U.S. patent application number 11/194776 was filed with the patent office on 2006-02-02 for antisense modulation of ptp1b expression.
Invention is credited to Sanjay Bhanot, Madelline M. Butler, Lex M. Cowsert, Kenneth W. Dobie, Susan M. Freier, Robert McKay, Brett P. Monia, Jacqueline R. Wyatt.
Application Number | 20060025372 11/194776 |
Document ID | / |
Family ID | 32867944 |
Filed Date | 2006-02-02 |
United States Patent
Application |
20060025372 |
Kind Code |
A1 |
Bhanot; Sanjay ; et
al. |
February 2, 2006 |
Antisense modulation of PTP1B expression
Abstract
Compounds, compositions and methods are provided for modulating
the expression of PTP1B. The compositions comprise antisense
compounds, particularly antisense oligonucleotides, targeted to
nucleic acids encoding PTP1B. Methods of using these compounds for
modulation of PTP1B expression and for treatment of diseases
associated with expression of PTP1B are provided.
Inventors: |
Bhanot; Sanjay; (Carlsbad,
CA) ; Cowsert; Lex M.; (Pittsburgh, PA) ;
Wyatt; Jacqueline R.; (Sundance, WY) ; Monia; Brett
P.; (Encinitas, CA) ; Butler; Madelline M.;
(Rancho Santa Fe, CA) ; McKay; Robert; (Poway,
CA) ; Freier; Susan M.; (San Diego, CA) ;
Dobie; Kenneth W.; (Del Mar, CA) |
Correspondence
Address: |
ELMORE PATENT LAW GROUP
209 MAIN STREET
N. CHELMSFORD
MA
01863
US
|
Family ID: |
32867944 |
Appl. No.: |
11/194776 |
Filed: |
August 1, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US04/02003 |
Feb 6, 2004 |
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11194776 |
Aug 1, 2005 |
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10360510 |
Feb 7, 2003 |
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PCT/US04/02003 |
Feb 6, 2004 |
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Current U.S.
Class: |
514/44A ;
536/23.1 |
Current CPC
Class: |
C12N 2310/321 20130101;
Y02P 20/582 20151101; A61P 3/04 20180101; C12N 2310/315 20130101;
C12N 15/1137 20130101; C12N 2310/345 20130101; C12N 15/113
20130101; A61P 3/10 20180101; A61K 38/00 20130101; C12Y 301/03048
20130101; C12N 2310/3341 20130101; C12N 2310/341 20130101; A61K
48/00 20130101; A61P 35/00 20180101; C12N 2310/346 20130101; C12N
2310/11 20130101; C12N 2310/321 20130101; C12N 2310/3525 20130101;
C12N 2310/14 20130101 |
Class at
Publication: |
514/044 ;
536/023.1 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07H 21/02 20060101 C07H021/02 |
Claims
1. A compound 8 to 50 nucleobases in length targeted to a nucleic
acid molecule encoding PTP1B (SEQ ID NO: 3 or 243), wherein said
compound comprises a sense region and an antisense region, said
antisense region comprising comprising a stretch of at least eight
(8) consecutive nucleobases of SEQ ID NO: 18, 19, 20, 21, 22, 23,
24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 40, 42, 45, 46, 47, 48,
49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 69, 70, 72, 73, 75, 78, 79, 80, 81, 83, 84, 86, 87, 89, 90, 92,
93, 94, 95, 96, 97, 99, 100, 101, 102, 103, 104, 106, 107, 108,
109, 110, 112, 113, 114, 115, 117, 120, 121, 122, 123, 124, 126,
127, 128, 130, 131, 133, 134, 135, 136, 137, 138, 139, 140, 141,
142, 144, 145, 146, 147, 148, 151, 152, 153, 154, 155, 156, 157,
158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,
171, 172, 173, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,
187, 188, 189, 191, 193, 195, 196, 198, 201, 202, 204, 205, 206,
211, 215, 217, 219, 223, 225, 226, 228, 229, 230, 232, 233, 235,
236, 237, 239, 240, 244, 245, 247, 248, 249, 250, 251, 252, 254,
255, 256, 257, 258, 259, 260, 261, 262, 263, 267, 268, 269, 271,
275, 276, 277, 278, 279, 281, 282, 283, 288, 290, 291, 292, 294,
296, 297, 298, 299, 300, 302, 303, 307, 310, 311, 313, 315, 317,
318, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333,
334, 335, 337, 340, 341, 342, 343, 344, 345, 347, 349, 350, 351,
352, 353, 354, 355, 356, 357, 358, 360, 361, 362, 363, 364, 365,
366, 368, 369, 371, 372, 373, 374, 375, 377, 378, 380, 381, 384,
385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, or
398 wherein said compound specifically hybridizes with said nucleic
acid molecule encoding PTP1B and inhibits the expression of
PTP1B.
2. The compound of claim 1 wherein said antisense region comprises
a stretch of at least eight (8) consecutive nucleobases of SEQ ID
NO: 390, 391, 392, 393, 394, 395, 396, 397, or 398.
3. The compound of claim 2 wherein said sense region comprises a
sequence complementary to said antisense region selected from
sequences comprising a stretch of at least eight (8) consecutive
nucleobases of SEQ ID NO: 403, 404, 405, 406, 407, 408, 409, 410,
or 411
4. The compound of claim 1 wherein said antisense region comprises
a stretch of at least eight (8) consecutive nucleobases of
nucleobases 1 to 19 of SEQ ID NO: 390, 391, 392, 393, 394, or
395.
5. The compound of claim 4 wherein said sense region comprises a
sequence complementary to said antisense region selected from
sequences comprising a stretch of at least eight (8) consecutive
nucleobases of nucleobases 1 to 19 of SEQ ID NO: 403, 404, 405,
406, 407, or 408
6. The compound of claim 1 wherein said antisense region comprises
a stretch of at least eight (8) consecutive nucleobases of SEQ ID
NO: 396, 397, or 398.
7. The compound of claim 6 wherein said sense region comprises a
sequence complementary to said antisense region selected from
sequences comprising a stretch of at least eight (8) consecutive
nucleobases of SEQ ID NO: 409, 410, or 411.
8. The compound of claim 1 wherein the compound is a RNA
oligonucleotide.
9. The compound of claim 2 wherein the compound is a RNA
oligonucleotide.
10. The compound of claim 4 wherein the compound is a RNA
oligonucleotide.
11. The compound of claim 6 wherein the compound is a RNA
oligonucleotide.
12. The compound of claim 1 wherein said antisense strand further
comprises an overhang of two deoxynucleotides at one or both
termini.
13. The compound of claim 1 wherein said sense strand further
comprises an overhang of two deoxynucleotides at one or both
termini.
14. The compound of claim 2 wherein said antisense strand further
comprises an overhang of two deoxynucleotides at one or both
termini.
15. The compound of claim 2 wherein said sense strand further
comprises an overhang of two deoxynucleotides at one or both
termini.
16. The compound of claim 1 further comprising at least one
modified internucleoside linkage.
17. The compound of claim 16 wherein the modified internucleoside
linkage is a phosphorothioate linkage.
18. The compound of claim 1 further comprising at least one
modified sugar moiety.
19. The compound of claim 1 further comprising at least one
modified nucleobase.
20. The compound of claim 1, wherein said compound is chimeric.
21. The compound of claim 1 wherein said antisense region and said
sense region are part of a single molecule.
22. The compound of claim 1 wherein said antisense region and said
sense region are on separate molecules.
23. A composition comprising the compound of claim 1 and a
pharmaceutically acceptable carrier, diluent, enhancer or
excipient.
24. A method of inhibiting the expression of PTP1B in cells or
tissues comprising contacting said cells or tissues with the
compound of claim 1 so that expression of PTP1B is inhibited.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of PCT Application No.
PCT/US04/002003, filed Feb. 6, 2004, which is a
continuation-in-part of U.S. application Ser. No. 10/360,510 filed
Feb. 7, 2003. The entire contents of each is herein incorporated by
reference.
INCORPORATION OF SEQUENCE LISTING
[0002] Three computer-readable form copies of the sequence listing
(Seq. Listing Copy 1, Seq. Listing Copy 2, and Seq. Listing Copy
3), all on CD-ROMs, each containing the file named
BIOL0001US.C2_SequenceListing.txt, which is 202,752 bytes (measured
in MS-DOS) and was created on Aug. 1, 2005, are herein incorporated
by reference.
BACKGROUND OF THE INVENTION
[0003] The process of phosphorylation, defined as the attachment of
a phosphate moiety to a biological molecule through the action of
enzymes called kinases, represents one course by which
intracellular signals are propagated resulting finally in a
cellular response. Within the cell, proteins can be phosphorylated
on serine, threonine or tyrosine residues and the extent of
phosphorylation is regulated by the opposing action of
phosphatases, which remove the phosphate moieties. While the
majority of protein phosphorylation within the cell is on serine
and threonine residues, tyrosine phosphorylation is modulated to
the greatest extent during oncogenic transformation and growth
factor stimulation (Zhang, Crit. Rev. Biochem. Mol. Biol., 1998,
33, 1-52).
[0004] Because phosphorylation is such a ubiquitous process within
cells and because cellular phenotypes are largely influenced by the
activity of these pathways, it is currently believed that a number
of disease states and/or disorders are a result of either aberrant
activation of, or functional mutations in, kinases and
phosphatases. Consequently, considerable attention has been devoted
recently to the characterization of tyrosine kinases and tyrosine
phosphatases.
[0005] PTP1B (also known as protein phosphatase 1B and PTPN1) is an
endoplasmic reticulum (ER)-associated enzyme originally isolated as
the major protein tyrosine phosphatase of the human placenta (Tonks
et al., J. Biol. Chem., 1988, 263, 6731-6737; Tonks et al., J.
Biol. Chem., 1988, 263, 6722-6730).
[0006] An essential regulatory role in signaling mediated by the
insulin receptor has been established for PTP1B. PTP1B interacts
with and dephosphorylates the activated insulin receptor both in
vitro and in intact cells resulting in the downregulation of the
signaling pathway (Goldstein et al., Mol. Cell. Biochem., 1998,
182, 91-99; Seely et al., Diabetes, 1996, 45, 1379-1385). In
addition, PTP1B modulates the mitogenic actions of insulin
(Goldstein et al., Mol. Cell. Biochem., 1998, 182, 91-99). In rat
adipose cells overexpressing PTP1B, the translocation of the GLUT4
glucose transporter was inhibited, implicating PTP1B as a negative
regulator of glucose transport as well (Chen et al., J. Biol.
Chem., 1997, 272, 8026-8031).
[0007] Mouse knockout models lacking the PTP1B gene also point
toward the negative regulation of insulin signaling by PTP1B. Mice
harboring a disrupted PTP1B gene showed increased insulin
sensitivity, increased phosphorylation of the insulin receptor and
when placed on a high-fat diet, PTP B -/- mice were resistant to
weight gain and remained insulin sensitive (Elchebly et al.,
Science, 1999, 283, 1544-1548). These studies clearly establish
PTP1B as a therapeutic target in the treatment of diabetes and
obesity.
[0008] PTP1B, which is differentially regulated during the cell
cycle (Schievella et al., Cell. Growth Differ., 1993, 4, 239-246),
is expressed in insulin sensitive tissues as two different isoforms
that arise from alternate splicing of the pre-mRNA (Shifrin and
Neel, J. Biol. Chem., 1993, 268, 25376-25384). It was recently
demonstrated that the ratio of the alternatively spliced products
is affected by growth factors such as insulin and differs in
various tissues examined (Sell and Reese, Mol. Genet. Metab., 1999,
66, 189-192). In these studies it was also found that the levels of
the variants correlated with the plasma insulin concentration and
percentage body fat and may therefore be used as a biomarker for
patients with chronic hyperinsulinemia or type 2 diabetes.
[0009] Liu and Chernoff have shown that PTP1B binds to and serves
as a substrate for the epidermal growth factor receptor (EGFR) (Liu
and Chernoff, Biochem. J, 1997, 327, 139-145). Furthermore, in A431
human epidermoid carcinoma cells, PT1B was found to be inactivated
by the presence of H.sub.2O.sub.2 generated by the addition of EGF.
These studies indicate that PTP1B can be negatively regulated by
the oxidation state of the cell, which is often deregulated during
tumorigenesis (Lee et al., J. Biol. Chem., 1998, 273,
15366-15372).
[0010] Overexpression of PTP1B has been demonstrated in malignant
ovarian cancers and this correlation was accompanied by a
concomitant increase in the expression of the associated growth
factor receptor (Wiener et al., Am. J. Obstet. Gynecol., 1994, 170,
1177-1183).
[0011] PTP1B has been shown to suppress transformation in NIH3T3
cells induced by the neu oncogene (Brown-Shimer et al., Cancer
Res., 1992, 52, 478-482), as well as in rat 3Y1 fibroblasts induced
by v-srk, v-src, and v-ras (Liu et al., Mol. Cell. Biol., 1998, 18,
250-259) and rat-1 fibroblasts induced by bcr-abl (LaMontagne et
al., Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 14094-14099). It has
also been shown that PTP1B promotes differentiation of K562 cells,
a chronic myelogenous leukemia cell line, in a similar manner as
does an inhibitor of the bcr-abl oncoprotein. These studies
describe the possible role of PTP1B in controlling the pathogenesis
of chronic myeloid leukemia (LaMontagne et al., Proc. Natl. Acad.
Sci. U.S.A., 1998, 95, 14094-14099).
[0012] PTP1B negatively regulates integrin signaling by interacting
with one or more adhesion-dependent signaling components and
repressing integrin-mediated MAP kinase activation (Liu et al.,
Curr. Biol., 1998, 8, 173-176). Other studies designed to study
integrin signaling, using a catalytically inactive form of PTP1B,
have shown that PTP1B regulates cadherin-mediated cell adhesion
(Balsamo et al., J. Cell. Biol., 1998, 143, 523-532) as well as
cell spreading, focal adhesion and stress fiber formation and
tyrosine phosphorylation (Arregui et al., J. Cell. Biol., 1998,
143, 861-873).
[0013] Currently, therapeutic agents designed to inhibit the
synthesis or action of PTP 1 B include small molecules (Ham et al.,
Bioorg. Med. Chem. Lett., 1999, 9, 185-186; Skorey et al., J. Biol.
Chem., 1997, 272, 22472-22480; Taing et al., Biochemistry, 1999,
38, 3793-3803; Taylor et al., Bioorg. Med. Chem., 1998, 6,
1457-1468; Wang et al., Bioorg. Med. Chem. Lett., 1998, 8, 345-350;
Wang et al., Biochem. Pharmacol., 1997, 54, 703-711; Yao et al.,
Bioorg. Med. Chem., 1998, 6, 1799-1810) and peptides (Chen et al.,
Biochemistry, 1999, 38, 384-389; Desmarais et al., Arch. Biochem.
Biophys., 1998, 354, 225-231; Roller et al., Bioorg. Med. Chem.
Lett., 1998, 8, 2149-2150). In addition, disclosed in the PCT
publication WO 97/32595 (Olefsky, 1997) are phosphopeptides and
antibodies that inhibit the association of PTP1B with the activated
insulin receptor for the treatment of disorders associated with
insulin resistance. Antisense nucleotides against PTP1B are also
generally disclosed.
[0014] There remains a long felt need for additional agents capable
of effectively inhibiting PTP1B function.
SUMMARY OF THE INVENTION
[0015] Contemplated herein are compounds comprising a sense region
and an antisense region, said antisense region comprising an
antisense compound targeted to PTP1b. Also contemplated are
compounds comprising a sense region and an antisense region, said
antisense region comprising a sequence exemplified herein. In a
preferred embodiment, the compounds specifically hybridize with a
nucleic acid molecule encoding PTP1B and inhibit the expression of
PTP1B. In some embodiments, the antisense region and the sense
region are separate molecules. In some embodiments, the antisense
region and the sense region are part of a single molecule.
[0016] Further contemplated are antisense regions comprising a
stretch of at least eight (8) consecutive nucleobases of sequences
described herein, preferably of SEQ ID NO: 18, 19, 20, 21, 22, 23,
24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 40, 42, 45, 46, 47, 48,
49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 69, 70, 72, 73, 75, 78, 79, 80, 81, 83, 84, 86, 87, 89, 90, 92,
93, 94, 95, 96, 97, 99, 100, 101, 102, 103, 104, 106, 107, 108,
109, 110, 112, 113, 114, 115, 117, 120, 121, 122, 123, 124, 126,
127, 128, 130, 131, 133, 134, 135, 136, 137, 138, 139, 140, 141,
142, 144, 145, 146, 147, 148, 151, 152, 153, 154, 155, 156, 157,
158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,
171, 172, 173, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,
187, 188, 189, 191, 193, 195, 196, 198, 201, 202, 204, 205, 206,
211, 215, 217, 219, 223, 225, 226, 228, 229, 230, 232, 233, 235,
236, 237, 239, 240, 244, 245, 247, 248, 249, 250, 251, 252, 254,
255, 256, 257, 258, 259, 260, 261, 262, 263, 267, 268, 269, 271,
275, 276, 277, 278, 279, 281, 282, 283, 288, 290, 291, 292, 294,
296, 297, 298, 299, 300, 302, 303, 307, 310, 311, 313, 315, 317,
318, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333,
334, 335, 337, 340, 341, 342, 343, 344, 345, 347, 349, 350, 351,
352, 353, 354, 355, 356, 357, 358, 360, 361, 362, 363, 364, 365,
366, 368, 369, 371, 372, 373, 374, 375, 377, 378, 380, 381, 384,
385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, or
398 wherein said compound specifically hybridizes with said nucleic
acid molecule encoding PTP1B and inhibits the expression of
PTP1B.
