U.S. patent application number 16/243926 was filed with the patent office on 2019-05-02 for multiple exon skipping compositions for dmd.
This patent application is currently assigned to Sarepta Therapeutics, Inc.. The applicant listed for this patent is Sarepta Therapeutics, Inc.. Invention is credited to Ryszard KOLE, Peter SAZANI.
Application Number | 20190127738 16/243926 |
Document ID | / |
Family ID | 41510869 |
Filed Date | 2019-05-02 |
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United States Patent
Application |
20190127738 |
Kind Code |
A1 |
SAZANI; Peter ; et
al. |
May 2, 2019 |
MULTIPLE EXON SKIPPING COMPOSITIONS FOR DMD
Abstract
Provided are antisense molecules capable of binding to a
selected target site in the human dystrophin gene to induce exon
skipping, and methods of use thereof to treat muscular
dystrophy.
Inventors: |
SAZANI; Peter; (Bothell,
WA) ; KOLE; Ryszard; (Bellevue, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sarepta Therapeutics, Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
Sarepta Therapeutics, Inc.
Cambridge
MA
|
Family ID: |
41510869 |
Appl. No.: |
16/243926 |
Filed: |
January 9, 2019 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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16146328 |
Sep 28, 2018 |
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16243926 |
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15789862 |
Oct 20, 2017 |
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16146328 |
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15349778 |
Nov 11, 2016 |
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15789862 |
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14523610 |
Oct 24, 2014 |
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15349778 |
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12605276 |
Oct 23, 2009 |
8871918 |
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14523610 |
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61108416 |
Oct 24, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/3233 20130101;
C12N 2310/3513 20130101; A61P 21/04 20180101; C12N 2310/11
20130101; C12N 2310/3341 20130101; C12N 15/111 20130101; C12N
15/113 20130101; A61P 21/00 20180101; C12N 2310/321 20130101; C12N
2310/331 20130101; C12N 2320/30 20130101; C12N 2320/33
20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; C12N 15/11 20060101 C12N015/11 |
Claims
1-65. (canceled)
66. An antisense oligonucleotide of 21 bases comprising a base
sequence that is 100% complementary to 21 consecutive bases of exon
53 of the human dystrophin pre-mRNA, wherein the base sequence
comprises 19 consecutive bases of TABLE-US-00009 (SEQ ID NO: 431)
CTGTTGCCTCCGGTTCTGAAGGTGT,
wherein the antisense oligonucleotide is a morpholino oligomer, and
wherein the antisense oligonucleotide induces exon 53 skipping; or
a pharmaceutically acceptable salt thereof.
67. A pharmaceutical composition comprising an antisense
oligonucleotide of 21 bases comprising a base sequence that is 100%
complementary to 21 consecutive bases of exon 53 of the human
dystrophin pre-mRNA, wherein the base sequence comprises 19
consecutive bases of TABLE-US-00010 (SEQ ID NO: 431)
CTGTTGCCTCCGGTTCTGAAGGTGT,
wherein the antisense oligonucleotide is a morpholino oligomer, and
wherein the antisense oligonucleotide induces exon 53 skipping; or
a pharmaceutically acceptable salt thereof; and a pharmaceutically
acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. Provisional Patent Application No. 61/108,416 filed
Oct. 24, 2008; wherein this provisional application is incorporated
herein by reference in its entirety.
STATEMENT REGARDING SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
120178_410_SEQUENCE_LISTING.txt. The text file is 157 KB, was
created on Oct. 23, 2009 and is being submitted electronically via
EFS-Web.
FIELD OF THE INVENTION
[0003] The present invention relates to novel antisense compounds
and compositions suitable for facilitating exon skipping in the
human dystrophin gene. It also provides methods for inducing exon
skipping using the antisense compositions adapted for use in the
methods of the invention.
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 bind the various nuclear
splicing factors that are then involved in the splicing reactions.
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.
[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 (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] 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.
[0011] Deletions of an exon or exons which do not alter the reading
frame of a dystrophin protein 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.
[0012] Although antisense molecules may provide a tool in the
treatment of Duchenne Muscular Dystrophy (DMD), attempts to induce
exon skipping using antisense molecules have had mixed success.
Successful skipping of dystrophin exon 19 from the dystrophin
pre-mRNA was achieved using a variety of antisense molecules
directed at the flanking splice sites or motifs within the exon
involved in exon definition as described by Errington et al.,
(Errington, Mann et al. 2003).
[0013] The first example of specific and reproducible exon skipping
in the mdx mouse model was reported by Wilton et ael (Wilton, Lloyd
et al. 1999). By directing an antisense molecule to the donor
splice site, 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 exon skipping in primary cultured myoblasts, this
compound was found to be much less efficient in immortalized cell
cultures expressing higher levels of dystrophin.
[0014] Despite these efforts, there remains a need for improved
antisense oligomers targeted to multiple dystrophin exons and
improved muscle delivery compositions and methods for DMD
therapeutic applications.
BRIEF SUMMARY OF THE INVENTION
[0015] Embodiments of the present invention relate generally to
antisense compounds capable of binding to a selected target to
induce exon skipping, and methods of use thereof to induce exon
skipping. In certain embodiments, it is possible to combine two or
more antisense oligonucleotides of the present invention together
to induce single or multiple exon skipping.
[0016] In certain embodiments, it is possible to improve exon
skipping of a single or multiple exons by covalently linking
together two or more antisense oligonucleotide molecules (see,
e.g., Aartsma-Rus, Janson et al. 2004).
[0017] In certain embodiments, the antisense compounds of the
present invention induce exon skipping in the human dystrophin
gene, and thereby allow muscle cells to produce a functional
dystrophin protein.
[0018] The antisense oligonucleotide compounds (also referred to
herein as oligomers) of the present invention typically: (i)
comprise morpholino subunits and phosphorus-containing intersubunit
linkages joining a morpholino nitrogen of one subunit to a 5'
exocyclic carbon of an adjacent subunit, (ii) contain between 10-40
nucleotide bases, preferably 20-35 bases (iii) comprise a base
sequence effective to hybridize to at least 12 contiguous bases of
a target sequence in dystrophin pre-mRNA and induce exon
skipping.
[0019] In certain embodiments, the antisense compounds of the
present invention may comprise phosphorus-containing intersubunit
linkages joining a morpholino nitrogen of one subunit to a 5'
exocyclic carbon of an adjacent subunit, in accordance with the
following structure (I):
##STR00001##
wherein:
[0020] Y.sub.1 is --O--, --S--, --NH--, or --CH.sub.2--;
[0021] Z is O or S;
[0022] Pj is a purine or pyrimidine base-pairing moiety effective
to bind, by base-specific hydrogen bonding, to a base in a
polynucleotide; and
[0023] X is fluoro, optionally substituted alkyl, optionally
substituted alkoxy, optionally substituted thioalkoxy, amino,
optionally substituted alkylamino, or optionally substituted
heterocyclyl.
[0024] In certain embodiments, the above intersubunit linkages,
which are uncharged, may be interspersed with linkages that are
positively charged at physiological pH, where the total number of
positively charged linkages is between 2 and no more than half of
the total number of linkages. For example, the positively charged
linkages may have the above structure in which X is optionally
substituted 1-piperazinyl. In other embodiments, the positively
charged linkages may have the above structure in which X is
substituted 1-piperazynyl, wherein the 1-piperazynyl is substituted
at the 4-position with an optionally substituted alkyl guanidynyl
moiety.
[0025] Where the antisense compound administered is effective to
target a splice site of preprocessed human dystrophin, it may have
a base sequence complementary to a target region containing at
least 12 contiguous bases in a preprocessed messenger RNA (mRNA)
human dystrophin transcript. Exemplary antisense sequences include
those identified by SEQ ID NOS: 1 to 569 and 612 to 633.
[0026] In certain embodiments, an antisense sequence of the present
invention is contained within:
[0027] (a) any of the sequences identified by SEQ ID NOS: 1-20,
preferably SEQ ID NOS: 4, 8, 11 and 12, and more preferably SEQ ID
NO:12 for use in producing skipping of exon 44 in the processing of
human dystrophin pre-processed mRNA;
[0028] (b) any of the sequences identified by SEQ ID NOS: 21-76 and
612 to 624, preferably SEQ ID NOS: 27, 29, 34 and 39, and more
preferably SEQ ID NO: 34 for use in producing skipping of exon 45
in the processing of human dystrophin pre-processed mRNA;
[0029] (c) any of the sequences identified by SEQ ID NOS: 77-125,
preferably SEQ ID NOS: 21 to 53, and more preferably SEQ ID NOS:
82, 84-87, 90 96, 98, 99 and 101, for use in producing skipping of
exon 46 in the processing of human dystrophin pre-processed
mRNA;
[0030] (d) any of the sequences identified by SEQ ID NOS: 126-169,
preferably SEQ ID NOS: 126-149, and more preferably SEQ ID NOS:
126, 128-130, 132, 144 and 146-149, for use in producing skipping
of exon 47 in the processing of human dystrophin pre-processed
mRNA;
[0031] (e) any of the sequences identified by SEQ ID NOS: 170-224
and 634, preferably SEQ ID NOS: 170-201 and 634, and more
preferably SEQ ID NOS: 176, 178, 181-183, 194 and 198-201, for use
in producing skipping of exon 48 in the processing of human
dystrophin pre-processed mRNA;
[0032] (f) any of the sequences identified by SEQ ID NOS: 225-266,
preferably SEQ ID NOS: 225-248, and more preferably SEQ ID NOS:
227, 229, 234, 236, 237 and 244-248, for use in producing skipping
of exon 49 in the processing of human dystrophin pre-processed
mRNA;
[0033] (g) any of the sequences identified by SEQ ID NOS: 267-308,
preferably SEQ ID NOS: 277, 287 and 290, and more preferably SEQ ID
NO: 287, for use in producing skipping of exon 50 in the processing
of human dystrophin pre-processed mRNA; [0034] (h) any of the
sequences identified by SEQ ID NOS: 309-371, preferably SEQ ID NOS:
324, 326 and 327, and more preferably SEQ ID NO: 327 for use in
producing skipping of exon 51 in the processing of human dystrophin
pre-processed mRNA; [0035] (i) any of the sequences identified by
SEQ ID NOS: 372-415, preferably SEQ ID NOS: 372-397, and more
preferably SEQ ID NOS: 379-382, 384, 390 and 392-395 for use in
producing skipping of exon 52 in the processing of human dystrophin
pre-processed mRNA;
[0036] (j) any of the sequences identified by SEQ ID NOS: 416-475
and 625-633, preferably SEQ ID NOS: 428, 429 and 431, and more
preferably SEQ ID NO: 429, for use in producing skipping of exon 53
in the processing of human dystrophin pre-processed mRNA;
[0037] (k) any of the sequences identified by SEQ ID NOS: 476-519,
preferably SEQ ID NOS: 476-499, and more preferably SEQ ID NOS:
479-482, 484, 489 and 491-493, for use in producing skipping of
exon 54 in the processing of human dystrophin pre-processed mRNA;
and
[0038] (l) any of the sequences identified by SEQ ID NOS: 520-569
and 635, preferably SEQ ID NOS: 520-546 and 635, and more
preferably SEQ ID NOS: 524-528, 537, 539, 540, 542 and 544, for use
in producing skipping of exon 55 in the processing of human
dystrophin pre-processed mRNA;
[0039] In certain embodiments, the compound may be conjugated to an
arginine-rich polypeptide effective to promote uptake of the
compound into cells. Exemplary peptides include those identified by
SEQ ID NOS: 570 to 578, among others described herein.
[0040] In one exemplary embodiment, the arginine-rich polypeptide
is covalently coupled at its N-terminal or C-terminal residue to
the 3' or 5' end of the antisense compound. Also in an exemplary
embodiment, the antisense compound is composed of morpholino
subunits and phosphorus-containing intersubunit linkages joining a
morpholino nitrogen of one subunit to a 5' exocyclic carbon of an
adjacent subunit.
[0041] In general, the peptide-oligomer conjugate may further
comprise a homing peptide which is selective for a selected
mammalian tissue, i.e., the same tissue being targeted by the
cell-penetrating peptide. The conjugate may be of the form: cell
penetrating peptide--homing peptide--antisense oligomer, or, more
preferably, of the form: homing peptide--cell penetrating
peptide--antisense oligomer. For example, a peptide conjugate
compound for use in treating Duchenne muscular dystrophy, as
described above, can further comprise a homing peptide which is
selective for muscle tissue, such as the peptide having the
sequence identified as SEQ ID NO: 579, conjugated to the
cell-penetrating peptide. Exemplary conjugates of this type include
those represented herein as CP06062-MSP-PMO (cell penetrating
peptide--homing peptide--antisense oligomer) and as MSP-CP06062-PMO
(homing peptide--cell penetrating peptide--antisense oligomer) (see
SEQ ID NOs: 580-583).
[0042] In some embodiments, the peptide is conjugated to the
oligomer via a linker moiety. In certain embodiments the linker
moiety may comprise an optionally substituted piperazynyl moiety.
In other embodiments, the linker moiety may further comprise a beta
alanine and/or a 6-aminohexanoic acid subunit. In yet other
embodiments, the peptide is conjugated directly to the oligomer
without a linker moiety.
[0043] Conjugation of the peptide to the oligomer may be at any
position suitable for forming a covalent bond between the peptide
and the oligomer or between the linker moiety and the oligomer. For
example, in some embodiments conjugation of the peptide may be at
the 3' end of the oligomer. In other embodiments, conjugation of
the peptide to the oligomer may be at the 5' end of the oligomer.
In yet other embodiments, the peptide may be conjugated to the
oligomer through any of the intersubunit linkages.
[0044] In some embodiments, the peptide is conjugated to the
oligomer at the 5' end of the oligomer. In embodiments comprising
phosphorus-containing intersubunit linkages, the peptide may be
conjugated to the oligomer via a covalent bond to the phosphorous
of the terminal linkage group. Conjugation in this manner may be
with or without the linker moiety described above.
[0045] In yet other embodiments, the peptide may be conjugated to
the oligomer at the 3' end of the oligomer. In some further
embodiments, the peptide may be conjugated to the nitrogen atom of
the 3' terminal morpolino group of the oligomer. In this respect,
the peptide may be conjugated to the oligomer directly or via the
linker moiety described above.
[0046] In some embodiments, the oligomer may be conjugated to a
moiety that enhances the solubility of the oligomer in aqueous
medium. In some embodiments, the moiety that enhances solubility of
the oligomer in aqueous medium is a polyethyleneglycol. In yet
further embodiments, the moiety that enhances solubility of the
oligomer in aqueous medium is triethylene glycol. For example, in
some embodiments the moiety that enhances solubility in aqueous
medium may be conjugated to the oligomer at the 5' end of the
oligomer. Conjugation of the moiety that enhances solubility of the
oligomer in aqueous medium to the oligomer may be either directly
or through the linker moiety described above.
[0047] Certain embodiments of the present invention provide
antisense molecules selected and or adapted to aid in the
prophylactic or therapeutic treatment of a genetic disorder
comprising at least an antisense molecule in a form suitable for
delivery to a patient.
[0048] Certain embodiments of the invention provide methods for
treating a patient suffering from a genetic disease wherein there
is a mutation in a gene encoding a particular protein and the
affect of the mutation can be abrogated by exon skipping,
comprising the steps of: (a) selecting an antisense molecule in
accordance with the methods described herein; and (b) administering
the molecule to a patient in need of such treatment. The present
invention also includes the use of purified and isolated antisense
oligonucleotides of the invention, for the manufacture of a
medicament for treatment of a genetic disease.
[0049] Certain embodiments provide a method of treating muscular
dystrophy, such as a condition characterized by Duchenne muscular
dystrophy, which method comprises administering to a patient in
need of treatment an effective amount of an appropriately designed
antisense oligonucleotide, as described herein, relevant to the
particular genetic lesion in that patient. Further, certain
embodiments provide a method for prophylactically treating a
patient to prevent or at least minimize muscular dystrophy,
including Duchene muscular dystrophy, comprising the step of:
administering to the patient an effective amount of an antisense
oligonucleotide or a pharmaceutical composition comprising one or
more of these biological molecules.
[0050] Certain embodiments relate to methods of treating muscular
dystrophy in a subject, comprising administering to the subject an
effective amount of a substantially uncharged antisense compound
containing 20-35 morpholino subunits linked by
phosphorus-containing intersubunit linkages joining a morpholino
nitrogen of one subunit to a 5' exocyclic carbon of an adjacent
subunit, comprising a sequence selected from the group consisting
SEQ ID NOS:1 to 569 and 612 to 635, and capable of forming with the
complementary mRNA sequence in a dystrophin-gene exon a
heteroduplex structure between said compound and mRNA having a Tm
of at least 45.degree. C., wherein the exon is selected from the
group consisting of exons 44-55.
[0051] In certain embodiments, the muscular dystrophy is Duchenne's
muscular dystrophy (DMD). In certain embodiments, the muscular
dystrophy is Becker muscular dystrophy (BMD).
[0052] In certain embodiments, the sequence is selected from the
group consisting SEQ ID NOS: 1-20, and the exon is exon 44. In
certain embodiments, the sequence is selected from the group
consisting SEQ ID NOS: 21-76 and 612 to 624, and the exon is exon
45.
[0053] In certain embodiments, the sequence is selected from the
group consisting SEQ ID NOS: 77-125, and the exon is exon 46. In
certain embodiments, the sequence selected from the group
consisting SEQ ID NOS: 126-169, and the exon is exon 47.
[0054] In certain embodiments, the sequence is selected from the
group consisting SEQ ID NOS: 170-224 and 634, and the exon is exon
48. In certain embodiments, the sequence selected from the group
consisting SEQ ID NOS: 225-266, and the exon is exon 49.
[0055] In certain embodiments, the sequence is selected from the
group consisting SEQ ID NOS: 267-308, and the exon is exon 50. In
certain embodiments, the sequence is selected from the group
consisting SEQ ID NOS: 309-371, and the exon is exon 51.
[0056] In certain embodiments, the sequence is selected from the
group consisting SEQ ID NOS: 372-415, and the exon is exon 52. In
certain embodiments, the sequence is selected from the group
consisting SEQ ID NOS: 416-475 and 625-633, and the exon is exon
53. In certain embodiments, the sequence is selected from the group
consisting SEQ ID NOS: 476-519, and the exon is exon 54. In certain
embodiments, the sequence is selected from the group consisting SEQ
ID NOS: 520-569 and 635, and the exon is exon 55. In certain
embodiments, the sequence comprises or consists essentially of SEQ
ID NO:287.
[0057] Certain embodiments provide kits for treating a genetic
disease, which kits comprise at least an antisense oligonudeotide
of the present invention, packaged in a suitable container and
instructions for its use.
[0058] These and other objects and features will be more fully
understood when the following detailed description of the invention
is read in conjunction with the figures.
BRIEF DESCRIPTION OF THE FIGURES
[0059] FIG. 1A shows an exemplary morpholino oligomer structure
with a phosphorodiamidate linkage;
[0060] FIG. 1B shows a conjugate of an arginine-rich peptide and an
antisense oligomer, in accordance with an embodiment of the
invention;
[0061] FIG. 1C shows a conjugate as in FIG. 1B, wherein the
backbone linkages contain one or more positively charged
groups;
[0062] FIGS. 1D-G show the repeating subunit segment of exemplary
morpholino oligonucleotides, designated D through G.
[0063] FIG. 2A shows the relative location and results of an
antisense oligomer exon 51 scan designed to induce skipping of
human dystrophin exon 51.
[0064] FIG. 2B-C shows the relative activity in cultured human
rhabdomyosarcoma (RD) cells and human primary skeletal muscle cells
of the three best oligomers selected from the exon 51 scan (SEQ ID
NOs: 324, 326 and 327) relative to sequences (AVI-5658; SEQ ID NO:
588 and h51AON1; SEQ ID NO:594) that are effective at inducing exon
51 skipping. FIG. 2D shows the relative location within exon 51 of
three selected oligomers compared to certain sequences.
[0065] FIG. 3A shows the relative location and results of an
antisense oligomer exon 50 scan designed to induce skipping of
human dystrophin exon 50 compared to other sequences that induce
exon 50 skipping.
[0066] FIG. 3B shows the relative location and activity of
antisense sequences selected from the exon 50 scan (SEQ ID NOS:
277, 287, 290 and 291) compared to other sequences (SEQ ID NOS: 584
and 585).
[0067] FIG. 4A shows the relative location and results of an
antisense oligomer exon 53 scan designed to induce skipping of
human dystrophin exon 53.
[0068] FIG. 4B shows the relative location of certain sequences
used to compare the exon-skipping activity of those oligomers
selected as being most active in the exon 53 scan.
[0069] FIGS. 4C-F show the results of dose-ranging studies,
summarized in FIG. 4G, using the oligomers selected as being most
efficacious in the exon 53 scan (SEQ ID NOS:422, 428, 429 and
431).
[0070] FIGS. 4H and 4I show the relative activity of certain
sequences (SEQ ID NOS: 608-611) compared to the activity of the
most active exon 53-skipping oligomer (SEQ ID NO:429) in both RD
cells and human primary skeletal muscle cells.
[0071] FIG. 5A shows the relative location and results of an
antisense oligomer exon 44 scan designed to induce skipping of
human dystrophin exon 44.
[0072] FIG. 5B shows the relative location within exon 44 of
certain sequences used to compare the exon-skipping activity to
those oligomers selected as being most active in the exon 44
scan.
[0073] FIGS. 5C-G show the results of dose-ranging studies,
summarized in FIG. 5H, using the oligomers selected as being most
efficacious in the exon 44 scan (SEQ ID NOS: 4, 8, 11, 12 and
13).
[0074] FIGS. 5I and 5J show the relative activity of certain
sequences (SEQ ID NOS: 600-603) compared to the activity of the
most active exon 53-skipping oligomer (SEQ ID NO:12) in both RD
cells and human primary skeletal muscle cells.
[0075] FIG. 6A shows the relative location and results of an
antisense oligomer exon 45 scan designed to induce skipping of
human dystrophin exon 45. FIG. 6B shows the relative location
within exon 45 of certain sequences used to compare the
exon-skipping activity to those oligomers selected as being most
active in the exon 45 scan.
[0076] FIGS. 6C-F show the results of dose-ranging studies,
summarized in FIG. 6H, using the oligomers selected as being most
efficacious in the exon 45 scan (SEQ ID NOS: 27, 29, 34 and 39).
FIG. 6G uses a relatively inactive oligomer (SEQ ID NO: 49) as a
negative control.
[0077] FIGS. 6I and 6J show the relative activity of certain
sequences (SEQ ID NOS: 604-607) compared to the activity of the
most active exon 53-skipping oligomer (SEQ ID NO: 34) in both RD
cells and human primary skeletal muscle cells.
DETAILED DESCRIPTION OF THE INVENTION
[0078] Embodiments of the present invention relate generally to
improved antisense compounds, and methods of use thereof, which are
specifically designed to induce exon skipping in the 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 antisense
compounds described herein induce exon skipping in mutated forms of
the human dystrophin gene, such as the mutated dystrophin genes
found in Duchenne's muscular dystrophy (DMD) and Becker's muscular
dystrophy (BMD).
[0079] 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 typically 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.
[0080] By increasing the levels of functional dystrophin protein in
muscle cells, these and related embodiments may be 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 specific oligomers described
herein further provide improved, dystrophin-exon-specific targeting
over other oligomers in use, and thereby offer significant and
practical advantages over alternate methods of treating relevant
forms of muscular dystrophy.
[0081] 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.
