test, pulmonary
function testing (PFT), timed function tests, and assessment of
quality of life.
Statistical Analysis
[0229] SAS version 9.3 (Cary, N.C.) was used for all statistical
analyses. Mixed model with treatment as fixed effect, subject
nested within treatment as random effect, with the baseline value
and time since DMD diagnosis as covariates for the analysis of
muscle biopsy data was used. Mixed model repeated measures (MMRM)
with treatment, time, and treatment-by-time interaction terms as
fixed effect, subject nested within treatment as random effect, and
with the baseline value and time since DMD diagnosis as covariates
for analysis of the 6MWT data was used. Safety and muscle biopsy
analyses were performed on the intent-to-treat population; analysis
of ambulation-related outcomes, including the 6MWT, used a modified
intent-to-treat (mITT) population that excluded two patients in
Cohort 1 who showed signs of disease progression and significant
decline on the 6MWT within weeks of enrollment and could not
perform measures of ambulation at week 24 or beyond.
Example 1: Subject Characteristics
[0230] Baseline characteristics of the 12 patients in this study
are summarized in Table 2. Five different genotypes amenable to
exon 51 skipping were represented in the study population. Mean
distances on the 6-Minute Walk Test (6MWT) at baseline were similar
to those in other studies of children with DMD, and as expected,
were well below the 600 plus meters typically observed in
age-matched healthy children. Due to the stochastic nature of the
sampling, the 30 mg/kg cohort was slightly older, heavier, and
taller, relative to the other cohorts, and had a lower mean 6MWT
distance at baseline. All patients received all infusions of study
medication as planned and completed all assessments.
TABLE-US-00002 TABLE 2 Baseline Demography and Disease
Characteristics Placebo/ Eteplirsen Eteplirsen Delayed Eteplirsen
30 mg/kg 50 mg/kg Treatment Arm N = 4 N = 4 N = 4 Mutation 45-50 n
(%) 0 2 (50) 1 (25) 48-50 n (%) 0 1 (25) 0 49-50 n (%) 3 (75) 0 2
(50) 50 n (%) 1 (25) 0 0 52 n (%) 0 1 (25) 1 (25) Gender n (%) Male
4 (100) 4 (100) 4 (100) Age, years Mean 8.5 9.3 8.5 SD 1.73 0.50
1.29 Min, Max 7, 10 9, 10 7, 10 Height, cm Mean 119.3 130.5 121.3
SD 3.40 9.47 7.85 Min, Max 116, 124 117, 138 117, 133 Weight, kg
Mean 30.6 34.8 29.0 SD 6.04 7.05 6.38 Min, Max 22.1, 36.2 24.8,
39.8 23.7, 38.3 Race, n (%) Asian 0 1 (25) 0 White 4 (100) 3 (75) 4
(100) 6MWT*, meters Mean 394.5 355.3 396.0 SD 42.25 74.78 26.61
Min, Max 364, 456 261, 442 365, 429 *6MWT results are the maximum
observed value of two tests administered on two consecutive days
during screening. Abbreviations: 6MWT = 6-Minute Walk Test; min =
minimum; max = maximum; SD = standard deviation.
Example 2: Safety and Lack of Adverse Events
[0231] Eteplirsen was well tolerated with no treatment-related
adverse events, serious adverse events, discontinuations or missed
doses through 48 weeks of treatment. Moreover, no clinically
significant changes were observed on physical examination or in
vital signs. Electrocardiograms, echocardiograms, and PFTs remained
stable, and chemistries showed no clinically significant changes in
hematologic, renal, coagulation or liver functions. Mild and
transient proteinuria was observed in a single placebo-treated
subject.
Example 3: Pharmacokinetic Profile
[0232] Analysis of PK parameters at week 12 revealed rapid
absorption. Plasma clearance averaged 339.+-.75.8 mL/hr/kg for 30
mg/kg and 319.+-.125 mL/hr/kg for 50 mg/kg. Half-life averaged
3.30.+-.0.341 hr for 30 mg/kg and 3.17.+-.0.249 hr for 50 mg/kg,
with renal clearance accounting for approximately 65-70% of total
systemic clearance.
Example 4: Efficacy
[0233] At week 48, eteplirsen produced robust increases in the
number and intensity of dystrophin-positive fibers. As shown in
FIG. 3, patients who received 30 or 50 mg/kg eteplirsen without
interruption for 48 weeks showed a mean increase in the percentage
of dystrophin-positive fibers to 47% of normal (p.ltoreq.0.001),
relative to baseline. Increases were similar when the 30 (52%;
p.ltoreq.0.001) and 50 (43%; p.ltoreq.0.008) mg/kg cohorts were
analyzed separately, suggesting that eteplirsen's effect on the
production of novel dystrophin is independent of dose within this
range of doses.
[0234] Biopsies were taken at staggered time points (see FIG. 2) to
evaluate the impact of treatment duration on novel dystrophin
production. At week 12, the 50 mg/kg cohort had undetectable levels
of novel dystrophin. At week 24, the 30 mg/kg cohort demonstrated
an increase in the percentage of dystrophin-positive fibers to 23%
of normal (p.ltoreq.0.002), and at week 48, after 24 weeks of
treatment with 30 or 50 mg/kg eteplirsen, the 4 patients in the
placebo/delayed eteplirsen cohort showed an increase to 38% of
normal, relative to baseline (p.ltoreq.0.009). Together these data
suggest that treatment duration plays an important role in
eteplirsen's ability to uniformly restore novel dystrophin
production. Consistent with these findings, eteplirsen also
significantly increased mean fluorescence signal intensity at week
48 in all three treatment groups (all p-values.ltoreq.0.023).
[0235] FIG. 4 illustrates eteplirsen's time-dependent effect on the
percentage of dystrophin-positive fibers (Panel A), which was
accompanied by restoration of .beta.- and .gamma.-sarcoglycan and
nNOS.mu. at the sarcolemma (Panel B). Dystrophin expression and
exon skipping were confirmed by Western blot and RT-PCR in all
patients. RT-PCR results from a representative patient are shown in
Panel C. These data confirmed the increase in functional dystrophin
in the patients.
Example 5: Functional Outcomes
[0236] The progressive loss of walking ability is a universal
hallmark of DMD, with most patients showing functional compromise
by 7 or 8 years of age and becoming wheelchair dependent by 10 to
14 years of age. Consistent with this, boys assigned to the
placebo/delayed eteplirsen cohort in this study showed a decline in
walking ability after week 12 at a rate predicted by prior studies,
culminating in a loss of approximately 60 meters by week 48 (FIG.
5). In marked contrast, eteplirsen-treated patients maintained a
stable walking distance over the duration of the study, with a mean
increase from baseline of about 7 meters by week 48. The difference
between the eteplirsen-treated patients and those in the
placebo/delayed eteplirsen cohort first became statistically
significant at week 32 (39-meter difference; p.ltoreq.0.05).
Interestingly, patients in the placebo/delayed eteplirsen cohort
appeared to stabilize after week 36, i.e., between 12 and 24 weeks
after initiating treatment with eteplirsen at week 25. As
previously noted, two boys who showed signs of rapid disease
progression and significant decline on the 6MWT within weeks of
enrollment and were unable to perform measures of ambulation at 24
weeks or beyond, were excluded from this analysis. However, both
remained on eteplirsen through week 48 with no treatment-related
adverse events and maintained stable pulmonary and upper limb
function as measured by PFT and the 9-Hole Peg Test,
respectively.
[0237] Notably, patients receiving eteplirsen for 48 weeks,
evaluable on the 6MWT (n=6), significantly (p.ltoreq.0.001)
improved on the 6MWT (67.3 m) compared to the placebo/delayed
cohort.
Example 6: Immune Response
[0238] There were no differences between the eteplirsen- and
placebo-treated patients in the number of
interferon-.gamma.-induced spot forming colonies to dystrophin
peptide pools (extended over the entire protein) at any time point
assessed, including week 24, indicating that the newly expressed
dystrophin in the eteplirsen-treated patients did not elicit a
T-cell response.
[0239] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
REFERENCES
[0240] 1. Emery A E H. Population frequencies of inherited
neuromuscular diseases--a world survey. Neuromuscul Disord 1991;
1:19-29. [0241] 2. Mendell J R, Shilling C, Leslie N D, Flanigan K
M, al-Dahhak R, Gastier-Foster, J, et al. Evidence-based path to
newborn screening for Duchenne muscular dystrophy. Ann Neurol 2012;
71:304-13. [0242] 3. McDonald C M, Abresch R T, Carter G T, Fowler
W M Jr, Johnson E R, Kilmer D D, et al. Profiles of neuromuscular
diseases. Duchenne muscular dystrophy. Am J Phys Med Rehabil 1995;
74:S70-S92. [0243] 4. Bushby K, Finkel R, Birnkrant D J, Case L E,
Clemens P R, Cripe L, et al. Diagnosis and management of Duchenne
muscular dystrophy, part 1: diagnosis, and pharmacological and
psychosocial management. Lancet Neurol 2010; 9:77-93. [0244] 5.
Kohler M, Clarenbach C F, Boni L, Brack T, Russi E W, Bloch K E.
Quality of life, physical disability and respiratory impairment in
Duchenne muscular dystrophy. Am J Respir Crit Care Med 2005;
172:1032-6. [0245] 6. Mendell J R, Moxley R T, Griggs R C, Brooke M
E I, Fenichel G M, Miller J P, et al. Randomized, double-blind
six-month trial of prednisone in Duchenne's muscular dystrophy. N
Engl J Med 1989; 320:1592-97. [0246] 7. Manzur A Y, Kuntzer T, Pike
M, Swan A. Glucocorticoid corticosteroids for Duchenne muscular
dystrophy. Cochrane Database Syst Rev. 2004; (2):CD003725. [0247]
8. van Deutekom J C, Janson A A, Ginjaar I B, Frankhuizen W S,
Aartsma-Rus A, Bremmer-Bout M, et al. Local dystrophin restoration
with antisense oligonucleotide PRO051. N Engl J Med 2007;
357:2677-86. [0248] 9. Kinali M, Arechavala-Gomeza V, Feng L, Cirak
S, Hunt D, Adkin C, et al. Local restoration of dystrophin
expression with the morpholino oligomer AVI-4658 in Duchenne
muscular dystrophy: a single-blind, placebo-controlled,
dose-escalation, proof-of-concept study. Lancet Neurol 2009;
8:918-28. [0249] 10. Goemans N M, Tulinius M, van den Akker J T,
Burm B E, Ekhart P F, Heuvelmans N, et al. Systemic administration
of PRO051 in Duchenne's muscular dystrophy. N Engl J Med 2011;
364:1513-22. [0250] 11. Cirak S, Arechavala-Gomeza V, Guglieri M,
Feng L, Torelli S, Anthony K, et al. Exon skipping and dystrophin
restoration in patients with Duchenne muscular dystrophy after
systemic phosphorodiamidate morpholino oligomer treatment: an
open-label, phase 2, dose-escalation study. Lancet 2011;
378:595-605. [0251] 12. Aartsma-Rus A, Fokkema I, Verschuuren J,
Ginjaar I, van Deutekom J, van Ommen G J, et al. Theoretic
applicability of antisense-mediated exon skipping for Duchenne
muscular dystrophy mutations. Hum Mutat 2009; 30:293-99. [0252] 13.
Muntoni F, Torelli S, Ferlini A. Dystrophin and mutations: one
gene, several proteins, multiple phenotypes. Lancet Neurol. 2003;
2:731-40. [0253] 14. Bushby K M, Gardner-Medwin D. The clinical,
genetic and dystrophin characteristics of Becker muscular
dystrophy. I. Natural history. J Neurol 1993; 240:98-104. [0254]
15. Arechavala-Gomeza V, Graham I R, Popplewell L J, Adams A M,
Aartsma-Rus A, Kinali M, et al. Comparative analysis of antisense
oligonucleotide sequences for targeted skipping of exon 51 during
dystrophin pre-mRNA splicing in human muscle. Hum Gene Ther 2007;
18:798-810. [0255] 16. Mendell J R, Campbell K, Rodino-Klapac L,
Sahenk Z, Shilling C, Lewis S, et al. Dystrophin immunity revealed
by gene therapy in Duchenne muscular dystrophy. N Engl J Med 2010;
363:1429-37. [0256] 17. Nguyen T M, Morris G E. Use of epitope
libraries to identify exon-specific monoclonal antibodies for
characterization of altered dystrophins in muscular dystrophy. Am J
Hum Genet 1993; 52:1057-66. [0257] 18. Arechavala-Gomeza V, Kinali
M, Feng L, Brown S C, Sewry C, Morgan J E, et al.
Immunohistological intensity measurements as a tool to assess
sarcolemma-associated protein expression. Neuropathol Appl
Neurobiol 2010; 36: 265-74. [0258] 19. McDonald C M, Henricson E K,
Han J J, Abresch R T, Nicorici A, Elfring G L, et al. The 6-minute
walk test as a new outcome measure in Duchenne muscular dystrophy.
Muscle Nerve 2010; 41:500-10. [0259] 20. Mazzone E, Vasco G,
Sormani M P, Torrente Y, Berardinelli A, Messina S, et al.
Functional changes in Duchenne muscular dystrophy: a 12-month
longitudinal cohort study. Neurology 2011; 77(3):250-6. [0260] 21.
McDonald C M, Henricson E K, Han J J, Abresch R T, Nicorici A,
Atkinson L, et al. The 6-minute walk test in Duchenne/Becker
muscular dystrophy: longitudinal observations. Muscle Nerve 2010;
42: 966-74. [0261] 22. Strober J B. Therapeutics in Duchenne
muscular dystrophy. NeuroRX 2006; 3:225-34. [0262] 23. Hoffman E P,
Fischbeck K H, Brown R H, Johnson M, Medori R, Loike J D, et al.
Characterization of dystrophin in muscle-biopsy specimens from
patients with Duchenne's or Becker's muscular dystrophy. N Engl J
Med 1988; 318:1363-68. [0263] 24. Azofeifa J, Voit T, Hubner C,
Cremer M. X-chromosome methylation in manifesting and healthy
carriers of dystrophinopathies: concordance of activation ratios
among first degree female relatives and skewed inactivation as
cause of the affected phenotypes. Hum Genet 1995; 96:167-76. [0264]
25. van Putten M, Hulsker M, Nadarajah V D, van Heiningen S H, van
Huizen E, van Iterson M, et al. The Effects of Low Levels of
Dystrophin on Mouse Muscle Function and Pathology. PLoS ONE 2012;
7:e31937. [0265] 26. Brooke M H, Fenichel G M, Griggs R C, Mendell
J R, Moxley R, Miller J P, et al. Clinical investigation in
Duchenne dystrophy: 2. Determination of the "power" of therapeutic
trials based on the natural history. Muscle Nerve. 1983; 6:91-103.
[0266] 27. Ahmad A, Brinson M, Hodges B L, Chamberlain J S,
Amalfitano A. Mdx mice inducibly expressing dystrophin provide
insights into the potential of gene therapy for Duchenne muscular
dystrophy. Hum Mol Genet 2000; 9:2507-15. [0267] 28. Hoffman E P,
Bronson A, Levin A A, Takeda S, Yokota T, Baudy A R, Connor E M.
Restoring dystrophin expression in Duchenne muscular dystrophy
muscle: Progress in exon skipping and stop codon read through. Am J
Path 2011; 179:12-22. [0268] 29. Merlini L, Gennari M, Malaspina E,
Cecconi I, Armaroli A, Gnudi S, et al. Early corticosteroid
treatment in 4 Duchenne muscular dystrophy patients: 14-year
follow-up. Muscle Nerve 2012; 45:796-802. [0269] 30. Fletcher S,
Honeyman K, Fall A M, Harding P L, Johnsen R D, Steinhaus J P, et
al. Morpholino oligomer-mediated exon skipping averts the onset of
dystrophic pathology in the mdx mouse. Mol Ther 2007; 15:1587-92.
[0270] 31. Yokota T, Lu Q L, Partridge T, Kobayashi M, Nakamura A,
Takeda S, et al. Efficacy of systemic morpholino exon-skipping in
Duchenne dystrophy dogs. Ann Neurol 2009; 65:667-76. [0271] 32.
Aartsma-Rus A, Janson A A, Kaman W E, Bremmer-Bout M, van Ommen G
J, den Dunnen J T, et al. Antisense-induced multiexon skipping for
Duchenne muscular dystrophy makes more sense. Am J Hum Genet 2004;
74:83-92.
SEQUENCE LISTING
[0272] With respect to the nucleic acid sequences provided in the
application, persons skilled in the art will appreciate that
depending on the use of the oligomers, Ts and Us are
interchangeable.
TABLE-US-00003 TABLE 3 EXON SEQ ID SEQUENCE NUCLEOTIDE SEQUENCE
(5'-3') 51 1 eteplirsen CTC CAA CAT CAA GGA AGA TGG CAT H51A(+66 +
95) TTC TAG 51 2 H51A(+66 + 90) ACA UCA AGG APG AUG GCA UUU CUA G
51 3 H51A(+61 + 90) ACA UCA AGG AAG AUG GCA UUU CUA GUU UGG 51 4
Hu.DMD.exon51.25.001.2 GAG CAG GTA CCT CCA ACA TCA AGG AA 50 5
H50D(+07 - 18) GGG AUC CAG UAU ACU UAC AGG CUC C 50 6 AVI-4038/5038
CTT ACA GGC TCC AAT AGT GGT CAG T 53 7 H53A27(+30 + 56) CCT CCG GTT
CTG APG GTG TTC TTG TAC 53 8 H53A(+36 + 60) GTT GCC TCC GGT TCT GAA
GGT GTT C 45 9 H45A (-03 + 19) CAA TGC CAT CCT GGA GTT CCT G
TABLE-US-00004 TABLE 4 SEQ ID SEQUENCE NUCLEOTIDE SEQUENCE (5'-3')
10 H8A(-06 + 18) GAU AGG UGG UAU CAA CAU CUG UAA 11 H8A (-03 + 18)
GAU AGG UGG UAU CAA CAU CUG 12 H8A(-07 + 18) GAU AGG UGG UAU CAA
CAU CUG UAA G 13 H8A(-06 + 14) GGU GGU AUC AAC AUC UGU AA 14
H8A(-10 + 10) GUA UCA ACA UCU GUA AGC AC 15 H7A(+45 + 67) UGC AUG
UUC CAG UCG UUG UGU GG 16 H7A(+02 + 26) CAC UAU UCC AGU CAA AUA GGU
CUG G 17 H7D(+15 - 10) AUU UAC CAA CCU UCA GGA UCG AGU A 18 H7A(-18
+ 03) GGC CUA AAA CAC AUA CAC AUA 19 C6A(-10 + 10) CAU UUU UGA CCU
ACA UGU GG 20 C6A(-14 + 06) UUU GAC CUA CAU GUG GAA AG 21 C6A(-14 +
12) UAC AUU UUU GAC CUA CAU GUG GAA AG 22 C6A(-13 + 09) AUU UUU GAC
CUA CAU GGG AAA G 23 CH6A(+69 + 91) UAC GAG UUG AUU GUC GGA CCC AG
24 C6D(+12 - 13) GUG GUC UCC UUA CCU AUG ACU GUG G 25 C6D(+06 - 11)
GGU CUC CUU ACC UAU GA 26 H6D(+04 - 21) UGU CUC AGU AAU CUU CUU ACC
UAU 27 H6D(+18 - 04) UCU UAC CUA UGA CUA UGG AUG AGA 28 H4A(+13 +
32) GCA UGA ACU CUU GUG GAU CC 29 H4D(+04 - 16) CCA GGG UAC UAC UUA
CAU UA 30 H4D(-24 - 44) AUC GUG UGU CAC AGC AUC CAG 31 H4A(+11 +
40) UGU UCA GGG CAU GAA CUC UUG UGG AUC CUU 32 H3A(+30 + 60) UAG
GAG GCG CCU CCC AUC CUG UAG GUC ACU G 33 H3A(+35 + 65) AGG UCU AGG
AGG CGC CUC CCA UCC UGU AGG U 34 H3A(+30 + 54) GCG CCU CCC AUC CUG
UAG GUC ACU G 35 H3D(+46 - 21) CUU CGA GGA GGU CUA GGA GGC GCC UC
36 H3A(+30 + 50) CUC CCA UCC UGU AGG UCA CUG 37 H3D(+19 - 03) UAC
CAG UUU UUG CCC UGU CAG G 38 H3A(-06 + 20) UCA AUA UGC UGC UUC CCA
AAC UGA AA 39 H3A(+37 + 61) CUA GGA GGC GCC UCC CAU CCU GUA G 40
H5A(+20 + 50) UUA UGA UUU CCA UCU ACG AUG UCA GUA CUU C 41 H5D(+25
- 05) CUU ACC UGC CAG UGG AGG AUU AUA UUC CAA A 42 H5D(+10 - 15)
CAU CAG GAU UCU UAC CUG CCA GUG G 43 H5A(+10 + 34) CGA UGU CAG UAC
UUC CAA UAU UCA C 44 H5D(-04 - 21) ACC AUU CAU CAG GAU UCU 45
H5D(+16 - 02) ACC UGC CAG UGG AGG AUU 46 H5A(-07 + 20) CCA AUA UUC
ACU AAA UCA ACC UGU UAA 47 H5D(+18 - 12) CAG GAU UGU UAC CUG CCA
GUG GAG GAU UAU 48 H5A(+05 + 35) ACG AUG UCA GUA CUU CCA AUA UUC
ACU AAA U 49 H5A(+15 + 45) AUU UCC AUC UAC GAU GUC AGU ACU UCC AAU
A 50 H10A(-05 + 16) CAG GAG CUU CCA AAU GCU GCA 51 H10A(-05 + 24)
CUU GUC UUC AGG AGC UUC CAA AUG CUG CA 52 H10A(+98 + 119) UCC UCA
GCA GAA AGA AGC CAC G 53 H10A(+130 + 149) UUA GAA AUC UCU CCU UGU
GC 54 H10A(-33 - 14) UAA AUU GGG UGU UAC ACA AU 55 H11D(+26 + 49)
CCC UGA GGC AUU CCC AUC UUG AAU 56 H11D(+11 - 09) AGG ACU UAC UUG
CUU UGU UU 57 H11A(+118 + 140) CUU GAA UUU AGG AGA UUC AUC UG 58
H11A(+75 + 97) CAU CUU CUG AUA AUU UUC CUG UU 59 H12A(+52 + 75) UCU
UCU GUU UUU GUU AGC CAG UCA 60 H12A(-10 + 10) UCU AUG UAA ACU GAA