[0017] Further provided are compounds 8 to 50 nucleobases in length
targeted to a nucleic acid molecule encoding PTP1B, wherein said
compound comprises a sense region and an antisense region, said
antisense region comprising a stretch of at least eight (8)
consecutive nucleobases of SEQ ID NO: 390, 391, 392, 393, 394, 395,
396, 397, or 398 or comprising a stretch of at least eight (8)
consecutive nucleobases of nucleobases 1 to 19 of SEQ ID NO: 390,
391, 392, 393, 394, or 395. In one embodiment, said sense region
comprises a sequence complementary to the antisense region selected
from sequences comprising a stretch of at least eight (8)
consecutive nucleobases of SEQ ID NO: 403, 404, 405, 406, 407, 408,
409, 410, or 411 or comprising a stretch of at least eight (8)
consecutive nucleobases of nucleobases 1 to 19 of SEQ ID NO: 403,
404, 405, 406, 407, or 408 Further provided are compounds 8 to 50
nucleobases in length targeted to a nucleic acid molecule encoding
PTP1B, wherein said compound comprises a sense region and an
antisense region, said antisense region comprising a stretch of at
least eight (8) consecutive nucleobases of SEQ ID NO: 396, 397, or
398. In some embodiments, said sense region comprises a sequence
complementary to said antisense region selected from sequences
comprising a stretch of at least eight (8) consecutive nucleobases
of SEQ ID NO: 409, 410, or 411.
[0018] Another aspect of the present invention are compounds 8 to
50 nucleobases in length targeted to a nucleic acid molecule
encoding PTP1B, wherein said compound comprises a sense region and
an antisense region, said antisense region comprising a stretch of
at least eight (8) consecutive nucleobases of SEQ ID NO: 166.
[0019] In some embodiments, the antisense region and/or the sense
region further comprise an overhang of two deoxynucleotides.
[0020] In some embodiments, the compound comprises at least one
modified internucleoside linkage, such as a phosphorothioate
linkage, a modified sugar moiety, and/or modified nucleobase. The
compounds may be chimeric compounds in some embodiments.
[0021] Further contemplated are pharmaceutical compositions
comprising the compounds of the invention and a pharmaceutically
acceptable carrier, diluent, enhancer or excipient. Also
contemplated is a method of inhibiting the expression of PTP1B in
cells or tissues comprising contacting said cells or tissues with a
compound of the invention so that expression of PTP1B is
inhibited.
[0022] Another aspect of the present invention is a method of
treating an animal having a disease or condition associated with
PTP1B comprising administering to said animal a therapeutically or
prophylactically effective amount of an antisense compound of the
invention so that PTP1B is inhibited. In some embodiments, diseases
or conditions include metabolic diseases or conditions. In some
embodiments, diseases or conditions include diabetes, obesity, or
hyperproliferative disorders including cancer. In some embodiments,
the diabetes is Type 2 diabetes.
[0023] Other embodiments of the present invention are methods of
decreasing blood glucose levels or plasma insulin levels or
improving insulin sensitivity in an animal. In some embodiments,
the animal is diabetic, hyperinsulinemic, insulin-resistant or
obese. Also contemplated herein is the use of a compound of the
present invention in the preparation of a medicament for a
metabolic disease or condition, wherein said disease or condition
is diabetes, including Type 2 diabetes, obesity, or a
hyperproliferative condition.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention employs oligomeric antisense
compounds, particularly oligonucleotides, for use in modulating the
function of nucleic acid molecules encoding PTP1B, ultimately
modulating the amount of PTP1B produced. This is accomplished by
providing antisense compounds which specifically hybridize with one
or more nucleic acids encoding PTP1B. As used herein, the terms
"target nucleic acid" and "nucleic acid encoding PTP1B" encompass
DNA encoding PTP1B, RNA (including pre-mRNA and mRNA) transcribed
from such DNA, and also cDNA derived from such RNA. The specific
hybridization of an oligomeric compound with its target nucleic
acid interferes with the normal function of the nucleic acid. This
modulation of function of a target nucleic acid by compounds which
specifically hybridize to it is generally referred to as
"antisense".
[0025] The functions of DNA to be interfered with include
replication and transcription. The functions of RNA to be
interfered with include all vital functions such as, for example,
translocation of the RNA to the site of protein translation,
translation of protein from the RNA, splicing of the RNA to yield
one or more mRNA species, and catalytic activity which may be
engaged in or facilitated by the RNA. The overall effect of such
interference with target nucleic acid function is modulation of the
expression of PTP1B. In the context of the present invention,
"modulation" means either an increase (stimulation) or a decrease
(inhibition) in the expression of a gene. In the context of the
present invention, inhibition is the preferred form of modulation
of gene expression and mRNA is a preferred target.
[0026] It is preferred to target specific nucleic acids for
antisense. "Targeting" an antisense compound to a particular
nucleic acid, in the context of this invention, is a multistep
process. The process usually begins with the identification of a
nucleic acid sequence whose function is to be modulated. This may
be, for example, a cellular gene (or mRNA transcribed from the
gene) whose expression is associated with a particular disorder or
disease state, or a nucleic acid molecule from an infectious agent.
In the present invention, the target is a nucleic acid molecule
encoding PTP1B. The targeting process also includes determination
of a site or sites within this gene for the antisense interaction
to occur such that the desired effect, e.g., detection or
modulation of expression of the protein, will result. Within the
context of the present invention, a preferred intragenic site is
the region encompassing the translation initiation or termination
codon of the open reading frame (ORF) of the gene. Since, as is
known in the art, the translation initiation codon is typically
5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding
DNA molecule), the translation initiation codon is also referred to
as the "AUG codon," the "start codon" or the "AUG start codon". A
minority of genes has a translation initiation codon having the RNA
sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG
have been shown to function in vivo. Thus, the terms "translation
initiation codon" and "start codon" can encompass many codon
sequences, even though the initiator amino acid in each instance is
typically methionine (in eukaryotes) or formylmethionine (in
prokaryotes). It is also known in the art that eukaryotic and
prokaryotic genes may have two or more alternative start codons,
any one of which may be preferentially utilized for translation
initiation in a particular cell type or tissue, or under a
particular set of conditions. In the context of the invention,
"start codon" and "translation initiation codon" refer to the codon
or codons that are used in vivo to initiate translation of an mRNA
molecule transcribed from a gene encoding PTP1B, regardless of the
sequence(s) of such codons.
[0027] It is also known in the art that a translation termination
codon (or "stop codon") of a gene may have one of three sequences,
i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences
are 5'-TAA, 5'-TAG and 5'-TGA, respectively). The terms "start
codon region" and "translation initiation codon region" refer to a
portion of such an mRNA or gene that encompasses from about 25 to
about 50 contiguous nucleotides in either direction (i.e., 5' or
3') from a translation initiation codon. Similarly, the terms "stop
codon region" and "translation termination codon region" refer to a
portion of such an mRNA or gene that encompasses from about 25 to
about 50 contiguous nucleotides in either direction (i.e., 5' or
3') from a translation termination codon.
[0028] The open reading frame (ORF) or "coding region," which is
known in the art to refer to the region between the translation
initiation codon and the translation termination codon, is also a
region which may be targeted effectively. Other target regions
include the 5' untranslated region (5'UTR), known in the art to
refer to the portion of an mRNA in the 5' direction from the
translation initiation codon, and thus including nucleotides
between the 5' cap site and the translation initiation codon of an
mRNA or corresponding nucleotides on the gene, and the 3'
untranslated region (3'UTR), known in the art to refer to the
portion of an mRNA in the 3' direction from the translation
termination codon, and thus including nucleotides between the
translation termination codon and 3' end of an mRNA or
corresponding nucleotides on the gene. The 5' cap of an mRNA
comprises an N7-methylated guanosine residue joined to the 5'-most
residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap
region of an mRNA is considered to include the 5' cap structure
itself as well as the first 50 nucleotides adjacent to the cap. The
5' cap region may also be a preferred target region.
[0029] Although some eukaryotic mRNA transcripts are directly
translated, many contain one or more regions, known as "introns,"
which are excised from a transcript before it is translated. The
remaining (and therefore translated) regions are known as "exons"
and are spliced together to form a continuous mRNA sequence. mRNA
splice sites, i.e., intron-exon junctions, may also be preferred
target regions, and are particularly useful in situations where
aberrant splicing is implicated in disease, or where an
overproduction of a particular mRNA splice product is implicated in
disease. Aberrant fusion junctions due to rearrangements or
deletions are also preferred targets. It has also been found that
introns can also be effective, and therefore preferred, target
regions for antisense compounds targeted, for example, to DNA or
pre-mRNA.
[0030] Once one or more target sites have been identified,
oligonucleotides are chosen which are sufficiently complementary to
the target, i.e., hybridize sufficiently well and with sufficient
specificity, to give the desired effect.
[0031] In the context of this invention, "hybridization" means
hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed
Hoogsteen hydrogen bonding, between complementary nucleoside or
nucleotide bases. For example, adenine and thymine are
complementary nucleobases which pair through the formation of
hydrogen bonds. "Complementary," as used herein, refers to the
capacity for precise pairing between two nucleotides. For example,
if a nucleotide at a certain position of an oligonucleotide is
capable of hydrogen bonding with a nucleotide at the same position
of a DNA or RNA molecule, then the oligonucleotide and the DNA or
RNA are considered to be complementary to each other at that
position. The oligonucleotide and the DNA or RNA are complementary
to each other when a sufficient number of corresponding positions
in each molecule are occupied by nucleotides which can hydrogen
bond with each other. Thus, "specifically hybridizable" and
"complementary" are terms which are used to indicate a sufficient
degree of complementarity or precise pairing such that stable and
specific binding occurs between the oligonucleotide and the DNA or
RNA target. It is understood in the art that the sequence of an
antisense compound need not be 100% complementary to that of its
target nucleic acid to be specifically hybridizable. An antisense
compound is specifically hybridizable when binding of the compound
to the target DNA or RNA molecule interferes with the normal
function of the target DNA or RNA to cause a loss of utility, and
there is a sufficient degree of complementarity to avoid
non-specific binding of the antisense compound to non-target
sequences under conditions in which specific binding is desired,
i.e., under physiological conditions in the case of in vivo assays
or therapeutic treatment, and in the case of in vitro assays, under
conditions in which the assays are performed.
[0032] The PTP1B inhibitors of the present invention effectively
inhibit the activity of the PTP1B protein or inhibit the expression
of the PTP1B protein. In one embodiment, the activity or expression
of PTP1B is inhibited by about 10%. Preferably, the activity or
expression of PTP1B is inhibited by about 30%. More preferably, the
activity or expression of PTP1B is inhibited by 50% or more. Thus,
the oligomeric antisense compounds modulate expression of PTP1B
mRNA by at least 10%, by at least 20%, by at least 25%, by at least
30%, by at least 40%, by at least 50%, by at least 60%, by at least
70%, by at least 75%, by at least 80%, by at least 85%, by at least
90%, by at least 95%, by at least 98%, by at least 99%, or by
100%.
[0033] The compounds of the present inventions are inhibitors of
PTP1B expression. Thus, the compounds of the present invention are
believed to be useful for treating metabolic diseases and
conditions, particularly diabetes, obesity, hyperlipidemia or
metabolic syndrome X. The compounds of the invention are also
believed to be useful for preventing or delaying the onset of
metabolic diseases and conditions, particularly diabetes, obesity,
hyperlipidemia or metabolic syndrome X. Metabolic syndrome,
metabolic syndrome X or simply Syndrome X refers to a cluster of
risk factors that include obesity, dyslipidemia, particularly high
blood triglycerides, glucose intolerance, high blood sugar and high
blood pressure (Scott, C. L., Am. J. Cardiol. 2003 Jul. 3;
92(1A):35i-42i). Antisense inhibitors of PTP1B have surprisingly
been found to be effective for lowering blood glucose, including
plasma glucose, and for lowering blood lipids, including serum
lipids, particularly serum cholesterol and serum triglycerides. The
compounds of the invention are therefore particularly useful in
medicaments for the treatment, prevention and delay of onset of
type 2 diabetes, high blood glucose and hyperlipidemia.
[0034] It is understood in the art that the sequence of an
antisense compound need not be 100% complementary to that of its
target nucleic acid to be specifically hybridizable. Moreover, an
oligonucleotide may hybridize over one or more segments such that
intervening or adjacent segments are not involved in the
hybridization event (e.g., a loop structure or hairpin structure).
It is preferred that the antisense compounds of the present
invention comprise at least 70%, or at least 75%, or at least 80%,
or at least 85% sequence complementarity to a target region within
the target nucleic acid, more preferably that they comprise at
least 90% sequence complementarity and even more preferably
comprise at least 95% or at least 99% sequence complementarity to
the target region within the target nucleic acid sequence to which
they are targeted. For example, an antisense compound in which 18
of 20 nucleobases of the antisense compound are complementary to a
target region, and would therefore specifically hybridize, would
represent 90 percent complementarity. In this example, the
remaining noncomplementary nucleobases may be clustered or
interspersed with complementary nucleobases and need not be
contiguous to each other or to complementary nucleobases. As such,
an antisense compound which is 18 nucleobases in length having 4
(four) noncomplementary nucleobases which are flanked by two
regions of complete complementarity with the target nucleic acid
would have 77.8% overall complementarity with the target nucleic
acid and would thus fall within the scope of the present
invention.
[0035] Percent complementarity of an antisense compound with a
region of a target nucleic acid can be determined routinely using
BLAST programs (basic local alignment search tools) and PowerBLAST
programs known in the art (Altschul et al., J. Mol. Biol., 1990,
215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
[0036] Percent homology, sequence identity or complementarity, can
be determined by, for example, the Gap program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, Madison Wis.), using default settings,
which uses the algorithm of Smith and Waterman (Adv. Appl. Math.,
1981, 2, 482-489). In some preferred embodiments, homology,
sequence identity or complementarity, between the oligomeric and
target is between about 50% to about 60%. In some embodiments,
homology, sequence identity or complementarity, is between about
60% to about 70%. In preferred embodiments, homology, sequence
identity or complementarity, is between about 70% and about 80%. In
more preferred embodiments, homology, sequence identity or
complementarity, is between about 80% and about 90%. In some
preferred embodiments, homology, sequence identity or
complementarity, is about 90%, about 92%, about 94%, about 95%,
about 96%, about 97%, about 98%, about 99% or about 100%.
[0037] According to the present invention, compounds include
antisense oligomeric compounds, antisense oligonucleotides,
ribozymes, external guide sequence (EGS) oligonucleotides,
alternate splicers, primers, probes, and other oligomeric compounds
which hybridize to at least a portion of the target nucleic acid.
As such, these compounds may be introduced in the form of
single-stranded, double-stranded, circular or hairpin oligomeric
compounds and may contain structural elements such as internal or
terminal bulges or loops. Once introduced to a system, the
compounds of the invention may elicit the action of one or more
enzymes or structural proteins to effect modification of the target
nucleic acid.
[0038] One non-limiting example of such an enzyme is RNAse H, a
cellular endonuclease which cleaves the RNA strand of an RNA:DNA
duplex. It is known in the art that single-stranded antisense
compounds which are "DNA-like" elicit RNAse H. Activation of RNase
H, therefore, results in cleavage of the RNA target, thereby
greatly enhancing the efficiency of oligonucleotide-mediated
inhibition of gene expression. Similar roles have been postulated
for other ribonucleases such as those in the RNase III and
ribonuclease L family of enzymes.
[0039] While a suitable form of antisense compound is a
single-stranded antisense oligonucleotide, in many species the
introduction of double-stranded structures, such as double-stranded
RNA (dsRNA) molecules, has been shown to induce potent and specific
antisense-mediated reduction of the function of a gene or its
associated gene products. This phenomenon occurs in both plants and
animals and is believed to have an evolutionary connection to viral
defense and transposon silencing.
[0040] The first evidence that dsRNA could lead to gene silencing
in animals came in 1995 from work in the nematode, Caenorhabditis
elegans (Guo and Kempheus, Cell, 1995, 81, 611-620).
[0041] Montgomery et al. have shown that the primary interference
effects of dsRNA are posttranscriptional (Montgomery et al., Proc.
Natl. Acad. Sci. USA, 1998, 95, 15502-15507). The
posttranscriptional antisense mechanism defined in Caenorhabditis
elegans resulting from exposure to double-stranded RNA (dsRNA) has
since been designated RNA interference (RNAi). This term has been
generalized to mean antisense-mediated gene silencing involving the
introduction of dsRNA leading to the sequence-specific reduction of
endogenous targeted mRNA levels (Fire et al., Nature, 1998, 391,
806-811). Recently, it has been shown that it is, in fact, the
single-stranded RNA oligomers of antisense polarity of the dsRNAs
which are the potent inducers of RNAi (Tijsterman et al., Science,
2002, 295, 694-697).
[0042] The antisense compounds of the present invention also
include modified compounds in which a different base is present at
one or more of the nucleotide positions in the compound. For
example, if the first nucleotide is an adenosine, modified
compounds may be produced which contain thymidine, guanosine or
cytidine at this position. This may be done at any of the positions
of the antisense compound.
[0043] The antisense compounds of the present invention can be
utilized for diagnostics, therapeutics, prophylaxis and as research
reagents and kits. Furthermore, antisense oligonucleotides, which
are able to inhibit gene expression with exquisite specificity, are
often used by those of ordinary skill to elucidate the function of
particular genes or to distinguish between functions of various
members of a biological pathway. Antisense modulation has,
therefore, been harnessed for research use.
[0044] For use in kits and diagnostics, the compounds of the
present invention, either alone or in combination with other
compounds or therapeutics, can be used as tools in differential
and/or combinatorial analyses to elucidate expression patterns of a
portion or the entire complement of genes expressed within cells
and tissues.
