U.S. patent application number 14/026699 was filed with the patent office on 2014-03-20 for identification of small molecules that facilitate therapeutic exon skipping.
This patent application is currently assigned to The Regents of the University of california. The applicant listed for this patent is Carrie Miceli, Miriana Moran, Stanley F. Nelson. Invention is credited to Carrie Miceli, Miriana Moran, Stanley F. Nelson.
Application Number | 20140080896 14/026699 |
Document ID | / |
Family ID | 50275100 |
Filed Date | 2014-03-20 |
United States Patent
Application |
20140080896 |
Kind Code |
A1 |
Nelson; Stanley F. ; et
al. |
March 20, 2014 |
IDENTIFICATION OF SMALL MOLECULES THAT FACILITATE THERAPEUTIC EXON
SKIPPING
Abstract
This invention relates, e.g., to a method for enhancing exon
skipping in a pre-mRNA of interest, comprising contacting the
pre-mRNA with an effective amount of a small molecule selected from
the compounds shown in Table 1, or a pharmaceutically acceptable
salt, hydrate, solvate, or isomer thereof, and, optionally, with an
antisense oligonucleotide that is specific for a splicing sequence
in the pre-mRNA Methods for treating Duchenne muscular dystrophy
(DMD) are disclosed.
Inventors: |
Nelson; Stanley F.; (Los
Angeles, CA) ; Miceli; Carrie; (Los Angeles, CA)
; Moran; Miriana; (Los Angeles, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Nelson; Stanley F.
Miceli; Carrie
Moran; Miriana |
Los Angeles
Los Angeles
Los Angeles |
CA
CA
CA |
US
US
US |
|
|
Assignee: |
The Regents of the University of
california
Oakland
CA
|
Family ID: |
50275100 |
Appl. No.: |
14/026699 |
Filed: |
September 13, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2012/053157 |
Aug 30, 2012 |
|
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14026699 |
|
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61529041 |
Aug 30, 2011 |
|
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61700661 |
Sep 13, 2012 |
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Current U.S.
Class: |
514/44A ;
435/375; 435/6.11; 435/6.12; 435/6.13; 506/10; 506/9; 514/392 |
Current CPC
Class: |
A61K 31/122 20130101;
A61K 31/475 20130101; A61K 31/4178 20130101; A61K 31/5377 20130101;
A61K 31/554 20130101; A61K 31/166 20130101; A61K 31/4025 20130101;
A61K 31/7105 20130101; A61K 31/609 20130101; A61K 31/713 20130101;
C12Q 1/6806 20130101; A61K 31/609 20130101; A61K 31/713 20130101;
C12N 2310/3233 20130101; A61K 31/7064 20130101; A61K 31/7088
20130101; C12N 15/113 20130101; A61K 31/5415 20130101; C12N
2310/113 20130101; A61K 31/277 20130101; C12Q 1/6897 20130101; A61K
31/454 20130101; A61K 31/5415 20130101; A61K 31/549 20130101; A61K
31/404 20130101; C12N 2320/33 20130101; A61K 31/475 20130101; A61K
31/7105 20130101; A61K 31/138 20130101; A61K 31/407 20130101; A61K
45/06 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; C12N 2320/31 20130101; A61K 31/4184
20130101; A61K 31/4178 20130101 |
Class at
Publication: |
514/44.A ;
435/375; 514/392; 506/10; 435/6.13; 435/6.12; 506/9; 435/6.11 |
International
Class: |
A61K 31/4178 20060101
A61K031/4178; C12Q 1/68 20060101 C12Q001/68; A61K 31/7105 20060101
A61K031/7105 |
Goverment Interests
[0002] This invention was made with Government support of Grant No.
W81XWH-05-1-0616, awarded by the Department of Defense, and
5RC1AR058333, awarded by the National Institutes of Health. The
Government has certain rights in this invention.
Claims
1. A method for enhancing exon skipping in an mRNA of interest,
comprising contacting the mRNA with an effective amount of a small
molecule compound selected from one or more of furaltadone
hydrochloride, 5-iodotubericidin, bendroflumethiazide,
cyclopiazonic acid, GW 5074, indirubin, rescinnamin, U-0126,
acetopromazine maleate salt, Ro 31-8220, dantrolene,
dichlorobenzamil, ellipticine, fenbendazole, GF 109203.times.,
halofantrine, niclosamide, pimozide, reserpine, syringospine, a
RyCal, piperacetazine, fluphenazine dihydrochloride,
trifluorperazine dihydrochloride, yohimbinic acid, or menadione, or
a pharmaceutically acceptable salt, hydrate, solvate, or isomer
thereof.
2. The method of claim 1, wherein an antisense oligonucleotide (AO)
which is specific for a splicing sequence in the mRNA is
administered in conjunction with the compound.
3. The method of claim 1, wherein no AO is introduced in
conjunction with the compound.
4. The method of claim 1, wherein the mRNA is from the muscle
dystrophin (DMD) gene, which encodes a muscle dystrophin
protein.
5. The method of claim 4, wherein the exon which is skipped is exon
23, 44, 45, 50, 51, 52 and/or 53 of the DMD gene.
6. The method of claim 1, which is carried out in vitro.
7. The method of claim 1, which is carried out in a subject that
has Duchenne Muscular Dystrophy (DMD), is an animal model of DMD,
or is another animal in which the exon skipping can be assayed.
8. The method of claim 7, wherein the subject is human.
9. The method of claim 1, wherein the compound is dantrolene or an
active variant thereof.
10. The method of claim 9, wherein the compound is dantrolene.
11. A method for treating a subject that has Duchenne Muscular
Dystrophy (DMD), or is a non-human model of DMD, comprising
administering to the subject an effective amount of a small
molecule compound selected from one or more of furaltadone
hydrochloride, 5-iodotubericidin, bendroflumethiazide,
cyclopiazonic acid, GW 5074, indirubin, rescinnamin, U-0126,
acetopromazine maleate salt, Ro 31-8220, dantrolene,
dichlorobenzamil, ellipticine, fenbendazole, GF 109203.times.,
halofantrine, niclosamide, pimozide, reserpine, syringospine,
Ryanodine, RyCal S107, piperacetazine, fluphenazine
dihydrochloride, trifluorperazine dihydrochloride, yohimbinic acid,
or menadione, or a pharmaceutically acceptable salt, hydrate,
solvate, or isomer thereof, in conjunction with an AO which is
specific for a splicing sequence of exon 23, 45, 44, 50, 51, 52
and/or 53 of the DMD gene.
12. A method for identifying a small molecule compound that
enhances exon skipping in an mRNA of interest, comprising testing
small molecule candidates for their ability to enhance exon
skipping in the mRNA, and selecting compounds which exhibit greater
enhancement of exon skipping than one of the molecules in Table
1.
13. The method of claim 12, wherein the small molecule candidates
tested in conjunction with an AO specific for a splicing sequence
of the exon that is to be skipped.
14. The method of claim 12, wherein the small molecule candidate is
a variant of furaltadone hydrochloride, 5-iodotubericidin,
bendroflumethiazide, cyclopiazonic acid, GW 5074, indirubin,
rescinnamin, U-0126, acetopromazine maleate salt, Ro 31-8220,
dantrolene, dichlorobenzamil, ellipticine, fenbendazole, GF
109203.times., halofantrine, niclosamide, pimozide, reserpine,
syringospine, Ryanodine, RyCal S107, piperacetazine, fluphenazine
dihydrochloride, trifluorperazine dihydrochloride, yohimbinic acid,
or menadione, or a pharmaceutically acceptable salt, hydrate,
solvate, or isomer thereof.
15. The method of claim 13, wherein the small molecule candidate is
a variant of furaltadone hydrochloride, 5-iodotubericidin,
bendroflumethiazide, cyclopiazonic acid, GW 5074, indirubin,
rescinnamin, U-0126, acetopromazine maleate salt, Ro 31-8220,
dantrolene, dichlorobenzamil, ellipticine, fenbendazole, GF
109203.times., halofantrine, niclosamide, pimozide, reserpine,
syringospine, Ryanodine, RyCal S107, piperacetazine, fluphenazine
dihydrochloride, trifluorperazine dihydrochloride, yohimbinic acid,
or menadione, or a pharmaceutically acceptable salt, hydrate,
solvate, or isomer thereof.
16. A combination for enhancing exon skipping in an mRNA of
interest, comprising a small molecule compound selected from one or
more of furaltadone hydrochloride, 5-iodotubericidin,
bendroflumethiazide, cyclopiazonic acid, GW 5074, indirubin,
rescinnamin, U-0126, acetopromazine maleate salt, Ro 31-8220,
dantrolene, dichlorobenzamil, ellipticine, fenbendazole, GF
109203.times., halofantrine, niclosamide, pimozide, reserpine,
syringospine, Ryanodine, RyCal S107, piperacetazine, fluphenazine
dihydrochloride, trifluorperazine dihydrochloride, yohimbinic acid,
or menadione, or a a pharmaceutically acceptable salt, hydrate,
solvate, or isomer thereof, and an AO that is specific for an exon
that is to be skipped, and, optionally, a pharmaceutically
acceptable carrier.
17. A kit for enhancing exon skipping in an mRNA of interest,
comprising a compound from Table 1, or a a pharmaceutically
acceptable salt, hydrate, solvate, or isomer thereof, and an AO
that is specific for an exon splicing sequence in the mRNA of
interest, wherein the compound and the AO are optionally packaged
in containers, separately or together.
18. A kit for enhancing exon skipping in a muscle dystrophin mRNA
in a subject that has Duchenne Muscular Dystrophy (DMD), or is an
animal model of DMD, comprising a dosage form of a compound of
Table 1, or a pharmaceutically acceptable salt, hydrate, solvate,
or isomer thereof, and of an AO that is specific for the exon which
is to be skipped, and a pharmaceutically acceptable carrier,
wherein the compound and the AO are optionally packaged in
containers, separately or together, wherein the compound of Table 1
is one or more of Furaltadone hydrochloride, 5-iodotubericidin,
bendroflumethiazide, cyclopiazonic acid, GW 5074, indirubin,
rescinnamin, U-0126, acetopromazine maleate salt, Ro 31-8220,
dantrolene, dichlorobenzamil, ellipticine, fenbendazole, GF
109203.times., halofantrine, niclosamide, pimozide, reserpine,
syringospine, Ryanodine, RyCal S107, piperacetazine, fluphenazine
dihydrochloride, trifluorperazine dihydrochloride, yohimbinic acid,
or menadione, or a pharmaceutically acceptable salt, hydrate,
solvate, or isomer thereof.
19. The method of claim 1, wherein the RyCal is Ryanodine or RyCal
S 107.
Description
[0001] This application claims the benefit of the filing date of
U.S. Provisional Application No. 61/700,661, filed Sep. 13, 2012,
and is a CIP of PCT Application No. PCT/US2012/053157 filed Aug.
30, 2012, which claims the benefit of the filing date of U.S.
Provisional Application No. 61/529,041, filed Aug. 30, 2011, all of
which are incorporated by reference herein in their entireties.
SEQUENCE LISTING
Sequence Listing
[0003] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Sep. 13, 2013, is named 58086-354490_SL.txt and is 37,016 bytes
in size.
BACKGROUND INFORMATION
[0004] Duchenne muscular dystrophy (DMD) is a lethal X-linked
recessive disease characterized by progressive muscle weakness over
a patient's lifetime [1]. It is the most common childhood form of
muscular dystrophy affecting about 1 out of 3500 live male births
worldwide [2]. DMD is primarily caused by out of frame multi-exon
deletions in the DMD gene that ablate dystrophin protein production
[3]. Dystrophin is an essential component of the dystrophin
glycoprotein complex (DGC), which functions in linking the actin
cytoskeleton to extracellular matrix to provide sarcolemmal
stability in the context of muscle contraction. The DGC also plays
a role recruiting and organizing signal transducers at the
sarcolemmal membrane. Both of these activities are required for
muscle cell health, and thus the absence of dystrophin leads to
progressive loss of muscle function. Dystrophin binds to actin via
N-terminal sequences and to b dystroglycan within the DGC via
carboxyl terminal domains, whereas the central portion of the
protein consists of a rod domain containing multiple spectrin
repeats. Deletions within the central rod domain that preserve the
reading frame can produce an internally deleted dystrophin protein
that retains some functionality and localizes to the membrane
within the DGC [4]. Typically, the more mild allelic disorder,
Becker muscular dystrophy, results from DMD mutations in the rod
domain which remain in-frame 3' of the deletion and produce a
functional dystrophin protein [5]. There are no curative therapies
for DMD, and the only demonstrated pharmacological treatment is
corticosteroids, which may prolong ambulation for up to 3 years,
but with substantial side effects [6]. An emerging therapy, exon
skipping, targets individual exons with antisense oligos (AOs) for
exclusion from mRNA based on an individual's known genomic DNA
mutation with the goal to change out-of-frame mutations into
in-frame DMD deletions that restore the reading frame in dystrophin
mRNA and allow translation of dystrophin protein. FIG. 14 is a
schematic illustration of antisense-mediated therapeutic exon
skipping. AOs have been successfully demonstrated to promote DMD
exon skipping and restore dystrophin protein expression in mice,
dogs and humans in recent clinical trials [7-12]. High dose,
chronic administration of an exon 23 directed AO in the mdx mouse
demonstrated substantial disease reduction highlighting the
tremendous promise of this therapy for DMD in humans [13]. A series
of AOs are under development for human use and about half of all
DMD patients could be treated with the targeting of 6 different
exons (51, 45, 53, 44, 52, 50) in the most frequently deleted
portion of the gene between exons 45-53 [14]. For instance, DMD
exon 51 skipping will be appropriate for about 13% of all DMD
patients, and is the first in clinical trials with two different
backbone chemistries, 2'-O-methyl phosphorothioate and morpholino
phosphorodiamidate (PMO), both of which have shown promising
results [8-10]. These studies are paving the way in personalized
genetic medicine.
[0005] Recent phase 1-2a clinical trial results utilizing systemic
2'-O-methyl modified AO directed against DMD exon 51 (Pro051)
rescued dystrophin protein at levels ranging from 1.8-15.6% of
normal [8]. A modest improvement in the 6 minute walk test at 48
weeks was observed with weekly subcutaneous dosing of 6 mg/kg in a
non-placebo controlled extension trail, but it remains to be
determined if the levels of dystophin produced are sufficient to
impart substantial functional utility or longterm protection of
muscle [15]. Morpholino AO directed against exon 51 (AVI-4658)
resulted in dystrophin rescue with up to 55% of myofibers induced
to be dystrophin positive after 12 weeks of therapy in humans.
However, the total amount of dystrophin induced was generally low,
at 0-27% of normal [16]. Further, DMD exon skipping efficacy and
dystrophin expression varies across patients, and muscle types.
[0006] There is a need for an improvement in exon skipping therapy
that would result in more total dystrophin expression and broader
effect in multiple muscle groups. For example, synergistic
treatments that would permit equal efficacy with reduced AO dose,
accompanied by lower toxicity, could substantially impact the
practicality of the chronic administration of expensive to produce
oligonucleotides [17].
DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 shows that high throughput screening identifies
dantrolene as a modulator of antisense oligo (AO) mediated human
DMD exon 50 skipping. Small molecule libraries were screened for
exon skipping promoting activity in C2C12 myoblasts expressing a
human DMD exon 50 GFP based reporter [18]. Using an automated and
quantifiable system the BioMol chemical library (n=503) was
screened at 10 uM concentration both in the presence and absence of
2'-O-methyl 27-mer AO [5' AACUUCCUCUUUAACAGAAAAGCAUAC 3', (SEQ ID
NO:1)] targeting the splice donor site of human DMD exon 50. In the
reporter cells, successful skipping of DMD exon 50 creates in-frame
GFP expression. Number of cells that were fluorescing was
quantified using a high content cell imager in 384 well plate
format. Fluorescence was normalized by subtracting the average
fluorescence value of the carrier (DMSO) controls. Fluorescence
readouts are plotted for the BioMol library screen both with (+AO)
and without (-AO) from Source Plate 1 (containing the Orphan
Ligand, Ion Channel, Enzyme Inhibitor, and Endocannabanoid
libraries, n=300). Each point for the DMSO controls, all compounds,
and the Top 5% from Source Plate 1 (n=15) represents the average
normalized fluorescence of 6 replicates in the -AO screen and 3
replicates in the +AO screen. In the with AO screen, dantrolene had
three fluorescence measurements that were averaged, and this
average compared to the average fluorescence of the other compounds
in the top 15 (top 5%) of the screen. Dantrolene was identified to
have enhanced exon skipping activity in the screen +AO, while its
activity was indistinguishable from the DMSO controls in the -AO
screen. Individual points for dantrolene are plotted with the bold
horizontal line indicating the median. The short horizontal lines
interspersed among the data points indicate 1, 2, and 3 standard
deviations away from the DMSO treatment mean fluorescence.
[0008] FIG. 2 shows that Dantrolene synergizes with AO to increase
DMD exon skipping in mouse and human DMD mutant cells. (A)
Differentiated primary mouse myotubes were transfected either with
100 nM 2'-O-methyl M23D, which targets exon 23 splice donor region
or mock transfected for 24 hours after which the transfection
reagent was removed, and myotubes were treated with different
concentrations of Dantrolene for 48 hours. Nested RT-PCR was
performed on cDNA between exons 20-26, as previously described [6].
The 901 bp band is the full-length mRNA product, the 688 bp mRNA
product is a single skip of exon 23, and the 550 bp mRNA product is
a double skip of exons 22 and 23. (B) Mouse Dmd exon 23-skipped
transcript levels were quantified using a taqman assay with
primer-probe sets spanning the splice junction created by an exon
23 skip (22-24 join) relative to primers amplifying the splice
junction of exons 22 and 23 (representing the full length mRNA), as
previously described [25]. Data from each primer-probe set was
normalized to the ribosomal gene 36B4, and the ratios are displayed
as the fold change of the skip/full length mRNA levels relative to
the mock treated controls. Error bars represent standard deviation
of qRT-PCR triplicates. (C) Patient derived fibroblasts with exon
45-50 deletion (confirmed by microarray, see FIG. 6) were
immortalized with lentiviral hTERT and transduced with inducible
lentiviral MyoD [26]. Cells were grown to confluence, induced for
MyoD activity, and fused for 10 days in low serum differentiation
media. On Day 7, h51AON [5' UCAAGGAAGAUGGCAUUUCU 3'] (SEQ ID NO:2)
(same sequence as Pro051) within human exon 51 was added at
concentrations indicated. Twenty-four hours later Dantrolene or
vehicle was added. Cells were harvested 2 days later and total RNA
isolated. RT-PCR amplified a fragment of cDNA from exons 42-53
which was followed by a nested PCR to generate a fragment spanning
exons 43-52. The 540 bp product is the full length DMD mRNA species
and the 307 bp product indicates the exon 51 skip isoform.
Quantitation was performed using the Agilent Bioanalyzer and
represented as the skip/full-length mRNA ratio.
[0009] FIG. 3 shows that Dantrolene enhances intramuscularly
injected AO DMD mRNA skipping and dystrophin protein expression in
mdx mice. One dose of either bug or 2 ug of morpholino M23D in 25
ul PBS was injected into the tibialis anterior (TA) of 6 week old
mdx mice on day 1 (n=3 per group). Dantrolene was administered by
intraperitoneal injection at either 10 or 20 mg/kg/day for 9 days
in a volume of 200 ul every 12 hours. Dantrolene was solubilized in
20% DMSO in normal saline. TA muscle was harvested on Day 11 and
immediately frozen in Optimal Cutting Temperature compound (O.C.T.)
embedding medium. Serial sections along the TA with intervals 800
microns apart were processed to perform assessments within the TA
region with maximal AO delivery. There were 6-7 intervals per TA,
and the middle 4 intervals demonstrated exon skipping in treated
TA. (A) For Western blot analysis, half of each of the 4 middle
intervals were pooled. Dystrophin protein was detected using
MANDYS8 (exons 31 and 32) antibody. Control C57 was loaded at 5
ug/well, and 50 ug/well was loaded for other mdx samples. (B)
Western blot was quantified by densitometry and plotted as
arbitrary densitometry units normalized to vinculin loading
control. (C) Immunostaining for dystrophin localization was
performed using MANDYS8 of 10 um sections from representative
middle sections of the TA, and is consistent with sarcolemmal
staining (D) Data from whole muscle cross sections from C were
quantified as total fluorescence, without inclusion of edges with
artifactual staining, normalized to surface area scanned. Data are
plotted as percent of C57 as control (set at 100). Images were
analyzed using Ariol SL-50 (Applied Imaging Corp., San Jose,
Calif.). Error bars in B and D represent the standard deviation of
3 mice per group.
[0010] FIG. 4 shows that Dantrolene enhances intravascularly
delivered exon 23 AO to promote exon 23 skipping of Dmd mRNA in mdx
mice and rescues dystrophin protein and other DGC components. A
systemic dose of 100 mg/kg or sub-optimal 10 mg/kg of morpholino
M23D (+07-18) was administered by tail vein injection on Day 1.
From Day 2-7 Dantrolene was administered intraperitoneally at a
dose of 10 mg/kg/day in two divided doses. On Day 8 multiple
muscles were harvested for analysis. (A) DMD exon 23 skipping was
assessed as in FIG. 3. Skip/full-length mRNA ratio data were
combined for all mice and for all initial muscle groups tested
(quadricep, gastrocnemius, tibialis anterior and diaphragm). (B)
Dystrophin protein was assessed by Western blot (Mandys8)
quantitative densitometry for all muscle groups and individual mice
(quadricep, gastrocnemius, tibialis anterior and diaphragm). (C)
Quantitative immunohistochemistry is plotted as arbitrary units
normalized to surface area for each section for all muscle groups
and mice using one whole muscle cross section per animal per
muscle. (D) Representative Western blot from the gastrocnemius
demonstrating appropriate size of dystrophin. C57 was loaded at one
tenth the protein concentration of the other lanes. (E)
Immunostaining of serial sections of treated mdx quadricep detects
sarcolemmal localization of dystrophin (MANDYS8), alpha-sarcoglycan
(NCL-a-sarc) and beta-dystroglycan (NCL-b-DG). Additional
immunostain photomicrographs are shown in supplemental FIG. 8 of
individual muscle types. Error bars in A-C represent the standard
deviation among mice and muscles in each group (n=3 animals or n=12
total observations in saline+dantrolene and 100 mg/kg AO+DMSO; n=4
animals or n=16 observations in 10 mg/kg AO with Dantrolene or
DMSO).
[0011] FIG. 5 shows the identification of positive compounds after
12 or 16 point titrations on the DMD exon 50 reporter cell line.
Secondary screening was performed on 8/15 compounds to evaluate
synergy with AON6 to enhance human exon 50 skipping. 12 or 16 point
titrations of compounds were added to the Ex50-GFP and C2C12
myoblasts either with or without AON6. To be considered positive
compounds must exhibit a dose response and >10% increased
fluorescence in the Ex50-GFP reporter line with AO as compared to
the without AO condition.
[0012] FIG. 6 shows that a custom CGH array confirms deletion
breakpoints in GM05017. A custom CGH array was designed with 14022
probes tiling the DMD gene with a resolution of approximately 1
probe every 160 bp. Probe number one maps to genomic position
chrX:31047266 and probe 14022 maps to genomic position
chrX:33267570. Genomic DNA from the GM05017 was labeled with Cy3,
and non-mutated human genomic DNA was labeled with Cy5 and were
co-hybridized to the custom designed array. Arrays were scanned
with the DNA Microarray Scanner with Surescan High-Resolution
Technology (Agilent) and data was extracted with Feature Extraction
Software version 10.5.1.1. The values were extracted from the
software and analyzed in R. The log ratio of the Cy3/Cy5 intensity
for all probes is given in Panel B. Probes 4409 to 5615
demonstrated lower Cy3 signal and are consistent with a deletion
from intron 44 to intron 50, which includes exons 45-50 of DMD.
[0013] FIG. 7 shows that reprogrammed fibroblasts temporally
express muscle markers at the RNA and protein level during the
fusion process. Reprogrammed GM05017 patient fibroblasts were
seeded onto laminin coated dishes in growth media (DMEM with 15%
FBS, 1% nonessential amino acids, 1% pen/strep). The following day
MyoD was induced with 5 uM tamoxifen in growth media for 24 hours.
On day 3 the growth media was removed and replaced with fusion
media (2% horse serum, 2% insulin-transferrin-selenium (Sigma), 1:1
serum free DMEM to Ham's F-10) that contained 1 uM tamoxifen. The
cells were incubated in fusion media with 1 uM tamoxifen remained
on the cells until harvesting at day 10. (A) During the fusion
process cells temporally expressed indicated genes as detected by
RT-PCR. (B) Myosin heavy chain (MHC) is expressed in multinucleated
elongated cells consistent with differentiation into myotubes
(lower panel). Cells remaining in growth media without tamoxifen
failed to express myosin heavy chain (upper panel).
[0014] FIG. 8 shows that Dantrolene enhances Dmd exon 23 skipping
with intramuscular PMOE23. (A) Exon 23 skipping was determined
using a nested RT-PCR of RNA isolated from the tibialis anterior
muscle, between Dmd exons 20-26. The 901 bp product is the
unskipped mRNA species whereas the 688 bp product represents the
exclusion of exon 23, and the 550 bp product is skipping both exons
22 and 23 [Mann et al 2001]. (B) Quantitative taqman assay to
assess Dmd exon 23 skipping. The ratio of mRNA species (exon 23
skipped vs. full length) from total RNA, derived from two central
intervals spanning 400 microns within the tibialis anterior muscle,
was compared for 3 mice per treatment group. The skip to
full-length mRNA ratios are represented as their fold change with
respect to mdx untreated controls, with error bars indicating the
standard deviation of measurements from 3 mice per group.