AAU UU 61 H12A(+11 + 30) UUC UGG AGA UCC AUU AAA AC 62 H13A(+77 +
100) CAG CAG UUG CGU GAU CUC CAC UAG 63 H13A(+55 + 75) UUC AUC AAC
UAC CAC CAC CAU 64 H13D(+06 - 19) CUA AGC AAA AUA AUC UGA CCU UAA G
65 H14A(+37 + 64) CUU GUA AAA GAA CCC AGC GGU CUU CUG U 66 H14A(+14
+ 35) CAU CUA CAG AUG UUU GCC CAU C 67 H14A(+51 + 73) GAA GGA UGU
CUU GUA AAA GAA CC 68 H14D(-02 + 18) ACC UGU UCU UCA GUA AGA CG 69
H14D(+14 - 10) CAU GAC ACA CCU GUU CUU CAG UAA 70 H14A(+61 + 80)
CAU UUG AGA AGG AUG UCU UG 71 H14A(-12 + 12) AUC UCC CAA UAC CUG
GAG AAG AGA 72 H15A(-12 + 19) GCC AUG CAC UAA AAA GGC ACU GCA AGA
CAU U 73 H15A(+48 + 71) UCU UUA AAG CCA GUU GUG UGA AUC 74 H15A(+08
+ 28) UUU CUG AAA GCC AUG CAC UAA 75 H15D(+17 - 08) GUA CAU ACG GCC
AGU UUU UGA AGA C 76 H16A(-12 + 19) CUA GAU CCG CUU UUA AAA CCU GUU
AAA ACA A 77 H16A(-06 + 25) UCU UUU CUA GAU CCG CUU UUA AAA CCU GUU
A 78 H16A(-06 + 19) CUA GAU CCG CUU UUA AAA CCU GUU A 79 H16A(+87 +
109) CCG UCU UCU GGG UCA CUG ACU UA 80 H16A(-07 + 19) CUA GAU CCG
CUU UUA AAA CCU GUU AA 81 H16A(-07 + 13) CCG CUU UUA AAA CCU GUU AA
82 H16A(+12 + 7) UGG AUU GCU UUU UCU UUU CUA GAU CC 83 H16A(+92 +
116) CAU GCU UCC GUC UUC UGG GUC ACU G 84 H16A(+45 + 67) G AUC UUG
UUU GAG UGA AUA CAG U 85 H16A(+105 + 126) GUU AUC CAG CCA UGC UUC
CGU C 86 H16D(+05 - 20) UGA UAA UUG GUA UCA CUA ACC UGU G 87
H16D(+12 - 11) GUA UCA CUA ACC UGU GCU GUA C 88 H19A(+35 + 53) CUG
CUG GCA UCU UGC AGU U 89 H19A(+35 + 65) GCC UGA GCU GAU CUG CUG GCA
UCU UGC AGU U 90 H20A(+44 + 71) CUG GCA GAA UUC GAU CCA CCG GCU GUU
C 91 H20A(+147 + 168) CAG CAG UAG UUG UCA UCU GCU C 92 H20A(+185 +
203) UGA UGG GGU GGU GGG UUG G 93 H20A(-08 + 17) AUC UGC AUU AAC
ACC CUC UAG AAA G 94 H20A(+30 + 53) CCG GCU GUU CAG UUG UUC UGA GGC
95 H20A(-11 + 17) AUC UGC AUU AAC ACC CUC UAG AAA GAA A 96 H20D(+08
- 20) GAA GGA GAA GAG AUU CUU ACC UUA CAA A 97 H20A(+44 + 63) AUU
CGA UCC ACC GGC UGU UC 98 H20A(+149 + 168) CAG CAG UAG UUG UCA UCU
GC 99 H21A(-06 + 16) GCC GGU UGA CUU CAU CCU GUG C 100 H21A(+85 +
106) CUG CAU CCA GGA ACA UGG GUC C 101 H21A(+85 + 108) GUC UGC AUC
CAG GAA CAU GGG UC 102 H21A(+08 + 31) GUU GAA GAU CUG AUA GCC GGU
UGA 103 H21D(+18 - 07) UAC UUA CUG UCU GUA GCU CUU UCU 104 H22A(+22
+ 45) CAC UCA UGG UCU CCU GAU AGC GCA 105 H22A(+125 + 146) CUG CAA
UUC CCC GAG UCU CUG C 106 H22A(+47 + 69) ACU GCU GGA CCC AUG UCC
UGA UG 107 H22A(+80 + 101) CUA AGU UGA GGU AUG GAG AGU 108 H22D(+13
- 11) UAU UCA CAG ACC UGC AAU UCC CC 109 H23A(+34 + 59) ACA GUG GUG
CUG AGA UAG UAU AGG CC 110 H23A(+18 + 39) UAG GCC ACU UUG UUG CUC
UUG C 111 H23A(+72 + 90) UUC AGA GGG CGC UUU CUU C 112 H24A(+48 +
70) GGG CAG GCC AUU CCU CCU UCA GA 113 H24A(-02 + 22) UCU UCA GGG
UUU GUA UGU GAU UCU 114 H25A(+9 + 36) CUG GGC UGA AUU GUC UGA AUA
UCA CUG 115 H25A(+131 + 156) CUG UUG GCA CAU GUG AUC CCA CUG AG 116
H25D(+16 - 08) GUC UAU ACC UGU UGG CAC AUG UGA 117 H26A(+132 + 156)
UGC UUU CUG UAA UUC AUC UGG AGU U 118 H26A(-07 + 19) CCU CCU UUC
UGG CAU AGA CCU UCC AC 119 H26A(+68 + 92) UGU GUC AUC CAU UCG UGC
AUC UCU G 120 H27A(+82 + 106) UUA AGG CCU CUU GUG CUA CAG GUG G 121
H27A(-4 + 19) GGG GCU CUU CUU UAG CUC UCU GA 122 H27D(+19 - 03) GAC
UUC CAA AGU CUU GCA UUU C 123 H28A(-05 + 19) GCC AAC AUG CCC AAA
CUU CCU AAG 124 H28A(+99 + 124) CAG AGA UUU CCU CAG CUC CGC CAG GA
125 H28D(+16 - 05) CUU ACA UCU AGC ACC UCA GAG 126 H29A(+57 + 81)
UCC GCC AUC UGU UAG GGU CUG UGC C 127 H29A(+18 + 42) AUU UGG GUU
AUC CUC UGA AUG UCG C 128 H29D(+17 - 05) CAU ACC UCU UCA UGU AGU
UCC C 129 H30A(+122 + 147) CAU UUG AGC UGC GUC CAC CUU GUC UG 130
H30A(+25 + 50) UCC UGG GCA GAC UGG AUG CUC UGU UC 131 H30D(+19 -
04) UUG CCU GGG CUU CCU GAG GCA UU 132 H31D(+06 - 18) UUC UGA AAU
AAC AUA UAC CUG UGC 133 H31D(+03 - 22) UAG UUU CUG AAA UAA CAU AUA
CCU G 134 H31A(+05 + 25) GAC UUG UCA AAU CAG AUU GGA 135 H31D(+04 -
20) GUU UCU GAA AUA ACA UAU ACC UGU 136 H32D(+04 - 16) CAC CAG AAA
UAC AUA CCA CA 137 H32A(+151 + 170) CAA UGA UUU AGC UGU GAC UG 138
H32A(+10 + 32) CGA AAC UUC AUG GAG ACA UCU UG 139 H32A(+49 + 73)
CUU GUA GAC GCU GCU CAA AAU UGG C 140 H33D(+09 - 11) CAU GCA CAC
ACC UUU GCU CC 141 H33A(+53 + 76) UCU GUA CAA UCU GAC GUC CAG UCU
142 H33A(+30 + 56) GUC UUU AUC ACC AUU UCC ACU UCA GAC 143 H33A(+64
+ 88) CCG UCU GCU UUU UCU GUA CAA UCU G 144 H34A(+83 + 104) UCC AUA
UCU GUA GCU GCC AGC C 145 H34A(+143 + 165) CCA GGC AAC UUC AGA AUC
CAA AU 146 H34A(-20 + 10) UUU CUG UUA CCU GAA AAG AAU UAU AAU GAA
147 H34A(+46 + 70) CAU UCA UUU CCU UUC GCA UCU UAC G 148 H34A(+95 +
120) UGA UCU CUU UGU CAA UUC CAU AUC UG 149 H34D(+10 - 20) UUC AGU
GAU AUA GGU UUU ACC UUU CCC CAG 150 H34A(+72 + 96) CUG UAG CUG CCA
GCC AUU CUG UCA AG 151 H35A(+141 + 161) UCU UCU GCU CGG GAG GUG ACA
152 H35A(+116 + 135) CCA GUU ACU AUU CAG AAG AC 153 H35A(+24 + 43)
UCU UCA GGU GCA CCU UCU GU 154 H36A(+26 + 50) UGU GAU GUG GUC CAC
AUU CUG GUC A 155 H36A(-02 + 18) CCA UGU GUU UCU GGU AUU CC 156
H37A(+26 + 50) CGU GUA GAG UCC ACC UUU GGG CGU A 157 H37A(+82 +
105) UAC UAA UUU CCU GCA GUG GUC ACC 158 H37A(+134 + 157) UUC UGU
GUG AAA UGG CUG CAA AUC 159 H38A(-01 + 19) CCU UCA AAG GAA UGG AGG
CC 160 H38A(+59 + 83) UGC UGA AUU UCA GCC UCC AGU GGU U 161
H38A(+88 + 112) UGA AGU CUU CCU CUU UCA GAU UCA C 162 H39A(+62 +
85) CUG GCU UUC UCU CAU CUG UGA UUC 163 H39A(+39 + 58) GUU GUA AGU
UGU CUC CUC UU 164 H39A(+102 + 121) UUG UCU GUA ACA GCU GCU GU 165
H39D(+10 - 10) GCU CUA AUA CCU UGA GAG CA 166 H40A(-05 + 17) CUU
UGA GAC CUC AAA UCC UGU U 167 H40A(+129 + 153) CUU UAU UUU CCU UUC
AUC UCU GGG C 168 H42A(-04 + 23) AUC GUU UCU UCA CGG ACA GUG UGC
UGG 169 H42A(+86 + 109) GGG CUU GUG AGA CAU GAG UGA UUU 170
H42D(+19 - 02) A CCU UCA GAG GAC UCC UCU UGC 171 H43D(+10 - 15) UAU
GUG UUA CCU ACC CUU GUC GGU C 172 H43A(+101 + 120) GGA GAG AGC UUC
CUG UAG CU 173 H43A(+78 + 100) UCA CCC UUU CCA CAG GCG UUG CA 174
H44A(+85 + 104) UUU GUG UCU UUC UGA GAA AC 175 H44D(+10 - 10) AAA
GAC UUA CCU UAA GAU AC 176 H44A(-06 + 14) AUC UGU CAA AUC GCC UGC
AG 177 H46D(+16 - 04) UUA CCU UGA CUU GCU CAA GC 178 H46A(+90 +
109) UCC AGG UUC AAG UGG GAU AC 179 H47A(+76 + 100) GCU CUU CUG GGC
UUA UGG GAG CAC U 180 H47D(+25 - 02) ACC UUU AUC CAC UGG AGA UUU
GUC UGC 181 H47A(-9 + 12) UUC CAC CAG UAA CUG AAA CAG 182 H50A(+02
+ 30) CCA CUC AGA GCU CAG AUC UUC UAA CUU CC 183 H50A(+07 + 33) CUU
CCA CUC AGA GCU CAG AUC UUC UAA 184 H51A(-01 + 25) ACC AGA GUA ACA
GUC UGA GUA GGA GC 185 H51D(+16 - 07) CUC AUA CCU UCU GCU UGA UGA
UC 186 H51A(+111 + 134) UUC UGU CCA AGC CCG GUU GAA AUC 187
H51A(+66 + 95) CUC CAA CAU CAA GGA AGA UGG CAU UUC UAG 188 H51D(+08
- 17) AUC AUU UUU UCU CAU ACC UUC UGC U 189 H51A/D(+08 - 17) AUC
AUU UUU UCU CAU ACC UUC UGC UAG & (-15 +) GAG CUA AAA 190
H51A(+175 + 195) CAC CCA CCA UCA CCC UCU GUG 191 H51A(+199 + 220)
AUC AUC UCG UUG AUA UCC UCA A 192 H52A(-07 + 14) UCC UGC AUU GUU
GCC UGU AAG 193 H52A(+12 + 41) UCC AAC UGG GGA CGC CUC UGU UCC AAA
UCC 194 H52A(+17 + 37) ACU GGG GAC GCC UCU GUU CCA 195 H52A(+93 +
112) CCG UAA UGA UUG UUC UAG CC 196 H52D(+05 - 15) UGU UAA AAA ACU
UAC UUC GA 197 H53A(+45 + 69) CAU UCA ACU GUU GCC UCC GGU UCU G 198
H53A(+39 + 62) CUG UUG CCU CCG GUU CUG AAG GUG 199 H53A(+39 + 69)
CAU UCA ACU GUU GCC UCC GGU UCU GAA GGU G 200 H53D(+14 - 07) UAC
UAA CCU UGG UUU CUG UGA 201 H53A(+23 + 47) CUG AAG GUG UUC UUG UAC
UUC AUC C 202 H53A(+150 + 176) UGU AUA GGG ACC CUC CUU CCA UGA CUC
203 H53D(+20 - 05) CUA ACC UUG GUU UCU GUG AUU UUC U 204 H53D(+09 -
18) GGU AUC UUU GAU ACU AAC CUU GGU UUC 205 H53A(-12 + 10) AUU CUU
UCA ACU AGA AUA AAA G 206 H53A(-07 + 18) GAU UCU GAA UUC UUU CAA
CUA GAA U 207 H53A(+07 + 26) AUC CCA CUG AUU CUG AAU UC 208
H53A(+124 + 145) UUG GCU CUG GCC UGU CCU AAG A 209 H46A(+86 + 115)
CUC UUU UCC AGG UUC AAG UGG GAU ACU AGC 210 H46A(+107 + 137) CAA
GCU UUU CUU UUA GUU GCU GCU CUU UUC C 211 H46A(-10 + 20) UAU UCU
UUU GUU CUU CUA GCC UGG AGA AAG 212 H46A(+50 + 77) CUG CUU CCU CCA
ACC AUA AAA CAA AUU C 213 H45A(-06 + 20) CCA AUG CCA UCC UGG AGU
UCC UGU AA 214 H45A(+91 + 110) UCC UGU AGA AUA CUG GCA UC 215
H45A(+125 + 151) UGC AGA CCU CCU GCC ACC GCA GAU UCA 216 H45D(+16 -
04) CUA CCU CUU UUU UCU GUC UG 217 H45A(+71 + 90) UGU UUU UGA GGA
UUG CUG AA Description Sequence SEQ ID NO H53A(+33 + 60)
GTTGCCTCCGGTTCTGAAGGTGTTCTTG 218 H53A(+23 + 47)
CTGAAGGTGTTCTTGTACTTCATCC 219 H53A(+33 + 62)
CTGTTGCCTCCGGTTCTGAAGGTGTTCTTG 220 H53A(+33 + 65)
CAACTGTTGCCTCCGGTTCTGAAGGTGTTCTTG 221 H53A(+31 + 55)
CTCCGGTTCTGAAGGTGTTCTTGTA 222 H53A(+46 + 73)
ATTTCATTCAACTGTTGCCTCCGGTTCT 223 H53A(+22 + 46)
TGAAGGTGTTCTTGTACTTCATCCC 224
H53A(+46 + 69) CATTCAACTGTTGCCTCCGGTTCT 225 H53A(+40 + 61)
TGTTGCCTCCGGTTCTGAAGGT 226 H53A(+30 + 60) GTTGCCTCCGGTTCTGAAGGTGTTC
227 H53A(+30 + 57) GCCTCCGGTTCTGAAGGTGTTCTTGTAC 228 H53A(+30 + 56)
CCTCCGGTTCTGAAGGTGTTCTTGTAC 229 H53A(+30 + 55)
CTCCGGTTCTGAAGGTGTTCTTGTAC 230 H53A(+33 + 57)
GCCTCCGGTTCTGAAGGTGTTCTTG 231 H44A(-07 + 17)
CAGATCTGTCAAATCGCCTGCAGG 232 H44A(-07 + 20)
CAACAGATCTGTCAAATCGCCTGCAGG 233 H44A(-07 + 22)
CTCAACAGATCTGTCAAATCGCCTGCAGG 234 H44A(+77 + 101)
GTGTCTTTCTGAGAAACTGTTCAGC 235 H44A(+64 + 91)
GAGAAACTGTTCAGCTTCTGTTAGCCAC 236 H44A(+62 + 89)
GAAACTGTTCAGCTTCTGTTAGCCACTG 237 H44A(+62 + 85)
CTGTTCAGCTTCTGTTAGCCACTG 238 H44A(-06 + 14) ATCTGTCAAATCGCCTGCAG
239 H44A(+85 + 104) TTTGTGTCTTTCTGAGAAAC 240 H44A(+61 + 84)
TGTTCAGCTTCTGTTAGCCACTGA 241 H44A(-10 + 15)
GATCTGTCAAATCGCCTGCAGGTAA 242 H44A(+64 + 88)
AAACTGTTCAGCTTCTGTTAGCCAC 243 H44A(+79 + 103)
TTGTGTCTTTCTGAGAAACTGTTCA 244 H44A(-06 + 20)
CAACAGATCTGTCAAATCGCCTGCAG 245 H44A(-09 + 17)
CAGATCTGTCAAATCGCCTGCAGGTA 246 H44A(+59 + 85)
CTGTTCAGCTTCTGTTAGCCACTGATT 247 H44A(+59 + 89)
GAAACTGTTCAGCTTCTGTTAGCCACTGATT 248 H44A(+65 + 90)
AGAAACTGTTCAGCTTCTGTTAGCCA 249 Name Sequences SEQ ID NO. Oligomer
Targeting Sequences (5'-3'): Hu.DMD.Exon44.25.001
CTGCAGGTAAAAGCATATGGATCAA 250 Hu.DMD.Exon44.25.002
ATCGCCTGCAGGTAAAAGCATATGG 251 Hu.DMD.Exon44.25.003
GTCAAATCGCCTGCAGGTAAAAGCA 252 Hu.DMD.Exon44.25.005
CAACAGATCTGTCAAATCGCCTGCA 253 Hu.DMD.Exon44.25.006
TTTCTCAACAGATCTGTCAAATCGC 254 Hu.DMD.Exon44.25.007
CCATTTCTCAACAGATCTGTCAAAT 255 Hu.DMD.Exon44.25.008
ATAATGAAAACGCCGCCATTTCTCA 256 Hu.DMD.Exon44.25.009
AAATATCTTTATATCATAATGAAAA 257 Hu.DMD.Exon44.25.010
TGTTAGCCACTGATTAAATATCTTT 258 Hu.DMD.Exon44.25.013
CCAATTCTCAGGAATTTGTGTCTTT 259 Hu.DMD.Exon44.25.014
GTATTTAGCATGTTCCCAATTCTCA 260 Hu.DMD.Exon44.25.015
CTTAAGATACCATTTGTATTTAGCA 261 Hu.DMD.Exon44.25.016
CTTACCTTAAGATACCATTTGTATT 262 Hu.DMD.Exon44.25.017
AAAGACTTACCTTAAGATACCATTT 263 Hu.DMD.Exon44.25.018
AAATCAAAGACTTACCTTAAGATAC 264 Hu.DMD.Exon44.25.019
AAAACAAATCAAAGACTTACCTTAA 265 Hu.DMD.Exon44.25.020
TCGAAAAAACAAATCAAAGACTTAC 266 Hu.DMD.Exon45.25.001
CTGTAAGATACCAAAAAGGCAAAAC 267 Hu.DMD.Exon45.25.002
CCTGTAAGATACCAAAAAGGCAAAA 268 Hu.DMD.Exon45.25.002.2
AGTICCIGTAAGATACCAAAAAGGC 269 Hu.DMD.Exon45.25.003
GAGTTCCTGTAAGATACCAAAAAGG 270 Hu.DMD.Exon45.25.003.2
CCTGGAGTTCCTGTAAGATACCAAA 271 Hu.DMD.Exon45.25.004
TCCTGGAGTTCCTGTAAGATACCAA 272 Hu.DMD.Exon45.25.004.2
GCCATCCTGGAGTTCCTGTAAGATA 273 Hu.DMD.Exon45.25.005
TGCCATCCTGGAGTTCCTGTAAGAT 274 Hu.DMD.Exon45.25.005.2
CCAATGCCATCCTGGAGTTCCTGTA 275 Hu.DMD.Exon45.25.006
CCCAATGCCATCCTGGAGTTCCTGT 276 Hu.DMD.Exon45.25.006.2
GCTGCCCAATGCCATCCTGGAGTTC 277 Hu.DMD.Exon45.25.007
CGCTGCCCAATGCCATCCTGGAGTT 278 Hu.DMD.Exon45.25.008
AACAGTTTGCCGCTGCCCAATGCCA 279 Hu.DMD.Exon45.25.008.2
CTGACAACAGTTTGCCGCTGCCCAA 280 Hu.DMD.Exon45.25.009
GTTGCATTCAATGTTCTGACAACAG 281 Hu.DMD.Exon45.25.010
GCTGAATTATTTCTTCCCCAGTTGC 282 Hu.DMD.Exon45.25.010.2
ATTATTTCTTCCCCAGTTGCATTCA 283 Hu.DMD.Exon45.25.011
GGCATCTGTTTTTGAGGATTGCTGA 284 Hu.DMD.Exon45.25.011.2
TTTGAGGATTGCTGAATTATTTCTT 285 Hu.DMD.Exon45.25.012
AATTTTTCCTGTAGAATACTGGCAT 286 Hu.DMD.Exon45.25.012.2
ATACTGGCATCTGTTTTTGAGGATT 287 Hu.DMD.Exon45.25.013
ACCGCAGATTCAGGCTTCCCAATTT 288 Hu.DMD.Exon45.25.014
CTGTTTGCAGACCTCCTGCCACCGC 289 Hu.DMD.Exon45.25.014.2
AGATTCAGGCTTCCCAATTTTTCCT 290 Hu.DMD.Exon45.25.015
CTCTTTTTTCTGTCTGACAGCTGTT 291 Hu.DMD.Exon45.25.015.2
ACCTCCTGCCACCGCAGATTCAGGC 292 Hu.DMD.Exon45.25.016
CCTACCTCTTTTTTCTGTCTGACAG 293 Hu.DMD.Exon45.25.016.2
GACAGCTGTTTGCAGACCTCCTGCC 294 Hu.DMD.Exon45.25.017
GTCGCCCTACCTCTTTTTTCTGTCT 295 Hu.DMD.Exon45.25.018
GATCTGTCGCCCTACCTCTTTTTTC 296 Hu.DMD.Exon45.25.019
TATTAGATCTGTCGCCCTACCTCTT 297 Hu.DMD.Exon45.25.020
ATTCCTATTAGATCTGTCGCCCTAC 298 Hu.DMD.Exon45.20.001
AGATACCAAAAAGGCAAAAC 299 Hu.DMD.Exon45.20.002 AAGATACCAAAAAGGCAAAA
300 Hu.DMD.Exon45.20.003 CCTGTAAGATACCAAAAAGG 301
Hu.DMD.Exon45.20.004 GAGTTCCTGTAAGATACCAA 302 Hu.DMD.Exon45.20.005
TCCTGGAGTTCCTGTAAGAT 303 Hu.DMD.Exon45.20.006 TGCCATCCTGGAGTTCCTGT
304 Hu.DMD.Exon45.20.007 CCCAATGCCATCCTGGAGTT 305
Hu.DMD.Exon45.20.008 CGCTGCCCAATGCCATCCTG 306 Hu.DMD.Exon45.20.009
CTGACAACAGTTTGCCGCTG 307 Hu.DMD.Exon45.20.010 GTTGCATTCAATGTTCTGAC
308 Hu.DMD.Exon45.20.011 ATTATTTCTTCCCCAGTTGC 309
Hu.DMD.Exon45.20.012 TTTGAGGATTGCTGAATTAT 310 Hu.DMD.Exon45.20.013
ATACTGGCATCTGTTTTTGA 311 Hu.DMD.Exon45.20.014 AATTTTTCCTGTAGAATACT
312 Hu.DMD.Exon45.20.015 AGATTCAGGCTTCCCAATTT 313
Hu.DMD.Exon45.20.016 ACCTCCTGCCACCGCAGATT 314 Hu.DMD.Exon45.20.017
GACAGCTGTTTGCAGACCTC 315 Hu.DMD.Exon45.20.018 CTCTTTTTTCTGTCTGACAG
316 Hu.DMD.Exon45.20.019 CCTACCTCTTTTTTCTGTCT 317
Hu.DMD.Exon45.20.020 GTCGCCCTACCTCTTTTTTC 318 Hu.DMD.Exon45.20.021
GATCTGTCGCCCTACCTCTT 319 Hu.DMD.Exon45.20.022 TATTAGATCTGTCGCCCTAC
320 Hu.DMD.Exon45.20.023 ATTCCTATTAGATCTGTCGC 321
Hu.DMD.Exon46.25.001 GGGGGATTTGAGAAAATAAAATTAC 322
Hu.DMD.Exon46.25.002 ATTTGAGAAAATAAAATTACCTTGA 323
Hu.DMD.Exon46.25.002.2 CTAGCCTGGAGAAAGAAGAATAAAA 324
Hu.DMD.Exon46.25.003 AGAAAATAAAATTACCTTGACTTGC 325
Hu.DMD.Exon46.25.003.2 TTCTTCTAGCCTGGAGAAAGAAGAA 326
Hu.DMD.Exon46.25.004 ATAAAATTACCTTGACTTGCTCAAG 327
Hu.DMD.Exon46.25.004.2 TTTTGTTCTTCTAGCCTGGAGAAAG 328
Hu.DMD.Exon46.25.005 ATTACCTTGACTTGCTCAAGCTTTT 329
Hu.DMD.Exon46.25.005.2 TATTCTTTTGTTCTTCTAGCCTGGA 330
Hu.DMD.Exon46.25.006 CTTGACTTGCTCAAGCTTTTCTTTT 331
Hu.DMD.Exon46.25.006.2 CAAGATATTCTTTTGTTCTTCTAGC 332
Hu.DMD.Exon46.25.007 CiTTTAGTTGCTGCTCTiiiCCAGG 333
Hu.DMD.Exon46.25.008 CCAGGTTCAAGTGGGATACTAGCAA 334
Hu.DMD.Exon46.25.008.2 ATCTCTTTGAAATTCTGACAAGATA 335
Hu.DMD.Exon46.25.009 AGCAATGTTATCTGCTTCCTCCAAC 336
Hu.DMD.Exon46.25.009.2 AACAAATTCATTTAAATCTCTTTGA 337
Hu.DMD.Exon46.25.010 CCAACCATAAAACAAATTCATTTAA 338
Hu.DMD.Exon46.25.010.2 TTCCTCCAACCATAAAACAAATTCA 339
Hu.DMD.Exon46.25.011 TTTAAATCTCTTTGAAATTCTGACA 340
Hu.DMD.Exon46.25.012 TGACAAGATATTCTTTTGTTCTTCT 341
Hu.DMD.Exon46.25.012.2 TTCAAGTGGGATACTAGCAATGTTA 342
Hu.DMD.Exon46.25.013 AGATATTCTTTTGTTCTTCTAGCCT 343
Hu.DMD.Exon46.25.013.2 CTGCTCT+32CCAGGTTCAAGTGGG 344
Hu.DMD.Exon46.25.014 TTCTTTTGTTCTTCTAGCCTGGAGA 345
Hu.DMD.Exon46.25.014.2 CTTTTCTTTTAGTTGCTGCTCTTTT 346
Hu.DMD.Exon46.25.015 TTGTTCTTCTAGCCTGGAGAAAGAA 347
Hu.DMD.Exon46.25.016 CTTCTAGCCTGGAGAAAGAAGAATA 348
Hu.DMD.Exon46.25.017 AGCCTGGAGAAAGAAGAATAAAATT 349
Hu.DMD.Exon46.25.018 CTGGAGAAAGAAGAATAAAATTGTT 350
Hu.DMD.Exon46.20.001 GAAAGAAGAATAAAATTGTT 351 Hu.DMD.Exon46.20.002
GGAGAAAGAAGAATAAAATT 352 Hu.DMD.Exon46.20.003 AGCCTGGAGAAAGAAGAATA
353 Hu.DMD.Exon46.20.004 CTTCTAGCCTGGAGAAAGAA 354
Hu.DMD.Exon46.20.005 TTGTTCTTCTAGCCTGGAGA 355 Hu.DMD.Exon46.20.006
TTCTTTTGTTCTTCTAGCCT 356 Hu.DMD.Exon46.20.007 TGACAAGATATTCTTTTGTT
357 Hu.DMD.Exon46.20.008 ATCTCTTTGAAATTCTGACA 358
Hu.DMD.Exon46.20.009 AACAAATTCATTTAAATCTC 359 Hu.DMD.Exon46.20.010
TTCCTCCAACCATAAAACAA 360 Hu.DMD.Exon46.20.011 AGCAATGTTATCTGCTTCCT
361 Hu.DMD.Exon46.20.012 TTCAAGTGGGATACTAGCAA 362
Hu.DMD.Exon46.20.013 CTGCTCTTTTCCAGGTTCAA 363 Hu.DMD.Exon46.20.014
CTTTTCTTTTAGTTGCTGCT 364 Hu.DMD.Exon46.20.015 CTTGACTTGCTCAAGCTTTT
365 Hu.DMD.Exon46.20.016 ATTACCTTGACTTGCTCAAG 366
Hu.DMD.Exon46.20.017 ATAAAATTACCTTGACTTGC 367 Hu.DMD.Exon46.20.018
AGAAAATAAAATTACCTTGA 368 Hu.DMD.Exon46.20.019 ATTTGAGAAAATAAAATTAC
369 Hu.DMD.Exon46.20.020 GGGGGATTTGAGAAAATAAA 370
Hu.DMD.Exon47.25.001 CTGAAACAGACAAATGCAACAACGT 371
Hu.DMD.Exon47.25.002 AGTAACTGAAACAGACAAATGCAAC 372
Hu.DMD.Exon47.25.003 CCACCAGTAACTGAAACAGACAAAT 373
Hu.DMD.Exon47.25.004 CTCTTCCACCAGTAACTGAAACAGA 374
Hu.DMD.Exon47.25.005 GGCAACTCTTCCACCAGTAACTGAA 375
Hu.DMD.Exon47.25.006 GCAGGGGCAACTCTTCCACCAGTAA 376
Hu.DMD.Exon47.25.007 CTGGCGCAGGGGCAACTCTTCCACC 377
Hu.DMD.Exon47.25.008 TTTAATTGTTTGAGAATTCCCTGGC 378
Hu.DMD.Exon47.25.008.2 TTGTTTGAGAATTCCCTGGCGCAGG 379
Hu.DMD.Exon47.25.009 GCACGGGTCCTCCAGTTTCATTTAA 380
Hu.DMD.Exon47.25.009.2 TCCAGTTTCATTTAATTGTTTGAGA 381
Hu.DMD.Exon47.25.010 GCTTATGGGAGCACTTACAAGCACG 382
Hu.DMD.Exon47.25.010.2 TACAAGCACGGGTCCTCCAGTTTCA 383
Hu.DMD.Exon47.25.011 AGTTTATCTTGCTCTTCTGGGCTTA 384
Hu.DMD.Exon47.25.012 TCTGCTTGAGCTTATTTTCAAGTTT 385
Hu.DMD.Exon47.25.012.2 ATCTTGCTCTTCTGGGCTTATGGGA 386
Hu.DMD.Exon47.25.013 CTTTATCCACTGGAGATTTGTCTGC 387
Hu.DMD.Exon47.25.013.2 CTTATTTTCAAGTTTATCTTGCTCT 388
Hu.DMD.Exon47.25.014 CTAACCTTTATCCACTGGAGATTTG 389
Hu.DMD.Exon47.25.014.2 ATTTGTCTGCTTGAGCTTATTTTCA 390
Hu.DMD.Exon47.25.015 AATGTCTAACCTTTATCCACTGGAG 391
Hu.DMD.Exon47.25.016 TGGTTAATGTCTAACCTTTATCCAC 392
Hu.DMD.Exon47.25.017 AGAGATGGTTAATGTCTAACCTTTA 393
Hu.DMD.Exon47.25.018 ACGGAAGAGATGGTTAATGTCTAAC 394
Hu.DMD.Exon47.20.001 ACAGACAAATGCAACAACGT 395 Hu.DMD.Exon47.20.002
CTGAAACAGACAAATGCAAC 396 Hu.DMD.Exon47.20.003 AGTAACTGAAACAGACAAAT
397 Hu.DMD.Exon47.20.004 CCACCAGTAACTGAAACAGA 398
Hu.DMD.Exon47.20.005 CTCTTCCACCAGTAACTGAA 399 Hu.DMD.Exon47.20.006
GGCAACTCTTCCACCAGTAA 400 Hu.DMD.Exon47.20.007 CTGGCGCAGGGGCAACTCTT
401 Hu.DMD.Exon47.20.008 TTGTTTGAGAATTCCCTGGC 402
Hu.DMD.Exon47.20.009 TCCAGTTTCATTTAATTGTT 403 Hu.DMD.Exon47.20.010
TACAAGCACGGGTCCTCCAG 404 Hu.DMD.Exon47.20.011 GCTTATGGGAGCACTTACAA
405 Hu.DMD.Exon47.20.012 ATCTTGCTCTTCTGGGCTTA 406
Hu.DMD.Exon47.20.013 CTTATTTTCAAGTTTATCTT 407 Hu.DMD.Exon47.20.014
ATTTGTCTGCTTGAGCTTAT 408 Hu.DMD.Exon47.20.015 CTTTATCCACTGGAGATTTG
409 Hu.DMD.Exon47.20.016 CTAACCTTTATCCACTGGAG 410
Hu.DMD.Exon47.20.017 AATGTCTAACCTTTATCCAC 411 Hu.DMD.Exon47.20.018
TGGTTAATGTCTAACCTTTA 412 Hu.DMD.Exon47.20.019 AGAGATGGTTAATGTCTAAC
413 Hu.DMD.Exon47.20.020 ACGGAAGAGATGGTTAATGT 414
Hu.DMD.Exon48.25.001 CTGAAAGGAAAATACATTTTAAAAA 415
Hu.DMD.Exon48.25.002 CCTGAAAGGAAAATACATTTTAAAA 416
Hu.DMD.Exon48.25.002.2 GAAACCTGAAAGGAAAATACATITT 417
Hu.DMD.Exon48.25.003 GGAAACCTGAAAGGAAAATACATTT 418
Hu.DMD.Exon48.25.003.2 CTCTGGAAACCTGAAAGGAAAATAC 419
Hu.DMD.Exon48.25.004 GCTCTGGAAACCTGAAAGGAAAATA 420