Definitions
[0082] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0083] 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, 25, 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.
[0084] By "coding sequence" is meant any nucleic acid sequence that
contributes to the code for the polypeptide product of a gene. By
contrast, the term "non-coding sequence" refers to any nucleic acid
sequence that does not contribute to the code for the polypeptide
product of a gene.
[0085] Throughout this specification, unless the context requires
otherwise, the words "comprise," "comprises," and "comprising" will
be understood to imply the inclusion of a stated step or element or
group of steps or elements but not the exclusion of any other step
or element or group of steps or elements.
[0086] By "consisting of" is meant including, and limited to,
whatever follows the phrase "consisting of." Thus, the phrase
"consisting of" indicates that the listed elements are required or
mandatory, and that no other elements may be present. By
"consisting essentially of" is meant including any elements listed
after the phrase, and limited to other elements that do not
interfere with or contribute to the activity or action specified in
the disclosure for the listed elements. Thus, the phrase
"consisting essentially of" indicates that the listed elements are
required or mandatory, but that other elements are optional and may
or may not be present depending upon whether or not they materially
affect the activity or action of the listed elements.
[0087] The terms "complementary" and "complementarity" refer to
polynucleotides (i.e., a sequence of nucleotides) related by the
base-pairing rules. For example, the sequence "A-G-T," is
complementary to the sequence "T-C-A." Complementarity may be
"partial," in which only some of the nucleic acids' bases are
matched according to the 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.
[0088] The terms "cell penetrating peptide" or "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 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.
[0089] The terms "antisense oligomer" or "antisense compound" 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).
[0090] 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. In certain
embodiments, the target sequence includes a region including an AUG
start codon of an mRNA, a 3' or 5' splice site of a pre-processed
mRNA, or a branch point. The target sequence may be within an exon
or within an intron. 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 for a splice 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. Included are antisense
oligomers that comprise, consist essentially of, or consist of one
or more of SEQ ID NOS:1 to 569 and 612 to 635. Also included are
variants of these antisense oligomers, including variant oligomers
having 80%, 85%, 90%, 95%, 97%, 98%, or 99% (including all integers
in between) sequence identity or sequence homology to any one of
SEQ ID NOS:1 to 569 and 612 to 635, and/or variants that differ
from these sequences by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
nucleotides, preferably those variants that induce exon skipping of
one or more selected human dystrophin exons. Also included are
oligomers of any on or more of SEQ ID NOS:584-611 and 634-635,
which comprise a suitable number of charged linkages, as described
herein, e.g. up to about 1 per every 2-5 uncharged linkages, such
as about 4-5 per every 10 uncharged linkages, and/or which comprise
an Arg-rich peptide attached thereto, as also described herein.
[0091] 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, e.g., 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. See also
the discussion of cationic linkages below. 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, and 5,506,337, and PCT Appn. No.
PCT/US07/11435 (cationic linkages), all of which are incorporated
herein by reference.
[0092] The purine or pyrimidine base pairing moiety is typically
adenine, cytosine, guanine, uracil, thymine or inosine. Also
included are bases such as pyridin-4-one, pyridin-2-one, phenyl,
pseudouracil, 2,4,6-trime115thoxy benzene, 3-methyl uracil,
dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g.,
5-methylcytidine), 5-alkyluridines (e.g., ribothymidine),
5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or
6-alkylpyrimidines (e.g. 6-methyluridine), propyne, quesosine,
2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine,
4-acetyltidine, 5-(carboxyhydroxymethyl)uridine,
5'-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluridine, .beta.-D-galactosylqueosine,
1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,
3-methylcytidine, 2-methyladenosine, 2-methylguanosine,
N6-methyladenosine, 7-methylguanosine,
5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,
5-methylcarbonyhnethyluridine, 5-methyloxyuridine,
5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,
R-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,
threonine derivatives and others (Burgin et al., 1996,
Biochemistry, 35, 14090; Uhlman & Peyman, supra). By "modified
bases" in this aspect is meant nucleotide bases other than adenine
(A), guanine (G), cytosine (C), thymine (T), and uracil (U), as
illustrated above; such bases can be used at any position in the
antisense molecule. Persons skilled in the art will appreciate that
depending on the uses of the oligomers, Ts and Us are
interchangeable. For instance, with other antisense chemistries
such as 2'-O-methyl antisense oligonucleotides that are more
RNA-like, the T bases may be shown as U (see, e.g., Sequence ID
Listing).
[0093] An "amino acid subunit" or "amino acid residue" can refer to
an .alpha.-amino acid residue (e.g., --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.
[0094] The term "naturally occurring amino acid" refers to an amino
acid present in proteins found in nature, such as the 20 (L)-amino
acids utilized during protein biosynthesis as well as others such
as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine,
homocysteine, citrulline and omithine. The term "non-natural amino
acids" refers to those amino acids not present in proteins found in
nature, examples include beta-alanine (.beta.-Ala; or B),
6-aminohexanoic acid (Ahx) and 6-aminopentanoic acid. Additional
examples of "non-natural amino acids" include, without limitation,
(D)-amino acids, norleucine, norvaline, p-fluorophenylalanine,
ethionine and the like, which are known to a person skilled in the
art.
[0095] An "effective amount" or "therapeutically effective amount"
refers to an amount of therapeutic compound, such as an antisense
oligomer, administered to a mammalian subject, either as a single
dose or as part of a series of doses, which is effective to produce
a desired physiological response or therapeutic effect in the
subject. One example of a desired physiological response includes
increased expression of a relatively functional or biologically
active form of the dystrophin protein, mainly in muscle tissues or
cells that contain a defective dystrophin protein or no dystrophin,
as compared no antisense oligomer or a control oligomer. Examples
of desired therapeutic effects include, without limitation,
improvements in the symptoms or pathology of muscular dystrophy,
reducing the progression of symptoms or pathology of muscular
dystrophy, and slowing the onset of symptoms or pathology of
muscular dystrophy, among others. Examples of such symptoms include
fatigue, mental retardation, muscle weakness, difficulty with motor
skills (e.g., running, hopping, jumping), frequent falls, and
difficulty walking. The pathology of muscular dystrophy can be
characterized, for example, by muscle fibre damage and membrane
leakage. For an antisense oligomer, this effect is typically
brought about by altering the splice-processing of a selected
target sequence (e.g., dystrophin), such as to induce exon
skipping.
[0096] 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 75 exons.
[0097] 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.
[0098] "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 1-75 of the
dystrophin gene, though any one or more of exons 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, and/or 55 of the human dystrophin gene
are preferred.
[0099] "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.
[0100] 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.
[0101] Table A provides an illustration of the various dystrophin
domains, the amino acid residues that encompass these domains, and
the exons that encode them.
TABLE-US-00001 TABLE A Residue Domain Sub Domain Nos Exons actin
binding 14-240 2-8 domain central rod 253-3040 8-61 domain hinge 1
253-327 (8)-9 repeat 1 337-447 10-11 repeat 2 448-556 12-14 repeat
3 557-667 14-16 hinge 2 668-717 17 repeat 4 718-828 (17)-20 repeat
5 829-934 20-21 repeat 6 935-1045 22-23 repeat 7 1046-1154
(23)-(26) repeat 8 1155-1263 26-27 repeat 9 1264-1367 28-(30)
repeat 10 1368-1463 30-32 repeat 11 1464-1568 32-(34) repeat 12
1569-1676 34-35 repeat 13 1677-1778 36-37 repeat 14 1779-1874
38-(40) repeat 15 1875-1973 40-41 interruption 1974-1991 42 repeat
16 1992-2101 42-43 repeat 17 2102-2208 44-45 repeat 18 2209-2318
46-48 repeat 19 2319-2423 48-50 hinge 3 2424-2470 50-51 repeat 20
2471-2577 51-53 repeat 21 2578-2686 53-(55) repeat 22 2687-2802
55-(57) repeat 23 2803-2931 57-59 repeat 24 2932-3040 59-(61) hinge
4 3041-3112 61-64 Cysteine-rich 3080-3360 63-69 domain dystroglycan
binding site 3080-3408 63-70 WW domain 3056-3092 62-63 EF-hand 1
3130-3157 65 EF-hand 2 3178-3206 65-66 ZZ domain 3307-3354 68-69
Carboxy-terminal 3361-3685 70-79 domain alpha1-syntrophin binding
3444-3494 73-74 site .beta.1-syntrophin binding site 3495-3535
74-75 (Leu)6-heptad repeat 3558-3593 75
[0102] As used herein, the terms "function" and "functional" and
the like refer to a biological, enzymatic, or therapeutic
function.
[0103] 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.
[0104] By "gene" is meant a unit of inheritance that occupies a
specific locus on a chromosome and consists of transcriptional
and/or translational regulatory sequences and/or a coding region
and/or non-translated sequences (i.e., introns, 5' and 3'
untranslated sequences).
[0105] 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.
[0106] 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
fibres that express a functional dystrophin can also be measured,
including increased dystrophin expression in 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% of muscle fibres. 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.
[0107] 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.
[0108] "Homology" refers to the percentage number of amino acids
that are identical or constitute conservative substitutions.
Homology may be determined using sequence comparison programs such
as GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395).
In this way sequences of a similar or substantially different
length to those cited herein could be compared by insertion of gaps
into the alignment, such gaps being determined, for example, by the
comparison algorithm used by GAP.
[0109] The recitations "sequence identity" or, for example,
comprising a "sequence 50% identical to," as used herein, refer to
the extent that sequences are identical on a
nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis
over a window of comparison. Thus, a "percentage of sequence
identity" may be calculated by comparing two optimally aligned
sequences over the window of comparison, determining the number of
positions at which the identical nucleic acid base (e.g., A, T, C,
G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,
Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu,
Asn, Gin, Cys and Met) occurs in both sequences to yield the number
of matched positions, dividing the number of matched positions by
the total number of positions in the window of comparison (i.e.,
the window size), and multiplying the result by 100 to yield the
percentage of sequence identity.
[0110] Terms used to describe sequence relationships between two or
more polynucleotides or polypeptides include "reference sequence,"
"comparison window," "sequence identity," "percentage of sequence
identity," and "substantial identity". A "reference sequence" is at
least 8 or 10 but frequently 15 to 18 and often at least 25 monomer
units, inclusive of nucleotides and amino acid residues, in length.
Because two polynucleotides may each comprise (1) a sequence (i.e.,
only a portion of the complete polynucleotide sequence) that is
similar between the two polynucleotides, and (2) a sequence that is
divergent between the two polynucleotides, sequence comparisons
between two (or more) polynucleotides are typically performed by
comparing sequences of the two polynucleotides over a "comparison
window" to identify and compare local regions of sequence
similarity. A "comparison window" refers to a conceptual segment of
at least 6 contiguous positions, usually about 50 to about 100,
more usually about 100 to about 150 in which a sequence is compared
to a reference sequence of the same number of contiguous positions
after the two sequences are optimally aligned. The comparison
window may comprise additions or deletions (i.e., gaps) of about
20% or less as compared to the reference sequence (which does not
comprise additions or deletions) for optimal alignment of the two
sequences. Optimal alignment of sequences for aligning a comparison
window may be conducted by computerized implementations of
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group, 575
Science Drive Madison, Wis., USA) or by inspection and the best
alignment (i.e., resulting in the highest percentage homology over
the comparison window) generated by any of the various methods
selected. Reference also may be made to the BLAST family of
programs as for example disclosed by Altschul et al., 1997, Nucl.
Acids Res. 25:3389. A detailed discussion of sequence analysis can
be found in Unit 19.3 of Ausubel et al., "Current Protocols in
Molecular Biology," John Wiley & Sons Inc, 1994-1998, Chapter
15.
[0111] "Treatment" or "treating" of an individual (e.g., a mammal,
such as a human) or a cell may include 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.
[0112] Hence, included are methods of treating muscular dystrophy,
such as DMD and BMD, by administering one or more antisense
oligomers of the present invention (e.g., SEQ ID NOS: 1 to 569 and
612 to 635, and variants thereof), optionally as part of a
pharmaceutical formulation or dosage form, to a subject in need
thereof. Also included are methods of inducing exon-skipping in a
subject by administering one or more antisense oligomers, in which
the exon is one of exons 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, and/or 55 from the dystrophin gene, preferably the human
dystrophin gene. 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.
[0113] 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 SEQ ID NOS: 1 to 569 and 612
to 635, or 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 integrable 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.
[0114] A vector or nucleic acid construct system can comprise a
single vector or plasmid, two or more vectors or plasmids, which
together contain the total DNA to be introduced into the genome of
the host cell, or a transposon. The choice of the vector will
typically depend on the compatibility of the vector with the host
cell into which the vector is to be introduced. In the present
case, the vector or nucleic acid construct is preferably one which
is operably functional in a mammalian cell, such as a muscle cell.
The vector can also include a selection marker such as an
antibiotic or drug resistance gene, or a reporter gene (i.e., green
fluorescent protein, luciferase), that can be used for selection or
identification of suitable transformants or transfectants.
Exemplary delivery systems may include viral vector systems (i.e.,
viral-mediated transduction) including, but not limited to,
retroviral (e.g., lentiviral) vectors, adenoviral vectors,
adeno-associated viral vectors, and herpes viral vectors, among
others known in the art.
[0115] The term "operably linked" as used herein means placing an
oligomer-encoding sequence under the regulatory control of a
promoter, which then controls the transcription of the
oligomer.
[0116] A wild-type gene or gene product is that which is most
frequently observed in a population and is thus arbitrarily
designed the "normal" or "wild-type" form of the gene.
[0117] "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.
[0118] "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.
[0119] "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.
[0120] "Cycloalkyl" refers to a mono- or poly-cyclic alkyl radical.
Examples include without limitation cyclobutyl, cycopentyl,
cyclohexyl, cycloheptyl and cyclooctyl.
[0121] "Aryl" refers to a cyclic aromatic hydrocarbon moiety
containing from 5 to 18 carbons having one or more closed ring(s).
Examples include without limitation phenyl, benzyl, naphthyl,
anthracenyl, phenanthracenyl and biphenyl.
[0122] "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.
[0123] "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.
[0124] "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.
[0125] "Alkoxyalkyl" refers to an alkyl group substituted with an
alkoxy group.
[0126] "Carbonyl" refers to the --C(.dbd.O)-- radical.
[0127] "Guanidynyl" refers to the H.sub.2N(C.dbd.NH.sub.2)--NH--
radical.
[0128] "Amidinyl" refers to the H.sub.2N(C.dbd.NH.sub.2)CH--
radical.
[0129] "Amino" refers to the --NH.sub.2 radical.
[0130] "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.
[0131] "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, piperizynyl,
hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,
tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothiophenyl,
tetrahydrothiopyranyl, tetrahydropyrimidinyl,
tetrahydrothiopyranyl, and the like.
[0132] "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.
[0133] 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.
Constructing Antisense Oligonucleotides
[0134] Examples of morpholino oligonucleotides having
phosphorus-containing backbone linkages are illustrated in FIGS.
1A-1C. Especially 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 and their preparation, 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, and
5,506,337, all of which are expressly incorporated by reference
herein.
[0135] 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 oligonudeotide to be actively or passively
transported into mammalian cells; and 4) the ability of the
antisense oligonucleotide:RNA heteroduplex to resist RNAse and
RNaseH degradation, respectively.
[0136] 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, wherein 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.
[0137] 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=NH.sub.2, N(CH.sub.3).sub.2, optionally
substituted 1-piperazinyl, or other charged group, Y.dbd.O, and
Z.dbd.O.
[0138] As noted above, the uncharged or 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. Optimal improvement in antisense activity may
be seen when about 25% of the backbone linkages are cationic,
including about 20% to about 30%. Also included are oligomers in
which about 35%, 40%, 45%, 50%, 55%, 60% (including all integers in
between), or more of the backbone linkages are cationic.
Enhancement is also seen with a small number, e.g., 5% or 10-20%,
of cationic linkages.
[0139] A substantially uncharged, phosphorus containing backbone in
an oligonucleotide analog is typically one in which a majority of
the subunit linkages, e.g., between 50%-100%, typically at least
60% to 100% or 75% or 80% of its linkages, are uncharged at
physiological pH and contain a single phosphorous atom.
[0140] Additional experiments conducted in support of the present
invention indicate that the enhancement seen with added cationic
backbone charges may, in some cases, be further enhanced by
distributing the bulk of the charges close to the "center-region"
backbone linkages of the antisense oligonucleotide, e.g., in a
20mer oligonucleotide with 8 cationic backbone linkages, having at
least 70% of these charged linkages localized in the 10 centermost
linkages.
[0141] 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 of 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, typically
to a terminus of the oligomer, according to standard synthetic
methods. For example, addition of a polyethyleneglycol moiety or
other hydrophilic polymer, e.g., one having 10-100 monomeric
subunits, may be useful in enhancing solubility. One or more
charged groups, e.g., anionic charged groups such as an organic
acid, may enhance cell uptake. 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.
[0142] As noted above, the antisense compound can be constructed to
contain a selected number of cationic linkages interspersed with
uncharged linkages of the type described above. The intersubunit
linkages, both uncharged and cationic, preferably are
phosphorus-containing linkages, having the structure (II):
##STR00002##
wherein:
[0143] W is --S-- or --O--, and is preferably --O--,
[0144] X=--NR.sup.1R.sup.2 or --OR.sup.6,
[0145] Y=--O-- or --NR.sup.7, and
[0146] each said linkage in the oligomer is selected from:
[0147] (a) an uncharged linkage (a), wherein each of R.sup.1,
R.sup.2, R.sup.6 and R.sup.7 is independently selected from
hydrogen and lower alkyl;
[0148] (b1) a cationic linkage (b1), wherein X=--NR.sup.1R.sup.2
and Y=--O--, and --NR.sup.1R.sup.2 represents an optionally
substituted piperazinyl moiety, such that
R.sup.1R.sup.2=--CHRCHRN(R.sup.3)(R.sup.4)CHRCHR--, wherein:
[0149] each R is independently H or --CH.sub.3,
[0150] R.sup.4 is H, --CH.sub.3, or an electron pair, and
[0151] R.sup.3 is selected from H, optionally substituted lower
alkyl, --C(.dbd.NH)NH.sub.2, --Z-L-NHC(.dbd.NH)NH.sub.2, and
[--C(.dbd.O)CHR'NH].sub.mH, where: Z is --C(.dbd.O)-- or a direct
bond, L is an optional linker up to 18 atoms in length, preferably
up to 12 atoms, and more preferably up to 8 atoms in length, having
bonds selected from optionally substituted alkyl, optionally
substituted alkoxy, and optionally substituted alkylamino, R' is a
side chain of a naturally occurring amino acid or a one- or
two-carbon homolog thereof, and m is 1 to 6, preferably 1 to 4;
[0152] (b2) a cationic linkage (b2), wherein X=--NR.sup.1R.sup.2
and Y=--O--, R.sup.1=H or --CH.sub.3, and
R.sup.2=LNR.sup.3R.sup.4R.sup.5, wherein L, R.sup.3, and R.sup.4
are as defined above, and R.sup.5 is H, optionally substituted
lower alkyl, or optionally substituted lower (alkoxy)alkyl; and
[0153] (b3) a cationic linkage (b3), wherein Y=--NR.sup.7 and
X=--OR.sup.6, and R.sup.7=-LNR.sup.3R.sup.4R.sup.5, wherein L,
R.sup.3, R.sup.4 and R.sup.5 are as defined above, and R.sup.6 is H
or optionally substituted lower alkyl; and
[0154] at least one said linkage is selected from cationic linkages
(b1), (b2), and (b3).
[0155] Preferably, the oligomer includes at least two consecutive
linkages of type (a) (i.e. uncharged linkages). In further
embodiments, at least 5% of the linkages in the oligomer are
cationic linkages (i.e. type (b1), (b2), or (b3)); for example, 10%
to 60%, and preferably 20-50% linkages may be cationic
linkages.
[0156] In one embodiment, at least one linkage is of type (b1),
where, preferably, each R is H, R.sup.4 is H, --CH.sub.3, or an
electron pair, and R.sup.3 is selected from H, optionally
substituted lower alkyl, --C(.dbd.NH)NH.sub.2, and
--C(.dbd.O)-L-NHC(.dbd.NH)NH.sub.2. The latter two embodiments of
R.sup.3 provide a guanidino moiety, either attached directly to the
piperazine ring, or pendant to a linker group L, respectively. For
ease of synthesis, the variable Z in R.sup.3 is preferably
--C(.dbd.O)--, as shown.
[0157] The linker group L, as noted above, contains bonds in its
backbone selected from optionally substituted alkyl, optionally
substituted alkoxy, and optionally substituted alkylamino, wherein
the terminal atoms in L (e.g., those adjacent to carbonyl or
nitrogen) are carbon atoms. Although branched linkages are
possible, the linker is preferably unbranched. In one embodiment,
the linker is a linear alkyl linker. Such a linker may have the
structure --(CH.sub.2)--, where n is 1-12, preferably 2-8, and more
preferably 2-6.
[0158] The morpholino subunits have the following structure
(III):
##STR00003##
wherein Pi is a base-pairing moiety, and the linkages depicted
above connect the nitrogen atom of (III) to the 5' carbon of an
adjacent subunit. The base-pairing moieties Pi may be the same or
different, and are generally designed to provide a sequence which
binds to a target nucleic acid.
[0159] The use of embodiments of linkage types (b1), (b2) and (b3)
above to link morpholino subunits (III) may be illustrated
graphically as follows:
##STR00004##
[0160] Preferably, all cationic linkages in the oligomer are of the
same type; i.e. all of type (b1), all of type (b2), or all of type
(b3).
[0161] In further embodiments, the cationic linkages are selected
from linkages (b1') and (b1'') as shown below, where (b1') is
referred to herein as a "Pip" linkage and (b1'') is referred to
herein as a "GuX" linkage:
##STR00005##
[0162] In the structures above, W is S or O, and is preferably O;
each of R.sup.1 and R.sup.2 is independently selected from hydrogen
and optionally substituted lower alkyl, and is preferably methyl;
and A represents hydrogen or a non-interfering substituent (i.e. a
substituent that does not adversely affect the ability of an
oligomer to bind to its intended target) on one or more carbon
atoms in (b1') and (b1''). Preferably, the ring carbons in the
piperazine ring are unsubstituted; however, the ring carbons of the
piperazine ring may include non-interfering substituents, such as
methyl or fluorine. Preferably, at most one or two carbon atoms is
so substituted.
[0163] In further embodiments, at least 10% of the linkages are of
type (b1') or (b1''); for example, 10%-60% and preferably 20% to
50%, of the linkages may be of type (b1') or (b1'').
[0164] In other embodiments, the oligomer contains no linkages of
the type (b1') above. Alternatively, the oligomer contains no
linkages of type (b1) where each R is H, R.sup.3 is H or
--CH.sub.3, and R.sup.4 is H, --CH.sub.3, or an electron pair.
[0165] The morpholino subunits may also be linked by
non-phosphorus-based intersubunit linkages, as described further
below, where at least one linkage is modified with a pendant
cationic group as described above.
[0166] Other oligonucleotide analog linkages which are uncharged in
their unmodified state but which could also bear a pendant amine
substituent could be used. For example, a 5'nitrogen atom on a
morpholino ring could be employed in a sulfamide linkage or a urea
linkage (where phosphorus is replaced with carbon or sulfur,
respectively) and modified in a manner analogous to the 5'-nitrogen
atom in structure (b3) above.
[0167] 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.
[0168] 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.
[0169] 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%.