[0015] FIG. 9 shows that Dantrolene rescues dystrophin protein in
the tibialis anterior muscle after intramuscular injection of
PMOE23. (A) Western blot showing dystrophin expression (MANDYS8) in
isolated muscle samples from the tibialis anterior muscle. Vinculin
is shown as a relative loading control. Protein isolates from C57
mice were loaded at one-tenth the levels of samples from mdx mice.
(B) Quantitation of dystrophin expression in the tibialis anterior
as determined by densitometry analysis of western blot bands. (C)
Quantitative fluorescence of dystrophin expression from muscle
cross-sections as described in FIG. 3d.
[0016] FIG. 10 shows that Dantrolene enhances systemic PMOE23
directed Dmd exon 23 skipping and dystrophin protein rescue in the
mdx mouse. (A) Quantitative qRT-PCR for the detection of Dmd exon
23 skipping represented as the Dmd exon 23 skip/full-length mRNA
ratio. The increase in the proportion of exon 23 skipped mRNA
species in the 10 mg/kg AO+Dantrolene as compared to the carrier
control is given followed by the p value for each skeletal muscle.
(B) Quantitative fluorescence as described in FIG. 3d for each
skeletal muscle. The increase in the proportion of dystrophin
protein in the 10 mg/kg AO+Dantrolene as compared to the carrier
control is given followed by the p value for each skeletal muscle.
(C) Densitometry analysis of Dystrophin protein detected for each
treatment group from the quadriceps, gastrocnemius, tibialis
anterior, and diaphragm. Dystrophin signal was normalized to the
vinculin loading control before comparisons across treatment
groups.
[0017] FIG. 11 shows individual Western blots for dystrophin in the
quadriceps, tibialis anterior, and diaphragm. Dystrophin expression
in the muscles of treated mice is shown as detected using the
Mandys8 antibody. 40 ug of protein was loaded for each muscle for
the mdx mice and 4 ug of protein was loaded for the C57 control.
The percentage above each lane depicts the relative dystrophin
expression when comparing to a wildtype (C57) control, as
determined by optical density measurements of the indicated
bands.
[0018] FIG. 12 shows individual micrographs from all skeletal
muscles depicting dystrophin protein and DNA. Whole muscle cross
sections from mice are shown with cell nuclei labeled using DAPI
(light stain) and dystrophin expression detected using the Mandys8
antibody (more intense (whiter) stain).
[0019] FIG. 13 shows that Dantrolene rescues full-length dystrophin
protein in combination with AO that is correctly localized to the
sarcolemma. Serial sections of the quadriceps muscle from 1 mouse
per treatment group were stained for dystrophin with antibodies
corresponding to 3 different protein domains; the rod domain, N
terminus, and C terminus Dystrophin is detected and correctly
localized to the sarcolemma with all 3 antibodies.
[0020] FIG. 14 illustrates the general concept of antisense
mediated therapeutic exon skipping for DMD. Shown is Exon 51
skipping (with the antisense oligo PRO051).
[0021] FIG. 15 shows a schematic of enhancing PMO exon skipping in
the mdx mouse protocol. At day 11, the effect of the small molecule
compound being tested is assessed at the RNA, protein and
subcellular levels.
[0022] FIG. 16 shows that structurally similar phenothiazines
enhance AO directed DMD exon 51 skipping. Patient fibroblasts with
a DMD exon 45-50 deletion were immortalized and transduced with a
lenti-viral vector expressing inducible MyoD to create iDRMs
(inducibly directly reprogrammable myotubes, specifically
iDRM05017s). iDRM05017s were induced for MyoD activity and then
cultured for 10 days in fusion media. On Day 7, AO was added for
twenty-four hours then removed and Piperacetazine, Trifluoperazine
Dihydrochloride, Fluphenazine Dihydrochloride or vehicle (Dimethyl
sulfoxide; DMSO) were added in fresh media. After two days total
RNA was harvested, cDNA reverse transcribed with a DMD specific
primer in exon 54, and exon 51 skipping was detected by nested
RT-PCR spanning exons 43-52. Quantitation of exon 51 skipping was
performed using the Agilent Bioanalyzer and is represented as the
proportion of exon 51 skipping. Error bars represent the standard
deviation (SD) of 3 independent wells.
[0023] FIG. 17 shows that rauwolscine hydrochloride and yohimbinic
acid monohydrate enhance AO directed DMD exon 51 skipping. As in
FIG. 16, on day 7 of fusion, AO was added to iDRM05017s followed by
the addition of Rauwolscine HCl, Yohimbinic acid monohydrate or
vehicle (Dimethyl sulfoxide; DMSO) on Day 8. After two days total
RNA was harvested, cDNA reverse transcribed and exon 51 skipping
was detected by nested RT-PCR spanning exons 43-52. Quantitation of
exon 51 skipping was performed using the Agilent Bioanalyzer and is
represented as the proportion of exon 51 skipping. Error bars
represent the SD of 3 independent wells.
[0024] FIG. 18 shows that menadione enhances AO directed DMD exon
51 skipping. On day 7 of fusion, AO was added to iDRM05017s and
twenty-four hours later removed and Menadione or vehicle (Dimethyl
sulfoxide; DMSO) were added in fresh media. After two days total
RNA was harvested, cDNA reverse transcribed with a DMD specific
primer in exon 54, and exon 51 skipping was detected by nested
RT-PCR spanning exons 43-52. Quantitation of exon 51 skipping was
performed using the Agilent Bioanalyzer and is represented as the
proportion of exon 51 skipping. Error bars represent the SD of 3
independent wells.
[0025] FIG. 19 shows that water-soluble dantrolene enhances AO
directed DMD exon 51 skipping. On day 7 of fusion, AO was added to
iDRM05017s and twenty-four hours later removed and water-soluble
dantrolene (Revonto) or vehicle (6.7% Mannitol) were added in fresh
media. After two days total RNA was harvested, cDNA reverse
transcribed with a DMD specific primer in exon 54, and exon 51
skipping was detected by nested RT-PCR spanning exons 43-52.
Quantitation of exon 51 skipping was performed using the Agilent
Bioanalyzer and is represented as the proportion of exon 51
skipping. Error bars represent the SD of 3 independent wells.
[0026] FIG. 20 shows that ryanodine receptor antagonists enhance AO
directed DMD exon 51 skipping in a reprogrammed patient cell line.
On day 7 of fusion, AO was added to iDRM05017s and twenty-four
hours later removed and Dantrolene, Ryanodine, S107 or vehicle
(Dimethyl sulfoxide; DMSO) were added in fresh media. After two
days total RNA was harvested, cDNA reverse transcribed with a DMD
specific primer in exon 54, and exon 51 skipping was detected by
nested RT-PCR spanning exons 43-52. Quantitation of exon 51
skipping was performed using the Agilent Bioanalyzer and is
represented as the proportion of exon 51 skipping. Error bars
represent the SD of 3 independent wells.
[0027] FIG. 21 shows that dantrolene synergizes with
intravascularly delivered AO to increase muscle strength in mdx
mice. Weekly systemic doses of saline, 10 mg/kg of morpholino M23D
(+07-18) was administered intravascularly on Day 1, 8, and 15.
Dantrolene or carrier (20% DMSO in saline) was administered
intraperitoneally at a dose of 10 mg/kg/day in two divided doses
daily. On Day 18 functional improvement was blindly assessed by
using the taut wire test. Latency to fall (in seconds) was recorded
for five consecutive trials, with a one minute break occurring in
between each trial. Plotted is first the average across five
trials, and then the normalized average (seconds/grams) across
experimental groups. Error bars represent the s.e.m. There was a
significantly increased ability of mdx mice to hang on wire
(p=0.022).
DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0028] The present inventors identify herein low molecular weight
compounds (sometimes referred to herein as "small molecules" or
"small molecule compounds" or "compounds" of the invention) which
block some forms of mRNA splicing and/or enhance (facilitate,
augment) other forms of mRNA splicing. The types of splicing that
can be regulated by a method of the invention include alternative
splicing, in particular exon skipping. Depending on factors such as
the splicing sequence and the gene or exon involved, this
modulation of splicing can be accomplished in the presence of, or
in the absence of, antisense oligonucleotides (AOs) that are
specific for splicing sequences of interest. In embodiments of the
invention, a small molecule and an AO of the invention act
synergistically. The antisense molecules used in a method of the
invention are sometimes referred to herein as antisense "splice
switching oligonucleotides (SSO's)." Table 1 lists 27
representative small molecules which can be used in a method of the
invention. It is to be understood that references herein to the 27
small molecules in Table 1 include pharmaceutically acceptable
salts, hydrates, solvates or isomers thereof.
[0029] As shown in the Examples herein, the inventors performed a
small molecule cell based screen using a human exon 50 (of the DMD
gene) reporter cell line, which is activated when exon 50 is
skipped. The cell line, which was adapted to allow the screening of
thousands of compounds in multiple replicates, was obtained from
Dr. Qi Lu. The compounds which were screened were selected from FDA
approved libraries or known biologically active molecule libraries.
Lead hits (shown in Table 1) were further validated using
assessment of RNA sequence and with various dose titrations in
mouse cells, and demonstrate synergy with antisense
oligonucleotide. Each of the compounds was validated in
counterscreens to rule out toxicity and autofluorescence, and
demonstrated to have activity in 16 point titrations of the
compound, either alone or in synergy with anti-sense
oligonucleotide. The aggregate group of compounds defines new
classes of drugs which induce (enhance) exon skipping. Some of the
compounds are shown to increase the amount of skipped exon 50
dystrophin mRNA when applied externally to cells growing in culture
either alone or in synergy with anti-sense oligonucleotide. One of
the tested compounds, dantrolene, was demonstrated to affect mdx
mice in vivo with systemic administration. Other studies presented
herein also demonstate exon skipping of, e.g., exon 23 and exon 50
of DMD. It is expected that at least some of the compounds will
induce (enhance) exon skipping and create alternate splice forms of
proteins that are relevant to a variety of disease states.
[0030] Compounds that were identified in the counter screens
include, e.g., Furaltadone hydrochloride, 5-iodotubericidin,
bendroflumethiazide, cyclopiazonic acid, GW 5074, indirubin,
rescinnamin, U-0126, acetopromazine maleate salt, Ro 31-8220.
Additional compounds showing efficacy in counter screen and on mdx
mouse myotubes include, e.g., dantrolene, dichlorobenzamil,
ellipticine, fenbendazole, GF 109203.times., halofantrine,
niclosamide, pimozide, reserpine, syringospine. Other compounds
shown or expected to show exon skipping activity include, e.g.,
Ryanodine, RyCal S107, piperacetazine, fluphenazine
dihydrochloride, trifluorperazine dihydrochloride, yohimbinic acid,
and menadione. Pharmaceutically acceptable salts, hydrates,
solvates or isomers of these or other compounds of the invention
are also included. For example, sodium ions in the formulas can be
substituted with any of a variety of other pharmaceutically
acceptable cations. Suitable such salts, hydrates, solvates or
isomers will be evident to a skilled worker. See, e.g., Remington's
Pharmaceutical Sciences, 18.sup.th edition (1990, Mack Publishing
Co., Easton, Pa.).
TABLE-US-00001 TABLE 1 General Routes Linear FDA Molecular Chemical
of Chemical Compound Name Approved Weight Type Known Activity
Admission Structure Chemical Structure Furaltadone hydrochloride N
(as of 1991) but is in the FDA library 324.29 Antibiotic
Characterized by the Nitrofuran ring. Effective antibiotic when all
others fail against extremely drug resistant bacterial infections
but has many side effects. PO only C.sub.13H.sub.16N.sub.4O.sub.6
##STR00001## 5- IODOTUBERCIDIN N 392.15 Kinase Inhibitor Inhibits
ERK2 (Ki = 525 nM) also inhibits adenosine kinase (Ki = 30 nM) CK1
and CK2 and insulin receptor kinase2. --
C.sub.11H.sub.13IN.sub.4O.sub.4 ##STR00002## Benroflum- thiazide Y
421.41 Antihypertensive Agents, Diuretics, Sodium Chloride
Symporter Inhibitors Inhibits active chloride reabsorption at the
early distal tubule via the Na--Cl cotransporter, resulting in an
increase in the excretion of sodium, chloride, and water. Also
inhibits sodium ion transport across the renal tubular epithelium
through binding to the thiazide sensitive sodium-chloride
transporter. The antihypertensive mechanism of bendroflumethiazide
is less well understood although it may be mediated through its
action on carbonic anhydrases in the smooth muscle or through its
action on the large- conductance calcium-activated potassium (KCa)
channel. PO C.sub.15H.sub.14F.sub.3N.sub.3O.sub.4S.sub.2
##STR00003## CYCLOPIAZONIC ACID N 336.38 Fungal secondary
metabolite Induces the release of intracellular stored Ca2+,
without increasing IP3 levels, via inhibition of endoplasmic
reticulum Ca2+-ATPase. It is a highly specific inhibitor of the
Ca2+-ATPase of sarcoplasmic reticulum, completely inhibiting the
enzyme at 6-8 nmol/mg protein (at 0.5-2 .mu.M ATP). --
C.sub.20H.sub.20N.sub.2O.sub.3 ##STR00004## GW 5074 N 520.94 Enzyme
Inhibitor Potent and selective cell permeable inhibitor of cRAF1
kinase (IC50 = 9 nM) with 100- fold selectivity over CDK1, CDK2,
c-src, ERK2, MEK, p38, Tie2, VEGFR2 and c-fm. --
C.sub.15H.sub.8Br.sub.2INO.sub.2 ##STR00005## INDIRUBIN Y 277.28
Kinase Inhibitor Cyclin-dependent kinase inhibitor which functions
by competing with ATP for binding to the catalytic subunit. Inhbits
CDK1, CDK2, CDK4, and CDK5. IV, IP C.sub.16H.sub.11N.sub.3O.sub.2
##STR00006## Rescinnamin Y 634.72 Antihypertensive agent
Angiotensin- converting enzyme inhibitor used as an
antihypertensive drug. Also is a reserpine analog. PO
C.sub.35H.sub.42N.sub.2O.sub.9 ##STR00007## U-0126 N (in pre-
clinical trials currentyl) 426.56 Enzyme Inhibitor A novel, potent
and selective MEK inhibitor, MEK1 IC50 = 72 nM, MEK2 IC50 = 58 nM.
Also inhibits MAPKK. In pre clinical trials for cancer treatments.
IV C.sub.18H.sub.16N.sub.6S.sub.2.cndot.C.sub.2H.sub.5OH
##STR00008## Acetopromazine maleate salt Y 442.53 Antipsychotic
Agents Dopamine antagonist. IM, SC
C.sub.19H.sub.22N.sub.2OS.cndot.C.sub.4H.sub.4O.sub.4 ##STR00009##
Ro 31-8220 N 553.65 Enzyme Inhibitor Inhibitor of GRK-5 (G protein-
coupled receptor kinase); PKC (protein kinase C); MAPKAP kinase
1.beta. and p70 S6 kinase. PO
C.sub.25H.sub.23N.sub.5O.sub.2S.cndot.CH.sub.3SO.sub.3H
##STR00010## DANTROLENE Y 336.23 Muscle relaxant, Intracellular
calcium channel modulator Inhibitor of Ca.sup.2+ release from
sarcoplasmic reticulum; muscle relaxant. Dantrolene depresses
excitation- contraction coupling in skeletal muscle by binding the
ryanodine receptor and decreasing intracellular calcium
concentration. PO, IV IM C.sub.14H.sub.9N.sub.4NaO.sub.5
##STR00011## DICHLO- ROBENZAMIL N 425.1 Calcium Channel Modulator
Inhibits cyclic nucleotide-gated Ca + 2 channels (IC50 = 38- 50
.mu.M). Inhibits plasmalemmal Na+/Ca + 2 and Na+/H+ exchange (IC50
= 10 .mu.M). Blocks caffeine-induced current (by blocking Na+/ Ca +
2 exchange) at 50-100 .mu.M). Nonselective cation channel blocker
(25 .mu.M). -- C.sub.13H.sub.12N.sub.7OCl.sub.3.cndot.HCl
##STR00012## Ellipticine Y 246.31 Antineoplastic Agent, Uncoupling
Agent Antitumor alkaloid isolated from Ochrosia sp. It inhibits
cytochrome P450 (CYP1A1) and DNA topoisomerase II activities. PO,
IP C.sub.17H.sub.14N.sub.2 ##STR00013## Fenbendazole Y 299.35
Antinemoatodal agent Inhibits cytoplasmic microtubules in the
intestinal or absorptive cells of worms, thus inhibiting glucose
uptake and glycogen storage depletion, leading to death of the
worms within days. PO, IV C.sub.15H.sub.13N.sub.3O.sub.2S
##STR00014## GF 109203X N 412.48 Kinase Inhibitor Inhibitor of
protein kinase C; potent inhibitor of GSK-3. --
C.sub.25H.sub.24N.sub.4O.sub.2 ##STR00015## Halofantrine
hydrochloride Y 536.88 Antimalarial agent Halofantrine is a blocker
of delayed rectifier potassium current via the inhibition of hERG
channel. It is a blood schizontocide that is active against
chloroquine- resistant falciparum and vivax malaria. It can destroy
asexual blood forms and inhibit the proton pump. PO
C.sub.26H.sub.30Cl.sub.2F.sub.3NO.cndot.HCl ##STR00016##
Niclosamide Y 327.12 Anticestodal, Antinematodal, Molluscacidades
Niclosamide uncouples oxidative phosphorylation in mitochondria of
the tapeworm. It belongs to the class of alicylic acid derivative
agents used as anticestodals. PO
C.sub.13H.sub.8Cl.sub.2N.sub.2O.sub.4 ##STR00017## PIMOZIDE Y
461.55 Antipsychotic D.sub.2 dopamine receptor antagonist; binds
with high affinity to the cloned 5-HT.sub.7 receptor; Ca.sup.2+
channel antagonist; antipsychotic. PO
C.sub.28H.sub.29F.sub.2N.sub.3O ##STR00018## Reserpine Y 608.68
Antihypertensive, Antypsychotic Reserpine is an antihypertensive
drug that causes depletion of noradrenaline, catecholamine and
serotonin stores resulting in a reduction in BP, bradycardia and
CNS depression. It belongs to the class of rauwolfia alkaloids,
centrally-acting antiadrenergic agents. Used in the treatment of
hypertension. Reserpine can also be utilized in the relief of
symptoms in agitated psychotic states (e.q. schizophrenia). PO, or
injectable C.sub.33H.sub.40N.sub.2O.sub.9 ##STR00019##
Syrosingopine Y 666.71 Antihypertensive agent Syrosingopine is
prepared from reserpine by hydrolysis and reesterification; an
antihypertensive agent with actions similar to those of reserpine
PO, or injectable C.sub.35H.sub.42N.sub.2O.sub.11 ##STR00020##
Ryanodine ##STR00021## RyCal S107 ##STR00022## piperacetazine
##STR00023## Fluphenazine dihydrochloride ##STR00024##
Trifluoperazine dihydrochloride ##STR00025## Yohimbinic acid
##STR00026## Menadione ##STR00027## ##STR00028##
[0031] Each of the identified compounds has a different known
effect on cells and has been used for different therapeutic
purposes. How each of the compounds affects the RNA splicing
machinery to alter the efficiency of exclusion of targeted exons is
not known at this time. While the detailed molecular mechanisms are
not yet established, several of the compounds identified, for
instance Dantrolene, have well-characterized effects in cells and
in humans. Dantrolene's known effect is to block the ryanodine
receptor which prevents release of calcium that is needed for
muscle cell contraction when excited. This drug is used clinically
to mitigate the effects of malignant hyperthermia. The use of
Dantrolene in combination with antisense oligonucleotide to induce
an inframe transcript is a novel use for this compound. Dantrolene
has been tried as a single agent to treat Duchenne muscular
dystrophy without significant beneficial effect and without
significant deleterious effects. None of the identified compounds
has been used in order to alter exon splicing therapeutically. For
example, some of these compounds are known vermicidals,
anti-hypertensive, anti-malarial, anti-psychotic or anti-cancer
agents.
[0032] It is expected that endogenously generated antisense
oligonucleotides (for instance from gene delivery) will augment
exon skipping in a similar manner as exogenously administered AOs.
For example, endogenously generated small nuclear RNA (sRNA)
carrying appropriate antisense sequences and transcribed from,
e.g., a U7 snRNA-based gene construct can be used in a method of
the invention.
[0033] Advantages of methods and combinations of the invention
include that they augment the efficiency of exon skipping (e.g.,
when performed in the presence of AO) and thus allow a sufficient
amount of skipping to be therapeutically relevant and/or reduce the
cost resulting from high doses and repeated administration of
expensive AOs.
[0034] "Antisense-mediated exon skipping," as used herein, refers
to an approach that uses antisense oligonucleotides (AOs) to
modulate splicing by blocking (hiding) specific sequence motifs in
the pre-mRNA (sometimes referred to herein as "splicing sequences")
essential for exon inclusion from the splicing machinery. AOs that
block aberrant splice sites can restore normal splicing.
Alternatively, AOs targeting certain splicing sequences can switch
splicing patterns from detrimental to beneficial isoforms or can
convert at least partially non-functional mRNAs into functional
mRNA. An example of the latter approach is the restoration of a
disrupted reading frame, thereby generating semi-functional
proteins instead of non-functional proteins.
[0035] A compound of the invention can be used to block splicing at
a site of interest by specifically interacting with (e.g., binding
to) a splicing sequence at that site, either directly or
indirectly. By a "splicing sequence" is meant a sequence that
regulates and/or is required for splicing out of a particular
intron and/or the retention of a particular exon. The splicing
sequence can be, for example, a splice donor site, a splice
acceptor site, a branch site, an intronic splicing enhancer (ISE),
an exonic splicing enhancer (ESE), an intronic splicing silencer or
an exonic splicing silencer.
[0036] An AO used in a method of the invention can bind directly
and specifically to a target splicing sequence of interest. By
"specific binding" is meant that the AO binds preferentially to the
target sequence of interest, but not to non-target sequences under
conditions in which specific binding is desired. The conditions can
be, e.g., physiological conditions in the case of in vivo assays or
therapeutic treatment, and for in vitro assays, conditions in which
the assays are performed. Because the mechanism by which small
molecule compounds of the invention block splicing (e.g., enhance
exon skipping) is not known for all of the compounds, it is not
known whether the compound binds directly to a splice site or acts
indirectly (e.g., by binding to another RNA or protein element of a
spliceosome). Regardless of the mechanism, a compound of the
invention that "specifically" blocks a splicing event of interest
is one that preferentially blocks the particular splicing event but
does not block non-targeted splicing events, under conditions in
which specific blocking is desired.
[0037] As used herein, the term "antisense oligonucleotide (AO)"
refers to a single-stranded oligonucleotide that is specific for,
and complementary to, a splicing sequence of interest, and
accordingly is capable of hydrogen bonding to the sequence. One of
skill in the art can readily design AOs to be specific for suitable
target sequences, many of which are well-known in the art. For
example, one can access pre-mRNA sequences comprising suitable
splicing sequences in publications or in annotated, publically
available databases, such as the GenBank database operated by the
NCBI. A skilled worker will be able to design, make and use
suitable antisense oligonucleotides, based on these or other
sequences, without undue experimentation. A number of AO's have
been designed for enhancing exon skipping and some are currently in
preclinical or clinical trials. Any of these AOs is suitable for
use in a method of the invention.
[0038] An antisense nucleic acid may be, e.g., an oligonucleotide,
or a nucleic acid comprising an antisense sequence that is operably
linked to an expression control sequence and that is expressed in a
cell.
[0039] Antisense oligonucleotides may have a variety of different
backbone chemistries, such as morpholino phosphorodiamidate (PMO)
or 2'-O-methyl' or peptide nucleic acids, etc., which stabilize
them. For example, it can be DNA, RNA, PNA or LNA, or chimeric
mixtures or derivatives or modified versions thereof. The nucleic
acid can be modified at the base moiety, sugar moiety, or phosphate
backbone, using conventional procedures and modifications.
Modifications of the bases include, e.g., methylated versions of
purines or pyrimidines. Modifications may include other appending
groups that will be evident to a skilled worker.
[0040] Antisense oligonucleotides can be constructed using chemical
synthesis procedures known in the art. An AO can be chemically
synthesized using naturally occurring nucleotides or variously
modified nucleotides designed to increase the biological stability
of the molecules or to increase the physical stability of the
duplex formed between the antisense and sense nucleic acids, e.g.
phosphorothioate derivatives and acridine substituted nucleotides
can be used. For guidance in methods of synthesizing AOs used in
methods of the present invention, see, e.g.:
[0041] For guidance in methods of synthesizing morpholino AO's for
use in the present invention, see, e.g., US patent application
2009/0131624 ("Synthesis of morpholino oligomers using double
protecte guanine morpholino subunits").
[0042] For guidance in synthesizing oligonucleotides, see, e.g.,
Gough et al. (1979) Nucleic Acids Research 7, 1955-1964; Hata et
al. (1983) Tetrahedron Lett. 24, 2775-2778; Jones et al. (1982A
Tetrahedron Lett. 23, 2253-2256; Jones et al. (1982) Tetrahedron
Lett. 23, 2257-2260; O. Mitsunobu (1981) Synthesis 1, 1-28; Reese
et al. (1981) Tetrahedron Lett. 22, 4755-4758; Reese et al. (1984)
J. Chem. Soc., Perkin Trans. 11263-1270; Summerton et al. (1993)
U.S. Pat. No. 5,185,444; Summerton et al. (1997) Antisense Nucl.
Acid Drug Dev. 7(3), 187-195.