Hu.DMD.Exon48.25.004.2 TAAAGCTCTGGAAACCTGAAAGGAA 421
Hu.DMD.Exon48.25.005 GTAAAGCTCTGGAAACCTGAAAGGA 422
Hu.DMD.Exon48.25.005.2 TCAGGTAAAGCTCTGGAAACCTGAA 423
Hu.DMD.Exon48.25.006 CTCAGGTAAAGCTCTGGAAACCTGA 424
Hu.DMD.Exon48.25.006.2 GTTTCTCAGGTAAAGCTCTGGAAAC 425
Hu.DMD.Exon48.25.007 TGTTTCTCAGGTAAAGCTCTGGAAA 426
Hu.DMD.Exon48.25.007.2 AATTTCTCCTTGTTTCTCAGGTAAA 427
Hu.DMD.Exon48.25.008 TTTGAGCTTCAATTTCTCCTTGTTT 428
Hu.DMD.Exon48.25.008 TTTTATTTGAGCTTCAATTTCTCCT 429
Hu.DMD.Exon48.25.009 AAGCTGCCCAAGGTCTTTTATTTGA 430
Hu.DMD.Exon48.25.010 AGGTCTTCAAGCTTTTTTTCAAGCT 431
Hu.DMD.Exon48.25.010.2 TTCAAGCTTTTTTTCAAGCTGCCCA 432
Hu.DMD.Exon48.25.011 GATGATTTAACTGCTCTTCAAGGTC 433
Hu.DMD.Exon48.25.011.2 CTGCTCTTCAAGGTCTTCAAGCTTT 434
Hu.DMD.Exon48.25.012 AGGAGATAACCACAGCAGCAGATGA 435
Hu.DMD.Exon48.25.012.2 CAGCAGATGATTTAACTGCTCTTCA 436
Hu.DMD.Exon48.25.013 ATTTCCAACTGATTCCTAATAGGAG 437
Hu.DMD.Exon48.25.014 CTTGGTTTGGTTGGTTATAAATTTC 438
Hu.DMD.Exon48.25.014.2 CAACTGATTCCTAATAGGAGATAAC 439
Hu.DMD.Exon48.25.015 CTTAACGTCAAATGGTCCTTCTTGG 440
Hu.DMD.Exon48.25.015.2 TTGGTTATAAATTTCCAACTGATTC 441
Hu.DMD.Exon48.25.016 CCTACCTTAACGTCAAATGGTCCTT 442
Hu.DMD.Exon48.25.016.2 TCCTTCTTGGTTTGGTTGGTTATAA 443
Hu.DMD.Exon48.25.017 AGTTCCCTACCTTAACGTCAAATGG 444
Hu.DMD.Exon48.25.018 CAAAAAGTTCCCTACCTTAACGTCA 445
Hu.DMD.Exon48.25.019 TAAAGCAAAAAGTTCCCTACCTTAA 446
Hu.DMD.Exon48.25.020 ATATTTAAAGCAAAAAGTTCCCTAC 447
Hu.DMD.Exon48.20.001 AGGAAAATACATTTTAAAAA 448 Hu.DMD.Exon48.20.002
AAGGAAAATACATTTTAAAA 449 Hu.DMD.Exon48.20.003 CCTGAAAGGAAAATACATTT
450 Hu.DMD.Exon48.20.004 GGAAACCTGAAAGGAAAATA 451
Hu.DMD.Exon48.20.005 GCTCTGGAAACCTGAAAGGA 452 Hu.DMD.Exon48.20.006
GTAAAGCTCTGGAAACCTGA 453 Hu.DMD.Exon48.20.007 CTCAGGTAAAGCTCTGGAAA
454 Hu.DMD.Exon48.20.008 AATTTCTCCTTGTTTCTCAG 455
Hu.DMD.Exon48.20.009 TTTTATTTGAGCTTCAATTT 456 Hu.DMD.Exon48.20.010
AAGCTGCCCAAGGTCTTTTA 457 Hu.DMD.Exon48.20.011 TTCAAGCTTTTTTTCAAGCT
458 Hu.DMD.Exon48.20.012 CTGCTCTTCAAGGTCTTCAA 459
Hu.DMD.Exon48.20.013 CAGCAGATGATTTAACTGCT 460 Hu.DMD.Exon48.20.014
AGGAGATAACCACAGCAGCA 461 Hu.DMD.Exon48.20.015 CAACTGATTCCTAATAGGAG
462 Hu.DMD.Exon48.20.016 TTGGTTATAAATTTCCAACT 463
Hu.DMD.Exon48.20.017 TCCTTCTTGGTTTGGTTGGT 464 Hu.DMD.Exon48.20.018
CTTAACGTCAAATGGTCCTT 465 Hu.DMD.Exon48.20.019 CCTACCTTAACGTCAAATGG
466 Hu.DMD.Exon48.20.020 AGTTCCCTACCTTAACGTCA 467
Hu.DMD.Exon48.20.021 CAAAAAGTTCCCTACCTTAA 468 Hu.DMD.Exon48.20.022
TAAAGCAAAAAGTTCCCTAC 469 Hu.DMD.Exon48.20.023 ATATTTAAAGCAAAAAGTTC
470 Hu.DMD.Exon49.25.001 CTGGGGAAAAGAACCCATATAGTGC 471
Hu.DMD.Exon49.25.002 TCCTGGGGAAAAGAACCCATATAGT 472
Hu.DMD.Exon49.25.002.2 GTTTCCTGGGGAAAAGAACCCATAT 473
Hu.DMD.Exon49.25.003 CAGTTTCCTGGGGAAAAGAACCCAT 474
Hu.DMD.Exon49.25.003.2 TTTCAGTTTCCTGGGGAAAAGAACC 475
Hu.DMD.Exon49.25.004 TATTTCAGTTTCCTGGGGAAAAGAA 476
Hu.DMD.Exon49.25.004.2 TGCTATTTCAGTTTCCTGGGGAAAA 477
Hu.DMD.Exon49.25.005 ACTGCTATTTCAGTTTCCTGGGGAA 478
Hu.DMD.Exon49.25.005.2 TGAACTGCTATTTCAGTTTCCTGGG 479
Hu.DMD.Exon49.25.006 CTTGAACTGCTATTTCAGTTTCCTG 480
Hu.DMD.Exon49.25.006.2 TAGCTTGAACTGCTATTTCAGTTTC 481
Hu.DMD.Exon49.25.007 TTTAGCTTGAACTGCTATTTCAGTT 482
Hu.DMD.Exon49.25.008 TTCCACATCCGGTTGTTTAGCTTGA 483
Hu.DMD.Exon49.25.009 TGCCCTTTAGACAAAATCTCTTCCA 484
Hu.DMD.Exon49.25.009.2 TTTAGACAAAATCTCTTCCACATCC 485
Hu.DMD.Exon49.25.010 GTTTTTCCTTGTACAAATGCTGCCC 486
Hu.DMD.Exon49.25.010.2 GTACAAATGCTGCCCTTTAGACAAA 487
Hu.DMD.Exon49.25.011 CTTCACTGGCTGAGTGGCTGGTTTT 488
Hu.DMD.Exon49.25.011.2 GGCTGGTTTTTCCTTGTACAAATGC 489
Hu.DMD.Exon49.25.012 ATTACCTTCACTGGCTGAGTGGCTG 490
Hu.DMD.Exon49.25.013 GCTTCATTACCTTCACTGGCTGAGT 491
Hu.DMD.Exon49.25.014 AGGTTGCTTCATTACCTTCACTGGC 492
Hu.DMD.Exon49.25.015 GCTAGAGGTTGCTTCATTACCTTCA 493
Hu.DMD.Exon49.25.016 ATATTGCTAGAGGTTGCTTCATTAC 494
Hu.DMD.Exon49.20.001 GAAAAGAACCCATATAGTGC 495 Hu.DMD.Exon49.20.002
GGGAAAAGAACCCATATAGT 496 Hu.DMD.Exon49.20.003 TCCTGGGGAAAAGAACCCAT
497 Hu.DMD.Exon49.20.004 CAGTTTCCTGGGGAAAAGAA 498
Hu.DMD.Exon49.20.005 TATTTCAGTTTCCTGGGGAA 499 Hu.DMD.Exon49.20.006
ACTGCTATTTCAGTTTCCTG 500 Hu.DMD.Exon49.20.007 CTTGAACTGCTATTTCAGTT
501 Hu.DMD.Exon49.20.008 TTTAGCTTGAACTGCTATTT 502
Hu.DMD.Exon49.20.009 TTCCACATCCGGTTGTTTAG 503 Hu.DMD.Exon49.20.010
TTTAGACAAAATCTCTTCCA 504 Hu.DMD.Exon49.20.011 GTACAAATGCTGCCCTTTAG
505 Hu.DMD.Exon49.20.012 GGCTGGTTTTTCCTTGTACA 506
Hu.DMD.Exon49.20.013 CTTCACTGGCTGAGTGGCTG 507 Hu.DMD.Exon49.20.014
ATTACCTTCACTGGCTGAGT 508 Hu.DMD.Exon49.20.015 GCTTCATTACCTiCACTGGC
509 Hu.DMD.Exon49.20.016 AGGTTGCTTCATTACCTTCA 510
Hu.DMD.Exon49.20.017 GCTAGAGGTTGCTTCATTAC 511 Hu.DMD.Exon49.20.018
ATATTGCTAGAGGTTGCTTC 512 Hu.DMD.Exon50.25.001
CTTTAACAGAAAAGCATACACATTA 513 Hu.DMD.Exon50.25.002
TCCTCTTTAACAGAAAAGCATACAC 514 Hu.DMD.Exon50.25.002.2
TTCCTCTTTAACAGAAAAGCATACA 515 Hu.DMD.Exon50.25.003
TAACTTCCTCTTTAACAGAAAAGCA 516 Hu.DMD.Exon50.25.003.2
CTAACTTCCTCTTTAACAGAAAAGC 517 Hu.DMD.Exon50.25.004
TCTTCTAACTTCCTCTTTAACAGAA 518 Hu.DMD.Exon50.25.004.2
ATCTTCTAACTTCCTCTTTAACAGA 519 Hu.DMD.Exon50.25.005
TCAGATCTTCTAACTTCCTCTTTAA 520 Hu.DMD.Exon50.25.005.2
CTCAGATCTTCTAACTTCCTCTTTA 521 Hu.DMD.Exon50.25.006
AGAGCTCAGATCTTCTAACTTCCTC 522 Hu.DMD.Exon50.25.006.2
CAGAGCTCAGATCTICTAACTICCT 523 NG-08-0731 Hu.DMD.Exon50.25.007
CACTCAGAGCTCAGATCTTCTACT 524 Hu.DMD.Exon50.25.007.2
CCTTCCACTCAGAGCTCAGATCTTC 525 Hu.DMD.Exon50.25.008
GTAAACGGTTTACCGCCTiCCACTC 526 Hu.DMD.Exon50.25.009
CTTTGCCCTCAGCTCTTGAAGTAAA 527 Hu.DMD.Exon50.25.009.2
CCCTCAGCTCTTGAAGTAAACGGTT 528 Hu.DMD.Exon50.25.010
CCAGGAGCTAGGTCAGGCTGCTTTG 529 Hu.DMD.Exon50.25.010.2
GGTCAGGCTGCTTTGCCCTCAGCTC 530 Hu.DMD.Exon50.25.011
AGGCTCCAATAGTGGTCAGTCCAGG 531 Hu.DMD.Exon50.25.011.2
TCAGTCCAGGAGCTAGGICAGGCTG 532 Hu.DMD.Exon50.25.012
CTTACAGGCTCCAATAGTGGTCAGT 533 AVI-5038 Hu.DMD.Exon50.25.013
GTATACTTACAGGCTCCAATAGTGG 534 Hu.DMD.Exon50.25.014
ATCCAGTATACTTACAGGCTCCAAT 535 Hu.DMD.Exon50.25.015
ATGGGATCCAGTATACTTACAGGCT 536 NG-08-0741 Hu.DMD.Exon50.25.016
AGAGAATGGGATCCAGTATACTTAC 537 NG-08-0742 Hu.DMD.Exon50.20.001
ACAGAAAAGCATACACATTA 538 Hu.DMD.Exon50.20.002 TTTAACAGAAAAGCATACAC
539 Hu.DMD.Exon50.20.003 TCCTCTTTAACAGAAAAGCA 540
Hu.DMD.Exon50.20.004 TAACTTCCTCTTTAACAGAA 541 Hu.DMD.Exon50.20.005
TCTTCTAACTTCCTCTTTAA 542 Hu.DMD.Exon50.20.006 TCAGATCTTCTAACTTCCTC
543 Hu.DMD.Exon50.20.007 CCTTCCACTCAGAGCTCAGA 544
Hu.DMD.Exon50.20.008 GTAAACGGTTTACCGCCTTC 545 Hu.DMD.Exon50.20.009
CCCTCAGCTCTTGAAGTAAA 546 Hu.DMD.Exon50.20.010 GGTCAGGCTGCTTTGCCCTC
547 Hu.DMD.Exon50.20.011 TCAGTCCAGGAGCTAGGTCA 548
Hu.DMD.Exon50.20.012 AGGCTCCAATAGTGGTCAGT 549 Hu.DMD.Exon50.20.013
CTTACAGGCTCCAATAGTGG 550 Hu.DMD.Exon50.20.014 GTATACTTACAGGCTCCAAT
551 Hu.DMD.Exon50.20.015 ATCCAGTATACTTACAGGCT 552
Hu.DMD.Exon50.20.016 ATGGGATCCAGTATACTTAC 553 Hu.DMD.Exon50.20.017
AGAGAATGGGATCCAGTATA 554 Hu.DMD.Exon51.25.001-44
CTAAAATATTTTGGGTTTTTGCAAAA 555 Hu.DMD.Exon51.25.002-45
GCTAAAATATTTTGGGTTTTTGCAAA 556 Hu.DMD.Exon51.25.002.2-46
TAGGAGCTAAAATATTTTGGGTTTTT 557 Hu.DMD.Exon51.25.003
AGTAGGAGCTAAAATATTTTGGGTT 558 Hu.DMD.Exon51.25.003.2
TGAGTAGGAGCTAAAATATITTGGG 559 Hu.DMD.Exon51.25.004
CTGAGTAGGAGCTAAAATATTTTGGG 560 Hu.DMD.Exon51.25.004.2
CAGTCTGAGTAGGAGCTAAAATATT 561 Hu.DMD.Exon51.25.005
ACAGTCTGAGTAGGAGCTAAAATATT 562 Hu.DMD.Exon51.25.005.2
GAGTAACAGTCTGAGTAGGAGCTAAA 563 Hu.DMD.Exon51.25.006
CAGAGTAACAGTCTGAGTAGGAGCT 564 Hu.DMD.Exon51.25.006.2
CACCAGAGTAACAGTCTGAGTAGGAG 565 Hu.DMD.Exon51.25.007
GTCACCAGAGTAACAGTCTGAGTAG 566 Hu.DMD.Exon51.25.007.2
AACCACAGGTTGTGTCACCAGAGTAA 567 Hu.DMD.Exon51.25.008
GTTGTGTCACCAGAGTAACAGTCTG 568 Hu.DMD.Exon51.25.009
TGGCAGTTTCCTTAGTAACCACAGGT 569 Hu.DMD.Exon51.25.010
ATTTCTAGTTTGGAGATGGCAGTTTC 570 Hu.DMD.Exon51.25.010.2
GGAAGATGGCATTTCTAGTTTGGAG 571 Hu.DMD.Exon51.25.011
CATCAAGGAAGATGGCATTTCTAGTT 572 Hu.DMD.Exon51.25.011.2
GAGCAGGTACCTCCAACATCAAGGAA 573 Hu.DMD.Exon51.25.012
ATCTGCCAGAGCAGGTACCTCCAAC 574 Hu.DMD.Exon51.25.013
AAGTTCTGTCCAAGCCCGGTTGAAAT 575 Hu.DMD.Exon51.25.013.2
CGGTTGAAATCTGCCAGAGCAGGTAC 576 Hu.DMD.Exon51.25.014
GAGAAAGCCAGTCGGTAAGTTCTGTC 577 Hu.DMD.Exon51.25.014.2
GTCGGTAAGTTCTGTCCAAGCCCGG 578 Hu.DMD.Exon51.25.015
ATAACTTGATCAAGCAGAGAAAGCCA 579 Hu.DMD.Exon51.25.015.2
AAGCAGAGAAAGCCAGTCGGTAAGT 580 Hu.DMD.Exon51.25.016
CACCCTCTGTGATTTTATAACTTGAT 581 Hu.DMD.Exon51.25.017
CAAGGTCACCCACCATCACCCTCTGT 582 Hu.DMD.Exon51.25.017.2
CATCACCCTCTGTGATTTTATAACT 583 Hu.DMD.Exon51.25.018
CTTCTGCTTGATGATCATCTCGTTGA 584 Hu.DMD.Exon51.25.019
CCTTCTGCTTGATGATCATCTCGTTG 585 Hu.DMD.Exon51.25.019.2
ATCTCGTTGATATCCTCAAGGTCACC 586 Hu.DMD.Exon51.25.020
TCATACCTTCTGCTTGATGATCATCT 587 Hu.DMD.Exon51.25.020.2
TCATTTTTTCTCATACCTTCTGCTTG 588 Hu.DMD.Exon51.25.021
TTTTCTCATACCTTCTGCTTGATGAT 589 Hu.DMD.Exon51.25.022
TTTTATCATTTTTTCTCATACCTTCT 590 Hu.DMD.Exon51.25.023
CCAACTTTTATCATTTTTTCTCATAC 591 Hu.DMD.Exon51.20.001
ATATTTTGGGTTTTTGCAAA 592 Hu.DMD.Exon51.20.002 AAAATATTTTGGGTTTTTGC
593 Hu.DMD.Exon51.20.003 GAGCTAAAATATTTTGGGTT 594
Hu.DMD.Exon51.20.004 AGTAGGAGCTAAAATATTTT 595 Hu.DMD.Exon51.20.005
GTCTGAGTAGGAGCTAAAAT 596 Hu.DMD.Exon51.20.006 TAACAGTCTGAGTAGGAGCT
597 Hu.DMD.Exon51.20.007 CAGAGTAACAGTCTGAGTAG 598
Hu.DMD.Exon51.20.008 CACAGGTTGTGTCACCAGAG 599 Hu.DMD.Exon51.20.009
AGTTTCCTTAGTAACCACAG 600 Hu.DMD.Exon51.20.010 TAGTTTGGAGATGGCAGTTT
601 Hu.DMD.Exon51.20.011 GGAAGATGGCATTTCTAGTT 602
Hu.DMD.Exon51.20.012 TACCTCCAACATCAAGGAAG 603 Hu.DMD.Exon51.20.013
ATCTGCCAGAGCAGGTACCT 604 Hu.DMD.Exon51.20.014 CCAAGCCCGGTTGAAATCTG
605 Hu.DMD.Exon51.20.015 GTCGGTAAGTTCTGTCCAAG 606
Hu.DMD.Exon51.20.016 AAGCAGAGAAAGCCAGTCGG 607 Hu.DMD.Exon51.20.017
TTTTATAACTTGATCAAGCA 608 Hu.DMD.Exon51.20.018 CATCACCCTCTGTGATTTTA
609 Hu.DMD.Exon51.20.019 CTCAAGGTCACCCACCATCA 610
Hu.DMD.Exon51.20.020 CATCTCGTTGATATCCTCAA 611 Hu.DMD.Exon51.20.021
CTTCTGCTTGATGATCATCT 612 Hu.DMD.Exon51.20.022 CATACCTTCTGCTTGATGAT
613 Hu.DMD.Exon51.20.023 TTTCTCATACCTTCTGCTTG 614
Hu.DMD.Exon51.20.024 CATTTTTTCTCATACCTTCT 615 Hu.DMD.Exon51.20.025
TTTATCATTTTTTCTCATAC 616 Hu.DMD.Exon51.20.026 CAACTTTTATCATTTTTTCT
617 Hu.DMD.Exon52.25.001 CTGTAAGAACAAATATCCCTTAGTA 618
Hu.DMD.Exon52.25.002 TGCCTGTAAGAACAAATATCCCTTA 619
Hu.DMD.Exon52.25.002.2 GTTGCCTGTAAGAACAAATATCCCT 620
Hu.DMD.Exon52.25.003 ATTGTTGCCTGTAAGAACAAATATC 621
Hu.DMD.Exon52.25.003.2 GCATTGTTGCCTGTAAGAACAAATA 622
Hu.DMD.Exon52.25.004 CCTGCATTGTTGCCTGTAAGAACAA 623
Hu.DMD.Exon52.25.004.2 ATCCTGCATTGTTGCCTGTAAGAAC 624
Hu.DMD.Exon52.25.005 CAAATCCTGCATTGTTGCCTGTAAG 625
Hu.DMD.Exon52.25.005.2 TCCAAATCCTGCATTGTTGCCTGTA 626
Hu.DMD.Exon52.25.006 TGTTCCAAATCCTGCATTGTTGCCT 627
Hu.DMD.Exon52.25.006.2 TCTGTTCCAAATCCTGCATTGTTGC 628
Hu.DMD.Exon52.25.007 AACTGGGGACGCCTCTGTTCCAAAT 629
Hu.DMD.Exon52.25.007.2 GCCTCTGTTCCAAATCCTGCATTGT 630
Hu.DMD.Exon52.25.008 CAGCGGTAATGAGTTCTTCCAACTG 631
Hu.DMD.Exon52.25.008.2 CTTCCAACTGGGGACGCCTCTGTTC 632
Hu.DMD.Exon52.25.009 CTTGTTTTTCAAATTTTGGGCAGCG 633
Hu.DMD.Exon52.25.010 CTAGCCTCTTGATTGCTGGTCTTGT 634
Hu.DMD.Exon52.25.010.2 TTTTCAAATTTTGGGCAGCGGTAAT 635
Hu.DMD.Exon52.25.011 TTCGATCCGTAATGATTGTTCTAGC 636
Hu.DMD.Exon52.25.011.2 GATTGCTGGTCTTGTTTTTCAAATT 637
Hu.DMD.Exon52.25.012 CTTACTTCGATCCGTAATGATTGTT 638
Hu.DMD.Exon52.25.012.2 TTGTTCTAGCCTCTTGATTGCTGGT 639
Hu.DMD.Exon52.25.013 AAAAACTTACTTCGATCCGTAATGA 640
Hu.DMD.Exon52.25.014 TGTTAAAAAACTTACTTCGATCCGT 641
Hu.DMD.Exon52.25.015 ATGCTTGTTAAAAAACTTACTTCGA 642
Hu.DMD.Exon52.25.016 GTCCCATGCTTGTTAAAAAACTTAC 643
Hu.DMD.Exon52.20.001 AGAACAAATATCCCTTAGTA 644 Hu.DMD.Exon52.20.002
GTAAGAACAAATATCCCTTA 645 Hu.DMD.Exon52.20.003 TGCCTGTAAGAACAAATATC
646 Hu.DMD.Exon52.20.004 ATTGTTGCCTGTAAGAACAA 647
Hu.DMD.Exon52.20.005 CCTGCATTGTTGCCTGTAAG 648 Hu.DMD.Exon52.20.006
CAAATCCTGCATTGTTGCCT 649 Hu.DMD.Exon52.20.007 GCCTCTGTTCCAAATCCTGC
650 Hu.DMD.Exon52.20.008 CTTCCAACTGGGGACGCCTC 651
Hu.DMD.Exon52.20.009 CAGCGGTAATGAGTTCTTCC 652 Hu.DMD.Exon52.20.010
TTTTCAAATTTTGGGCAGCG 653 Hu.DMD.Exon52.20.011 GATTGCTGGTCTTGTTTTTC
654 Hu.DMD.Exon52.20.012 TTGTTCTAGCCTCTTGATTG 655
Hu.DMD.Exon52.20.013 TTCGATCCGTAATGATTGTT 656 Hu.DMD.Exon52.20.014
CTTACTTCGATCCGTAATGA 657 Hu.DMD.Exon52.20.015 AAAAACTTACTTCGATCCGT
658 Hu.DMD.Exon52.20.016 TGTTAAAAAACTTACTTCGA 659
Hu.DMD.Exon52.20.017 ATGCTTGTTAAAAAACTTAC 660 Hu.DMD.Exon52.20.018
GTCCCATGCTTGTTAAAAAA 661 Hu.DMD.Exon53.25.001
CTAGAATAAAAGGAAAAATAAATAT 662 Hu.DMD.Exon53.25.002
AACTAGAATAAAAGGAAAAATAAAT 663 Hu.DMD.Exon53.25.002.2
TTCAACTAGAATAAAAGGAAAAATA 664 Hu.DMD.Exon53.25.003
CTTTCAACTAGAATAAAAGGAAAAA 665 Hu.DMD.Exon53.25.003.2
ATTCTTTCAACTAGAATAAAAGGAA 666 Hu.DMD.Exon53.25.004
GAATTCTTTCAACTAGAATAAAAGG 667 Hu.DMD.Exon53.25.004.2
TCTGAATTCTITCAACTAGAATAAA 668 Hu.DMD.Exon53.25.005
ATTCTGAATTCTTTCAACTAGAATA 669 Hu.DMD.Exon53.25.005.2
CTGATTCTGAATTCTITCAACTAGA 670 Hu.DMD.Exon53.25.006
CACTGATTCTGAATTCTTTCAACTA 671 Hu.DMD.Exon53.25.006.2
TCCCACTGATTCTGAATTCTTTCAA 672 Hu.DMD.Exon53.25.007
CATCCCACTGATTCTGAATTCTTTC 673 Hu.DMD.Exon53.25.008
TACTTCATCCCACTGATTCTGAATT 674 Hu.DMD.Exon53.25.009
CGGTTCTGAAGGTGTTCTTGTACT 675 Hu.DMD.Exon53.25.009.2
CTGTTGCCTCCGGTTCTGAAGGTGT 676 Hu.DMD.Exon53.25.010
TTTCATTCAACTGTTGCCTCCGGTT 677 Hu.DMD.Exon53.25.010.2
TAACATTTCATTCAACTGTTGCCTC 678 Hu.DMD.Exon53.25.011
TTGTGTTGAATCCTTTAACATTTCA 679 Hu.DMD.Exon53.25.012
TCTTCCTTAGCTTCCAGCCATTGTG 680 Hu.DMD.Exon53.25.012.2
CTTAGCTTCCAGCCATTGTGTTGAA 681 Hu.DMD.Exon53.25.013
GTCCTAAGACCTGCTCAGCTTCTTC 682 Hu.DMD.Exon53.25.013.2
CTGCTCAGCTTCTTCCTTAGCTTCC 683 Hu.DMD.Exon53.25.014
CTCAAGCTTGGCTCTGGCCTGTCCT 684 Hu.DMD.Exon53.25.014.2
GGCCTGTCCTAAGACCTGCTCAGCT 685 Hu.DMD.Exon53.25.015
TAGGGACCCTCCTTCCATGACTCAA 686 Hu.DMD.Exon53.25.016
TTTGGATTGCATCTACTGTATAGGG 687 Hu.DMD.Exon53.25.016.2
ACCCTCCTTCCATGACTCAAGCTTG 688 Hu.DMD.Exon53.25.017
CTTGGTTTCTGTGATTTTCTTTTGG 689 Hu.DMD.Exon53.25.017.2
ATCTACTGTATAGGGACCCTCCTTC 690 Hu.DMD.Exon53.25.018
CTAACCTTGGTTTCTGTGATTTTCT 691 Hu.DMD.Exon53.25.018.2
TTTCTTTTGGATTGCATCTACTGTA 692 Hu.DMD.Exon53.25.019
TGATACTAACCTTGGTTTCTGTGAT 693 Hu.DMD.Exon53.25.020
ATCTTTGATACTAACCTTGGTTTCT 694 Hu.DMD.Exon53.25.021
AAGGTATCTTTGATACTAACCTTGG 695 Hu.DMD.Exon53.25.022
TTAAAAAGGTATCTTTGATACTAAC 696 Hu.DMD.Exon53.20.001
ATAAAAGGAAAAATAAATAT 697 Hu.DMD.Exon53.20.002 GAATAAAAGGAAAAATAAAT
698 Hu.DMD.Exon53.20.003 AACTAGAATAAAAGGAAAAA 699
Hu.DMD.Exon53.20.004 CTTTCAACTAGAATAAAAGG 700 Hu.DMD.Exon53.20.005
GAATTCTTTCAACTAGAATA 701 Hu.DMD.Exon53.20.006 ATTCTGAATTCTTTCAACTA
702 Hu.DMD.Exon53.20.007 TACTTCATCCCACTGATTCT 703
Hu.DMD.Exon53.20.008 CTGAAGGTGTTCTTGTACT 704 Hu.DMD.Exon53.20.009
CTGTTGCCTCCGGTTCTGAA 705 Hu.DMD.Exon53.20.010 TAACATTTCATTCAACTGTT
706 Hu.DMD.Exon53.20.011 TTGTGTTGAATCCTTTAACA 707
Hu.DMD.Exon53.20.012 CTTAGCTTCCAGCCATTGTG 708 Hu.DMD.Exon53.20.013
CTGCTCAGCTTCTTCCTTAG 709 Hu.DMD.Exon53.20.014 GGCCTGTCCTAAGACCTGCT
710 Hu.DMD.Exon53.20.015 CTCAAGCTTGGCTCTGGCCT 711
Hu.DMD.Exon53.20.016 ACCCTCCTTCCATGACTCAA 712 Hu.DMD.Exon53.20.017
ATCTACTGTATAGGGACCCT 713 Hu.DMD.Exon53.20.018 TTTCTTTTGGATTGCATCTA
714 Hu.DMD.Exon53.20.019 CTTGGTTTCTGTGATTTTCT 715
Hu.DMD.Exon53.20.020 CTAACCTTGGTTTCTGTGAT 716 Hu.DMD.Exon53.20.021
TGATACTAACCTTGGTTTCT 717 Hu.DMD.Exon53.20.022 ATCTTTGATACTAACCTTGG
718
Hu.DMD.Exon53.20.023 AAGGTATCTTTGATACTAAC 719 Hu.DMD.Exon53.20.024
TTAAAAAGGTATCTTTGATA 720 Hu.DMD.Exon54.25.001
CTATAGATTTTTATGAGAAAGAGA 721 Hu.DMD.Exon54.25.002
AACTGCTATAGATTTTTATGAGAAA 722 Hu.DMD.Exon54.25.003
TGGCCAACTGCTATAGATTTTTATG 723 Hu.DMD.Exon54.25.004
GTCTTTGGCCAACTGCTATAGATTT 724 Hu.DMD.Exon54.25.005
CGGAGGTCTTTGGCCAACTGCTATA 725 Hu.DMD.Exon54.25.006
ACTGGCGGAGGTCTTTGGCCAACTG 726 Hu.DMD.Exon54.25.007
TTTGTCTGCCACTGGCGGAGGTCTT 727 Hu.DMD.Exon54.25.008
AGTCATTTGCCACATCTACATTTGT 728 Hu.DMD.Exon54.25.008.2
TTTGCCACATCTACATTTGTCTGCC 729 Hu.DMD.Exon54.25.009
CCGGAGAAGTTTCAGGGCCAAGTCA 730 Hu.DMD.Exon54.25.010
GTATCATCTGCAGAATAATCCCGGA 731 Hu.DMD.Exon54.25.010.2
TAATCCCGGAGAAGTTTCAGGGCCA 732 Hu.DMD.Exon54.25.011
TTATCATGTGGACTTTTCTGGTATC 733 Hu.DMD.Exon54.25.012
AGAGGCATTGATATTCTCTGTTATC 734 Hu.DMD.Exon54.25.012.2
ATGTGGACTTTTCTGGTATCATCTG 735 Hu.DMD.Exon54.25.013
CTTTTATGAATGCTTCTCCAAGAGG 736 Hu.DMD.Exon54.25.013.2
ATATTCTCTGTTATCATGTGGACTT 737 Hu.DMD.Exon54.25.014
CATACCTTTTATGAATGCTTCTCCA 738 Hu.DMD.Exon54.25.014.2
CTCCAAGAGGCATTGATATTCTCTG 739 Hu.DMD.Exon54.25.015
TAATTCATACCTTTTATGAATGCTT 740 Hu.DMD.Exon54.25.016
TAATGTAATTCATACCTTTTATGAA 741 Hu.DMD.Exon54.25.017
AGAAATAATGTAATTCATACCTTTT 742 Hu.DMD.Exon54.25.018
GTTTTAGAAATAATGTAATTCATAC 743 Hu.DMD.Exon54.20.001
GATTTTTATGAGAAAGAGA 744 Hu.DMD.Exon54.20.002 CTATAGATTTTTATGAGAAA
745 Hu.DMD.Exon54.20.003 AACTGCTATAGATTTTTATG 746
Hu.DMD.Exon54.20.004 TGGCCAACTGCTATAGATTT 747 Hu.DMD.Exon54.20.005
GTCTTTGGCCAACTGCTATA 748 Hu.DMD.Exon54.20.006 CGGAGGTCTTTGGCCAACTG
749 Hu.DMD.Exon54.20.007 TTTGTCTGCCACTGGCGGAG 750
Hu.DMD.Exon54.20.008 TTTGCCACATCTACATTTGT 751 Hu.DMD.Exon54.20.009
TTCAGGGCCAAGTCATTTGC 752 Hu.DMD.Exon54.20.010 TAATCCCGGAGAAGTTTCAG
753 Hu.DMD.Exon54.20.011 GTATCATCTGCAGAATAATC 754
Hu.DMD.Exon54.20.012 ATGTGGACTTTTCTGGTATC 755 Hu.DMD.Exon54.20.013
ATATTCTCTGTTATCATGTG 756 Hu.DMD.Exon54.20.014 CTCCAAGAGGCATTGATATT
757 Hu.DMD.Exon54.20.015 CTTTTATGAATGCTTCTCCA 758
Hu.DMD.Exon54.20.016 CATACCTTTTATGAATGCTT 759 Hu.DMD.Exon54.20.017
TAATTCATACCTTTTATGAA 760 Hu.DMD.Exon54.20.018 TAATGTAATTCATACCTTTT
761 Hu.DMD.Exon54.20.019 AGAAATAATGTAATTCATAC 762
Hu.DMD.Exon54.20.020 GTTTTAGAAATAATGTAATT 763 Hu.DMD.Exon55.25.001
CTGCAAAGGACCAAATGTTCAGATG 764 Hu.DMD.Exon55.25.002
TCACCCTGCAAAGGACCAAATGTTC 765 Hu.DMD.Exon55.25.003
CTCACTCACCCTGCAAAGGACCAAA 766 Hu.DMD.Exon55.25.004
TCTCGCTCACTCACCCTGCAAAGGA 767 Hu.DMD.Exon55.25.005
CAGCCTCTCGCTCACTCACCCTGCA 768 Hu.DMD.Exon55.25.006
CAAAGCAGCCTCTCGCTCACTCACC 769 Hu.DMD.Exon55.25.007
TCTTCCAAAGCAGCCTCTCGCTCAC 770 Hu.DMD.Exon55.25.007.2
TCTATGAGTTTCTTCCAAAGCAGCC 771 Hu.DMD.Exon55.25.008
GTTGCAGTAATCTATGAGTTTCTTC 772 Hu.DMD.Exon55.25.008.2
GAACTGTTGCAGTAATCTATGAGTT 773 Hu.DMD.Exon55.25.009
TTCCAGGTCCAGGGGGAACTGTTGC 774 Hu.DMD.Exon55.25.010
GTAAGCCAGGCAAGAAACTTTTCCA 775 Hu.DMD.Exon55.25.010.2
CCAGGCAAGAAACT+32CCAGGTCC 776 Hu.DMD.Exon55.25.011
TGGCAGTTGTTTCAGCTTCTGTAAG 777 Hu.DMD.Exon55.25.011.2
TTCAGCTTCTGTAAGCCAGGCAAGA 778 Hu.DMD.Exon55.25.012
GGTAGCATCCTGTAGGACATTGGCA 779 Hu.DMD.Exon55.25.012.2
GACATTGGCAGTTGTTTCAGCTTCT 780 Hu.DMD.Exon55.25.013
TCTAGGAGCCTTTCCTTACGGGTAG 781 Hu.DMD.Exon55.25.014
CTTTTACTCCCTTGGAGTCTTCTAG 782 Hu.DMD.Exon55.25.014.2
GAGCC+32CCTTACGGGTAGCATCC 783 Hu.DMD.Exon55.25.015
TTGCCATTGTTTCATCAGCTCTTTT 784 Hu.DMD.Exon55.25.015.2
CTTGGAGTCTTCTAGGAGCCTTTCC 785 Hu.DMD.Exon55.25.016