[0170] Oligomers for use in antisense applications generally range
in length from about 10 to about 40 subunits, more preferably about
10 to 30 subunits, and typically 15-25 bases, including those
having 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, or 40
bases. In certain embodiments, 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 to 5, cationic
linkages, and the remainder uncharged linkages.
[0171] 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.
Peptide Transporters
[0172] The antisense compounds of the invention may include an
oligonucleotide moiety conjugated to 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 peptide transport moiety preferably comprises 6 to 16
subunits selected from X' subunits, Y subunits, and Z' subunits,
wherein:
[0173] (a) each X' subunit independently represents lysine,
arginine or an arginine analog, said analog being a cationic
.alpha.-amino acid comprising a side chain of the structure
R'N=C(NH.sub.2)R.sup.2, where R.sup.1 is H or R; R.sup.2 is R,
--NH.sub.2, --NHR, or --NR.sub.2, where R is optionally substituted
lower alkyl or optionally substituted lower alkenyl; R.sup.1 and
R.sup.2 may join together to form a ring; and the side chain is
linked to said amino acid via R.sup.1 or R.sup.2;
[0174] (b) each Y' subunit independently represents a neutral amino
acid --C(.dbd.O)--(CHR).sub.n--NH--, where n is 2 to 7 and each R
is independently H or methyl; and
[0175] (c) each Z' subunit independently represents an
.alpha.-amino acid having a neutral aralkyl side chain;
wherein the peptide comprises a sequence represented by at least
one of (X'Y'X').sub.p, (X'Y').sub.m, and/or (X'Z'Z'), where p is 2
to 5 and m is 2 to 8. Certain embodiments include various
combinations selected independently from (X'Y'X'), (X'Y').sub.m.
and/or (X'Z'Z').sub.p, including, for example, peptides having the
sequence (X'Y'X')(X'Z'Z')(X'Y'X'X'Z'Z') (SEQ ID NO:637).
[0176] In selected embodiments, for each X', the side chain moiety
is guanidyl, as in the amino acid subunit arginine (Arg). In
certain embodiments, each Y' is independently
--C(.dbd.O)--(CH.sub.2).sub.n--CHR--NH--, where n is 2 to 7 and R
is H. For example, when n is 5 and R is H, Y' is a 6-aminohexanoic
acid subunit, abbreviated herein as Ahx; when n is 2 and R is H, Y'
is a .beta.-alanine subunit, abbreviated herein as B. Certain
embodiments relate to carrier peptides having a combination of
different neutral amino acids, including, for example, peptides
comprising the sequence -RahxRRBRRAhxRRBRAhxB- (SEQ ID NO:578),
which contains both .beta.-alanine and 6-aminohexanoic acid.
[0177] Preferred peptides of this type include those comprising
arginine dimers alternating with single Y' subunits, where Y' is
preferably Ahx or B or both. Examples include peptides having the
formula (RY'R).sub.p and/or the formula (RRY').sub.p, where p is 1
to 2 to 5 and where Y' is preferably Ahx. In one embodiment, Y' is
a 6-aminohexanoic acid subunit, R is arginine and p is 4. Certain
embodiments include various linear combinations of at least two of
(RY'R).sub.p and (RRY').sub.p, including, for example, illustrative
peptides having the sequence (RY'R)(RRY')(RY'R)(RRY') (SEQ ID
NO:638), or (RRY')(RY'R)(RRY') (SEQ ID NO:639). Other combinations
are contemplated. In a further illustrative embodiment, each Z' is
phenylalanine, and m is 3 or 4.
[0178] The conjugated peptide is preferably linked to a terminus of
the oligomer via a linker Ahx-B, where Ahx is a 6-aminohexanoic
acid subunit and B is a .beta.-alanine subunit, as shown, for
example, in FIGS. 1B and 1C.
[0179] In selected embodiments, for each X', the side chain moiety
is independently selected from the group consisting of guanidyl
(HN.dbd.C(NH.sub.2)NH--), amidinyl (HN.dbd.C(NH.sub.2)CH--),
2-aminodihydropyrimidyl, 2-aminotetrahydropyrimidyl,
2-aminopyridinyl, and 2-aminopyrimidonyl, and it is preferably
selected from guanidyl and amidinyl. In one embodiment, the side
chain moiety is guanidyl, as in the amino acid subunit arginine
(Arg).
[0180] In certain embodiments, the Y' subunits may be contiguous,
in that no X' subunits intervene between Y' subunits, or
interspersed singly between X' subunits. In certain embodiments,
the linking subunit may be between Y' subunits. In one embodiment,
the Y' subunits are at a terminus of the transporter; in other
embodiments, they are flanked by X' subunits. In further preferred
embodiments, each Y' is --C(.dbd.O)--(CH.sub.2).sub.nCHR--NH--,
where n is 2 to 7 and R is H. For example, when n is 5 and R is H,
Y is a 6-aminohexanoic acid subunit, abbreviated herein as Ahx. In
selected embodiments of this group, each X' comprises a guanidyl
side chain moiety, as in an arginine subunit. Preferred peptides of
this type include those comprising arginine dimers alternating with
single Y' subunits, where Y is preferably Ahx. Examples include
peptides having the formula (RY'R).sub.4 or the formula
(RRY').sub.4 where Y' is preferably Ahx. In the latter case, the
nucleic acid analog is preferably linked to a terminal Y' subunit,
preferably at the C-terminus, as shown, for example, in FIGS. 1B
and 1C. The preferred linker is of the structure AhxB, where Ahx is
a 6-aminohexanoic acid subunit and B is a .beta.-alanine
subunit.
[0181] 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, and relative to uptake by an attached transport moiety
lacking the hydrophobic subunits Y'. Such enhanced uptake is
preferably evidenced by at least a two-fold increase, and
preferably a four-fold increase, in the uptake of the compound into
mammalian cells relative to uptake of the agent by an attached
transport moiety lacking the hydrophobic subunits Y'. Uptake is
preferably enhanced at least twenty fold, and more preferably forty
fold, relative to the unconjugated compound.
[0182] A further benefit of the transport moiety is its expected
ability to stabilize a duplex between an antisense compound and its
target nucleic acid sequence, presumably by virtue of electrostatic
interaction between the positively charged transport moiety and the
negatively charged nucleic acid. The number of charged subunits in
the transporter is less than 14, as noted above, and preferably
between 8 and 11, since too high a number of charged subunits may
lead to a reduction in sequence specificity.
[0183] The use of arginine-rich peptide transporters (i.e.,
cell-penetrating peptides) is 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;
Jearawiriyapaisam, Moulton et al. 2008; Wu, Moulton et al. 2008).
Furthermore, compared to other 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). Especially preferred are the P007,
CP06062 and CP04057 transport peptides listed below in Table 3 (SEQ
ID NOS: 573, 578 and 577, respectively).
[0184] Exemplary peptide transporters, including linkers (B or
AhxB) are given below in Table B below. Preferred sequences are
those designated CP06062 (SEQ ID NO: 578), P007 (SEQ ID NO: 573)
and CP04057 (SEQ ID NO: 577).
TABLE-US-00002 TABLE B Exemplary Peptide Transporters for
intracellular Delivery of PMO SEQ Sequence (N-terminal to ID
Peptide C-terminal) NO: rTAT RRRQRRKKRC 570 R.sub.9F.sub.2
RRRRRRRRRFFC 571 (RRAhx).sub.4B RRAhxRRAhxRRAhxRRAhxB 572
(RAhxR).sub.4AhxB; RAhxRRAhxRRAhxRRAhxRAhxB 573 (P007)
(AhxRR).sub.4AhxB AhxRRAhxRRAhxRRAhxRRAhxB 574 (RAhx).sub.6B
RAhxRAhxRAhxRAhxRAhxRAhxB 575 (RAhx).sub.8B
RAhxRAhxRAhxRAhxRAhxRAhxRAhx 576 B (RAhxR).sub.5AhxB
RAhxRRAhxRRAhxRRAhxRRAhxRAhx 577 (CP05057) B (RAhxRRBR).sub.2AhxB;
RAhxRRBRRAhxRRBRAhxB 578 (CP06062) MSP ASSLNIA 579
Formulations
[0185] 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).
[0186] 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.
[0187] 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.
[0188] 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.
[0189] 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.
[0190] 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.
[0191] 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).
[0192] 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.
[0193] 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.
[0194] 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).
[0195] 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.
[0196] 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).
[0197] 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.
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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.
[0206] 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.
[0207] 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.
[0208] 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.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] 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.
[0213] 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.
[0214] 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.
[0215] 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.
[0216] 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.
[0217] 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.
[0218] As noted above, the formulations or preparations of the
present invention may be given orally, parenterally, topically, or
rectally. 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.
[0219] 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, intraarticulare, subcapsular,
subarachnoid, intraspinal and intrastemal injection and
infusion.
[0220] 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.
[0221] 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.
[0222] 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.
[0223] 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 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.
[0224] 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.
[0225] 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.
[0226] 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.
[0227] 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.
[0228] 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).
[0229] 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).
[0230] 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.
[0231] 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).
[0232] 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.
[0233] 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 copolymers
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.
[0234] 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).
[0235] 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-cydodextrin) to 147% soluble (w/v)
(G-2-beta-cydodextrin). 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.
[0236] 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).
[0237] 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.
[0238] 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.
[0239] 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.
[0240] 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
(LPCs) 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 CMCs in the
micromolar range; higher CMC surfactants may be utilized to prepare
micelles entrapped within liposomes of the present invention.
[0241] 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 D D, 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.
[0242] 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.
[0243] 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.
[0244] 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).
[0245] 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).
[0246] 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.
[0247] 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).
[0248] 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.
[0249] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to one of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims. The
following examples are provided by way of illustration only and not
by way of limitation. Those of skill in the art will readily
recognize a variety of noncritical parameters that could be changed
or modified to yield essentially similar results.
REFERENCES
[0250] Aartsma-Rus, A., A. A. Janson, et al. (2004).
"Antisense-induced multiexon skipping for Duchenne muscular
dystrophy makes more sense." Am J Hum Genet 74(1): 83-92. [0251]
Dunckley, M. G., I. C. Eperon, et al. (1997). "Modulation of
splicing in the DMD gene by antisense oligoribonucleotides."
Nucleosides & Nucleotides 16(7-9): 1665-1668. [0252] Dunckley,
M. G., M. Manoharan, et al. (1998). "Modification of splicing in
the dystrophin gene in cultured Mdx muscle cells by antisense
oligoribonudeotides." Hum Mol Genet 7(7): 1083-90. [0253]
Errington, S. J., C. J. Mann, et al. (2003). "Target selection for
antisense oligonucleotide induced exon skipping in the dystrophin
gene." J Gene Med 5(6): 518-27. [0254] Jearawiriyapaisam, N., H. M.
Moulton, et al. (2008). "Sustained Dystrophin Expression Induced by
Peptide-conjugated Morpholino Oligomers in the Muscles of mdx
Mice." Mol Ther. [0255] Lu, Q. L., C. J. Mann, et al. (2003).
"Functional amounts of dystrophin produced by skipping the mutated
exon in the mdx dystrophic mouse." Nat Med 9(8): 1009-14. [0256]
Mann, C. J., K. Honeyman, et al. (2002). "Improved antisense
oligonucleotide induced exon skipping in the mdx mouse model of
muscular dystrophy." J Gene Med 4(6): 644-54. [0257] Marshall, N.
B., S. K. Oda, et al. (2007). "Arginine-rich cell-penetrating
peptides facilitate delivery of antisense oligomers into murine
leukocytes and alter pre-mRNA splicing." Journal of Immunological
Methods 325(1-2): 114-126. [0258] Matsuo, M., T. Masumura, et al.
(1991). "Exon skipping during splicing of dystrophin mRNA precursor
due to an intraexon deletion in the dystrophin gene of Duchenne
muscular dystrophy kobe." J Clin Invest 87(6): 2127-31. [0259]
Monaco, A. P., C. J. Bertelson, et al. (1988). "An explanation for
the phenotypic differences between patients bearing partial
deletions of the DMD locus." Genomics 2(1): 90-5. [0260] Pramono,
Z. A., Y. Takeshima, et al. (1996). "Induction of exon skipping of
the dystrophin transcript in lymphoblastoid cells by transfecting
an antisense oligodeoxynucleotide complementary to an exon
recognition sequence." Biochem Biophys Res Commun 226(2): 445-9.
[0261] Sazani, P., R. Kole, et al. (2007). Splice switching
oligomers for the TNF superfamily receptors and their use in
treatment of disease. PCT WO2007058894, University of North
Carolina [0262] Sierakowska, H., M. J. Sambade, et al. (1996).
"Repair of thalassemic human beta-globin mRNA in mammalian cells by
antisense oligonucleotides." Proc Natl Acad Sci USA 93(23):
12840-4. [0263] Summerton, J. and D. Weller (1997). "Morpholino
antisense oligomers: design, preparation, and properties."
Antisense Nucleic Acid Drug Dev 7(3): 187-95. [0264] Takeshima, Y.,
H. Nishio, et al. (1995). "Modulation of in vitro splicing of the
upstream intron by modifying an intra-exon sequence which is
deleted from the dystrophin gene in dystrophin Kobe." J Clin Invest
95(2): 515-20. [0265] van Deutekom, J. C., M. Bremmer-Bout, et al.
(2001). "Antisense-induced exon skipping restores dystrophin
expression in DMD patient derived muscle cells." Hum Mol Genet
10(15): 1547-54. [0266] van Deutekom, J. C., A. A. Janson, et al.
(2007). "Local dystrophin restoration with antisense
oligonucleotide PRO051." N Enal J Med 357(26): 2677-86. [0267]
Wilton, S. D., A. M. Fall, et al. (2007). "Antisense
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EXAMPLES
Materials and Methods
[0271] Cells and Tissue Culture Treatment Conditions
[0272] Human Rhabdomyosarcoma cells (ATCC, CCL-136; RD cells)
preserved in a 5% DMSO solution (Sigma) at a low passage number
were thawed in a 37.degree. C. water bath until the ice sliver was
no longer visible. Cells were seeded into tissue culture-treated
T75 flasks (Nunc) at 1.5.times.10.sup.6 cells/flask in 24 mL of
warmed DMEM with L-Glutamine (HyClone), 10% fetal bovine serum, and
1% Penicillin-Streptomycin antibiotic solution (CelGro); after 24
hours, media was aspirated, cells were washed once in warmed PBS,
and fresh media was added. Cells were grown to 80% confluence in a
37.degree. C. incubator at 5.0% CO2.
[0273] Media was aspirated from T75 flasks; cells were washed once
in warmed PBS and aspirated. 3 mL of Trypsin/EDTA, warmed in a
37.degree. C. water bath, was added to each T75. Cells were
incubated at 37.degree. C. 5 2-5 minutes until, with gentle
agitation, they released from the flask. Cell suspension was
transferred to a 15.0 mL conical tube; flasks were rinsed with 1.0
mL of Trypsin/EDTA solution to gather remaining cells. Cells were
counted with a Vi-Cell XR cell counter (Beckman Coulter). Cells
were seeded into tissue culture-treated 12-well plates (Falcon) at
2.0.times.10.sup.5 viable cells per well in 1.0 mL media. Cells
were incubated overnight in a 37.degree. C. incubator at 5.0%
CO.sub.2.
[0274] Twelve-well seeded plates were examined for even cellular
distribution and plate adherence. Lyophilized peptide conjugated
phosphorodiamidate morpholino oligomers (PPMOs) were re-suspended
at 2.0 mM in nuclease-free water (Ambion), and kept on ice during
cell treatment; to verify molarity, PPMOs were measured using a
NanoDrop 2000 spectrophotometer (Thermo Scientific). Immediately
prior to PPMO treatment, media was aspirated, and cells were rinsed
in warmed PBS. PPMOs were diluted in warmed media to the desired
molarity; cells were treated in a total of 1.0 mL PPMO per well.
PPMOs were tested in triplicate. For no-treatment controls, fresh,
warmed media was added in 1.0 mL total volume. Cells were incubated
for 48 hours in a 37.degree. C. incubator at 5.0% C02.
[0275] RNA Extraction
[0276] Media was aspirated, and cells were rinsed in warmed PBS.
RNA was extracted with the QuickGene-Mini80 system, QuickGene RNA
cultured cell HC kit S, and MagNAlyser with ceramic bead
homogenization using the manufacturers' recommended protocols.
Briefly, cells were lysed in treatment plates with 350 uL LRP (10
uL .beta.-Mercaptoethanol added per 100 uL LRP) lysis buffer;
homogenate was gently triturated to ensure full lysis, and
transferred to MagNAlyser tubes. Tubes were spun at 2800 rpm for 30
seconds in the MagNAlyser to ensure full homogenization, and iced
briefly. 50 uL SRP solubilization buffer was added and homogenate
was vortexed for 15 seconds. 170 uL >99% ethanol was added to
each tube, and homogenate was vortexed for 60 seconds. Homogenate
was flash-spun and transferred to Mini80 RNA cartridges, samples
were pressurized and flow-through was discarded. Cartridges were
washed in 750 uL WRP wash buffer and pressurized. 40 uL of DNase
solution (1.25 uL Qiagen DNasel, 35 uL RDD Buffer, 3.75 uL
nuclease-free water) was added directly to the cartridge membrane;
cartridges were incubated four minutes at room temperature.
Cartridges were washed twice with 750 uL WRP, pressurizing after
each wash. Cartridges were placed over nuclease-free tubes. 50 uL
CRP elution buffer was added to each membrane; membranes were
incubated for five minutes at room-temperature. Cartridges were
pressurized and eluate was collected. RNA was stored at -80.degree.
C. pending quantification. RNA was quantified using the
NanoDrop.TM. 2000 spectrophotometer.
[0277] Nested RT-PCR
[0278] Primer-specific, exon-specific, optimized nested RT-PCR
amplification was performed using the primer pair sets for each
dystrophin exon as shown below in Table 1.
TABLE-US-00003 TABLE 1 Primer pair sets used to PCR amplify human
dystrophin mRNA to detect exon-skipping SEQ Name F/R I/O Sequence
(5'-3') Exon Purpose ID NO: PS170 F O CCAGAGCTTTACCTGAGAAACAAG 48
Detection 640 PS172 F I CCAGCCACTCAGCCAGTGAAG 49 of Exon 50 641
PS174 R I CGATCCGTAATGATTGTTCTAGCC 52 and 51 642 PS176 R O
CATTTCATTCAACTGTTGCCTCCG 53 Skipping in 643 Human Dystrophin PS186
F O CAATGCTCCTGACCTCTGTGC 42 Detection 644 PS187 F I
GTCTACAACAAAGCTCAGGTCG 43 of Exon 44 645 PS189 F I
GCAATGTTATCTGCTTCCTCCAACC 46 and 45 646 PS190 R O
GCTCTTTTCCAGGTTCAAGTGG 46 Skipping in 647 Human Dystrophin PS192 F
O CTTGGACAGAACTTACCGACTGG 51 Detection 648 PS193 F I
GCAGGATTTGGAACAGAGGCG 52 of Exon 53 649 PS195 R I
CATCTACATTTGTCTGCCACTGG 54 Skipping in 650 PS197 R O
GTTTCTTCCAAAGCAGCCTCTCG 55 Human 651 Dystrophin
[0279] The indicated primer pairs are shown as either forward or
reverse (F/R) and either outer or inner primer pairs (I/O)
corresponding to primary or secondary amplifications, respectively.
The location of the primer target is indicated in the Exon column
and the Purpose indicates the exon-skipping events can be detected.
For example, PS170 and PS176 primers amplify a region from exon 48
to 53 in the primary amplification. Primers PS172 and PS174 then
amplify a region from exon 49 to 52 in the secondary amplification.
This nested PCR reaction will detect exon skipping of both exons 50
and/or exon 51. The specific nested RT-PCR reaction conditions are
provided below.
[0280] RNA extracted from treated cells (described above) was
diluted to 20 ng/ul for all samples.
TABLE-US-00004 TABLE 2 Reaction setup for RT-PCR and primary
amplification (50 .mu.l reaction): 2x Reaction mix 25 .mu.l PS XXX
Forward Primer (30 .mu.M) 0.5 .mu.l (see Table 1) PS XXX Reverse
Primer (30 .mu.M) 0.5 .mu.l (see Table 1) Superscript III Platinum
Taq mix 2 .mu.l Template RNA (20 ng/.mu.l) 10 .mu.l Nuclease-Free
Water (50 .mu.l total 12 .mu.l volume)
TABLE-US-00005 TABLE 3 RT-PCR and primary amplification program:
Temperature Time Reverse 55.degree. C. 30 minutes Transcription RT
Inactivation 94.degree. C. 2 minutes Denaturing 94.degree. C. 1
minute 8 Cycles Annealing 59.degree. C. 1 minute Extension
68.degree. C. 1 minute 4.degree. C. .infin.
TABLE-US-00006 TABLE 4 Reaction setup for nested secondary
amplification (50 .mu.l reaction): 10x PCR Buffer 5 .mu.l dNTP
solution (10 mM) 0.5 .mu.l 50 mM MgCl 1.5 .mu.l PS XXX Forward
Primer (30 .mu.M) 0.33 .mu.l (see Table 1) PS XXX Reverse Primer
(30 .mu.M) 0.33 .mu.l (see Table 1) Platinum Taq DNA polymerase 0.2
.mu.l 0.1 mM Cy5-dCTP 1 .mu.l RT-PCR product (from Step 1) 1 .mu.l
Nuclease-Free Water (50 .mu.l total 40.15 .mu.l volume)
TABLE-US-00007 TABLE 5 Nested secondary amplification program:
Temperature Time Primary 94.degree. C. 3 minutes Denature
Denaturing 94.degree. C. 45 seconds 28-30 Annealing 59.degree. C.
30 seconds Cycles Extension 68.degree. C. 1 minute 4.degree. C.
.infin.
[0281] Gel Electrophoresis Analysis
[0282] Ten microliters of 5.times. Ficoll loading dye was added to
each 50 microliter nested RT-PCR reaction. Fifteen microliters of
PCR/dye mixture was run on a 10% TBE gel at 300 volts for 30
minutes. After electrophoresis, the gel was washed in diH2O for at
least one hour, changing the water every 30 minutes. The gel was
then scanned on a Typhoon Trio Variable Mode Imager (GE
Healthcare). For exon 44 skipping, the nested RT-PCR product from
full-length dystrophin transcript is 571 bp, and 423 bp from Exon
44-skipped mRNA (exon 44 is 148 bp). For exon 45, the nested RT-PCR
product from full-length dystrophin transcript is 571 bp, and 395
bp from Exon 45-skipped mRNA (exon 45 is 176 bp). For exon 53, the
PCR product from full-length dystrophin transcript is 365 bp, and
153 bp from exon 53-skipped mRNA (exon 53 is 212 bp).
[0283] The gel images were subjected to quantitative analysis by
measuring the band intensities of the full-length PCR product
compared to the exon-skipped product. In some cases, the percent
skipping at a fixed PPMO concentration (e.g., 3 micromolar) was
used to determine the relative activity of a series of PPMO to
induce exon skipping of a given exon. In other situations, a PPMO
dose-range was used to treat cells (e.g., 0.1, 0.3, 1.0, 3.0 and 10
micromolar) and an EC.sub.50 was calculated based on the percent
skipping induced at each concentration.