[0043] For guidance in synthesizing 2-O-methyl' oligos, see e.g.
Verma et al. (1998) MODIFIED OLIGONUCLEOTIDES: Synthesis and
Strategy for Users, Annu. Rev. Biochem. 67, 99-134
[0044] For guidance in synthesizing dantrolene, see e.g. Oleinik et
al. (1984) Pharmaceutical Chemistry Journal 18 (5), 310-312.
[0045] To enhance exon skipping in cells in culture, AO's can be
added to cells in culture media. Typically, synthetic
oligonucleotides are added to a final concentration of about 10 nM
to about 10 microM, e.g., about 50 nM to about 1000 nM (e.g., at
increments of 10 nM within the indicated ranges). The term "about"
a particular value, as used herein, means plus or minus 10% of the
indicated value.
[0046] Effective doses of AOs for in vivo administration can be
determined, e.g., on the basis of the amounts used for exon
skipping in the absence of a small molecule of the present
invention. Many AO's have been administered to subjects in the
absence of small molecule compounds of the invention, and doses
have been established which are at least partically effective and
are non-toxic to the subjects. In general, doses of AOs ranging
from about 5-100 mg/kg/wk IV (intravenous) (or comparable amounts
for other modes of admin) are effective for inducing at least a
detectable amount of dystrophin expression with targeted removal of
a given exon.
[0047] Alternatively, an antisense oligonucleotide can be produced
biologically using an expression vector into which a nucleic acid
has been subcloned in an antisense orientation (i.e., nucleic acid
transcribed from the inserted nucleic acid will be of an antisense
orientation to a target sequence of interest). Expression control
sequences (e.g., regulatory sequences) operatively linked to a
nucleic acid cloned in the antisense orientation can be chosen
which direct the expression of the antisense RNA molecule in a cell
of interest. For instance, promoters and/or enhancers or other
regulatory sequences can be chosen which direct constitutive,
tissue specific or inducible expression of an AO. Inducible
expression of antisense RNA, regulated by an inducible eukaryotic
regulatory system, such as the Tet system (e.g., as described in
Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551;
Gossen et al. (1995) Science 268, 1766-1769; PCT Publication No. WO
94/29442; and PCT Publication No. WO 96/01313) can be used. The
antisense expression vector can be in the form of, for example, a
recombinant plasmid, phagemid or attenuated virus. Suitable viral
vectors include, e.g., adeno-associated virus (AAV) or lentivirus
vectors. The antisense expression vector can be introduced into
cells using standard techniques well known in the art. For guidance
in using AAV vectors for introducing antisense molecules into mdx
mice, see e.g. Denti et al. (2008) Hum Gene Ther 19, 601-608 or
Incitti et al. (2010) Mol. Ther. 18, 1675-1682.
[0048] In one embodiment of the invention, an RNA molecule that
comprises the sequence antisense to a splicing sequence in, e.g.,
the dystrophin pre-mRNA, is produced biologically by using an
expression vector into which a nucleic acid has been subcloned.
Expression control sequences (e.g. regulatory sequences) operably
linked to the cloned nucleic acid can be chosen which direct the
expression of the antisense RNA molecule comprising the sequence
antisense to a splicing sequence in, e.g., dystrophin pre-mRNA, in
a cell of interest. The RNA molecule may comprise, e.g., a U1
snRNA, U2 snRNA, U6 snRNA or U7 snRNA. Without wishing to be
limited by any particular mechanism, it is suggested that
expression of the snRNA generates an snRNP particle which then
binds to the target sequence in dystrophin pre-mRNA via the
complementary fragment of snRNA. Any of the types of expression
control sequences described in the previous paragraph can be used
to direct the expression of the desired RNA in this embodiment.
[0049] In one embodiment of the invention, an AO comprises a strand
that is completely complementary (100% identical in sequence) to a
splicing sequence that it is designed to inhibit. That is, every
contiguous nucleotide in the AO is hybridized to every nucleotide
in a splicing sequence. However, 100% sequence identity between the
AO and the target splicing sequence is not required to practice the
present invention. Thus, the invention has the advantage of being
able to tolerate naturally occurring sequence variations that might
be expected due to genetic mutation, strain polymorphism, or
evolutionary divergence. Alternatively, the variants may be
artificially generated. Nucleic acid sequences with, e.g., small
insertions, deletions, and single point mutations relative to the
target sequence can be effective for inhibition. The degree of
sequence identity can be, e.g., 95%, 98%, 99%, or 100%. Such a
variant AO must, of course, retain the relevant activity of the AO
from which it is derived. (e.g., the ability to suppress splicing
at a site of interest). Such variants are sometimes referred to
herein as "active variants."
[0050] The length of an AO may vary, provided that it is capable of
binding selectively to the intended splicing sequence within the
pre-mRNA molecule. A skilled worker can readily determine a
satisfactory length. Generally, an AO is from about 10 nt in length
to about 50 nt in length. Any length of nucleotides within this
range, including the endpoints, can be used in a method of the
invention. In one embodiment, the length of the AO is about 17-30
nt in length.
[0051] For further guidance for designing suitable antisense
molecules that are complementary to a region of a pre-mRNA involved
in splicing (thereby blocking splicing), and for methods for making
and delivering such molecules to a cell or a subject, see, e.g., US
2008/0200409 or U.S. Pat. No. 7,973,015, 7,960,541, 7,902,160,
7,888,012, 7,879,992 or 7,737,110.
[0052] A method of the invention can be carried out in vitro (e.g.,
to elucidate the mechanism by which splicing occurs, such as to
reveal novel molecular interactions in the processing of mRNA; or
to screen for compounds that can block a splicing event and thus,
for example, enhance exon skipping).
[0053] In another embodiment of the invention, the method is
carried out in a subject, in vivo. A "subject," as used herein, can
refer to any animal which is subject to a disease or condition that
can be treated by a method of the invention. Suitable subjects
include, e.g., a mammal, such as an experimental animal or disease
model, a farm animal, pet, or the like. In some embodiments, the
animal is a primate, for example a human.
[0054] In some embodiments of the invention, a subject is treated
with an effective amount of a compound of the invention, or with a
combination of a compound of the invention and a suitable AO, each
of which is designed to block a splicing event of interest. An
"effective amount" of a compound (or combination) of the invention
is an amount that is effective to elicit a measurable amount of
biological activity, e.g. a measurable amount of enhancement of
exon skipping (in some embodiments in the absence of AOs, and in
some embodiments in the presence of a suitable AO). Preferably, an
effective amount of a compound or combination of the invention does
not elicit substantial amounts of undesirable (e.g., toxic)
effects. The enhancement can occur prophylactically (e.g.
preventively, to inhibit the development of the disorder), or in a
subject who already has the condition. For example, treatment by a
method of the invention can ameliorate one or more symptoms of the
condition.
[0055] A skilled worker will recognize a variety of conditions that
can be treated by a method of the invention. A probabilistic
analysis indicated that over 60% of human disease-causing mutations
affect splicing rather than directly affecting coding sequences
(Lopez-Bigas et al. (2005) FEBS Letters 579, 1900-3). See also Wang
et al. (2007), Splicing in disease: disruption of the splicing code
and the decoding machinery, Nature Reviews Genetics 8, 749-761 and
Singh et al. (2012), Pre-mRNA splicing in disease and therapeutics,
Trends in Molecular Medicine 18, (8), 472-482. Diseases associated
with aberrant splicing or missplicing that can be inhibited by a
method of the invention include e.g. beta-thalassemia and certain
forms of cancers. Alternatively, exon skipping by a method of the
invention can remove exons that contain mutations which are
associated with diseases, such as mutations that alter the reading
frame of the protein encoded by an mRNA. These conditions include,
e.g., DMD, as described above (changing DMD dystrophin to a more
functional form of dystrophin, in effect converting Duchenne MD
into Becker MD). One embodiment of the invention is a method for
treating a subject that has Duchenne muscular dystrophy (DMD), or
is a non-human model of DMD, comprising administering to the
subject an effective amount of small molecule selected from the
compounds shown in Table 1, in conjunction with an AO specific for
modulating splicing of dystrophin pre-mRNA, such as one for exon
23, 44, 45, 50, 51, 52, or 53 of the DMD gene. The exon skipping
can be either single or multi-exon skipping (e.g., skipping of many
possible 2-10 exon combinations that will be evident to a skilled
worker).
[0056] Some suitable exons that can be skipped by a method of the
invention are summarized in Table 6 below. Listed are human DMD
coding sequences with 50 intronic nucleotides at the exon
boundaries. mRNA sequences are in upper case, and intronic
sequences in lower case. On the basis of these sequences, a skilled
worker can readily design AO's specific for blocking the relevant
splice sites.
TABLE-US-00002 TABLE 6 Exon 1 (SEQ ID NO: 18) 1 ATGCTTTGGT
GGGAAGAAGT AGAGGACTGT Tgtaagtaca aagtaactaa aaatatattt tactgtggca
taacgtttag t Exon 2 (SEQ ID NO: 19) 1 ttatatttaa agttgcttcc
taacttttat ttttttattt tgcattttag ATGAAAGAGA AGATGTTCAA AAGAAAACAT
TCACAAAATG GGTAAATGCA 101 CAATTTTCTA AGgtaagaat ggttt ttac
tttactttta agatctaagt tgtgaaattt tc Exon 3 (SEQ ID NO: 20) 1
atcattggaa gtgtgctttg ttaaattgag tgtatttttt ttaatttcag TTTGGGAAGC
AGCATATTGA GAACCTCTTC AGTGACCTAC AGGATGGGAG 101 GCGCCTCCTA
GACCTCCTCG AAGGCCTGAC AGGGCAAAAA CTGgtatgtg acttattttt aagaaagtta
actttaaact tagtagaatt tca Exon 4 (SEQ ID NO: 21) 1 attgtcggtc
tctctgctgg tcagtgaaca ctcttttgtt ttgttctcag CCAAAAGAAA AAGGATCCAC
AAGAGTTCAT GCCCTGAACA ATGTCAACAA 101 GGCACTGCGG GTTTTGCAGA
ACAATAATgt aagtagtacc ctggacaagg tctggatgct gtgacacagc atgcttca
Exon 5 (SEQ ID NO: 22) 1 ctaggcattt ggtctcttac cttcaaatgt
tttacccctt tctttaacag GTTGATTTAG TGAATATTGG AAGTACTGAC ATCGTAGATG
GAAATCATAA 101 ACTGACTCTT GGTTTGATTT GGAATATAAT CCTCCACTGG
CAGgtaagaa tcctgatgaa tggtttcctt ttgggtaaca ttaatcttgt ttt Exon 6
(SEQ ID NO: 23) 1 ttcttgctca aggaatgcat tttcttatga aaatttattt
ccacatgtag GTCAAAAATG TAATGAAAAA TATCATGGCT GGATTGCAAC AAACCAACAG
101 TGAAAAGATT CTCCTGAGCT GGGTCCGACA ATCAACTCGT AATTATCCAC
AGGTTAATGT AATCAACTTC ACCACCAGCT GGTCTGATGG CCTGGCTTTG 201
AATGCTCTCA TCCATAGTCA TAGgtaagaa gattactgag acattaaata acttgtaaaa
gtggtgattt aga Exon 7 (SEQ ID NO: 24) 1 gattgattta tatttgtctt
tgtgtatgtg tgtatgtgta tgtgttttag GCCAGACCTA TTTGACTGGA ATAGTGTGGT
TTGCCAGCAG TCAGCCACAC 101 AACGACTGGA ACATGCATTC AACATCGCCA
GATATCAATT AGGCATAGAG AAACTACTCG ATCCTGAAGg ttggtaaatt tctggactac
cactgctttt 201 agtatggtag agtttaatg Exon 8 (SEQ ID NO: 25) 1
tctcaaatat agaaaccaaa aattgatgtg tagtgttaat gtgcttacag ATGTTGATAC
CACCTATCCA GATAAGAAGT CCATCTTAAT GTACATCACA 101 TCACTCTTCC
AAGTTTTGCC TCAACAAGTG AGCATTGAAG CCATCCAGGA AGTGGAAATG TTGCCAAGGC
CACCTAAAGT GACTAAAGAA GAACATTTTC 201 AGTTACATCA TCAAATGCAC
TATTCTCAAC AGgtaaagtg tgtaaaggac agctactatt caagatgttt tctgttttat
at Exon 9 (SEQ ID NO: 26) 1 atggtttttc cccctcctct ctatccactc
ccccaaaccc ttctctgcag ATCACGGTCA GTCTAGCACA GGGATATGAG AGAACTTCTT
CCCCTAAGCC 101 TCGATTCAAG AGCTATGCCT ACACACAGGC TGCTTATGTC
ACCACCTCTG ACCCTACACG GAGCCCATTT CCTTCACAGg tctgtcaaca tttactctct
201 gttgtacaaa ccagagaact gcttccaag Exon 10 (SEQ ID NO: 27) 1
aatctgcaaa gacattaatt gtgtaacacc caatttattt tattgtgcag CATTTGGAAG
CTCCTGAAGA CAAGTCATTT GGCAGTTCAT TGATGGAGAG 101 TGAAGTAAAC
CTGGACCGTT ATCAAACAGC TTTAGAAGAA GTATTATCGT GGCTTCTTTC TGCTGAGGAC
ACATTGCAAG CACAAGGAGA GATTTCTAAT 201 GATGTGGAAG TGGTGAAAGA
CCAGTTTCAT ACTCATGAGg taaactaaaa cgttaattta caaaacaaaa catatgactt
gttataatg Exon 11 (SEQ ID NO: 28) 1 ccgatttacc tagagttcta
attacaattg ttaacttcct tctttgtcag GGGTACATGA TGGATTTGAC AGCCCATCAG
GGCCGGGTTG GTAATATTCT 101 ACAATTGGGA AGTAAGCTGA TTGGAACAGG
AAAATTATCA GAAGATGAAG AAACTGAAGT ACAAGAGCAG ATGAATCTCC TAAATTCAAG
ATGGGAATGC 201 CTCAGGGTAG CTAGCATGGA AAAACAAAGC AAgtaagtcc
ttatttgttt ttaattaaga agactaacaa gttttggaag ct Exon 12 (SEQ ID NO:
29) 1 taataagttg ctttcaaaga ggtcataata ggcttctttc aaattttcag
TTTACATAGA GTTTTAATGG ATCTCCAGAA TCAGAAACTG AAAGAGTTGA 101
ATGACTGGCT AACAAAAACA GAAGAAAGAA CAAGGAAAAT GGAGGAAGAG CCTCTTGGAC
CTGATCTTGA AGACCTAAAA CGCCAAGTAC AACAACATAA 201 Ggtaggtgta
tcttatgttg cgtgctttct actagaaagc aaactctgtg t Exon 13 (SEQ ID NO:
30) 1 cacatgtaag aatatcattt taatttcctt taaaacattt tatctttcag
GTGCTTCAAG AAGATCTAGA ACAAGAACAA GTCAGGGTCA ATTCTCTCAC 101
TCACATGGTG GIGGTAGTTG ATGAATCTAG TGGAGATCAC GCAACTGCTG CTTTGGAAGA
ACAACTTAAG gtcagattat tttgcttagt aaactaaata 201 tgtcctttaa
aagaactata Exon 14 (SEQ ID NO: 31) 1 cgtagttacc aattgtttgc
tgatgctgtg cttgattgtc tcttctccag GTATTGGGAG ATCGATGGGC AAACATCTGT
AGATGGACAG AAGACCGCTG 101 GGTTCTTTTA CAAGACATCC TTCTCAAATG
GCAACGTCTT ACTGAAGAAC AGgtgtgtca tgtgtgagaa actagctgta aaagacacgg
ggggatatta 201 Aa Exon 15 (SEQ ID NO: 32) 1 agtaaagatt tatgtttatt
tattccttgg aattctttaa tgtcttgcag TGCCTTTTTA GTGCATGGCT TTCAGAAAAA
GAAGATGCAG TGAACAAGAT 101 TCACACAACT GGCTTTAAAG ATCAAAATGA
AATGTTATCA AGTCTTCAAA AACTGGCCgt atgtactttc tagctttcaa tggtcttata
aaaacccagt 201 Actgtata Exon 16 (SEQ ID NO: 33) 1 tgtatggaat
gcaacccagg cttattctgt gatctttctt gttttaacag GTTTTAAAAG CGGATCTAGA
AAAGAAAAAG CAATCCATGG GCAAACTGTA 101 TTCACTCAAA CAAGATCTTC
TTTCAACACT GAAGAATAAG TCAGTGACCC AGAAGACGGA AGCATGGCTG GATAACTTTG
CCCGGTGTTG GGATAATTTA 201 GTCCAAAAAC TTGAAAAGAG TACAGCACAG
gttagtgata ccaattatca tgctacagac tatctcagag attttttaaa Exon 17 (SEQ
ID NO: 34) 1 actgaagtct ttctagcaat gtctgacctc tgtttcaata cttctcacag
ATTTCACAGG CTGTCACCAC CACTCAGCCA TCACTAACAC AGACAACTGT 101
AATGGAAACA GTAACTACGG TGACCACAAG GGAACAGATC CTGGTAAAGC ATGCTCAAGA
GGAACTTCCA CCACCACCTC CCCAAAAGAA GAGGCAGATT 201 ACTGTGGATT
CTGAAATTAG GAAAAGgtga gagcatctta agcttttatc tgcaaatgaa gtggagaaaa
ctcatt Exon 18 (SEQ ID NO: 35) 1 gaagaaagag ataatcaaga aataatgact
tttatttttt gctgtcttag GTTGGATGTT GATATAACTG AACTTCACAG CTGGATTACT
CGCTCAGAAG 101 CTGTGTTGCA GAGTCCTGAA TTTGCAATCT TTCGGAAGGA
AGGCAACTTC TCAGACTTAA AAGAAAAAGT CAATgtaggt tatgcattaa tttttatatc
201 tgtactcatt ttgtgctgct tgta Exon 19 (SEQ ID NO: 36) 1 agattcacag
tccttgtatt gaattactca tctttgctct catgctgcag GCCATAGAGC GAGAAAAAGC
TGAGAAGTTC AGAAAACTGC AAGATGCCAG 101 CAGATCAGCT CAGGCCCTGG
TGGAACAGAT GGTGAATGgt aattacacga gttgatttag ataatcttct tagggatttg
ataaacac Exon 20 (SEQ ID NO: 37) 1 tttcagtctg tgggttcagg ggatatattt
aattattttt ttctttctag AGGGTGTTAA TGCAGATAGC ATCAAACAAG CCTCAGAACA
ACTGAACAGC 101 CGGTGGATCG AATTCTGCCA GTTGCTAAGT GAGAGACTTA
ACTGGCTGGA GTATCAGAAC AACATCATCG CTTTCTATAA TCAGCTACAA CAATTGGAGC
201 AGATGACAAC TACTGCTGAA AACTGGTTGA AAATCCAACC CACCACCCCA
TCAGAGCCAA CAGCAATTAA AAGTCAGTTA AAAATTTGTA AGgtaagaat 301
ctcttctcct tccatttgga gcataatcaa taggtatttc tt Exon 21 (SEQ ID NO:
38) 1 aatgtatgca aagtaaacgt gttacttact ttccatactc tatggcacag
GATGAAGTCA ACCGGCTATC AGATCTTCAA CCTCAAATTG AACGATTAAA 101
AATTCAAAGC ATAGCCCTGA AAGAGAAAGG ACAAGGACCC ATGTTCCTGG ATGCAGACTT
TGTGGCCTTT ACAAATCATT TTAAGCAAGT CTTTTCTGAT 201 GTGCAGGCCA
GAGAGAAAGA GCTACAGACA Agtaagtaaa aagcctaaaa tggctaactt gacattttcc
aaaatggtta t Exon 22 (SEQ ID NO: 39) 1 aagtgtgaaa caattaagtg
attctcattc ttttttccct tttgataaag TTTTTGACAC TTTGCCACCA ATGCGCTATC
AGGAGACCAT GAGTGCCATC 101 AGGACATGGG TCCAGCAGTC AGAAACCAAA
CTCTCCATAC CTCAACTTAG TGTCACCGAC TATGAAATCA TGGAGCAGAG ACTCGGGGAA
TTGCAGgtct 201 gtgaatattt gaatgtcaaa acaataaagc acgcttatca agcatt
Exon 23 (SEQ ID NO: 40) 1 aattattatt catcaattag ggtaaatgta
tttaaaaaat tgttttttag GCTTTACAAA GTTCTCTGCA AGAGCAACAA AGTGGCCTAT
ACTATCTCAG 101 CACCACTGTG AAAGAGATGT CGAAGAAAGC GCCCTCTGAA
ATTAGCCGGA AATATCAATC AGAATTTGAA GAAATTGAGG GACGCTGGAA GAAGCTCTCC
201 TCCCAGCTGG TTGAGCATTG TCAAAAGCTA GAGGAGCAAA TGAATAAACT
CCGAAAAATT CAGgtaattc aagattttac tttctaccct catttttatt 301
tacttgtttt ttc Exon 24 (SEQ ID NO: 41) 1 ttaaaagtaa tcagcacacc
agtaatgcct tataacgggt ctcgtttcag AATCACATAC AAACCCTGAA GAAATGGATG
GCTGAAGTTG ATGTTTTTCT 101 GAAGGAGGAA TGGCCTGCCC TTGGGGATTC
AGAAATTCTA AAAAAGCAGC TGAAACAGTG CAGAgtaaga tttttatatg atgcctttaa
tatgaataat 201 tttgtatgaa tatt Exon 25 (SEQ ID NO: 42) 1 tatgtggcag