CTTACTTGCCATTGTTTCATCAGCT 786 Hu.DMD.Exon55.25.016.2
CAGCTCTTTTACTCCCTTGGAGTCT 787 Hu.DMD.Exon55.25.017
CCTGACTTACTTGCCATTGTTTCAT 788 Hu.DMD.Exon55.25.018
AAATGCCTGACTTACTTGCCATTGT 789 Hu.DMD.Exon55.25.019
AGCGGAAATGCCTGACTTACTTGCC 790 Hu.DMD.Exon55.25.020
GCTAAAGCGGAAATGCCTGACTTAC 791 Hu.DMD.Exon55.20.001
AAGGACCAAATGTTCAGATG 792 Hu.DMD.Exon55.20.002 CTGCAAAGGACCAAATGTTC
793 Hu.DMD.Exon55.20.003 TCACCCTGCAAAGGACCAAA 794
Hu.DMD.Exon55.20.004 CTCACTCACCCTGCAAAGGA 795 Hu.DMD.Exon55.20.005
TCTCGCTCACTCACCCTGCA 796 Hu.DMD.Exon55.20.006 CAGCCTCTCGCTCACTCACC
797 Hu.DMD.Exon55.20.007 CAAAGCAGCCTCTCGCTCAC 798
Hu.DMD.Exon55.20.008 TCTATGAGTTTCTTCCAAAG 799 Hu.DMD.Exon55.20.009
GAACTGTTGCAGTAATCTAT 800 Hu.DMD.Exon55.20.010 TTCCAGGTCCAGGGGGAACT
801 Hu.DMD.Exon55.20.011 CCAGGCAAGAAACTTTTCCA 802
Hu.DMD.Exon55.20.012 TTCAGCTTCTGTAAGCCAGG 803 Hu.DMD.Exon55.20.013
GACATTGGCAGTTGTTTCAG 804 Hu.DMD.Exon55.20.014 GGTAGCATCCTGTAGGACAT
805 Hu.DMD.Exon55.20.015 GAGCCTTTCCTTACGGGTAG 806
Hu.DMD.Exon55.20.016 CTTGGAGTCTTCTAGGAGCC 807 Hu.DMD.Exon55.20.017
CAGCTCTTTTACTCCCTTGG 808 Hu.DMD.Exon55.20.018 TTGCCATTGTTTCATCAGCT
809 Hu.DMD.Exon55.20.019 CTTACTTGCCATTGTTTCAT 810
Hu.DMD.Exon55.20.020 CCTGACTTACTTGCCATTGT 811 Hu.DMD.Exon55.20.021
AAATGCCTGACTTACTTGCC 812 Hu.DMD.Exon55.20.022 AGCGGAAATGCCTGACTTAC
813 Hu.DMD.Exon55.20.023 GCTAAAGCGGAAATGCCTGA 814 H50A(+02 +
30)-AVI-5656 CCACTCAGAGCTCAGATCTTCTAACTTCC 815 H5 0D(+07 -
18)-AVI-5915 GGGATCCAGTATACTTACAGGCTCC 816 H50A(+07 + 33)
CTTCCACTCAGAGCTCAGATCTTCTAA 817 H51A(+61 + 90)-AVI-4657
ACATCAAGGAAGATGGCATTTCTAGTTTGG 818 H51A(+66 + 95)-AVI-4658
CTCCAACATCAAGGAAGATGGCATTTCTAG 819 H51A(+111 + 134)
TTCTGTCCAAGCCCGGTTGAAATC 820 H51A(+175 + 195) CACCCACCATCACCCTCYGTG
821 H51A(+199 + 220) ATCATCTCGTTGATATCCTCAA 822 H51A(+66 + 90)
ACATCAAGGAAGATGGCATTTCTAG 823 H51A(-01 + 25)
ACCAGAGTAACAGTCTGAGTAGGAGC 824 h51A0N1 TCAAGGAAGATGGCATTTCT 825
h51A0N2 CCTCTGTGATTTTATAACTTGAT 826 H51D(+08 - 17)
ATCATTTTTTCTCATACCTTCTGCT 827 H51D(+16 - 07)
CTCATACCTTCTGCTTGATGATC 828 hAON#23 TGGCATTTCTAGTTTGG 829 hAON#24
CCAGAGCAGGTACCTCCAACATC 830 h44A0N1 CGCCGCCATTTCTCAACAG 831
H45A(+71 + 90) TGTTTTTGAGGATTGCTGAA 832 h45A0N1 GCTGAATTATTTCTTCCCC
833 h45A0N5 GCCCAATGCCATCCTGG 834 H45A(-06 + 20)
CCAATGCCATCCTGGAGTTCCTGTAA 835 H53A(+39 + 69)
CATTCAACTGTTGCCTCCGGTTCTGAAGGTG 836 h53A0N1 CTGTTGCCTCCGGTTCTG 837
H53A(-12 + 10) ATTCTTTCAACTAGAATAAAAG 838 huEx45.30.66
GCCATCCTGGAGTTCCTGTAAGATACCAAA 839 huEx45.30.71
CCAATGCCATCCTGGAGTTCCTGTAAGATA 840 huEx45.30.79
GCCGCTGCCCAATGCCATCCTGGAGTTCCT 841 huEx45.30.83
GTTTGCCGCTGCCCAATGCCATCCTGGAGT 842 huEx45.30.88
CAACAGTTTGCCGCTGCCCAATGCCATCCT 843 huEx45.30.92
CTGACAACAGTTTGCCGCTGCCCAATGCCA 844 huEx45.30.96
TGTTCTGACAACAGTTTGCCGCTGCCCAAT 845 huEx45.30.99
CAATGTTCTGACAACAGTTTGCCGCTGCCC 846 huEx45.30.103
CATTCAATGTTCTGACAACAGTTTGCCGCT 847 huEx45.30.120
TATTTCTTCCCCAGTTGCATTCAATGTTCT 848 huEx45.30.127
GCTGAATTATTTCTTCCCCAGTTGCATTCA 849 huEx45.30.132
GGATTGCTGAATTATTTCTTCCCCAGTTGC 850 huEx45.30.137
TTTGAGGATTGCTGAATTATTTCTTCCCCA 851 huEx53.30.84
GTACTTCATCCCACTGATTCTGAATTCTTT 852 huEx53.30.88
TCTTGTACTTCATCCCACTGATTCTGAATT 853 huEx53.30.91
TGTTCTTGTACTTCATCCCACTGATTCTGA 854 huEx53.30.103
CGGTTCTGAAGGTGTTCTTGTACTTCATCC 855 huEx53.30.106
CTCCGGTTCTGAAGGTGTTCTTGTACTTCA 856 huEx53.30.109
TGCCTCCGGTTCTGAAGGTGTTCTTGTACT 857 huEx53.30.112
TGTTGCCTCCGGTTCTGAAGGTGTTCTTGT 858 huEx53.30.115
AACTGTTGCCTCCGGTTCTGAAGGTGTTCT 859 huEx53.30.118
TTCAACTGTTGCCTCCGGTTCTGAAGGTGT 860 h50AON1 h50AON2 Peptide
Transporters (NH.sub.2 to COOH)*: rTAT RRRQRRKKRC 861
R.sub.9F.sub.2 RRRRRRRRRFFC 862 (RRAhx).sub.4B
RRAhxRRAhxRRAhxRRAhxB 863 (RAhxR).sub.4AhxB; (P007)
RAhxRRAhxRRAhxRRAhxRAhxB 864 (AhxRR).sub.4AhxB
AhxRRAhxRRAhxRRAhxRRAhxB 865 (RAhx).sub.6B
RAhxRAhxRAhxRAhxRAhxRAhxB 866 (RAhx).sub.8B
RAhxRAhxRAhxRAhxRAhxRAhxRAhxB 867 (RAhxR).sub.5AhxB
RAhxRRAhxRRAhxRRAhxRRAhxRAhxB 868 (RAhxRRBR).sub.2AhxB;
RAhxRRBRRAhxRRBRAhxB 869 (CPO6062) MSP ASSLNIA 870 Cell Penetrating
Peptide/Homing Peptide/PMO Conjugates (NH.sub.2 to COOH and 5' to
3') MSP-PMO ASSLNIA-XB- 871 GGCCAAACCTCGGCTTACCTGAAAT 875
CP06062-MSP-PMO RXRRBRRXRRBR-XB-ASSLNIA-X- 872
GGCCAAACCTCGGCTTACCTGAAAT 875 MSP-CP06062-PMO
ASSLNIA-X-RXRRBRRXRRBR-B- 873 GGCCAAACCTCGGCTTACCTGAAAT 875
CP06062-PMO RXRRBRRXRRBR-XB- 874 GGCCAAACCTCGGCTTACCTGAAAT 875 *Ahx
is 6-aminohexanoic acid and B is beta-alanine
Sequence CWU 1
1
891130DNAArtificial SequenceSynthetic (eteplirsen H51A(+66+95))
1ctccaacatc aaggaagatg gcatttctag 30225RNAArtificial
SequenceSynthetic (H51A(+66+90)) 2acaucaagga agauggcauu ucuag
25330RNAArtificial SequenceSynthetic (H51A(+61+90)) 3acaucaagga
agauggcauu ucuaguuugg 30426DNAArtificial SequenceSynthetic
(Hu.DMD.exon51.25.001.2) 4gagcaggtac ctccaacatc aaggaa
26525RNAArtificial SequenceSynthetic (H50D(+07-18)) 5gggauccagu
auacuuacag gcucc 25625DNAArtificial SequenceSynthetic
(AVI-4038/5038 ) 6cttacaggct ccaatagtgg tcagt 25727DNAArtificial
SequenceSynthetic (H53A27(+30+56)) 7cctccggttc tgaaggtgtt cttgtac
27825DNAArtificial SequenceSynthetic (H53A(+36+60) ) 8gttgcctccg
gttctgaagg tgttc 25922DNAArtificial SequenceSynthetic (H45A
(-03+19)) 9caatgccatc ctggagttcc tg 221024RNAArtificial
SequenceSynthetic (H8A(-06+18)) 10gauagguggu aucaacaucu guaa
241121RNAArtificial SequenceSynthetic (H8A (-03+18)) 11gauagguggu
aucaacaucu g 211225RNAArtificial SequenceSynthetic (H8A(-07+18))
12gauagguggu aucaacaucu guaag 251320RNAArtificial SequenceSynthetic
(H8A(-06+14)) 13ggugguauca acaucuguaa 201420RNAArtificial
SequenceSynthetic (H8A(-10+10)) 14guaucaacau cuguaagcac
201523RNAArtificial SequenceSynthetic (H7A(+45+67)) 15ugcauguucc
agucguugug ugg 231625RNAArtificial SequenceSynthetic (H7A(+02+26))
16cacuauucca gucaaauagg ucugg 251725RNAArtificial SequenceSynthetic
(H7D(+15-10)) 17auuuaccaac cuucaggauc gagua 251821RNAArtificial
SequenceSynthetic (H7A(-18+03)) 18ggccuaaaac acauacacau a
211920RNAArtificial SequenceSynthetic (C6A(-10+10)) 19cauuuuugac
cuacaugugg 202020RNAArtificial SequenceSynthetic (C6A(-14+06))
20uuugaccuac auguggaaag 202126RNAArtificial SequenceSynthetic
(C6A(-14+12)) 21uacauuuuug accuacaugu ggaaag 262222RNAArtificial
SequenceSynthetic (C6A(-13+09)) 22auuuuugacc uacaugggaa ag
222323RNAArtificial SequenceSynthetic (CH6A(+69+91)) 23uacgaguuga
uugucggacc cag 232425RNAArtificial SequenceSynthetic (C6D(+12-13))
24guggucuccu uaccuaugac ugugg 252517RNAArtificial SequenceSynthetic
(C6D(+06-11)) 25ggucuccuua ccuauga 172624RNAArtificial
SequenceSynthetic (H6D(+04-21)) 26ugucucagua aucuucuuac cuau
242724RNAArtificial SequenceSynthetic (H6D(+18-04)) 27ucuuaccuau
gacuauggau gaga 242820RNAArtificial SequenceSynthetic (H4A(+13+32))
28gcaugaacuc uuguggaucc 202920RNAArtificial SequenceSynthetic
(H4D(+04-16)) 29ccaggguacu acuuacauua 203021RNAArtificial
SequenceSynthetic (H4D(-24-44)) 30aucguguguc acagcaucca g
213130RNAArtificial SequenceSynthetic (H4A(+11+40)) 31uguucagggc
augaacucuu guggauccuu 303231RNAArtificial SequenceSynthetic
(H3A(+30+60)) 32uaggaggcgc cucccauccu guaggucacu g
313331RNAArtificial SequenceSynthetic (H3A(+35+65)) 33aggucuagga
ggcgccuccc auccuguagg u 313425RNAArtificial SequenceSynthetic
(H3A(+30+54)) 34gcgccuccca uccuguaggu cacug 253526RNAArtificial
SequenceSynthetic (H3D(+46-21)) 35cuucgaggag gucuaggagg cgccuc
263621RNAArtificial SequenceSynthetic (H3A(+30+50)) 36cucccauccu
guaggucacu g 213722RNAArtificial SequenceSynthetic (H3D(+19-03))
37uaccaguuuu ugcccuguca gg 223826RNAArtificial SequenceSynthetic
(H3A(-06+20)) 38ucaauaugcu gcuucccaaa cugaaa 263925RNAArtificial
SequenceSynthetic (H3A(+37+61)) 39cuaggaggcg ccucccaucc uguag
254031RNAArtificial SequenceSynthetic (H5A(+20+50)) 40uuaugauuuc
caucuacgau gucaguacuu c 314131RNAArtificial SequenceSynthetic
(H5D(+25-05)) 41cuuaccugcc aguggaggau uauauuccaa a
314225RNAArtificial SequenceSynthetic (H5D(+10-15)) 42caucaggauu
cuuaccugcc agugg 254325RNAArtificial SequenceSynthetic
(H5A(+10+34)) 43cgaugucagu acuuccaaua uucac 254418RNAArtificial
SequenceSynthetic (H5D(-04-21)) 44accauucauc aggauucu
184518RNAArtificial SequenceSynthetic (H5D(+16-02)) 45accugccagu
ggaggauu 184627RNAArtificial SequenceSynthetic (H5A(-07+20))
46ccaauauuca cuaaaucaac cuguuaa 274730RNAArtificial
SequenceSynthetic (H5D(+18-12)) 47caggauuguu accugccagu ggaggauuau
304831RNAArtificial SequenceSynthetic (H5A(+05+35)) 48acgaugucag
uacuuccaau auucacuaaa u 314931RNAArtificial SequenceSynthetic
(H5A(+15+45)) 49auuuccaucu acgaugucag uacuuccaau a
315021RNAArtificial SequenceSynthetic (H10A(-05+16)) 50caggagcuuc
caaaugcugc a 215129RNAArtificial SequenceSynthetic (H10A(-05+24))
51cuugucuuca ggagcuucca aaugcugca 295222RNAArtificial
SequenceSynthetic (H10A(+98+119)) 52uccucagcag aaagaagcca cg
225320RNAArtificial SequenceSynthetic (H10A(+130+149)) 53uuagaaaucu
cuccuugugc 205420RNAArtificial SequenceSynthetic (H10A(-33-14))
54uaaauugggu guuacacaau 205524RNAArtificial SequenceSynthetic
(H11D(+26+49)) 55cccugaggca uucccaucuu gaau 245620RNAArtificial
SequenceSynthetic (H11D(+11-09)) 56aggacuuacu ugcuuuguuu
205723RNAArtificial SequenceSynthetic (H11A(+118+140)) 57cuugaauuua
ggagauucau cug 235823RNAArtificial SequenceSynthetic (H11A(+75+97))
58caucuucuga uaauuuuccu guu 235924RNAArtificial SequenceSynthetic
(H12A(+52+75)) 59ucuucuguuu uuguuagcca guca 246020RNAArtificial
SequenceSynthetic (H12A(-10+10)) 60ucuauguaaa cugaaaauuu
206120RNAArtificial SequenceSynthetic (H12A(+11+30)) 61uucuggagau
ccauuaaaac 206224RNAArtificial SequenceSynthetic (H13A(+77+100))
62cagcaguugc gugaucucca cuag 246321RNAArtificial SequenceSynthetic
(H13A(+55+75)) 63uucaucaacu accaccacca u 216425RNAArtificial
SequenceSynthetic (H13D(+06-19)) 64cuaagcaaaa uaaucugacc uuaag
256528RNAArtificial SequenceSynthetic (H14A(+37+64)) 65cuuguaaaag
aacccagcgg ucuucugu 286622RNAArtificial SequenceSynthetic
(H14A(+14+35)) 66caucuacaga uguuugccca uc 226723RNAArtificial
SequenceSynthetic (H14A(+51+73)) 67gaaggauguc uuguaaaaga acc
236820RNAArtificial SequenceSynthetic (H14D(-02+18)) 68accuguucuu
caguaagacg 206924RNAArtificial SequenceSynthetic (H14D(+14-10))
69caugacacac cuguucuuca guaa 247020RNAArtificial SequenceSynthetic
(H14A(+61+80)) 70cauuugagaa ggaugucuug 207124RNAArtificial
SequenceSynthetic (H14A(-12+12)) 71aucucccaau accuggagaa gaga
247231RNAArtificial SequenceSynthetic (H15A(-12+19)) 72gccaugcacu
aaaaaggcac ugcaagacau u 317324RNAArtificial SequenceSynthetic
(H15A(+48+71)) 73ucuuuaaagc caguugugug aauc 247421RNAArtificial
SequenceSynthetic (H15A(+08+28)) 74uuucugaaag ccaugcacua a
217525RNAArtificial SequenceSynthetic (H15D(+17-08)) 75guacauacgg
ccaguuuuug aagac 257631RNAArtificial SequenceSynthetic
(H16A(-12+19)) 76cuagauccgc uuuuaaaacc uguuaaaaca a
317731RNAArtificial SequenceSynthetic (H16A(-06+25)) 77ucuuuucuag
auccgcuuuu aaaaccuguu a 317825RNAArtificial SequenceSynthetic
(H16A(-06+19)) 78cuagauccgc uuuuaaaacc uguua 257923RNAArtificial
SequenceSynthetic (H16A(+87+109)) 79ccgucuucug ggucacugac uua
238026RNAArtificial SequenceSynthetic (H16A(-07+19)) 80cuagauccgc
uuuuaaaacc uguuaa 268120RNAArtificial SequenceSynthetic
(H16A(-07+13)) 81ccgcuuuuaa aaccuguuaa 208226RNAArtificial
SequenceSynthetic (H16A(+12+37)) 82uggauugcuu uuucuuuucu agaucc
268325RNAArtificial SequenceSynthetic (H16A(+92+116)) 83caugcuuccg
ucuucugggu cacug 258423RNAArtificial SequenceSynthetic
(H16A(+45+67)) 84gaucuuguuu gagugaauac agu 238522RNAArtificial
SequenceSynthetic (H16A(+105+126)) 85guuauccagc caugcuuccg uc
228625RNAArtificial SequenceSynthetic (H16D(+05-20)) 86ugauaauugg
uaucacuaac cugug 258722RNAArtificial SequenceSynthetic
(H16D(+12-11)) 87guaucacuaa ccugugcugu ac 228819RNAArtificial
SequenceSynthetic (H19A(+35+53)) 88cugcuggcau cuugcaguu
198931RNAArtificial SequenceSynthetic (H19A(+35+65)) 89gccugagcug
aucugcuggc aucuugcagu u 319028RNAArtificial SequenceSynthetic
(H20A(+44+71)) 90cuggcagaau ucgauccacc ggcuguuc 289122RNAArtificial
SequenceSynthetic (H20A(+147+168)) 91cagcaguagu ugucaucugc uc
229219RNAArtificial SequenceSynthetic (H20A(+185+203)) 92ugauggggug
guggguugg 199325RNAArtificial SequenceSynthetic (H20A(-08+17))
93aucugcauua acacccucua gaaag 259424RNAArtificial SequenceSynthetic
(H20A(+30+53)) 94ccggcuguuc aguuguucug aggc 249528RNAArtificial
SequenceSynthetic (H20A(-11+17)) 95aucugcauua acacccucua gaaagaaa
289628RNAArtificial SequenceSynthetic (H20D(+08-20)) 96gaaggagaag
agauucuuac cuuacaaa 289720RNAArtificial SequenceSynthetic
(H20A(+44+63)) 97auucgaucca ccggcuguuc 209820RNAArtificial
SequenceSynthetic (H20A(+149+168)) 98cagcaguagu ugucaucugc
209922RNAArtificial SequenceSynthetic (H21A(-06+16)) 99gccgguugac
uucauccugu gc 2210022RNAArtificial SequenceSynthetic
(H21A(+85+106)) 100cugcauccag gaacaugggu cc 2210123RNAArtificial
SequenceSynthetic (H21A(+85+108)) 101gucugcaucc aggaacaugg guc
2310224RNAArtificial SequenceSynthetic (H21A(+08+31)) 102guugaagauc
ugauagccgg uuga 2410324RNAArtificial SequenceSynthetic
(H21D(+18-07)) 103uacuuacugu cuguagcucu uucu 2410424RNAArtificial
SequenceSynthetic (H22A(+22+45)) 104cacucauggu cuccugauag cgca
2410522RNAArtificial SequenceSynthetic (H22A(+125+146))
105cugcaauucc ccgagucucu gc 2210623RNAArtificial SequenceSynthetic
(H22A(+47+69)) 106acugcuggac ccauguccug aug 2310721RNAArtificial
SequenceSynthetic (H22A(+80+101)) 107cuaaguugag guauggagag u
2110823RNAArtificial SequenceSynthetic (H22D(+13-11)) 108uauucacaga
ccugcaauuc ccc 2310926RNAArtificial SequenceSynthetic
(H23A(+34+59)) 109acaguggugc ugagauagua uaggcc 2611022RNAArtificial
SequenceSynthetic (H23A(+18+39)) 110uaggccacuu uguugcucuu gc
2211119RNAArtificial SequenceSynthetic (H23A(+72+90)) 111uucagagggc
gcuuucuuc 1911223RNAArtificial SequenceSynthetic (H24A(+48+70))
112gggcaggcca uuccuccuuc aga 2311324RNAArtificial SequenceSynthetic
(H24A(-02+22)) 113ucuucagggu uuguauguga uucu 2411427RNAArtificial
SequenceSynthetic (H25A(+9+36)) 114cugggcugaa uugucugaau aucacug
2711526RNAArtificial SequenceSynthetic (H25A(+131+156))
115cuguuggcac augugauccc acugag 2611624RNAArtificial
SequenceSynthetic (H25D(+16-08)) 116gucuauaccu guuggcacau guga
2411725RNAArtificial SequenceSynthetic (H26A(+132+156))
117ugcuuucugu aauucaucug gaguu 2511826RNAArtificial
SequenceSynthetic (H26A(-07+19)) 118ccuccuuucu ggcauagacc uuccac
2611925RNAArtificial SequenceSynthetic (H26A(+68+92)) 119ugugucaucc
auucgugcau cucug 2512025RNAArtificial SequenceSynthetic
(H27A(+82+106)) 120uuaaggccuc uugugcuaca ggugg 2512123RNAArtificial
SequenceSynthetic (H27A(-4+19)) 121ggggcucuuc uuuagcucuc uga
2312222RNAArtificial SequenceSynthetic (H27D(+19-03)) 122gacuuccaaa
gucuugcauu uc 2212324RNAArtificial SequenceSynthetic (H28A(-05+19))
123gccaacaugc ccaaacuucc uaag 2412426RNAArtificial
SequenceSynthetic (H28A(+99+124)) 124cagagauuuc cucagcuccg ccagga
2612521RNAArtificial SequenceSynthetic (H28D(+16-05)) 125cuuacaucua
gcaccucaga g 2112625RNAArtificial SequenceSynthetic (H29A(+57+81))
126uccgccaucu guuagggucu gugcc 2512725RNAArtificial
SequenceSynthetic (H29A(+18+42)) 127auuuggguua
uccucugaau gucgc 2512822RNAArtificial SequenceSynthetic
(H29D(+17-05)) 128cauaccucuu cauguaguuc cc 2212926RNAArtificial
SequenceSynthetic (H30A(+122+147)) 129cauuugagcu gcguccaccu ugucug
2613026RNAArtificial SequenceSynthetic (H30A(+25+50)) 130uccugggcag
acuggaugcu cuguuc 2613123RNAArtificial SequenceSynthetic
(H30D(+19-04)) 131uugccugggc uuccugaggc auu 2313224RNAArtificial
SequenceSynthetic (H31D(+06-18)) 132uucugaaaua acauauaccu gugc
2413325RNAArtificial SequenceSynthetic (H31D(+03-22)) 133uaguuucuga
aauaacauau accug 2513421RNAArtificial SequenceSynthetic
(H31A(+05+25)) 134gacuugucaa aucagauugg a 2113524RNAArtificial
SequenceSynthetic (H31D(+04-20)) 135guuucugaaa uaacauauac cugu
2413620RNAArtificial SequenceSynthetic (H32D(+04-16)) 136caccagaaau
acauaccaca 2013720RNAArtificial SequenceSynthetic (H32A(+151+170))
137caaugauuua gcugugacug 2013823RNAArtificial SequenceSynthetic
(H32A(+10+32)) 138cgaaacuuca uggagacauc uug 2313925RNAArtificial
SequenceSynthetic (H32A(+49+73)) 139cuuguagacg cugcucaaaa uuggc
2514020RNAArtificial SequenceSynthetic (H33D(+09-11)) 140caugcacaca
ccuuugcucc 2014124RNAArtificial SequenceSynthetic (H33A(+53+76))
141ucuguacaau cugacgucca gucu 2414227RNAArtificial
SequenceSynthetic (H33A(+30+56)) 142gucuuuauca ccauuuccac uucagac
2714325RNAArtificial SequenceSynthetic (H33A(+64+88)) 143ccgucugcuu
uuucuguaca aucug 2514422RNAArtificial SequenceSynthetic
(H34A(+83+104)) 144uccauaucug uagcugccag cc 2214523RNAArtificial
SequenceSynthetic (H34A(+143+165)) 145ccaggcaacu ucagaaucca aau
2314630RNAArtificial SequenceSynthetic (H34A(-20+10)) 146uuucuguuac
cugaaaagaa uuauaaugaa 3014725RNAArtificial SequenceSynthetic
(H34A(+46+70)) 147cauucauuuc cuuucgcauc uuacg 2514826RNAArtificial
SequenceSynthetic (H34A(+95+120)) 148ugaucucuuu gucaauucca uaucug
2614930RNAArtificial SequenceSynthetic (H34D(+10-20)) 149uucagugaua
uagguuuuac cuuuccccag 3015026RNAArtificial SequenceSynthetic
(H34A(+72+96)) 150cuguagcugc cagccauucu gucaag 2615121RNAArtificial
SequenceSynthetic (H35A(+141+161)) 151ucuucugcuc gggaggugac a
2115220RNAArtificial SequenceSynthetic (H35A(+116+135))
152ccaguuacua uucagaagac 2015320RNAArtificial SequenceSynthetic
(H35A(+24+43)) 153ucuucaggug caccuucugu 2015425RNAArtificial
SequenceSynthetic (H36A(+26+50)) 154ugugaugugg uccacauucu gguca
2515520RNAArtificial SequenceSynthetic (H36A(-02+18)) 155ccauguguuu
cugguauucc 2015625RNAArtificial SequenceSynthetic (H37A(+26+50))
156cguguagagu ccaccuuugg gcgua 2515724RNAArtificial
SequenceSynthetic (H37A(+82+105)) 157uacuaauuuc cugcaguggu cacc
2415824RNAArtificial SequenceSynthetic (H37A(+134+157))
158uucuguguga aauggcugca aauc 2415920RNAArtificial
SequenceSynthetic (H38A(-01+19)) 159ccuucaaagg aauggaggcc
2016025RNAArtificial SequenceSynthetic (H38A(+59+83)) 160ugcugaauuu
cagccuccag ugguu 2516125RNAArtificial SequenceSynthetic
(H38A(+88+112)) 161ugaagucuuc cucuuucaga uucac 2516224RNAArtificial
SequenceSynthetic (H39A(+62+85)) 162cuggcuuucu cucaucugug auuc
2416320RNAArtificial SequenceSynthetic (H39A(+39+58)) 163guuguaaguu
gucuccucuu 2016420RNAArtificial SequenceSynthetic (H39A(+102+121))
164uugucuguaa cagcugcugu 2016520RNAArtificial SequenceSynthetic
(H39D(+10-10)) 165gcucuaauac cuugagagca 2016622RNAArtificial
SequenceSynthetic (H40A(-05+17)) 166cuuugagacc ucaaauccug uu
2216725RNAArtificial SequenceSynthetic (H40A(+129+153))