[0045] As one nonlimiting example, expression patterns within cells
or tissues treated with one or more antisense compounds are
compared to control cells or tissues not treated with antisense
compounds and the patterns produced are analyzed for differential
levels of gene expression as they pertain, for example, to disease
association, signaling pathway, cellular localization, expression
level, size, structure or function of the genes examined. These
analyses can be performed on stimulated or unstimulated cells and
in the presence or absence of other compounds which affect
expression patterns.
[0046] Examples of methods of gene expression analysis known in the
art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett.,
2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE
(serial analysis of gene expression)(Madden, et al., Drug Discov.
Today, 2000, 5, 415-425), READS (restriction enzyme amplification
of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999,
303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et
al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein
arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16;
Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed
sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000,
480, 2-16; Larsson, et al., J Biotechnol., 2000, 80, 143-57),
subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.
Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,
203-208), subtractive cloning, differential display (DD) (Jurecic
and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative
genomic hybridization (Carulli, et al., J Cell Biochem. Suppl.,
1998, 31, 286-96), FISH (fluorescent in situ hybridization)
techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35,
1895-904) and mass spectrometry methods (To, Comb. Chem. High
Throughput Screen, 2000, 3, 235-41).
[0047] The antisense compounds of the invention are useful for
research and diagnostics, because these compounds hybridize to
nucleic acids encoding PTP1B. For example, oligonucleotides that
are shown to hybridize with such efficiency and under such
conditions as disclosed herein as to be effective PTP1B inhibitors
are effective primers or probes under conditions favoring gene
amplification or detection, respectively. These primers and probes
are useful in methods requiring the specific detection of nucleic
acid molecules encoding PTP1B and in the amplification of said
nucleic acid molecules for detection or for use in further studies
of PTP1B.
[0048] Hybridization of the antisense oligonucleotides,
particularly the primers and probes, of the invention with a
nucleic acid encoding PTP1B can be detected by means known in the
art. Such means may include conjugation of an enzyme to the
oligonucleotide, radiolabelling of the oligonucleotide or any other
suitable detection means. Kits using such detection means for
detecting the level of PTP1B in a sample may also be prepared.
[0049] The specificity and sensitivity of antisense are also
harnessed by those of skill in the art for therapeutic uses.
Antisense oligonucleotides have been employed as therapeutic
moieties in the treatment of disease states in animals and man.
Antisense oligonucleotides have been safely and effectively
administered to humans and numerous clinical trials are presently
underway. It is thus established that oligonucleotides can be
useful therapeutic modalities that can be configured to be useful
in treatment regimes for treatment of cells, tissues and animals,
especially humans.
[0050] In the context of this invention, the term "oligonucleotide"
refers to an oligomer or polymer of ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA) or mimetics thereof. This term includes
oligonucleotides composed of naturally-occurring nucleobases,
sugars and covalent internucleoside (backbone) linkages as well as
oligonucleotides having non-naturally-occurring portions which
function similarly. Such modified or substituted oligonucleotides
are often preferred over native forms because of desirable
properties such as, for example, enhanced cellular uptake, enhanced
affinity for nucleic acid target and increased stability in the
presence of nucleases.
[0051] While antisense oligonucleotides are a preferred form of
antisense compound, the present invention comprehends other
oligomeric antisense compounds, including but not limited to
oligonucleotide mimetics such as are described below. The antisense
compounds in accordance with this invention preferably comprise
from about 8 to about 50 nucleobases (i.e. from about 8 to about 50
linked nucleosides). One having ordinary skill in the art will
appreciate that this embodies compounds of 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, or 50 nucleobases in length.
[0052] In another embodiment, the antisense compounds of the
invention are 15 to 30 nucleobases in length. One having ordinary
skill in the art will appreciate that this embodies compounds of
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30
nucleobases in length.
[0053] Antisense compounds 8-50 nucleobases in length comprising a
stretch of at least eight (8) consecutive nucleobases selected from
within the illustrative antisense compounds are considered to be
suitable antisense compounds as well. Exemplary preferred antisense
compounds include oligonucleotide sequences that comprise at least
the 8 consecutive nucleobases from the 5'-terminus of one of the
illustrative preferred antisense compounds (the remaining
nucleobases being a consecutive stretch of the same oligonucleotide
beginning immediately upstream of the 5'-terminus of the antisense
compound which is specifically hybridizable to the target nucleic
acid and continuing until the oligonucleotide contains about 8 to
about 50 nucleobases). Similarly preferred antisense compounds are
represented by oligonucleotide sequences that comprise at least the
8 consecutive nucleobases from the 3'-terminus of one of the
illustrative preferred antisense compounds (the remaining
nucleobases being a consecutive stretch of the same oligonucleotide
beginning immediately downstream of the 3'-terminus of the
antisense compound which is specifically hybridizable to the target
nucleic acid and continuing until the oligonucleotide contains
about 8 to about 50 nucleobases). It is also understood that
preferred antisense compounds may be represented by oligonucleotide
sequences that comprise at least 8 consecutive nucleobases from an
internal portion of the sequence of an illustrative preferred
antisense compound, and may extend in either or both directions
until the oligonucleotide contains about 8 to about 50 nucleobases.
One having skill in the art armed with the preferred antisense
compounds illustrated herein will be able, without undue
experimentation, to identify further preferred antisense
compounds.
[0054] As is known in the art, a nucleoside is a base-sugar
combination. The base portion of the nucleoside is normally a
heterocyclic base. The two most common classes of such heterocyclic
bases are the purines and the pyrimidines. Nucleotides are
nucleosides that further include a phosphate group covalently
linked to the sugar portion of the nucleoside. For those
nucleosides that include a pentofuranosyl sugar, the phosphate
group can be linked to either the 2', 3' or 5' hydroxyl moiety of
the sugar. In forming oligonucleotides, the phosphate groups
covalently link adjacent nucleosides to one another to form a
linear polymeric compound. In turn the respective ends of this
linear polymeric structure can be further joined to form a circular
structure, however, open linear structures are generally preferred.
Within the oligonucleotide structure, the phosphate groups are
commonly referred to as forming the internucleoside backbone of the
oligonucleotide. The normal linkage or backbone of RNA and DNA is a
3' to 5' phosphodiester linkage.
[0055] Specific examples of preferred antisense compounds useful in
this invention include oligonucleotides containing modified
backbones or non-natural internucleoside linkages. As defined in
this specification, oligonucleotides having modified backbones
include those that retain a phosphorus atom in the backbone and
those that do not have a phosphorus atom in the backbone. For the
purposes of this specification, and as sometimes referenced in the
art, modified oligonucleotides that do not have a phosphorus atom
in their internucleoside backbone can also be considered to be
oligonucleosides.
[0056] Preferred modified oligonucleotide backbones include, for
example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Various salts, mixed salts and free acid forms are also
included.
[0057] Representative United States patents that teach the
preparation of the above phosphorus-containing linkages include,
but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863;
4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;
5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;
5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;
5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, certain
of which are commonly owned with this application, and each of
which is herein incorporated by reference.
[0058] Preferred modified oligonucleotide backbones that do not
include a phosphorus atom therein have backbones that are formed by
short chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; alkene containing backbones; sulfamate backbones;
methyleneimino and methylenehydrazino backbones; sulfonate and
sulfonamide backbones; amide backbones; and others having mixed N,
O, S and CH.sub.2 component parts.
[0059] Representative United States patents that teach the
preparation of the above oligonucleosides include, but are not
limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;
5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;
5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;
5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;
5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and
5,677,439, certain of which are commonly owned with this
application, and each of which is herein incorporated by
reference.
[0060] In other preferred oligonucleotide mimetics, both the sugar
and the internucleoside linkage, i.e., the backbone, of the
nucleotide units are replaced with novel groups. The base units are
maintained for hybridization with an appropriate nucleic acid
target compound. One such oligomeric compound, an oligonucleotide
mimetic that has been shown to have excellent hybridization
properties, is referred to as a peptide nucleic acid (PNA). In PNA
compounds, the sugar-backbone of an oligonucleotide is replaced
with an amide containing backbone, in particular an
aminoethylglycine backbone. The nucleobases are retained and are
bound directly or indirectly to aza nitrogen atoms of the amide
portion of the backbone. Representative United States patents that
teach the preparation of PNA compounds include, but are not limited
to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of
which is herein incorporated by reference. Further teaching of PNA
compounds can be found in Nielsen et al., Science, 1991, 254,
1497-1500.
[0061] Most preferred embodiments of the invention are
oligonucleotides with phosphorothioate backbones and
oligonucleosides with heteroatom backbones, and in particular
--CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- ((known as a methylene
(methylimino) or MMI backbone)),
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- (wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2--) of
the above referenced U.S. Pat. No. 5,489,677, and the amide
backbones of the above referenced U.S. Pat. No. 5,602,240. Also
preferred are oligonucleotides having morpholino backbone
structures of the above-referenced U.S. Pat. No. 5,034,506.
[0062] Modified oligonucleotides may also contain one or more
substituted sugar moieties. Preferred oligonucleotides comprise one
of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-,
S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein
the alkyl, alkenyl and alkynyl may be substituted or unsubstituted
C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and
alkynyl. Particularly preferred are
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m
are from 1 to about 10. Other preferred oligonucleotides comprise
one of the following at the 2' position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or
O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3,
SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3,
NH.sub.2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. A preferred
modification includes 2'-methoxyethoxy
(2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta,
1995, 78, 486-504) i.e., an alkoxyalkoxy group.
[0063] A further preferred modification includes
2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples hereinbelow, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2, also described in
examples hereinbelow.
[0064] Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2'-fluoro (2'-F).
Similar modifications may also be made at other positions on the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide or in 2'-5' linked oligonucleotides and the
5' position of 5' terminal nucleotide. Oligonucleotides may also
have sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl sugar. Representative United States patents that
teach the preparation of such modified sugar structures include,
but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800;
5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;
5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;
5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and
5,700,920, certain of which are commonly owned with the instant
application, and each of which is herein incorporated by reference
in its entirety.
[0065] Oligonucleotides may also include nucleobase (often referred
to in the art simply as "base") modifications or substitutions. As
used herein, "unmodified" or "natural" nucleobases include the
purine bases adenine (A) and guanine (G), and the pyrimidine bases
thymine (T), cytosine (C) and uracil (U). Modified nucleobases
include other synthetic and natural nucleobases such as
5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,
hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives
of adenine and guanine, 2-propyl and other alkyl derivatives of
adenine and guanine, 2-thiouracil, 2-thiothymine and
2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and
cytosine, 6-azo uracil, cytosine and thymine, 5-uracil
(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,
8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and
guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other
5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and
7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further
nucleobases include those disclosed in U.S. Pat. No. 3,687,808,
those disclosed in The Concise Encyclopedia Of Polymer Science And
Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley &
Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie,
International Edition, 1991, 30, 613, and those disclosed by
Sanghvi, Y. S., Chapter 15, Antisense Research and Applications,
pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993.
Certain of these nucleobases are particularly useful for increasing
the binding affinity of the oligomeric compounds of the invention.
These include 5-substituted pyrimidines, 6-azapyrimidines and N-2,
N-6 and O-6 substituted purines, including 2-aminopropyladenine,
5-propynyluracil and 5-propynylcytosine. 5-methylcytosine
substitutions have been shown to increase nucleic acid duplex
stability by 0.6-1.2 C (Sanghvi, Y. S., Crooke, S. T. and Lebleu,
B., eds., Antisense Research and Applications, CRC Press, Boca
Raton, 1993, pp. 276-278) and are presently preferred base
substitutions, even more particularly when combined with
2'-O-methoxyethyl sugar modifications.
[0066] Representative United States patents that teach the
preparation of certain of the above noted modified nucleobases as
well as other modified nucleobases include, but are not limited to,
the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.
4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;
5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;
5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, certain
of which are commonly owned with the instant application, and each
of which is herein incorporated by reference, and U.S. Pat. No.
5,750,692, which is commonly owned with the instant application and
also herein incorporated by reference.
[0067] Another modification of the oligonucleotides of the
invention involves chemically linking to the oligonucleotide one or
more moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. Such
moieties include but are not limited to lipid moieties such as a
cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,
1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med.
Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,
hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,
660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3,
2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,
1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or
undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10,
1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;
Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,
e.g., di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923-937.
[0068] Representative United States patents that teach the
preparation of such oligonucleotide conjugates include, but are not
limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;
5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;
5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,
5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;
5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;
5,599,928 and 5,688,941, certain of which are commonly owned with
the instant application, and each of which is herein incorporated
by reference.
[0069] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within an oligonucleotide.
The present invention also includes antisense compounds which are
chimeric compounds. "Chimeric" antisense compounds or "chimeras,"
in the context of this invention, are antisense compounds,
particularly oligonucleotides, which contain two or more chemically
distinct regions, each made up of at least one monomer unit, i.e.,
a nucleotide in the case of an oligonucleotide compound. These
oligonucleotides typically contain at least one region wherein the
oligonucleotide is modified so as to confer upon the
oligonucleotide increased resistance to nuclease degradation,
increased cellular uptake, and/or increased binding affinity for
the target nucleic acid. An additional region of the
oligonucleotide may serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is
a cellular endonuclease which cleaves the RNA strand of an RNA:DNA
duplex. Activation of RNase H, therefore, results in cleavage of
the RNA target, thereby greatly enhancing the efficiency of
oligonucleotide inhibition of gene expression. Consequently,
comparable results can often be obtained with shorter
oligonucleotides when chimeric oligonucleotides are used, compared
to phosphorothioate deoxyoligonucleotides hybridizing to the same
target region. Cleavage of the RNA target can be routinely detected
by gel electrophoresis and, if necessary, associated nucleic acid
hybridization techniques known in the art.
[0070] Chimeric antisense compounds of the invention may be formed
as composite structures of two or more oligonucleotides, modified
oligonucleotides, oligonucleosides and/or oligonucleotide mimetics
as described above. Such compounds have also been referred to in
the art as hybrids or gapmers. Representative United States patents
that teach the preparation of such hybrid structures include, but
are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007;
5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;
5,652,355; 5,652,356; and 5,700,922, certain of which are commonly
owned with the instant application, and each of which is herein
incorporated by reference in its entirety.
[0071] The antisense compounds used in accordance with this
invention may be conveniently and routinely made through the
well-known technique of solid phase synthesis. Equipment for such
synthesis is sold by several vendors including, for example,
Applied Biosystems (Foster City, Calif.). Any other means for such
synthesis known in the art may additionally or alternatively be
employed. It is well known to use similar techniques to prepare
oligonucleotides such as the phosphorothioates and alkylated
derivatives.
[0072] The antisense compounds of the invention are synthesized in
vitro and do not include antisense compositions of biological
origin, or genetic vector constructs designed to direct the in vivo
synthesis of antisense molecules.
[0073] The compounds of the invention may also be admixed,
encapsulated, conjugated or otherwise associated with other
molecules, molecule structures or mixtures of compounds, as for
example, liposomes, receptor targeted molecules, oral, rectal,
topical or other formulations, for assisting in uptake,
distribution and/or absorption. Representative United States
patents that teach the preparation of such uptake, distribution
and/or absorption assisting formulations include, but are not
limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;
5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;
4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;
5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;
5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;
5,580,575; and 5,595,756, each of which is herein incorporated by
reference.
[0074] The antisense compounds of the invention encompass any
pharmaceutically acceptable salts, esters, or salts of such esters,
or any other compound which, upon administration to an animal
including a human, is capable of providing (directly or indirectly)
the biologically active metabolite or residue thereof. Accordingly,
for example, the disclosure is also drawn to prodrugs and
pharmaceutically acceptable salts of the compounds of the
invention, pharmaceutically acceptable salts of such prodrugs, and
other bioequivalents.
[0075] The term "prodrug" indicates a therapeutic agent that is
prepared in an inactive form that is converted to an active form
(i.e., drug) within the body or cells thereof by the action of
endogenous enzymes or other chemicals and/or conditions. In
particular, prodrug versions of the oligonucleotides of the
invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate]
derivatives according to the methods disclosed in WO 93/24510 to
Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 to Imbach
et al.
[0076] The term "pharmaceutically acceptable salts" refers to
physiologically and pharmaceutically acceptable salts of the
compounds of the invention: i.e., salts that retain the desired
biological activity of the parent compound and do not impart
undesired toxicological effects thereto.
[0077] Pharmaceutically acceptable base addition salts are formed
with metals or amines, such as alkali and alkaline earth metals or
organic amines. Examples of metals used as cations are sodium,
potassium, magnesium, calcium, and the like. Examples of suitable
amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, dicyclohexylamine, ethylenediamine,
N-methylglucamine, and procaine (see, for example, Berge et al.,
"Pharmaceutical Salts," J. of Pharma Sci., 1977, 66, 1-19). The
base addition salts of said acidic compounds are prepared by
contacting the free acid form with a sufficient amount of the
desired base to produce the salt in the conventional manner. The
free acid form may be regenerated by contacting the salt form with
an acid, and isolating the free acid in the conventional manner.
The free acid forms differ from their respective salt forms
somewhat in certain physical properties such as solubility in polar
solvents, but otherwise the salts are equivalent to their
respective free acid for purposes of the present invention. As used
herein, a "pharmaceutical addition salt" includes a
pharmaceutically acceptable salt of an acid form of one of the
components of the compositions of the invention. These include
organic or inorganic acid salts of the amines. Preferred acid salts
are the hydrochlorides, acetates, salicylates, nitrates and
phosphates. Other suitable pharmaceutically acceptable salts are
well known to those skilled in the art and include basic salts of a
variety of inorganic and organic acids, such as, for example, with
inorganic acids, such as for example hydrochloric acid, hydrobromic
acid, sulfuric acid or phosphoric acid; with organic carboxylic,
sulfonic, sulfo or phospho acids or N-substituted sulfamic acids,
for example acetic acid, propionic acid, glycolic acid, succinic
acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric
acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic
acid, glucaric acid, glucuronic acid, citric acid, benzoic acid,
cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic
acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid,
nicotinic acid or isonicotinic acid; and with amino acids, such as
the 20 alpha-amino acids involved in the synthesis of proteins in
nature, for example glutamic acid or aspartic acid, and also with
phenylacetic acid, methanesulfonic acid, ethanesulfonic acid,
2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,
benzenesulfonic acid, 4-methylbenzenesulfoc acid,
naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or
3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid
(with the formation of cyclamates), or with other acid organic
compounds, such as ascorbic acid. Pharmaceutically acceptable salts
of compounds may also be prepared with a pharmaceutically
acceptable cation. Suitable pharmaceutically acceptable cations are
well known to those skilled in the art and include alkaline,
alkaline earth, ammonium and quaternary ammonium cations.