taattttttt cagctggctt aaattgattt attttcttag CTTTTAGTCA GTGATATTCA
GACAATTCAG CCCAGTCTAA ACAGTGTCAA 101 TGAAGGTGGG CAGAAGATAA
AGAATGAAGC AGAGCCAGAG TTTGCTTCGA GACTTGAGAC AGAACTCAAA GAACTTAACA
CTCAGTGGGA TCACATGTGC 201 CAACAGgtat agacaatctc tttcactgtg
gcttgcctca acgtacttaa ctaaga Exon 26 (SEQ ID NO: 43) 1 atgtttcatc
actgtcaata atcgtgtttt gtttgtttgt tttgtggaag GTCTATGCCA GAAAGGAGGC
CTTGAAGGGA GGTTTGGAGA AAACTGTAAG 101 CCTCCAGAAA GATCTATCAG
AGATGCACGA ATGGATGACA CAAGCTGAAG AAGAGTATCT TGAGAGAGAT TTTGAATATA
AAACTCCAGA TGAATTACAG 201 AAAGCAGTTG AAGAGATGAA Ggtaaaaaaa
aaaaaagaaa aactaagtaa aacaaaggaa ataaatggaa a Exon 27 (SEQ ID NO:
44) 1 ggatgtaaag ttattttcat gctattaaga gagcattctt tatttttcag
AGAGCTAAAG AAGAGGCCCA ACAAAAAGAA GCGAAAGTGA AACTCCTTAC 101
TGAGTCTGTA AATAGTGTCA TAGCTCAAGC TCCACCTGTA GCACAAGAGG CCTTAAAAAA
GGAACTTGAA ACTCTAACCA CCAACTACCA GTGGCTCTGC 201 ACTAGGCTGA
ATGGGAAATG CAAGACTTTG GAAgtcagtt gcttttcttg gtctttgtca atgatatgtc
aatacatggt at Exon 28 (SEQ ID NO: 45) 1 tttacttttc taccataata
tttaatctgt gatatatatt tctttcttag GAAGTTTGGG CATGTTGGCA TGAGTTATTG
TCATACTTGG AGAAAGCAAA 101 CAAGTGGCTA AATGAAGTAG AATTTAAACT
TAAAACCACT GAAAACATTC CTGGCGGAGC TGAGGAAATC TCTGAGGTGC TAGATgtaag
ttgtaaatta 201 agccaaatga tgataattta tatgcagtat taaaa Exon 29 (SEQ
ID NO: 46) 1 tgtatttaga aaaaaaagga gaaatagtaa ttattgcaaa tgtgtttcag
TCACTTGAAA ATTTGATGCG ACATTCAGAG GATAACCCAA ATCAGATTCG 101
CATATTGGCA CAGACCCTAA CAGATGGCGG AGTCATGGAT GAGCTAATCA ATGAGGAACT
TGAGACATTT AATTCTCGTT GGAGGGAACT ACATGAAGAG 201 gtatgaagat
aagtgaaaaa tctctttaat ctaatttgca ttaatgtata Exon 30 (SEQ ID NO: 47)
1 gctatcaaga gtaaacattt aactgataca ctcttattcc ttctttttag GCTGTAAGGA
GGCAAAAGTT GCTTGAACAG AGCATCCAGT CTGCCCAGGA 101 GACTGAAAAA
TCCTTACACT TAATCCAGGA GTCCCTCACA TTCATTGACA AGCAGTTGGC AGCTTATATT
GCAGACAAGG TGGACGCAGC TCAAATGCCT 201 CAGGAAGCCC AGgcaagtac
atctgggaat cagcttccat tcttttgttt ttattacttc aa Exon 31 (SEQ ID NO:
48) 1 tagttgttct ttgtagagca tgctgactaa taatgctatc ctcccaacag
AAAATCCAAT CTGATTTGAC AAGTCATGAG ATCAGTTTAG AAGAAATGAA 101
GAAACATAAT CAGGGGAAGG AGGCTGCCCA AAGAGTCCTG TCTCAGATTG ATGTTGCACA
Ggtatatgtt atttcagaaa ctaaggaacg tgttttcgtt 201 gggcattata c Exon
32 (SEQ ID NO: 49) 1 ttgtttgaaa ggcaaaatta aatcagtgcc tttttacact
gtccttacag AAAAAATTAC AAGATGTCTC CATGAAGTTT CGATTATTCC AGAAACCAGC
101 CAATTTTGAG CAGCGTCTAC AAGAAAGTAA GATGATTTTA GATGAAGTGA
AGATGCACTT GCCTGCATTG GAAACAAAGA GTGTGGAACA GGAAGTAGTA 201
CAGTCACAGC TAAATCATTG TGTGgtatgt atttctggtg gcaaatacgc aggtacccct
tgactttcct catt Exon 33 (SEQ ID NO: 50) 1 Aataatttaa ctctactgat
tatcatgttt tgttttatgt ttaaacttag AACTTGTATA AAAGTCTGAG TGAAGTGAAG
TCTGAAGTGG AAATGGTGAT 101 AAAGACTGGA CGTCAGATTG TACAGAAAAA
GCAGACGGAA AATCCCAAAG AACTTGATGA AAGAGTAACA GCTTTGAAAT TGCATTATAA
TGAGCTGGGA 201 GCAAAGgtgt gtgcatgctg agaccacaaa cacttctttc
cactttcctt ataaat Exon 34 (SEQ ID NO: 51) 1 atttgaatta aagagtaaac
taaattacat ttcattataa ttcttttcag GTAACAGAAA GAAAGCAACA GTTGGAGAAA
TGCTTGAAAT TGTCCCGTAA 101 GATGCGAAAG GAAATGAATG TCTTGACAGA
ATGGCTGGCA GCTACAGATA TGGAATTGAC AAAGAGATCA GCAGTTGAAG GAATGCCTAG
TAATTTGGAT 201 TCTGAAGTTG CCTGGGGAAA Ggtaaaacct atatcactga
aggttatttt gaacatacgt gaaaacacat a Exon 35 (SEQ ID NO: 52) 1
tcttaagact acaagacatt acttgaaggt caatgctctc cttttcacag GCTACTCAAA
AAGAGATTGA GAAACAGAAG GTGCACCTGA AGAGTATCAC 101 AGAGGTAGGA
GAGGCCTTGA AAACAGTTTT GGGCAAGAAG GAGACGTTGG TGGAAGATAA
ACTCAGTCTT CTGAATAGTA ACTGGATAGC TGTCACCTCC 201 CGAGCAGAAG
AGTGGTTAAA TCTTTTGTTG gtaagagaaa aggctagaag cttttacacc cttctctgtc
acgagaaaaa Exon 36 (SEQ ID NO: 53) 1 aagaatattg tctaaccaat
aatgccatgg tatgtctctg tacaattaag GAATACCAGA AACACATGGA AACTTTTGAC
CAGAATGTGG ACCACATCAC 101 AAAGTGGATC ATTCAGGCTG ACACACTTTT
GGATGAATCA GAGAAAAAGA AACCCCAGCA AAAAGAAGAC GTGCTTAAGg tagcaaataa
aatatgaaaa 201 gtaatgtcca aattgtacac cagttactt Exon 37 (SEQ ID NO:
54) 1 ccttcattaa ttactaactt caagtcctat ctcttgctca tggaatatag
CGTTTAAAGG CAGAACTGAA TGACATACGC CCAAAGGTGG ACTCTACACG 101
TGACCAAGCA GCAAACTTGA TGGCAAACCG CGGTGACCAC TGCAGGAAAT TAGTAGAGCC
CCAAATCTCA GAGCTCAACC ATCGATTTGC AGCCATTTCA 201 CACAGAATTA
AGACTGGAAA Ggtaggaaga tctactccaa ggtggaaact tgtgctaaat ggtctcttgc g
Exon 38 (SEQ ID NO: 55) 1 ttctaataaa aagtaatttt gatttaaagt
agcactatct ttttttttag GCCTCCATTC CTTTGAAGGA ATTGGAGCAG TTTAACTCAG
ATATACAAAA 101 ATTGCTTGAA CCACTGGAGG CTGAAATTCA GCAGGGGGTG
AATCTGAAAG AGGAAGACTT CAATAAAGAT ATGgtaaatt ggttgtgata aaagtgtgaa
201 tgaactagga gtggaaataa ata Exon 39 (SEQ ID NO: 56) 1 acagcttttt
aaaaaccaaa atgaagactg tacttgttgt ttttgatcag AATGAAGACA ATGAGGGTAC
TGTAAAAGAA TTGTTGCAAA GAGGAGACAA 101 CTTACAACAA AGAATCACAG
ATGAGAGAAA GCGAGAGGAA ATAAAGATAA AACAGCAGCT GTTACAGACA AAACATAATG
CTCTCAAGgt attagagcta 201 aaattataat ataccttgcc tgtggttttt ttttaata
Exon 40 (SEQ ID NO: 57) 1 tgcactatac atatatattg atattttaat
aatgtctgca ccatgaacag GATTTGAGGT CTCAAAGAAG AAAAAAGGCT CTAGAAATTT
CTCATCAGTG 101 GTATCAGTAC AAGAGGCAGG CTGATGATCT CCTGAAATGC
TTGGATGACA TTGAAAAAAA ATTAGCCAGC CTACCTGAGC CCAGAGATGA AAGGAAAATA
201 AAGgtaatgt tgttttagaa tgtcaatacc agattttatt atacagttta att Exon
41 (SEQ ID NO: 58) 1 tgatgtggtt agctaactgc cctgggccct gtattggttt
tgctcaatag GAAATTGATC GGGAATTGCA GAAGAAGAAA GAGGAGCTGA ATGCAGTGCG
101 TAGGCAAGCT GAGGGCTTGT CTGAGGATGG GGCCGCAATG GCAGTGGAGC
CAACTCAGAT CCAGCTCAGC AAGCGCTGGC GGGAAATTGA GAGCAAATTT 201
GCTCAGTTTC GAAGACTCAA CTTTGCACAA ATTgtgagtt gttactggca aacccacgta
tgtgtttgca actactactc tat Exon 42 (SEQ ID NO: 59) 1 ttcactgtta
ggaagctaaa aaaaattgtt cttttgtata tctataccag CACACTGTCC GTGAAGAAAC
GATGATGGTG ATGACTGAAG ACATGCCTTT 101 GGAAATTTCT TATGTGCCTT
CTACTTATTT GACTGAAATC ACTCATGTCT CACAAGCCCT ATTAGAAGTG GAACAACTTC
TCAATGCTCC TGACCTCTGT 201 GCTAAGGACT TTGAAGATCT CTTTAAGCAA
GAGGAGTCTC TGAAGgtaaa accaaagcac tttcattcgt attttacaag gtgatcatac
tgatc Exon 43 (SEQ ID NO: 60) 1 tatagacagc taattcattt ttttactgtt
ttaaaatttt tatattacag AATATAAAAG ATAGTCTACA ACAAAGCTCA GGTCGGATTG
ACATTATTCA 101 TAGCAAGAAG ACAGCAGCAT TGCAAAGTGC AACGCCTGTG
GAAAGGGTGA AGCTACAGGA AGCTCTCTCC CAGCTTGATT TCCAATGGGA AAAAGTTAAC
201 AAAATGTACA AGGACCGACA AGGgtaggta acacatatat ttttcttgat
acttgcagaa atgatttgtt ttc Exon 44 (SEQ ID NO: 61) 1 gttttacata
atccatctat ttttcttgat ccatatgctt ttacctgcag GCGATTTGAC AGATCTGTTG
AGAAATGGCG GCGTTTTCAT TATGATATAA 101 AGATATTTAA TCAGTGGCTA
ACAGAAGCTG AACAGTTTCT CAGAAAGACA CAAATTCCTG AGAATTGGGA ACATGCTAAA
TACAAATGGT ATCTTAAGgt 201 aagtctttga tttgtttttt cgaaattgta
tttatcttca gcacatct Exon 45 (SEQ ID NO: 62) 1 taaaaagaca tggggcttca
ttttt tttt gcctttttgg tatcttacag GAACTCCAGG ATGGCATTGG GCAGCGGCAA
ACTGTTGTCA GAACATTGAA 101 TGCAACTGGG GAAGAAATAA TTCAGCAATC
CTCAAAAACA GATGCCAGTA TTCTACAGGA AAAATTGGGA AGCCTGAATC TGCGGTGGCA
GGAGGTCTGC 201 AAACAGCTGT CAGACAGAAA AAAGAGgtag ggcgacagat
ctaataggaa tgaaaacatt ttagcagact ttttaa Exon 46 (SEQ ID NO: 63) 1
tgagaactat gttggaaaaa aaaataacaa ttttattctt ctttctccag GCTAGAAGAA
CAAAAGAATA TCTTGTCAGA ATTTCAAAGA GATTTAAATG 101 AATTTGTTTT
ATGGTTGGAG GAAGCAGATA ACATTGCTAG TATCCCACTT GAACCTGGAA AAGAGCAGCA
ACTAAAAGAA AAGCTTGAGC AAGTCAAGgt 201 aattttattt tctcaaatcc
cccagggcct gcttgcataa agaagtat Exon 47 (SEQ ID NO: 64) 1 ggaattgtgc
tgtaattcat tttaaacgtt gttgcatttg tctgtttcag TTACTGGTGG AAGAGTTGCC
CCTGCGCCAG GGAATTCTCA AACAATTAAA 101 TGAAACTGGA GGACCCGTGC
TTGTAAGTGC TCCCATAAGC CCAGAAGAGC AAGATAAACT TGAAAATAAG CTCAAGCAGA
CAAATCTCCA GTGGATAAAG 201 gttagacatt aaccatctct tccgtcacat
gtgttaaatg ttgcaagtat Exon 48 (SEQ ID NO: 65) 1 gcttatgcct
tgagaattat ttaccttttt aaaatgtatt ttcctttcag GTTTCCAGAG CTTTACCTGA
GAAACAAGGA GAAATTGAAG CTCAAATAAA 101 AGACCTTGGG CAGCTTGAAA
AAAAGCTTGA AGACCTTGAA GAGCAGTTAA ATCATCTGCT GCTGTGGTTA TCTCCTATTA
GGAATCAGTT GGAAATTTAT 201 AACCAACCAA ACCAAGAAGG ACCATTTGAC
GTTAAGgtag ggaacttttt gctttaaata tttttgtctt ttttaagaaa aatggc Exon
49 (SEQ ID NO: 66) 1 ttattgctaa ctgtgaagtt aatctgcact atatgggttc
ttttccccag GAAACTGAAA TAGCAGTTCA AGCTAAACAA CCGGATGTGG AAGAGATTTT
101 GTCTAAAGGG CAGCATTTGT ACAAGGAAAA ACCAGCCACT CAGCCAGTGA
AGgtaatgaa gcaacctcta gcaatatcca ttacctcata atgggttatg 201 Ct Exon
50 (SEQ ID NO: 67) 1 atcttcaaag tgttaatcga ataagtaatg tgtatgcttt
tctgttaaag AGGAAGTTAG AAGATCTGAG CTCTGAGTGG AAGGCGGTAA ACCGTTTACT
101 TCAAGAGCTG AGGGCAAAGC AGCCTGACCT AGCTCCTGGA CTGACCACTA
TTGGAGCCTg taagtatact ggatcccatt ctctttggct ctagctattt 201
Gttcaaaag Exon 51 (SEQ ID NO: 68) 1 tttttctttt tcttcttttt
tcctttttgc aaaaacccaa aatattttag CTCCTACTCA GACTGTTACT CTGGTGACAC
AACCTGTGGT TACTAAGGAA 101 ACTGCCATCT CCAAACTAGA AATGCCATCT
TCCTTGATGT TGGAGGTACC TGCTCTGGCA GATTTCAACC GGGCTTGGAC AGAACTTACC
GACTGGCTTT 201 CTCTGCTTGA TCAAGTTATA AAATCACAGA GGGTGATGGT
GGGTGACCTT GAGGATATCA ACGAGATGAT CATCAAGCAG AAGgtatgag aaaaaatgat
301 aaaagttggc agaagttttt ctttaaaatg aag Exon 52 (SEQ ID NO: 69) 1
aatacacaac gctgaagaac cctgatacta agggatattt gttcttacag GCAACAATGC
AGGATTTGGA ACAGAGGCGT CCCCAGTTGG AAGAACTCAT 101 TACCGCTGCC
CAAAATTTGA AAAACAAGAC CAGCAATCAA GAGGCTAGAA CAATCATTAC GGATCGAAgt
aagtttttta acaagcatgg gacacacaaa 201 gcaagatgca tgacaagt Exon 53
(SEQ ID NO: 70) 1 cctccagact agcatttact actatatatt tatttttcct
tttattctag TTGAAAGAAT TCAGAATCAG TGGGATGAAG TACAAGAACA CCTTCAGAAC
101 CGGAGGCAAC AGTTGAATGA AATGTTAAAG GATTCAACAC AATGGCTGGA
AGCTAAGGAA GAAGCTGAGC AGGTCTTAGG ACAGGCCAGA GCCAAGCTTG 201
AGTCATGGAA GGAGGGTCCC TATACAGTAG ATGCAATCCA AAAGAAAATC ACAGAAACCA
AGgttagtat caaagatacc tttttaaaat aaaatactgg 301 ttacatttga ta Exon
54 (SEQ ID NO: 71) 1 atttcataaa aaaaactgac attcattctc tttctcataa
aaatctatag CAGTTGGCCA AAGACCTCCG CCAGTGGCAG ACAAATGTAG ATGTGGCAAA
101 TGACTTGGCC CTGAAACTTC TCCGGGATTA TTCTGCAGAT GATACCAGAA
AAGTCCACAT GATAACAGAG AATATCAATG CCTCTTGGAG AAGCATTCAT 201
AAAAGgtatg aattacatta tttctaaaac tactgttggc tgtaataatg gggtg Exon
55 (SEQ ID NO: 72) 1 gcaccattct gatatttaat aattgcatct gaacatttgg
tcctttgcag GGTGAGTGAG CGAGAGGCTG CTTTGGAAGA AACTCATAGA TTACTGCAAC
101 AGTTCCCCCT GGACCTGGAA AAGTTTCTTG CCTGGCTTAC AGAAGCTGAA
ACAACTGCCA ATGTCCTACA GGATGCTACC CGTAAGGAAA GGCTCCTAGA 201
AGACTCCAAG GGAGTAAAAG AGCTGATGAA ACAATGGCAA gtaagtcagg catttccgct
ttagcactct tgtggatcca attgaacaat Exon 56 (SEQ ID NO: 73) 1
ttcttttgtt tggtaattct gcacatattc ttcttcctgc tgtcctgtag GACCTCCAAG
GTGAAATTGA AGCTCACACA GATGTTTATC ACAACCTGGA 101 TGAAAACAGC
CAAAAAATCC TGAGATCCCT GGAAGGTTCC GATGATGCAG TCCTGTTACA AAGACGTTTG
GATAACATGA ACTTCAAGTG GAGTGAACTT 201 CGGAAAAAGT CTCTCAACAT
TAGgtaggaa aagatgtgga gcaaaaaggc cacaaatgaa ttaaaatggc caa Exon 57
(SEQ ID NO: 74) 1 caattacact tctagatatt ctgacatggt acgctgctgt
tctttttcag GTCCCATTTG GAAGCCAGTT CTGACCAGTG GAAGCGTCTG CACCTTTCTC
101 TGCAGGAACT TCTGGTGTGG CTACAGCTGA AAGATGATGA ATTAAGCCGG
CAGGCACCTA TTGGAGGCGA CTTTCCAGCA GTTCAGAAGC AGAACGATGT 201
ACATAGGgta ggacattttt aagcctcgtg ccttgcacat gttaagcaca tagtaat Exon
58 (SEQ ID NO: 75) 1 agaagaatgc cacaagccaa ataagcactt cttttcatct
catttcacag GCCTTCAAGA GGGAATTGAA AACTAAAGAA CCTGTAATCA TGAGTACTCT
101 TGAGACTGTA CGAATATTTC TGACAGAGCA GCCTTTGGAA GGACTAGAGA
AACTCTACCA GGAGCCCAGA Ggtaattgaa tgtggaacta taataacata 201
ttgatagaag gatcagtggt g Exon 59 (SEQ ID NO: 76) 1 gtttaaaaaa
aaagaatgtg gcctaaaacc ttgtcatatt gccaatttag AGCTGCCTCC TGAGGAGAGA
GCCCAGAATG TCACTCGGCT TCTACGAAAG 101 CAGGCTGAGG AGGTCAATAC
TGAGTGGGAA AAATTGAACC TGCACTCCGC TGACTGGCAG AGAAAAATAG ATGAGACCCT
TGAAAGACTC CGGGAACTTC 201 AAGAGGCCAC GGATGAGCTG GACCTCAAGC
TGCGCCAAGC TGAGGTGATC AAGGGATCCT GGCAGCCCGT GGGCGATCTC CTCATTGACT
CTCTCCAAGA 301 TCACCTCGAG AAAGTCAAGg taccgtctac ttctttgctt
cagggccctt tgagagactc aaaagagct Exon 60 (SEQ ID NO: 77) 1
ttgttttaaa tattctcatc ttccaatttg cttttgacta ttgcacacag GCACTTCGAG
GAGAAATTGC GCCTCTGAAA GAGAACGTGA GCCACGTCAA 101 TGACCTTGCT
CGCCAGCTTA CCACTTTGGG CATTCAGCTC TCACCGTATA ACCTCAGCAC TCTGGAAGAC
CTGAACACCA GATGGAAGCT TCTGCAGgta 201 agcacattgt aaacattgtt
gtcctttgtt acagtaaaat aatatac Exon 61 (SEQ ID NO: 78) 1 tcctcattat
atagaatgag agaacatcat ttctctcctt ttcctcccag GTGGCCGTCG AGGACCGAGT
CAGGCAGCTG CATGAAGCCC ACAGGGACTT 101 TGGTCCAGCA TCTCAGCACT
TTCTTTCCAg taagtcattt tcagctttta tcacttaact ttattgcatc ttgattaat
Exon 62 (SEQ ID NO: 79) 1 gcgatgaatt tgacctcctt gcctttcttt
ttttcctccc ttcttttcag CGTCTGTCCA GGGTCCCTGG GAGAGAGCCA TCTCGCCAAA
CAAAGTGCCC 101 TACTATATCA Agtaagttgg aagtatcaca tttttaaaag
agcatttatt gtgactaacc t Exon 63 (SEQ ID NO: 80) 1 tgactactca
ttgtaaatgc taaagtcttt ctttatgttt tgtgttttag CCACGAGACT CAAACAACTT
GCTGGGACCA TCCCAAAATG ACAGAGCTCT 101 ACCAGTCTTT AGgtaaggac
atggccatgt ttcctccaag ttaaatgaca ggtgaccttt ag Exon 64 (SEQ ID NO:
81) 1 ctgttatttc tgatggaata acaaatgctc tttgttttcc ctcttttcag
CTGACCTGAA TAATGTCAGA TTCTCAGCTT ATAGGACTGC CATGAAACTC 101
CGAAGACTGC AGAAGGCCCT TTGCTgtaag tattggccag tatttgaaga tcttgatact
atgtctttgc ttaga Exon 65 (SEQ ID NO: 82) 1 aggaaggttt tactctttga
gtcatttgtg attttatttg ttttttgcag TGGATCTCTT GAGCCTGTCA GCTGCATGTG
ATGCCTTGGA CCAGCACAAC 101 CTCAAGCAAA ATGACCAGCC CATGGATATC
CTGCAGATTA TTAATTGTTT GACCACTATT TATGACCGCC TGGAGCAAGA GCACAACAAT
TTGGTCAACG 201 TCCCTCTCTG CGTGGATATG TGTCTGAACT GGCTGCTGAA
TGTTTATGAT ACgtacgtat ggcatgtttt tatttcccgg gctctgtcac aggaggctta
301 Gc Exon 66 (SEQ ID NO: 83) 1 cctctaggaa agggtcagta attgttttct
gctttgattc ttcataatag GGGACGAACA GGGAGGATCC GTGTCCTGTC TTTTAAAACT
GGCATCATTT 101 CCCTGTGTAA AGCACATTTG GAAGACAAGT ACAGATgtaa
gtcgtgtata ttaatgctgt attcttttat taatgttggc taatta Exon 67 (SEQ ID
NO: 84) 1 atccatgggt gctgtgtttt gactgttgca attttcttct tcctttgtag
ACCTTTTCAA GCAAGTGGCA AGTTCAACAG GATTTTGTGA CCAGCGCAGG 101
CTGGGCCTCC TTCTGCATGA TTCTATCCAA ATTCCAAGAC AGTTGGGTGA AGTTGCATCC
TTTGGGGGCA GTAACATTGA GCCAAGTGTC CGGAGCTGCT 201 TCCAATTTgt
aagttattca ccttctaggt aacatattta ttctttcata ttttagaa Exon 68 (SEQ
ID NO: 85) 1 ctttcctttc atccttttgc cctccttctc tctccctcct gtctttgcag
GCTAATAATA AGCCAGAGAT CGAAGCGGCC CTCTTCCTAG ACTGGATGAG 101
ACTGGAACCC CAGTCCATGG TGTGGCTGCC CGTCCTGCAC AGAGTGGCTG CTGCAGAAAC
TGCCAAGCAT CAGGCCAAAT GTAACATCTG CAAAGAGTGT 201 CCAATCATTG
GATTCAGgta ttaggaacca aaaaaaaaat gtcatttttt tctcatcatt tttcacc Exon
69 (SEQ ID NO: 86) 1 ggaatttgat tcgaagaaat acatacgtgt ttgtttttgc
tctttatcag GTACAGGAGT CTAAAGCACT TTAATTATGA CATCTGCCAA AGCTGCTTTT
101 TTTCTGGTCG AGTTGCAAAA GGCCATAAAA TGCACTATCC CATGGTGGAA
TATTGCACTC CGgtaagttt gacgccagcc tgacgtgaga gttagttcac
201 ctgggataaa tt Exon 70 (SEQ ID NO: 87) 1 tttgaaatca tcctgtccta
aatctgatct caccatgatc tcccttttag ACTACATCAG GAGAAGATGT TCGAGACTTT
GCCAAGGTAC TAAAAAACAA 101 ATTTCGAACC AAAAGGTATT TTGCGAAGCA
TCCCCGAATG GGCTACCTGC CAGTGCAGAC TGTCTTAGAG GGGGACAACA TGGAAACtgt
agtagtagca 201 aaagcagaac acactcttgt ttgatgtata tttgaac Exon 71
(SEQ ID NO: 88) 1 cggctgagtt tgcgtgtgtc tccttcacca cctcattttt
tgttttgcag TCCCGTTACT CTGATCAACT TCTGGCCAGT AGATTCTGCg tgagtacttt
101 ttttgctgaa gggtgctgct accaccaaca cattcgctc Exon 72 (SEQ ID NO:
89) 1 tctccattaa tggatggtat ctgtgactaa tcacattttc tgccttatag
GCCTGCCTCG TCCCCTCAGC TTTCACACGA TGATACTCAT TCACGCATTG 101
AACATTATGC TAGCAGgtat gagactagtt gtatgccagg caaatattga ttgaaataac
taacca Exon 73 (SEQ ID NO: 90) 1 gattctaaga cgtcacataa gttttaatga
gcttttacgt tttttatcag GCTAGCAGAA ATGGAAAACA GCAATGGATC TTATCTAAAT
GATAGCATCT 101 CTCCTAATGA GAGCATgtaa gtatcccatc tctttttaca
aaatgttcct gacaatgaaa ttgctt Exon 74 (SEQ ID NO: 91) 1 aagcaaaata
agggggggaa aaaaccaaaa cctttgattt tattttccag AGATGATGAA CATTTGTTAA
TCCAGCATTA CTGCCAAAGT TTGAACCAGG 101 ACTCCCCCCT GAGCCAGCCT
CGTAGTCCTG CCCAGATCTT GATTTCCTTA GAGAGTGAGG AAAGAGGGGA GCTAGAGAGA
ATCCTAGCAG ATCTTGAGGA 201 AGAAAACAGg tgagttttct ttctagcttt
gtcattggta tgcagagtgc atacacttg Exon 75 (SEQ ID NO: 92) 1
ttttcttttt ctttcttttt ttttcttttt tacttttttg atgccaatag GAATCTGCAA
GCAGAATATG ACCGTCTAAA GCAGCAGCAC GAACATAAAG 101 GCCTGTCCCC
ACTGCCGTCC CCTCCTGAAA TGATGCCCAC CTCTCCCCAG AGTCCCCGGG ATGCTGAGCT
CATTGCTGAG GCCAAGCTAC TGCGTCAACA 201 CAAAGGCCGC CTGGAAGCCA
GGATGCAAAT CCTGGAAGAC CACAATAAAC AGCTGGAGTC ACAGTTACAC AGGCTAAGGC
AGCTGCTGGA GCAAgtgagg 301 agagagatgg gatttttaca aacattcatt
tttccctctt aaac Exon 76 (SEQ ID NO: 93) 1 tttgtatgtt tattatgaaa
agtaattctg ttttcttttg gatgacttag CCCCAGGCAG AGGCCAAAGT GAATGGCACA
ACGGTGTCCT CTCCTTCTAC 101 CTCTCTACAG AGGTCCGACA GCAGTCAGCC
TATGCTGCTC CGAGTGGTTG GCAGTCAAAC TTCGGACTCC ATGGgtaagt gtcctagcta
ctctcagatt 201 ttgttgtctg aagaaaggta gagt Exon 77 (SEQ ID NO: 94) 1
ctgttttcta taaatgtaat tttccattat ttgtttttgc ttttattaag GTGAGGAAGA
TCTTCTCAGT CCTCCCCAGG ACACAAGCAC AGGGTTAGAG 101 GAGGTGATGG
AGCAACTCAA CAACTCCTTC CCTAGTTCAA GAGgtaagct ccaataccta gaagggactc
agatttgctg ggatcaggcc at Exon 78 (SEQ ID NO: 95) 1 tttttttccc
tttctgatat ctctgcctct tcctctctct attattaaag GAAGAAATAC CCCTGGAAAG
CCAATGAGAG AGgttagtga gattcaggct 101 cacggccatg gcttctgtct
gtctcatcct gc Exon 79 (SEQ ID NO: 96) 1 tctatctgca ccttttgtaa
agtctgtctt tctttctctt tgttttccag GACACAATGT AG
[0057] Exons for which exon skipping can be therapeutic, for the
treatment of muscular dystrophy and other conditions, will be
evident to a skilled worker. There is a substantial literature on
the design of specific exons in DMD and many thousands of other
exons in the human genome potentially amenable to exon skipping.