167cuuuauuuuc cuuucaucuc ugggc 2516827RNAArtificial
SequenceSynthetic (H42A(-04+23)) 168aucguuucuu cacggacagu gugcugg
2716924RNAArtificial SequenceSynthetic (H42A(+86+109))
169gggcuuguga gacaugagug auuu 2417022RNAArtificial
SequenceSynthetic (H42D(+19-02)) 170accuucagag gacuccucuu gc
2217125RNAArtificial SequenceSynthetic (H43D(+10-15)) 171uauguguuac
cuacccuugu cgguc 2517220RNAArtificial SequenceSynthetic
(H43A(+101+120)) 172ggagagagcu uccuguagcu 2017323RNAArtificial
SequenceSynthetic (H43A(+78+100)) 173ucacccuuuc cacaggcguu gca
2317420RNAArtificial SequenceSynthetic (H44A(+85+104))
174uuugugucuu ucugagaaac 2017520RNAArtificial SequenceSynthetic
(H44D(+10-10)) 175aaagacuuac cuuaagauac 2017620RNAArtificial
SequenceSynthetic (H44A(-06+14)) 176aucugucaaa ucgccugcag
2017720RNAArtificial SequenceSynthetic (H46D(+16-04)) 177uuaccuugac
uugcucaagc 2017820RNAArtificial SequenceSynthetic (H46A(+90+109))
178uccagguuca agugggauac 2017925RNAArtificial SequenceSynthetic
(H47A(+76+100)) 179gcucuucugg gcuuauggga gcacu 2518027RNAArtificial
SequenceSynthetic (H47D(+25-02)) 180accuuuaucc acuggagauu ugucugc
2718121RNAArtificial SequenceSynthetic (H47A(-9+12)) 181uuccaccagu
aacugaaaca g 2118229RNAArtificial SequenceSynthetic (H50A(+02+30))
182ccacucagag cucagaucuu cuaacuucc 2918327RNAArtificial
SequenceSynthetic (H50A(+07+33)) 183cuuccacuca gagcucagau cuucuaa
2718426RNAArtificial SequenceSynthetic (H51A(-01+25)) 184accagaguaa
cagucugagu aggagc 2618523RNAArtificial SequenceSynthetic
(H51D(+16-07)) 185cucauaccuu cugcuugaug auc 2318624RNAArtificial
SequenceSynthetic (H51A(+111 +134)) 186uucuguccaa gcccgguuga aauc
2418730RNAArtificial SequenceSynthetic (H51A(+66+95)) 187cuccaacauc
aaggaagaug gcauuucuag 3018825RNAArtificial SequenceSynthetic
(H51D(+08-17)) 188aucauuuuuu cucauaccuu cugcu 2518936RNAArtificial
SequenceSynthetic (H51A/D(+08-17)& (-15+)) 189aucauuuuuu
cucauaccuu cugcuaggag cuaaaa 3619021RNAArtificial SequenceSynthetic
(H51A(+175+195)) 190cacccaccau cacccucugu g 2119122RNAArtificial
SequenceSynthetic (H51A(+199+220)) 191aucaucucgu ugauauccuc aa
2219221RNAArtificial SequenceSynthetic (H52A(-07+14)) 192uccugcauug
uugccuguaa g 2119330RNAArtificial SequenceSynthetic (H52A(+12+41))
193uccaacuggg gacgccucug uuccaaaucc 3019421RNAArtificial
SequenceSynthetic (H52A(+17+37)) 194acuggggacg ccucuguucc a
2119520RNAArtificial SequenceSynthetic (H52A(+93+112))
195ccguaaugau uguucuagcc 2019620RNAArtificial SequenceSynthetic
(H52D(+05-15)) 196uguuaaaaaa cuuacuucga 2019725RNAArtificial
SequenceSynthetic (H53A(+45+69)) 197cauucaacug uugccuccgg uucug
2519824RNAArtificial SequenceSynthetic (H53A(+39+62)) 198cuguugccuc
cgguucugaa ggug 2419931RNAArtificial SequenceSynthetic
(H53A(+39+69)) 199cauucaacug uugccuccgg uucugaaggu g
3120021RNAArtificial SequenceSynthetic (H53D(+14-07)) 200uacuaaccuu
gguuucugug a 2120125RNAArtificial SequenceSynthetic (H53A(+23+47))
201cugaaggugu ucuuguacuu caucc 2520227RNAArtificial
SequenceSynthetic (H53A(+150+176)) 202uguauaggga cccuccuucc augacuc
2720325RNAArtificial SequenceSynthetic (H53D(+20-05)) 203cuaaccuugg
uuucugugau uuucu 2520427RNAArtificial SequenceSynthetic
(H53D(+09-18)) 204gguaucuuug auacuaaccu ugguuuc
2720522RNAArtificial SequenceSynthetic (H53A(-12+10)) 205auucuuucaa
cuagaauaaa ag 2220625RNAArtificial SequenceSynthetic (H53A(-07+18))
206gauucugaau ucuuucaacu agaau 2520720RNAArtificial
SequenceSynthetic (H53A(+07+26)) 207aucccacuga uucugaauuc
2020822RNAArtificial SequenceSynthetic (H53A(+124+145))
208uuggcucugg ccuguccuaa ga 2220930RNAArtificial SequenceSynthetic
(H46A(+86+115)) 209cucuuuucca gguucaagug ggauacuagc
3021031RNAArtificial SequenceSynthetic (H46A(+107+137))
210caagcuuuuc uuuuaguugc ugcucuuuuc c 3121130RNAArtificial
SequenceSynthetic (H46A(-10+20)) 211uauucuuuug uucuucuagc
cuggagaaag 3021228RNAArtificial SequenceSynthetic (H46A(+50+77))
212cugcuuccuc caaccauaaa acaaauuc 2821326RNAArtificial
SequenceSynthetic (H45A(-06+20)) 213ccaaugccau ccuggaguuc cuguaa
2621420RNAArtificial SequenceSynthetic (H45A(+91 +110))
214uccuguagaa uacuggcauc 2021527RNAArtificial SequenceSynthetic
(H45A(+125+151)) 215ugcagaccuc cugccaccgc agauuca
2721620RNAArtificial SequenceSynthetic (H45D(+16 -04))
216cuaccucuuu uuucugucug 2021720RNAArtificial SequenceSynthetic
(H45A(+71+90)) 217uguuuuugag gauugcugaa 2021828DNAArtificial
SequenceSynthetic (H53A(+33+60)) 218gttgcctccg gttctgaagg tgttcttg
2821925DNAArtificial SequenceSynthetic (H53A(+23+47)) 219ctgaaggtgt
tcttgtactt catcc 2522030DNAArtificial SequenceSynthetic
(H53A(+33+62)) 220ctgttgcctc cggttctgaa ggtgttcttg
3022133DNAArtificial SequenceSynthetic (H53A(+33+65)) 221caactgttgc
ctccggttct gaaggtgttc ttg 3322225DNAArtificial SequenceSynthetic
(H53A(+31+55)) 222ctccggttct gaaggtgttc ttgta 2522328DNAArtificial
SequenceSynthetic (H53A(+46+73)) 223atttcattca actgttgcct ccggttct
2822425DNAArtificial SequenceSynthetic (H53A(+22+46)) 224tgaaggtgtt
cttgtacttc atccc 2522524DNAArtificial SequenceSynthetic
(H53A(+46+69)) 225cattcaactg ttgcctccgg ttct 2422622DNAArtificial
SequenceSynthetic (H53A(+40+61)) 226tgttgcctcc ggttctgaag gt
2222725DNAArtificial SequenceSynthetic (H53A(+30+60)) 227gttgcctccg
gttctgaagg tgttc 2522828DNAArtificial SequenceSynthetic
(H53A(+30+57)) 228gcctccggtt ctgaaggtgt tcttgtac
2822927DNAArtificial SequenceSynthetic (H53A(+30+56)) 229cctccggttc
tgaaggtgtt cttgtac 2723026DNAArtificial SequenceSynthetic
(H53A(+30+55)) 230ctccggttct gaaggtgttc ttgtac 2623125DNAArtificial
SequenceSynthetic (H53A(+33+57)) 231gcctccggtt ctgaaggtgt tcttg
2523224DNAArtificial SequenceSynthetic (H44A(-07+17)) 232cagatctgtc
aaatcgcctg cagg 2423327DNAArtificial SequenceSynthetic
(H44A(-07+20)) 233caacagatct gtcaaatcgc ctgcagg
2723429DNAArtificial SequenceSynthetic (H44A(-07+22)) 234ctcaacagat
ctgtcaaatc gcctgcagg 2923525DNAArtificial SequenceSynthetic
(H44A(+77+101)) 235gtgtctttct gagaaactgt tcagc 2523628DNAArtificial
SequenceSynthetic (H44A(+64+91)) 236gagaaactgt tcagcttctg ttagccac
2823728DNAArtificial SequenceSynthetic (H44A(+62+89)) 237gaaactgttc
agcttctgtt agccactg 2823824DNAArtificial SequenceSynthetic
(H44A(+62+85)) 238ctgttcagct tctgttagcc actg 2423920DNAArtificial
SequenceSynthetic (H44A(-06+14)) 239atctgtcaaa tcgcctgcag
2024020DNAArtificial SequenceSynthetic (H44A(+85+104))
240tttgtgtctt tctgagaaac 2024124DNAArtificial SequenceSynthetic
(H44A(+61+84)) 241tgttcagctt ctgttagcca ctga 2424225DNAArtificial
SequenceSynthetic (H44A(-10+15)) 242gatctgtcaa atcgcctgca ggtaa
2524325DNAArtificial SequenceSynthetic (H44A(+64+88)) 243aaactgttca
gcttctgtta gccac 2524425DNAArtificial SequenceSynthetic
(H44A(+79+103)) 244ttgtgtcttt ctgagaaact gttca 2524526DNAArtificial
SequenceSynthetic (H44A(-06+20)) 245caacagatct gtcaaatcgc ctgcag
2624626DNAArtificial SequenceSynthetic (H44A(-09+17)) 246cagatctgtc
aaatcgcctg caggta 2624727DNAArtificial SequenceSynthetic
(H44A(+59+85)) 247ctgttcagct tctgttagcc actgatt
2724831DNAArtificial SequenceSynthetic (H44A(+59+89)) 248gaaactgttc
agcttctgtt agccactgat t 3124926DNAArtificial SequenceSynthetic
(H44A(+65+90)) 249agaaactgtt cagcttctgt tagcca 2625025DNAArtificial
SequenceSynthetic (Hu.DMD.Exon44.25.001) 250ctgcaggtaa aagcatatgg
atcaa 2525125DNAArtificial SequenceSynthetic (Hu.DMD.Exon44.25.002)
251atcgcctgca ggtaaaagca tatgg 2525225DNAArtificial
SequenceSynthetic (Hu.DMD.Exon44.25.003) 252gtcaaatcgc ctgcaggtaa
aagca
2525325DNAArtificial SequenceSynthetic (Hu.DMD.Exon44.25.005)
253caacagatct gtcaaatcgc ctgca 2525425DNAArtificial
SequenceSynthetic (Hu.DMD.Exon44.25.006) 254tttctcaaca gatctgtcaa
atcgc 2525525DNAArtificial SequenceSynthetic (Hu.DMD.Exon44.25.007)
255ccatttctca acagatctgt caaat 2525625DNAArtificial
SequenceSynthetic (Hu.DMD.Exon44.25.008) 256ataatgaaaa cgccgccatt
tctca 2525725DNAArtificial SequenceSynthetic (Hu.DMD.Exon44.25.009)
257aaatatcttt atatcataat gaaaa 2525825DNAArtificial
SequenceSynthetic (Hu.DMD.Exon44.25.010) 258tgttagccac tgattaaata
tcttt 2525925DNAArtificial SequenceSynthetic (Hu.DMD.Exon44.25.013)
259ccaattctca ggaatttgtg tcttt 2526025DNAArtificial
SequenceSynthetic (Hu.DMD.Exon44.25.014) 260gtatttagca tgttcccaat
tctca 2526125DNAArtificial SequenceSynthetic (Hu.DMD.Exon44.25.015)
261cttaagatac catttgtatt tagca 2526225DNAArtificial
SequenceSynthetic (Hu.DMD.Exon44.25.016) 262cttaccttaa gataccattt
gtatt 2526325DNAArtificial SequenceSynthetic (Hu.DMD.Exon44.25.017)
263aaagacttac cttaagatac cattt 2526425DNAArtificial
SequenceSynthetic (Hu.DMD.Exon44.25.018) 264aaatcaaaga cttaccttaa
gatac 2526525DNAArtificial SequenceSynthetic (Hu.DMD.Exon44.25.019)
265aaaacaaatc aaagacttac cttaa 2526625DNAArtificial
SequenceSynthetic (Hu.DMD.Exon44.25.020) 266tcgaaaaaac aaatcaaaga
cttac 2526725DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.25.001)
267ctgtaagata ccaaaaaggc aaaac 2526825DNAArtificial
SequenceSynthetic (Hu.DMD.Exon45.25.002) 268cctgtaagat accaaaaagg
caaaa 2526925DNAArtificial SequenceSynthetic
(Hu.DMD.Exon45.25.002.2) 269agttcctgta agataccaaa aaggc
2527025DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.25.003)
270gagttcctgt aagataccaa aaagg 2527125DNAArtificial
SequenceSynthetic (Hu.DMD.Exon45.25.003.2) 271cctggagttc ctgtaagata
ccaaa 2527225DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.25.004)
272tcctggagtt cctgtaagat accaa 2527325DNAArtificial
SequenceSynthetic (Hu.DMD.Exon45.25.004.2) 273gccatcctgg agttcctgta
agata 2527425DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.25.005)
274tgccatcctg gagttcctgt aagat 2527525DNAArtificial
SequenceSynthetic (Hu.DMD.Exon45.25.005.2) 275ccaatgccat cctggagttc
ctgta 2527625DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.25.006)
276cccaatgcca tcctggagtt cctgt 2527725DNAArtificial
SequenceSynthetic (Hu.DMD.Exon45.25.006.2) 277gctgcccaat gccatcctgg
agttc 2527825DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.25.007)
278cgctgcccaa tgccatcctg gagtt 2527925DNAArtificial
SequenceSynthetic (Hu.DMD.Exon45.25.008) 279aacagtttgc cgctgcccaa
tgcca 2528025DNAArtificial SequenceSynthetic
(Hu.DMD.Exon45.25.008.2) 280ctgacaacag tttgccgctg cccaa
2528125DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.25.009)
281gttgcattca atgttctgac aacag 2528225DNAArtificial
SequenceSynthetic (Hu.DMD.Exon45.25.010) 282gctgaattat ttcttcccca
gttgc 2528325DNAArtificial SequenceSynthetic
(Hu.DMD.Exon45.25.010.2) 283attatttctt ccccagttgc attca
2528425DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.25.011)
284ggcatctgtt tttgaggatt gctga 2528525DNAArtificial
SequenceSynthetic (Hu.DMD.Exon45.25.011.2) 285tttgaggatt gctgaattat
ttctt 2528625DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.25.012)
286aatttttcct gtagaatact ggcat 2528725DNAArtificial
SequenceSynthetic (Hu.DMD.Exon45.25.012.2) 287atactggcat ctgtttttga
ggatt 2528825DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.25.013)
288accgcagatt caggcttccc aattt 2528925DNAArtificial
SequenceSynthetic (Hu.DMD.Exon45.25.014) 289ctgtttgcag acctcctgcc
accgc 2529025DNAArtificial SequenceSynthetic
(Hu.DMD.Exon45.25.014.2) 290agattcaggc ttcccaattt ttcct
2529125DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.25.015)
291ctcttttttc tgtctgacag ctgtt 2529225DNAArtificial
SequenceSynthetic (Hu.DMD.Exon45.25.015.2) 292acctcctgcc accgcagatt
caggc 2529325DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.25.016)
293cctacctctt ttttctgtct gacag 2529425DNAArtificial
SequenceSynthetic (Hu.DMD.Exon45.25.016.2) 294gacagctgtt tgcagacctc
ctgcc 2529525DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.25.017)
295gtcgccctac ctcttttttc tgtct 2529625DNAArtificial
SequenceSynthetic (Hu.DMD.Exon45.25.018) 296gatctgtcgc cctacctctt
ttttc 2529725DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.25.019)
297tattagatct gtcgccctac ctctt 2529825DNAArtificial
SequenceSynthetic (Hu.DMD.Exon45.25.020) 298attcctatta gatctgtcgc
cctac 2529920DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.20.001)
299agataccaaa aaggcaaaac 2030020DNAArtificial SequenceSynthetic
(Hu.DMD.Exon45.20.002) 300aagataccaa aaaggcaaaa
2030120DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.20.003)
301cctgtaagat accaaaaagg 2030220DNAArtificial SequenceSynthetic
(Hu.DMD.Exon45.20.004) 302gagttcctgt aagataccaa
2030320DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.20.005)
303tcctggagtt cctgtaagat 2030420DNAArtificial SequenceSynthetic
(Hu.DMD.Exon45.20.006) 304tgccatcctg gagttcctgt
2030520DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.20.007)
305cccaatgcca tcctggagtt 2030620DNAArtificial SequenceSynthetic
(Hu.DMD.Exon45.20.008) 306cgctgcccaa tgccatcctg
2030720DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.20.009)
307ctgacaacag tttgccgctg 2030820DNAArtificial SequenceSynthetic
(Hu.DMD.Exon45.20.010) 308gttgcattca atgttctgac
2030920DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.20.011)
309attatttctt ccccagttgc 2031020DNAArtificial SequenceSynthetic
(Hu.DMD.Exon45.20.012) 310tttgaggatt gctgaattat
2031120DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.20.013)
311atactggcat ctgtttttga 2031220DNAArtificial SequenceSynthetic
(Hu.DMD.Exon45.20.014) 312aatttttcct gtagaatact
2031320DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.20.015)
313agattcaggc ttcccaattt 2031420DNAArtificial SequenceSynthetic
(Hu.DMD.Exon45.20.016) 314acctcctgcc accgcagatt
2031520DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.20.017)
315gacagctgtt tgcagacctc 2031620DNAArtificial SequenceSynthetic
(Hu.DMD.Exon45.20.018) 316ctcttttttc tgtctgacag
2031720DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.20.019)
317cctacctctt ttttctgtct 2031820DNAArtificial SequenceSynthetic
(Hu.DMD.Exon45.20.020) 318gtcgccctac ctcttttttc
2031920DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.20.021)
319gatctgtcgc cctacctctt 2032020DNAArtificial SequenceSynthetic
(Hu.DMD.Exon45.20.022) 320tattagatct gtcgccctac
2032120DNAArtificial SequenceSynthetic (Hu.DMD.Exon45.20.023)
321attcctatta gatctgtcgc 2032225DNAArtificial SequenceSynthetic
(Hu.DMD.Exon46.25.001) 322gggggatttg agaaaataaa attac
2532325DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.25.002)
323atttgagaaa ataaaattac cttga 2532425DNAArtificial
SequenceSynthetic (Hu.DMD.Exon46.25.002.2) 324ctagcctgga gaaagaagaa
taaaa 2532525DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.25.003)
325agaaaataaa attaccttga cttgc 2532625DNAArtificial
SequenceSynthetic (Hu.DMD.Exon46.25.003.2) 326ttcttctagc ctggagaaag
aagaa 2532725DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.25.004)
327ataaaattac cttgacttgc tcaag 2532825DNAArtificial
SequenceSynthetic (Hu.DMD.Exon46.25.004.2) 328ttttgttctt ctagcctgga
gaaag 2532925DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.25.005)
329attaccttga cttgctcaag ctttt 2533025DNAArtificial
SequenceSynthetic (Hu.DMD.Exon46.25.005.2) 330tattcttttg ttcttctagc
ctgga 2533125DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.25.006)
331cttgacttgc tcaagctttt ctttt 2533225DNAArtificial
SequenceSynthetic (Hu.DMD.Exon46.25.006.2) 332caagatattc ttttgttctt
ctagc 2533325DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.25.007)
333cttttagttg ctgctctttt ccagg 2533425DNAArtificial
SequenceSynthetic (Hu.DMD.Exon46.25.008) 334ccaggttcaa gtgggatact
agcaa 2533525DNAArtificial SequenceSynthetic
(Hu.DMD.Exon46.25.008.2) 335atctctttga aattctgaca agata
2533625DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.25.009)
336agcaatgtta tctgcttcct ccaac 2533725DNAArtificial
SequenceSynthetic (Hu.DMD.Exon46.25.009.2) 337aacaaattca tttaaatctc
tttga 2533825DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.25.010)
338ccaaccataa aacaaattca tttaa 2533925DNAArtificial
SequenceSynthetic (Hu.DMD.Exon46.25.010.2) 339ttcctccaac cataaaacaa
attca 2534025DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.25.011)
340tttaaatctc tttgaaattc tgaca 2534125DNAArtificial
SequenceSynthetic (Hu.DMD.Exon46.25.012) 341tgacaagata ttcttttgtt
cttct 2534225DNAArtificial SequenceSynthetic
(Hu.DMD.Exon46.25.012.2) 342ttcaagtggg atactagcaa tgtta
2534325DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.25.013)
343agatattctt ttgttcttct agcct 2534425DNAArtificial
SequenceSynthetic (Hu.DMD.Exon46.25.013.2) 344ctgctctttt ccaggttcaa
gtggg 2534525DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.25.014)
345ttcttttgtt cttctagcct ggaga 2534625DNAArtificial
SequenceSynthetic (Hu.DMD.Exon46.25.014.2) 346cttttctttt agttgctgct
ctttt 2534725DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.25.015)
347ttgttcttct agcctggaga aagaa 2534825DNAArtificial
SequenceSynthetic (Hu.DMD.Exon46.25.016) 348cttctagcct ggagaaagaa
gaata 2534925DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.25.017)
349agcctggaga aagaagaata aaatt 2535025DNAArtificial
SequenceSynthetic (Hu.DMD.Exon46.25.018) 350ctggagaaag aagaataaaa
ttgtt 2535120DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.20.001)
351gaaagaagaa taaaattgtt 2035220DNAArtificial SequenceSynthetic
(Hu.DMD.Exon46.20.002) 352ggagaaagaa gaataaaatt
2035320DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.20.003)
353agcctggaga aagaagaata 2035420DNAArtificial SequenceSynthetic
(Hu.DMD.Exon46.20.004) 354cttctagcct ggagaaagaa
2035520DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.20.005)
355ttgttcttct agcctggaga 2035620DNAArtificial SequenceSynthetic
(Hu.DMD.Exon46.20.006) 356ttcttttgtt cttctagcct
2035720DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.20.007)
357tgacaagata ttcttttgtt 2035820DNAArtificial SequenceSynthetic
(Hu.DMD.Exon46.20.008) 358atctctttga aattctgaca
2035920DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.20.009)
359aacaaattca tttaaatctc 2036020DNAArtificial SequenceSynthetic
(Hu.DMD.Exon46.20.010) 360ttcctccaac cataaaacaa
2036120DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.20.011)
361agcaatgtta tctgcttcct 2036220DNAArtificial SequenceSynthetic
(Hu.DMD.Exon46.20.012) 362ttcaagtggg atactagcaa
2036320DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.20.013)
363ctgctctttt ccaggttcaa 2036420DNAArtificial SequenceSynthetic
(Hu.DMD.Exon46.20.014) 364cttttctttt agttgctgct
2036520DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.20.015)
365cttgacttgc tcaagctttt 2036620DNAArtificial SequenceSynthetic
(Hu.DMD.Exon46.20.016) 366attaccttga cttgctcaag
2036720DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.20.017)