Carbonates or hydrogen carbonates are also possible.
[0078] For oligonucleotides, preferred examples of pharmaceutically
acceptable salts include but are not limited to (a) salts formed
with cations such as sodium, potassium, ammonium, magnesium,
calcium, polyamines such as spermine and spermidine, etc.; (b) acid
addition salts formed with inorganic acids, for example
hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric
acid, nitric acid and the like; (c) salts formed with organic acids
such as, for example, acetic acid, oxalic acid, tartaric acid,
succinic acid, maleic acid, fumaric acid, gluconic acid, citric
acid, malic acid, ascorbic acid, benzoic acid, tannic acid,
palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic
acid, methanesulfonic acid, p-toluenesulfonic acid,
naphthalenedisulfonic acid, polygalacturonic acid, and the like;
and (d) salts formed from elemental anions such as chlorine,
bromine, and iodine.
[0079] The antisense compounds of the present invention can be
utilized for diagnostics, therapeutics, prophylaxis and as research
reagents and kits. For therapeutics, an animal, preferably a human,
suspected of having a disease or disorder, which can be treated by
modulating the expression of PTP1B, is treated by administering
antisense compounds in accordance with this invention. The
compounds of the invention can be utilized in pharmaceutical
compositions by adding an effective amount of an antisense compound
to a suitable pharmaceutically acceptable diluent or carrier. Use
of the antisense compounds and methods of the invention may also be
useful prophylactically, e.g., to prevent or delay infection,
inflammation or tumor formation, for example.
[0080] The antisense compounds of the invention are useful for
research and diagnostics, because these compounds hybridize to
nucleic acids encoding PTP1B, enabling sandwich and other assays to
easily be constructed to exploit this fact. Hybridization of the
antisense oligonucleotides of the invention with a nucleic acid
encoding PTP1B can be detected by means known in the art. Such
means may include conjugation of an enzyme to the oligonucleotide,
radiolabelling of the oligonucleotide or any other suitable
detection means. Kits using such detection means for detecting the
level of PTP1B in a sample may also be prepared.
[0081] The present invention also includes pharmaceutical
compositions and formulations which include the antisense compounds
of the invention. The pharmaceutical compositions of the present
invention may be administered in a number of ways depending upon
whether local or systemic treatment is desired and upon the area to
be treated. Administration may be topical (including ophthalmic and
to mucous membranes including vaginal and rectal delivery),
pulmonary, e.g., by inhalation or insufflation of powders or
aerosols, including by nebulizer; intratracheal, intranasal,
epidermal and transdermal), oral or parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal or intramuscular injection or infusion; or
intracranial, e.g., intrathecal or intraventricular,
administration. Oligonucleotides with at least one
2'-O-methoxyethyl modification are believed to be particularly
useful for oral administration.
[0082] Pharmaceutical compositions and formulations for topical
administration may include transdermal patches, ointments, lotions,
creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily
bases, thickeners and the like may be necessary or desirable.
Coated condoms, gloves and the like may also be useful.
[0083] Compositions and formulations for oral administration
include powders or granules, suspensions or solutions in water or
non-aqueous media, capsules, sachets or tablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable.
[0084] Compositions and formulations for parenteral, intrathecal or
intraventricular administration may include sterile aqueous
solutions which may also contain buffers, diluents and other
suitable additives such as, but not limited to, penetration
enhancers, carrier compounds and other pharmaceutically acceptable
carriers or excipients.
[0085] Pharmaceutical compositions of the present invention
include, but are not limited to, solutions, emulsions, and
liposome-containing formulations. These compositions may be
generated from a variety of components that include, but are not
limited to, preformed liquids, self-emulsifying solids and
self-emulsifying semisolids.
[0086] The pharmaceutical formulations of the present invention,
which may conveniently be presented in unit dosage form, may be
prepared according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0087] The compositions of the present invention may be formulated
into any of many possible dosage forms such as, but not limited to,
tablets, capsules, liquid syrups, soft gels, suppositories, and
enemas. The compositions of the present invention may also be
formulated as suspensions in aqueous, non-aqueous or mixed media.
Aqueous suspensions may further contain substances which increase
the viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
[0088] In one embodiment of the present invention the
pharmaceutical compositions may be formulated and used as foams.
Pharmaceutical foams include formulations such as, but not limited
to, emulsions, microemulsions, creams, jellies and liposomes. While
basically similar in nature these formulations vary in the
components and the consistency of the final product. The
preparation of such compositions and formulations is generally
known to those skilled in the pharmaceutical and formulation arts
and may be applied to the formulation of the compositions of the
present invention.
Emulsions
[0089] The compositions of the present invention may be prepared
and formulated as emulsions. Emulsions are typically heterogenous
systems of one liquid dispersed in another in the form of droplets
usually exceeding 0.1 m in diameter. (Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p.
335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often
biphasic systems comprising of two immiscible liquid phases
intimately mixed and dispersed with each other. In general,
emulsions may be either water-in-oil (w/o) or of the oil-in-water
(o/w) variety. When an aqueous phase is finely divided into and
dispersed as minute droplets into a bulk oily phase the resulting
composition is called a water-in-oil (w/o) emulsion. Alternatively,
when an oily phase is finely divided into and dispersed as minute
droplets into a bulk aqueous phase the resulting composition is
called an oil-in-water (o/w) emulsion. Emulsions may contain
additional components in addition to the dispersed phases and the
active drug which may be present as a solution in either the
aqueous phase, oily phase or itself as a separate phase.
Pharmaceutical excipients such as emulsifiers, stabilizers, dyes,
and anti-oxidants may also be present in emulsions as needed.
Pharmaceutical emulsions may also be multiple emulsions that are
comprised of more than two phases such as, for example, in the case
of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w)
emulsions. Such complex formulations often provide certain
advantages that simple binary emulsions do not. Multiple emulsions
in which individual oil droplets of an o/w emulsion enclose small
water droplets constitute a w/o/w emulsion. Likewise a system of
oil droplets enclosed in globules of water stabilized in an oily
continuous provides an o/w/o emulsion.
[0090] Emulsions are characterized by little or no thermodynamic
stability. Often, the dispersed or discontinuous phase of the
emulsion is well dispersed into the external or continuous phase
and maintained in this form through the means of emulsifiers or the
viscosity of the formulation. Either of the phases of the emulsion
may be a semisolid or a solid, as is the case of emulsion-style
ointment bases and creams. Other means of stabilizing emulsions
entail the use of emulsifiers that may be incorporated into either
phase of the emulsion. Emulsifiers may broadly be classified into
four categories: synthetic surfactants, naturally occurring
emulsifiers, absorption bases, and finely dispersed solids (Idson,
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
199).
[0091] Synthetic surfactants, also known as surface active agents,
have found wide applicability in the formulation of emulsions and
have been reviewed in the literature (Rieger, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).
Surfactants are typically amphiphilic and comprise a hydrophilic
and a hydrophobic portion. The ratio of the hydrophilic to the
hydrophobic nature of the surfactant has been termed the
hydrophile/lipophile balance (HLB) and is a valuable tool in
categorizing and selecting surfactants in the preparation of
formulations. Surfactants may be classified into different classes
based on the nature of the hydrophilic group: nonionic, anionic,
cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 285).
[0092] Naturally occurring emulsifiers used in emulsion
formulations include lanolin, beeswax, phosphatides, lecithin and
acacia. Absorption bases possess hydrophilic properties such that
they can soak up water to form w/o emulsions yet retain their
semisolid consistencies, such as anhydrous lanolin and hydrophilic
petrolatum. Finely divided solids have also been used as good
emulsifiers especially in combination with surfactants and in
viscous preparations. These include polar inorganic solids, such as
heavy metal hydroxides, nonswelling clays such as bentonite,
attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum
silicate and colloidal magnesium aluminum silicate, pigments and
nonpolar solids such as carbon or glyceryl tristearate.
[0093] A large variety of non-emulsifying materials are also
included in emulsion formulations and contribute to the properties
of emulsions. These include fats, oils, waxes, fatty acids, fatty
alcohols, fatty esters, humectants, hydrophilic colloids,
preservatives and antioxidants (Block, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 199).
[0094] Hydrophilic colloids or hydrocolloids include naturally
occurring gums and synthetic polymers such as polysaccharides (for
example, acacia, agar, alginic acid, carrageenan, guar gum, karaya
gum, and tragacanth), cellulose derivatives (for example,
carboxymethylcellulose and carboxypropylcellulose), and synthetic
polymers (for example, carbomers, cellulose ethers, and
carboxyvinyl polymers). These disperse or swell in water to form
colloidal solutions that stabilize emulsions by forming strong
interfacial films around the dispersed-phase droplets and by
increasing the viscosity of the external phase.
[0095] Since emulsions often contain a number of ingredients such
as carbohydrates, proteins, sterols and phosphatides that may
readily support the growth of microbes, these formulations often
incorporate preservatives. Commonly used preservatives included in
emulsion formulations include methyl paraben, propyl paraben,
quaternary ammonium salts, benzalkonium chloride, esters of
p-hydroxybenzoic acid, and boric acid. Antioxidants are also
commonly added to emulsion formulations to prevent deterioration of
the formulation. Antioxidants used may be free radical scavengers
such as tocopherols, alkyl gallates, butylated hydroxyanisole,
butylated hydroxytoluene, or reducing agents such as ascorbic acid
and sodium metabisulfite, and antioxidant synergists such as citric
acid, tartaric acid, and lecithin.
[0096] The application of emulsion formulations via dermatological,
oral and parenteral routes and methods for their manufacture have
been reviewed in the literature (Idson, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for
oral delivery have been very widely used because of reasons of ease
of formulation, efficacy from an absorption and bioavailability
standpoint. (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,
Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York,
N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 199). Mineral-oil base laxatives,
oil-soluble vitamins and high fat nutritive preparations are among
the materials that have commonly been administered orally as o/w
emulsions.
[0097] In one embodiment of the present invention, the compositions
of oligonucleotides and nucleic acids are formulated as
microemulsions. A microemulsion may be defined as a system of
water, oil and amphiphile which is a single optically isotropic and
thermodynamically stable liquid solution (Rosoff, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically
microemulsions are systems that are prepared by first dispersing an
oil in an aqueous surfactant solution and then adding a sufficient
amount of a fourth component, generally an intermediate
chain-length alcohol to form a transparent system. Therefore,
microemulsions have also been described as thermodynamically
stable, isotropically clear dispersions of two immiscible liquids
that are stabilized by interfacial films of surface-active
molecules (Leung and Shah, in: Controlled Release of Drugs:
Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH
Publishers, New York, pages 185-215). Microemulsions commonly are
prepared via a combination of three to five components that include
oil, water, surfactant, cosurfactant and electrolyte. Whether the
microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w)
type is dependent on the properties of the oil and surfactant used
and on the structure and geometric packing of the polar heads and
hydrocarbon tails of the surfactant molecules (Schott, in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., 1985, p. 271).
[0098] The phenomenological approach utilizing phase diagrams has
been extensively studied and has yielded a comprehensive knowledge,
to one skilled in the art, of how to formulate microemulsions
(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,
p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger
and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,
volume 1, p. 335). Compared to conventional emulsions,
microemulsions offer the advantage of solubilizing water-insoluble
drugs in a formulation of thermodynamically stable droplets that
are formed spontaneously.
[0099] Surfactants used in the preparation of microemulsions
include, but are not limited to, ionic surfactants, non-ionic
surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol
fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol
monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol
pentaoleate (PO500), decaglycerol monocaprate (MCA750),
decaglycerol monooleate (M0750), decaglycerol sequioleate (S0750),
decaglycerol decaoleate (DA0750), alone or in combination with
cosurfactants. The cosurfactant, usually a short-chain alcohol such
as ethanol, 1-propanol, and 1-butanol, serves to increase the
interfacial fluidity by penetrating into the surfactant film and
consequently creating a disordered film because of the void space
generated among surfactant molecules. Microemulsions may, however,
be prepared without the use of cosurfactants and alcohol-free
self-emulsifying microemulsion systems are known in the art. The
aqueous phase may typically be, but is not limited to, water, an
aqueous solution of the drug, glycerol, PEG300, PEG400,
polyglycerols, propylene glycols, and derivatives of ethylene
glycol. The oil phase may include, but is not limited to, materials
such as Captex 300, Captex 355, Capmul MCM, fatty acid esters,
medium chain (C.sub.8-C.sub.12) mono, di, and tri-glycerides,
polyoxyethylated glyceryl fatty acid esters, fatty alcohols,
polyglycolized glycerides, saturated polyglycolized
C.sub.8-C.sub.10 glycerides, vegetable oils and silicone oil.
[0100] Microemulsions are particularly of interest from the
standpoint of drug solubilization and the enhanced absorption of
drugs. Lipid based microemulsions (both o/w and w/o) have been
proposed to enhance the oral bioavailability of drugs, including
peptides (Constantinides et al., Pharmaceutical Research, 1994, 11,
1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13,
205). Microemulsions afford advantages of improved drug
solubilization, protection of drug from enzymatic hydrolysis,
possible enhancement of drug absorption due to surfactant-induced
alterations in membrane fluidity and permeability, ease of
preparation, ease of oral administration over solid dosage forms,
improved clinical potency, and decreased toxicity (Constantinides
et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J.
Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form
spontaneously when their components are brought together at ambient
temperature. This may be particularly advantageous when formulating
thermolabile drugs, peptides or oligonucleotides. Microemulsions
have also been effective in the transdermal delivery of active
components in both cosmetic and pharmaceutical applications. It is
expected that the microemulsion compositions and formulations of
the present invention will facilitate the increased systemic
absorption of oligonucleotides and nucleic acids from the
gastrointestinal tract, as well as improve the local cellular
uptake of oligonucleotides and nucleic acids within the
gastrointestinal tract, vagina, buccal cavity and other areas of
administration.
[0101] Microemulsions of the present invention may also contain
additional components and additives such as sorbitan monostearate
(Grill 3), Labrasol, and penetration enhancers to improve the
properties of the formulation and to enhance the absorption of the
oligonucleotides and nucleic acids of the present invention.
Penetration enhancers used in the microemulsions of the present
invention may be classified as belonging to one of five broad
categories--surfactants, fatty acids, bile salts, chelating agents,
and non-chelating non-surfactants (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these
classes has been discussed above.
Liposomes
[0102] There are many organized surfactant structures besides
microemulsions that have been studied and used for the formulation
of drugs. These include monolayers, micelles, bilayers and
vesicles. Vesicles, such as liposomes, have attracted great
interest because of their specificity and the duration of action
they offer from the standpoint of drug delivery. As used in the
present invention, the term "liposome" means a vesicle composed of
amphiphilic lipids arranged in a spherical bilayer or bilayers.
[0103] Liposomes are unilamellar or multilamellar vesicles which
have a membrane formed from a lipophilic material and an aqueous
interior. The aqueous portion contains the composition to be
delivered. Cationic liposomes possess the advantage of being able
to fuse to the cell wall. Non-cationic liposomes, although not able
to fuse as efficiently with the cell wall, are taken up by
macrophages in vivo.
[0104] In order to cross intact mammalian skin, lipid vesicles must
pass through a series of fine pores, each with a diameter less than
50 nm, under the influence of a suitable transdermal gradient.
Therefore, it is desirable to use a liposome which is highly
deformable and able to pass through such fine pores.
[0105] Further advantages of liposomes include; liposomes obtained
from natural phospholipids are biocompatible and biodegradable;
liposomes can incorporate a wide range of water and lipid soluble
drugs; liposomes can protect encapsulated drugs in their internal
compartments from metabolism and degradation (Rosoff, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
[0106] 245). Important considerations in the preparation of
liposome formulations are the lipid surface charge, vesicle size
and the aqueous volume of the liposomes.
[0107] Liposomes are useful for the transfer and delivery of active
ingredients to the site of action. Because the liposomal membrane
is structurally similar to biological membranes, when liposomes are
applied to a tissue, the liposomes start to merge with the cellular
membranes. As the merging of the liposome and cell progresses, the
liposomal contents are emptied into the cell where the active agent
may act.
[0108] Liposomal formulations have been the focus of extensive
investigation as the mode of delivery for many drugs. There is
growing evidence that for topical administration, liposomes present
several advantages over other formulations. Such advantages include
reduced side-effects related to high systemic absorption of the
administered drug, increased accumulation of the administered drug
at the desired target, and the ability to administer a wide variety
of drugs, both hydrophilic and hydrophobic, into the skin.
[0109] Several reports have detailed the ability of liposomes to
deliver agents including high-molecular weight DNA into the skin.
Compounds including analgesics, antibodies, hormones and
high-molecular weight DNAs have been administered to the skin. The
majority of applications resulted in the targeting of the upper
epidermis.
[0110] Liposomes fall into two broad classes. Cationic liposomes
are positively charged liposomes which interact with the negatively
charged DNA molecules to form a stable complex. The positively
charged DNA/liposome complex binds to the negatively charged cell
surface and is internalized in an endosome. Due to the acidic pH
within the endosome, the liposomes are ruptured, releasing their
contents into the cell cytoplasm (Wang et al., Biochem. Biophys.