For instance, a nonsense mutation within an exon which if deleted
would not alter the reading frame, may be able to be removed from
the mature RNA by targeted removal by exon skipping. The possible
exons in the human genome are too numerous to list. In the DMD gene
alone, there are 79 exons and many sequences that can be used to
partially block inclusion of a given exon (from exon 2-exon 78)
that are therapeutically relevant. For example, in 2007, Wilton et
al described a series of oligos that can skip single exons across
the DMD gene. (Wilton et al (2007) Mol. Ther. 15, 1288-1296). Other
work by Pramono et al demonstrates oligo design principles (Pramono
et al. (2012) Hum Gene Ther 23(7), 781-90). Malueka et al describe
a decision metric for oligo targeting in DMD (Malueka et al (2012)
BMC Genet. 13, 23). Popplewell, et al also describe design
principles for the oligo component of the combined therapeutic
described in the present invention (Popplewell, et al (2012)
Methods Mol. Biol. 867, 143-67). Further, recently published work
by Aoki, et al describe the skipping of multiple exons from exon
45-55 in mouse (Aoki, et al (2012) Proc Natl Acad Sci USA. 109
(34), 13763-8). This is therapeutically relevant for human Duchenne
therapy as well as up to 65% of all DMD affected individuals could
be treated by this cocktail. Since the described invention works on
multiple independent exons, it is expected that the chemical
entities described herein will improve the removal of specific
individual and sets of exons from the mature transcript in vivo and
in vitro. The general field of AO design for DMD is described in
Aarstma-Rus, 2012 and Lu, 2011. Further, the removal of exonic
duplications (see Aartsma-Rus (2007), BMC Med. Genet. 5, 8:43)
commonly observed in DMD may also be improved by combination use
with the compounds described herein.
[0058] For reviews of conditions or diseases that can be treated by
a method of the invention, see, e.g., Bauman et al. (2011) Bioeng.
Bugs. 2, 125-8, Hammond et al. (2011) Trends Genet. 27, 196-205,
Wood et al. (2010) Brain 133, 957-72 or Sazani et al.,
"Splice-switching oligonucleotides as potential therapeutics"
(2007) in Antisense Drug Technology: Principles, Strategies, and
Applications, Second Edition (Ed. S. T. Crooke) 89-114 (CRC Press,
Boca Raton). Among the diseases treatable by modulation of exon
skipping are, e.g., spinal muscle atrophy (SMA), Hutchinson-Gilford
progeria syndrome (HGPS), beta-thalassemia, Ataxia telangiectasia
(ATM), dysferlinopathies, frontotemporal dementia and cystic
fibrosis.
[0059] In embodiments of the invention, a compound of the invention
is administered to a subject, e.g. as part of an adjuvant
treatment, or is contacted (e.g., in vitro) with a pre-mRNA target
of interest, in conjunction with a suitable AO that is designed to
specifically block a splicing event of interest. "In conjunction
with" means that the AO can be administered before, or at the same
time as, or after, the compound, and that the two components can be
administered in separate delivery vehicles or in the same delivery
vehicle. The two agents can be administered with the same, or
different, dosage regimens. As used herein, the singular forms "a,"
"an," and "the" include plural referents unless the context clearly
dictates otherwise. For example, "an" AO, as used above, means one
or more AO molecules, which can be the same or different.
[0060] A number of considerations are generally taken into account
in designing delivery systems, routes of administration, and
formulations for compounds or combinations of compounds and an AO
of the invention. The appropriate delivery system for an agent of
the invention will depend upon its particular nature, the
particular clinical application, and the site of drug action. One
skilled in the art can easily determine the appropriate dose,
schedule, and method of administration for the exact formulation of
the composition being used, in order to achieve the desired
response in the individual patient.
[0061] Any of a variety of conventional methods can be used to
introduce AOs and/or small molecules of the invention into cells,
in vitro or in vivo. These methods include, for example,
transfection, electroporation, hydrodynamic "high pressure"
delivery, nanoparticle delivery, liposomes, colloidal dispersal
systems, or other methods known in the art.
[0062] Intracellular AO delivery can be enhanced by conjugating
cell penetrating peptides to the AO using methods and compounds
known in the art. See, e.g., U.S. Pat. No. 7,468,418 and PCT
publications WO2009/005793 and WO2009/147368.
[0063] Compounds and AO's can be administered (delivered) to a
subject by the same or by different modes of administration.
Suitable modes of administration include, e.g., subcutaneous,
intramuscular, intravenous, oral, intranasal, cutaneous, or
suppository routes, depending on the formulation, the compound, and
the condition to be treated. Compounds and AO's of the invention
may be delivered via a variety of routes including all of the above
routes, in dosing patterns that can be optimized with routine,
conventional methods. In one embodiment, the compounds are
administered chronically to subjects (patients) in conjunction with
therapeutic antisense oligonucleodies. In some embodiments, a
compound of the invention is administered frequently (e.g., daily
or more frequently) to augment less frequent (e.g., monthly or
weekly) administration, such as by intravenous or subcutaneous
injection, of AO.
[0064] Formulations for delivery by a particular method (e.g.,
solutions, buffers, and preservatives) can be optimized by routine,
conventional methods that are well-known in the art. See, e.g.,
Remington's Pharmaceutical Sciences, 18.sup.th edition (1990, Mack
Publishing Co., Easton, Pa.). for guidance in suitable
formulations.
[0065] An "effective" dose of an agent of the invention (either a
compound, or a compound in conjunction with an AO, or the AO), or
composition thereof, is a dose that, when administered to an
animal, particularly a human, in the context of the present
invention, is sufficient to effect at least a detectable amount of
a therapeutic response in the individual over a reasonable time
frame.
[0066] The exact amount of the dose (of a small molecule of the
invention, used alone or in conjunction with an AO, or of the AO),
will vary from subject to subject, depending on the species, age,
weight and general condition of the subject, the severity or
mechanism of any disorder being treated, the particular agent or
vehicle used, its mode of administration and the like. The dose
will also be a function of the exon that is being skipped/removed
from the mature RNA and the sequence of the AO. The dose used to
achieve a desired effect in vivo will be determined by the potency
of the particular agent employed, the pharmacodynamics associated
with the agent in the host, the severity of the disease state of
infected individuals, as well as, in the case of systemic
administration, the body weight and age of the individual. The size
of the dose also will be determined by the existence of any adverse
side effects that may accompany the particular inhibitory agent, or
composition thereof, employed. It is generally desirable, whenever
possible, to keep adverse side effects to a minimum.
[0067] For example, a dose of a small molecule of the invention can
range from about 4-10 mg/kg/day, or can be higher or lower. In
general, the dose of a small molecule of the invention is one, or
close to one, which has been shown to be safe for subjects, such as
human patients. Dantrolene, for example, has been shown to be safe
when administered to humans up to 8 mg/kg/day during long term
administration. Suitable oral doses of Dantrolene include doses of
about 4-10, e.g. about 6-8, mg/kg/day. An example herein shows a
functional benefit (wire hang test in mdx mice) using 10 mg/kg/week
of the oligo AON23 and dantrolene at 10 mg/kg/day compared to 10
mg/kg/week of the AON23 alone (p=0.022).
[0068] Dosages for administration of a therapeutic agent of the
invention can be in unit dosage form, such as a tablet or capsule.
The term "unit dosage form" as used herein refers to physically
discrete units suitable as unitary dosages for human and animal
subjects, each unit containing a predetermined quantity of an
inhibitor of the invention, alone or in combination with other
therapeutic agents, calculated in an amount sufficient to produce
the desired effect in association with a pharmaceutically
acceptable diluent, carrier, or vehicle.
[0069] One embodiment of the invention is a method for identifying
a small molecule compound that enhances exon skipping in an mRNA of
interest, comprising testing candidate small molecules, such as
variants of a compound in Table 1, for their ability to enhance
exon skipping in the mRNA, and selecting compounds which exhibit
greater enhancement activity than the compound from Table 1. The
screening method can be carried out in the absence of, or in
conjunction with, an AO specific for a splicing sequence of the
exon that is to be skipped.
[0070] In one embodiment, the method comprises contacting a
suitable cell (in vitro or in vivo) with a putative small molecule
compound, such as a variant of one of the compounds of Table 1, and
measuring the amount of splicing or, in one embodiment, of exon
skipping, of interest. Any of the assays discussed herein can be
adapted to such a screen. The amount of splicing or exon skipping
can be compared to a control value. For example, for an assay which
is conducted in the absence of an AO, the control can be a cell
that has not been contacted with the compound. For an assay which
is conducted in the presence of a suitable AO, the control can be a
cell that is contacted with the AO but not the putative compound. A
statistically significant decrease in the amount of splicing or
increase in the amount of exon skipping in the test cells compared
to the control is indicative that the putative compound is superior
to the compound from which it has been derived, or to a suitable
arbitrarily selected control compound.
[0071] As Dantrolene has a known molecular target, the ryanodine
receptor which it binds directly, other agents that modify the
activity of the ryanodine receptor are likely to have the same
effect. For instance, a class of agents called `RyCals` or `calcium
channel stabilizers` which stabilize the interaction of calstabin
with ryanodine receptor and effectively block ryanodine receptor
calcium leak are expected to have a similar effect as Dantrolene.
See, e.g., Andersson et al. (2010) Drug Discov Today Dis Mech 7,
3151-e157 or Wehrens et al. (20050 Proc Natl Acad Sci USA 102,
9607-12.
[0072] Suitable variant compounds that can be tested will be
evident to a skilled worker. For example, a substituent on, e.g.,
an aromatic or non-aromatic carbon can be substituted with H,
alkyl, alkoxy, hydroxyalkyl, thioalkyl, haloalkyl, aminoalkyl,
alkoxyalkyl, alkylaminoalkyl, etc. Some suitable variants are
discussed below. Others will be evident to a skilled worker.
Suitable (e.g., improved) variant compounds that are identified by
such a screen are also included in the invention, and are sometimes
referred to herein as "active variants" of the compounds. An
"active variant," as used herein, refers to a compound which
retains at least one activity of the compound of which it is a
variant, e.g. the ability to block splicing of an exon of
interest.
[0073] The terms "alkyl" used alone or as part of a larger moiety
(i.e. "alkoxy," "hydroxyalkyl," "alkoxyalkyl," and
"alkoxycarbonyl") include both straight and branched chains
containing one to ten carbon atoms (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 carbon atoms), as well as cyclic structures such as
cyclopropyl and cyclobutyl. Examples of alkyl groups include methyl
(Me), ethyl (Et), propyl (Pr) (including n-propyl (.sup.nPr or
n-Pr), isopropyl (.sup.iPr or i-Pr) and cyclopropyl (.sup.cPr or
c-Pr)), butyl (Bu) (including n-butyl (.sup.nBu or n-Bu), isobutyl
(.sup.iBu or i-Bu), tert-butyl (Bu or t-Bu) and cyclobutyl
(.sup.cBu or c-Bu)), pentyl (Pe) (including n-pentyl) and so forth.
Alkyl groups also include mixed cyclic and linear alkyl groups,
such as cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl,
etc., so long as the total number of carbon atoms is not exceeded.
The term "alkoxy" refers to an --O-alkyl radical, such as, for
example --O-Me, --O-Et, --O-Pr, and so on. The term "hydroxyalkyl"
refers to an alkyl group substituted with one or more hydroxyl,
such as, for example, hydroxymethyl, 1-hydroxyethyl,
2-hydroxyethyl, 1,2-dihydroxyethyl, and so forth. The term
"thioalkyl" refers to an --S-alkyl group, such as, for example,
example --S-Me, --S-Et, --S--Pr. The term "haloalkyl" means alkyl,
substituted with one or more halogen atoms, such as
trifluoromethyl, chloromethyl, 2,2,2-trifluoroethyl,
1,1,2,2,2,-petanfluoroethyl, and so on. The term "aminoalkyl" means
alkyl, substituted with an amine group (NH.sub.2), such as, for
example, aminomethyl, 1-aminoethyl, 2-aminoethyl, 3-aminopropyl and
so forth. The term "alkoxyalkyl" refers to an alkyl group,
substituted with an alkoxy group, such as, for example,
methoxymethyl, ethoxymethyl, methoxyethyl, and so forth. As used
herein, the term "alkylaminoalkyl" refers to an alkyl group
substituted with an alkylamine group, such as, for example,
N-methylaminomethyl, N,N-dimethylaminomethyl,
N,N-methylpentylaminomethyl, 2-(N-methylamino)ethyl,
2-(N,N-dimethylamino)ethyl, and so forth.
[0074] The term "halogen" or "halo" means F, Cl, Br, or I.
[0075] The term "nitro" means (--NO.sub.2).
[0076] The term "amine" or "amino" used alone or as part of a
larger moiety refers to unsubstituted (--NH.sub.2). The term
"alkylamine" refers to mono- (--NRH) or di-substituted (--NR.sub.2)
amine where at least one R group is an alkyl substituent, as
defined above. Examples include methylamino (--NHCH.sub.3),
dimethylamino (--N(CH.sub.3).sub.2). The term "arylamine" refers to
a mono (--NRH) or di-substituted (--NR.sub.2) amine, where at least
one R group is an aryl group as defined below, including, for
example, phenylamino, diphenylamino, and so forth. The term
"heteroarylamine" refers to a mono (--NRH) or di-substituted
(--NR.sub.2) amine, where at least one R group is a heteroaryl
group as defined below, including, for example, 2-pyridylamino,
3-pyridylamino and so forth. The term "aralkylamine" refers to a
mono (--NRH) or di-substituted (--NR.sub.2) amine, where at least
one R group is an aralkyl group, including, for example,
benzylamino, phenethylamino, and so forth. The term
"heteroaralkylamine" refers to a mono (--NRH) or di-substituted
(--NR.sub.2) amine, where at least one R group is a heteroaralkyl
group. As used herein, the term "alkylaminoalkyl" refers to an
alkyl group substituted with an alkylamine group. Analogously,
"arylaminoalkyl" refers to an alkyl group substituted with an
arylamine, and so forth, for any substituted amine described
herein.
[0077] The term "alkenyl" used alone or as part of a larger moiety
include both straight and branched chains containing at least one
double bond and two to ten carbon atoms (i.e. 2, 3, 4, 5, 6, 7, 8,
9, or 10 carbon atoms), as well as cyclic, non-aromatic alkenyl
groups such as cyclopropenyl, cyclobutenyl, cyclopentenyl,
cyclopentadienyl, cyclohexenyl, cyclohexadienyl, etc. As used
herein, alkenyl groups also include mixed cyclic and linear alkyl
groups, such as cyclopentenylmethyl, cyclopentenylethyl,
cyclohexenylmethyl, etc., so long as the total number of carbon
atoms is not exceeded. When the total number of carbons allows
(i.e. more than 4 carbons), an alkenyl group may have multiple
double bonds, whether conjugated or non-conjugated, but do not
include aromatic structures. Examples of alkenyl groups include
ethenyl, propenyl, butenyl, butadienyl, isoprenyl, dimethylallyl,
geranyl and so forth.
[0078] The term "aryl" used alone or as part of a larger moiety,
refers to mono-, bi-, or tricyclic aromatic hydrocarbon ring
systems having five to fourteen members, such as phenyl,
1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl. The term
"aryl" may be used interchangeably with the term "aryl ring".
"Aryl" also includes fused polycyclic aromatic ring systems in
which an aromatic ring is fused to one or more rings. Examples
include 1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl. Also
included within the scope of the term "aryl", as it is used herein,
is a group in which an aromatic ring is fused to one or more
non-aromatic rings, such as in an indanyl, phenanthridinyl or
tetrahydronaphthyl, where the radical or point of attachment is on
the aromatic ring. The term "aralkyl" refers to an alkyl
substituent substituted by an aryl group. The term "aryloxy" refers
to an --O-aryl group, such as, for example phenoxy, 4-chlorophenoxy
and so forth. The term "arylthio" refers to an --S-aryl group such
as, for example phenylthio, 4-chlorophenylthio, and so forth. The
term "aryl" used alone or as part of a larger moiety also refers to
aryl rings that are substituted such as, for example,
4-chlorophenyl, 3,4-dibromophenyl and so forth. An aryl group may
have more than one substituent, up to the total number of free
substitution positions. For example, an aryl group may have 1, 2,
3, 4, or 5 substituents. The substituents may the same or
different. Substituents on an aryl group include hydrogen, halogen,
alkyl, alkenyl, nitro, hydroxyl, amino, alkylamino, alkoxy, and
alkylthio, O-acyl, N-acyl, S-acyl as defined herein.
[0079] The term "heteroaryl", used alone or as part of a larger
moiety, refers to heteroaromatic ring groups having five to
fourteen members, preferably five to ten, in which one or more ring
carbons, preferably one to four, are each replaced by a heteroatom
such as N, O, or S. Examples of heteroaryl rings include 2-furanyl,
3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl,
3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxadiazolyl,
5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 1-pyrrolyl,
2-pyrrolyl, 3-pyrrolyl, 1-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl,
2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl,
5-pyrimidyl, 3-pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,
5-tetrazolyl, 2-triazolyl, 5-triazolyl, 2-thienyl, 3-thienyl,
carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl,
quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl,
benzimidazolyl, isoquinolinyl, indazolyl, isoindolyl, acridinyl, or
benzoisoxazolyl. Also included within the scope of the term
"heteroaryl", as it is used herein, is a group in which a
heteroaromatic ring is fused to one or more aromatic or nonaromatic
rings where the radical or point of attachment is on the
heteroaromatic ring. Examples include tetrahydroquinolinyl,
tetrahydroisoquinolinyl, and pyrido[3,4-d]pyrimidinyl. The term
"heteroaryl" may be used interchangeably with the term "heteroaryl
ring" or the term "heteroaromatic." The term "heteroaralkyl" refers
to an alkyl group substituted by a heteroaryl, such as, for
example, 2-pyridylmethyl, 3-pyridylmethyl, 1-imidazolomethyl,
2-imidazolomethyl and so forth. The term "heteroaryloxy" refers to
an --O-heteroaryl group. The term "heteroarylthio" refers to an
--S-aryl group. A heteroaryl group may have more than one
substituent, up to the total number of free substitution positions.
For example, a heteroaryl group may have 1, 2, 3, 4, or 5
substituents. The substituents may the same or different.
Substituents on a heteroaryl group include hydrogen, halogen,
alkyl, alkenyl, nitro, hydroxyl, amino, alkylamino, alkoxy, and
alkylthio, O-acyl, N-acyl, S-acyl as defined herein.
[0080] The term "O-acyl" refers to an "--O--C(O)-alkyl,"
"--O--C(O)-aryl," or "--O--C(O)-heteroaryl" group. The term
"N-acyl" refers to an "--NR--C(O)-alkyl," "--NR--C(O)-aryl," or
"--NR--C(O)-heteroaryl" where R is an alkyl, hydroxyl, or alkoxy
group. The term "S-acyl" refers to "--S--C(O)-alkyl,"
"--S--C(O)-aryl," or "--S--C(O)-heteroaryl." The term "N--O-acyl"
refers to an "N--O--C(O)-alkyl," "N--O--C(O)-aryl," or
"N--O--C(O)-heteroaryl" group.
[0081] As used herein, a "substituted" structure refers to a
chemical structure where a hydrogen atom has been replaced by a
substituent. A "substituent" is a chemical structure that replaces
a hydrogen atom on the substituted structure. The term
"substituent" does not imply that the substituent is smaller than
the substituted structure.
[0082] Another embodiment of the invention is a combination for
enhancing exon skipping in an mRNA of interest, comprising a
compound from Table 1 and an AO that is specific for an exon that
is to be skipped, and, optionally, a pharmaceutically acceptable
carrier. In one embodiment, the combination comprises a dosage form
of a compound of Table 1 and a dosage form of an AO that is
specific for the exon which is to be skipped.
[0083] Suitable pharmaceutically acceptable carriers will be
evident to a skilled worker. For guidance, see, e.g., Remington's
Pharmaceutical Sciences (supra).
[0084] Another embodiment of the invention is a kit for carrying
out one of the methods of the invention. For example, a kit for
enhancing exon skipping in a pre-mRNA of interest can comprise a
compound from Table 1 and an AO that is specific for an exon
splicing sequence in the mRNA of interest. A kit for enhancing exon
skipping in a muscle dystrophin mRNA in a subject that has Duchenne
Muscular Dystrophy (DMD), in an animal model of DMD, or in an
animal that is not necessarily an animal model for DMD, such as a
monkey, can comprise a dosage form of a compound of Table 1 and a
dosage form of an AO that is specific for the exon which is to be
skipped.
[0085] A kit of the invention can comprise a device, composition,
or other means for administering the agents of the invention to a
subject. A kit suitable for a therapeutic treatment in a subject
may further comprise a pharmaceutically acceptable carrier and,
optionally, a container or packaging material.
[0086] Optionally, the kits comprise instructions for performing
the method, and/or a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products (such as the FDA), which
notice reflects approval by the agency of manufacture, use or sale
for human administration. In addition, agents in a kit of the
invention may comprise other therapeutic compounds, for combination
therapy. Other optional elements of a kit of the invention include
suitable buffers, pharmaceutically acceptable carriers, or the
like, containers, or packaging materials. The reagents of the kit
may be in containers in which the reagents are stable, e.g., in
lyophilized form or stabilized liquids. The reagents may also be in
single use form, e.g., in single dosage form for use as
therapeutics, or in single reaction form for diagnostic use.
[0087] Methods for making and using antisense and/or small molecule
reagents, and for testing them for desirable properties, are
conventional and well-known in the art. Guidance in performing some
of the methods of the invention is provided, for example, in
Sambrook et al., Molecular Cloning, A Laboratory Manual (volumes
Cold Spring Harbor Laboratory Press, USA or Harlowe and Lane,
Antibodies a Laboratory Manual 1988 and 1998, Cold Spring Harbor
Laboratory Press, USA. These and other references cited herein
which provide guidance for performing methods related to the
present invention are incorporated by reference herein in their
entirety.
[0088] In the foregoing and in the following examples, all
temperatures are set forth in uncorrected degrees Celsius; and,
unless otherwise indicated, all parts and percentages are by
weight.
EXAMPLES
Example I
Identification of Small Molecule Enhancers of Antisense Mediated
Exon Skipping
[0089] In this Example, we describe the implementation of a
strategy to identify compounds that synergize with AO to promote
exon skipping and the follow-up of a lead hit, dantrolene, in
mutation repair of specific mouse and human models of Duchenne
muscular dystrophy in vitro and in vivo. In contrast to prior
screens aimed at identifying small molecules which impact exon
skipping, our screen is unique at least because it relies on robust
quantitation of a skipping reporter in the context of a muscle
lineage cell in the presence and absence of suboptimal AO. These
screens were performed using a mouse muscle cell line (C2C12)
expressing a human DMD exon 50 GFP based reporter [18] selected to
minimize experimental variation and sensitivity in the context of
an automated and quantitative fluorescent scanning system.
[0090] The BioMol small molecule library (n=503) was screened at an
effective concentration of 10 uM (dissolved in DMSO) for all
compounds both in the presence and absence of 2'-O-methyl AO (AON6)
targeting the splice donor site of human DMD exon 50. AO was added
prior to small molecule incubation in an effort to identify
molecules that facilitate AO skipping rather than AO delivery. The
fluorescence induced by each compound was normalized to a vehicle
controls per plate to correct for plate to plate variability.
Fluorescence was averaged across replicate plates (n=6 without AO
screen, n=3 with AO screen). Compounds were rank ordered based on
average intensity of fluorescence and difference between the
without AO and with AO screen (FIG. 1 and Table 2).
[0091] The lipid library component of the BioMol library had high
variability and these compounds were not analyzed. Within the top
5% of compounds from the remaining BioMol library screened in the
context of AO, there was a significant over-representation of
compounds modulating intracellular calcium, including dantrolene
and ryanodine, both known to target the ryanodine receptor.
(Z=5.49; Table 3). We found this intriguing given that calcium
signaling has been previously identified as a modulator of mRNA
splicing machinery in other settings [19].