Example 1
Exon 51 Scan
[0284] A series of overlapping antisense PPMOs that target human
dystrophin exon 51 were designed, synthesized and used to treat
either human rhabdomyosarcoma cells (RD cells) or primary human
skeletal muscle cells. This strategy is termed an "exon scan" and
was used similarly for several other dystrophin exons as described
below. All the PPMOs were synthesized as peptide-conjugated PMO
(PPMO) using the CP06062 peptide (SEQ ID NO: 578) and a 3' terminal
PMO linkage. For exon 51, a series of 26 PPMOs, each 26 bases in
length, were made (SEQ ID NOS: 309-311, 314, 316, 317, 319, 321,
323, 324, 326, 327, 329-331, 333, 335, 336, 338-345) as shown in
FIG. 2A. The PPMOs were evaluated for exon skipping efficacy by
treating RD cells at various concentrations as described above in
the Materials and Methods. Three PPMOs (SEQ ID NOS: 324, 326 and
327) were identified as effective in inducing exon-skipping and
selected for additional evaluation. Dose-ranging experiments in RD
cells and primary human skeletal muscle cells were used to confirm
the relative efficacy of these three PPMO sequences. SEQ ID NO: 327
was shown to be most effective at inducing exon 51 skipping as
shown in FIGS. 2B and 2C.
[0285] A comparison of the relative effectiveness of SEQ ID NO: 327
to other exon 51-targeted antisense sequences was performed in RD
cells and primary human skeletal muscle cells, as described above.
All the evaluated sequences were made as peptide-conjugated PMOs
using the CP06062 peptide (SEQ ID NO: 578). This allowed direct
comparison of the relative effectiveness of the antisense sequences
without regard to antisense chemistry or cell delivery. The
relative location of the certain exon 51-targeted oligos compared
to SEQ ID NO: 327 is shown in FIG. 2D. As shown in FIG. 2C, there
is a ranked hierarchy of exon-skipping effectiveness, with SEQ ID
NO: 327 being the most effective by at least a factor of
several-fold compared to other sequences.
Example 2
Exon 50 Scan
[0286] A series of overlapping antisense PPMOs that target human
dystrophin exon 50 were designed and synthesized. For exon 50, a
series of 17 PPMOs, each 25 bases in length, were made (SEQ ID
NOS:267, 269, 271, 273, 275, 277, 279, 280, 282 and 284-291) as
shown in FIG. 3A. The PPMOs were evaluated for exon skipping
efficacy by treating RD cells at various concentrations as
described above in the Materials and Methods. Four PPMOs (SEQ ID
NOS: 277, 287, 290 and 291) were identified as effective in
inducing exon-skipping and selected for additional evaluation.
Dose-ranging experiments in RD cells were used to confirm the
relative efficacy of these four PMO sequences. SEQ ID NOs: 584
(AVI-5656) and 287 (AVI-5038) were shown to be most effective at
inducing exon 50 skipping as shown in FIG. 3B. The EC.sub.50 values
were derived from the dose-ranging experiments and represent the
calculated concentration where 50% of the PCR product is from the
mRNA lacking exon 50 relative to the PCR product produced from the
mRNA containing exon 50. Compared to other sequences (see, e.g.,
SEQ ID NOs: 584 and 585 correspond to SEQ ID NOs: 173 and 175 in
WO2006/000057, respectively) AVI-5038 (SEQ ID NO: 287) is
equivalent or better at inducing exon-skipping activity in the RD
cell assay as shown in FIG. 3B.
Example 3
Exon 53 Scan
[0287] A series of overlapping antisense PPMOs that target human
dystrophin exon 53 were designed and synthesized. For exon 53, a
series of 24 PPMOs, each 25 bases in length, were made (SEQ ID
NOS:416, 418, 420, 422, 424, 426, 428, 429, 431, 433, 434, 436,
438-440 and 443-451) as shown in FIG. 4A. The PPMOs were evaluated
for exon skipping efficacy by treating RD cells and primary human
skeletal muscle cells at various concentrations as described above
in the Materials and Methods. Three PPMOs (SEQ ID NOS: 428, 429 and
431) were identified as effective in inducing exon-skipping and
selected for additional evaluation. Dose-ranging experiments in RD
cells were used to confirm the relative efficacy of these three PMO
sequences. SEQ ID NO: 429 was shown to be most effective at
inducing exon 53 skipping as shown in FIGS. 4B-F. However, when
compared to other exon 53 antisense sequences, SEQ ID NO: 429
proved identical to H53A(+23+47) which is listed as SEQ ID NO: 195
in WO2006/000057 and SEQ ID NO: 609 in the present application.
Other sequences were compared to SEQ ID NO: 429 including
H53A(+39+69) and H53A(-12+10) (listed as SEQ ID NOs:193 and 199 in
WO2006/000057, respectively) and h53AON1 (listed as SEQ ID NO:39 in
U.S. application Ser. No. 11/233,507) and listed as SEQ ID NOs:
608, 611 and 610, respectively, in the present application. All the
evaluated sequences were made as peptide-conjugated PMOs using the
CP06062 peptide (SEQ ID NO: 578). This allowed direct comparison of
the relative effectiveness of the antisense sequences without
regard to antisense chemistry or cell delivery. As shown in FIGS.
4I and 4G-H, SEQ ID NO: 429 was shown to be superior to each of
these four sequences.
Example 4
Exon 44 Scan
[0288] A series of overlapping antisense PPMOs that target human
dystrophin exon 44 were designed and synthesized. For exon 44, a
series of PPMOs, each 25 bases in length, were made (SEQ ID
NOS:1-20) as shown in FIG. 5A. The PPMOs were evaluated for exon
skipping efficacy by treating RD cells at various concentrations as
described above in the Materials and Methods. Five PPMOs (SEQ ID
NOS:4, 8, 11, 12 and 13) were identified as effective in inducing
exon-skipping and selected for additional evaluation. Dose-ranging
experiments in RD cells were used to confirm the relative efficacy
of these five PPMO sequences as shown in FIGS. 5C to 5H. SEQ ID
NOs: 8, 11 and 12 were shown to be most effective at inducing exon
44 skipping as shown in FIG. 5H with SEQ ID NO:12 proving the most
efficacious.
[0289] Comparison of SEQ ID NO: 12 to other exon 44 antisense
sequences was done in both RD cells and human primary skeletal
muscle cells. All the evaluated sequences were made as
peptide-conjugated PMOs using the CP06062 peptide (SEQ ID NO: 578).
This allowed direct comparison of the relative effectiveness of the
antisense sequences without regard to antisense chemistry or cell
delivery.
[0290] The alignment of the sequences (SEQ ID NOS: 600, 601, 602
and 603) with SEQ ID NOS: 4, 8, 11 and 12 is shown in FIG. 5B. SEQ
ID NOS: 601 and 603 are listed as SEQ ID NOS: 165 and 167 in
WO2006/000057. SEQ ID NO:602 is listed in WO2004/083446 and as SEQ
ID NO: 21 in U.S. application Ser. No. 11/233,507. SEQ ID NO:600
was published in 2007 (Wilton, Fall et al. 2007). The comparison in
RD cells showed that both SEQ ID NOS: 602 and 603 were superior to
SEQ ID NO:12 (FIG. 5I). However, as shown in FIG. 5J, in human
primary skeletal muscle cells SEQ ID NO:12 was superior (8.86% exon
skipping) to SEQ ID NO:602 (6.42%). Similar experiments are
performed with SEQ ID NO:603.
Example 5
Exon 45 Scan
[0291] A series of overlapping antisense PPMOs that target human
dystrophin exon 45 were designed and synthesized. For exon 45, a
series of 22 PPMOs, each 25 bases in length, were made (SEQ ID NOS:
21, 23, 25, 27, 29, 31, 32, 34, 35, 37, 39, 41, 43 and 45-53) as
shown in FIG. 6A. The PPMOs were evaluated for exon skipping
efficacy by treating RD cells and human primary skeletal muscle
cells at various concentrations as described above in the Materials
and Methods. Five PPMOs (SEQ ID NOS:27, 29, 34, and 39) were
identified as effective in inducing exon-skipping and selected for
additional evaluation. Dose-ranging experiments in RD cells were
used to confirm the relative efficacy of these four PMO sequences
as shown in FIGS. 6C-G and summarized in FIG. 6H. SEQ ID NO: 49 was
used as a negative control in these experiments. SEQ ID NOs: 29 and
34 were shown to be most effective at inducing exon 45 skipping as
shown in FIG. 6H.
[0292] Comparison of SEQ ID NO: 34 to other exon 45 antisense
sequences was done in both RD cells and human primary skeletal
muscle cells. All the evaluated sequences were made as
peptide-conjugated PMOs using the CP06062 peptide (SEQ ID NO: 578).
This allowed direct comparison of the relative effectiveness of the
antisense sequences without regard to antisense chemistry or cell
delivery. The alignment of the sequences (SEQ ID NOS: 604, 605, 606
and 607) with SEQ ID NOS: 27, 29, 34 and 39 is shown in FIG. 6B.
SEQ ID NOS: 604 and 607 are listed as SEQ ID NOS: 211 and 207 in
WO2006/000057, respectively. SEQ ID NOS:605 and 606 are listed in
U.S. application Ser. No. 11/233,507 as SEQ ID NOS: 23 and 1,
respectively. The comparison in RD cells showed that SEQ ID NO: 34
was superior to all four sequences evaluated as shown in FIG. 6I.
Testing of these compounds in different populations of human
primary skeletal muscle cells is performed as described above.
SEQUENCE ID LISTING
[0293] Sequences are shown using the nucleotide base symbols common
for DNA: A, G, C and T. Other antisense chemistries such as
2'-O-methyl use U in place of T. Any of the bases may be
substituted with inosine (I) especially in stretches of three or
more G residues.
TABLE-US-00008 Name Sequences SEQ ID NO. Oligomer Targeting
Sequences (5' to 3'); Hu.DMD.Exon44.25.001
CTGCAGGTAAAAGCATATGGATCAA 1 Hu.DMD.Exon44.25.002
ATCGCCTGCAGGTAAAAGCATATGG 2 Hu.DMD.Exon44.25.003
GTCAAATCGCCTGCAGGTAAAAGCA 3 Hu.DMD.Exon44.25.004
GATCTGTCAAATCGCCTGCAGGTAA 4 Hu.DMD.Exon44.25.005
CAACAGATCTGTCAAATCGCCTGCA 5 Hu.DMD.Exon44.25.006
TTTCTCAACAGATCTGTCAAATCGC 6 Hu.DMD.Exon44.25.007
CCATTTCTCAACAGATCTGTCAAAT 7 Hu.DMD.Exon44.25.008
ATAATGAAAACGCCGCCATTTCTCA 8 Hu.DMD.Exon44.25.009
AAATATCTTTATATCATAATGAAAA 9 Hu.DMD.Exon44.25.010
TGTTAGCCACTGATTAAATATCTTT 10 Hu.DMD.Exon44.25.011
AAACTGTTCAGCTTCTGTTAGCCAC 11 Hu.DMD.Exon44.25.012
TTGTGTCTTTCTGAGAAACTGTTCA 12 Hu.DMD.Exon44.25.013
CCAATTCTCAGGAATTTGTGTCTTT 13 Hu.DMD.Exon44.25.014
GTATTTAGCATGTTCCCAATTCTCA 14 Hu.DMD.Exon44.25.015
CTTAAGATACCATTTGTATTTAGCA 15 Hu.DMD.Exon44.25.016
CTTACCTTAAGATACCATTTGTATT 16 Hu.DMD.Exon44.25.017
AAAGACTTACCTTAAGATACCATTT 17 Hu.DMD.Exon44.25.018
AAATCAAAGACTTACCTTAAGATAC 18 Hu.DMD.Exon44.25.019
AAAACAAATCAAAGACTTACCTTAA 19 Hu.DMD.Exon44.25.020
TCGAAAAAACAAATCAAAGACTTAC 20 Hu.DMD.Exon45.25.001
CTGTAAGATACCAAAAAGGCAAAAC 21 Hu.DMD.Exon45.25.002
CCTGTAAGATACCAAAAAGGCAAAA 22 Hu.DMD.Exon45.25.002.
AGTTCCTGTAAGATACCAAAAAGGC 23 2 Hu.DMD.Exon45.25.003
GAGTTCCTGTAAGATACCAAAAAGG 24 Hu.DMD.Exon45.25.003.
CCTGGAGTTCCTGTAAGATACCAAA 25 2 Hu.DMD.Exon45.25.004
TCCTGGAGTTCCTGTAAGATACCAA 26 Hu.DMD.Exon45.25.004.
GCCATCCTGGAGTTCCTGTAAGATA 27 2 Hu.DMD.Exon45.25.005
TGCCATCCTGGAGTTCCTGTAAGAT 28 Hu.DMD.Exon45.25.005.
CCAATGCCATCCTGGAGTTCCTGTA 29 2 Hu.DMD.Exon45.25.006
CCCAATGCCATCCTGGAGTTCCTGT 30 Hu.DMD.Exon45.25.006.
GCTGCCCAATGCCATCCTGGAGTTC 31 2 Hu.DMD.Exon45.25.007
CGCTGCCCAATGCCATCCTGGAGTT 32 Hu.DMD.Exon45.25.008
AACAGTTTGCCGCTGCCCAATGCCA 33 Hu.DMD.Exon45.25.008.
CTGACAACAGTTTGCCGCTGCCCAA 34 2 Hu.DMD.Exon45.25.009
GTTGCATTCAATGTTCTGACAACAG 35 Hu.DMD.Exon45.25.010
GCTGAATTATTTCTTCCCCAGTTGC 36 Hu.DMD.Exon45.25.010.
ATTATTTCTTCCCCAGTTGCATTCA 37 2 Hu.DMD.Exon45.25.011
GGCATCTGTTTTTGAGGATTGCTGA 38 Hu.DMD.Exon45.25.011.
TTTGAGGATTGCTGAATTATTTCTT 39 2 Hu.DMD.Exon45.25.012
AATTTTTCCTGTAGAATACTGGCAT 40 Hu.DMD.Exon45.25.012.
ATACTGGCATCTGTTTTTGAGGATT 41 2 Hu.DMD.Exon45.25.013
ACCGCAGATTCAGGCTTCCCAATTT 42 Hu.DMD.Exon45.25.013.
AATTTTTCCTGTAGAATACTGGCAT 43 2 Hu.DMD.Exon45.25.014
CTGTTTGCAGACCTCCTGCCACCGC 44 Hu.DMD.Exon45.25.014.
AGATTCAGGCTTCCCAATTTTTCCT 45 2 Hu.DMD.Exon45.25.015
CTCTTTTTTCTGTCTGACAGCTGTT 46 Hu.DMD.Exon45.25.015.
ACCTCCTGCCACCGCAGATTCAGGC 47 2 Hu.DMD.Exon45.25.016
CCTACCTCTTTTTTCTGTCTGACAG 48 Hu.DMD.Exon45.25.016.
GACAGCTGTTTGCAGACCTCCTGCC 49 2 Hu.DMD.Exon45.25.017
GTCGCCCTACCTCTTTTTTCTGTCT 50 Hu.DMD.Exon45.25.018
GATCTGTCGCCCTACCTCTTTTTTC 51 Hu.DMD.Exon45.25.019
TATTAGATCTGTCGCCCTACCTCTT 52 Hu.DMD.Exon45.25.020
ATTCCTATTAGATCTGTCGCCCTAC 53 Hu.DMD.Exon45.20.001
AGATACCAAAAAGGCAAAAC 54 Hu.DMD.Exon45.20.002 AAGATACCAAAAAGGCAAAA
55 Hu.DMD.Exon45.20.003 CCTGTAAGATACCAAAAAGG 56
Hu.DMD.Exon45.20.004 GAGTTCCTGTAAGATACCAA 57 Hu.DMD.Exon45.20.005
TCCTGGAGTTCCTGTAAGAT 58 Hu.DMD.Exon45.20.006 TGCCATCCTGGAGTTCCTGT
59 Hu.DMD.Exon45.20.007 CCCAATGCCATCCTGGAGTT 60
Hu.DMD.Exon45.20.008 CGCTGCCCAATGCCATCCTG 61 Hu.DMD.Exon45.20.009
CTGACAACAGTTTGCCGCTG 62 Hu.DMD.Exon45.20.010 GTTGCATTCAATGTTCTGAC
63 Hu.DMD.Exon45.20.011 ATTATTTCTTCCCCAGTTGC 64
Hu.DMD.Exon45.20.012 TTTGAGGATTGCTGAATTAT 65 Hu.DMD.Exon45.20.013
ATACTGGCATCTGTTTTTGA 66 Hu.DMD.Exon45.20.014 AATTTTTCCTGTAGAATACT
67 Hu.DMD.Exon45.20.015 AGATTCAGGCTTCCCAATTT 68
Hu.DMD.Exon45.20.016 ACCTCCTGCCACCGCAGATT 69 Hu.DMD.Exon45.20.017
GACAGCTGTTTGCAGACCTC 70 Hu.DMD.Exon45.20.018 CTCTTTTTTCTGTCTGACAG
71 Hu.DMD.Exon45.20.019 CCTACCTCTTTTTTCTGTCT 72
Hu.DMD.Exon45.20.020 GTCGCCCTACCTCTTTTTTC 73 Hu.DMD.Exon45.20.021
GATCTGTCGCCCTACCTCTT 74 Hu.DMD.Exon45.20.022 TATTAGATCTGTCGCCCTAC
75 Hu.DMD.Exon45.20.023 ATTCCTATTAGATCTGTCGC 76
Hu.DMD.Exon46.25.001 GGGGGATTTGAGAAAATAAAATTAC 77
Hu.DMD.Exon46.25.002 ATTTGAGAAAATAAAATTACCTTGA 78
Hu.DMD.Exon46.25.002. CTAGCCTGGAGAAAGAAGAATAAAA 79 2
Hu.DMD.Exon46.25.003 AGAAAATAAAATTACCTTGACTTGC 80
Hu.DMD.Exon46.25.003. TTCTTCTAGCCTGGAGAAAGAAGAA 81 2
Hu.DMD.Exon46.25.004 ATAAAATTACCTTGACTTGCTCAAG 82
Hu.DMD.Exon46.25.004. TTTTGTTCTTCTAGCCTGGAGAAAG 83 2
Hu.DMD.Exon46.25.005 ATTACCTTGACTTGCTCAAGCTTTT 84
Hu.DMD.Exon46.25.005. TATTCTTTTGTTCTTCTAGCCTGGA 85 2
Hu.DMD.Exon46.25.006 CTTGACTTGCTCAAGCTTTTCTTTT 86
Hu.DMD.Exon46.25.006. CAAGATATTCTTTTGTTCTTCTAGC 87 2
Hu.DMD.Exon46.25.007 CTTTTAGTTGCTGCTCTTTTCCAGG 88 Hu.DMD.Exon46
25.008 CCAGGTTCAAGTGGGATACTAGCAA 89 Hu.DMD.Exon46.25.008.
ATCTCTTTGAAATTCTGACAAGATA 90 2 Hu.DMD.Exon46.25.009
AGCAATGTTATCTGCTTCCTCCAAC 91 Hu.DMD.Exon46.25.009.
AACAAATTCATTTAAATCTCTTTGA 92 2 Hu.DMD.Exon46.25.010
CCAACCATAAAACAAATTCATTTAA 93 Hu.DMD.Exon46.25.010.
TTCCTCCAACCATAAAACAAATTCA 94 2 Hu.DMD.Exon46.25.011
TTTAAATCTCTTTGAAATTCTGACA 95 Hu.DMD.Exon46.25.012
TGACAAGATATTCTTTTGTTCTTCT 96 Hu.DMD.Exon46.25.012.
TTCAAGTGGGATACTAGCAATGTTA 97 2 Hu.DMD.Exon46.25.013
AGATATTCTTTTGTTCTTCTAGCCT 98 Hu.DMD.Exon46.25.013.
CTGCTCTTTTCCAGGTTCAAGTGGG 99 2 Hu.DMD.Exon46.25.014
TTCTTTTGTTCTTCTAGCCTGGAGA 100 Hu.DMD.Exon46.25.014.
CTTTTCTTTTAGTTGCTGCTCTTTT 101 2 Hu.DMD.Exon46.25.015
TTGTTCTTCTAGCCTGGAGAAAGAA 102 Hu.DMD.Exon46.25.016
CTTCTAGCCTGGAGAAAGAAGAATA 103 Hu.DMD.Exon46.25.017
AGCCTGGAGAAAGAAGAATAAAATT 104 Hu.DMD.Exon46.25.018
CTGGAGAAAGAAGAATAAAATTGTT 105 Hu.DMD.Exon46.20.001
GAAAGAAGAATAAAATTGTT 106 Hu.DMD.Exon46.20.002 GGAGAAAGAAGAATAAAATT
107 Hu.DMD.Exon46.20.003 AGCCTGGAGAAAGAAGAATA 108
Hu.DMD.Exon46.20.004 CTTCTAGCCTGGAGAAAGAA 109 Hu.DMD.Exon46.20.005
TTGTTCTTCTAGCCTGGAGA 110 Hu.DMD.Exon46.20.006 TTCTTTTGTTCTTCTAGCCT
111
Hu.DMD.Exon46.20.007 TGACAAGATATTCTTTTGTT 112 Hu.DMD.Exon46.20.008
ATCTCTTTGAAATTCTGACA 113 Hu.DMD.Exon46.20.009 AACAAATTCATTTAAATCTC
114 Hu.DMD.Exon46.20.010 TTCCTCCAACCATAAAACAA 115
Hu.DMD.Exon46.20.011 AGCAATGTTATCTGCTTCCT 116 Hu.DMD.Exon46.20.012
TTCAAGTGGGATACTAGCAA 117 Hu.DMD.Exon46.20.013 CTGCTCTTTTCCAGGTTCAA
118 Hu.DMD.Exon46.20.014 CTTTTCTTTTAGTTGCTGCT 119
Hu.DMD.Exon46.20.015 CTTGACTTGCTCAAGCTTTT 120 Hu.DMD.Exon46.20.016
ATTACCTTGACTTGCTCAAG 121 Hu.DMD.Exon46.20.017 ATAAAATTACCTTGACTTGC
122 Hu.DMD.Exon46.20.018 AGAAAATAAAATTACCTTGA 123
Hu.DMD.Exon46.20.019 ATTTGAGAAAATAAAATTAC 124 Hu.DMD.Exon46.20.020
GGGGGATTTGAGAAAATAAA 125 Hu.DMD.Exon47.25.001
CTGAAACAGACAAATGCAACAACGT 126 Hu.DMD.Exon47.25.002
AGTAACTGAAACAGACAAATGCAAC 127 Hu.DMD.Exon47.25.003
CCACCAGTAACTGAAACAGACAAAT 128 Hu.DMD.Exon47.25.004
CTCTTCCACCAGTAACTGAAACAGA 129 Hu.DMD.Exon47.25.005
GGCAACTCTTCCACCAGTAACTGAA 130 Hu.DMD.Exon47.25.006
GCAGGGGCAACTCTTCCACCAGTAA 131 Hu.DMD.Exon47.25.007
CTGGCGCAGGGGCAACTCTTCCACC 132 Hu.DMD.Exon47.25.008
TTTAATTGTTTGAGAATTCCCTGGC 133 Hu.DMD.Exon47.25.008.
TTGTTTGAGAATTCCCTGGCGCAGG 134 2 Hu.DMD.Exon47.25.009
GCACGGGTCCTCCAGTTTCATTTAA 135 Hu.DMD.Exon47.25.009.
TCCAGTTTCATTTAATTGTTTGAGA 136 2 Hu.DMD.Exon47.25.010
GCTTATGGGAGCACTTACAAGCACG 137 Hu.DMD.Exon47.25.010.