Res. Commun., 1987, 147, 980-985).
[0111] Liposomes which are pH-sensitive or negatively-charged,
entrap DNA rather than complex with it. Since both the DNA and the
lipid are similarly charged, repulsion rather than complex
formation occurs. Nevertheless, some DNA is entrapped within the
aqueous interior of these liposomes. pH-sensitive liposomes have
been used to deliver DNA encoding the thymidine kinase gene to cell
monolayers in culture. Expression of the exogenous gene was
detected in the target cells (Zhou et al., Journal of Controlled
Release, 1992, 19, 269-274).
[0112] One major type of liposomal composition includes
phospholipids other than naturally-derived phosphatidyl-choline.
Neutral liposome compositions, for example, can be formed from
dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl
phosphatidylcholine (DPPC). Anionic liposome compositions generally
are formed from dimyristoyl phosphatidylglycerol, while anionic
fusogenic liposomes are formed primarily from dioleoyl
phosphatidylethanol-amine (DOPE). Another type of liposomal
composition is formed from phosphatidylcholine (PC) such as, for
example, soybean PC, and egg PC. Another type is formed from
mixtures of phospholipid and/or phosphatidylcholine and/or
cholesterol.
[0113] Several studies have assessed the topical delivery of
liposomal drug formulations to the skin. Application of liposomes
containing interferon to guinea pig skin resulted in a reduction of
skin herpes sores while delivery of interferon via other means
(e.g. as a solution or as an emulsion) were ineffective (Weiner et
al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an
additional study tested the efficacy of interferon administered as
part of a liposomal formulation to the administration of interferon
using an aqueous system, and concluded that the liposomal
formulation was superior to aqueous administration (du Plessis et
al., Antiviral Research, 1992, 18, 259-265).
[0114] Non-ionic liposomal systems have also been examined to
determine their utility in the delivery of drugs to the skin, in
particular systems comprising non-ionic surfactant and cholesterol.
Non-ionic liposomal formulations comprising Novasomem I (glyceryl
dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and
Novasomem II (glyceryl
distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used
to deliver cyclosporin-A into the dermis of mouse skin. Results
indicated that such non-ionic liposomal systems were effective in
facilitating the deposition of cyclosporin-A into different layers
of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).
[0115] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids that, when incorporated into liposomes,
result in enhanced circulation lifetimes relative to liposomes
lacking such specialized lipids. Examples of sterically stabilized
liposomes are those in which part of the vesicle-forming lipid
portion of the liposome (A) comprises one or more glycolipids, such
as monosialoganglioside G.sub.M1, or (B) is derivatized with one or
more hydrophilic polymers, such as a polyethylene glycol (PEG)
moiety. While not wishing to be bound by any particular theory, it
is thought in the art that, at least for sterically stabilized
liposomes containing gangliosides, sphingomyelin, or
PEG-derivatized lipids, the enhanced circulation half-life of these
sterically stabilized liposomes derives from a reduced uptake into
cells of the reticuloendothelial system (RES) (Allen et al., FEBS
Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53,
3765).
[0116] Various liposomes comprising one or more glycolipids are
known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci.,
1987, 507, 64) reported the ability of monosialoganglioside
G.sub.M1, galactocerebroside sulfate and phosphatidylinositol to
improve blood half-lives of liposomes. These findings were
expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A.,
1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to
Allen et al., disclose liposomes comprising (1) sphingomyelin and
(2) the ganglioside G.sub.M1 or a galactocerebroside sulfate ester.
U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes
comprising sphingomyelin. Liposomes comprising
1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499
(Lim et al.).
[0117] Many liposomes comprising lipids derivatized with one or
more hydrophilic polymers, and methods of preparation thereof, are
known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53,
2778) described liposomes comprising a nonionic detergent,
2C.sub.1215G, that contains a PEG moiety. Illum et al. (FEBS Lett.,
1984, 167, 79) noted that hydrophilic coating of polystyrene
particles with polymeric glycols results in significantly enhanced
blood half-lives. Synthetic phospholipids modified by the
attachment of carboxylic groups of polyalkylene glycols (e.g., PEG)
are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899).
Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments
demonstrating that liposomes comprising phosphatidyl-ethanolamine
(PE) derivatized with PEG or PEG stearate have significant
increases in blood circulation half-lives. Blume et al. (Biochimica
et Biophysica Acta, 1990, 1029, 91) extended such observations to
other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from
the combination of distearoylphosphatidylethanolamine (DSPE) and
PEG. Liposomes having covalently bound PEG moieties on their
external surface are described in European Patent No. EP 0 445 131
B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20
mole percent of PE derivatized with PEG, and methods of use
thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556
and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and
European Patent No. EP 0 496 813 B1). Liposomes comprising a number
of other lipid-polymer conjugates are disclosed in WO 91/05545 and
U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073
(Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids
are described in WO 96/10391 (Choi et al.). U.S. Pat. No. 5,540,935
(Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.)
describe PEG-containing liposomes that can be further derivatized
with functional moieties on their surfaces.
[0118] A limited number of liposomes comprising nucleic acids are
known in the art. WO 96/40062 to Thierry et al. discloses methods
for encapsulating high molecular weight nucleic acids in liposomes.
U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded
liposomes and asserts that the contents of such liposomes may
include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al.
describes certain methods of encapsulating oligodeoxynucleotides in
liposomes. WO 97/04787 to Love et al. discloses liposomes
comprising antisense oligonucleotides targeted to the raf gene.
[0119] Transfersomes are yet another type of liposomes, and are
highly deformable lipid aggregates which are attractive candidates
for drug delivery vehicles. Transfersomes may be described as lipid
droplets which are so highly deformable that they are easily able
to penetrate through pores which are smaller than the droplet.
Transfersomes are adaptable to the environment in which they are
used, e.g. they are self-optimizing (adaptive to the shape of pores
in the skin), self-repairing, frequently reach their targets
without fragmenting, and often self-loading. To make transfersomes
it is possible to add surface edge-activators, usually surfactants,
to a standard liposomal composition. Transfersomes have been used
to deliver serum albumin to the skin. The transfersome-mediated
delivery of serum albumin has been shown to be as effective as
subcutaneous injection of a solution containing serum albumin.
[0120] Surfactants find wide application in formulations such as
emulsions (including microemulsions) and liposomes. The most common
way of classifying and ranking the properties of the many different
types of surfactants, both natural and synthetic, is by the use of
the hydrophile/lipophile balance (HLB). The nature of the
hydrophilic group (also known as the "head") provides the most
useful means for categorizing the different surfactants used in
formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel
Dekker, Inc., New York, N.Y., 1988, p. 285).
[0121] If the surfactant molecule is not ionized, it is classified
as a nonionic surfactant. Nonionic surfactants find wide
application in pharmaceutical and cosmetic products and are usable
over a wide range of pH values. In general their HLB values range
from 2 to about 18 depending on their structure. Nonionic
surfactants include nonionic esters such as ethylene glycol esters,
propylene glycol esters, glyceryl esters, polyglyceryl esters,
sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic
alkanolamides and ethers such as fatty alcohol ethoxylates,
propoxylated alcohols, and ethoxylated/propoxylated block polymers
are also included in this class. The polyoxyethylene surfactants
are the most popular members of the nonionic surfactant class.
[0122] If the surfactant molecule carries a negative charge when it
is dissolved or dispersed in water, the surfactant is classified as
anionic. Anionic surfactants include carboxylates such as soaps,
acyl lactylates, acyl amides of amino acids, esters of sulfuric
acid such as alkyl sulfates and ethoxylated alkyl sulfates,
sulfonates such as alkyl benzene sulfonates, acyl isethionates,
acyl taurates and sulfosuccinates, and phosphates. The most
important members of the anionic surfactant class are the alkyl
sulfates and the soaps.
[0123] If the surfactant molecule carries a positive charge when it
is dissolved or dispersed in water, the surfactant is classified as
cationic. Cationic surfactants include quaternary ammonium salts
and ethoxylated amines. The quaternary ammonium salts are the most
used members of this class.
[0124] If the surfactant molecule has the ability to carry either a
positive or negative charge, the surfactant is classified as
amphoteric. Amphoteric surfactants include acrylic acid
derivatives, substituted alkylamides, N-alkylbetaines and
phosphatides.
[0125] The use of surfactants in drug products, formulations and in
emulsions has been reviewed (Rieger, in Pharmaceutical Dosage
Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
Penetration Enhancers
[0126] In one embodiment, the present invention employs various
penetration enhancers to accomplish the efficient delivery of
nucleic acids, particularly oligonucleotides, to the skin of
animals. Most drugs are present in solution in both ionized and
nonionized forms. However, usually only lipid soluble or lipophilic
drugs readily cross cell membranes. It has been discovered that
even non-lipophilic drugs may cross cell membranes if the membrane
to be crossed is treated with a penetration enhancer. In addition
to aiding the diffusion of non-lipophilic drugs across cell
membranes, penetration enhancers also enhance the permeability of
lipophilic drugs.
[0127] Penetration enhancers may be classified as belonging to one
of five broad categories, i.e., surfactants, fatty acids, bile
salts, chelating agents, and non-chelating non-surfactants (Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.
92). Each of the above mentioned classes of penetration enhancers
are described below in greater detail.
Surfactants
[0128] In connection with the present invention, surfactants (or
"surface-active agents") are chemical entities which, when
dissolved in an aqueous solution, reduce the surface tension of the
solution or the interfacial tension between the aqueous solution
and another liquid, with the result that absorption of
oligonucleotides through the mucosa is enhanced. In addition to
bile salts and fatty acids, these penetration enhancers include,
for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether
and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews
in Therapeutic Drug Carrier Systems, 1991, p. 92); and
perfluorochemical emulsions, such as FC-43. Takahashi et al., J.
Pharm. Pharmacol., 1988, 40, 252).
Fatty Acids
[0129] Various fatty acids and their derivatives which act as
penetration enhancers include, for example, oleic acid, lauric
acid, capric acid (n-decanoic acid), myristic acid, palmitic acid,
stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,
monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid,
arachidonic acid, glycerol 1-monocaprate,
1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines,
C.sub.1-10 alkyl esters thereof (e.g., methyl, isopropyl and
t-butyl), and mono- and di-glycerides thereof (i.e., oleate,
laurate, caprate, myristate, palmitate, stearate, linoleate, etc.)
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,
1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug
Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm.
Pharmacol., 1992, 44, 651-654).
Bile Salts
[0130] The physiological role of bile includes the facilitation of
dispersion and absorption of lipids and fat-soluble vitamins
(Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological
Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill,
New York, 1996, pp. 934-935). Various natural bile salts, and their
synthetic derivatives, act as penetration enhancers. Thus the term
"bile salts" includes any of the naturally occurring components of
bile as well as any of their synthetic derivatives. The bile salts
of the invention include, for example, cholic acid (or its
pharmaceutically acceptable sodium salt, sodium cholate),
dehydrocholic acid (sodium dehydrocholate), deoxycholic acid
(sodium deoxycholate), glucholic acid (sodium glucholate),
glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium
glycodeoxycholate), taurocholic acid (sodium taurocholate),
taurodeoxycholic acid (sodium taurodeoxy-cholate), chenodeoxycholic
acid (sodium chenodeoxy-cholate), ursodeoxycholic acid (UDCA),
sodium tauro-24,25-dihydro-fusidate (STDHF), sodium
glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee
et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical
Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,
1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic
Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm.
Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990,
79, 579-583).
Chelating Agents
[0131] Chelating agents, as used in connection with the present
invention, can be defined as compounds that remove metallic ions
from solution by forming complexes therewith, with the result that
absorption of oligonucleotides through the mucosa is enhanced. With
regards to their use as penetration enhancers in the present
invention, chelating agents have the added advantage of also
serving as DNase inhibitors, as most characterized DNA nucleases
require a divalent metal ion for catalysis and are thus inhibited
by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339).
Chelating agents of the invention include but are not limited to
disodium ethylenediaminetetraacetate (EDTA), citric acid,
salicylates (e.g., sodium salicylate, 5-methoxysalicylate and
homovanilate), N-acyl derivatives of collagen, laureth-9 and
N-amino acyl derivatives of beta-diketones (enamines)(Lee et al.,
Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page
92; Muranishi, Critical Reviews in Therapeutic Drug Carrier
Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14,
43-51).
Non-Chelating Non-Surfactants
[0132] As used herein, non-chelating non-surfactant penetration
enhancing compounds can be defined as compounds that demonstrate
insignificant activity as chelating agents or as surfactants but
that nonetheless enhance absorption of oligonucleotides through the
alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug
Carrier Systems, 1990, 7, 1-33). This class of penetration
enhancers include, for example, unsaturated cyclic ureas, 1-alkyl-
and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical
Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and
non-steroidal anti-inflammatory agents such as diclofenac sodium,
indomethacin and phenylbutazone (Yamashita et al., J. Pharm.
Pharmacol., 1987, 39, 621-626).
[0133] Agents that enhance uptake of oligonucleotides at the
cellular level may also be added to the pharmaceutical and other
compositions of the present invention. For example, cationic
lipids, such as lipofectin (Junichi et al, U.S. Pat. No.
5,705,188), cationic glycerol derivatives, and polycationic
molecules, such as polylysine (Lollo et al., PCT Application WO
97/30731), are also known to enhance the cellular uptake of
oligonucleotides.
[0134] Other agents may be utilized to enhance the penetration of
the administered nucleic acids, including glycols such as ethylene
glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and
terpenes such as limonene and menthone.
Carriers
[0135] Certain compositions of the present invention also
incorporate carrier compounds in the formulation. As used herein,
"carrier compound" or "carrier" can refer to a nucleic acid, or
analog thereof, which is inert (i.e., does not possess biological
activity per se) but is recognized as a nucleic acid by in vivo
processes that reduce the bioavailability of a nucleic acid having
biological activity by, for example, degrading the biologically
active nucleic acid or promoting its removal from circulation. The
coadministration of a nucleic acid and a carrier compound,
typically with an excess of the latter substance, can result in a
substantial reduction of the amount of nucleic acid recovered in
the liver, kidney or other extracirculatory reservoirs, presumably
due to competition between the carrier compound and the nucleic
acid for a common receptor. For example, the recovery of a
partially phosphorothioate oligonucleotide in hepatic tissue can be
reduced when it is coadministered with polyinosinic acid, dextran
sulfate, polycytidic acid or
4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et
al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al.,
Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).
Excipients
[0136] In contrast to a carrier compound, a "pharmaceutical
carrier" or "excipient" is a pharmaceutically acceptable solvent,
suspending agent or any other pharmacologically inert vehicle for
delivering one or more nucleic acids to an animal. The excipient
may be liquid or solid and is selected, with the planned manner of
administration in mind, so as to provide for the desired bulk,
consistency, etc., when combined with a nucleic acid and the other
components of a given pharmaceutical composition. Typical
pharmaceutical carriers include, but are not limited to, binding
agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and
other sugars, microcrystalline cellulose, pectin, gelatin, calcium
sulfate, ethyl cellulose, polyacrylates or calcium hydrogen
phosphate, etc.); lubricants (e.g., magnesium stearate, talc,
silica, colloidal silicon dioxide, stearic acid, metallic
stearates, hydrogenated vegetable oils, corn starch, polyethylene
glycols, sodium benzoate, sodium acetate, etc.); disintegrants
(e.g., starch, sodium starch glycolate, etc.); and wetting agents
(e.g., sodium lauryl sulphate, etc.).
[0137] Pharmaceutically acceptable organic or inorganic excipient
suitable for non-parenteral administration which do not
deleteriously react with nucleic acids can also be used to
formulate the compositions of the present invention. Suitable
pharmaceutically acceptable carriers include, but are not limited
to, water, salt solutions, alcohols, polyethylene glycols, gelatin,
lactose, amylose, magnesium stearate, talc, silicic acid, viscous
paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the
like.
[0138] Formulations for topical administration of nucleic acids may
include sterile and non-sterile aqueous solutions, non-aqueous
solutions in common solvents such as alcohols, or solutions of the
nucleic acids in liquid or solid oil bases. The solutions may also
contain buffers, diluents and other suitable additives.
Pharmaceutically acceptable organic or inorganic excipients
suitable for non-parenteral administration which do not
deleteriously react with nucleic acids can be used.
[0139] Suitable pharmaceutically acceptable excipients include, but
are not limited to, water, salt solutions, alcohol, polyethylene
glycols, gelatin, lactose, amylose, magnesium stearate, talc,
silicic acid, viscous paraffin, hydroxymethylcellulose,
polyvinylpyrrolidone and the like.
Other Components
[0140] The compositions of the present invention may additionally
contain other adjunct components conventionally found in
pharmaceutical compositions, at their art-established usage levels.
Thus, for example, the compositions may contain additional,
compatible, pharmaceutically-active materials such as, for example,
antipruritics, astringents, local anesthetics or anti-inflammatory
agents, or may contain additional materials useful in physically
formulating various dosage forms of the compositions of the present
invention, such as dyes, flavoring agents, preservatives,
antioxidants, opacifiers, thickening agents and stabilizers.
However, such materials, when added, should not unduly interfere
with the biological activities of the components of the
compositions of the present invention. The formulations can be
sterilized and, if desired, mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
colorings, flavorings and/or aromatic substances and the like which
do not deleteriously interact with the nucleic acid(s) of the
formulation.