TABLE-US-00003 TABLE 2 Top 5% of compounds from the BioMol high
throughput screen either with or without AON6. Compounds were rank
ordered based on the average normalized fluorescence and the top 5%
(n = 15) from source plate 1 are given for the with and without AO
high throughput screen. BioMol Screen Results: Top 5% of Compounds
in the With AON6 Screen Fluorescence Fluorescence Fluorescence
Average Standard Compound Plate 1 Plate 2 Plate 3 Fluorescence (n =
3) Deviation Library Target 6-FORMYLINDOLO [3,2-b] 30.05 30.08
32.59 30.90 1.46 Orphan Ligand Endogenous CARBAZOLE CYCLOPIAZONIC
ACID 29.57 33.40 17.24 26.74 8.44 Ion Channel Intracellular Calcium
H-7 29.03 30.45 12.11 23.87 10.20 Enzyme Inhibitor Inhibits PKA,
PKG, MLCK, and PKC. U-0126 17.87 35.65 16.65 23.39 10.63 Enzyme
Inhibitor MEK inhibitor. AG-494 19.38 25.36 25.25 23.33 3.42 Enzyme
Inhibitor Tyrosine kinase inhibitor. HARMALINE HCl 15.00 42.55 0.22
19.26 21.48 Orphan Ligand Possible endogenous beta- carboline
derivative DANTROLENE 16.32 11.76 26.51 18.20 7.55 Ion Channel
Intracellular Calcium PINACIDIL 15.64 27.20 11.56 18.13 8.11 Ion
Channel Potassium Channels PROCAINAMIDE 13.80 19.66 20.81 18.09
3.76 Ion Channel Sodium Channels 1,1'-ETHYLIDENE-bis-L- 20.72 11.15
22.18 18.02 5.99 Orphan Ligand Bioactive tryptophan derivative
TRYPTOPHAN TYRPHOSTIN 46 8.61 30.70 14.09 17.80 11.50 Enzyme
Inhibitor EGF receptor kinase, p56, and PDGF receptor kinase
inhibitor. AG-825 17.24 23.38 11.53 17.38 5.93 Enzyme Inhibitor
HER1-2 tyrosine kinase inhibitor. AG-490 7.17 30.27 13.54 16.99
11.93 Enzyme Inhibitor JAK-2 tyrosine kinase inhibitor. H-9 18.09
19.17 13.25 16.84 3.16 Enzyme Inhibitor Protein kinase inhibitor
RYANODINE 7.93 28.86 13.31 16.70 10.87 Ion Channel Intracellutar
Calcium BioMol Screen Results: Top 5% of Compounds in the Without
AON6 Screen Fluorescence Fluorescence Fluorescence Fluorescence
Fluorescence Fluorescence Average Standard Compound Plate 1 Plate 2
Plate 3 Plate 4 Plate 5 Plate 6 Fluorescence (n = 6) Deviation
Library Target GF 109203X 22.19 15.01 13.67 66.78 64.46 68.48 41.76
27.36 Enzyme Inhibitor Protein kinase C inhibitor. Ro 31-8220 17.56
50.61 46.79 38.49 31.21 34.94 36.60 11.81 Enzyme Inhibitor Protein
kinase C inhibitor. HARMALINE HCl 31.97 27.56 27.06 22.50 16.09
19.75 24.15 5.80 Orphan Ligand Possible endogenous beta-carboline
derivative INDIRUBIN 4.64 -0.42 6.43 89.35 3.42 3.88 17.88 35.08
Enzyme Inhibitor GSK-3beta and CDK5 inhibitor. 5-IODOTUBERCIDIN
-0.41 6.65 9.13 18.27 30.70 6.10 11.74 11.08 Enzyme Inhibitor
Inhibits ERK2, adenosine kinase, CK1, CK2, and insulin receptor
kinase. DICHLOROBENZAMIL 22.19 15.01 13.67 4.78 7.44 6.66 11.63
6.58 Ion Channel Calcium Channels INDIRUBIN 24.66 2.02 6.44 8.35
6.60 1.51 8.26 8.48 Orphan Ligand Endogenous WORTMANNIN 4.99 3.07
4.23 5.92 6.44 3.78 4.74 1.29 Enzyme Inhibitor Phosphatidylinositol
3- kinase inhibitor, Docosatetra- -1.65 5.21 7.47 2.30 9.50 5.44
4.71 3.94 Endocannabinoid -- 7Z,10Z,13Z,16Z-enoyl dopamine
TETRANDRINE 3.15 2.41 5.39 4.64 5.03 6.64 4.54 1.54 Ion Channel
Calcium Channels AG-879 1.23 3.24 3.46 6.94 8.93 2.08 4.31 2.99
Enzyme Inhibitor Tyrosine kinase inhibitor. CANTHARIDIN 11.40 -1.04
0.63 5.55 4.34 4.45 4.22 4.33 Enzyme Inhibitor PP1 and PP2A
inhibitor. AG-494 5.92 1.23 1.65 7.58 3.54 5.26 4.20 2.50 Enzyme
Inhibitor Tyrosine kinase inhibitor. NIGULDIPINE 5.07 1.34 3.34
8.06 6.06 0.84 4.12 2.80 Ion Channel Calcium Channels Oleoyl
dopamine 2.78 1.51 12.34 3.21 3.30 1.32 4.08 4.14 Endocannabinoid
--
TABLE-US-00004 TABLE 3 Compounds that affect intracellular calcium
levels are overrepresented in the top 5% in the BioMol with AON6
high throughput screen. Library subsets are overrepresented in the
Top 5% of the BioMol screen both with and without AO as determined
by analyzing the rate of appearance in randomly selected subsets (N
= 20; 15 elements per subset). Biomol with AO screen has an
enrichment of intracellular calcium channels (5 standard deviations
above what is expected given random sampling) whereas BioMol
without AO screen has a slight over- representation of the enzyme
inhibitor library (2 standard deviations above what is expected
given random sampling). Rate of BioMol Screen Results Appearance in
Randomly for Top 5% Z-Score Selected Subsets (N = 20; (N = 15
compounds) [(N.sub.O - A.sub..epsilon.)/.sigma..sub..epsilon.)]
BioMol IntraLibrary 15 elements per subset) +AO -AO +AO -AO
Composition Average Standard Deviation Distribution Distribution
Z-Score Z-Score Orphan Ligand Library 84 4.19 1.72 3 2 -0.69 -1.27
Intracellular Calcium Channels 7 0.19 0.51 3 0 5.49{circumflex over
( )}* -0.37 Calcium Channels 25 1.05 1.07 0 3 -0.98 1.82 Potassium
Channels 23 1.29 0.96 1 0 -0.30 -1.34 Sodium Channels 11 0.90 1.14
1 0 0.08 -0.80 Misc. Channels 6 0.62 1.36 0 0 -0.46 -0.46 Enzyme
Inhibitor Library 84 4.29 1.62 7 8 1.68 2.30* Endocannabinoid
Library 60 3.48 1.97 0 2 -1.77 -0.75 Total # of Compounds 300 -- --
15 15 N.sub.O - # of observed compounds in each group for BioMol
Top 5% (either with or without AO) A.sub..epsilon. - Average # of
compounds expected per group over 20 randomly selected subsets
.sigma..sub..epsilon. - Standard deviation of # compounds expected
in each group over 20 randomly selected subsets
[0092] Eight of the top nine top hits from the screen with AO were
screened in a secondary assay with 12 or 16 point titrations using
the Ex50-GFP reporter C2C12 cells in the presence or absence of
AON6. Of the 8 compounds that were selected for secondary screening
only 3 exhibited: 1) 10% increase in fluorescence in the
Ex50-GFP+AO compared to the reporter line without AO and 2)
evidence of a dose response. These three compounds were
cyclopiazonic acid, dantrolene, and H-7 (FIG. 5). Dantrolene was of
high interest given that it is the only FDA approved drug of the 3
and is still currently being used as a chronic treatment for
malignant hyperthermia and muscle spasticity [20, 21].
Additionally, a previous study investigated oral dantrolene as a
potential therapy for DMD patients, based on its potential to
rectify calcium signaling defects in DMD muscle, and found that
after 2 years of daily treatment creatine kinase levels slightly
reduced, and there was a modest improvement on the manual muscle
test without substantial harmful side effects [22]. Dantrolene
treatment of mdx mice has similarly been reported to lower CK
values [23]. Therefore, dantrolene was an attractive first
candidate to evaluate the effects on exon skipping in vivo in mdx
mouse and in the context of human DMD mutations. Variants or
alternative formulations of dantrolene are also shown herein to be
effective, or would be expected by skilled workers to be effective.
These include, e.g., Revonto, azumolene (which is more water
soluble than dantrolene), and others.
[0093] Dantrolene enhancement of AO directed DMD exon skipping was
assessed in both mouse and human primary muscle cell systems in
vitro. In primary fused mouse myotubes dantrolene synergized with a
2'-O-methyl AO M23D (overlapping splice donor site from +02-18) to
enhance Dmd exon 23 skipping. Increasing concentrations of M23D
shifted the Dmd mRNA species from an unskipped form to either exon
23 skipped or dual 22 and 23 skipped forms, both of which are in
frame and lack exon 23 which contains the mdx premature stop
mutation. A sub-optimal dose of M23D AO was established as 100 nM
in order to approximate a dose that generates 20% of optimal
skipping in fused myotubes, and used to measure potentiation of
exon 23 skipping. Optimal skipping was typically achieved in a dose
range of 200-600 nM M23D. After incubation with suboptimal M23D,
the complex was removed, and 25-50 uM dantrolene was added for a
sufficient time to allow for the complete transcription of new Dmd
mRNA species [24]. RNA was extracted and the mRNA from exons 20-26
was assessed for exon 23 skipping by RT-PCR. Dantrolene increased
the amount of exon 23 skipped product at both 25 and 50 uM
concentrations (FIG. 2a). Dmd exon 23 skipping was quantitated in
these same RNA samples in a taqman based assay with primer-probe
sets spanning the Dmd splice junctions of exons 22-24 (exon skip
specific junction) and exons 22-23 (full length specific junction).
Data from each primer-probe set were normalized to the ribosomal
gene 36B4, and the ratios are displayed as the fold change of the
skip/full length mRNA levels relative to the mock treated controls
[25]. Dantrolene increased Dmd exon 23 skipping 3 fold in
combination with the suboptimal dose of 100 nM AO M23D as compared
to mouse myotubes treated with AO alone (FIG. 2b). Addition of
dantrolene in the absence of AO failed to cause exon 23 skipping,
consistent with it acting synergistically with AO to promote exon
23 skipping.
[0094] Dantrolene treatment also increased DMD exon skipping in a
disease relevant human mutational context using reprogrammed
primary DMD patient fibroblasts fused to differentiated myotubes.
The patient DMD mutation was confirmed as an exon 45-50 deletion
predicted to be rendered in frame by skipping DMD exon 51, using a
custom 15,000 probe CGH array (FIG. 6). Patient fibroblasts were
transduced with HTERT and an inducible MyoD vector [26]. Following
tamoxifen induction of MyoD activity and subsequent fusion, the DMD
patient derived cells became multi-nucleated and expressed multiple
muscle differentiation markers including MHC, myogenin, RyR1 and
(mutant) dystrophin at the RNA and/or protein level within six days
(FIG. 7), further validating this human DMD culture model. Exon 51
skipping activity was assessed in the context of transfecting an
exon 51 2'-O-methyl AO with equivalent sequence to Pro051 seven
days after fusion. This AO is directed at an exonic splicing
enhancer (ESE) sequence within exon 51. The AO was removed prior to
addition of dantrolene and cultures were harvested two days later.
A nested RT-PCR was performed between DMD exons 43-52 and levels of
exon 51 skipping were determined by quantitating capillary
electrophoresis separated fragments representing skipped and
unskipped DMD mRNA. We found that dantrolene enhanced exon 51
skipping in the presence of the suboptimal dose of AO by up to 8
fold as compared to the vehicle control (FIG. 2c). Therefore,
dantrolene exhibits synergy with two different AOs, targeting
differing regions of the DMD mRNA transcript consisting of a splice
donor site or potential ESE sequence, in both human and mouse
myotube cell culture. Dantrolene's effectiveness regardless of
sequence specificity of the AO could be potentially useful given
the wide spectrum of treatable mutations that require various AO
sequences. Thus, the versatility of dantrolene gives it a wide
range of applicability in a clinical setting.
[0095] To assess the efficacy of dantrolene as a potentiator of AO
mediated exon skipping in an art-recognized in vivo mouse model of
DMD, we utilized two separate experimental protocols in which
dantrolene was administered systemically in the context of either a
single intramuscular or single intravenous injection of AO in mdx
mice. See FIG. 15 for a schematic representation of one such
protocol. Initially drug synergy was assessed with local
intramuscular injections of morpholino AO PMOE23 (overlapping with
exon 23 splice donor site +07-18) into the tibialis anterior muscle
(TA) of mdx mice. Previous studies indicate that Mug of PMOE23
rescues up to 70% of dystrophin positive fibers as assessed by
dystrophin immunostain [7]. Therefore, Mug PMOE23 was used as a
positive control and 2 ug selected as a sub-optimal dose of AO. To
evaluate if dantrolene could facilitate exon 23 skipping and
restore dystrophin protein expression by synergizing with PMOE23,
dantrolene was administered at doses of 10 mg/kg/day or 20
mg/kg/day by intraperotineal injection for nine days following a
single intramuscular PMOE23 injection (n=3 mice per group (Table
4).
TABLE-US-00005 TABLE 4 Treatment groups for the local
administration of PMOE23 in combination with systemic dantrolene.
IM PMOE23 IP Dantrolene IP 20% Group (ug) in (mg/kg) in 20% DMSO #
# saline DMSO/saline in saline Mice Sex Age 1 Saline 10 - 3 F 15
weeks 2 Saline 20 - 3 F 15 weeks 3 10 ug -- + 3 M 15 weeks 4 2 ug
-- + 3 F 15 weeks 5 2 ug 10 - 3 M 15 weeks 6 2 ug 20 - 3 F 15
weeks
[0096] The entire TA was harvested on the tenth day and divided for
analysis into 6-7 intervals, each of 800 .mu.m length. One half of
each of the middle four intervals were pooled to prepare sufficient
protein for Western blot analysis (total of 1600 um length). Four
central sections from the other half were use for
immunofluorescence staining Western blotting demonstrated that
treatment with dantrolene at either dose in combination with 2 ug
of PMOE23 increased expression of dystrophin protein to levels
observed with the higher Mug dose of PMOE23. The induced levels of
dystrophin observed represent about 20% of C57 dystrophin levels.
Western blots from representative mice are shown in FIG. 3a,
whereas average densitometry measurements obtained by quantitating
western blots from of all of the mice in each experimental group is
shown in FIG. 3b.
[0097] Representative immunoflourescence images of TA cross
sections stained with anti-dystrophin antibody are shown in FIG. 3c
and demonstrate proper localization of dystrophin protein at the
sarcolemma in treated samples. Total fluorescence was quantitated
from TA sections by scanning four entire cross sections from each
of the mice for each experimental group. Quantitation of dystrophin
immunofluorescence was highly concordant with western blot
quantification. Again, equivalency between the Mug dose of PMOE23
and the 2 ug dose of PMOE23 in combination with dantrolene dose was
observed. Dantrolene only rescued protein expression in the
presence of PMOE23 reflecting synergistic activity of dantrolene
with exon skipping PMOE23 in vivo. Quantitation of
skipped/unskipped Dmd mRNA using taqman PCR assay in an independent
experiment similarly demonstrated that dantrolene synergizes with
1M injection of PMOE23 to facilitate exon skipping (FIGS. 8 and
9).
[0098] Results from local PMOE23 administration prompted further
exploration of dantrolene's efficacy in the context of systemic
PMOE23 delivery to mdx mice. This enabled us to assess whether
dantrolene in combination with systemic morpholino PMOE23 could
enhance Dmd exon 23 skipping and induce dystrophin protein
expression in multiple skeletal muscles. A single intravenous dose
of 100 mg/kg PMOE23 was used as a positive control. A single
intravenous dose of 10 mg/kg AO was used as a sub-optimal dose
alone or in combination with twice daily dosing of 10 mg/kg/day of
dantrolene intraperitoneally for the subsequent 6 days [7, 27] (n=3
in control groups and n=4 in experimental groups; Table 5).
TABLE-US-00006 TABLE 5 Treatment groups for the systemic
administration of PMOE23 in combination with systemic dantrolene.
IP Dantrolene Systemic PMOE23 (mg/kg) in 20% IP 20% DMSO Group #
(ug) in saline DMSO/saline in saline # Mice Sex Age 1 Saline 10 - 3
F 6 weeks 2 100 mg/kg (2 mg) -- + 3 M 6 weeks 3 10 mg/kg (.2 mg) --
+ 3 M 6 weeks 4 10 mg/kg (.2 mg) 10 - 3 F 6 weeks
[0099] Multiple skeletal muscles were harvested for analysis on day
7 including the quadriceps, gastrocnemius, tibialis anterior,
diaphragm, triceps and heart. Muscles were assessed for: 1)
increased amounts of skipped Dmd exon 23 mRNA species 2) Dystrophin
protein rescue by Western blot 3) Dystrophin protein expression by
quantitative immunostain, 4) appropriate subcellular localization,
and 5) restoration of other components of the dystroglycan complex
to the sarcolemmal membrane. To determine if dantrolene enhanced
Dmd exon 23 skipping, the quantitative taqman assay was performed
on RNA from each skeletal muscle. Dantrolene significantly
increased Dmd exon 23 mRNA skipping in an aggregate analysis of all
skeletal muscle groups (excluding heart) (FIG. 4A). Analysis of
individual muscle groups demonstrated that dantrolene enhanced
skipping in the gastrocnemius, TA, diaphragm and quadriceps (FIG.
4a and FIG. 10a). Enhancement was not apparent in the triceps,
often targeted less well by AOs. No appreciable skipping was
observed in heart muscle under any experimental condition. Western
blot analysis for dystrophin protein was concordant with mRNA
skipping in all muscle groups analyzed (FIG. 4b and FIGS. 10c,11).
Pooled densitometry quantitation of western blots across the
quadricep, gastrocnemius, TA and diaphragm for all mice indicates a
mean fold increase of 3.1 in dystrophin protein expression when
PMOE23 is combined with dantrolene relative to PMOE23 with vehicle
(FIG. 4b). Quantitative immunofluorescence supports qRT-PCR and
Western blot quantitation (FIG. 4c), and further demonstrates
dantrolene enhancement of dystrophin protein expression (FIG. 4d
and FIG. 12). Immuno-staining with several anti-dystrophin
antibodies demonstrate full N and C terminal expression of
dystrophin and correct sarcolemmal localization (FIG. 4d and FIG.
13). In addition, sequential serial sections of the quadricep
muscle indicate that dystrophin expression reestablishes other
components of the DGC: .beta.-dystroglycan and .alpha.-sarcoglycan
(FIG. 4d, FIG. 10). The levels of .alpha.-sarcoglycan and
.beta.-dystroglycan expression with 2 ug of PMOE23 and dantrolene
are similar to that induced by bug from the higher systemic dose of
PMOE23. The ability of rescued dystrophin to recruit other members
of the DGC is suggestive of its ultimate functionality in vivo.
Taken together these data demonstrate that dantrolene synergizes
with suboptimal dosing of systemic PMOE23 to facilitate exon
skipping and rescue of dystrophin protein and sarcolemmal DGC
expression in multiple muscles.
[0100] Our results suggest a model in which dantrolene synergizes
with AOs, regardless of sequence specificity and chemistry, to
enhance targeted DMD exon skipping. This has been demonstrated both
in vitro in mouse and human cell systems, as well as in multiple
skeletal muscles with intramuscular and intravenous delivery of
PMOE in the mdx mouse. Given the timing of addition of AO and drug,
it is unlikely that dantrolene is enhancing uptake of AO. Without
wishing to be bound by any particular mechanism, we suggest that it
is enhancing exon skipping through interaction with a specific
molecular target that is modulating DMD splicing activity. The
concept of utilizing small molecules to increase exon skipping
efficiency has been demonstrated in a patient with a rare point
mutation in DMD exon 31 that disrupts an ESE binding site for the
SRp30c splicing factor. The addition of TG003, a specific inhibitor
for Clks known to phosphorylate SR proteins increased mutant exon
31 skipping and facilitated dystrophin protein rescue [28]. However
this therapeutic strategy is unlikely to be generalizable to broad
treatment of DMD patients.
[0101] Without wishing to be bound by any particular mechanism, we
propose that the mechanism by which dantrolene facilitates exon
skipping may be that it functions by targeting the ryanodine
receptor, its known molecular target. Ryanodine receptor regulates
calcium release from the sarcoplasmic reticulum during
excitation-contraction coupling in skeletal muscle. Because calcium
signaling is a known regulator of splicing activity, dantrolene
modulation of RyR1 mediated calcium flux in muscle is a plausible
mechanism of its activity, which we are currently investigating.
Further RyR expression on the nucleoplasmic reticulum has been
implicated in regulating calcium signaling in the nucleus [29].
Hypermitrosylation of RyR in DMD has been attributed to calcium
leak and downstream DMD pathology, possibly from calium regulated
protein degradation [30]. A more recently developed class of drugs,
called `rycals` stabilize the cardiac RyR2/calstabin interactions,
and are under active development for heart failure treatment to
prevent a chronic leak of calcium through RyR2 [31]. Thus,
dantrolene and rycals which prevent chronic calcium leak have been
proposed as potential therapeutics for DMD. While it is possible
that the synergistic action of dantrolene in mdx muscle is
secondary to stabilization of proteins necessary for regulating the
splicing machinery that were previously being degraded, this is
unlikely, as we have observed effects of dantrolene on exon 23
skipping in cultured myotubes from C57BL6 as well as mdx mice.
Nonetheless, potential activity of dantrolene resulting from
non-splicing related effects of calcium modulation may provide
another level of synergy in protecting DMD muscle function.
[0102] Studies of long-term dantrolene efficacy in the context of
multiple AP injections and functional redouts, in the models
presented herein as well as in humans, will confirm the results
presented herein, demonstrating that the optimized administration
of the agents of the invention improves DMD disease
progression.
Example II
Supplementary Studies
A. Materials and Methods
High-Throughput Screen and Secondary Screening in the Reporter Cell
Line
[0103] A stable clone was generated from C2C12 cells transfected
with a human exon 50 DMD GFP reporter (ex50GFP) that has been
previously described [18]. Ex50GFP reporter myoblasts were seeded
into uncoated 384 well plates and were incubated for 4 hours either
with or without 300 nM of 2'-O-methyl phosphorothioate AON6 [5'
AACUUCCUCUUUAACAGAAAAGCAUAC 3' (SEQ ID NO:1)] targeting the human
exon 50 splice donor site. Cells were transfected with AON6 using
the FUGENE (Roche) transfection reagent per manufacturer's
instructions. Following AON6 incubation, each component of the
BioMol library (n=503) was screened at 10 uM concentration with a
final concentration of the DMSO carrier being 1%. Forty-eight hours
later fluorescence was measured using the MicroXpress high content
imager and analyzed using MetaXpress. Immediately preceding
imaging, DNA was stained with Hoescht for 30 min. The BioMol screen
without AO (-AO) was performed in 6 replicates, and the with AO
(+AO) screen was performed in 3 replicates. For the screen analysis
raw fluorescence values were normalized to carrier controls present
on each plate by subtracting the values. Negative fluorescence
values were set to 0. The data from each compound were averaged
from all replicates. For secondary screening by 12 or 16 point dose
response, Ex50GFP myoblasts or C2C12 cells without the reporter
were seeded on uncoated 384 well plates. A sub-optimal dose of AON6
targeting DMD exon 50 was added for 4 hours. Following the 4 hour
incubation, a compound dilution was added (beginning at 100 uM with
1:1 dilutions) for either 12 or 16 points. After a 48 hour
incubation with compounds, DNA was stained with Hoescht, and
fluorescence determined using the high content imager and analyzed
with MetaXpress.
Primary Cell Culture and Antisense Oligonucleotide Transfection
[0104] Primary mouse myoblasts were isolated from the quadriceps of
a C57/B16 mouse and were carried in culture with 20% FBS in DMEM
and 2 ng/uL FGF. For Dmd exon 23 skipping assays cells were seeded
onto extracellular matrix (ECM) (Sigma) coated plates in growth
media. On day 2 growth media was removed and fusion media (2% horse
serum in phenol red free DMEM) was added. On day 3 a 2-O'-methyl
phosphorothioate AO M23D(+02-18) [5' GGCCAAACCUCGGCUUACCU 3' (SEQ
ID NO:3)] was transfected into cells using FUGENE per manufacturers
instructions. M23D concentrations ranged from 100 nM to 600 nM,
with 100 nM M23D representing the sub-optimal dose. On day 4 cells
were washed in PBS, and dantrolene (dissolved in DMSO) was added in
fresh fusion media. After 48 hours cells were harvested for
analysis.
[0105] Primary human dermal fibroblasts (GM05017) from a DMD
patient were obtained from Coriell and were maintained in growth
media (DMEM with 15% FBS, 1% nonessential amino acids, 1%
pen/strep). Prior to experiments the genomic DMD deletion between
exons 45-50 was confirmed using a custom CGH array with 14022
probes tiling the DMD gene (FIG. 6). Fibroblasts were then
immortalized with a lentiviral hTERT and subsequently transduced
with a previously described tamoxifen inducible lentiviral MyoD
[26] (Kind gift from J. Chamberlain). For exon 51 skipping
experiments, reprogrammed fibroblasts were seeded onto laminin
coated plates in growth media. On Day 2, 5 uM tamoxifen (Sigma) was
added in growth media. On day 3 fusion media (2% horse serum, 2%
insulin-transferrin-selenium (Sigma), 1:1 serum free DMEM to Ham's
F-10) with 1 uM tamoxifen was added to the cells. A DMD exon 51
2'-O-methyl phosphorothioate AO [5' UCAAGGAAGAUGGCAUUUCU 3' (SEQ ID
NO:2)] (MWG Operon) at position +68 to +88 was transfected into
cells on Day 7 with ExGen500 (Fermentas) per manufacturer's
instructions. AO concentrations ranged from 25-200 nM and the
sub-optimal dose of AO was 100 nM. On Day 8 the AO complex was
removed and titrations of drug or carrier (DMSO) were added to
wells for 48 hours. On day 10 cells were harvested for
analysis.
RNA Isolation, RT-PCR and qRT-PCR
[0106] RNA was isolated from cell culture using TRIZOL (mouse) and
the QIAGEN RNAeasy Microkit (human). RNA was isolated from snap
frozen skeletal muscle using the QIAGEN RNAeasy Fibrous Tissue Kit.