TACAAGCACGGGTCCTCCAGTTTCA 138 2 Hu.DMD.Exon47.25.011
AGTTTATCTTGCTCTTCTGGGCTTA 139 Hu.DMD.Exon47.25.012
TCTGCTTGAGCTTATTTTCAAGTTT 140 Hu.DMD.Exon47.25.012.
ATCTTGCTCTTCTGGGCTTATGGGA 141 2 Hu.DMD.Exon47.25.013
CTTTATCCACTGGAGATTTGTCTGC 142 Hu.DMD.Exon47.25.013.
CTTATTTTCAAGTTTATCTTGCTCT 143 2 Hu.DMD.Exon47.25.014
CTAACCTTTATCCACTGGAGATTTG 144 Hu.DMD.Exon47.25.014.
ATTTGTCTGCTTGAGCTTATTTTCA 145 2 Hu.DMD.Exon47.25.015
AATGTCTAACCTTTATCCACTGGAG 146 Hu.DMD.Exon47.25.016
TGGTTAATGTCTAACCTTTATCCAC 147 Hu.DMD.Exon47.25.017
AGAGATGGTTAATGTCTAACCTTTA 148 Hu.DMD.Exon47.25.018
ACGGAAGAGATGGTTAATGTCTAAC 149 Hu.DMD.Exon47.20.001
ACAGACAAATGCAACAACGT 150 Hu.DMD.Exon47.20.002 CTGAAACAGACAAATGCAAC
151 Hu.DMD.Exon47.20.003 AGTAACTGAAACAGACAAAT 152
Hu.DMD.Exon47.20.004 CCACCAGTAACTGAAACAGA 153 Hu.DMD.Exon47.20.005
CTCTTCCACCAGTAACTGAA 154 Hu.DMD.Exon47.20.006 GGCAACTCTTCCACCAGTAA
155 Hu.DMD.Exon47.20.007 CTGGCGCAGGGGCAACTCTT 156
Hu.DMD.Exon47.20.008 TTGTTTGAGAATTCCCTGGC 157 Hu.DMD.Exon47.20.009
TCCAGTTTCATTTAATTGTT 158 Hu.DMD.Exon47.20.010 TACAAGCACGGGTCCTCCAG
159 Hu.DMD.Exon47.20.011 GCTTATGGGAGCACTTACAA 160
Hu.DMD.Exon47.20.012 ATCTTGCTCTTCTGGGCTTA 161 Hu.DMD.Exon47.20.013
CTTATTTTCAAGTTTATCTT 162 Hu.DMD.Exon47.20.014 ATTTGTCTGCTTGAGCTTAT
163 Hu.DMD.Exon47.20.015 CTTTATCCACTGGAGATTTG 164
Hu.DMD.Exon47.20.016 CTAACCTTTATCCACTGGAG 165 Hu.DMD.Exon47.20.017
AATGTCTAACCTTTATCCAC 166 Hu.DMD.Exon47.20.018 TGGTTAATGTCTAACCTTTA
167 Hu.DMD.Exon47.20.019 AGAGATGGTTAATGTCTAAC 168
Hu.DMD.Exon47.20.020 ACGGAAGAGATGGTTAATGT 169 Hu.DMD.Exon48.25.001
CTGAAAGGAAAATACATTTTAAAAA 170 Hu.DMD.Exon48.25.002
CCTGAAAGGAAAATACATTTTAAAA 171 Hu.DMD.Exon48.25.002.
GAAACCTGAAAGGAAAATACATTTT 172 2 Hu.DMD.Exon48.25.003
GGAAACCTGAAAGGAAAATACATTT 173 Hu.DMD.Exon48.25.003.
CTCTGGAAACCTGAAAGGAAAATAC 174 2 Hu.DMD.Exon48.25.004
GCTCTGGAAACCTGAAAGGAAAATA 175 Hu.DMD.Exon48.25.004.
TAAAGCTCTGGAAACCTGAAAGGAA 634 2 Hu.DMD.Exon48.25.005
GTAAAGCTCTGGAAACCTGAAAGGA 176 Hu.DMD.Exon48 25.005.
TCAGGTAAAGCTCTGGAAACCTGAA 177 2 Hu.DMD.Exon48.25.006
CTCAGGTAAAGCTCTGGAAACCTGA 178 Hu.DMD.Exon48.25.006.
GTTTCTCAGGTAAAGCTCTGGAAAC 179 2 Hu.DMD.Exon48.25.007
TGTTTCTCAGGTAAAGCTCTGGAAA 180 Hu.DMD.Exon48.25.007.
AATTTCTCCTTGTTTCTCAGGTAAA 181 2 Hu.DMD.Exon48.25.008
TTTGAGCTTCAATTTCTCCTTGTTT 182 Hu.DMD.Exon48.25.008
TTTTATTTGAGCTTCAATTTCTCCT 183 Hu.DMD.Exon48.25.009
AAGCTGCCCAAGGTCTTTTATTTGA 184 Hu.DMD.Exon48.25.010
AGGTCTTCAAGCTTTTTTTCAAGCT 185 Hu.DMD.Exon48.25.010.
TTCAAGCTTTTTTTCAAGCTGCCCA 186 2 Hu.DMD.Exon48.25.011
GATGATTTAACTGCTCTTCAAGGTG 187 Hu.DMD.Exon48.25.011.
CTGCTCTTCAAGGTCTTCAAGCTTT 188 2 Hu.DMD.Exon48.25.012
AGGAGATAACCACAGCAGCAGATGA 189 Hu.DMD.Exon48.25.012.
CAGCAGATGATTTAACTGCTCTTCA 190 2 Hu.DMD.Exon48.25.013
ATTTCCAACTGATTCCTAATAGGAG 191 Hu.DMD.Exon48.25.014
CTTGGTTTGGTTGGTTATAAATTTC 192 Hu.DMD.Exon48.25.014.
CAACTGATTCCTAATAGGAGATAAC 193 2 Hu.DMD.Exon48.25.015
CTTAACGTCAAATGGTCCTTCTTGG 194 Hu.DMD.Exon48.25.015.
TTGGTTATAAATTTCCAACTGATTC 195 2 Hu.DMD.Exon48.25.016
CCTACCTTAACGTCAAATGGTCCTT 196 Hu.DMD.Exon48.25.016.
TCCTTCTTGGTTTGGTTGGTTATAA 197 2 Hu.DMD.Exon48.25.017
AGTTCCCTACCTTAACGTCAAATGG 198 Hu.DMD.Exon48.25.018
CAAAAAGTTCCCTACCTTAACGTCA 199 Hu.DMD.Exon48.25.019
TAAAGCAAAAAGTTCCCTACCTTAA 200 Hu.DMD.Exon48.25.020
ATATTTAAAGCAAAAAGTTCCCTAC 201 Hu.DMD.Exon48.20.001
AGGAAAATACATTTTAAAAA 202 Hu.DMD.Exon48.20.002 AAGGAAAATACATTTTAAAA
203 Hu.DMD.Exon48.20.003 CCTGAAAGGAAAATACATTT 204
Hu.DMD.Exon48.20.004 GGAAACCTGAAAGGAAAATA 205 Hu.DMD.Exon48.20.005
GCTCTGGAAACCTGAAAGGA 206 Hu.DMD.Exon48.20.006 GTAAAGCTCTGGAAACCTGA
207 Hu.DMD.Exon48.20.007 CTCAGGTAAAGCTCTGGAAA 208
Hu.DMD.Exon48.20.008 AATTTCTCCTTGTTTCTCAG 209 Hu.DMD.Exon48.20.009
TTTTATTTGAGCTTCAATTT 210 Hu.DMD.Exon48.20.010 AAGCTGCCCAAGGTCTTTTA
211 Hu.DMD.Exon48.20.011 TTCAAGCTTTTTTTCAAGCT 212
Hu.DMD.Exon48.20.012 CTGCTCTTCAAGGTCTTCAA 213 Hu.DMD.Exon48.20.013
CAGCAGATGATTTAACTGCT 214 Hu.DMD.Exon48.20.014 AGGAGATAACCACAGCAGCA
215 Hu.DMD.Exon48.20.015 CAACTGATTCCTAATAGGAG 216
Hu.DMD.Exon48.20.016 TTGGTTATAAATTTCCAACT 217 Hu.DMD.Exon48.20.017
TCCTTCTTGGTTTGGTTGGT 218 Hu.DMD.Exon48.20.018 CTTAACGTCAAATGGTCCTT
219 Hu.DMD.Exon48.20.019 CCTACCTTAACGTCAAATGG 220
Hu.DMD.Exon48.20.020 AGTTCCCTACCTTAACGTCA 221 Hu.DMD.Exon48.20.021
CAAAAAGTTCCCTACCTTAA 222 Hu.DMD.Exon48.20.022 TAAAGCAAAAAGTTCCCTAC
223 Hu.DMD.Exon48.20.023 ATATTTAAAGCAAAAAGTTC 224
Hu.DMD.Exon49.25.001 CTGGGGAAAAGAACCCATATAGTGC 225
Hu.DMD.Exon49.25.002 TCCTGGGGAAAAGAACCCATATAGT 226
Hu.DMD.Exon49.25.002. GTTTCCTGGGGAAAAGAACCCATAT 227 2
Hu.DMD.Exon49.25.003 CAGTTTCCTGGGGAAAAGAACCCAT 228
Hu.DMD.Exon49.25.003. TTTCAGTTTCCTGGGGAAAAGAACC 229 2
Hu.DMD.Exon49.25.004 TATTTCAGTTTCCTGGGGAAAAGAA 230
Hu.DMD.Exon49.25.004. TGCTATTTCAGTTTCCTGGGGAAAA 231 2
Hu.DMD.Exon49.25.005 ACTGCTATTTCAGTTTCCTGGGGAA 232
Hu.DMD.Exon49.25.005. TGAACTGCTATTTCAGTTTCCTGGG 233 2
Hu.DMD.Exon49.25.006 CTTGAACTGCTATTTCAGTTTCCTG 234 Hu.DMD.Exon49
25.006. TAGCTTGAACTGCTATTTCAGTTTC 235 2 Hu.DMD.Exon49.25.007
TTTAGCTTGAACTGCTATTTCAGTT 236 Hu.DMD.Exon49.25.008
TTCCACATCCGGTTGTTTAGCTTGA 237 Hu.DMD.Exon49.25.009
TGCCCTTTAGACAAAATCTCTTCCA 238 Hu.DMD.Exon49.25.009.
TTTAGACAAAATCTCTTCCACATCC 239 2 Hu.DMD.Exon49.25.010
GTTTTTCCTTGTACAAATGCTGCCC 240 Hu.DMD.Exon49.25.010
GTACAAATGCTGCCCTTTAGACAAA 241 2 Hu.DMD.Exon49.25.011
CTTCACTGGCTGAGTGGCTGGTTTT 242 Hu.DMD.Exon49.25.011.
GGCTGGTTTTTCCTTGTACAAATGC 243 2 Hu.DMD.Exon49.25.012
ATTACCTTCACTGGCTGAGTGGCTG 244 Hu.DMD.Exon49.25.013
GCTTCATTACCTTCACTGGCTGAGT 245 Hu.DMD.Exon49.25.014
AGGTTGCTTCATTACCTTCACTGGC 246 Hu.DMD.Exon49.25.015
GCTAGAGGTTGCTTCATTACCTTCA 247 Hu.DMD.Exon49.25.016
ATATTGCTAGAGGTTGCTTCATTAC 248 Hu.DMD.Exon49.20.001
GAAAAGAACCCATATAGTGC 249 Hu.DMD.Exon49.20.002 GGGAAAAGAACCCATATAGT
250 Hu.DMD.Exon49.20.003 TCCTGGGGAAAAGAACCCAT 251
Hu.DMD.Exon49.20.004 CAGTTTCCTGGGGAAAAGAA 252 Hu.DMD.Exon49.20.005
TATTTCAGTTTCCTGGGGAA 253 Hu.DMD.Exon49.20.006 ACTGCTATTTCAGTTTCCTG
254 Hu.DMD.Exon49.20.007 CTTGAACTGCTATTTCAGTT 255
Hu.DMD.Exon49.20.008 TTTAGCTTGAACTGCTATTT 256 Hu.DMD.Exon49.20.009
TTCCACATCCGGTTGTTTAG 257 Hu.DMD.Exon49.20.010 TTTAGACAAAATCTCTTCCA
258 Hu.DMD.Exon49.20.011 GTACAAATGCTGCCCTTTAG 259
Hu.DMD.Exon49.20.012 GGCTGGTTTTTCCTTGTACA 260 Hu.DMD.Exon49.20.013
CTTCACTGGCTGAGTGGCTG 261 Hu.DMD.Exon49.20.014 ATTACCTTCACTGGCTGAGT
262 Hu.DMD.Exon49.20.015 GCTTCATTACCTTCACTGGC 263
Hu.DMD.Exon49.20.016 AGGTTGCTTCATTACCTTCA 264 Hu.DMD.Exon49.20.017
GCTAGAGGTTGCTTCATTAC 265 Hu.DMD.Exon49.20.018 ATATTGCTAGAGGTTGCTTC
266 Hu.DMD.Exon50.25.001 CTTTAACAGAAAAGCATACACATTA 267
Hu.DMD.Exon50.25.002 TCCTCTTTAACAGAAAAGCATACAC 268
Hu.DMD.Exon50.25.002. TTCCTCTTTAACAGAAAAGCATACA 269 2
Hu.DMD.Exon50.25.003 TAACTTCCTCTTTAACAGAAAAGCA 270
Hu.DMD.Exon50.25.003. CTAACTTCCTCTTTAACAGAAAAGC 271 2
Hu.DMD.Exon50.25.004 TCTTCTAACTTCCTCTTTAACAGAA 272
Hu.DMD.Exon50.25.004. ATCTTCTAACTTCCTCTTTAACAGA 273 2
Hu.DMD.Exon50.25.005 TCAGATCTTCTAACTTCCTCTTTAA 274
Hu.DMD.Exon50.25.005. CTCAGATCTTCTAACTTCCTCTTTA 275 2
Hu.DMD.Exon50.25.006 AGAGCTCAGATCTTCTAACTTCCTC 276
Hu.DMD.Exon50.25.006. CAGAGCTCAGATCTTCTAACTTCCT 277 2 NG-08-0731
Hu.DMD.Exon50.25.007 CACTCAGAGCTCAGATCTTCTACT 278
Hu.DMD.Exon50.25.007. CCTTCCACTCAGAGCTCAGATCTTC 279 2
Hu.DMD.Exon50.25.008 GTAAACGGTTTACCGCCTTCCACTC 280
Hu.DMD.Exon50.25.009 CTTTGCCCTCAGCTCTTGAAGTAAA 281
Hu.DMD.Exon50.25.009. CCCTCAGCTCTTGAAGTAAACGGTT 282 2
Hu.DMD.Exon50.25.010 CCAGGAGCTAGGTCAGGCTGCTTTG 283
Hu.DMD.Exon50.25.010. GGTCAGGCTGCTTTGCCCTCAGCTC 284 2
Hu.DMD.Exon50.25.011 AGGCTCCAATAGTGGTCAGTCCAGG 285
Hu.DMD.Exon50.25.011. TCAGTCCAGGAGCTAGGTCAGGCTG 286 2
Hu.DMD.Exon50.25.012 CTTACAGGCTCCAATAGTGGTCAGT 287 AVI-5038
Hu.DMD.Exon50.25.013 GTATACTTACAGGCTCCAATAGTGG 288
Hu.DMD.Exon50.25.014 ATCCAGTATACTTACAGGCTCCAAT 289
Hu.DMD.Exon50.25.015 ATGGGATCCAGTATACTTACAGGCT 290 NG-08-0741
Hu.DMD.Exon50.25.016 AGAGAATGGGATCCAGTATACTTAC 291 NG-08-0742
Hu.DMD.Exon50.20.001 ACAGAAAAGCATACACATTA 292 Hu.DMD.Exon50.20.002
TTTAACAGAAAAGCATACAC 293 Hu.DMD.Exon50.20.003 TCCTCTTTAACAGAAAAGCA
294 Hu.DMD.Exon50.20.004 TAACTTCCTCTTTAACAGAA 295
Hu.DMD.Exon50.20.005 TCTTCTAACTTCCTCTTTAA 296 Hu.DMD.Exon50.20.006
TCAGATCTTCTAACTTCCTC 297 Hu.DMD.Exon50.20.007 CCTTCCACTCAGAGCTCAGA
298 Hu.DMD.Exon50.20.008 GTAAACGGTTTACCGCCTTC 299
Hu.DMD.Exon50.20.009 CCCTCAGCTCTTGAAGTAAA 300 Hu.DMD.Exon50.20.010
GGTCAGGCTGCTTTGCCCTC 301 Hu.DMD.Exon50.20.011 TCAGTCCAGGAGCTAGGTCA
302 Hu.DMD.Exon50.20.012 AGGCTCCAATAGTGGTCAGT 303
Hu.DMD.Exon50.20.013 CTTACAGGCTCCAATAGTGG 304 Hu.DMD.Exon50.20.014
GTATACTTACAGGCTCCAAT 305 Hu.DMD.Exon50.20.015 ATCCAGTATACTTACAGGCT
306 Hu.DMD.Exon50.20.016 ATGGGATCCAGTATACTTAC 307
Hu.DMD.Exon50.20.017 AGAGAATGGGATCCAGTATA 308 Hu.DMD.Exon51.25.001-
CTAAAATATTTTGGGTTTTTGCAAAA 309 44 Hu.DmD.Exon51.25.002-
GCTAAAATATTTTGGGTTTTTGCAAA 310 45 Hu.DMD.Exon51.25.002.
TAGGAGCTAAAATATTTTGGGTTTTT 311 2-46 Hu.DMD.Exon51.25.003
AGTAGGAGCTAAAATATTTTGGGTT 312 Hu.DMD.Exon51.25.003.
TGAGTAGGAGCTAAAATATTTTGGG 313 2 Hu.DMD.Exon51.25.004
CTGAGTAGGAGCTAAAATATTTTGGG 314 Hu.DMD.Exon51.25.004.
CAGTCTGAGTAGGAGCTAAAATATT 315 2 Hu.DMD.Exon51.25.005
ACAGTCTGAGTAGGAGCTAAAATATT 316 Hu.DMD.Exon51.25.005.
GAGTAACAGTCTGAGTAGGAGCTAAA 317 2 Hu.DMD.Exon51.25.006
CAGAGTAACAGTCTGAGTAGGAGCT 318 Hu.DMD.Exon51.25.006.
CACCAGAGTAACAGTCTGAGTAGGAG 319 2 Hu.DMD.Exon51.25.007
GTCACCAGAGTAACAGTCTGAGTAG 320 Hu.DMD.Exon51.25.007.
AACCACAGGTTGTGTCACCAGAGTAA 321 2 Hu.DMD.Exon51.25.008
GTTGTGTCACCAGAGTAACAGTCTG 322 Hu.DMD.Exon51.25.009
TGGCAGTTTCCTTAGTAACCACAGGT 323 Hu.DMD.Exon51.25.010
ATTTCTAGTTTGGAGATGGCAGTTTC 324 Hu.DMD.Exon51.25.010.
GGAAGATGGCATTTCTAGTTTGGAG 325 2 Hu.DMD.Exon51.25.011
CATCAAGGAAGATGGCATTTCTAGTT 326 Hu.DMD.Exon51.25.011.
GAGCAGGTACCTCCAACATCAAGGAA 327 2 Hu.DMD.Exon51.25.012
ATCTGCCAGAGCAGGTACCTCCAAC 328 Hu.DMD.Exon51.25.013
AAGTTCTGTCCAAGCCCGGTTGAAAT 329 Hu.DMD.Exon51.25.013.
CGGTTGAAATCTGCCAGAGCAGGTAC 330 2 Hu.DMD.Exon51.25.014
GAGAAAGCCAGTCGGTAAGTTCTGTC 331 Hu.DMD.Exon51.25.014.
GTCGGTAAGTTCTGTCCAAGCCCGG 332 2 Hu.DMD.Exon51.25.015
ATAACTTGATCAAGCAGAGAAAGCCA 333 Hu.DMD.Exon51.25.015.
AAGCAGAGAAAGCCAGTCGGTAAGT 334 2 Hu.DMD.Exon51.25.016
CACCCTCTGTGATTTTATAACTTGAT 335
Hu.DMD.Exon51.25.017 CAAGGTCACCCACCATCACCCTCTGT 336
Hu.DMD.Exon51.25.017. CATCACCCTCTGTGATTTTATAACT 337 2
Hu.DMD.Exon51.25.018 CTTCTGCTTGATGATCATCTCGTTGA 338
Hu.DMD.Exon51.25.019 CCTTCTGCTTGATGATCATCTCGTTG 339
Hu.DMD.Exon51.25.019. ATCTCGTTGATATCCTCAAGGTCACC 340 2
Hu.DMD.Exon51.25.020 TCATACCTTCTGCTTGATGATCATCT 341
Hu.DMD.Exon51.25.020. TCATTTTTTCTCATACCTTCTGCTTG 342 2
Hu.DMD.Exon51.25.021 TTTTCTCATACCTTCTGCTTGATGAT 343
Hu.DMD.Exon51.25.022 TTTTATCATTTTTTCTCATACCTTCT 344
Hu.DMD.Exon51.25.023 CCAACTTTTATCATTTTTTCTCATAC 345
Hu.DMD.Exon51.20.001 ATATTTTGGGTTTTTGCAAA 346 Hu.DMD.Exon51 20.002
AAAATATTTTGGGTTTTTGC 347 Hu.DMD.Exon51.20.003 GAGCTAAAATATTTTGGGTT
348 Hu.DMD.Exon51.20.004 AGTAGGAGCTAAAATATTTT 349
Hu.DMD.Exon51.20.005 GTCTGAGTAGGAGCTAAAAT 350 Hu.DMD.Exon51.20.006
TAACAGTCTGAGTAGGAGCT 351 Hu.DMD.Exon51.20.007 CAGAGTAACAGTCTGAGTAG
352 Hu.DMD.Exon51.20.008 CACAGGTTGTGTCACCAGAG 353
Hu.DMD.Exon51.20.009 AGTTTCCTTAGTAACCACAG 354 Hu.DMD.Exon51.20.010
TAGTTTGGAGATGGCAGTTT 355 Hu.DMD.Exon51.20.011 GGAAGATGGCATTTCTAGTT
356 Hu.DMD.Exon51.20.012 TACCTCCAACATCAAGGAAG 357
Hu.DMD.Exon51.20.013 ATCTGCCAGAGCAGGTACCT 358 Hu.DMD.Exon51.20.014
CCAAGCCCGGTTGAAATCTG 359 Hu.DMD.Exon51.20.015 GTCGGTAAGTTCTGTCCAAG
360 Hu.DMD.Exon51.20.016 AAGCAGAGAAAGCCAGTCGG 361
Hu.DMD.Exon51.20.017 TTTTATAACTTGATCAAGCA 362 Hu.DMD.Exon51.20.018
CATCACCCTCTGTGATTTTA 363 Hu DMD.Exon51.20.019 CTCAAGGTCACCCACCATCA
364 Hu.DMD.Exon51.20.020 CATCTCGTTGATATCCTCAA 365
Hu.DMD.Exon51.20.021 CTTCTGCTTGATGATCATCT 366 Hu.DMD.Exon51.20.022
CATACCTTCTGCTTGATGAT 367 Hu.DMD.Exon51.20.023 TTTCTCATACCTTCTGCTTG
368 Hu.DMD.Exon51.20.024 CATTTTTTCTCATACCTTCT 369
Hu.DMD.Exon51.20.025 TTTATCATTTTTTCTCATAC 370 Hu.DMD.Exon51.20.026
CAACTTTTATCATTTTTTCT 371 Hu.DMD.Exon52.25.001
CTGTAAGAACAAATATCCCTTAGTA 372 Hu.DMD.Exon52.25.002
TGCCTGTAAGAACAAATATCCCTTA 373 Hu.DMD.Exon52 25.002.