[0141] Aqueous suspensions may contain substances which increase
the viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
[0142] Certain embodiments of the invention provide pharmaceutical
compositions containing (a) one or more antisense compounds and (b)
one or more other chemotherapeutic agents which function by a
non-antisense mechanism. Examples of such chemotherapeutic agents
include, but are not limited to, anticancer drugs such as
daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin,
nitrogen mustard, chlorambucil, melphalan, cyclophosphamide,
6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil
(5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine,
vincristine, vinblastine, etoposide, teniposide, cisplatin and
diethylstilbestrol (DES). See, generally, The Merck Manual of
Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,
N.J., pages 1206-1228). Anti-inflammatory drugs, including but not
limited to nonsteroidal anti-inflammatory drugs and
corticosteroids, and antiviral drugs, including but not limited to
ribivirin, vidarabine, acyclovir and ganciclovir, may also be
combined in compositions of the invention. See, generally, The
Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al.,
eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively).
Other non-antisense chemotherapeutic agents are also within the
scope of this invention. Two or more combined compounds may be used
together or sequentially.
[0143] In another related embodiment, compositions of the invention
may contain one or more antisense compounds, particularly
oligonucleotides, targeted to a first nucleic acid and one or more
additional antisense compounds targeted to a second nucleic acid
target. Numerous examples of antisense compounds are known in the
art. Two or more combined compounds may be used together or
sequentially.
[0144] The formulation of therapeutic compositions and their
subsequent administration is believed to be within the skill of
those in the art. Dosing is dependent on severity and
responsiveness of the disease state to be treated, with the course
of treatment lasting from several days to several months, or until
a cure is effected or a diminution of the disease state is
achieved. Optimal dosing schedules can be calculated from
measurements of drug accumulation in the body of the patient.
Persons of ordinary skill can easily determine optimum dosages,
dosing methodologies and repetition rates. Optimum dosages may vary
depending on the relative potency of individual oligonucleotides,
and can generally be estimated based on EC.sub.50s found to be
effective in in vitro and in vivo animal models. In general, dosage
is from 0.01 .mu.g to 100 g per kg of body weight, and may be given
once or more daily, weekly, monthly or yearly, or even once every 2
to 20 years. Persons of ordinary skill in the art can easily
estimate repetition rates for dosing based on measured residence
times and concentrations of the drug in bodily fluids or tissues.
Following successful treatment, it may be desirable to have the
patient undergo maintenance therapy to prevent the recurrence of
the disease state, wherein the oligonucleotide is administered in
maintenance doses, ranging from 0.01 .mu.g to 100 g per kg of body
weight, once or more daily, to once every 20 years.
[0145] While the present invention has been described with
specificity in accordance with certain of its preferred
embodiments, the following examples serve only to illustrate the
invention and are not intended to limit the same.
EXAMPLES
Example 1
Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and
2'-alkoxy amidites
[0146] 2'-Deoxy and 2'-methoxy beta-cyanoethyldiisopropyl
phosphoramidites were purchased from commercial sources (e.g.
Chemgenes, Needham, Mass. or Glen Research, Inc., Sterling, Va.).
Other 2'-O-alkoxy substituted nucleoside amidites are prepared as
described in U.S. Pat. No. 5,506,351, herein incorporated by
reference. For oligonucleotides synthesized using 2'-alkoxy
amidites, the standard cycle for unmodified oligonucleotides was
utilized, except the wait step after pulse delivery of tetrazole
and base was increased to 360 seconds.
[0147] Oligonucleotides containing 5-methyl-2'-deoxycytidine
(5-Me-C) nucleotides were synthesized according to published
methods [Sanghvi, et. al., Nucleic Acids Research, 1993, 21,
3197-3203] using commercially available phosphoramidites (Glen
Research, Sterling, Va., or ChemGenes, Needham, Mass.).
2'-Fluoro amidites
2'-Fluorodeoxyadenosine amidites
[0148] 2'-fluoro oligonucleotides were synthesized as described
previously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841]
and U.S. Pat. No. 5,670,633, herein incorporated by reference.
Briefly, the protected nucleoside
N6-benzoyl-2'-deoxy-2'-fluoroadenosine was synthesized utilizing
commercially available 9-beta-D-arabinofuranosyladenine as starting
material and by modifying literature procedures whereby the
2'-alpha-fluoro atom is introduced by a S.sub.N2-displacement of a
2'-beta-trityl group. Thus
N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively
protected in moderate yield as the 3',5'-ditetrahydropyranyl (THP)
intermediate. Deprotection of the THP and N6-benzoyl groups was
accomplished using standard methodologies and standard methods were
used to obtain the 5'-dimethoxytrityl-(DMT) and
5'-DMT-3'-phosphoramidite intermediates.
2'-Fluorodeoxyguanosine
[0149] The synthesis of 2'-deoxy-2'-fluoroguanosine was
accomplished using tetraisopropyldisiloxanyl (TPDS) protected
9-beta-D-arabinofuranosylguanine as starting material, and
conversion to the intermediate
diisobutyrylarabinofuranosylguanosine. Deprotection of the TPDS
group was followed by protection of the hydroxyl group with THP to
give diisobutyryl di-THP protected arabinofuranosylguanine.
Selective O-deacylation and triflation was followed by treatment of
the crude product with fluoride, then deprotection of the THP
groups. Standard methodologies were used to obtain the 5'-DMT- and
5'-DMT-3'-phosphoramidites.
2'-Fluorouridine
[0150] Synthesis of 2'-deoxy-2'-fluorouridine was accomplished by
the modification of a literature procedure in which
2,2'-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%
hydrogen fluoride-pyridine. Standard procedures were used to obtain
the 5'-DMT and 5'-DMT-3phosphoramidites.
2'-Fluorodeoxycytidine
[0151] 2'-deoxy-2'-fluorocytidine was synthesized via amination of
2'-deoxy-2'-fluorouridine, followed by selective protection to give
N4-benzoyl-2'-deoxy-2'-fluorocytidine. Standard procedures were
used to obtain the 5'-DMT and 5'-DMT-3'phosphoramidites.
2'-O-(2-Methoxyethyl) modified amidites
[0152] 2'-O-Methoxyethyl-substituted nucleoside amidites are
prepared as follows, or alternatively, as per the methods of
Martin, P., Helvetica Chimica Acta, 1995, 78, 486-504.
2,2'-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]
[0153] 5-Methyluridine (ribosylthymine, commercially available
through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate
(90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were
added to DMF (300 .mu.L). The mixture was heated to reflux, with
stirring, allowing the evolved carbon dioxide gas to be released in
a controlled manner. After 1 hour, the slightly darkened solution
was concentrated under reduced pressure. The resulting syrup was
poured into diethylether (2.5 L), with stirring. The product formed
a gum. The ether was decanted and the residue was dissolved in a
minimum amount of methanol (ca. 400 mL). The solution was poured
into fresh ether (2.5 L) to yield a stiff gum. The ether was
decanted and the gum was dried in a vacuum oven (60.degree. C. at 1
mm Hg for 24 h) to give a solid that was crushed to a light tan
powder (57 g, 85% crude yield). The NMR spectrum was consistent
with the structure, contaminated with phenol as its sodium salt
(ca. 5%). The material was used as is for further reactions (or it
can be purified further by column chromatography using a gradient
of methanol in ethyl acetate (10-25%) to give a white solid, mp
222-4.degree. C.).
2'-O-Methoxyethyl-5-methyluridine
[0154] 2,2'-Anhydro-5-methyluridine (195 g, 0.81 M),
tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol
(1.2 L) were added to a 2 L stainless steel pressure vessel and
placed in a pre-heated oil bath at 160.degree. C. After heating for
48 hours at 155-16 C, the vessel was opened and the solution
evaporated to dryness and triturated with MeOH (200 mL). The
residue was suspended in hot acetone (1 L). The insoluble salts
were filtered, washed with acetone (150 mL) and the filtrate
evaporated. The residue (280 g) was dissolved in CH.sub.3CN (600
mL) and evaporated. A silica gel column (3 kg) was packed in
CH.sub.2Cl.sub.2/acetone/MeOH (20:5:3) containing 0.5% Et.sub.3NH.
The residue was dissolved in CH.sub.2Cl.sub.2 (250 mL) and adsorbed
onto silica (150 g) prior to loading onto the column. The product
was eluted with the packing solvent to give 160 g (63%) of product.
Additional material was obtained by reworking impure fractions.
2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine
[0155] 2'-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was
co-evaporated with pyridine (250 mL) and the dried residue
dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl
chloride (94.3 g, 0.278 M) was added and the mixture stirred at
room temperature for one hour. A second aliquot of dimethoxytrityl
chloride (94.3 g, 0.278 M) was added and the reaction stirred for
an additional one hour. Methanol (170 mL) was then added to stop
the reaction. HPLC showed the presence of approximately 70%
product. The solvent was evaporated and triturated with CH.sub.3CN
(200 mL). The residue was dissolved in CHCl.sub.3 (1.5 L) and
extracted with 2.times.500 mL of saturated NaHCO.sub.3 and
2.times.500 mL of saturated NaCl. The organic phase was dried over
Na.sub.2SO.sub.4, filtered and evaporated. 275 g of residue was
obtained. The residue was purified on a 3.5 kg silica gel column,
packed and eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5%
Et.sub.3NH. The pure fractions were evaporated to give 164 g of
product. Approximately 20 g additional was obtained from the impure
fractions to give a total yield of 183 g (57%).
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine
[0156] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (106
g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from
562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38
mL, 0.258 M) were combined and stirred at room temperature for 24
hours. The reaction was monitored by TLC by first quenching the TLC
sample with the addition of MeOH. Upon completion of the reaction,
as judged by TLC, MeOH (50 mL) was added and the mixture evaporated
at 35 C. The residue was dissolved in CHCl.sub.3 (800 mL) and
extracted with 2.times.200 mL of saturated sodium bicarbonate and
2.times.200 mL of saturated NaCl. The water layers were back
extracted with 200 mL of CHCl.sub.3. The combined organics were
dried with sodium sulfate and evaporated to give 122 g of residue
(approx. 90% product). The residue was purified on a 3.5 kg silica
gel column and eluted using EtOAc/hexane (4:1). Pure product
fractions were evaporated to yield 96 g (84%). An additional 1.5 g
was recovered from later fractions.
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleurid-
ine
[0157] A first solution was prepared by dissolving
3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine
(96 g, 0.144 M) in CH.sub.3CN (700 mL) and set aside. Triethylamine
(189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M)
in CH.sub.3CN (1 L), cooled to -5.degree. C. and stirred for 0.5 h
using an overhead stirrer. POCl.sub.3 was added dropwise, over a 30
minute period, to the stirred solution maintained at 0-10C, and the
resulting mixture stirred for an additional 2 hours. The first
solution was added dropwise, over a 45 minute period, to the latter
solution. The resulting reaction mixture was stored overnight in a
cold room. Salts were filtered from the reaction mixture and the
solution was evaporated. The residue was dissolved in EtOAc (1 L)
and the insoluble solids were removed by filtration. The filtrate
was washed with 1.times.300 mL of NaHCO.sub.3 and 2.times.300 mL of
saturated NaCl, dried over sodium sulfate and evaporated. The
residue was triturated with EtOAc to give the title compound.
2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
[0158] A solution of
3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuri-
dine (103 g, 0.141 M) in dioxane (500 mL) and NH.sub.4OH (30 mL)
was stirred at room temperature for 2 hours. The dioxane solution
was evaporated and the residue azeotroped with MeOH (2.times.200
mL). The residue was dissolved in MeOH (300 mL) and transferred to
a 2 L stainless steel pressure vessel. MeOH (400 mL) saturated with
NH.sub.3 gas was added and the vessel heated to 100.degree. C. for
2 hours (TLC showed complete conversion). The vessel contents were
evaporated to dryness and the residue was dissolved in EtOAc (500
mL) and washed once with saturated NaCl (200 mL). The organics were
dried over sodium sulfate and the solvent was evaporated to give 85
g (95%) of the title compound.
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
[0159] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (85
g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride
(37.2 g, 0.165 M) was added with stirring. After stirring for 3
hours, TLC showed the reaction to be approximately 95% complete.
The solvent was evaporated and the residue azeotroped with MeOH
(200 mL). The residue was dissolved in CHCl.sub.3 (700 mL) and
extracted with saturated NaHCO.sub.3 (2.times.300 mL) and saturated
NaCl (2.times.300 mL), dried over MgSO.sub.4 and evaporated to give
a residue (96 g). The residue was chromatographed on a 1.5 kg
silica column using EtOAc/hexane (1:1) containing 0.5% Et.sub.3NH
as the eluting solvent. The pure product fractions were evaporated
to give 90 g (90%) of the title compound.
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine-3'-amid-
ite
[0160]
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
(74 g, 0.10 M) was dissolved in CH.sub.2Cl.sub.2 (1 L). Tetrazole
diisopropylamine (7.1 g) and
2-cyanoethoxy-tetra(iso-propyl)phosphite (40.5 mL, 0.123 M) were
added with stirring, under a nitrogen atmosphere. The resulting
mixture was stirred for 20 hours at room temperature (TLC showed
the reaction to be 95% complete). The reaction mixture was
extracted with saturated NaHCO.sub.3 (1.times.300 mL) and saturated
NaCl (3.times.300 mL). The aqueous washes were back-extracted with
CH.sub.2Cl.sub.2 (300 mL), and the extracts were combined, dried
over MgSO.sub.4 and concentrated. The residue obtained was
chromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1)
as the eluting solvent. The pure fractions were combined to give
90.6 g (87%) of the title compound.
2'-O-(Aminooxyethyl) nucleoside amidites and
2'-O-(dimethylaminooxyethyl) nucleoside amidites
2'-(Dimethylaminooxyethoxy) nucleoside amidites
[0161] 2'-(Dimethylaminooxyethoxy) nucleoside amidites (also known
in the art as 2'-O-(dimethylaminooxyethyl) nucleoside amidites) are
prepared as described in the following paragraphs. Adenosine,
cytidine and guanosine nucleoside amidites are prepared similarly
to the thymidine (5-methyluridine) except the exocyclic amines are
protected with a benzoyl moiety in the case of adenosine and
cytidine and with isobutyryl in the case of guanosine.
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-5-methyluridine
[0162] O.sup.2-2'-anhydro-5-methyluridine (Pro. Bio. Sint., Varese,
Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013
eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient
temperature under an argon atmosphere and with mechanical stirring.
tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458
mmol) was added in one portion. The reaction was stirred for 16 h
at ambient temperature. TLC (Rf 0.22, ethyl acetate) indicated a
complete reaction. The solution was concentrated under reduced
pressure to a thick oil. This was partitioned between
dichloromethane (1 L) and saturated sodium bicarbonate (2.times.1
L) and brine (1 L). The organic layer was dried over sodium sulfate
and concentrated under reduced pressure to a thick oil. The oil was
dissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600
mL) and the solution was cooled to -10.degree. C. The resulting
crystalline product was collected by filtration, washed with ethyl
ether (3.times.200 mL) and dried (40.degree. C., 1 mm Hg, 24 h) to
149 g (74.8%) of white solid. TLC and NMR were consistent with pure
product.
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine
[0163] In a 2 L stainless steel, unstirred pressure reactor was
added borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the
fume hood and with manual stirring, ethylene glycol (350 mL,
excess) was added cautiously at first until the evolution of
hydrogen gas subsided.
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-5-methyluridine
(149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were
added with manual stirring. The reactor was sealed and heated in an
oil bath until an internal temperature of 160.degree. C. was
reached and then maintained for 16 h (pressure <100 psig). The
reaction vessel was cooled to ambient and opened. TLC (Rf 0.67 for
desired product and Rf 0.82 for ara-T side product, ethyl acetate)
indicated about 70% conversion to the product. In order to avoid
additional side product formation, the reaction was stopped,
concentrated under reduced pressure (10 to 1 mm Hg) in a warm water
bath (40-100.degree. C.) with the more extreme conditions used to
remove the ethylene glycol. Alternatively, once the low boiling
solvent is gone, the remaining solution can be partitioned between
ethyl acetate and water. The product will be in the organic phase.
The residue was purified by column chromatography (2 kg silica gel,
ethyl acetate-hexanes gradient 1:1 to 4:1). The appropriate
fractions were combined, stripped and dried to product as a white
crisp foam (84 g, 50%), contaminated starting material (17.4 g) and
pure reusable starting material 20 g. The yield based on starting
material less pure recovered starting material was 58%. TLC and NMR
were consistent with 99% pure product.
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine
[0164]
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine
(20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g,
44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was
then dried over P.sub.2O.sub.5 under high vacuum for two days at
40.degree. C. The reaction mixture was flushed with argon and dry
THF (369.8 mL, Aldrich, sure seal bottle) was added to get a clear
solution. Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added
dropwise to the reaction mixture. The rate of addition is
maintained such that resulting deep red coloration is just
discharged before adding the next drop. After the addition was
complete, the reaction was stirred for 4 hrs. By that time TLC
showed the completion of the reaction (ethylacetate:hexane, 60:40).
The solvent was evaporated in vacuum. Residue obtained was placed
on a flash column and eluted with ethyl acetate:hexane (60:40), to
get
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenyl-silyl-5-methyluridine
as white foam (21.819 g, 86%).
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methylurid-
ine
[0165]
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methylurid-
ine (3.1 g, 4.5 mmol) was dissolved in dry CH.sub.2Cl.sub.2 4.5 mL)
and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at
-10.degree. C. to 0.degree. C. After 1 h the mixture was filtered,
the filtrate was washed with ice cold CH.sub.2Cl.sub.2 and the
combined organic phase was washed with water, brine and dried over
anhydrous Na.sub.2SO.sub.4. The solution was concentrated to get
2'-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH
(67.5 mL). To this formaldehyde (20% aqueous solution, w/w, 1.1
eq.) was added and the resulting mixture was strirred for 1 h.
Solvent was removed under vacuum; residue chromatographed to get
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximi-nooxy)
ethyl]-5-methyluridine as white foam (1.95 g, 78%).