367ataaaattac cttgacttgc 2036820DNAArtificial SequenceSynthetic
(Hu.DMD.Exon46.20.018) 368agaaaataaa attaccttga
2036920DNAArtificial SequenceSynthetic (Hu.DMD.Exon46.20.019)
369atttgagaaa ataaaattac 2037020DNAArtificial SequenceSynthetic
(Hu.DMD.Exon46.20.020) 370gggggatttg agaaaataaa
2037125DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.25.001)
371ctgaaacaga caaatgcaac aacgt 2537225DNAArtificial
SequenceSynthetic (Hu.DMD.Exon47.25.002) 372agtaactgaa acagacaaat
gcaac 2537325DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.25.003)
373ccaccagtaa ctgaaacaga caaat 2537425DNAArtificial
SequenceSynthetic (Hu.DMD.Exon47.25.004) 374ctcttccacc agtaactgaa
acaga 2537525DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.25.005)
375ggcaactctt ccaccagtaa ctgaa 2537625DNAArtificial
SequenceSynthetic (Hu.DMD.Exon47.25.006) 376gcaggggcaa ctcttccacc
agtaa 2537725DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.25.007)
377ctggcgcagg ggcaactctt ccacc 2537825DNAArtificial
SequenceSynthetic (Hu.DMD.Exon47.25.008)
378tttaattgtt tgagaattcc ctggc 2537925DNAArtificial
SequenceSynthetic (Hu.DMD.Exon47.25.008.2) 379ttgtttgaga attccctggc
gcagg 2538025DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.25.009)
380gcacgggtcc tccagtttca tttaa 2538125DNAArtificial
SequenceSynthetic (Hu.DMD.Exon47.25.009.2) 381tccagtttca tttaattgtt
tgaga 2538225DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.25.010)
382gcttatggga gcacttacaa gcacg 2538325DNAArtificial
SequenceSynthetic (Hu.DMD.Exon47.25.010.2) 383tacaagcacg ggtcctccag
tttca 2538425DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.25.011)
384agtttatctt gctcttctgg gctta 2538525DNAArtificial
SequenceSynthetic (Hu.DMD.Exon47.25.012) 385tctgcttgag cttattttca
agttt 2538625DNAArtificial SequenceSynthetic
(Hu.DMD.Exon47.25.012.2) 386atcttgctct tctgggctta tggga
2538725DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.25.013)
387ctttatccac tggagatttg tctgc 2538825DNAArtificial
SequenceSynthetic (Hu.DMD.Exon47.25.013.2) 388cttattttca agtttatctt
gctct 2538925DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.25.014)
389ctaaccttta tccactggag atttg 2539025DNAArtificial
SequenceSynthetic (Hu.DMD.Exon47.25.014.2) 390atttgtctgc ttgagcttat
tttca 2539125DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.25.015)
391aatgtctaac ctttatccac tggag 2539225DNAArtificial
SequenceSynthetic (Hu.DMD.Exon47.25.016) 392tggttaatgt ctaaccttta
tccac 2539325DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.25.017)
393agagatggtt aatgtctaac cttta 2539425DNAArtificial
SequenceSynthetic (Hu.DMD.Exon47.25.018) 394acggaagaga tggttaatgt
ctaac 2539520DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.20.001)
395acagacaaat gcaacaacgt 2039620DNAArtificial SequenceSynthetic
(Hu.DMD.Exon47.20.002) 396ctgaaacaga caaatgcaac
2039720DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.20.003)
397agtaactgaa acagacaaat 2039820DNAArtificial SequenceSynthetic
(Hu.DMD.Exon47.20.004) 398ccaccagtaa ctgaaacaga
2039920DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.20.005)
399ctcttccacc agtaactgaa 2040020DNAArtificial SequenceSynthetic
(Hu.DMD.Exon47.20.006) 400ggcaactctt ccaccagtaa
2040120DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.20.007)
401ctggcgcagg ggcaactctt 2040220DNAArtificial SequenceSynthetic
(Hu.DMD.Exon47.20.008) 402ttgtttgaga attccctggc
2040320DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.20.009)
403tccagtttca tttaattgtt 2040420DNAArtificial SequenceSynthetic
(Hu.DMD.Exon47.20.010) 404tacaagcacg ggtcctccag
2040520DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.20.011)
405gcttatggga gcacttacaa 2040620DNAArtificial SequenceSynthetic
(Hu.DMD.Exon47.20.012) 406atcttgctct tctgggctta
2040720DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.20.013)
407cttattttca agtttatctt 2040820DNAArtificial SequenceSynthetic
(Hu.DMD.Exon47.20.014) 408atttgtctgc ttgagcttat
2040920DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.20.015)
409ctttatccac tggagatttg 2041020DNAArtificial SequenceSynthetic
(Hu.DMD.Exon47.20.016) 410ctaaccttta tccactggag
2041120DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.20.017)
411aatgtctaac ctttatccac 2041220DNAArtificial SequenceSynthetic
(Hu.DMD.Exon47.20.018) 412tggttaatgt ctaaccttta
2041320DNAArtificial SequenceSynthetic (Hu.DMD.Exon47.20.019)
413agagatggtt aatgtctaac 2041420DNAArtificial SequenceSynthetic
(Hu.DMD.Exon47.20.020) 414acggaagaga tggttaatgt
2041525DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.25.001)
415ctgaaaggaa aatacatttt aaaaa 2541625DNAArtificial
SequenceSynthetic (Hu.DMD.Exon48.25.002) 416cctgaaagga aaatacattt
taaaa 2541725DNAArtificial SequenceSynthetic
(Hu.DMD.Exon48.25.002.2) 417gaaacctgaa aggaaaatac atttt
2541825DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.25.003)
418ggaaacctga aaggaaaata cattt 2541925DNAArtificial
SequenceSynthetic (Hu.DMD.Exon48.25.003.2) 419ctctggaaac ctgaaaggaa
aatac 2542025DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.25.004)
420gctctggaaa cctgaaagga aaata 2542125DNAArtificial
SequenceSynthetic (Hu.DMD.Exon48.25.004.2) 421taaagctctg gaaacctgaa
aggaa 2542225DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.25.005)
422gtaaagctct ggaaacctga aagga 2542325DNAArtificial
SequenceSynthetic (Hu.DMD.Exon48.25.005.2) 423tcaggtaaag ctctggaaac
ctgaa 2542425DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.25.006)
424ctcaggtaaa gctctggaaa cctga 2542525DNAArtificial
SequenceSynthetic (Hu.DMD.Exon48.25.006.2) 425gtttctcagg taaagctctg
gaaac 2542625DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.25.007)
426tgtttctcag gtaaagctct ggaaa 2542725DNAArtificial
SequenceSynthetic (Hu.DMD.Exon48.25.007.2) 427aatttctcct tgtttctcag
gtaaa 2542825DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.25.008)
428tttgagcttc aatttctcct tgttt 2542925DNAArtificial
SequenceSynthetic (Hu.DMD.Exon48.25.008) 429ttttatttga gcttcaattt
ctcct 2543025DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.25.009)
430aagctgccca aggtctttta tttga 2543125DNAArtificial
SequenceSynthetic (Hu.DMD.Exon48.25.010) 431aggtcttcaa gctttttttc
aagct 2543225DNAArtificial SequenceSynthetic
(Hu.DMD.Exon48.25.010.2) 432ttcaagcttt ttttcaagct gccca
2543325DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.25.011)
433gatgatttaa ctgctcttca aggtc 2543425DNAArtificial
SequenceSynthetic (Hu.DMD.Exon48.25.011.2) 434ctgctcttca aggtcttcaa
gcttt 2543525DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.25.012)
435aggagataac cacagcagca gatga 2543625DNAArtificial
SequenceSynthetic (Hu.DMD.Exon48.25.012.2) 436cagcagatga tttaactgct
cttca 2543725DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.25.013)
437atttccaact gattcctaat aggag 2543825DNAArtificial
SequenceSynthetic (Hu.DMD.Exon48.25.014) 438cttggtttgg ttggttataa
atttc 2543925DNAArtificial SequenceSynthetic
(Hu.DMD.Exon48.25.014.2) 439caactgattc ctaataggag ataac
2544025DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.25.015)
440cttaacgtca aatggtcctt cttgg 2544125DNAArtificial
SequenceSynthetic (Hu.DMD.Exon48.25.015.2) 441ttggttataa atttccaact
gattc 2544225DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.25.016)
442cctaccttaa cgtcaaatgg tcctt 2544325DNAArtificial
SequenceSynthetic (Hu.DMD.Exon48.25.016.2) 443tccttcttgg tttggttggt
tataa 2544425DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.25.017)
444agttccctac cttaacgtca aatgg 2544525DNAArtificial
SequenceSynthetic (Hu.DMD.Exon48.25.018) 445caaaaagttc cctaccttaa
cgtca 2544625DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.25.019)
446taaagcaaaa agttccctac cttaa 2544725DNAArtificial
SequenceSynthetic (Hu.DMD.Exon48.25.020) 447atatttaaag caaaaagttc
cctac 2544820DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.20.001)
448aggaaaatac attttaaaaa 2044920DNAArtificial SequenceSynthetic
(Hu.DMD.Exon48.20.002) 449aaggaaaata cattttaaaa
2045020DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.20.003)
450cctgaaagga aaatacattt 2045120DNAArtificial SequenceSynthetic
(Hu.DMD.Exon48.20.004) 451ggaaacctga aaggaaaata
2045220DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.20.005)
452gctctggaaa cctgaaagga 2045320DNAArtificial SequenceSynthetic
(Hu.DMD.Exon48.20.006) 453gtaaagctct ggaaacctga
2045420DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.20.007)
454ctcaggtaaa gctctggaaa 2045520DNAArtificial SequenceSynthetic
(Hu.DMD.Exon48.20.008) 455aatttctcct tgtttctcag
2045620DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.20.009)
456ttttatttga gcttcaattt 2045720DNAArtificial SequenceSynthetic
(Hu.DMD.Exon48.20.010) 457aagctgccca aggtctttta
2045820DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.20.011)
458ttcaagcttt ttttcaagct 2045920DNAArtificial SequenceSynthetic
(Hu.DMD.Exon48.20.012) 459ctgctcttca aggtcttcaa
2046020DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.20.013)
460cagcagatga tttaactgct 2046120DNAArtificial SequenceSynthetic
(Hu.DMD.Exon48.20.014) 461aggagataac cacagcagca
2046220DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.20.015)
462caactgattc ctaataggag 2046320DNAArtificial SequenceSynthetic
(Hu.DMD.Exon48.20.016) 463ttggttataa atttccaact
2046420DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.20.017)
464tccttcttgg tttggttggt 2046520DNAArtificial SequenceSynthetic
(Hu.DMD.Exon48.20.018) 465cttaacgtca aatggtcctt
2046620DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.20.019)
466cctaccttaa cgtcaaatgg 2046720DNAArtificial SequenceSynthetic
(Hu.DMD.Exon48.20.020) 467agttccctac cttaacgtca
2046820DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.20.021)
468caaaaagttc cctaccttaa 2046920DNAArtificial SequenceSynthetic
(Hu.DMD.Exon48.20.022) 469taaagcaaaa agttccctac
2047020DNAArtificial SequenceSynthetic (Hu.DMD.Exon48.20.023)
470atatttaaag caaaaagttc 2047125DNAArtificial SequenceSynthetic
(Hu.DMD.Exon49.25.001) 471ctggggaaaa gaacccatat agtgc
2547225DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.25.002)
472tcctggggaa aagaacccat atagt 2547325DNAArtificial
SequenceSynthetic (Hu.DMD.Exon49.25.002.2) 473gtttcctggg gaaaagaacc
catat 2547425DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.25.003)
474cagtttcctg gggaaaagaa cccat 2547525DNAArtificial
SequenceSynthetic (Hu.DMD.Exon49.25.003.2) 475tttcagtttc ctggggaaaa
gaacc 2547625DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.25.004)
476tatttcagtt tcctggggaa aagaa 2547725DNAArtificial
SequenceSynthetic (Hu.DMD.Exon49.25.004.2) 477tgctatttca gtttcctggg
gaaaa 2547825DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.25.005)
478actgctattt cagtttcctg gggaa 2547925DNAArtificial
SequenceSynthetic (Hu.DMD.Exon49.25.005.2) 479tgaactgcta tttcagtttc
ctggg 2548025DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.25.006)
480cttgaactgc tatttcagtt tcctg 2548125DNAArtificial
SequenceSynthetic (Hu.DMD.Exon49.25.006.2) 481tagcttgaac tgctatttca
gtttc 2548225DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.25.007)
482tttagcttga actgctattt cagtt 2548325DNAArtificial
SequenceSynthetic (Hu.DMD.Exon49.25.008) 483ttccacatcc ggttgtttag
cttga 2548425DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.25.009)
484tgccctttag acaaaatctc ttcca 2548525DNAArtificial
SequenceSynthetic (Hu.DMD.Exon49.25.009.2) 485tttagacaaa atctcttcca
catcc 2548625DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.25.010)
486gtttttcctt gtacaaatgc tgccc 2548725DNAArtificial
SequenceSynthetic (Hu.DMD.Exon49.25.010.2) 487gtacaaatgc tgccctttag
acaaa 2548825DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.25.011)
488cttcactggc tgagtggctg gtttt 2548925DNAArtificial
SequenceSynthetic (Hu.DMD.Exon49.25.011.2) 489ggctggtttt tccttgtaca
aatgc 2549025DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.25.012)
490attaccttca ctggctgagt ggctg 2549125DNAArtificial
SequenceSynthetic (Hu.DMD.Exon49.25.013) 491gcttcattac cttcactggc
tgagt 2549225DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.25.014)
492aggttgcttc attaccttca ctggc 2549325DNAArtificial
SequenceSynthetic (Hu.DMD.Exon49.25.015) 493gctagaggtt gcttcattac
cttca 2549425DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.25.016)
494atattgctag aggttgcttc attac 2549520DNAArtificial
SequenceSynthetic (Hu.DMD.Exon49.20.001) 495gaaaagaacc catatagtgc
2049620DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.20.002)
496gggaaaagaa cccatatagt 2049720DNAArtificial SequenceSynthetic
(Hu.DMD.Exon49.20.003) 497tcctggggaa aagaacccat
2049820DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.20.004)
498cagtttcctg gggaaaagaa 2049920DNAArtificial SequenceSynthetic
(Hu.DMD.Exon49.20.005) 499tatttcagtt tcctggggaa
2050020DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.20.006)
500actgctattt cagtttcctg 2050120DNAArtificial SequenceSynthetic
(Hu.DMD.Exon49.20.007) 501cttgaactgc tatttcagtt
2050220DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.20.008)
502tttagcttga actgctattt 2050320DNAArtificial SequenceSynthetic
(Hu.DMD.Exon49.20.009) 503ttccacatcc ggttgtttag
2050420DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.20.010)
504tttagacaaa atctcttcca 2050520DNAArtificial SequenceSynthetic
(Hu.DMD.Exon49.20.011) 505gtacaaatgc tgccctttag
2050620DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.20.012)
506ggctggtttt tccttgtaca 2050720DNAArtificial SequenceSynthetic
(Hu.DMD.Exon49.20.013) 507cttcactggc tgagtggctg
2050820DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.20.014)
508attaccttca ctggctgagt 2050920DNAArtificial SequenceSynthetic
(Hu.DMD.Exon49.20.015) 509gcttcattac cttcactggc
2051020DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.20.016)
510aggttgcttc attaccttca 2051120DNAArtificial SequenceSynthetic
(Hu.DMD.Exon49.20.017) 511gctagaggtt gcttcattac
2051220DNAArtificial SequenceSynthetic (Hu.DMD.Exon49.20.018)
512atattgctag aggttgcttc 2051325DNAArtificial SequenceSynthetic
(Hu.DMD.Exon50.25.001) 513ctttaacaga aaagcataca catta
2551425DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.25.002)
514tcctctttaa cagaaaagca tacac 2551525DNAArtificial
SequenceSynthetic (Hu.DMD.Exon50.25.002.2) 515ttcctcttta acagaaaagc
ataca 2551625DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.25.003)
516taacttcctc tttaacagaa aagca 2551725DNAArtificial
SequenceSynthetic (Hu.DMD.Exon50.25.003.2) 517ctaacttcct ctttaacaga
aaagc 2551825DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.25.004)
518tcttctaact tcctctttaa cagaa 2551925DNAArtificial
SequenceSynthetic (Hu.DMD.Exon50.25.004.2) 519atcttctaac ttcctcttta
acaga 2552025DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.25.005)
520tcagatcttc taacttcctc tttaa 2552125DNAArtificial
SequenceSynthetic (Hu.DMD.Exon50.25.005.2) 521ctcagatctt ctaacttcct
cttta 2552225DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.25.006)
522agagctcaga tcttctaact tcctc 2552325DNAArtificial
SequenceSynthetic (Hu.DMD.Exon50.25.006.2 NG-08-0731) 523cagagctcag
atcttctaac ttcct 2552424DNAArtificial SequenceSynthetic
(Hu.DMD.Exon50.25.007) 524cactcagagc tcagatcttc tact
2452525DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.25.007.2)
525ccttccactc agagctcaga tcttc 2552625DNAArtificial
SequenceSynthetic (Hu.DMD.Exon50.25.008) 526gtaaacggtt taccgccttc
cactc 2552725DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.25.009)
527ctttgccctc agctcttgaa gtaaa 2552825DNAArtificial
SequenceSynthetic (Hu.DMD.Exon50.25.009.2) 528ccctcagctc ttgaagtaaa
cggtt 2552925DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.25.010)
529ccaggagcta ggtcaggctg ctttg 2553025DNAArtificial
SequenceSynthetic (Hu.DMD.Exon50.25.010.2) 530ggtcaggctg ctttgccctc
agctc 2553125DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.25.011)
531aggctccaat agtggtcagt ccagg 2553225DNAArtificial
SequenceSynthetic (Hu.DMD.Exon50.25.011.2) 532tcagtccagg agctaggtca
ggctg 2553325DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.25.012
AVI-5038) 533cttacaggct ccaatagtgg tcagt 2553425DNAArtificial
SequenceSynthetic (Hu.DMD.Exon50.25.013) 534gtatacttac aggctccaat
agtgg 2553525DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.25.014)
535atccagtata cttacaggct ccaat 2553625DNAArtificial
SequenceSynthetic (Hu.DMD.Exon50.25.015 NG-08-0741) 536atgggatcca
gtatacttac aggct 2553725DNAArtificial SequenceSynthetic
(Hu.DMD.Exon50.25.016 NG-08-0742) 537agagaatggg atccagtata cttac
2553820DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.20.001)
538acagaaaagc atacacatta 2053920DNAArtificial SequenceSynthetic
(Hu.DMD.Exon50.20.002) 539tttaacagaa aagcatacac
2054020DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.20.003)
540tcctctttaa cagaaaagca 2054120DNAArtificial SequenceSynthetic
(Hu.DMD.Exon50.20.004) 541taacttcctc tttaacagaa
2054220DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.20.005)
542tcttctaact tcctctttaa 2054320DNAArtificial SequenceSynthetic
(Hu.DMD.Exon50.20.006) 543tcagatcttc taacttcctc
2054420DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.20.007)
544ccttccactc agagctcaga 2054520DNAArtificial SequenceSynthetic
(Hu.DMD.Exon50.20.008) 545gtaaacggtt taccgccttc
2054620DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.20.009)
546ccctcagctc ttgaagtaaa 2054720DNAArtificial SequenceSynthetic
(Hu.DMD.Exon50.20.010) 547ggtcaggctg ctttgccctc
2054820DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.20.011)
548tcagtccagg agctaggtca 2054920DNAArtificial SequenceSynthetic
(Hu.DMD.Exon50.20.012) 549aggctccaat agtggtcagt
2055020DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.20.013)
550cttacaggct ccaatagtgg 2055120DNAArtificial SequenceSynthetic
(Hu.DMD.Exon50.20.014) 551gtatacttac aggctccaat
2055220DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.20.015)
552atccagtata cttacaggct 2055320DNAArtificial SequenceSynthetic
(Hu.DMD.Exon50.20.016) 553atgggatcca gtatacttac
2055420DNAArtificial SequenceSynthetic (Hu.DMD.Exon50.20.017)
554agagaatggg atccagtata 2055526DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.25.001-44) 555ctaaaatatt ttgggttttt gcaaaa
2655626DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.25.002-45)
556gctaaaatat tttgggtttt tgcaaa 2655726DNAArtificial
SequenceSynthetic (Hu.DMD.Exon51.25.002.2-46) 557taggagctaa
aatattttgg gttttt 2655825DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.25.003) 558agtaggagct aaaatatttt gggtt