GTTGCCTGTAAGAACAAATATCCCT 374 2 Hu.DMD.Exon52.25.003
ATTGTTGCCTGTAAGAACAAATATC 375 Hu.DMD.Exon52.25.003.
GCATTGTTGCCTGTAAGAACAAATA 376 2 Hu.DMD.Exon52.25.004
CCTGCATTGTTGCCTGTAAGAACAA 377 Hu.DMD.Exon52.25.004.
ATCCTGCATTGTTGCCTGTAAGAAC 378 2 Hu.DMD.Exon52.25.005
CAAATCCTGCATTGTTGCCTGTAAG 379 Hu.DMD.Exon52.25.005.
TCCAAATCCTGCATTGTTGCCTGTA 380 2 Hu.DMD.Exon52.25.006
TGTTCCAAATCCTGCATTGTTGCCT 381 Hu.DMD.Exon52.25.006.
TCTGTTCCAAATCCTGCATTGTTGC 382 2 Hu.DMD.Exon52.25.007
AACTGGGGACGCCTCTGTTCCAAAT 383 Hu.DMD.Exon52.25.007.
GCCTCTGTTCCAAATCCTGCATTGT 384 2 Hu.DMD.Exon52.25.008
CAGCGGTAATGAGTTCTTCCAACTG 385 Hu.DMD.Exon52.25.008.
CTTCCAACTGGGGACGCCTCTGTTC 386 2 Hu.DMD.Exon52.25.009
CTTGTTTTTCAAATTTTGGGCAGCG 387 Hu.DMD.Exon52.25.010
CTAGCCTCTTGATTGCTGGTCTTGT 388 Hu.DMD.Exon52.25.010.
TTTTCAAATTTTGGGCAGCGGTAAT 389 2 Hu.DMD.Exon52.25.011
TTCGATCCGTAATGATTGTTCTAGC 390 Hu.DMD.Exon52.25.011.
GATTGCTGGTCTTGTTTTTCAAATT 391 2 Hu.DMD.Exon52.25.012
CTTACTTCGATCCGTAATGATTGTT 392 Hu.DMD.Exon52.25.012.
TTGTTCTAGCCTCTTGATTGCTGGT 393 2 Hu.DMD.Exon52.25.013
AAAAACTTACTTCGATCCGTAATGA 394 Hu.DMD.Exon52.25.014
TGTAAAAAACTTACTTCGATCCGT 395 Hu.DMD.Exon52.25.015
ATGCTTGTTAAAAAACTTACTTCGA 396 Hu.DMD.Exon52.25.016
GTCCCATGCTTGTTAAAAAACTTAC 397 Hu.DMD.Exon52.20.001
AGAACAAATATCCCTTAGTA 398 Hu.DMD.Exon52.20.002 GTAAGAACAAATATCCCTTA
399 Hu.DMD.Exon52.20.003 TGCCTGTAAGAACAAATATC 400
Hu.DMD.Exon52.20.004 ATTGTTGCCTGTAAGAACAA 401 Hu.DMD.Exon52.20.005
CCTGCATTGTTGCCTGTAAG 402 Hu.DMD.Exon52.20.006 CAAATCCTGCATTGTTGCCT
403 Hu.DMD.Exon52.20.007 GCCTCTGTTCCAAATCCTGC 404
Hu.DMD.Exon52.20.008 CTTCCAACTGGGGACGCCTC 405 Hu.DMD.Exon52.20.009
CAGCGGTAATGAGTTCTTCC 406 Hu.DMD.Exon52.20.010 TTTTCAAATTTTGGGCAGCG
407 Hu.DMD.Exon52.20.011 GATTGCTGGTCTTGTTTTTC 408
Hu.DMD.Exon52.20.012 TTGTTCTAGCCTCTTGATTG 409 Hu.DMD.Exon52.20.013
TTCGATCCGTAATGATTGTT 410 Hu.DMD.Exon52.20.014 CTTACTTCGATCCGTAATGA
411 Hu.DMD.Exon52.20.015 AAAAACTTACTTCGATCCGT 412
Hu.DMD.Exon52.20.016 TGTTAAAAAACTTACTTCGA 413 Hu.DMD.Exon52.20.017
ATGCTTGTTAAAAAACTTAC 414 Hu.DMD.Exon52.20.018 GTCCCATGCTTGTTAAAAAA
415 Hu.DMD.Exon53.25.001 CTAGAATAAAAGGAAAAATAAATAT 416
Hu.DMD.Exon53.25.002 AACTAGAATAAAAGGAAAAATAAAT 417
Hu.DMD.Exon53.25.002. TTCAACTAGAATAAAAGGAAAAATA 418 2
Hu.DMD.Exon53.25.003 CTTTCAACTAGAATAAAAGGAAAAA 419
Hu.DMD.Exon53.25.003. ATTCTTTCAACTAGAATAAAAGGAA 420 2
Hu.DMD.Exon53.25.004 GAATTCTTTCAACTAGAATAAAAGG 421
Hu.DMD.Exon53.25.004. TCTGAATTCTTTCAACTAGAATAAA 422 2
Hu.DMD.Exon53.25.005 ATTCTGAATTCTTTCAACTAGAATA 423
Hu.DMD.Exon53.25.005. CTGATTCTGAATTCTTTCAACTAGA 424 2
Hu.DMD.Exon53.25.006 CACTGATTCTGAATTCTTTCAACTA 425
Hu.DMD.Exon53.25.006. TCCCACTGATTCTGAAfTCTTTCAA 426 2
Hu.DMD.Exon53.25.007 CATCCCACTGATTCTGAATTCTTTC 427
Hu.DMD.Exon53.25.008 TACTTCATCCCACTGATTCTGAATT 428
Hu.DMD.Exon53.25.008. CTGAAGGTGTTCTTGTACTTCATCC 429 2
Hu.DMD.Exon53.25.009 CGGTTCTGAAGGTGTTCTTGTACT 430
Hu.DMD.Exon53.25.009. CTGTTGCCTCCGGTTCTGAAGGTGT 431 2
Hu.DMD.Exon53.25.010 TTTCATTCAACTGTTGCCTCCGGTT 432
Hu.DMD.Exon53.25.010. TAACATTTCATTCAACTGTTGCCTC 433 2
Hu.DMD.Exon53.25.011 TTGTGTTGAATCCTTTAACATTTCA 434
Hu.DMD.Exon53.25.012 TCTTCCTTAGCTTCCAGCCATTGTG 435
Hu.DMD.Exon53.25.012. CTTAGCTTCCAGCCATTGTGTTGAA 436 2
Hu.DMD.Exon53.25.013 GTCCTAAGACCTGCTCAGCTTCTTC 437
Hu.DMD.Exon53.25.013. CTGCTCAGCTTCTTCCTTAGCTTCC 438 2
Hu.DMD.Exon53.25.014 CTCAAGCTTGGCTCTGGCCTGTCCT 439
Hu.DMD.Exon53.25.014. GGCCTGTCCTAAGACCTGCTCAGCT 440 2
Hu.DMD.Exon53.25.015 TAGGGACCCTCCTTCCATGACTCAA 441
Hu.DMD.Exon53.25.016 TTTGGATTGCATCTACTGTATAGGG 442
Hu.DMD.Exon53.25.016. ACCCTCCTTCCATGACTCAAGCTTG 443 2
Hu.DMD.Exon53.25.017 CTTGGTTTCTGTGATTTTCTTTTGG 444
Hu.DMD.Exon53.25.017. ATCTACTGTATAGGGACCCTCCTTC 445 2
Hu.DMD.Exon53.25.018 CTAACCTTGGTTTCTGTGATTTTCT 446
Hu.DMD.Exon53.25.018. TTTCTTTTGGATTGCATCTACTGTA 447 2
Hu.DMD.Exon53.25.019 TGATACTAACCTTGGTTTCTGTGAT 448
Hu.DMD.Exon53.25.020 ATCTTTGATACTAACCTTGGTTTCT 449
Hu.DMD.Exon53.25.021 AAGGTATCTTTGATACTAACCTTGG 450
Hu.DMD.Exon53.25.022 TTAAAAAGGTATCTTTGATACTAAC 451
Hu.DMD.Exon53.20.001 ATAAAAGGAAAAATAAATAT 452 Hu.DMD.Exon53.20.002
GAATAAAAGGAAAAATAAAT 453 Hu.DMD.Exon53.20.003 AACTAGAATAAAAGGAAAAA
454 Hu.DMD.Exon53.20.004 CTTTCAACTAGAATAAAAGG 455
Hu.DMD.Exon53.20.005 GAATTCTTTCAACTAGAATA 456 Hu.DMD.Exon53.20.006
ATTCTGAATTCTTTCAACTA 457 Hu.DMD.Exon53.20.007 TACTTCATCCCACTGATTCT
458 Hu.DMD.Exon53.20.008 CTGAAGGTGTTCTTGTACT 459
Hu.DMD.Exon53.20.009 CTGTTGCCTCCGGTTCTGAA 460 Hu.DMD.Exon53.20.010
TAACATTTCATTCAACTGTT 461 Hu.DMD.Exon53.20.011 TTGTGTTGAATCCTTTAACA
462 Hu.DMD.Exon53.20.012 CTTAGCTTCCAGCCATTGTG 463
Hu.DMD.Exon53.20.013 CTGCTCAGCTTCTTCCTTAG 464 Hu.DMD.Exon53.20.014
GGCCTGTCCTAAGACCTGCT 465 Hu.DMD.Exon53.20.015 CTCAAGCTTGGCTCTGGCCT
466 Hu.DMD.Exon53.20.016 ACCCTCCTTCCATGACTCAA 467
Hu.DMD.Exon53.20.017 ATCTACTGTATAGGGACCCT 468 Hu.DMD.Exon53.20.018
TTTCTTTTGGATTGCATCTA 469 Hu.DMD.Exon53.20.019 CTTGGTTTCTGTGATTTTCT
470 Hu.DMD.Exon53.20.020 CTAACCTTGGTTTCTGTGAT 471
Hu.DMD.Exon53.20.021 TGATACTAACCTTGGTTTCT 472 Hu.DMD.Exon53.20.022
ATCTTTGATACTAACCTTGG 473 Hu.DMD.Exon53.20.023 AAGGTATCTTTGATACTAAC
474 Hu.DMD.Exon53.20.024 TTAAAAAGGTATCTTTGATA 475
Hu.DMD.Exon54.25.001 CTATAGATTTTTATGAGAAAGAGA 476
Hu.DMD.Exon54.25.002 AACTGCTATAGATTTTATGAGAAA 477
Hu.DMD.Exon54.25.003 TGGCCAACTGCTATAGATTTTTATG 478
Hu.DMD.Exon54.25.004 GTCTTTGGCCAACTGCTATAGATTT 479
Hu.DMD.Exon54.25.005 CGGAGGTCTTTGGCCAACTGCTATA 480
Hu.DMD.Exon54.25.006 ACTGGCGGAGGTCTTTGGCCAACTG 481
Hu.DMD.Exon54.25.007 TTTGTCTGCCACTGGCGGAGGTCTT 482
Hu.DMD.Exon54.25.008 AGTCATTTGCCACATCTACATTTGT 483
Hu.DMD.Exon54.25.008. TTTGCCACATCTACATTTGTCTGCC 484 2
Hu.DMD.Exon54.25.009 CCGGAGAAGTTTCAGGGCCAAGTCA 485
Hu.DMD.Exon54.25.010 GTATCATCTGCAGAATAATCCCGGA 486
Hu.DMD.Exon54.25.010. TAATCCCGGAGAAGTTTCAGGGCCA 487 2
Hu.DMD.Exon54.25.011 TTATCATGTGGACTTTTCTGGTATC 488
Hu.DMD.Exon54.25.012 AGAGGCATTGATATTCTCTGTTATC 489
Hu.DMD.Exon54.25.012. ATGTGGACTTTTCTGGTATCATCTG 490 2
Hu.DMD.Exon54.25.013 CTTTTATGAATGCTTCTCCAAGAGG 491
Hu.DMD.Exon54.25.013. ATATTCTCTGTTATCATGTGGACTT 492 2
Hu.DMD.Exon54.25.014 CATACCTTTTATGAATGCTTCTCCA 493
Hu.DMD.Exon54.25.014. CTCCAAGAGGCATTGATATTCTCTG 494 2
Hu.DMD.Exon54.25.015 TAATTCATACCTTTTATGAATGCTT 495
Hu.DMD.Exon54.25.015. CTTTTATGAATGCTTCTCCAAGAGG 496 2
Hu.DMD.Exon54.25.016 TAATGTAATTCATACCTTTTATGAA 497
Hu.DMD.Exon54.25.017 AGAAATAATGTAATTCATACCTTTT 498
Hu.DMD.Exon54.25.018 GTTTTAGAAATAATGTAATTCATAC 499
Hu.DMD.Exon54.20.001 GATTTTTATGAGAAAGAGA 500 Hu.DMD.Exon54.20.002
CTATAGATTTTTATGAGAAA 501 Hu.DMD.Exon54.20.003 AACTGCTATAGATTTTTATG
502 Hu.DMD.Exon54.20.004 TGGCCAACTGCTATAGATTT 503
Hu.DMD.Exon54.20.005 GTCTTTGGCCAACTGCTATA 504 Hu.DMD.Exon54.20.006
CGGAGGTCTTTGGCCAACTG 505 Hu.DMD.Exon54.20.007 TTTGTCTGCCACTGGCGGAG
506 Hu.DMD.Exon54.20.008 TTTGCCACATCTACATTTGT 507
Hu.DMD.Exon54.20.009 TTCAGGGCCAAGTCATTTGC 508 Hu.DMD.Exon54.20.010
TAATCCCGGAGAAGTTTCAG 509 Hu.DMD.Exon54.20.011 GTATCATCTGCAGAATAATC
510 Hu.DMD.Exon54.20.012 ATGTGGACTTTTCTGGTATC 511
Hu.DMD.Exon54.20.013 ATATTCTCTGTTATCATGTG 512 Hu.DMD.Exon54.20.014
CTCCAAGAGGCATTGATATT 513 Hu.DMD.Exon54.20.015 CTTTTATGAATGCTTCTCCA
514 Hu.DMD.Exon54.20.016 CATACCTTTTATGAATGCTT 515
Hu.DMD.Exon54.20.017 TAATTCATACCTTTTATGAA 516 Hu.DMD.Exon54.20.018
TAATGTAATTCATACCTTTT 517 Hu.DMD.Exon54.20.019 AGAAATAATGTAATTCATAC
518 Hu.DMD.Exon54.20.020 GTTTTAGAAATAATGTAATT 519
Hu.DMD.Exon55.25.001 CTGCAAAGGACCAAATGTTCAGATG 520
Hu.DMD.Exon55.25.002 TCACCCTGCAAAGGACCAAATGTTC 521
Hu.DMD.Exon55.25.003 CTCACTCACCCTGCAAAGGACCAAA 522
Hu.DMD.Exon55.25.004 TCTCGCTCACTCACCCTGCAAAGGA 523
Hu.DMD.Exon55.25.005 CAGCCTCTCGCTCACTCACCCTGCA 524
Hu.DMD.Exon55.25.006 CAAAGCAGCCTCTCGCTCACTCACC 525
Hu.DMD.Exon55.25.007 TCTTCCAAAGCAGCCTCTCGCTCAC 526
Hu.DMD.Exon55.25.007. TCTATGAGTTTCTTCCAAAGCAGCC 527 2
Hu.DMD.Exon55.25.008 GTTGCAGTAATCTATGAGTTTCTTC 528
Hu.DMD.Exon55.25.008. GAACTGTTGCAGTAATCTATGAGTT 529 2
Hu.DMD.Exon55.25.009 TTCCAGGTCCAGGGGGAACTGTTGC 530
Hu.DMD.Exon55.25.010 GTAAGCCAGGCAAGAAACTTTTCCA 531
Hu.DMD.Exon55.25.010. CCAGGCAAGAAACTTTTCCAGGTCC 532 2
Hu.DMD.Exon55.25.011 TGGCAGTTGTTTCAGCTTCTGTAAG 533
Hu.DMD.Exon55.25.011. TTCAGCTTCTGTAAGCCAGGCAAGA 635 2
Hu.DMD.Exon55.25.012 GGTAGCATCCTGTAGGACATTGGCA 534
Hu.DMD.Exon55.25.012. GACATTGGCAGTTGTTTCAGCTTCT 535 2
Hu.DMD.Exon55.25.013 TCTAGGAGCCTTTCCTTACGGGTAG 536
Hu.DMD.Exon55.25.014 CTTTTACTCCCTTGGAGTCTTCTAG 537
Hu.DMD.Exon55.25.014. GAGCCTTTCCTTACGGGTAGCATCC 538 2
Hu.DMD.Exon55.25.015 TTGCCATTGTTTCATCAGCTCTTTT 539
Hu.DMD.Exon55.25.015. CTTGGAGTCTTCTAGGAGCCTTTCC 540 2
Hu.DMD.Exon55.25 016 CTTACTTGCCATTGTTTCATCAGCT 541
Hu.DMD.Exon55.25.016. CAGCTCTTTTACTCCCTTGGAGTCT 542 2
Hu.DMD.Exon55.25.017 CCTGACTTACTTGCCATTGTTTCAT 543
Hu.DMD.Exon55.25.018 AAATGCCTGACTTACTTGCCATTGT 544
Hu.DMD.Exon55.25.019 AGCGGAAATGCCTGACTTACTTGCC 545
Hu.DMD.Exon55.25.020 GCTAAAGCGGAAATGCCTGACTTAC 546
Hu.DMD.Exon55.20.001 AAGGACCAAATGTTCAGATG 547 Hu.DMD.Exon55.20.002
CTGCAAAGGACCAAATGTTC 548 Hu.DMD.Exon55.20.003 TCACCCTGCAAAGGACCAAA
549 Hu.DMD.Exon55.20.004 CTCACTCACCCTGCAAAGGA 550
Hu.DMD.Exon55.20.005 TCTCGCTCACTCACCCTGCA 551 Hu.DMD.Exon55.20.006
CAGCCTCTCGCTCACTCACC 552 Hu.DMD.Exon55.20.007 CAAAGCAGCCTCTCGCTCAC
553 Hu.DMD.Exon55.20.008 TCTATGAGTTTCTTCCAAAG 554
Hu.DMD.Exon55.20.009 GAACTGTTGCAGTAATCTAT 555 Hu.DMD.Exon55.20.010
TTCCAGGTCCAGGGGGAACT 556 Hu.DMD.Exon55.20.011 CCAGGCAAGAAACTTTTCCA
557 Hu.DMD.Exon55.20.012 TTCAGCTTCTGTAAGCCAGG 558
Hu.DMD.Exon55.20.013 GACATTGGCAGTTGTTTCAG 559 Hu.DMD.Exon55.20.014
GGTAGCATCCTGTAGGACAT 560 Hu.DMD.Exon55.20.015 GAGCCTTTCCTTACGGGTAG
561 Hu.DMD.Exon55.20.016 CTTGGAGTCTTCTAGGAGCC 562
Hu.DMD.Exon55.20.017 CAGCTCTTTTACTCCCTTGG 563 Hu.DMD.Exon55.20.018
TTGCCATTGTTTCATCAGCT 564
Hu.DMD.Exon55.20.019 CTTACTTGCCATTGTTTCAT 565 Hu.DMD.Exon55.20.020
CCTGACTTACTTGCCATTGT 566 Hu.DMD.Exon55.20.021 AAATGCCTGACTTACTTGCC
567 Hu.DMD.Exon55.20.022 AGCGGAAATGCCTGACTTAC 568
Hu.DMD.Exon55.20.023 GCTAAAGCGGAAATGCCTGA 569 H50A(+02+30)-AVI-5656
CCACTCAGAGCTCAGATCTTCTAACTTC 584 C H50D(+07-18)-AVI-5915
GGGATCCAGTATACTTACAGGCTCC 585 H50A(+07+33)
CTTCCACTCAGAGCTCAGATCTTCTAA 586 H51A(+61+90)-AVI-4657
ACATCAAGGAAGATGGCATTTCTAGTTT 587 GG H51A(+66+95)-AVI-4658
CTCCAACATCAAGGAAGATGGCATTTCT 588 AG H51A(+111+134)
TTCTGTCCAAGCCCGGTTGAAATC 589 H51A(+175+195) CACCCACCATCACCCTCYGTG
590 H51A(+199+220) ATCATCTCGTTGATATCCTCAA 591 H51A(+66+90)
ACATCAAGGAAGATGGCATTTCTAG 592 H51A(-01+25)
ACCAGAGTAACAGTCTGAGTAGGAGC 593 h51AON1 TCAAGGAAGATGGCATTTCT 594
h51AON2 CCTCTGTGATTTTATAACTTGAT 595 H51D(+08-17)
ATCATTTTTTCTCATACCTTCTGCT 596 H51D(+16-07) CTCATACCTTCTGCTTGATGATC
597 hAON#23 TGGCATTTCTAGTTTGG 598 hAON#24 CCAGAGCAGGTACCTCCAACATC
599 H44A(+61+84) TGTTCAGCTTCTGTTAGCCACTGA 600 H44A(+85+104 )
TTTGTGTCTTTCTGAGAAAC 601 h44AON1 CGCCGCCATTTCTCAACAG 602
H44A(-06+14) ATCTGTCAAATCGCCTGCAG 603 H45A(+71+90)
TGTTTTTGAGGATTGCTGAA 604 h45AON1 GCTGAATTATTTCTTCCCC 605 h45AON5
GCCCAATGCCATCCTGG 606 H45A(-06+20) CCAATGCCATCCTGGAGTTCCTGTAA 607
H53A(+39+69) CATTCAACTGTTGCCTCCGGTTCTGAAG 608 GTG H53A(+23+47)
CTGAAGGTGTTCTTGTACTTCATCC 609 h53AON1 CTGTTGCCTCCGGTTCTG 610
F153A(-12+10) ATTCTTTCAACTAGAATAAAAG 611 huEx45.30.66
GCCATCCTGGAGTTCCTGTAAGATACC 612 AAA huEx45.30.71
CCAATGCCATCCTGGAGTTCCTGTAAG 613 ATA huEx45.30.79
GCCGCTGCCCAATGCCATCCTGGAGTT 614 CCT huEx45.30.83
GTTTGCCGCTGCCCAATGCCATCCTGG 615 AGT huEx45.30.88
CAACAGTTTGCCGCTGCCCAATGCCAT 616 CCT huEx45.30.92
CTGACAACAGTTTGCCGCTGCCCAATG 617 CCA huEx45.30.96
TGTTCTGACAACAGTTTGCCGCTGCCC 618 AAT huEx45.30.99
CAATGTTCTGACAACAGTTTGCCGCTG 619 CCC huEx45.30.103
CATTCAATGTTCTGACAACAGTTTGCCG 620 CT huEx45.30.120
TATTTCTTCCCCAGTTGCATTCAATGTT 621 CT huEx45.30.127
GCTGAATTATTTCTTCCCCAGTTGCATT 622 CA huEx45.30.132
GGATTGCTGAATTATTTCTTCCCCAGTT 623 GC huEx45.30.137
TTTGAGGATTGCTGAATTATTTCTTCCC 624 CA huEx53.30.84
GTACTTCATCCCACTGATTCTGAATTCT 625 TT huEx53.30.88
TCTTGTACTTCATCCCACTGATTCTGAA 626 TT huEx53.30.91
TGTTCTTGTACTTCATCCCACTGATTCT 627 GA huEx53.30.103
CGGTTCTGAAGGTGTTCTTGTACTTCAT 628 CC huEx53.30.106
CTCCGGTTCTGAAGGTGTTCTTGTACTT 629 CA huEx53.30.109
TGCCTCCGGTTCTGAAGGTGTTCTTGTA 630 CT huEx53.30.112
TGTTGCCTCCGGTTCTGAAGGTGTTCTT 631 GT huEx53.30.115
AACTGTTGCCTCCGGTTCTGAAGGTGT 632 TCT huEx53.30.118
TTCAACTGTTGCCTCCGGTTCTGAAGGT 633 GT h50AON1 h50AON2 Peptide
Transporters (NH.sub.2 to COOH)*: rTAT RRRQRRKKRC 570
R.sub.9F.sub.2 RRRRRRRRRFFC 571 (RRAnx).sub.4B
RRAhxRRAhxRRAhxRRAhxB 572 (RAhxR).sub.4AhxB; (P007)
RAhxRRAhxRRAhxRRAhxRAhxB 573 (AhxRR).sub.4AhxB
AhxRRAhxRRAhxRRAhxRRAhxB 574 (RAhx).sub.6B
RAhxRAhxRAhxRAhxRAhxRAhxB 575 (RAhx).sub.8B
RAhxRAhxRAhxRAhxRAhxRAhxRAhxRAhx 576 B (RAhxR).sub.5AhxB
RAhxRRAhxRRAhxRRAhxRRAhxRAhxB 577 (RAhxRRBR).sub.2AhxB;
RAhxRRBRRAhxRRBRAhxB 578 (CP06062) MSP ASSLNIA 579 Cell Penetrating
Peptide / Homing Peptide / PMO Conjugates (NH.sub.2 to COOH and 5'
to 3') MSP-PMO ASSLNIA-XB- 580 GGCCAAACCTCGGCTTACCTGAAAT 636
CP06062-MSP-PMO RXRRBRRXRRBR-XB-ASSLNIA-X- 581
GGCCAAACCTCGGCTTACCTGAAAT 636 MSP-CP06062-PMO
ASSLNIA-X-RXRRBRRXRRBR-B- 582 GGCCAAACCTCGGCTTACCTGAAAT 636
CP06062-PMO RXRRBRRXRRBR-XB- 583 GGCCAAACCTCGGCTTACCTGAAAT 636 *Ahx
is 6-aminohexanoic acid and B is beta-aianine.