5'-O-tert-Butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methylurid-
ine
[0166]
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-me-
thyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M
pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium
cyanoborohydride (0.39 g, 6.13 mmol) was added to this solution at
10.degree. C. under inert atmosphere. The reaction mixture was
stirred for 10 minutes at 10.degree. C. After that the reaction
vessel was removed from the ice bath and stirred at room
temperature for 2 h, the reaction monitored by TLC (5% MeOH in
CH.sub.2Cl.sub.2). Aqueous NaHCO.sub.3 solution (5%, 10 mL) was
added and extracted with ethyl acetate (2.times.20 mL). Ethyl
acetate phase was dried over anhydrous Na.sub.2SO.sub.4, evaporated
to dryness. Residue was dissolved in a solution of 1M PPTS in MeOH
(30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) was added and
the reaction mixture was stirred at room temperature for 10
minutes. Reaction mixture cooled to 10.degree. C. in an ice bath,
sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reaction
mixture stirred at 10 C for 10 minutes. After 10 minutes, the
reaction mixture was removed from the ice bath and stirred at room
temperature for 2 hrs. To the reaction mixture 5% NaHCO.sub.3 (25
mL) solution was added and extracted with ethyl acetate (2.times.25
mL). Ethyl acetate layer was dried over anhydrous Na.sub.2SO.sub.4
and evaporated to dryness. The residue obtained was purified by
flash column chromatography and eluted with 5% MeOH in
CH.sub.2Cl.sub.2 to get
5'-O-tert-butyldiphenylsilyl-2'-O-[N,N-dimethylamino-oxyethyl]-5-methylur-
idine as a white foam (14.6 g, 80%).
2'-O-(dimethylaminooxyethyl)-5-methyluridine
[0167] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was
dissolved in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept
over KOH). This mixture of triethylamine-2HF was then added to
5'-O-tert-butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluri-
dine (1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs.
Reaction was monitored by TLC (5% MeOH in CH.sub.2Cl.sub.2).
Solvent was removed under vacuum and the residue placed on a flash
column and eluted with 10% MeOH in CH.sub.2Cl.sub.2 to get
2'-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%).
5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine
[0168] 2'-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17
mmol) was dried over P.sub.2O.sub.5 under high vacuum overnight at
40.degree. C. It was then co-evaporated with anhydrous pyridine (20
mL). The residue obtained was dissolved in pyridine (11 mL) under
argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol),
4,4'-dimethoxytrityl chloride (880 mg, 2.60 mmol) was added to the
mixture and the reaction mixture was stirred at room temperature
until all of the starting material disappeared. Pyridine was
removed under vacuum and the residue chromatographed and eluted
with 10% MeOH in CH.sub.2Cl.sub.2 (containing a few drops of
pyridine) to get
5'-O-DMT-2'-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g,
80%).
5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoet-
hyl)-N,N-diisopropylphosphoramidite]
[0169] 5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine (1.08
g, 1.67 mmol) was co-evaporated with toluene (20 mL). To the
residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was
added and dried over P.sub.2O.sub.5 under high vacuum overnight at
40.degree. C. Then the reaction mixture was dissolved in anhydrous
acetonitrile (8.4 mL) and
2-cyanoethyl-N,N,N.sup.1,N.sup.1-tetraisopropylphosphoramidite
(2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at
ambient temperature for 4 hrs under inert atmosphere. The progress
of the reaction was monitored by TLC (hexane:ethyl acetate 1:1).
The solvent was evaporated, then the residue was dissolved in ethyl
acetate (70 mL) and washed with 5% aqueous NaHCO.sub.3 (40 mL).
Ethyl acetate layer was dried over anhydrous Na.sub.2SO.sub.4 and
concentrated. Residue obtained was chromatographed (ethyl acetate
as eluent) to get
5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoe-
thyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g,
74.9%).
2'-(Aminooxyethoxy) nucleoside amidites
[0170] 2'-(Aminooxyethoxy) nucleoside amidites (also known in the
art as 2'-O-(aminooxyethyl) nucleoside amidites) are prepared as
described in the following paragraphs. Adenosine, cytidine and
thymidine nucleoside amidites are prepared similarly.
N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimeth-
oxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]
[0171] The 2'-O-aminooxyethyl guanosine analog may be obtained by
selective 2'-O-alkylation of diaminopurine riboside. Multigram
quantities of diaminopurine riboside may be purchased from Schering
AG (Berlin) to provide 2'-O-(2-ethylacetyl) diaminopurine riboside
along with a minor amount of the 3'-O-isomer. 2'-O-(2-ethylacetyl)
diaminopurine riboside may be resolved and converted to
2'-O-(2-ethylacetyl)guanosine by treatment with adenosine
deaminase. (PCT Publication WO 94/02501). Standard protection
procedures should afford
2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxy-trityl)guanosine and
2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dime-
thoxytrityl)guanosine which may be reduced to provide
2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dime-
thoxytrityl)guanosine. As before the hydroxyl group may be
displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the
protected nucleoside may phosphitylated as usual to yield
2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dime-
thoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].
2'-dimethylaminoethoxyethoxy (2'-DMAEOE) nucleoside amidites
[0172] 2'-dimethylaminoethoxyethoxy nucleoside amidites (also known
in the art as 2'-O-dimethylaminoethoxyethyl, i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2, or 2'-DMAEOE
nucleoside amidites) are prepared as follows. Other nucleoside
amidites are prepared similarly.
2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine
[0173] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol)
is slowly added to a solution of borane in tetrahydrofuran (1 M, 10
mL, 10 mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves
as the solid dissolves. O.sup.2-,2'-anhydro-5-methyluridine (1.2 g,
5 mmol), and sodium bicarbonate (2.5 mg) are added and the bomb is
sealed, placed in an oil bath and heated to 155.degree. C. for 26
hours. The bomb is cooled to room temperature and opened. The crude
solution is concentrated and the residue partitioned between water
(200 mL) and hexanes (200 mL). The excess phenol is extracted into
the hexane layer. The aqueous layer is extracted with ethyl acetate
(3.times.200 mL) and the combined organic layers are washed once
with water, dried over anhydrous sodium sulfate and concentrated.
The residue is columned on silica gel using methanol/methylene
chloride 1:20 (which has 2% triethylamine) as the eluent. As the
column fractions are concentrated a colorless solid forms which is
collected to give the title compound as a white solid.
5'-O-dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl
uridine
[0174] To 0.5 g (1.3 mmol) of
2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine in
anhydrous pyridine (8 mL), triethylamine (0.36 mL) and
dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) are added and
stirred for 1 hour. The reaction mixture is poured into water (200
mL) and extracted with CH.sub.2Cl.sub.2 (2.times.200 mL). The
combined CH.sub.2Cl.sub.2 layers are washed with saturated
NaHCO.sub.3 solution, followed by saturated NaCl solution and dried
over anhydrous sodium sulfate. Evaporation of the solvent followed
by silica gel chromatography using MeOH:CH.sub.2Cl.sub.2:Et.sub.3N
(20:1, v/v, with 1% triethylamine) gives the title compound.
5'-O-Dimethoxytrityl-2'-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl
uridine-3'-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite
[0175] Diisopropylaminotetrazolide (0.6 g) and
2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are
added to a solution of
5'-O-dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methylur-
idine (2.17 g, 3 mmol) dissolved in CH.sub.2Cl.sub.2 (20 mL) under
an atmosphere of argon. The reaction mixture is stirred overnight
and the solvent evaporated. The resulting residue is purified by
silica gel flash column chromatography with ethyl acetate as the
eluent to give the title compound.
Example 2
Oligonucleotide Synthesis
[0176] Unsubstituted and substituted phosphodiester (P.dbd.O)
oligonucleotides are synthesized on an automated DNA synthesizer
(Applied Biosystems model 380B) using standard phosphoramidite
chemistry with oxidation by iodine.
[0177] Phosphorothioates (P.dbd.S) are synthesized as for the
phosphodiester oligonucleotides except the standard oxidation
bottle was replaced by 0.2 M solution of 3H-1,2-benzodithiole-3-one
1,1-dioxide in acetonitrile for the stepwise thiation of the
phosphite linkages. The thiation wait step was increased to 68 sec
and was followed by the capping step. After cleavage from the CPG
column and deblocking in concentrated ammonium hydroxide at 55 C
(18 h), the oligonucleotides were purified by precipitating twice
with 2.5 volumes of ethanol from a 0.5 M NaCl solution. Phosphinate
oligonucleotides are prepared as described in U.S. Pat. No.
5,508,270, herein incorporated by reference.
[0178] Alkyl phosphonate oligonucleotides are prepared as described
in U.S. Pat. No. 4,469,863, herein incorporated by reference.
[0179] 3'-Deoxy-3'-methylene phosphonate oligonucleotides are
prepared as described in U.S. Pat. No. 5,610,289 or U.S. Pat. No.
5,625,050, herein incorporated by reference.
[0180] Phosphoramnidite oligonucleotides are prepared as described
in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein
incorporated by reference.
[0181] Alkylphosphonothioate oligonucleotides are prepared as
described in PCT applications PCT/US94/00902 and PCT/US93/06976
(published as WO 94/17093 and WO 94/02499, respectively), herein
incorporated by reference. 3'-Deoxy-3'-amino phosphoramidate
oligonucleotides are prepared as described in U.S. Pat. No.
5,476,925, herein incorporated by reference.
[0182] Phosphotriester oligonucleotides are prepared as described
in U.S. Pat. No. 5,023,243, herein incorporated by reference.
[0183] Borano phosphate oligonucleotides are prepared as described
in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated
by reference.
Example 3
Oligonucleoside Synthesis
[0184] Methylenemethylimino linked oligonucleosides, also
identified as MMI linked oligonucleosides, methylenedimethylhydrazo
linked oligonucleosides, also identified as MDH linked
oligonucleosides, and methylenecarbonylamino linked
oligonucleosides, also identified as amide-3 linked
oligonucleosides, and methyleneaminocarbonyl linked
oligonucleosides, also identified as amide-4 linked
oligonucleosides, as well as mixed backbone compounds having, for
instance, alternating MMI and P.dbd.O or P.dbd.S linkages are
prepared as described in U.S. Pat. Nos. 5,378,825; 5,386,023;
5,489,677; 5,602,240; and 5,610,289, all of which are herein
incorporated by reference.
[0185] Formacetal and thioformacetal linked oligonucleosides are
prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564,
herein incorporated by reference.
[0186] Ethylene oxide linked oligonucleosides are prepared as
described in U.S. Pat. No. 5,223,618, herein incorporated by
reference.
Example 4
PNA Synthesis
[0187] Peptide nucleic acids (PNAs) are prepared in accordance with
any of the various procedures referred to in Peptide Nucleic Acids
(PNA): Synthesis, Properties and Potential Applications, Bioorganic
& Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared
in accordance with U.S. Pat. Nos. 5,539,082; 5,700,922; and
5,719,262, herein incorporated by reference.
Example 5
Synthesis of Chimeric Oligonucleotides
[0188] Chimeric oligonucleotides, oligonucleosides or mixed
oligonucleotides/oligonucleosides of the invention can be of
several different types. These include a first type wherein the
"gap" segment of linked nucleosides is positioned between 5' and 3'
"wing" segments of linked nucleosides and a second "open end" type
wherein the "gap" segment is located at either the 3' or the 5'
terminus of the oligomeric compound. Oligonucleotides of the first
type are also known in the art as "gapmers" or gapped
oligonucleotides. Oligonucleotides of the second type are also
known in the art as "hemimers" or "wingmers".
[2'-O-Me]-[2'-deoxy]--[2'-O-Me] Chimeric Phosphorothioate
Oligonucleotides
[0189] Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate
and 2'-deoxy phosphorothioate oligonucleotide segments are
synthesized using an Applied Biosystems automated DNA synthesizer
Model 380B, as above. Oligonucleotides are synthesized using the
automated synthesizer and
2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA
portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for
5' and 3' wings. The standard synthesis cycle is modified by
increasing the wait step after the delivery of tetrazole and base
to 600 s repeated four times for RNA and twice for 2'-O-methyl. The
fully protected oligonucleotide is cleaved from the support and the
phosphate group is deprotected in 3:1 ammonia/ethanol at room
temperature overnight then lyophilized to dryness. Treatment in
methanolic ammonia for 24 hrs at room temperature is then done to
deprotect all bases and sample was again lyophilized to dryness.
The pellet is resuspended in 1M TBAF in THF for 24 hrs at room
temperature to deprotect the 2' positions. The reaction is then
quenched with 1M TEAA and the sample is then reduced to 1/2 volume
by rotovac before being desalted on a G25 size exclusion column.
The oligo recovered is then analyzed spectrophotometrically for
yield and for purity by capillary electrophoresis and by mass
spectrometry.
[2'-O-(2-Methoxyethyl)]--[2'-deoxy]--[2'-O-(Methoxyethyl)] Chimeric
Phosphorothioate Oligonucleotides
[0190] [2'-O-(2-methoxyethyl)]--[2'-deoxy]--[-2'-O-(methoxyethyl)]
chimeric phosphorothioate oligonucleotides were prepared as per the
procedure above for the 2'-O-methyl chimeric oligonucleotide, with
the substitution of 2'-O-(methoxyethyl) amidites for the
2'-O-methyl amidites.
[2'-O-(2-Methoxyethyl)Phosphodiester]--[2'-deoxy
Phosphorothioate]--[2'-O-(2-Methoxyethyl) Phosphodiester] Chimeric
Oligonucleotides
[0191] [2'-O-(2-methoxyethyl phosphodiester]--[2'-deoxy
phosphorothioate]--[2'-O-(methoxyethyl) phosphodiester] chimeric
oligonucleotides are prepared as per the above procedure for the
2'-O-methyl chimeric oligonucleotide with the substitution of
2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites,
oxidization with iodine to generate the phosphodiester
internucleotide linkages within the wing portions of the chimeric
structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one
1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate
internucleotide linkages for the center gap.
[0192] Other chimeric oligonucleotides, chimeric oligonucleosides
and mixed chimeric oligonucleotides/oligonucleosides are
synthesized according to U.S. Pat. No. 5,623,065, herein
incorporated by reference.
Example 6
Oligonucleotide Isolation
[0193] After cleavage from the controlled pore glass column
(Applied Biosystems) and deblocking in concentrated ammonium
hydroxide at 55.degree. C. for 18 hours, the oligonucleotides or
oligonucleosides are purified by precipitation twice out of 0.5 M
NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were
analyzed by polyacrylamide gel electrophoresis on denaturing gels
and judged to be at least 85% full length material. The relative
amounts of phosphorothioate and phosphodiester linkages obtained in
synthesis were periodically checked by .sup.31P nuclear magnetic
resonance spectroscopy, and for some studies oligonucleotides were
purified by HPLC, as described by Chiang et al., J. Biol. Chem.
1991, 266, 18162-18171. Results obtained with HPLC-purified
material were similar to those obtained with non-HPLC purified
material.
Example 7
Oligonucleotide Synthesis--96 Well Plate Format
[0194] Oligonucleotides were synthesized via solid phase P(III)
phosphoramidite chemistry on an automated synthesizer capable of
assembling 96 sequences simultaneously in a standard 96 well
format. Phosphodiester internucleotide linkages were afforded by
oxidation with aqueous iodine. Phosphorothioate internucleotide
linkages were generated by sulfurization utilizing 3,H-1,2
benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous
acetonitrile. Standard base-protected beta-cyanoethyldiisopropyl
phosphoramidites were purchased from commercial vendors (e.g.
PE-Applied Biosystems, Foster City, Calif., or Pharmacia,
Piscataway, N.J.). Non-standard nucleosides are synthesized as per
known literature or patented methods. They are utilized as base
protected beta-cyanoethyldiisopropyl phosphoramidites.
[0195] Oligonucleotides were cleaved from support and deprotected
with concentrated NH.sub.4OH at elevated temperature (55-60.degree.
C.) for 12-16 hours and the released product then dried in vacuo.
The dried product was then re-suspended in sterile water to afford
a master plate from which all analytical and test plate samples are
then diluted utilizing robotic pipettors.
Example 8
Oligonucleotide Analysis--96 Well Plate Format
[0196] The concentration of oligonucleotide in each well was
assessed by dilution of samples and UV absorption spectroscopy. The
full-length integrity of the individual products was evaluated by
capillary electrophoresis (CE) in either the 96 well format
(Beckman P/ACE.TM. MDQ instrument) or, for individually prepared
samples, on a commercial CE apparatus (e.g., Beckman P/ACE.TM. 5000
instrument, ABI 270 instrument). Base and backbone composition was
confirmed by mass analysis of the compounds utilizing
electrospray-mass spectroscopy. All assay test plates were diluted
from the master plate using single and multi-channel robotic
pipettors. Plates were judged to be acceptable if at least 85% of
the compounds on the plate were at least 85% full length.
Example 9
Cell Culture and Oligonucleotide Treatment
[0197] The effect of antisense compounds on target nucleic acid
expression can be tested in any of a variety of cell types provided
that the target nucleic acid is present at measurable levels. This
can be routinely determined using, for example, PCR or Northern
blot analysis. The following 5 cell types are provided for
illustrative purposes, but other cell types can be routinely used,
provided that the target is expressed in the cell type chosen. This
can be readily determined by methods routine in the art, for
example Northern blot analysis, Ribonuclease protection assays, or
RT-PCR.
T-24 Cells
[0198] The human transitional cell bladder carcinoma cell line T-24
was obtained from the American Type Culture Collection (ATCC)
(Manassas, Va.). T-24 cells were routinely cultured in complete
McCoy's SA basal media (Gibco/Life Technologies, Gaithersburg, Md.)
supplemented with 10% fetal calf serum (Gibco/Life Technologies,
Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin
100 .mu.g/mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells
were routinely passaged by trypsinization and dilution when they
reached 90% confluence. Cells were seeded into 96-well plates
(Falcon-Primaria #3872) at a density of 7000 cells/well for use in
RT-PCR analysis.