2555925DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.25.003.2)
559tgagtaggag ctaaaatatt ttggg 2556026DNAArtificial
SequenceSynthetic (Hu.DMD.Exon51.25.004) 560ctgagtagga gctaaaatat
tttggg 2656125DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.25.004.2) 561cagtctgagt aggagctaaa atatt
2556226DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.25.005)
562acagtctgag taggagctaa aatatt 2656326DNAArtificial
SequenceSynthetic (Hu.DMD.Exon51.25.005.2) 563gagtaacagt ctgagtagga
gctaaa 2656425DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.25.006) 564cagagtaaca gtctgagtag gagct
2556526DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.25.006.2)
565caccagagta acagtctgag taggag 2656625DNAArtificial
SequenceSynthetic (Hu.DMD.Exon51.25.007) 566gtcaccagag taacagtctg
agtag 2556726DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.25.007.2) 567aaccacaggt tgtgtcacca gagtaa
2656825DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.25.008)
568gttgtgtcac cagagtaaca gtctg 2556926DNAArtificial
SequenceSynthetic (Hu.DMD.Exon51.25.009) 569tggcagtttc cttagtaacc
acaggt 2657026DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.25.010) 570atttctagtt tggagatggc agtttc
2657125DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.25.010.2)
571ggaagatggc atttctagtt tggag 2557226DNAArtificial
SequenceSynthetic (Hu.DMD.Exon51.25.011) 572catcaaggaa gatggcattt
ctagtt 2657326DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.25.011.2) 573gagcaggtac ctccaacatc aaggaa
2657425DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.25.012)
574atctgccaga gcaggtacct ccaac 2557526DNAArtificial
SequenceSynthetic (Hu.DMD.Exon51.25.013) 575aagttctgtc caagcccggt
tgaaat 2657626DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.25.013.2) 576cggttgaaat ctgccagagc aggtac
2657726DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.25.014)
577gagaaagcca gtcggtaagt tctgtc 2657825DNAArtificial
SequenceSynthetic (Hu.DMD.Exon51.25.014.2) 578gtcggtaagt tctgtccaag
cccgg 2557926DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.25.015)
579ataacttgat caagcagaga aagcca 2658025DNAArtificial
SequenceSynthetic (Hu.DMD.Exon51.25.015.2) 580aagcagagaa agccagtcgg
taagt 2558126DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.25.016)
581caccctctgt gattttataa cttgat 2658226DNAArtificial
SequenceSynthetic (Hu.DMD.Exon51.25.017) 582caaggtcacc caccatcacc
ctctgt 2658325DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.25.017.2) 583catcaccctc tgtgatttta taact
2558426DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.25.018)
584cttctgcttg atgatcatct cgttga 2658526DNAArtificial
SequenceSynthetic (Hu.DMD.Exon51.25.019) 585ccttctgctt gatgatcatc
tcgttg 2658626DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.25.019.2) 586atctcgttga tatcctcaag gtcacc
2658726DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.25.020)
587tcataccttc tgcttgatga tcatct 2658826DNAArtificial
SequenceSynthetic (Hu.DMD.Exon51.25.020.2) 588tcattttttc tcataccttc
tgcttg 2658926DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.25.021) 589ttttctcata ccttctgctt gatgat
2659026DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.25.022)
590ttttatcatt ttttctcata ccttct 2659126DNAArtificial
SequenceSynthetic (Hu.DMD.Exon51.25.023) 591ccaactttta tcattttttc
tcatac 2659220DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.20.001) 592atattttggg tttttgcaaa
2059320DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.20.002)
593aaaatatttt gggtttttgc 2059420DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.20.003) 594gagctaaaat attttgggtt
2059520DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.20.004)
595agtaggagct aaaatatttt 2059620DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.20.005) 596gtctgagtag gagctaaaat
2059720DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.20.006)
597taacagtctg agtaggagct 2059820DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.20.007) 598cagagtaaca gtctgagtag
2059920DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.20.008)
599cacaggttgt gtcaccagag 2060020DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.20.009) 600agtttcctta gtaaccacag
2060120DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.20.010)
601tagtttggag atggcagttt 2060220DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.20.011) 602ggaagatggc atttctagtt
2060320DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.20.012)
603tacctccaac atcaaggaag 2060420DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.20.013) 604atctgccaga gcaggtacct
2060520DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.20.014)
605ccaagcccgg ttgaaatctg 2060620DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.20.015) 606gtcggtaagt tctgtccaag
2060720DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.20.016)
607aagcagagaa agccagtcgg 2060820DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.20.017) 608ttttataact tgatcaagca
2060920DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.20.018)
609catcaccctc tgtgatttta 2061020DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.20.019) 610ctcaaggtca cccaccatca
2061120DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.20.020)
611catctcgttg atatcctcaa 2061220DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.20.021) 612cttctgcttg atgatcatct
2061320DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.20.022)
613cataccttct gcttgatgat 2061420DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.20.023) 614tttctcatac cttctgcttg
2061520DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.20.024)
615cattttttct cataccttct 2061620DNAArtificial SequenceSynthetic
(Hu.DMD.Exon51.20.025) 616tttatcattt tttctcatac
2061720DNAArtificial SequenceSynthetic (Hu.DMD.Exon51.20.026)
617caacttttat cattttttct 2061825DNAArtificial SequenceSynthetic
(Hu.DMD.Exon52.25.001) 618ctgtaagaac aaatatccct tagta
2561925DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.25.002)
619tgcctgtaag aacaaatatc cctta 2562025DNAArtificial
SequenceSynthetic (Hu.DMD.Exon52.25.002.2) 620gttgcctgta agaacaaata
tccct 2562125DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.25.003)
621attgttgcct gtaagaacaa atatc 2562225DNAArtificial
SequenceSynthetic (Hu.DMD.Exon52.25.003.2) 622gcattgttgc ctgtaagaac
aaata 2562325DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.25.004)
623cctgcattgt tgcctgtaag aacaa 2562425DNAArtificial
SequenceSynthetic (Hu.DMD.Exon52.25.004.2) 624atcctgcatt gttgcctgta
agaac 2562525DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.25.005)
625caaatcctgc attgttgcct gtaag 2562625DNAArtificial
SequenceSynthetic (Hu.DMD.Exon52.25.005.2) 626tccaaatcct gcattgttgc
ctgta 2562725DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.25.006)
627tgttccaaat cctgcattgt tgcct 2562825DNAArtificial
SequenceSynthetic (Hu.DMD.Exon52.25.006.2) 628tctgttccaa atcctgcatt
gttgc 2562925DNAArtificial SequenceSynthetic
(Hu.DMD.Exon52.25.007)
629aactggggac gcctctgttc caaat 2563025DNAArtificial
SequenceSynthetic (Hu.DMD.Exon52.25.007.2) 630gcctctgttc caaatcctgc
attgt 2563125DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.25.008)
631cagcggtaat gagttcttcc aactg 2563225DNAArtificial
SequenceSynthetic (Hu.DMD.Exon52.25.008.2) 632cttccaactg gggacgcctc
tgttc 2563325DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.25.009)
633cttgtttttc aaattttggg cagcg 2563425DNAArtificial
SequenceSynthetic (Hu.DMD.Exon52.25.010) 634ctagcctctt gattgctggt
cttgt 2563525DNAArtificial SequenceSynthetic
(Hu.DMD.Exon52.25.010.2) 635ttttcaaatt ttgggcagcg gtaat
2563625DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.25.011)
636ttcgatccgt aatgattgtt ctagc 2563725DNAArtificial
SequenceSynthetic (Hu.DMD.Exon52.25.011.2) 637gattgctggt cttgtttttc
aaatt 2563825DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.25.012)
638cttacttcga tccgtaatga ttgtt 2563925DNAArtificial
SequenceSynthetic (Hu.DMD.Exon52.25.012.2) 639ttgttctagc ctcttgattg
ctggt 2564025DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.25.013)
640aaaaacttac ttcgatccgt aatga 2564125DNAArtificial
SequenceSynthetic (Hu.DMD.Exon52.25.014) 641tgttaaaaaa cttacttcga
tccgt 2564225DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.25.015)
642atgcttgtta aaaaacttac ttcga 2564325DNAArtificial
SequenceSynthetic (Hu.DMD.Exon52.25.016) 643gtcccatgct tgttaaaaaa
cttac 2564420DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.20.001)
644agaacaaata tcccttagta 2064520DNAArtificial SequenceSynthetic
(Hu.DMD.Exon52.20.002) 645gtaagaacaa atatccctta
2064620DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.20.003)
646tgcctgtaag aacaaatatc 2064720DNAArtificial SequenceSynthetic
(Hu.DMD.Exon52.20.004) 647attgttgcct gtaagaacaa
2064820DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.20.005)
648cctgcattgt tgcctgtaag 2064920DNAArtificial SequenceSynthetic
(Hu.DMD.Exon52.20.006) 649caaatcctgc attgttgcct
2065020DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.20.007)
650gcctctgttc caaatcctgc 2065120DNAArtificial SequenceSynthetic
(Hu.DMD.Exon52.20.008) 651cttccaactg gggacgcctc
2065220DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.20.009)
652cagcggtaat gagttcttcc 2065320DNAArtificial SequenceSynthetic
(Hu.DMD.Exon52.20.010) 653ttttcaaatt ttgggcagcg
2065420DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.20.011)
654gattgctggt cttgtttttc 2065520DNAArtificial SequenceSynthetic
(Hu.DMD.Exon52.20.012) 655ttgttctagc ctcttgattg
2065620DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.20.013)
656ttcgatccgt aatgattgtt 2065720DNAArtificial SequenceSynthetic
(Hu.DMD.Exon52.20.014) 657cttacttcga tccgtaatga
2065820DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.20.015)
658aaaaacttac ttcgatccgt 2065920DNAArtificial SequenceSynthetic
(Hu.DMD.Exon52.20.016) 659tgttaaaaaa cttacttcga
2066020DNAArtificial SequenceSynthetic (Hu.DMD.Exon52.20.017)
660atgcttgtta aaaaacttac 2066120DNAArtificial SequenceSynthetic
(Hu.DMD.Exon52.20.018) 661gtcccatgct tgttaaaaaa
2066225DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.25.001)
662ctagaataaa aggaaaaata aatat 2566325DNAArtificial
SequenceSynthetic (Hu.DMD.Exon53.25.002) 663aactagaata aaaggaaaaa
taaat 2566425DNAArtificial SequenceSynthetic
(Hu.DMD.Exon53.25.002.2) 664ttcaactaga ataaaaggaa aaata
2566525DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.25.003)
665ctttcaacta gaataaaagg aaaaa 2566625DNAArtificial
SequenceSynthetic (Hu.DMD.Exon53.25.003.2) 666attctttcaa ctagaataaa
aggaa 2566725DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.25.004)
667gaattctttc aactagaata aaagg 2566825DNAArtificial
SequenceSynthetic (Hu.DMD.Exon53.25.004.2) 668tctgaattct ttcaactaga
ataaa 2566925DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.25.005)
669attctgaatt ctttcaacta gaata 2567025DNAArtificial
SequenceSynthetic (Hu.DMD.Exon53.25.005.2) 670ctgattctga attctttcaa
ctaga 2567125DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.25.006)
671cactgattct gaattctttc aacta 2567225DNAArtificial
SequenceSynthetic (Hu.DMD.Exon53.25.006.2) 672tcccactgat tctgaattct
ttcaa 2567325DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.25.007)
673catcccactg attctgaatt ctttc 2567425DNAArtificial
SequenceSynthetic (Hu.DMD.Exon53.25.008) 674tacttcatcc cactgattct
gaatt 2567524DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.25.009)
675cggttctgaa ggtgttcttg tact 2467625DNAArtificial
SequenceSynthetic (Hu.DMD.Exon53.25.009.2) 676ctgttgcctc cggttctgaa
ggtgt 2567725DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.25.010)
677tttcattcaa ctgttgcctc cggtt 2567825DNAArtificial
SequenceSynthetic (Hu.DMD.Exon53.25.010.2) 678taacatttca ttcaactgtt
gcctc 2567925DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.25.011)
679ttgtgttgaa tcctttaaca tttca 2568025DNAArtificial
SequenceSynthetic (Hu.DMD.Exon53.25.012) 680tcttccttag cttccagcca
ttgtg 2568125DNAArtificial SequenceSynthetic
(Hu.DMD.Exon53.25.012.2) 681cttagcttcc agccattgtg ttgaa
2568225DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.25.013)
682gtcctaagac ctgctcagct tcttc 2568325DNAArtificial
SequenceSynthetic (Hu.DMD.Exon53.25.013.2) 683ctgctcagct tcttccttag
cttcc 2568425DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.25.014)
684ctcaagcttg gctctggcct gtcct 2568525DNAArtificial
SequenceSynthetic (Hu.DMD.Exon53.25.014.2) 685ggcctgtcct aagacctgct
cagct 2568625DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.25.015)
686tagggaccct ccttccatga ctcaa 2568725DNAArtificial
SequenceSynthetic (Hu.DMD.Exon53.25.016) 687tttggattgc atctactgta
taggg 2568825DNAArtificial SequenceSynthetic
(Hu.DMD.Exon53.25.016.2) 688accctccttc catgactcaa gcttg
2568925DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.25.017)
689cttggtttct gtgattttct tttgg 2569025DNAArtificial
SequenceSynthetic (Hu.DMD.Exon53.25.017.2) 690atctactgta tagggaccct
ccttc 2569125DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.25.018)
691ctaaccttgg tttctgtgat tttct 2569225DNAArtificial
SequenceSynthetic (Hu.DMD.Exon53.25.018.2) 692tttcttttgg attgcatcta
ctgta 2569325DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.25.019)
693tgatactaac cttggtttct gtgat 2569425DNAArtificial
SequenceSynthetic (Hu.DMD.Exon53.25.020) 694atctttgata ctaaccttgg
tttct 2569525DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.25.021)
695aaggtatctt tgatactaac cttgg 2569625DNAArtificial
SequenceSynthetic (Hu.DMD.Exon53.25.022) 696ttaaaaaggt atctttgata
ctaac 2569720DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.20.001)
697ataaaaggaa aaataaatat 2069820DNAArtificial SequenceSynthetic
(Hu.DMD.Exon53.20.002) 698gaataaaagg aaaaataaat
2069920DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.20.003)
699aactagaata aaaggaaaaa 2070020DNAArtificial SequenceSynthetic
(Hu.DMD.Exon53.20.004) 700ctttcaacta gaataaaagg
2070120DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.20.005)
701gaattctttc aactagaata 2070220DNAArtificial SequenceSynthetic
(Hu.DMD.Exon53.20.006) 702attctgaatt ctttcaacta
2070320DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.20.007)
703tacttcatcc cactgattct 2070419DNAArtificial SequenceSynthetic
(Hu.DMD.Exon53.20.008) 704ctgaaggtgt tcttgtact 1970520DNAArtificial
SequenceSynthetic (Hu.DMD.Exon53.20.009) 705ctgttgcctc cggttctgaa
2070620DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.20.010)
706taacatttca ttcaactgtt 2070720DNAArtificial SequenceSynthetic
(Hu.DMD.Exon53.20.011) 707ttgtgttgaa tcctttaaca
2070820DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.20.012)
708cttagcttcc agccattgtg 2070920DNAArtificial SequenceSynthetic
(Hu.DMD.Exon53.20.013) 709ctgctcagct tcttccttag
2071020DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.20.014)
710ggcctgtcct aagacctgct 2071120DNAArtificial SequenceSynthetic
(Hu.DMD.Exon53.20.015) 711ctcaagcttg gctctggcct
2071220DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.20.016)
712accctccttc catgactcaa 2071320DNAArtificial SequenceSynthetic
(Hu.DMD.Exon53.20.017) 713atctactgta tagggaccct
2071420DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.20.018)
714tttcttttgg attgcatcta 2071520DNAArtificial SequenceSynthetic
(Hu.DMD.Exon53.20.019) 715cttggtttct gtgattttct
2071620DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.20.020)
716ctaaccttgg tttctgtgat 2071720DNAArtificial SequenceSynthetic
(Hu.DMD.Exon53.20.021) 717tgatactaac cttggtttct
2071820DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.20.022)
718atctttgata ctaaccttgg 2071920DNAArtificial SequenceSynthetic
(Hu.DMD.Exon53.20.023) 719aaggtatctt tgatactaac
2072020DNAArtificial SequenceSynthetic (Hu.DMD.Exon53.20.024)
720ttaaaaaggt atctttgata 2072124DNAArtificial SequenceSynthetic
(Hu.DMD.Exon54.25.001) 721ctatagattt ttatgagaaa gaga
2472225DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.25.002)
722aactgctata gatttttatg agaaa 2572325DNAArtificial
SequenceSynthetic (Hu.DMD.Exon54.25.003) 723tggccaactg ctatagattt
ttatg 2572425DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.25.004)
724gtctttggcc aactgctata gattt 2572525DNAArtificial
SequenceSynthetic (Hu.DMD.Exon54.25.005) 725cggaggtctt tggccaactg
ctata 2572625DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.25.006)
726actggcggag gtctttggcc aactg 2572725DNAArtificial
SequenceSynthetic (Hu.DMD.Exon54.25.007) 727tttgtctgcc actggcggag
gtctt 2572825DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.25.008)
728agtcatttgc cacatctaca tttgt 2572925DNAArtificial
SequenceSynthetic (Hu.DMD.Exon54.25.008.2) 729tttgccacat ctacatttgt
ctgcc 2573025DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.25.009)
730ccggagaagt ttcagggcca agtca 2573125DNAArtificial
SequenceSynthetic (Hu.DMD.Exon54.25.010) 731gtatcatctg cagaataatc
ccgga 2573225DNAArtificial SequenceSynthetic
(Hu.DMD.Exon54.25.010.2) 732taatcccgga gaagtttcag ggcca
2573325DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.25.011)
733ttatcatgtg gacttttctg gtatc 2573425DNAArtificial
SequenceSynthetic (Hu.DMD.Exon54.25.012) 734agaggcattg atattctctg
ttatc 2573525DNAArtificial SequenceSynthetic
(Hu.DMD.Exon54.25.012.2) 735atgtggactt ttctggtatc atctg
2573625DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.25.013)
736cttttatgaa tgcttctcca agagg 2573725DNAArtificial
SequenceSynthetic (Hu.DMD.Exon54.25.013.2) 737atattctctg ttatcatgtg
gactt 2573825DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.25.014)
738catacctttt atgaatgctt ctcca 2573925DNAArtificial
SequenceSynthetic (Hu.DMD.Exon54.25.014.2) 739ctccaagagg cattgatatt
ctctg 2574025DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.25.015)
740taattcatac cttttatgaa tgctt 2574125DNAArtificial
SequenceSynthetic (Hu.DMD.Exon54.25.016) 741taatgtaatt catacctttt
atgaa 2574225DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.25.017)
742agaaataatg taattcatac ctttt 2574325DNAArtificial
SequenceSynthetic (Hu.DMD.Exon54.25.018) 743gttttagaaa taatgtaatt
catac 2574419DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.20.001)
744gatttttatg agaaagaga 1974520DNAArtificial SequenceSynthetic
(Hu.DMD.Exon54.20.002) 745ctatagattt ttatgagaaa
2074620DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.20.003)
746aactgctata gatttttatg 2074720DNAArtificial SequenceSynthetic
(Hu.DMD.Exon54.20.004) 747tggccaactg ctatagattt
2074820DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.20.005)