Sequence CWU 1
1
651125DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 1ctgcaggtaa aagcatatgg atcaa
25225DNAArtificial SequenceAntisense sequence to target splice site
of proprocessed human dystrophin 2atcgcctgca ggtaaaagca tatgg
25325DNAArtificial SequenceAntisense sequence to target splice site
of proprocessed human dystrophin 3gtcaaatcgc ctgcaggtaa aagca
25425DNAArtificial SequenceAntisense sequence to target splice site
of proprocessed human dystrophin 4gatctgtcaa atcgcctgca ggtaa
25525DNAArtificial SequenceAntisense sequence to target splice site
of proprocessed human dystrophin 5caacagatct gtcaaatcgc ctgca
25625DNAArtificial SequenceAntisense sequence to target splice site
of proprocessed human dystrophin 6tttctcaaca gatctgtcaa atcgc
25725DNAArtificial SequenceAntisense sequence to target splice site
of proprocessed human dystrophin 7ccatttctca acagatctgt caaat
25825DNAArtificial SequenceAntisense sequence to target splice site
of proprocessed human dystrophin 8ataatgaaaa cgccgccatt tctca
25925DNAArtificial SequenceAntisense sequence to target splice site
of proprocessed human dystrophin 9aaatatcttt atatcataat gaaaa
251025DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 10tgttagccac tgattaaata tcttt
251125DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 11aaactgttca gcttctgtta gccac
251225DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 12ttgtgtcttt ctgagaaact gttca
251325DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 13ccaattctca ggaatttgtg tcttt
251425DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 14gtatttagca tgttcccaat tctca
251525DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 15cttaagatac catttgtatt tagca
251625DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 16cttaccttaa gataccattt gtatt
251725DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 17aaagacttac cttaagatac cattt
251825DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 18aaatcaaaga cttaccttaa gatac
251925DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 19aaaacaaatc aaagacttac cttaa
252025DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 20tcgaaaaaac aaatcaaaga cttac
252125DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 21ctgtaagata ccaaaaaggc aaaac
252225DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 22cctgtaagat accaaaaagg caaaa
252325DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 23agttcctgta agataccaaa aaggc
252425DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 24gagttcctgt aagataccaa aaagg
252525DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 25cctggagttc ctgtaagata ccaaa
252625DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 26tcctggagtt cctgtaagat accaa
252725DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 27gccatcctgg agttcctgta agata
252825DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 28tgccatcctg gagttcctgt aagat
252925DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 29ccaatgccat cctggagttc ctgta
253025DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 30cccaatgcca tcctggagtt cctgt
253125DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 31gctgcccaat gccatcctgg agttc
253225DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 32cgctgcccaa tgccatcctg gagtt
253325DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 33aacagtttgc cgctgcccaa tgcca
253425DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 34ctgacaacag tttgccgctg cccaa
253525DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 35gttgcattca atgttctgac aacag
253625DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 36gctgaattat ttcttcccca gttgc
253725DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 37attatttctt ccccagttgc attca
253825DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 38ggcatctgtt tttgaggatt gctga
253925DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 39tttgaggatt gctgaattat ttctt
254025DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 40aatttttcct gtagaatact ggcat
254125DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 41atactggcat ctgtttttga ggatt
254225DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 42accgcagatt caggcttccc aattt
254325DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 43aatttttcct gtagaatact ggcat
254425DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 44ctgtttgcag acctcctgcc accgc
254525DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 45agattcaggc ttcccaattt ttcct
254625DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 46ctcttttttc tgtctgacag ctgtt
254725DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 47acctcctgcc accgcagatt caggc
254825DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 48cctacctctt ttttctgtct gacag
254925DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 49gacagctgtt tgcagacctc ctgcc
255025DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 50gtcgccctac ctcttttttc tgtct
255125DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 51gatctgtcgc cctacctctt ttttc
255225DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 52tattagatct gtcgccctac ctctt
255325DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 53attcctatta gatctgtcgc cctac
255420DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 54agataccaaa aaggcaaaac
205520DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 55aagataccaa aaaggcaaaa
205620DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 56cctgtaagat accaaaaagg
205720DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 57gagttcctgt aagataccaa
205820DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 58tcctggagtt cctgtaagat
205920DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 59tgccatcctg gagttcctgt
206020DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 60cccaatgcca tcctggagtt
206120DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 61cgctgcccaa tgccatcctg
206220DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 62ctgacaacag tttgccgctg
206320DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 63gttgcattca atgttctgac
206420DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 64attatttctt ccccagttgc
206520DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 65tttgaggatt gctgaattat
206620DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 66atactggcat ctgtttttga
206720DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 67aatttttcct gtagaatact
206820DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 68agattcaggc ttcccaattt
206920DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 69acctcctgcc accgcagatt
207020DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 70gacagctgtt tgcagacctc
207120DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 71ctcttttttc tgtctgacag
207220DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 72cctacctctt ttttctgtct
207320DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 73gtcgccctac ctcttttttc
207420DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 74gatctgtcgc cctacctctt
207520DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 75tattagatct gtcgccctac
207620DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 76attcctatta gatctgtcgc
207725DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 77gggggatttg agaaaataaa attac
257825DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 78atttgagaaa ataaaattac cttga
257925DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 79ctagcctgga gaaagaagaa taaaa
258025DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 80agaaaataaa attaccttga cttgc
258125DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 81ttcttctagc ctggagaaag aagaa
258225DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 82ataaaattac cttgacttgc tcaag
258325DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 83ttttgttctt ctagcctgga gaaag
258425DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 84attaccttga cttgctcaag ctttt
258525DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 85tattcttttg ttcttctagc ctgga
258625DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 86cttgacttgc tcaagctttt ctttt
258725DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 87caagatattc ttttgttctt ctagc
258825DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 88cttttagttg ctgctctttt ccagg
258925DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 89ccaggttcaa gtgggatact agcaa
259025DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 90atctctttga aattctgaca agata
259125DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 91agcaatgtta tctgcttcct ccaac
259225DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 92aacaaattca tttaaatctc tttga
259325DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 93ccaaccataa aacaaattca tttaa
259425DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 94ttcctccaac cataaaacaa attca
259525DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 95tttaaatctc tttgaaattc tgaca
259625DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 96tgacaagata ttcttttgtt cttct
259725DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 97ttcaagtggg atactagcaa tgtta
259825DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 98agatattctt ttgttcttct agcct
259925DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 99ctgctctttt ccaggttcaa gtggg
2510025DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 100ttcttttgtt cttctagcct
ggaga 2510125DNAArtificial SequenceAntisense sequence to target
splice site of
proprocessed human dystrophin 101cttttctttt agttgctgct ctttt
2510225DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 102ttgttcttct agcctggaga
aagaa 2510325DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 103cttctagcct
ggagaaagaa gaata 2510425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 104agcctggaga
aagaagaata aaatt 2510525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 105ctggagaaag
aagaataaaa ttgtt 2510620DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 106gaaagaagaa
taaaattgtt 2010720DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 107ggagaaagaa
gaataaaatt 2010820DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 108agcctggaga
aagaagaata 2010920DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 109cttctagcct
ggagaaagaa 2011020DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 110ttgttcttct
agcctggaga 2011120DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 111ttcttttgtt
cttctagcct 2011220DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 112tgacaagata
ttcttttgtt 2011320DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 113atctctttga
aattctgaca 2011420DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 114aacaaattca
tttaaatctc 2011520DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 115ttcctccaac
cataaaacaa 2011620DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 116agcaatgtta
tctgcttcct 2011720DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 117ttcaagtggg
atactagcaa 2011820DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 118ctgctctttt
ccaggttcaa 2011920DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 119cttttctttt
agttgctgct 2012020DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 120cttgacttgc
tcaagctttt 2012120DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 121attaccttga
cttgctcaag 2012220DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 122ataaaattac
cttgacttgc 2012320DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 123agaaaataaa
attaccttga 2012420DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 124atttgagaaa
ataaaattac 2012520DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 125gggggatttg
agaaaataaa 2012625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 126ctgaaacaga
caaatgcaac aacgt 2512725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 127agtaactgaa
acagacaaat gcaac 2512825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 128ccaccagtaa
ctgaaacaga caaat 2512925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 129ctcttccacc
agtaactgaa acaga 2513025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 130ggcaactctt
ccaccagtaa ctgaa 2513125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 131gcaggggcaa
ctcttccacc agtaa 2513225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 132ctggcgcagg
ggcaactctt ccacc 2513325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 133tttaattgtt
tgagaattcc ctggc 2513425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 134ttgtttgaga
attccctggc gcagg 2513525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 135gcacgggtcc
tccagtttca tttaa 2513625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 136tccagtttca
tttaattgtt tgaga 2513725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 137gcttatggga
gcacttacaa gcacg 2513825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 138tacaagcacg
ggtcctccag tttca 2513925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 139agtttatctt
gctcttctgg gctta 2514025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 140tctgcttgag
cttattttca agttt 2514125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 141atcttgctct
tctgggctta tggga 2514225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 142ctttatccac
tggagatttg tctgc 2514325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 143cttattttca
agtttatctt gctct 2514425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 144ctaaccttta
tccactggag atttg 2514525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 145atttgtctgc
ttgagcttat tttca 2514625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 146aatgtctaac
ctttatccac tggag 2514725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 147tggttaatgt
ctaaccttta tccac 2514825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 148agagatggtt
aatgtctaac cttta 2514925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 149acggaagaga
tggttaatgt ctaac 2515020DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 150acagacaaat
gcaacaacgt 2015120DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 151ctgaaacaga
caaatgcaac 2015220DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 152agtaactgaa
acagacaaat 2015320DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 153ccaccagtaa
ctgaaacaga 2015420DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 154ctcttccacc
agtaactgaa 2015520DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 155ggcaactctt
ccaccagtaa 2015620DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 156ctggcgcagg
ggcaactctt 2015720DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 157ttgtttgaga
attccctggc 2015820DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 158tccagtttca
tttaattgtt 2015920DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 159tacaagcacg
ggtcctccag 2016020DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 160gcttatggga
gcacttacaa 2016120DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 161atcttgctct
tctgggctta 2016220DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 162cttattttca
agtttatctt 2016320DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 163atttgtctgc
ttgagcttat 2016420DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 164ctttatccac
tggagatttg 2016520DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 165ctaaccttta
tccactggag 2016620DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 166aatgtctaac
ctttatccac 2016720DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 167tggttaatgt
ctaaccttta 2016820DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 168agagatggtt
aatgtctaac 2016920DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 169acggaagaga
tggttaatgt 2017025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 170ctgaaaggaa
aatacatttt aaaaa 2517125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 171cctgaaagga
aaatacattt taaaa 2517225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 172gaaacctgaa
aggaaaatac atttt 2517325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 173ggaaacctga
aaggaaaata cattt 2517425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 174ctctggaaac
ctgaaaggaa aatac 2517525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 175gctctggaaa
cctgaaagga aaata 2517625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 176gtaaagctct
ggaaacctga aagga 2517725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 177tcaggtaaag
ctctggaaac ctgaa 2517825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 178ctcaggtaaa
gctctggaaa cctga 2517925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 179gtttctcagg
taaagctctg gaaac 2518025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 180tgtttctcag
gtaaagctct ggaaa 2518125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 181aatttctcct
tgtttctcag gtaaa 2518225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 182tttgagcttc
aatttctcct tgttt 2518325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 183ttttatttga
gcttcaattt ctcct 2518425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 184aagctgccca
aggtctttta tttga 2518525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 185aggtcttcaa
gctttttttc aagct 2518625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 186ttcaagcttt
ttttcaagct gccca 2518725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 187gatgatttaa
ctgctcttca aggtc 2518825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 188ctgctcttca
aggtcttcaa gcttt 2518925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 189aggagataac
cacagcagca gatga 2519025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 190cagcagatga
tttaactgct cttca 2519125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 191atttccaact
gattcctaat aggag 2519225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 192cttggtttgg
ttggttataa atttc 2519325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 193caactgattc
ctaataggag ataac 2519425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 194cttaacgtca
aatggtcctt cttgg 2519525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 195ttggttataa
atttccaact gattc 2519625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 196cctaccttaa
cgtcaaatgg tcctt 2519725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 197tccttcttgg
tttggttggt tataa 2519825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 198agttccctac
cttaacgtca aatgg 2519925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 199caaaaagttc
cctaccttaa cgtca 2520025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 200taaagcaaaa
agttccctac cttaa 2520125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 201atatttaaag
caaaaagttc cctac
2520220DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 202aggaaaatac attttaaaaa
2020320DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 203aaggaaaata cattttaaaa
2020420DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 204cctgaaagga aaatacattt
2020520DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 205ggaaacctga aaggaaaata
2020620DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 206gctctggaaa cctgaaagga
2020720DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 207gtaaagctct ggaaacctga
2020820DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 208ctcaggtaaa gctctggaaa
2020920DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 209aatttctcct tgtttctcag
2021020DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 210ttttatttga gcttcaattt
2021120DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 211aagctgccca aggtctttta
2021220DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 212ttcaagcttt ttttcaagct
2021320DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 213ctgctcttca aggtcttcaa
2021420DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 214cagcagatga tttaactgct
2021520DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 215aggagataac cacagcagca
2021620DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 216caactgattc ctaataggag
2021720DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 217ttggttataa atttccaact
2021820DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 218tccttcttgg tttggttggt
2021920DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 219cttaacgtca aatggtcctt
2022020DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 220cctaccttaa cgtcaaatgg
2022120DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 221agttccctac cttaacgtca
2022220DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 222caaaaagttc cctaccttaa
2022320DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 223taaagcaaaa agttccctac
2022420DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 224atatttaaag caaaaagttc
2022525DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 225ctggggaaaa gaacccatat
agtgc 2522625DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 226tcctggggaa
aagaacccat atagt 2522725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 227gtttcctggg
gaaaagaacc catat 2522825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 228cagtttcctg
gggaaaagaa cccat 2522925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 229tttcagtttc
ctggggaaaa gaacc 2523025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 230tatttcagtt
tcctggggaa aagaa 2523125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 231tgctatttca
gtttcctggg gaaaa 2523225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 232actgctattt
cagtttcctg gggaa 2523325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 233tgaactgcta
tttcagtttc ctggg 2523425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 234cttgaactgc
tatttcagtt tcctg 2523525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 235tagcttgaac
tgctatttca gtttc 2523625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 236tttagcttga
actgctattt cagtt 2523725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 237ttccacatcc
ggttgtttag cttga 2523825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 238tgccctttag
acaaaatctc ttcca 2523925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 239tttagacaaa
atctcttcca catcc 2524025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 240gtttttcctt
gtacaaatgc tgccc 2524125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 241gtacaaatgc
tgccctttag acaaa 2524225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 242cttcactggc
tgagtggctg gtttt 2524325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 243ggctggtttt
tccttgtaca aatgc 2524425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 244attaccttca
ctggctgagt ggctg 2524525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 245gcttcattac
cttcactggc tgagt 2524625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 246aggttgcttc
attaccttca ctggc 2524725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 247gctagaggtt
gcttcattac cttca 2524825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 248atattgctag
aggttgcttc attac 2524920DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 249gaaaagaacc
catatagtgc 2025020DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 250gggaaaagaa
cccatatagt 2025120DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 251tcctggggaa
aagaacccat 2025220DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 252cagtttcctg
gggaaaagaa 2025320DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 253tatttcagtt
tcctggggaa 2025420DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 254actgctattt
cagtttcctg 2025520DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 255cttgaactgc
tatttcagtt 2025620DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 256tttagcttga
actgctattt 2025720DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 257ttccacatcc
ggttgtttag 2025820DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 258tttagacaaa
atctcttcca 2025920DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 259gtacaaatgc
tgccctttag 2026020DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 260ggctggtttt
tccttgtaca 2026120DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 261cttcactggc
tgagtggctg 2026220DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 262attaccttca
ctggctgagt 2026320DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 263gcttcattac
cttcactggc 2026420DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 264aggttgcttc
attaccttca 2026520DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 265gctagaggtt
gcttcattac 2026620DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 266atattgctag
aggttgcttc 2026725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 267ctttaacaga
aaagcataca catta 2526825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 268tcctctttaa
cagaaaagca tacac 2526925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 269ttcctcttta
acagaaaagc ataca 2527025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 270taacttcctc
tttaacagaa aagca 2527125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 271ctaacttcct
ctttaacaga aaagc 2527225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 272tcttctaact
tcctctttaa cagaa 2527325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 273atcttctaac
ttcctcttta acaga 2527425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 274tcagatcttc
taacttcctc tttaa 2527525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 275ctcagatctt
ctaacttcct cttta 2527625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 276agagctcaga
tcttctaact tcctc 2527725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 277cagagctcag
atcttctaac ttcct 2527824DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 278cactcagagc
tcagatcttc tact 2427925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 279ccttccactc
agagctcaga tcttc 2528025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 280gtaaacggtt
taccgccttc cactc 2528125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 281ctttgccctc
agctcttgaa gtaaa 2528225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 282ccctcagctc
ttgaagtaaa cggtt 2528325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 283ccaggagcta
ggtcaggctg ctttg 2528425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 284ggtcaggctg
ctttgccctc agctc 2528525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 285aggctccaat
agtggtcagt ccagg 2528625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 286tcagtccagg
agctaggtca ggctg 2528725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 287cttacaggct
ccaatagtgg tcagt 2528825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 288gtatacttac
aggctccaat agtgg 2528925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 289atccagtata
cttacaggct ccaat 2529025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 290atgggatcca
gtatacttac aggct 2529125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 291agagaatggg
atccagtata cttac 2529220DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 292acagaaaagc
atacacatta 2029320DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 293tttaacagaa
aagcatacac 2029420DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 294tcctctttaa
cagaaaagca 2029520DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 295taacttcctc
tttaacagaa 2029620DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 296tcttctaact
tcctctttaa 2029720DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 297tcagatcttc
taacttcctc 2029820DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 298ccttccactc
agagctcaga 2029920DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 299gtaaacggtt
taccgccttc 2030020DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 300ccctcagctc
ttgaagtaaa 2030120DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 301ggtcaggctg
ctttgccctc 2030220DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
302tcagtccagg agctaggtca 2030320DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
303aggctccaat agtggtcagt 2030420DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
304cttacaggct ccaatagtgg 2030520DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
305gtatacttac aggctccaat 2030620DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
306atccagtata cttacaggct 2030720DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
307atgggatcca gtatacttac 2030820DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
308agagaatggg atccagtata 2030926DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
309ctaaaatatt ttgggttttt gcaaaa 2631026DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 310gctaaaatat tttgggtttt tgcaaa
2631126DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 311taggagctaa aatattttgg
gttttt 2631225DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 312agtaggagct
aaaatatttt gggtt 2531325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 313tgagtaggag
ctaaaatatt ttggg 2531426DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 314ctgagtagga
gctaaaatat tttggg 2631525DNAArtificial SequenceAntisense sequence
to target splice site of proprocessed human dystrophin
315cagtctgagt aggagctaaa atatt 2531626DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 316acagtctgag taggagctaa aatatt
2631726DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 317gagtaacagt ctgagtagga
gctaaa 2631825DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 318cagagtaaca
gtctgagtag gagct 2531926DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 319caccagagta
acagtctgag taggag 2632025DNAArtificial SequenceAntisense sequence
to target splice site of proprocessed human dystrophin
320gtcaccagag taacagtctg agtag 2532126DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 321aaccacaggt tgtgtcacca gagtaa
2632225DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 322gttgtgtcac cagagtaaca
gtctg 2532326DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 323tggcagtttc
cttagtaacc acaggt 2632426DNAArtificial SequenceAntisense sequence
to target splice site of proprocessed human dystrophin
324atttctagtt tggagatggc agtttc 2632525DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 325ggaagatggc atttctagtt tggag
2532626DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 326catcaaggaa gatggcattt
ctagtt 2632726DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 327gagcaggtac
ctccaacatc aaggaa 2632825DNAArtificial SequenceAntisense sequence
to target splice site of proprocessed human dystrophin
328atctgccaga gcaggtacct ccaac 2532926DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 329aagttctgtc caagcccggt tgaaat
2633026DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 330cggttgaaat ctgccagagc
aggtac 2633126DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 331gagaaagcca
gtcggtaagt tctgtc 2633225DNAArtificial SequenceAntisense sequence
to target splice site of proprocessed human dystrophin
332gtcggtaagt tctgtccaag cccgg 2533326DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 333ataacttgat caagcagaga aagcca
2633425DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 334aagcagagaa agccagtcgg
taagt 2533526DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 335caccctctgt
gattttataa cttgat 2633626DNAArtificial SequenceAntisense sequence
to target splice site of proprocessed human dystrophin
336caaggtcacc caccatcacc ctctgt 2633725DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 337catcaccctc tgtgatttta taact