In the mouse cells cDNA was reverse transcribed from total RNA with
OligodT20 (Invitrogen). In the non-quantitative RT-PCR assay a
nested PCR was performed between Dmd exons 20-26 as has been
previously described [12]. The quantitative taqman assay to assess
Dmd exon 23 skipping detection was performed as previously
described [25]. In human cells dystrophin cDNA was reverse
transcribed with a gene specific primer in DMD exon 54. A nested
RT-PCR between DMD exons 43-52 was then performed using previously
described primers [10]. For identifying muscle markers in
reprogrammed fusing myotubes cDNA was reverse transcribed with
OligodT20. Primers for muscle markers were as follows: MyoD (Fwd-5'
GCAGGTGTAACCGTAACC 3' (SEQ ID NO:4), Rev-5' ACGTACAAATTCCCTGTAGC 3'
(SEQ ID NO:5)), Myosin Heavy Chain (Fwd-5' CAGTAGCCCCATCACATTTG
3'(SEQ ID NO:6), Rev-5' ATAACGCAATGGACAAGTG 3' (SEQ ID NO:7)),
Desmin (Fwd-5' CCTACTCTGCCCTCAACTTC 3' (SEQ ID NO:8), Rev-5'
AGTATCCCAACACCCTGCTC 3' (SEQ ID NO:9)), Myogenin (Fwd-5'
GCCACAGATGCCACTACTTC 3'(SEQ ID NO:10) Rev-5' CAACTTCAGCACAGGAGACC
3'(SEQ ID NO:11)). GAPDH primers are as follows: Fwd-5'
GAGCCACATCGCTCAGACAC 3' (SEQ ID NO:12), Rev-5'
CATGTAGTTGAGGTCAATGAAGG 3'(SEQ ID NO:13). The thermocycler
conditions were 94 C for 2 min, followed by 33 cycles of 94 C for
30s, 62 C for 30s, and 72 C for 30s, with a final extension of 72 C
for 10 min. Amplification of the ryanodine receptor required a
nested PCR. For the initial PCR the primers were
Fwd-5'-CATCAACTATGTCACCAGCATCCG-3' (SEQ ID NO:14) and
Rev-5'-GGCTGAACCTTAGAAGAGTC-3' (SEQ ID NO:15) and for the nested
PCR the primers were Fwd-5' GAGACCTTCTATGATGCAGC 3' (SEQ ID NO:16)
and Rev-5' AGAGCTCGTGGATGTTCTC 3'. (SEQ ID NO:17). Conditions for
the initial ryanodine receptor PCR were 95 C for 5 min, 20 cycles
of 95 C for 30s, 56 C for 2 min, 72 C for 90s and a final extension
of 72 C for 10 min. The nested PCR conditions were 95 C for 5 min,
35 cycles of 95 C for 30s, 59 C for 2 min, 72 C for 90s and a final
extension of 72 C for 10 min.
Western Blot
[0107] Total protein was isolated from flash frozen skeletal muscle
from both the membrane and cytoplasmic fractions. Briefly, 1/2 of
each analyzed muscle were homogenized for 1 minute in 1 mL of
ice-cold mito-buffer (0.2 mM EDTA, 0.25 mM sucrose, 10 mM TrisHCl,
pH 7.4) with protease/phosphatase inhibitors cocktail (Pierce) and
DNAse/RNAse and subjected to low-speed (1500 g) centrifugation for
10 min at 4 C. The supernatant was centrifuged at 100000 g (high
speed centrifugation) for 1 hr for isolation of membrane fraction.
Isolated membranes and pellet after low speed centrifugation were
combined and re-suspended in 300 uL of extraction buffer (50 mM
Tris-HCl, pH 7.4, 7 M urea, 2 M thiourea, 4% CHAPS, 2% SDS, 50 mM
beta-mercaptoethanol). Protein concentration in solubilized pellet
and supernatant after high-speed centrifugation (cytoplasmic
fraction) was determined by 2-D Quant Kit (GE Healthcare Life
Sciences). 50 ug of total protein from dystrophic mice, or 5 ug
from wildtype, was run on a 6% polyacrylamide gel and transferred
onto a nitrocellulose membrane for 2 hours at 4 C. The membrane was
blocked for 1 hr in 5% milk and then incubated with MANDYS8
(Sigma)1:500 against dystrophin or 1:5000 anti-vinculin (Sigma), a
skeletal muscle membrane protein not associated with the DGC that
was utilized as a loading control. For analysis dystrophin protein
levels were normalized to the vinculin loading control and then
pooled across treatment groups or muscles to determine average
dystrophin rescue. Dystrophin and vinculin were detected in pellet
(miofibrillar/membrane fraction) but not in cytoplasmic
fraction.
Immunofluorescence
[0108] Unfixed frozen tissue sections were air dried and incubated
for 1 hr in MOM Mouse IgG blocking reagent. Sections were incubated
with MANDYS8 (Sigma) for dystrophin detection in the rod domain,
Ab15277 (Abcam) for dystrophin detection at the C terminus, and
Manex 1A (Developmental Studies Hybridoma Bank) for dystrophin
detection at the N terminus. Staining for other members of the
dystrophin-glycoprotein complex was performed with NCL-a-SARC
(Novocastra) for alpha-sarcoglycan and NCL-b-DG (Novocastra) for
beta-dystroglycan. Nuclear DNA was visualized with a DAPI stain.
Secondary labeling was performed with a FITC labeled anti-mouse or
anti-rabbit from Vector labs.
[0109] For immunofluorescence in human cell culture terminally
fused myotubes were fixed in 2% paraformaldehyde for 15 min and
then blocked in 20% goat serum for 1 hour. Cells were washed and
then incubated with 1:40 MF20 for detection of myosin heavy chain
(Developmental Studies Hybridoma Bank). Cells were then incubated
in Alexa488 (Invitrogen) at 1:400 and were mounted in ProLong Gold
Antifade Mounting Medium with DAPI (Invitrogen).
In Vivo Administration of Antisense Oligonucleotide and
Dantrolene
[0110] PMOE23 morpholino (GeneTools) was resuspended in 150 mM
sterile saline for intramuscular injections in a 25 uL volume into
the tibialis anterior muscle. Intravenous administration of PMOE23
was achieved by tail vein injection of morpholino resuspended in
200 uL sterile saline. Dantrolene sodium salt (Sigma) was
resusupended in 100% DMSO stock solutions and diluted in sterile
saline (final 20% DMSO) immediately prior to the twice daily
intraperitoneal injection in a 200 uL volume.
Statistical Analysis
[0111] All statistical analysis were a two-tailed student's t test
with unequal variance in EXCEL.
Example III
Further Data
[0112] The experiments described in the figures as summarized below
were carried out using methods described elsewhere herein, and/or
by conventional methods that are well-known by those of skill in
the art.
[0113] These experiments provide data showing, e.g., that 7
additional small molecule compounds can enhance exon skipping of
the DMD gene of human myotube which are exon 51 skippable. See
FIGS. 16, 17 and 18. It is noted that two of these molecules
(Ryanodine and S107 (called a RYCAL) target the ryanodine receptor,
which is also targeted by dantrolene. See FIGS. 19 and 20. Without
wishing to be bound by any particular mechanism, it is suggested
that this observation supports the conclusion that blocking the
ryanodine receptor is one of the mechanisms of this effect.
[0114] Furthermore, additional confirmatory tests (titrations) are
presented and functional testing of dantrolene is shown in a mouse
system. FIG. 21 shows that low dose AO (exon 23 in mouse that
repairs the mdx mouse gene) with dantrolene improves skeletal
muscle function in a three week experiment relative to a higher
dose of AO alone.
[0115] An alternative formulation of dantrolene--Revonto--is also
shown to be effective.
REFERENCES
[0116] 1. Emery, A. E., The muscular dystrophies. Lancet, 2002.
359(9307): p. 687-95. [0117] 2. Emery, A. E., Population
frequencies of inherited neuromuscular diseases--a world survey.
Neuromuscul Disord, 1991. 1(1): p. 19-29. [0118] 3. Monaco, A. P.,
et al., Detection of deletions spanning the Duchenne muscular
dystrophy locus using a tightly linked DNA segment. Nature, 1985.
316(6031): p. 842-5. [0119] 4. Monaco, A. P., et al., An
explanation for the phenotypic differences between patients bearing
partial deletions of the DMD locus. Genomics, 1988. 2(1): p. 90-5.
[0120] 5. Nakamura, A., et al., Follow-up of three patients with a
large in frame deletion of exons 45-55 in the Duchenne muscular
dystrophy (DMD) gene. J Clin Neurosci, 2008. 15(7): p. 757-63.
[0121] 6. King, W. M., et al., Orthopedic outcomes of long-term
daily corticosteroid treatment in Duchenne muscular dystrophy.
Neurology, 2007. 68(19): p. 1607-13. [0122] 7. Alter, J., et al.,
Systemic delivery of morpholino oligonucleotide restores dystrophin
expression bodywide and improves dystrophic pathology. Nat Med,
2006. 12(2): p. 175-7. [0123] 8. Goemans, N. M., et al., Systemic
administration of PRO051 in Duchenne's muscular dystrophy. N Engl J
Med, 2011. 364(16): p. 1513-22. [0124] 9. Kinali, M., et al., Local
restoration of dystrophin expression with the morpholino oligomer
AVI-4658 in Duchenne muscular dystrophy: a single-blind,
placebo-controlled, dose-escalation, proof-of-concept study. Lancet
Neurol, 2009. 8(10): p. 918-28. [0125] 10. van Deutekom, J. C., et
al., Local dystrophin restoration with antisense oligonucleotide
PRO051. N Engl J Med, 2007. 357(26): p. 2677-86. [0126] 11. Yokota,
T., et al., Efficacy of systemic morpholino exon-skipping in
Duchenne dystrophy dogs. Ann Neurol, 2009. 65(6): p. 667-76. [0127]
12. Lu, Q. L., et al., Functional amounts of dystrophin produced by
skipping the mutated exon in the mdx dystrophic mouse. Nat Med,
2003. 9(8): p. 1009-14. [0128] 13. Crisp, A., et al., Diaphragm
rescue alone prevents heart dysfunction in dystrophic mice. Hum Mol
Genet, 2011. 20(3): p. 413-21. [0129] 14. Aartsma-Rus, A., et al.,
Theoretic applicability of antisense-mediated exon skipping for
Duchenne muscular dystrophy mutations. Hum Mutat, 2009. 30(3): p.
293-9. [0130] 15. Goemans, N. M., et al., 24 week follow-up data
from a phase I/IIa extension study of PRO051/GSK240220968 in
subjects with Duchenne muscular dystrophy, in 15th International
Congress of The World Muscle Society. 2010, Neuromuscular
Disorders. p. 639. [0131] 16. Shrewsbury, S. B., et al., Current
progress and preliminary results with the systemic administration
trial of AVI-4658, a novel phosphorodiamidate morpholino oligomer
(PMO) skipping dystrophin exon 51 in Duchenne muscular dystrophy
(DMD), in 15th International Congress of the World Muscle Society.
2010, Neuromuscular Disorders. p. 639-640. [0132] 17. Neri, M., et
al., Dystrophin levels as low as 30% are sufficient to avoid
muscular dystrophy in the human. Neuromuscul Disord, 2007.
17(11-12): p. 913-8. [0133] 18. Hu, Y., et al., Guanine analogues
enhance antisense oligonucleotide-induced exon skipping in
dystrophin gene in vitro and in vivo. Mol Ther, 2010. 18(4): p.
812-8. [0134] 19. Krebs, J., The influence of calcium signaling on
the regulation of alternative splicing. Biochim Biophys Acta, 2009.
1793(6): p. 979-84. [0135] 20. Glahn, K. P., et al., Recognizing
and managing a malignant hyperthermia crisis: guidelines from the
European Malignant Hyperthermia Group. Br J. Anaesth. 105(4): p.
417-20. [0136] 21. Verrotti, A., et al., Pharmacotherapy of
spasticity in children with cerebral palsy. Pediatr Neurol, 2006.
34(1): p. 1-6. [0137] 22. Bertorini, T. E., et al., Effect of
dantrolene in Duchenne muscular dystrophy. Muscle Nerve, 1991.
14(6): p. 503-7. [0138] 23. Quinlan, J. G., S. R. Johnson, and F.
J. Samaha, Dantrolene normalizes serum creatine kinase in MDX mice.
Muscle Nerve, 1990. 13(3): p. 268-9. [0139] 24. Tennyson, C. N., H.
J. Klamut, and R. G. Worton, The human dystrophin gene requires 16
hours to be transcribed and is cotranscriptionally spliced. Nat
Genet, 1995. 9(2): p. 184-90. [0140] 25. O'Leary, D. A., et al.,
Identification of small molecule and genetic modulators of
AON-induced dystrophin exon skipping by high-throughput screening.
PLoS One, 2009. 4(12): p. e8348. [0141] 26. Kimura, E., et al.,
Cell-lineage regulated myogenesis for dystrophin replacement: a
novel therapeutic approach for treatment of muscular dystrophy. Hum
Mol Genet, 2008. 17(16): p. 2507-17. [0142] 27. Malerba, A., et
al., Dosing regimen has a significant impact on the efficiency of
morpholino oligomer-induced exon skipping in mdx mice. Hum Gene
Ther, 2009. 20(9): p. 955-65. [0143] 28. Nishida, A., et al.,
Chemical treatment enhances skipping of a mutated exon in the
dystrophin gene. Nat Commun, 2011. 2: p. 308. [0144] 29. Marius,
P., et al., Calcium release from ryanodine receptors in the
nucleoplasmic reticulum. Cell Calcium, 2006. 39(1): p. 65-73.
[0145] 30. Bellinger, A. M., et al., Hypermitrosylated ryanodine
receptor calcium release channels are leaky in dystrophic muscle.
Nat Med, 2009. 15(3): p. 325-30. [0146] 31. Shan, J., et al., Role
of chronic ryanodine receptor phosphorylation in heart failure and
beta-adrenergic receptor blockade in mice. J Clin Invest. 120(12):
p. 4375-87.
[0147] From the foregoing description, one skilled in the art can
easily ascertain the essential characteristics of this invention,
and without departing from the spirit and scope thereof, can make
changes and modifications of the invention to adapt it to various
usage and conditions and to utilize the present invention to its
fullest extent. The preceding preferred specific embodiments are to
be construed as merely illustrative, and not limiting of the scope
of the invention in any way whatsoever. The entire disclosure of
all applications, patents, and publications cited above, including
U.S. Provisional Application No. 61/529,041, filed Aug. 30, 2011,
and in the figures are hereby incorporated in their entirety by
reference, particularly with regard to the information for which
they are cited.
Sequence CWU 1
1
96127RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1aacuuccucu uuaacagaaa agcauac
27220RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 2ucaaggaaga uggcauuucu 20320RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 3ggccaaaccu cggcuuaccu 20418DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
4gcaggtgtaa ccgtaacc 18520DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 5acgtacaaat tccctgtagc
20620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 6cagtagcccc atcacatttg 20719DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
7ataacgcaat ggacaagtg 19820DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 8cctactctgc cctcaacttc
20920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 9agtatcccaa caccctgctc 201020DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
10gccacagatg ccactacttc 201120DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 11caacttcagc acaggagacc
201220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 12gagccacatc gctcagacac 201323DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
13catgtagttg aggtcaatga agg 231424DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 14catcaactat gtcaccagca
tccg 241520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 15ggctgaacct tagaagagtc 201620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
16gagaccttct atgatgcagc 201719DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 17agagctcgtg gatgttctc
191881DNAHomo sapiens 18atgctttggt gggaagaagt agaggactgt tgtaagtaca
aagtaactaa aaatatattt 60tactgtggca taacgtttag t 8119162DNAHomo
sapiens 19ttatatttaa agttgcttcc taacttttat ttttttattt tgcattttag
atgaaagaga 60agatgttcaa aagaaaacat tcacaaaatg ggtaaatgca caattttcta
aggtaagaat 120ggtttgttac tttactttta agatctaagt tgtgaaattt tc
16220193DNAHomo sapiens 20atcattggaa gtgtgctttg ttaaattgag
tgtatttttt ttaatttcag tttgggaagc 60agcatattga gaacctcttc agtgacctac
aggatgggag gcgcctccta gacctcctcg 120aaggcctgac agggcaaaaa
ctggtatgtg acttattttt aagaaagtta actttaaact 180tagtagaatt tca
19321178DNAHomo sapiens 21attgtcggtc tctctgctgg tcagtgaaca
ctcttttgtt ttgttctcag ccaaaagaaa 60aaggatccac aagagttcat gccctgaaca
atgtcaacaa ggcactgcgg gttttgcaga 120acaataatgt aagtagtacc
ctggacaagg tctggatgct gtgacacagc atgcttca 17822193DNAHomo sapiens
22ctaggcattt ggtctcttac cttcaaatgt tttacccctt tctttaacag gttgatttag
60tgaatattgg aagtactgac atcgtagatg gaaatcataa actgactctt ggtttgattt
120ggaatataat cctccactgg caggtaagaa tcctgatgaa tggtttcctt
ttgggtaaca 180ttaatcttgt ttt 19323273DNAHomo sapiens 23ttcttgctca
aggaatgcat tttcttatga aaatttattt ccacatgtag gtcaaaaatg 60taatgaaaaa
tatcatggct ggattgcaac aaaccaacag tgaaaagatt ctcctgagct
120gggtccgaca atcaactcgt aattatccac aggttaatgt aatcaacttc
accaccagct 180ggtctgatgg cctggctttg aatgctctca tccatagtca
taggtaagaa gattactgag 240acattaaata acttgtaaaa gtggtgattt aga
27324219DNAHomo sapiens 24gattgattta tatttgtctt tgtgtatgtg
tgtatgtgta tgtgttttag gccagaccta 60tttgactgga atagtgtggt ttgccagcag
tcagccacac aacgactgga acatgcattc 120aacatcgcca gatatcaatt
aggcatagag aaactactcg atcctgaagg ttggtaaatt 180tctggactac
cactgctttt agtatggtag agtttaatg 21925282DNAHomo sapiens
25tctcaaatat agaaaccaaa aattgatgtg tagtgttaat gtgcttacag atgttgatac
60cacctatcca gataagaagt ccatcttaat gtacatcaca tcactcttcc aagttttgcc
120tcaacaagtg agcattgaag ccatccagga agtggaaatg ttgccaaggc
cacctaaagt 180gactaaagaa gaacattttc agttacatca tcaaatgcac
tattctcaac aggtaaagtg 240tgtaaaggac agctactatt caagatgttt
tctgttttat at 28226229DNAHomo sapiens 26atggtttttc cccctcctct
ctatccactc ccccaaaccc ttctctgcag atcacggtca 60gtctagcaca gggatatgag
agaacttctt cccctaagcc tcgattcaag agctatgcct 120acacacaggc
tgcttatgtc accacctctg accctacacg gagcccattt ccttcacagg
180tctgtcaaca tttactctct gttgtacaaa ccagagaact gcttccaag
22927289DNAHomo sapiens 27aatctgcaaa gacattaatt gtgtaacacc
caatttattt tattgtgcag catttggaag 60ctcctgaaga caagtcattt ggcagttcat
tgatggagag tgaagtaaac ctggaccgtt 120atcaaacagc tttagaagaa
gtattatcgt ggcttctttc tgctgaggac acattgcaag 180cacaaggaga
gatttctaat gatgtggaag tggtgaaaga ccagtttcat actcatgagg
240taaactaaaa cgttaattta caaaacaaaa catatgactt gttataatg
28928282DNAHomo sapiens 28ccgatttacc tagagttcta attacaattg
ttaacttcct tctttgtcag gggtacatga 60tggatttgac agcccatcag ggccgggttg
gtaatattct acaattggga agtaagctga 120ttggaacagg aaaattatca
gaagatgaag aaactgaagt acaagagcag atgaatctcc 180taaattcaag
atgggaatgc ctcagggtag ctagcatgga aaaacaaagc aagtaagtcc
240ttatttgttt ttaattaaga agactaacaa gttttggaag ct 28229251DNAHomo
sapiens 29taataagttg ctttcaaaga ggtcataata ggcttctttc aaattttcag
tttacataga 60gttttaatgg atctccagaa tcagaaactg aaagagttga atgactggct
aacaaaaaca 120gaagaaagaa caaggaaaat ggaggaagag cctcttggac
ctgatcttga agacctaaaa 180cgccaagtac aacaacataa ggtaggtgta
tcttatgttg cgtgctttct actagaaagc 240aaactctgtg t 25130220DNAHomo
sapiens 30cacatgtaag aatatcattt taatttcctt taaaacattt tatctttcag
gtgcttcaag 60aagatctaga acaagaacaa gtcagggtca attctctcac tcacatggtg
gtggtagttg 120atgaatctag tggagatcac gcaactgctg ctttggaaga
acaacttaag gtcagattat 180tttgcttagt aaactaaata tgtcctttaa
aagaactata 22031202DNAHomo sapiens 31cgtagttacc aattgtttgc
tgatgctgtg cttgattgtc tcttctccag gtattgggag 60atcgatgggc aaacatctgt
agatggacag aagaccgctg ggttctttta caagacatcc 120ttctcaaatg
gcaacgtctt actgaagaac aggtgtgtca tgtgtgagaa actagctgta
180aaagacacgg ggggatatta aa 20232208DNAHomo sapiens 32agtaaagatt
tatgtttatt tattccttgg aattctttaa tgtcttgcag tgccttttta 60gtgcatggct
ttcagaaaaa gaagatgcag tgaacaagat tcacacaact ggctttaaag
120atcaaaatga aatgttatca agtcttcaaa aactggccgt atgtactttc
tagctttcaa 180tggtcttata aaaacccagt actgtata 20833280DNAHomo
sapiens 33tgtatggaat gcaacccagg cttattctgt gatctttctt gttttaacag
gttttaaaag 60cggatctaga aaagaaaaag caatccatgg gcaaactgta ttcactcaaa
caagatcttc 120tttcaacact gaagaataag tcagtgaccc agaagacgga
agcatggctg gataactttg 180cccggtgttg ggataattta gtccaaaaac
ttgaaaagag tacagcacag gttagtgata 240ccaattatca tgctacagac
tatctcagag attttttaaa 28034276DNAHomo sapiens 34actgaagtct
ttctagcaat gtctgacctc tgtttcaata cttctcacag atttcacagg 60ctgtcaccac
cactcagcca tcactaacac agacaactgt aatggaaaca gtaactacgg