748gtctttggcc aactgctata 2074920DNAArtificial SequenceSynthetic
(Hu.DMD.Exon54.20.006) 749cggaggtctt tggccaactg
2075020DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.20.007)
750tttgtctgcc actggcggag 2075120DNAArtificial SequenceSynthetic
(Hu.DMD.Exon54.20.008) 751tttgccacat ctacatttgt
2075220DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.20.009)
752ttcagggcca agtcatttgc 2075320DNAArtificial SequenceSynthetic
(Hu.DMD.Exon54.20.010) 753taatcccgga gaagtttcag
2075420DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.20.011)
754gtatcatctg cagaataatc
2075520DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.20.012)
755atgtggactt ttctggtatc 2075620DNAArtificial SequenceSynthetic
(Hu.DMD.Exon54.20.013) 756atattctctg ttatcatgtg
2075720DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.20.014)
757ctccaagagg cattgatatt 2075820DNAArtificial SequenceSynthetic
(Hu.DMD.Exon54.20.015) 758cttttatgaa tgcttctcca
2075920DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.20.016)
759catacctttt atgaatgctt 2076020DNAArtificial SequenceSynthetic
(Hu.DMD.Exon54.20.017) 760taattcatac cttttatgaa
2076120DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.20.018)
761taatgtaatt catacctttt 2076220DNAArtificial SequenceSynthetic
(Hu.DMD.Exon54.20.019) 762agaaataatg taattcatac
2076320DNAArtificial SequenceSynthetic (Hu.DMD.Exon54.20.020)
763gttttagaaa taatgtaatt 2076425DNAArtificial SequenceSynthetic
(Hu.DMD.Exon55.25.001) 764ctgcaaagga ccaaatgttc agatg
2576525DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.25.002)
765tcaccctgca aaggaccaaa tgttc 2576625DNAArtificial
SequenceSynthetic (Hu.DMD.Exon55.25.003) 766ctcactcacc ctgcaaagga
ccaaa 2576725DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.25.004)
767tctcgctcac tcaccctgca aagga 2576825DNAArtificial
SequenceSynthetic (Hu.DMD.Exon55.25.005) 768cagcctctcg ctcactcacc
ctgca 2576925DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.25.006)
769caaagcagcc tctcgctcac tcacc 2577025DNAArtificial
SequenceSynthetic (Hu.DMD.Exon55.25.007) 770tcttccaaag cagcctctcg
ctcac 2577125DNAArtificial SequenceSynthetic
(Hu.DMD.Exon55.25.007.2) 771tctatgagtt tcttccaaag cagcc
2577225DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.25.008)
772gttgcagtaa tctatgagtt tcttc 2577325DNAArtificial
SequenceSynthetic (Hu.DMD.Exon55.25.008.2) 773gaactgttgc agtaatctat
gagtt 2577425DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.25.009)
774ttccaggtcc agggggaact gttgc 2577525DNAArtificial
SequenceSynthetic (Hu.DMD.Exon55.25.010) 775gtaagccagg caagaaactt
ttcca 2577625DNAArtificial SequenceSynthetic
(Hu.DMD.Exon55.25.010.2) 776ccaggcaaga aacttttcca ggtcc
2577725DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.25.011)
777tggcagttgt ttcagcttct gtaag 2577825DNAArtificial
SequenceSynthetic (Hu.DMD.Exon55.25.011.2) 778ttcagcttct gtaagccagg
caaga 2577925DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.25.012)
779ggtagcatcc tgtaggacat tggca 2578025DNAArtificial
SequenceSynthetic (Hu.DMD.Exon55.25.012.2) 780gacattggca gttgtttcag
cttct 2578125DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.25.013)
781tctaggagcc tttccttacg ggtag 2578225DNAArtificial
SequenceSynthetic (Hu.DMD.Exon55.25.014) 782cttttactcc cttggagtct
tctag 2578325DNAArtificial SequenceSynthetic
(Hu.DMD.Exon55.25.014.2) 783gagcctttcc ttacgggtag catcc
2578425DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.25.015)
784ttgccattgt ttcatcagct ctttt 2578525DNAArtificial
SequenceSynthetic (Hu.DMD.Exon55.25.015.2) 785cttggagtct tctaggagcc
tttcc 2578625DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.25.016)
786cttacttgcc attgtttcat cagct 2578725DNAArtificial
SequenceSynthetic (Hu.DMD.Exon55.25.016.2) 787cagctctttt actcccttgg
agtct 2578825DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.25.017)
788cctgacttac ttgccattgt ttcat 2578925DNAArtificial
SequenceSynthetic (Hu.DMD.Exon55.25.018) 789aaatgcctga cttacttgcc
attgt 2579025DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.25.019)
790agcggaaatg cctgacttac ttgcc 2579125DNAArtificial
SequenceSynthetic (Hu.DMD.Exon55.25.020) 791gctaaagcgg aaatgcctga
cttac 2579220DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.20.001)
792aaggaccaaa tgttcagatg 2079320DNAArtificial SequenceSynthetic
(Hu.DMD.Exon55.20.002) 793ctgcaaagga ccaaatgttc
2079420DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.20.003)
794tcaccctgca aaggaccaaa 2079520DNAArtificial SequenceSynthetic
(Hu.DMD.Exon55.20.004) 795ctcactcacc ctgcaaagga
2079620DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.20.005)
796tctcgctcac tcaccctgca 2079720DNAArtificial SequenceSynthetic
(Hu.DMD.Exon55.20.006) 797cagcctctcg ctcactcacc
2079820DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.20.007)
798caaagcagcc tctcgctcac 2079920DNAArtificial SequenceSynthetic
(Hu.DMD.Exon55.20.008) 799tctatgagtt tcttccaaag
2080020DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.20.009)
800gaactgttgc agtaatctat 2080120DNAArtificial SequenceSynthetic
(Hu.DMD.Exon55.20.010) 801ttccaggtcc agggggaact
2080220DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.20.011)
802ccaggcaaga aacttttcca 2080320DNAArtificial SequenceSynthetic
(Hu.DMD.Exon55.20.012) 803ttcagcttct gtaagccagg
2080420DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.20.013)
804gacattggca gttgtttcag 2080520DNAArtificial SequenceSynthetic
(Hu.DMD.Exon55.20.014) 805ggtagcatcc tgtaggacat
2080620DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.20.015)
806gagcctttcc ttacgggtag 2080720DNAArtificial SequenceSynthetic
(Hu.DMD.Exon55.20.016) 807cttggagtct tctaggagcc
2080820DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.20.017)
808cagctctttt actcccttgg 2080920DNAArtificial SequenceSynthetic
(Hu.DMD.Exon55.20.018) 809ttgccattgt ttcatcagct
2081020DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.20.019)
810cttacttgcc attgtttcat 2081120DNAArtificial SequenceSynthetic
(Hu.DMD.Exon55.20.020) 811cctgacttac ttgccattgt
2081220DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.20.021)
812aaatgcctga cttacttgcc 2081320DNAArtificial SequenceSynthetic
(Hu.DMD.Exon55.20.022) 813agcggaaatg cctgacttac
2081420DNAArtificial SequenceSynthetic (Hu.DMD.Exon55.20.023)
814gctaaagcgg aaatgcctga 2081529DNAArtificial SequenceSynthetic
(H50A(+02+30)-AVI-5656) 815ccactcagag ctcagatctt ctaacttcc
2981625DNAArtificial SequenceSynthetic (H50D(+07-18)-AVI-5915)
816gggatccagt atacttacag gctcc 2581727DNAArtificial
SequenceSynthetic (H50A(+07+33)) 817cttccactca gagctcagat cttctaa
2781830DNAArtificial SequenceSynthetic (H51A(+61+90)-AVI-4657)
818acatcaagga agatggcatt tctagtttgg 3081930DNAArtificial
SequenceSynthetic (H51A(+66+95)-AVI-4658) 819ctccaacatc aaggaagatg
gcatttctag 3082024DNAArtificial SequenceSynthetic (H51A(+111+134))
820ttctgtccaa gcccggttga aatc 2482121DNAArtificial
SequenceSynthetic (H51A(+175+195)) 821cacccaccat caccctcygt g
2182222DNAArtificial SequenceSynthetic (H51A(+199+220))
822atcatctcgt tgatatcctc aa 2282325DNAArtificial SequenceSynthetic
(H51A(+66+90)) 823acatcaagga agatggcatt tctag 2582426DNAArtificial
SequenceSynthetic (H51A(-01+25)) 824accagagtaa cagtctgagt aggagc
2682520DNAArtificial SequenceSynthetic (h51AON1) 825tcaaggaaga
tggcatttct 2082623DNAArtificial SequenceSynthetic (h51AON2)
826cctctgtgat tttataactt gat 2382725DNAArtificial SequenceSynthetic
(H51D(+08-17)) 827atcatttttt ctcatacctt ctgct 2582823DNAArtificial
SequenceSynthetic (H51D(+16-07)) 828ctcatacctt ctgcttgatg atc
2382917DNAArtificial SequenceSynthetic (hAON#23) 829tggcatttct
agtttgg 1783023DNAArtificial SequenceSynthetic (hAON#24)
830ccagagcagg tacctccaac atc 2383119DNAArtificial SequenceSynthetic
(h44AON1) 831cgccgccatt tctcaacag 1983220DNAArtificial
SequenceSynthetic (H45A(+71+90)) 832tgtttttgag gattgctgaa
2083319DNAArtificial SequenceSynthetic (h45AON1) 833gctgaattat
ttcttcccc 1983417DNAArtificial SequenceSynthetic (h45AON5)
834gcccaatgcc atcctgg 1783526DNAArtificial SequenceSynthetic
(H45A(-06+20)) 835ccaatgccat cctggagttc ctgtaa 2683631DNAArtificial
SequenceSynthetic (H53A(+39+69)) 836cattcaactg ttgcctccgg
ttctgaaggt g 3183718DNAArtificial SequenceSynthetic (h53AON1)
837ctgttgcctc cggttctg 1883822DNAArtificial SequenceSynthetic
(H53A(-12+10)) 838attctttcaa ctagaataaa ag 2283930DNAArtificial
SequenceSynthetic (huEx45.30.66) 839gccatcctgg agttcctgta
agataccaaa 3084030DNAArtificial SequenceSynthetic (huEx45.30.71)
840ccaatgccat cctggagttc ctgtaagata 3084130DNAArtificial
SequenceSynthetic (huEx45.30.79) 841gccgctgccc aatgccatcc
tggagttcct 3084230DNAArtificial SequenceSynthetic (huEx45.30.83)
842gtttgccgct gcccaatgcc atcctggagt 3084330DNAArtificial
SequenceSynthetic (huEx45.30.88) 843caacagtttg ccgctgccca
atgccatcct 3084430DNAArtificial SequenceSynthetic (huEx45.30.92)
844ctgacaacag tttgccgctg cccaatgcca 3084530DNAArtificial
SequenceSynthetic (huEx45.30.96) 845tgttctgaca acagtttgcc
gctgcccaat 3084630DNAArtificial SequenceSynthetic (huEx45.30.99)
846caatgttctg acaacagttt gccgctgccc 3084730DNAArtificial
SequenceSynthetic (huEx45.30.103) 847cattcaatgt tctgacaaca
gtttgccgct 3084830DNAArtificial SequenceSynthetic (huEx45.30.120)
848tatttcttcc ccagttgcat tcaatgttct 3084930DNAArtificial
SequenceSynthetic (huEx45.30.127) 849gctgaattat ttcttcccca
gttgcattca 3085030DNAArtificial SequenceSynthetic (huEx45.30.132)
850ggattgctga attatttctt ccccagttgc 3085130DNAArtificial
SequenceSynthetic (huEx45.30.137) 851tttgaggatt gctgaattat
ttcttcccca 3085230DNAArtificial SequenceSynthetic (huEx53.30.84)
852gtacttcatc ccactgattc tgaattcttt 3085330DNAArtificial
SequenceSynthetic (huEx53.30.88) 853tcttgtactt catcccactg
attctgaatt 3085430DNAArtificial SequenceSynthetic (huEx53.30.91)
854tgttcttgta cttcatccca ctgattctga 3085530DNAArtificial
SequenceSynthetic (huEx53.30.103) 855cggttctgaa ggtgttcttg
tacttcatcc 3085630DNAArtificial SequenceSynthetic (huEx53.30.106)
856ctccggttct gaaggtgttc ttgtacttca 3085730DNAArtificial
SequenceSynthetic (huEx53.30.109) 857tgcctccggt tctgaaggtg
ttcttgtact 3085830DNAArtificial SequenceSynthetic (huEx53.30.112)
858tgttgcctcc ggttctgaag gtgttcttgt 3085930DNAArtificial
SequenceSynthetic (huEx53.30.115) 859aactgttgcc tccggttctg
aaggtgttct 3086030DNAArtificial SequenceSynthetic (huEx53.30.118)
860ttcaactgtt gcctccggtt ctgaaggtgt 3086110PRTArtificial
SequenceSynthetic (rTAT) 861Arg Arg Arg Gln Arg Arg Lys Lys Arg
Cys1 5 1086212PRTArtificial SequenceSynthetic (R9F2) 862Arg Arg Arg
Arg Arg Arg Arg Arg Arg Phe Phe Cys1 5 1086313PRTArtificial
SequenceSynthetic ((RRAhx)4B)misc_feature(3)..(3)Xaa is
6-aminohexanoic acid (Ahx)misc_feature(6)..(6)Xaa is
6-aminohexanoic acid (Ahx)misc_feature(9)..(9)Xaa is
6-aminohexanoic acid (Ahx)misc_feature(12)..(12)Xaa is
6-aminohexanoic acid (Ahx)misc_feature(13)..(13)Xaa is beta-alanine
863Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Xaa1 5
1086414PRTArtificial SequenceSynthetic ((RAhxR)4AhxB; (P007)
)misc_feature(2)..(2)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(5)..(5)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(8)..(8)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(11)..(11)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(13)..(13)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(14)..(14)Xaa is beta-alanine 864Arg Xaa Arg Arg
Xaa Arg Arg Xaa Arg Arg Xaa Arg Xaa Xaa1 5 1086514PRTArtificial
SequenceSynthetic ((AhxRR)4AhxB)misc_feature(1)..(1)Xaa is
6-aminohexanoic acid (Ahx)misc_feature(4)..(4)Xaa is
6-aminohexanoic acid (Ahx)misc_feature(7)..(7)Xaa is
6-aminohexanoic acid (Ahx)misc_feature(10)..(10)Xaa is
6-aminohexanoic acid (Ahx)misc_feature(13)..(13)Xaa is
6-aminohexanoic acid (Ahx)misc_feature(14)..(14)Xaa is beta-alanine
865Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Xaa1 5
1086613PRTArtificial SequenceSynthetic
((RAhx)6B)misc_feature(2)..(2)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(4)..(4)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(6)..(6)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(8)..(8)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(10)..(10)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(12)..(12)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(13)..(13)Xaa is beta-alanine 866Arg Xaa Arg Xaa
Arg Xaa Arg Xaa Arg Xaa Arg Xaa Xaa1 5 1086715PRTArtificial
SequenceSynthetic ((RAhx)8B)misc_feature(2)..(2)Xaa is
6-aminohexanoic acid (Ahx)misc_feature(4)..(4)Xaa is
6-aminohexanoic acid (Ahx)misc_feature(6)..(6)Xaa is
6-aminohexanoic acid (Ahx)misc_feature(8)..(8)Xaa is
6-aminohexanoic acid (Ahx)misc_feature(10)..(10)Xaa is
6-aminohexanoic acid (Ahx)misc_feature(12)..(12)Xaa is
6-aminohexanoic acid (Ahx)misc_feature(14)..(14)Xaa is
6-aminohexanoic acid (Ahx)misc_feature(15)..(15)Xaa is beta-alanine
867Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa Xaa1 5
10
1586817PRTArtificial SequenceSynthetic
((RAhxR)5AhxB)misc_feature(2)..(2)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(5)..(5)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(8)..(8)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(11)..(11)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(14)..(14)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(16)..(16)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(17)..(17)Xaa is beta-alanine 868Arg Xaa Arg Arg
Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Xaa1 5 10
15Xaa86914PRTArtificial SequenceSynthetic ((RAhxRRBR)2AhxB;
(CPO6062))misc_feature(2)..(2)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(5)..(5)Xaa is beta-alaninemisc_feature(8)..(8)Xaa
is 6-aminohexanoic acid (Ahx)misc_feature(11)..(11)Xaa is
beta-alaninemisc_feature(13)..(13)Xaa is 6-aminohexanoic acid
(Ahx)misc_feature(14)..(14)Xaa is beta-alanine 869Arg Xaa Arg Arg
Xaa Arg Arg Xaa Arg Arg Xaa Arg Xaa Xaa1 5 108707PRTArtificial
SequenceSynthetic (MSP) 870Ala Ser Ser Leu Asn Ile Ala1
58719PRTArtificial SequenceSynthetic
(MSP-PMO)misc_feature(8)..(9)Xaa can be any naturally occurring
amino acid 871Ala Ser Ser Leu Asn Ile Ala Xaa Xaa1
587222PRTArtificial SequenceSynthetic
(CP06062-MSP-PMO)misc_feature(2)..(2)Xaa can be any naturally
occurring amino acidmisc_feature(5)..(5)Xaa is
beta-alaninemisc_feature(8)..(8)Xaa can be any naturally occurring
amino acidmisc_feature(11)..(11)Xaa is
beta-alaninemisc_feature(13)..(13)Xaa can be any naturally
occurring amino acidmisc_feature(14)..(14)Xaa is
beta-alaninemisc_feature(22)..(22)Xaa can be any naturally
occurring amino acid 872Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa
Arg Xaa Xaa Ala Ser1 5 10 15Ser Leu Asn Ile Ala Xaa
2087321PRTArtificial SequenceSynthetic
(MSP-CP06062-PMO)misc_feature(8)..(8)Xaa can be any naturally
occurring amino acidmisc_feature(10)..(10)Xaa can be any naturally
occurring amino acidmisc_feature(13)..(13)Xaa is
beta-alaninemisc_feature(16)..(16)Xaa can be any naturally
occurring amino acidmisc_feature(19)..(19)Xaa is
beta-alaninemisc_feature(21)..(21)Xaa is beta-alanine 873Ala Ser
Ser Leu Asn Ile Ala Xaa Arg Xaa Arg Arg Xaa Arg Arg Xaa1 5 10 15Arg
Arg Xaa Arg Xaa 2087414PRTArtificial SequenceSynthetic
(CP06062-PMO)misc_feature(2)..(2)Xaa can be any naturally occurring
amino acidmisc_feature(5)..(5)Xaa is
beta-alaninemisc_feature(8)..(8)Xaa can be any naturally occurring
amino acidmisc_feature(11)..(11)Xaa is
beta-alaninemisc_feature(13)..(13)Xaa can be any naturally
occurring amino acidmisc_feature(14)..(14)Xaa is beta-alanine
874Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Xaa Xaa1 5
1087525DNAArtificial SequenceSynthetic (PMO) 875ggccaaacct
cggcttacct gaaat 258769PRTArtificial SequenceSynthetic (rTAT)
876Arg Arg Arg Gln Arg Arg Lys Lys Arg1 58779PRTArtificial
SequenceSynthetic (Tat) 877Arg Lys Lys Arg Arg Gln Arg Arg Arg1
587811PRTArtificial SequenceSynthetic (R9F2) 878Arg Arg Arg Arg Arg
Arg Arg Arg Arg Phe Phe1 5 1087911PRTArtificial SequenceSynthetic
(R5F2R4) 879Arg Arg Arg Arg Arg Phe Phe Arg Arg Arg Arg1 5
108804PRTArtificial SequenceSynthetic (R4) 880Arg Arg Arg
Arg18815PRTArtificial SequenceSynthetic (R5) 881Arg Arg Arg Arg
Arg1 58826PRTArtificial SequenceSynthetic (R6) 882Arg Arg Arg Arg
Arg Arg1 58837PRTArtificial SequenceSynthetic (R7) 883Arg Arg Arg
Arg Arg Arg Arg1 58848PRTArtificial SequenceSynthetic (R8) 884Arg
Arg Arg Arg Arg Arg Arg Arg1 58859PRTArtificial SequenceSynthetic
(R9) 885Arg Arg Arg Arg Arg Arg Arg Arg Arg1 588616PRTArtificial
SequenceSynthetic ((RX)8)misc_feature(2)..(2)Xaa can be any
naturally occurring amino acidmisc_feature(4)..(4)Xaa can be any
naturally occurring amino acidmisc_feature(6)..(6)Xaa can be any
naturally occurring amino acidmisc_feature(8)..(8)Xaa can be any
naturally occurring amino acidmisc_feature(10)..(10)Xaa can be any
naturally occurring amino acidmisc_feature(12)..(12)Xaa can be any
naturally occurring amino acidmisc_feature(14)..(14)Xaa can be any
naturally occurring amino acidmisc_feature(16)..(16)Xaa can be any
naturally occurring amino acid 886Arg Xaa Arg Xaa Arg Xaa Arg Xaa
Arg Xaa Arg Xaa Arg Xaa Arg Xaa1 5 10 1588712PRTArtificial
SequenceSynthetic ((RAhxR)4; (P007) )misc_feature(2)..(2)Xaa is
6-aminohexanoic acidmisc_feature(5)..(5)Xaa is 6-aminohexanoic
acidmisc_feature(8)..(8)Xaa is 6-aminohexanoic
acidmisc_feature(11)..(11)Xaa is 6-aminohexanoic acid 887Arg Xaa
Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg1 5 1088815PRTArtificial
SequenceSynthetic ((RAhxR)5; (CP04057))misc_feature(2)..(2)Xaa is
6-aminohexanoic acidmisc_feature(5)..(5)Xaa is 6-aminohexanoic
acidmisc_feature(8)..(8)Xaa is 6-aminohexanoic
acidmisc_feature(11)..(11)Xaa is 6-aminohexanoic
acidmisc_feature(14)..(14)Xaa is 6-aminohexanoic acid 888Arg Xaa
Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg1 5 10
1588912PRTArtificial SequenceSynthetic ((RAhxRRBR)2;
(CP06062))misc_feature(2)..(2)Xaa is 6-aminohexanoic
acidmisc_feature(5)..(5)Xaa is beta-alaninemisc_feature(8)..(8)Xaa
is 6-aminohexanoic acidmisc_feature(11)..(11)Xaa is beta-alanine
889Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg1 5
1089014PRTArtificial SequenceSynthetic ((RAR)4F2) 890Arg Ala Arg
Arg Ala Arg Arg Ala Arg Arg Ala Arg Phe Phe1 5 1089114PRTArtificial
SequenceSynthetic ((RGR)4F2)) 891Arg Gly Arg Arg Gly Arg Arg Gly
Arg Arg Gly Arg Phe Phe1 5 10
* * * * *