2533826DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 338cttctgcttg atgatcatct
cgttga 2633926DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 339ccttctgctt
gatgatcatc tcgttg 2634026DNAArtificial SequenceAntisense sequence
to target splice site of proprocessed human dystrophin
340atctcgttga tatcctcaag gtcacc 2634126DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 341tcataccttc tgcttgatga tcatct
2634226DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 342tcattttttc tcataccttc
tgcttg 2634326DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 343ttttctcata
ccttctgctt gatgat 2634426DNAArtificial SequenceAntisense sequence
to target splice site of proprocessed human dystrophin
344ttttatcatt ttttctcata ccttct 2634526DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 345ccaactttta tcattttttc tcatac
2634620DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 346atattttggg tttttgcaaa
2034720DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 347aaaatatttt gggtttttgc
2034820DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 348gagctaaaat attttgggtt
2034920DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 349agtaggagct aaaatatttt
2035020DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 350gtctgagtag gagctaaaat
2035120DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 351taacagtctg agtaggagct
2035220DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 352cagagtaaca gtctgagtag
2035320DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 353cacaggttgt gtcaccagag
2035420DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 354agtttcctta gtaaccacag
2035520DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 355tagtttggag atggcagttt
2035620DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 356ggaagatggc atttctagtt
2035720DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 357tacctccaac atcaaggaag
2035820DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 358atctgccaga gcaggtacct
2035920DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 359ccaagcccgg ttgaaatctg
2036020DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 360gtcggtaagt tctgtccaag
2036120DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 361aagcagagaa agccagtcgg
2036220DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 362ttttataact tgatcaagca
2036320DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 363catcaccctc tgtgatttta
2036420DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 364ctcaaggtca cccaccatca
2036520DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 365catctcgttg atatcctcaa
2036620DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 366cttctgcttg atgatcatct
2036720DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 367cataccttct gcttgatgat
2036820DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 368tttctcatac cttctgcttg
2036920DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 369cattttttct cataccttct
2037020DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 370tttatcattt tttctcatac
2037120DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 371caacttttat cattttttct
2037225DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 372ctgtaagaac aaatatccct
tagta 2537325DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 373tgcctgtaag
aacaaatatc cctta 2537425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 374gttgcctgta
agaacaaata tccct 2537525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 375attgttgcct
gtaagaacaa atatc 2537625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 376gcattgttgc
ctgtaagaac aaata 2537725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 377cctgcattgt
tgcctgtaag aacaa 2537825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 378atcctgcatt
gttgcctgta agaac 2537925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 379caaatcctgc
attgttgcct gtaag 2538025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 380tccaaatcct
gcattgttgc ctgta 2538125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 381tgttccaaat
cctgcattgt tgcct 2538225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 382tctgttccaa
atcctgcatt gttgc 2538325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 383aactggggac
gcctctgttc caaat 2538425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 384gcctctgttc
caaatcctgc attgt 2538525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 385cagcggtaat
gagttcttcc aactg 2538625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 386cttccaactg
gggacgcctc tgttc 2538725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 387cttgtttttc
aaattttggg cagcg 2538825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 388ctagcctctt
gattgctggt cttgt 2538925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 389ttttcaaatt
ttgggcagcg gtaat 2539025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 390ttcgatccgt
aatgattgtt ctagc 2539125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 391gattgctggt
cttgtttttc aaatt 2539225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 392cttacttcga
tccgtaatga ttgtt 2539325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 393ttgttctagc
ctcttgattg ctggt 2539425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 394aaaaacttac
ttcgatccgt aatga 2539525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 395tgttaaaaaa
cttacttcga tccgt 2539625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 396atgcttgtta
aaaaacttac ttcga 2539725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 397gtcccatgct
tgttaaaaaa cttac 2539820DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 398agaacaaata
tcccttagta 2039920DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 399gtaagaacaa
atatccctta 2040020DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 400tgcctgtaag
aacaaatatc 2040120DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 401attgttgcct
gtaagaacaa 2040220DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin
402cctgcattgt tgcctgtaag 2040320DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
403caaatcctgc attgttgcct 2040420DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
404gcctctgttc caaatcctgc 2040520DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
405cttccaactg gggacgcctc 2040620DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
406cagcggtaat gagttcttcc 2040720DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
407ttttcaaatt ttgggcagcg 2040820DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
408gattgctggt cttgtttttc 2040920DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
409ttgttctagc ctcttgattg 2041020DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
410ttcgatccgt aatgattgtt 2041120DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
411cttacttcga tccgtaatga 2041220DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
412aaaaacttac ttcgatccgt 2041320DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
413tgttaaaaaa cttacttcga 2041420DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
414atgcttgtta aaaaacttac 2041520DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
415gtcccatgct tgttaaaaaa 2041625DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
416ctagaataaa aggaaaaata aatat 2541725DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 417aactagaata aaaggaaaaa taaat
2541825DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 418ttcaactaga ataaaaggaa
aaata 2541925DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 419ctttcaacta
gaataaaagg aaaaa 2542025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 420attctttcaa
ctagaataaa aggaa 2542125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 421gaattctttc
aactagaata aaagg 2542225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 422tctgaattct
ttcaactaga ataaa 2542325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 423attctgaatt
ctttcaacta gaata 2542425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 424ctgattctga
attctttcaa ctaga 2542525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 425cactgattct
gaattctttc aacta 2542625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 426tcccactgat
tctgaattct ttcaa 2542725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 427catcccactg
attctgaatt ctttc 2542825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 428tacttcatcc
cactgattct gaatt 2542925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 429ctgaaggtgt
tcttgtactt catcc 2543024DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 430cggttctgaa
ggtgttcttg tact 2443125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 431ctgttgcctc
cggttctgaa ggtgt 2543225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 432tttcattcaa
ctgttgcctc cggtt 2543325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 433taacatttca
ttcaactgtt gcctc 2543425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 434ttgtgttgaa
tcctttaaca tttca 2543525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 435tcttccttag
cttccagcca ttgtg 2543625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 436cttagcttcc
agccattgtg ttgaa 2543725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 437gtcctaagac
ctgctcagct tcttc 2543825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 438ctgctcagct
tcttccttag cttcc 2543925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 439ctcaagcttg
gctctggcct gtcct 2544025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 440ggcctgtcct
aagacctgct cagct 2544125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 441tagggaccct
ccttccatga ctcaa 2544225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 442tttggattgc
atctactgta taggg 2544325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 443accctccttc
catgactcaa gcttg 2544425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 444cttggtttct
gtgattttct tttgg 2544525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 445atctactgta
tagggaccct ccttc 2544625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 446ctaaccttgg
tttctgtgat tttct 2544725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 447tttcttttgg
attgcatcta ctgta 2544825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 448tgatactaac
cttggtttct gtgat 2544925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 449atctttgata
ctaaccttgg tttct 2545025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 450aaggtatctt
tgatactaac cttgg 2545125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 451ttaaaaaggt
atctttgata ctaac 2545220DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 452ataaaaggaa
aaataaatat 2045320DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 453gaataaaagg
aaaaataaat 2045420DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 454aactagaata
aaaggaaaaa 2045520DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 455ctttcaacta
gaataaaagg 2045620DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 456gaattctttc
aactagaata 2045720DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 457attctgaatt
ctttcaacta 2045820DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 458tacttcatcc
cactgattct 2045919DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 459ctgaaggtgt
tcttgtact 1946020DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 460ctgttgcctc
cggttctgaa 2046120DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 461taacatttca
ttcaactgtt 2046220DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 462ttgtgttgaa
tcctttaaca 2046320DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 463cttagcttcc
agccattgtg 2046420DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 464ctgctcagct
tcttccttag 2046520DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 465ggcctgtcct
aagacctgct 2046620DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 466ctcaagcttg
gctctggcct 2046720DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 467accctccttc
catgactcaa 2046820DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 468atctactgta
tagggaccct 2046920DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 469tttcttttgg
attgcatcta 2047020DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 470cttggtttct
gtgattttct 2047120DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 471ctaaccttgg
tttctgtgat 2047220DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 472tgatactaac
cttggtttct 2047320DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 473atctttgata
ctaaccttgg 2047420DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 474aaggtatctt
tgatactaac 2047520DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 475ttaaaaaggt
atctttgata 2047624DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 476ctatagattt
ttatgagaaa gaga 2447725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 477aactgctata
gatttttatg agaaa 2547825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 478tggccaactg
ctatagattt ttatg 2547925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 479gtctttggcc
aactgctata gattt 2548025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 480cggaggtctt
tggccaactg ctata 2548125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 481actggcggag
gtctttggcc aactg 2548225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 482tttgtctgcc
actggcggag gtctt 2548325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 483agtcatttgc
cacatctaca tttgt 2548425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 484tttgccacat
ctacatttgt ctgcc 2548525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 485ccggagaagt
ttcagggcca agtca 2548625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 486gtatcatctg
cagaataatc ccgga 2548725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 487taatcccgga
gaagtttcag ggcca 2548825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 488ttatcatgtg
gacttttctg gtatc 2548925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 489agaggcattg
atattctctg ttatc 2549025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 490atgtggactt
ttctggtatc atctg 2549125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 491cttttatgaa
tgcttctcca agagg 2549225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 492atattctctg
ttatcatgtg gactt 2549325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 493catacctttt
atgaatgctt ctcca 2549425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 494ctccaagagg
cattgatatt ctctg 2549525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 495taattcatac
cttttatgaa tgctt 2549625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 496cttttatgaa
tgcttctcca agagg 2549725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 497taatgtaatt
catacctttt atgaa 2549825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 498agaaataatg
taattcatac ctttt 2549925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 499gttttagaaa
taatgtaatt catac 2550019DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 500gatttttatg
agaaagaga 1950120DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 501ctatagattt
ttatgagaaa 2050220DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 502aactgctata
gatttttatg
2050320DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 503tggccaactg ctatagattt
2050420DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 504gtctttggcc aactgctata
2050520DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 505cggaggtctt tggccaactg
2050620DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 506tttgtctgcc actggcggag
2050720DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 507tttgccacat ctacatttgt
2050820DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 508ttcagggcca agtcatttgc
2050920DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 509taatcccgga gaagtttcag
2051020DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 510gtatcatctg cagaataatc
2051120DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 511atgtggactt ttctggtatc
2051220DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 512atattctctg ttatcatgtg
2051320DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 513ctccaagagg cattgatatt
2051420DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 514cttttatgaa tgcttctcca
2051520DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 515catacctttt atgaatgctt
2051620DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 516taattcatac cttttatgaa
2051720DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 517taatgtaatt catacctttt
2051820DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 518agaaataatg taattcatac
2051920DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 519gttttagaaa taatgtaatt
2052025DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 520ctgcaaagga ccaaatgttc
agatg 2552125DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 521tcaccctgca
aaggaccaaa tgttc 2552225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 522ctcactcacc
ctgcaaagga ccaaa 2552325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 523tctcgctcac
tcaccctgca aagga 2552425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 524cagcctctcg
ctcactcacc ctgca 2552525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 525caaagcagcc
tctcgctcac tcacc 2552625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 526tcttccaaag
cagcctctcg ctcac 2552725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 527tctatgagtt
tcttccaaag cagcc 2552825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 528gttgcagtaa
tctatgagtt tcttc 2552925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 529gaactgttgc
agtaatctat gagtt 2553025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 530ttccaggtcc
agggggaact gttgc 2553125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 531gtaagccagg
caagaaactt ttcca 2553225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 532ccaggcaaga
aacttttcca ggtcc 2553325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 533tggcagttgt
ttcagcttct gtaag 2553425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 534ggtagcatcc
tgtaggacat tggca 2553525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 535gacattggca
gttgtttcag cttct 2553625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 536tctaggagcc
tttccttacg ggtag 2553725DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 537cttttactcc
cttggagtct tctag 2553825DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 538gagcctttcc
ttacgggtag catcc 2553925DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 539ttgccattgt
ttcatcagct ctttt 2554025DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 540cttggagtct
tctaggagcc tttcc 2554125DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 541cttacttgcc
attgtttcat cagct 2554225DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 542cagctctttt
actcccttgg agtct 2554325DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 543cctgacttac
ttgccattgt ttcat 2554425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 544aaatgcctga
cttacttgcc attgt 2554525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 545agcggaaatg
cctgacttac ttgcc 2554625DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 546gctaaagcgg
aaatgcctga cttac 2554720DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 547aaggaccaaa
tgttcagatg 2054820DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 548ctgcaaagga
ccaaatgttc 2054920DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 549tcaccctgca
aaggaccaaa 2055020DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 550ctcactcacc
ctgcaaagga 2055120DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 551tctcgctcac
tcaccctgca 2055220DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 552cagcctctcg
ctcactcacc 2055320DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 553caaagcagcc
tctcgctcac 2055420DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 554tctatgagtt
tcttccaaag 2055520DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 555gaactgttgc
agtaatctat 2055620DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 556ttccaggtcc
agggggaact 2055720DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 557ccaggcaaga
aacttttcca 2055820DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 558ttcagcttct
gtaagccagg 2055920DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 559gacattggca
gttgtttcag 2056020DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 560ggtagcatcc
tgtaggacat 2056120DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 561gagcctttcc
ttacgggtag 2056220DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 562cttggagtct
tctaggagcc 2056320DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 563cagctctttt
actcccttgg 2056420DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 564ttgccattgt
ttcatcagct 2056520DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 565cttacttgcc
attgtttcat 2056620DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 566cctgacttac
ttgccattgt 2056720DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 567aaatgcctga
cttacttgcc 2056820DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 568agcggaaatg
cctgacttac 2056920DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 569gctaaagcgg
aaatgcctga 2057010PRTArtificial SequencePeptide Transporter for
Intracellular Delivery of PMO 570Arg Arg Arg Gln Arg Arg Lys Lys
Arg Cys1 5 1057112PRTArtificial SequencePeptide Transporter for
Intracellular Delivery of PMO 571Arg Arg Arg Arg Arg Arg Arg Arg
Arg Phe Phe Cys1 5 1057213PRTArtificial SequencePeptide Transporter
for Intracellular Delivery of PMOMOD_RES3, 6, 9, 12X =
6-aminohexanoic acidMOD_RES13X = beta alanine 572Arg Arg Xaa Arg
Arg Xaa Arg Arg Xaa Arg Arg Xaa Xaa1 5 1057314PRTArtificial
SequencePeptide Transporter for Intracellular Delivery of
PMOMOD_RES2, 5, 8, 11, 13X = 6-aminohexanoic acidMOD_RES14X = beta
alanine 573Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Xaa Xaa1
5 1057414PRTArtificial SequencePeptide Transporter for
Intracellular Delivery of PMOMOD_RES1, 4, 7, 10, 13X =
6-aminohexanoic acidMOD_RES14X = beta alanine 574Xaa Arg Arg Xaa
Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Xaa1 5 1057513PRTArtificial
SequencePeptide Transporter for Intracellular Delivery of
PMOMOD_RES2, 4, 6, 8, 10, 12X = 6-aminohexanoic acidMOD_RES13X =
beta alanine 575Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa
Xaa1 5 1057617PRTArtificial SequencePeptide Transporter for
Intracellular Delivery of PMOMOD_RES2, 4, 6, 8, 10, 12, 14, 16X =
6-aminohexanoic acidMOD_RES17X = beta alanine 576Arg Xaa Arg Xaa
Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa1 5 10
15Xaa57717PRTArtificial SequencePeptide Transporter for
Intracellular Delivery of PMOMOD_RES2, 5, 8, 11, 14, 16X =
6-aminohexanoic acidMOD_RES17X = beta alanine 577Arg Xaa Arg Arg
Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Xaa1 5 10
15Xaa57814PRTArtificial SequencePeptide Transporter for
Intracellular Delivery of PMOMOD_RES2, 8, 13X = 6-aminohexanoic
acidMOD_RES5, 11, 14X = beta alanine 578Arg Xaa Arg Arg Xaa Arg Arg
Xaa Arg Arg Xaa Arg Xaa Xaa1 5 105797PRTArtificial SequencePeptide
Transporter for Intracellular Delivery of PMO 579Ala Ser Ser Leu
Asn Ile Ala1 55809PRTArtificial SequenceCell Penetrating Peptide /
Homing Peptide / PMO ConjugatesMOD_RES8X = a neutral amino acid
C(=O) (CHR)n NH-, where n is 2 to 7 and each R is independently H
or methylMOD_RES9X = Beta alanine 580Ala Ser Ser Leu Asn Ile Ala
Xaa Xaa1 558122PRTArtificial SequenceCell Penetrating Peptide /
Homing Peptide / PMO ConjugatesMOD_RES2, 8, 13, 22X = a neutral
amino acid C(=O) (CHR)n NH-, where n is 2 to 7 and each R is
independently H or methylMOD_RES5, 11, 14X = beta alanine 581Arg
Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Xaa Xaa Ala Ser1 5 10
15Ser Leu Asn Ile Ala Xaa 2058221PRTArtificial SequenceCell
Penetrating Peptide / Homing Peptide / PMO ConjugatesMOD_RES8, 10,
16X = a neutral amino acid C(=O) (CHR)n NH-, where n is 2 to 7 and
each R is independently H or methylMOD_RES13, 19, 21Xaa = beta
alanine 582Ala Ser Ser Leu Asn Ile Ala Xaa Arg Xaa Arg Arg Xaa Arg
Arg Xaa1 5 10 15Arg Arg Xaa Arg Xaa 2058314PRTArtificial
SequenceCell Penetrating Peptide / Homing Peptide / PMO
ConjugatesMOD_RES2, 8, 13X = a neutral amino acid C(=O) (CHR)n NH-,
where n is 2 to 7 and each R is independently H or methylMOD_RES5,
11, 14X = beta alanine 583Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg
Xaa Arg Xaa Xaa1 5 1058429DNAArtificial SequenceAntisense sequence
to target splice site of proprocessed human dystrophin
584ccactcagag ctcagatctt ctaacttcc 2958525DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 585gggatccagt atacttacag gctcc
2558627DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 586cttccactca gagctcagat
cttctaa 2758730DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 587acatcaagga
agatggcatt tctagtttgg 3058830DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
588ctccaacatc aaggaagatg gcatttctag 3058924DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 589ttctgtccaa gcccggttga aatc 2459021DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 590cacccaccat caccctcygt g 2159122DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 591atcatctcgt tgatatcctc aa 2259225DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 592acatcaagga agatggcatt tctag
2559326DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 593accagagtaa cagtctgagt
aggagc 2659420DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 594tcaaggaaga
tggcatttct 2059523DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 595cctctgtgat
tttataactt gat
2359625DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 596atcatttttt ctcatacctt
ctgct 2559723DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 597ctcatacctt
ctgcttgatg atc 2359817DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 598tggcatttct
agtttgg 1759923DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 599ccagagcagg
tacctccaac atc 2360024DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 600tgttcagctt
ctgttagcca ctga 2460120DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 601tttgtgtctt
tctgagaaac 2060219DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 602cgccgccatt
tctcaacag 1960320DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 603atctgtcaaa
tcgcctgcag 2060420DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 604tgtttttgag
gattgctgaa 2060519DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 605gctgaattat
ttcttcccc 1960617DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 606gcccaatgcc atcctgg
1760726DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 607ccaatgccat cctggagttc
ctgtaa 2660831DNAArtificial SequenceAntisense sequence to target
splice site of proprocessed human dystrophin 608cattcaactg
ttgcctccgg ttctgaaggt g 3160925DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
609ctgaaggtgt tcttgtactt catcc 2561018DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 610ctgttgcctc cggttctg 1861122DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 611attctttcaa ctagaataaa ag 2261230DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 612gccatcctgg agttcctgta agataccaaa
3061330DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 613ccaatgccat cctggagttc
ctgtaagata 3061430DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 614gccgctgccc
aatgccatcc tggagttcct 3061530DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
615gtttgccgct gcccaatgcc atcctggagt 3061630DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 616caacagtttg ccgctgccca atgccatcct
3061730DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 617ctgacaacag tttgccgctg
cccaatgcca 3061830DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 618tgttctgaca
acagtttgcc gctgcccaat 3061930DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
619caatgttctg acaacagttt gccgctgccc 3062030DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 620cattcaatgt tctgacaaca gtttgccgct
3062130DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 621tatttcttcc ccagttgcat
tcaatgttct 3062230DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 622gctgaattat
ttcttcccca gttgcattca 3062330DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
623ggattgctga attatttctt ccccagttgc 3062430DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 624tttgaggatt gctgaattat ttcttcccca
3062530DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 625gtacttcatc ccactgattc
tgaattcttt 3062630DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 626tcttgtactt
catcccactg attctgaatt 3062730DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
627tgttcttgta cttcatccca ctgattctga 3062830DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 628cggttctgaa ggtgttcttg tacttcatcc
3062930DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 629ctccggttct gaaggtgttc
ttgtacttca 3063030DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 630tgcctccggt
tctgaaggtg ttcttgtact 3063130DNAArtificial SequenceAntisense
sequence to target splice site of proprocessed human dystrophin
631tgttgcctcc ggttctgaag gtgttcttgt 3063230DNAArtificial
SequenceAntisense sequence to target splice site of proprocessed
human dystrophin 632aactgttgcc tccggttctg aaggtgttct
3063330DNAArtificial SequenceAntisense sequence to target splice
site of proprocessed human dystrophin 633ttcaactgtt gcctccggtt
ctgaaggtgt 3063425DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 634taaagctctg
gaaacctgaa aggaa 2563525DNAArtificial SequenceAntisense sequence to
target splice site of proprocessed human dystrophin 635ttcagcttct
gtaagccagg caaga 2563625DNAArtificial SequenceAntisense oligomer
636ggccaaacct cggcttacct gaaat 2563712PRTArtificial
Sequencearginine-rich peptide transportVARIANT1, 3, 4, 7, 9, 10Xaa
= K, R or arginine analogMOD_RES2, 8Xaa = a neutral amino acid
C(=O) (CHR)n NH-, where n is 2 to 7 and each R is independently H
or methylVARIANT5, 6, 11, 12Xaa = an alpha-amino acid having a
neutral aralkyl side chain 637Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa1 5 1063812PRTArtificial Sequencearginine-rich peptide
transportMOD_RES2, 6, 8, 12Xaa = a neutral amino acid C(=O) (CHR)n
NH-, where n is 2 to 7 and each R is independently H or methyl
638Arg Xaa Arg Arg Arg Xaa Arg Xaa Arg Arg Arg Xaa1 5
106399PRTArtificial Sequencearginine-rich peptide
transportMOD_RES3, 5, 9Xaa = a neutral amino acid C(=O) (CHR)n NH-,
where n is 2 to 7 and each R is independently H or methyl 639Arg
Arg Xaa Arg Xaa Arg Arg Arg Xaa1 564024DNAArtificial SequencePCR
Primer 640ccagagcttt acctgagaaa caag 2464121DNAArtificial
SequencePCR Primer 641ccagccactc agccagtgaa g 2164224DNAArtificial
SequencePCR Primer 642cgatccgtaa tgattgttct agcc
2464324DNAArtificial SequencePCR Primer 643catttcattc aactgttgcc
tccg 2464421DNAArtificial SequencePCR Primer 644caatgctcct
gacctctgtg c 2164522DNAArtificial SequencePCR Primer 645gtctacaaca
aagctcaggt cg 2264625DNAArtificial SequencePCR Primer 646gcaatgttat
ctgcttcctc caacc 2564722DNAArtificial SequencePCR Primer
647gctcttttcc aggttcaagt gg 2264823DNAArtificial SequencePCR Primer
648cttggacaga acttaccgac tgg 2364921DNAArtificial SequencePCR
Primer 649gcaggatttg gaacagaggc g 2165023DNAArtificial SequencePCR
Primer 650catctacatt tgtctgccac tgg 2365123DNAArtificial
SequencePCR Primer 651gtttcttcca aagcagcctc tcg 23
* * * * *