120tgaccacaag ggaacagatc ctggtaaagc atgctcaaga ggaacttcca
ccaccacctc 180cccaaaagaa gaggcagatt actgtggatt ctgaaattag
gaaaaggtga gagcatctta 240agcttttatc tgcaaatgaa gtggagaaaa ctcatt
27635224DNAHomo sapiens 35gaagaaagag ataatcaaga aataatgact
tttatttttt gctgtcttag gttggatgtt 60gatataactg aacttcacag ctggattact
cgctcagaag ctgtgttgca gagtcctgaa 120tttgcaatct ttcggaagga
aggcaacttc tcagacttaa aagaaaaagt caatgtaggt 180tatgcattaa
tttttatatc tgtactcatt ttgtgctgct tgta 22436188DNAHomo sapiens
36agattcacag tccttgtatt gaattactca tctttgctct catgctgcag gccatagagc
60gagaaaaagc tgagaagttc agaaaactgc aagatgccag cagatcagct caggccctgg
120tggaacagat ggtgaatggt aattacacga gttgatttag ataatcttct
tagggatttg 180ataaacac 18837342DNAHomo sapiens 37tttcagtctg
tgggttcagg ggatatattt aattattttt ttctttctag agggtgttaa 60tgcagatagc
atcaaacaag cctcagaaca actgaacagc cggtggatcg aattctgcca
120gttgctaagt gagagactta actggctgga gtatcagaac aacatcatcg
ctttctataa 180tcagctacaa caattggagc agatgacaac tactgctgaa
aactggttga aaatccaacc 240caccacccca tcagagccaa cagcaattaa
aagtcagtta aaaatttgta aggtaagaat 300ctcttctcct tccatttgga
gcataatcaa taggtatttc tt 34238281DNAHomo sapiens 38aatgtatgca
aagtaaacgt gttacttact ttccatactc tatggcacag gatgaagtca 60accggctatc
agatcttcaa cctcaaattg aacgattaaa aattcaaagc atagccctga
120aagagaaagg acaaggaccc atgttcctgg atgcagactt tgtggccttt
acaaatcatt 180ttaagcaagt cttttctgat gtgcaggcca gagagaaaga
gctacagaca agtaagtaaa 240aagcctaaaa tggctaactt gacattttcc
aaaatggtta t 28139246DNAHomo sapiens 39aagtgtgaaa caattaagtg
attctcattc ttttttccct tttgataaag tttttgacac 60tttgccacca atgcgctatc
aggagaccat gagtgccatc aggacatggg tccagcagtc 120agaaaccaaa
ctctccatac ctcaacttag tgtcaccgac tatgaaatca tggagcagag
180actcggggaa ttgcaggtct gtgaatattt gaatgtcaaa acaataaagc
acgcttatca 240agcatt 24640313DNAHomo sapiens 40aattattatt
catcaattag ggtaaatgta tttaaaaaat tgttttttag gctttacaaa 60gttctctgca
agagcaacaa agtggcctat actatctcag caccactgtg aaagagatgt
120cgaagaaagc gccctctgaa attagccgga aatatcaatc agaatttgaa
gaaattgagg 180gacgctggaa gaagctctcc tcccagctgg ttgagcattg
tcaaaagcta gaggagcaaa 240tgaataaact ccgaaaaatt caggtaattc
aagattttac tttctaccct catttttatt 300tacttgtttt ttc 31341214DNAHomo
sapiens 41ttaaaagtaa tcagcacacc agtaatgcct tataacgggt ctcgtttcag
aatcacatac 60aaaccctgaa gaaatggatg gctgaagttg atgtttttct gaaggaggaa
tggcctgccc 120ttggggattc agaaattcta aaaaagcagc tgaaacagtg
cagagtaaga tttttatatg 180atgcctttaa tatgaataat tttgtatgaa tatt
21442256DNAHomo sapiens 42tatgtggcag taattttttt cagctggctt
aaattgattt attttcttag cttttagtca 60gtgatattca gacaattcag cccagtctaa
acagtgtcaa tgaaggtggg cagaagataa 120agaatgaagc agagccagag
tttgcttcga gacttgagac agaactcaaa gaacttaaca 180ctcagtggga
tcacatgtgc caacaggtat agacaatctc tttcactgtg gcttgcctca
240acgtacttaa ctaaga 25643271DNAHomo sapiens 43atgtttcatc
actgtcaata atcgtgtttt gtttgtttgt tttgtggaag gtctatgcca 60gaaaggaggc
cttgaaggga ggtttggaga aaactgtaag cctccagaaa gatctatcag
120agatgcacga atggatgaca caagctgaag aagagtatct tgagagagat
tttgaatata 180aaactccaga tgaattacag aaagcagttg aagagatgaa
ggtaaaaaaa aaaaaagaaa 240aactaagtaa aacaaaggaa ataaatggaa a
27144283DNAHomo sapiens 44ggatgtaaag ttattttcat gctattaaga
gagcattctt tatttttcag agagctaaag 60aagaggccca acaaaaagaa gcgaaagtga
aactccttac tgagtctgta aatagtgtca 120tagctcaagc tccacctgta
gcacaagagg ccttaaaaaa ggaacttgaa actctaacca 180ccaactacca
gtggctctgc actaggctga atgggaaatg caagactttg gaagtcagtt
240gcttttcttg gtctttgtca atgatatgtc aatacatggt cat 28345235DNAHomo
sapiens 45tttacttttc taccataata tttaatctgt gatatatatt tctttcttag
gaagtttggg 60catgttggca tgagttattg tcatacttgg agaaagcaaa caagtggcta
aatgaagtag 120aatttaaact taaaaccact gaaaacattc ctggcggagc
tgaggaaatc tctgaggtgc 180tagatgtaag ttgtaaatta agccaaatga
tgataattta tatgcagtat taaaa 23546250DNAHomo sapiens 46tgtatttaga
aaaaaaagga gaaatagtaa ttattgcaaa tgtgtttcag tcacttgaaa 60atttgatgcg
acattcagag gataacccaa atcagattcg catattggca cagaccctaa
120cagatggcgg agtcatggat gagctaatca atgaggaact tgagacattt
aattctcgtt 180ggagggaact acatgaagag gtatgaagat aagtgaaaaa
tctctttaat ctaatttgca 240ttaatgtata 25047262DNAHomo sapiens
47gctatcaaga gtaaacattt aactgataca ctcttattcc ttctttttag gctgtaagga
60ggcaaaagtt gcttgaacag agcatccagt ctgcccagga gactgaaaaa tccttacact
120taatccagga gtccctcaca ttcattgaca agcagttggc agcttatatt
gcagacaagg 180tggacgcagc tcaaatgcct caggaagccc aggcaagtac
atctgggaat cagcttccat 240tcttttgttt ttattacttc aa 26248211DNAHomo
sapiens 48tagttgttct ttgtagagca tgctgactaa taatgctatc ctcccaacag
aaaatccaat 60ctgatttgac aagtcatgag atcagtttag aagaaatgaa gaaacataat
caggggaagg 120aggctgccca aagagtcctg tctcagattg atgttgcaca
ggtatatgtt atttcagaaa 180ctaaggaacg tgttttcgtt gggcattata c
21149274DNAHomo sapiens 49ttgtttgaaa ggcaaaatta aatcagtgcc
tttttacact gtccttacag aaaaaattac 60aagatgtctc catgaagttt cgattattcc
agaaaccagc caattttgag cagcgtctac 120aagaaagtaa gatgatttta
gatgaagtga agatgcactt gcctgcattg gaaacaaaga 180gtgtggaaca
ggaagtagta cagtcacagc taaatcattg tgtggtatgt atttctggtg
240gcaaatacgc aggtacccct tgactttcct catt 27450256DNAHomo sapiens
50aataatttaa ctctactgat tatcatgttt tgttttatgt ttaaacttag aacttgtata
60aaagtctgag tgaagtgaag tctgaagtgg aaatggtgat aaagactgga cgtcagattg
120tacagaaaaa gcagacggaa aatcccaaag aacttgatga aagagtaaca
gctttgaaat 180tgcattataa tgagctggga gcaaaggtgt gtgcatgctg
agaccacaaa cacttctttc 240cactttcctt ataaat 25651271DNAHomo sapiens
51atttgaatta aagagtaaac taaattacat ttcattataa ttcttttcag gtaacagaaa
60gaaagcaaca gttggagaaa tgcttgaaat tgtcccgtaa gatgcgaaag gaaatgaatg
120tcttgacaga atggctggca gctacagata tggaattgac aaagagatca
gcagttgaag 180gaatgcctag taatttggat tctgaagttg cctggggaaa
ggtaaaacct atatcactga 240aggttatttt gaacatacgt gaaaacacat a
27152280DNAHomo sapiens 52tcttaagact acaagacatt acttgaaggt
caatgctctc cttttcacag gctactcaaa 60aagagattga gaaacagaag gtgcacctga
agagtatcac agaggtagga gaggccttga 120aaacagtttt gggcaagaag
gagacgttgg tggaagataa actcagtctt ctgaatagta 180actggatagc
tgtcacctcc cgagcagaag agtggttaaa tcttttgttg gtaagagaaa
240aggctagaag cttttacacc cttctctgtc acgagaaaaa 28053229DNAHomo
sapiens 53aagaatattg tctaaccaat aatgccatgg tatgtctctg tacaattaag
gaataccaga 60aacacatgga aacttttgac cagaatgtgg accacatcac aaagtggatc
attcaggctg 120acacactttt ggatgaatca gagaaaaaga aaccccagca
aaaagaagac gtgcttaagg 180tagcaaataa aatatgaaaa gtaatgtcca
aattgtacac cagttactt 22954271DNAHomo sapiens 54ccttcattaa
ttactaactt caagtcctat ctcttgctca tggaatatag cgtttaaagg 60cagaactgaa
tgacatacgc ccaaaggtgg actctacacg tgaccaagca gcaaacttga
120tggcaaaccg cggtgaccac tgcaggaaat tagtagagcc ccaaatctca
gagctcaacc 180atcgatttgc agccatttca cacagaatta agactggaaa
ggtaggaaga tctactccaa 240ggtggaaact tgtgctaaat ggtctcttgc g
27155223DNAHomo sapiens 55ttctaataaa aagtaatttt gatttaaagt
agcactatct ttttttttag gcctccattc 60ctttgaagga attggagcag tttaactcag
atatacaaaa attgcttgaa ccactggagg 120ctgaaattca gcagggggtg
aatctgaaag aggaagactt caataaagat atggtaaatt 180ggttgtgata
aaagtgtgaa tgaactagga gtggaaataa ata 22356238DNAHomo sapiens
56acagcttttt aaaaaccaaa atgaagactg tacttgttgt ttttgatcag aatgaagaca
60atgagggtac tgtaaaagaa ttgttgcaaa gaggagacaa cttacaacaa agaatcacag
120atgagagaaa gcgagaggaa ataaagataa aacagcagct gttacagaca
aaacataatg 180ctctcaaggt attagagcta aaattataat ataccttgcc
tgtggttttt ttttaata 23857253DNAHomo sapiens 57tgcactatac atatatattg
atattttaat aatgtctgca ccatgaacag gatttgaggt 60ctcaaagaag aaaaaaggct
ctagaaattt ctcatcagtg gtatcagtac aagaggcagg 120ctgatgatct
cctgaaatgc ttggatgaca ttgaaaaaaa attagccagc ctacctgagc
180ccagagatga aaggaaaata aaggtaatgt tgttttagaa tgtcaatacc
agattttatt 240atacagttta att 25358283DNAHomo sapiens 58tgatgtggtt
agctaactgc cctgggccct gtattggttt tgctcaatag gaaattgatc 60gggaattgca
gaagaagaaa gaggagctga atgcagtgcg taggcaagct gagggcttgt
120ctgaggatgg ggccgcaatg gcagtggagc caactcagat ccagctcagc
aagcgctggc 180gggaaattga gagcaaattt gctcagtttc gaagactcaa
ctttgcacaa attgtgagtt 240gttactggca aacccacgta tgtgtttgca
actactactc tat 28359295DNAHomo sapiens 59ttcactgtta ggaagctaaa
aaaaattgtt cttttgtata tctataccag cacactgtcc 60gtgaagaaac gatgatggtg
atgactgaag acatgccttt ggaaatttct tatgtgcctt 120ctacttattt
gactgaaatc actcatgtct cacaagccct attagaagtg gaacaacttc
180tcaatgctcc tgacctctgt gctaaggact ttgaagatct ctttaagcaa
gaggagtctc 240tgaaggtaaa accaaagcac tttcattcgt attttacaag
gtgatcatac tgatc 29560273DNAHomo sapiens 60tatagacagc taattcattt
ttttactgtt ttaaaatttt tatattacag aatataaaag 60atagtctaca acaaagctca
ggtcggattg acattattca tagcaagaag acagcagcat 120tgcaaagtgc
aacgcctgtg gaaagggtga agctacagga agctctctcc cagcttgatt
180tccaatggga aaaagttaac aaaatgtaca aggaccgaca agggtaggta
acacatatat 240ttttcttgat acttgcagaa atgatttgtt ttc 27361248DNAHomo
sapiens 61gttttacata atccatctat ttttcttgat ccatatgctt ttacctgcag
gcgatttgac 60agatctgttg agaaatggcg gcgttttcat tatgatataa agatatttaa
tcagtggcta 120acagaagctg aacagtttct cagaaagaca caaattcctg
agaattggga acatgctaaa 180tacaaatggt atcttaaggt aagtctttga
tttgtttttt
cgaaattgta tttatcttca 240gcacatct 24862276DNAHomo sapiens
62taaaaagaca tggggcttca tttttgtttt gcctttttgg tatcttacag gaactccagg
60atggcattgg gcagcggcaa actgttgtca gaacattgaa tgcaactggg gaagaaataa
120ttcagcaatc ctcaaaaaca gatgccagta ttctacagga aaaattggga
agcctgaatc 180tgcggtggca ggaggtctgc aaacagctgt cagacagaaa
aaagaggtag ggcgacagat 240ctaataggaa tgaaaacatt ttagcagact ttttaa
27663248DNAHomo sapiens 63tgagaactat gttggaaaaa aaaataacaa
ttttattctt ctttctccag gctagaagaa 60caaaagaata tcttgtcaga atttcaaaga
gatttaaatg aatttgtttt atggttggag 120gaagcagata acattgctag
tatcccactt gaacctggaa aagagcagca actaaaagaa 180aagcttgagc
aagtcaaggt aattttattt tctcaaatcc cccagggcct gcttgcataa 240agaagtat
24864250DNAHomo sapiens 64ggaattgtgc tgtaattcat tttaaacgtt
gttgcatttg tctgtttcag ttactggtgg 60aagagttgcc cctgcgccag ggaattctca
aacaattaaa tgaaactgga ggacccgtgc 120ttgtaagtgc tcccataagc
ccagaagagc aagataaact tgaaaataag ctcaagcaga 180caaatctcca
gtggataaag gttagacatt aaccatctct tccgtcacat gtgttaaatg
240ttgcaagtat 25065286DNAHomo sapiens 65gcttatgcct tgagaattat
ttaccttttt aaaatgtatt ttcctttcag gtttccagag 60ctttacctga gaaacaagga
gaaattgaag ctcaaataaa agaccttggg cagcttgaaa 120aaaagcttga
agaccttgaa gagcagttaa atcatctgct gctgtggtta tctcctatta
180ggaatcagtt ggaaatttat aaccaaccaa accaagaagg accatttgac
gttaaggtag 240ggaacttttt gctttaaata tttttgtctt ttttaagaaa aatggc
28666202DNAHomo sapiens 66ttattgctaa ctgtgaagtt aatctgcact
atatgggttc ttttccccag gaaactgaaa 60tagcagttca agctaaacaa ccggatgtgg
aagagatttt gtctaaaggg cagcatttgt 120acaaggaaaa accagccact
cagccagtga aggtaatgaa gcaacctcta gcaatatcca 180ttacctcata
atgggttatg ct 20267209DNAHomo sapiens 67atcttcaaag tgttaatcga
ataagtaatg tgtatgcttt tctgttaaag aggaagttag 60aagatctgag ctctgagtgg
aaggcggtaa accgtttact tcaagagctg agggcaaagc 120agcctgacct
agctcctgga ctgaccacta ttggagcctg taagtatact ggatcccatt
180ctctttggct ctagctattt gttcaaaag 20968333DNAHomo sapiens
68tttttctttt tcttcttttt tcctttttgc aaaaacccaa aatattttag ctcctactca
60gactgttact ctggtgacac aacctgtggt tactaaggaa actgccatct ccaaactaga
120aatgccatct tccttgatgt tggaggtacc tgctctggca gatttcaacc
gggcttggac 180agaacttacc gactggcttt ctctgcttga tcaagttata
aaatcacaga gggtgatggt 240gggtgacctt gaggatatca acgagatgat
catcaagcag aaggtatgag aaaaaatgat 300aaaagttggc agaagttttt
ctttaaaatg aag 33369218DNAHomo sapiens 69aatacacaac gctgaagaac
cctgatacta agggatattt gttcttacag gcaacaatgc 60aggatttgga acagaggcgt
ccccagttgg aagaactcat taccgctgcc caaaatttga 120aaaacaagac
cagcaatcaa gaggctagaa caatcattac ggatcgaagt aagtttttta
180acaagcatgg gacacacaaa gcaagatgca tgacaagt 21870312DNAHomo
sapiens 70cctccagact agcatttact actatatatt tatttttcct tttattctag
ttgaaagaat 60tcagaatcag tgggatgaag tacaagaaca ccttcagaac cggaggcaac
agttgaatga 120aatgttaaag gattcaacac aatggctgga agctaaggaa
gaagctgagc aggtcttagg 180acaggccaga gccaagcttg agtcatggaa
ggagggtccc tatacagtag atgcaatcca 240aaagaaaatc acagaaacca
aggttagtat caaagatacc tttttaaaat aaaatactgg 300ttacatttga ta
31271255DNAHomo sapiens 71atttcataaa aaaaactgac attcattctc
tttctcataa aaatctatag cagttggcca 60aagacctccg ccagtggcag acaaatgtag
atgtggcaaa tgacttggcc ctgaaacttc 120tccgggatta ttctgcagat
gataccagaa aagtccacat gataacagag aatatcaatg 180cctcttggag
aagcattcat aaaaggtatg aattacatta tttctaaaac tactgttggc
240tgtaataatg gggtg 25572290DNAHomo sapiens 72gcaccattct gatatttaat
aattgcatct gaacatttgg tcctttgcag ggtgagtgag 60cgagaggctg ctttggaaga
aactcataga ttactgcaac agttccccct ggacctggaa 120aagtttcttg
cctggcttac agaagctgaa acaactgcca atgtcctaca ggatgctacc
180cgtaaggaaa ggctcctaga agactccaag ggagtaaaag agctgatgaa
acaatggcaa 240gtaagtcagg catttccgct ttagcactct tgtggatcca
attgaacaat 29073273DNAHomo sapiens 73ttcttttgtt tggtaattct
gcacatattc ttcttcctgc tgtcctgtag gacctccaag 60gtgaaattga agctcacaca
gatgtttatc acaacctgga tgaaaacagc caaaaaatcc 120tgagatccct
ggaaggttcc gatgatgcag tcctgttaca aagacgtttg gataacatga
180acttcaagtg gagtgaactt cggaaaaagt ctctcaacat taggtaggaa
aagatgtgga 240gcaaaaaggc cacaaatgaa ttaaaatggc caa 27374257DNAHomo
sapiens 74caattacact tctagatatt ctgacatggt acgctgctgt tctttttcag
gtcccatttg 60gaagccagtt ctgaccagtg gaagcgtctg cacctttctc tgcaggaact
tctggtgtgg 120ctacagctga aagatgatga attaagccgg caggcaccta
ttggaggcga ctttccagca 180gttcagaagc agaacgatgt acatagggta
ggacattttt aagcctcgtg ccttgcacat 240gttaagcaca tagtaat
25775221DNAHomo sapiens 75agaagaatgc cacaagccaa ataagcactt
cttttcatct catttcacag gccttcaaga 60gggaattgaa aactaaagaa cctgtaatca
tgagtactct tgagactgta cgaatatttc 120tgacagagca gcctttggaa
ggactagaga aactctacca ggagcccaga ggtaattgaa 180tgtggaacta
taataacata ttgatagaag gatcagtggt g 22176369DNAHomo sapiens
76gtttaaaaaa aaagaatgtg gcctaaaacc ttgtcatatt gccaatttag agctgcctcc
60tgaggagaga gcccagaatg tcactcggct tctacgaaag caggctgagg aggtcaatac
120tgagtgggaa aaattgaacc tgcactccgc tgactggcag agaaaaatag
atgagaccct 180tgaaagactc cgggaacttc aagaggccac ggatgagctg
gacctcaagc tgcgccaagc 240tgaggtgatc aagggatcct ggcagcccgt
gggcgatctc ctcattgact ctctccaaga 300tcacctcgag aaagtcaagg
taccgtctac ttctttgctt cagggccctt tgagagactc 360aaaagagct
36977247DNAHomo sapiens 77ttgttttaaa tattctcatc ttccaatttg
cttttgacta ttgcacacag gcacttcgag 60gagaaattgc gcctctgaaa gagaacgtga
gccacgtcaa tgaccttgct cgccagctta 120ccactttggg cattcagctc
tcaccgtata acctcagcac tctggaagac ctgaacacca 180gatggaagct
tctgcaggta agcacattgt aaacattgtt gtcctttgtt acagtaaaat 240aatatac
24778179DNAHomo sapiens 78tcctcattat atagaatgag agaacatcat
ttctctcctt ttcctcccag gtggccgtcg 60aggaccgagt caggcagctg catgaagccc
acagggactt tggtccagca tctcagcact 120ttctttccag taagtcattt
tcagctttta tcacttaact ttattgcatc ttgattaat 17979161DNAHomo sapiens
79gcgatgaatt tgacctcctt gcctttcttt ttttcctccc ttcttttcag cgtctgtcca
60gggtccctgg gagagagcca tctcgccaaa caaagtgccc tactatatca agtaagttgg
120aagtatcaca tttttaaaag agcatttatt gtgactaacc t 16180162DNAHomo
sapiens 80tgactactca ttgtaaatgc taaagtcttt ctttatgttt tgtgttttag
ccacgagact 60caaacaactt gctgggacca tcccaaaatg acagagctct accagtcttt
aggtaaggac 120atggccatgt ttcctccaag ttaaatgaca ggtgaccttt ag
16281175DNAHomo sapiens 81ctgttatttc tgatggaata acaaatgctc
tttgttttcc ctcttttcag ctgacctgaa 60taatgtcaga ttctcagctt ataggactgc
catgaaactc cgaagactgc agaaggccct 120ttgctgtaag tattggccag
tatttgaaga tcttgatact atgtctttgc ttaga 17582302DNAHomo sapiens
82aggaaggttt tactctttga gtcatttgtg attttatttg ttttttgcag tggatctctt
60gagcctgtca gctgcatgtg atgccttgga ccagcacaac ctcaagcaaa atgaccagcc
120catggatatc ctgcagatta ttaattgttt gaccactatt tatgaccgcc
tggagcaaga 180gcacaacaat ttggtcaacg tccctctctg cgtggatatg
tgtctgaact ggctgctgaa 240tgtttatgat acgtacgtat ggcatgtttt
tatttcccgg gctctgtcac aggaggctta 300gc 30283186DNAHomo sapiens
83cctctaggaa agggtcagta attgttttct gctttgattc ttcataatag gggacgaaca
60gggaggatcc gtgtcctgtc ttttaaaact ggcatcattt ccctgtgtaa agcacatttg
120gaagacaagt acagatgtaa gtcgtgtata ttaatgctgt attcttttat
taatgttggc 180taatta 18684258DNAHomo sapiens 84atccatgggt
gctgtgtttt gactgttgca attttcttct tcctttgtag accttttcaa 60gcaagtggca
agttcaacag gattttgtga ccagcgcagg ctgggcctcc ttctgcatga
120ttctatccaa attccaagac agttgggtga agttgcatcc tttgggggca
gtaacattga 180gccaagtgtc cggagctgct tccaatttgt aagttattca
ccttctaggt aacatattta 240ttctttcata ttttagaa 25885267DNAHomo
sapiens 85ctttcctttc atccttttgc cctccttctc tctccctcct gtctttgcag
gctaataata 60agccagagat cgaagcggcc ctcttcctag actggatgag actggaaccc
cagtccatgg 120tgtggctgcc cgtcctgcac agagtggctg ctgcagaaac
tgccaagcat caggccaaat 180gtaacatctg caaagagtgt ccaatcattg
gattcaggta ttaggaacca aaaaaaaaat 240gtcatttttt tctcatcatt tttcacc
26786212DNAHomo sapiens 86ggaatttgat tcgaagaaat acatacgtgt
ttgtttttgc tctttatcag gtacaggagt 60ctaaagcact ttaattatga catctgccaa
agctgctttt tttctggtcg agttgcaaaa 120ggccataaaa tgcactatcc
catggtggaa tattgcactc cggtaagttt gacgccagcc 180tgacgtgaga
gttagttcac ctgggataaa tt 21287237DNAHomo sapiens 87tttgaaatca
tcctgtccta aatctgatct caccatgatc tcccttttag actacatcag 60gagaagatgt
tcgagacttt gccaaggtac taaaaaacaa atttcgaacc aaaaggtatt
120ttgcgaagca tccccgaatg ggctacctgc cagtgcagac tgtcttagag
ggggacaaca 180tggaaacgtg agtagtagca aaagcagaac acactcttgt
ttgatgtata tttgaac 23788139DNAHomo sapiens 88cggctgagtt tgcgtgtgtc
tccttcacca cctcattttt tgttttgcag tcccgttact 60ctgatcaact tctggccagt
agattctgcg tgagtacttt ttttgctgaa gggtgctgct 120accaccaaca cattcgctc
13989166DNAHomo sapiens 89tctccattaa tggatggtat ctgtgactaa
tcacattttc tgccttatag gcctgcctcg 60tcccctcagc tttcacacga tgatactcat
tcacgcattg aacattatgc tagcaggtat 120gagactagtt gtatgccagg
caaatattga ttgaaataac taacca 16690166DNAHomo sapiens 90gattctaaga
cgtcacataa gttttaatga gcttttacgt tttttatcag gctagcagaa 60atggaaaaca
gcaatggatc ttatctaaat gatagcatct ctcctaatga gagcatgtaa
120gtatcccatc tctttttaca aaatgttcct gacaatgaaa ttgctt
16691259DNAHomo sapiens 91aagcaaaata agggggggaa aaaaccaaaa
cctttgattt tattttccag agatgatgaa 60catttgttaa tccagcatta ctgccaaagt
ttgaaccagg actcccccct gagccagcct 120cgtagtcctg cccagatctt
gatttcctta gagagtgagg aaagagggga gctagagaga 180atcctagcag
atcttgagga agaaaacagg tgagttttct ttctagcttt gtcattggta
240tgcagagtgc atacacttg 25992344DNAHomo sapiens 92ttttcttttt
ctttcttttt ttttcttttt tacttttttg atgccaatag gaatctgcaa 60gcagaatatg
accgtctaaa gcagcagcac gaacataaag gcctgtcccc actgccgtcc
120cctcctgaaa tgatgcccac ctctccccag agtccccggg atgctgagct
cattgctgag 180gccaagctac tgcgtcaaca caaaggccgc ctggaagcca
ggatgcaaat cctggaagac 240cacaataaac agctggagtc acagttacac
aggctaaggc agctgctgga gcaagtgagg 300agagagatgg gatttttaca
aacattcatt tttccctctt aaac 34493224DNAHomo sapiens 93tttgtatgtt
tattatgaaa agtaattctg ttttcttttg gatgacttag ccccaggcag 60aggccaaagt
gaatggcaca acggtgtcct ctccttctac ctctctacag aggtccgaca
120gcagtcagcc tatgctgctc cgagtggttg gcagtcaaac ttcggactcc
atgggtaagt 180gtcctagcta ctctcagatt ttgttgtctg aagaaaggta gagt
22494193DNAHomo sapiens 94ctgttttcta taaatgtaat tttccattat
ttgtttttgc ttttattaag gtgaggaaga 60tcttctcagt cctccccagg acacaagcac
agggttagag gaggtgatgg agcaactcaa 120caactccttc cctagttcaa
gaggtaagct ccaataccta gaagggactc agatttgctg 180ggatcaggcc act
19395132DNAHomo sapiens 95tttttttccc tttctgatat ctctgcctct
tcctctctct attattaaag gaagaaatac 60ccctggaaag ccaatgagag aggttagtga
gattcaggct cacggccatg gcttctgtct 120gtctcatcct gc 1329662DNAHomo
sapiens 96tctatctgca ccttttgtaa agtctgtctt tctttctctt tgttttccag
gacacaatgt 60ag 62
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