[0199] For Northern blotting or other analysis, cells may be seeded
onto 100 mm or other standard tissue culture plates and treated
similarly, using appropriate volumes of medium and
oligonucleotide.
A549 Cells
[0200] The human lung carcinoma cell line A549 was obtained from
the American Type Culture Collection (ATCC) (Manassas, Va.). A549
cells were routinely cultured in DMEM basal media (Gibco/Life
Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf
serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100
units per mL, and streptomycin 100 .mu.g/mL (Gibco/Life
Technologies, Gaithersburg, Md.). Cells were routinely passaged by
trypsinization and dilution when they reached 90% confluence.
NHDF Cells
[0201] Human neonatal dermal fibroblast (NHDF) were obtained from
the Clonetics Corporation (Walkersville Md.). NHDFs were routinely
maintained in Fibroblast Growth Medium (Clonetics Corporation,
Walkersville, Md.) supplemented as recommended by the supplier.
Cells were maintained for up to 10 passages as recommended by the
supplier.
HEK Cells
[0202] Human embryonic keratinocytes (HEK) were obtained from the
Clonetics Corporation (Walkersville, Md.). HEKs were routinely
maintained in Keratinocyte Growth Medium (Clonetics Corporation,
Walkersville Md.) formulated as recommended by the supplier. Cells
were routinely maintained for up to 10 passages as recommended by
the supplier.
PC-12 Cells
[0203] The rat neuronal cell line PC-12 was obtained from the
American Type Culture Collection (Manassas, Va.). PC-12 cells were
routinely cultured in DMEM, high glucose (Gibco/Life Technologies,
Gaithersburg, Md.) supplemented with 10% horse serum +5% fetal calf
serum (Gibco/Life Technologies, Gaithersburg, Md.). Cells were
routinely passaged by trypsinization and dilution when they reached
90% confluence. Cells were seeded into 96-well plates
(Falcon-Primaria #3872) at a density of 20000 cells/well for use in
RT-PCR analysis.
[0204] For Northern blotting or other analysis, cells may be seeded
onto 100 mm or other standard tissue culture plates and treated
similarly, using appropriate volumes of medium and
oligonucleotide.
Treatment with Antisense Compounds
[0205] When cells reached 80% confluency, they were treated with
oligonucleotide. For cells grown in 96-well plates, wells were
washed once with 200 .mu.L OPTI-MEM.TM.-1 reduced-serum medium
(Gibco BRL) and then treated with 130 .mu.L of OPTI-MEM.TM.-1
medium containing 3.75 .mu.g/mL LIPOFECTIN.TM. reagent (Gibco BRL)
and the desired concentration of oligonucleotide. After 4-7 hours
of treatment, the medium was replaced with fresh medium. Cells were
harvested 16-24 hours after oligonucleotide treatment.
[0206] The concentration of oligonucleotide used varies from cell
line to cell line. To determine the optimal oligonucleotide
concentration for a particular cell line, the cells are treated
with a positive control oligonucleotide at a range of
concentrations. For human cells the positive control
oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1,
a 2'-O-methoxyethyl gapmer (2'-O-methoxyethyls shown in bold) with
a phosphorothioate backbone which is targeted to human H-ras. For
mouse or rat cells the positive control oligonucleotide is ISIS
15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2'-O-methoxyethyl
gapmer (2'-O-methoxyethyls shown in bold) with a phosphorothioate
backbone which is targeted to both mouse and rat c-raf. The
concentration of positive control oligonucleotide that results in
80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS
15770) mRNA is then utilized as the screening concentration for new
oligonucleotides in subsequent experiments for that cell line. If
80% inhibition is not achieved, the lowest concentration of
positive control oligonucleotide that results in 60% inhibition of
H-ras or c-raf mRNA is then utilized as the oligonucleotide
screening concentration in subsequent experiments for that cell
line. If 60% inhibition is not achieved, that particular cell line
is deemed as unsuitable for oligonucleotide transfection
experiments.
Example 10
Analysis of Oligonucleotide Inhibition of PTP1B Expression
[0207] Antisense modulation of PTP1B expression can be assayed in a
variety of ways known in the art. For example, PTP1B mRNA levels
can be quantitated by, e.g., Northern blot analysis, competitive
polymerase chain reaction (PCR), or real-time PCR (RT-PCR).
Real-time quantitative PCR is presently preferred. RNA analysis can
be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA
isolation are taught in, for example, Ausubel, F. M. et al.,
Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9
and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot
analysis is routine in the art and is taught in, for example,
Ausubel, F. M. et al., Current Protocols in Molecular Biology,
Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996.
Real-time quantitative (PCR) can be conveniently accomplished using
the commercially available ABI PRISM.TM. 7700 Sequence Detection
System, available from PE-Applied Biosystems, Foster City, Calif.
and used according to manufacturer's instructions. Prior to
quantitative PCR analysis, primer-probe sets specific to the target
gene being measured are evaluated for their ability to be
"multiplexed" with a GAPDH amplification reaction. In multiplexing,
both the target gene and the internal standard gene GAPDH are
amplified concurrently in a single sample. In this analysis, mRNA
isolated from untreated cells is serially diluted. Each dilution is
amplified in the presence of primer-probe sets specific for GAPDH
only, target gene only ("single-plexing"), or both (multiplexing).
Following PCR amplification, standard curves of GAPDH and target
mRNA signal as a function of dilution are generated from both the
single-plexed and multiplexed samples. If both the slope and
correlation coefficient of the GAPDH and target signals generated
from the multiplexed samples fall within 10% of their corresponding
values generated from the single-plexed samples, the primer-probe
set specific for that target is deemed as multiplexable. Other
methods of PCR are also known in the art.
[0208] Protein levels of PTP1B can be quantitated in a variety of
ways well known in the art, such as immunoprecipitation, Western
blot analysis (immunoblotting), ELISA or fluorescence-activated
cell sorting (FACS). Antibodies directed to PTP1B can be identified
and obtained from a variety of sources, such as the MSRS catalog of
antibodies (Aerie Corporation, Birmingham, Mich.), or can be
prepared via conventional antibody generation methods. Methods for
preparation of polyclonal antisera are taught in, for example,
Ausubel, F. M. et al., Current Protocols in Molecular Biology,
Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997.
Preparation of monoclonal antibodies is taught in, for example,
Ausubel, F. M. et al., Current Protocols in Molecular Biology,
Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc.,
1997.
[0209] Immunoprecipitation methods are standard in the art and can
be found at, for example, Ausubel, F. M. et al., Current Protocols
in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley
& Sons, Inc., 1998. Western blot (immunoblot) analysis is
standard in the art and can be found at, for example, Ausubel, F.
M. et al., Current Protocols in Molecular Biology, Volume 2, pp.
10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked
immunosorbent assays (ELISA) are standard in the art and can be
found at, for example, Ausubel, F. M. et al., Current Protocols in
Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley &
Sons, Inc., 1991.
Example 11
Poly(A)+ mRNA Isolation
[0210] Poly(A)+mRNA was isolated according to Miura et al., Clin.
Chem., 1996, 42, 1758-1764. Other methods for poly(A)+mRNA
isolation are taught in, for example, Ausubel, F. M. et al.,
Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3,
John Wiley & Sons, Inc., 1993. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 .mu.L cold PBS. Sixty .mu.L lysis buffer
(10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM
vanadyl-ribonucleoside complex) was added to each well, the plate
was gently agitated and then incubated at room temperature for five
minutes. Fifty-five .mu.L of lysate was transferred to Oligo d(T)
coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were
incubated for 60 minutes at room temperature, washed 3 times with
200 .mu.L of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M
NaCl). After the final wash, the plate was blotted on paper towels
to remove excess wash buffer and then air-dried for 5 minutes. 60
.mu.L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to
70.degree. C. was added to each well, the plate was incubated on a
90.degree. C. hot plate for 5 minutes, and the eluate was then
transferred to a fresh 96-well plate.
[0211] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
Example 12
Total RNA Isolation
[0212] Total mRNA was isolated using an RNEASY 96.TM. kit and
buffers purchased from Qiagen Inc. (Valencia, Calif.) following the
manufacturer's recommended procedures. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 .mu.L cold PBS. One hundred .mu.L Buffer
RLT was added to each well and the plate vigorously agitated for 20
seconds. One hundred .mu.L of 70% ethanol was then added to each
well and the contents mixed by pipetting three times up and down.
The samples were then transferred to the RNEASY 96.TM. well plate
attached to a QIAVAC.TM. manifold fitted with a waste collection
tray and attached to a vacuum source. Vacuum was applied for 15
seconds. One mL of Buffer RW1 was added to each well of the RNEASY
96.TM. plate and the vacuum again applied for 15 seconds. One mL of
Buffer RPE was then added to each well of the RNEASY 96.TM. plate
and the vacuum applied for a period of 15 seconds. The Buffer RPE
wash was then repeated and the vacuum was applied for an additional
10 minutes. The plate was then removed from the QIAVAC.TM. manifold
and blotted dry on paper towels. The plate was then re-attached to
the QIAVAC.TM. manifold fitted with a collection tube rack
containing 1.2 mL collection tubes. RNA was then eluted by
pipetting 60 .mu.L water into each well, incubating 1 minute, and
then applying the vacuum for 30 seconds. The elution step was
repeated with an additional 60 .mu.L water.
[0213] The repetitive pipetting and elution steps may be automated
using a QIAGEN Bio-Robot 9604 apparatus (Qiagen, Inc., Valencia,
Calif.). Essentially, after lysing of the cells on the culture
plate, the plate is transferred to the robot deck where the
pipetting, DNase treatment and elution steps are carried out.
Example 13
Real-Time Quantitative PCR Analysis of PTP1B mRNA Levels
[0214] Quantitation of PTP1B mRNA levels was determined by
real-time quantitative PCR using the ABI PRISM.TM. 7700 Sequence
Detection System (PE-Applied Biosystems, Foster City, Calif.)
according to manufacturer's instructions. This is a closed-tube,
non-gel-based, fluorescence detection system which allows
high-throughput quantitation of polymerase chain reaction (PCR)
products in real-time. As opposed to standard PCR, in which
amplification products are quantitated after the PCR is completed,
products in real-time quantitative PCR are quantitated as they
accumulate. This is accomplished by including in the PCR reaction
an oligonucleotide probe that anneals specifically between the
forward and reverse PCR primers, and contains two fluorescent dyes.
A reporter dye (e.g., JOE, FAM, or VIC, obtained from either Operon
Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster
City, Calif.) is attached to the 5' end of the probe and a quencher
dye (e.g., TAMRA, obtained from either Operon Technologies Inc.,
Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is
attached to the 3' end of the probe. When the probe and dyes are
intact, reporter dye emission is quenched by the proximity of the
3' quencher dye. During amplification, annealing of the probe to
the target sequence creates a substrate that can be cleaved by the
5'-exonuclease activity of Taq polymerase. During the extension
phase of the PCR amplification cycle, cleavage of the probe by Taq
polymerase releases the reporter dye from the remainder of the
probe (and hence from the quencher moiety) and a sequence-specific
fluorescent signal is generated. With each cycle, additional
reporter dye molecules are cleaved from their respective probes,
and the fluorescence intensity is monitored at regular intervals by
laser optics built into the ABI PRISM.TM. 7700 Sequence Detection
System. In each assay, a series of parallel reactions containing
serial dilutions of mRNA from untreated control samples generates a
standard curve that is used to quantitate the percent inhibition
after antisense oligonucleotide treatment of test samples.
[0215] PCR reagents were obtained from PE-Applied Biosystems,
Foster City, Calif. RT-PCR reactions were carried out by adding 25
.mu.L PCR cocktail (1.times. TAQMAN.TM. buffer A, 5.5 mM
MgCl.sub.2, 300 .mu.M each of dATP, dCTP and dGTP, 600 .mu.M of
dUTP, 100 nM each of forward primer, reverse primer, and probe, 20
Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD.TM. reagent, and
12.5 Units MuLV reverse transcriptase) to 96 well plates containing
25 .mu.L poly(A) mRNA solution. The RT reaction was carried out by
incubation for 30 minutes at 48.degree. C. Following a 10 minute
incubation at 95.degree. C. to activate the AMPLITAQ GOLD.TM.
reagent, 40 cycles of a two-step PCR protocol were carried out:
95.degree. C. for 15 seconds (denaturation) followed by 60.degree.
C. for 1.5 minutes (annealing/extension).
[0216] Probes and primers to human PTP1B were designed to hybridize
to a human PTP1B sequence, using published sequence information
(GenBank accession number M31724, incorporated herein as SEQ ID
NO:3). For human PTP1B the PCR primers were: forward primer:
GGAGTTCGAGCAGATCGACAA (SEQ ID NO: 4) reverse primer:
GGCCACTCTACATGGGAAGTC (SEQ ID NO: 5) and the PCR probe was:
FAM-AGCTGGGCGGCCATTTACCAGGAT-TAMRA (SEQ ID NO: 6) where FAM
(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent
reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,
Calif.) is the quencher dye. For human GAPDH the PCR primers were:
forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7) reverse primer:
GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and the PCR probe was: 5'
JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3' (SEQ ID NO: 9) where JOE
(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent
reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,
Calif.) is the quencher dye.
[0217] Probes and primers to rat PTP1B were designed to hybridize
to a rat PTP1B sequence, using published sequence information
(GenBank accession number M33962, incorporated herein as SEQ ID
NO:10). For rat PTP1B the PCR primers were: [0218] forward primer:
CGAGGGTGCAAAGTTCATCAT (SEQ ID NO:11) [0219] reverse primer:
CCAGGTCTTCATGGGAAAGCT (SEQ ID NO: 12) and the PCR probe was:
FAM-CGACTCGTCAGTGCAGGATCAGTGGA-TAMRA [0220] (SEQ ID NO: 13) where
FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent
reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,
Calif.) is the quencher dye. For rat GAPDH the PCR primers were:
[0221] forward primer: TGTTCTAGAGACAGCCGCATCTT (SEQ ID NO: 14)
[0222] reverse primer: CACCGACCTTCACCATCTTGT (SEQ ID NO: 15) and
the PCR probe was: 5' JOE-TTGTGCAGTGCCAGCCTCGTCTCA-TAMRA 3' (SEQ ID
NO: 16) where JOE (PE-Applied Biosystems, Foster City, Calif.) is
the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems,
Foster City, Calif.) is the quencher dye.
Example 14
Northern Blot Analysis of PTP1B mRNA Levels
[0223] Eighteen hours after antisense treatment, cell monolayers
were washed twice with cold PBS and lysed in 1 mL RNAZOL.TM.
reagent (TEL-TEST "B" Inc., Friendswood, Tex.). Total RNA was
prepared following manufacturer's recommended protocols. Twenty
micrograms of total RNA was fractionated by electrophoresis through
1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer
system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the
gel to HYBOND.TM.-N+ nylon membranes (Amersham Pharmacia Biotech,
Piscataway, N.J.) by overnight capillary transfer using a
Northern/Southern Transfer buffer system (TEL-TEST "B" Inc.,
Friendswood, Tex.). RNA transfer was confirmed by UV visualization.
Membranes were fixed by UV cross-linking using a STRATALINKER.TM.
UV Crosslinker 2400 instrument (Stratagene, Inc, La Jolla, Calif.)
and then robed using QUICKHYB.TM. hybridization solution
(Stratagene, La Jolla, Calif.) using manufacturer's recommendations
for stringent conditions.
[0224] To detect human PTP1B, a human PTP1B specific probe was
prepared by PCR using the forward primer GGAGTTCGAGCAGATCGACAA (SEQ
ID NO: 4) and the reverse primer GGCCACTCTACATGGGAAGTC (SEQ ID NO:
5). To normalize for variations in loading and transfer efficiency
membranes were stripped and probed for human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,
Palo Alto, Calif.).
[0225] To detect rat PTP1B, a rat PTP1B specific probe was prepared
by PCR using the forward primer CGAGGGTGCAAAGTTCATCAT (SEQ ID NO:
11) and the reverse primer CCAGGTCTTCATGGGAAAGCT (SEQ ID NO: 12).
To normalize for variations in loading and transfer efficiency
membranes were stripped and probed for rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,
Palo Alto, Calif.).
[0226] Hybridized membranes were visualized and quantitated using a
PHOSPHORIMAGER.TM. instrument and IMAGEQUANT.TM. Software V3.3
(Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to
GAPDH levels in untreated controls.
Example 15
Antisense Inhibition of Human PTP1B Expression by Chimeric
Phosphorothioate Oligonucleotides Having 2'-MOE Wings and a Deoxy
Gap
[0227] In accordance with the present invention, a series of
oligonucleotides were designed to target different regions of the
human PTP1B RNA, using published sequences (GenBank accession
number M31724, incorporated herein as SEQ ID NO: 3). The
oligonucleotides are shown in Table 1. "Target site" indicates the
first (5'-most) nucleotide number on the particular target sequence
to which the oligonucleotide binds. All compounds in Table 1 are
chimeric oligonucleotides ("gapmers") 20 nucleotides in length,
composed of a central "gap" region consisting of ten
2'-deoxynucleotides, which is flanked on both sides (5' and 3'
directions) by five-nucleotide "wings". The wings are composed of
2'-methoxyethyl (2'-MOE) nucleotides. The internucleoside
(backbone) linkages are phosphorothioate (P.dbd.S) throughout the
oligonucleotide. All cytidine residues are 5-methylcytidines. The
compounds were analyzed for their effect on human PTP1B mRNA levels
by quantitative real-time PCR as described in other examples
herein. Data are averages from two experiments. If present, "N.D."
indicates "no data". TABLE-US-00001 TABLE 1 Inhibition of human
PTP1B mRNA levels by chimeric phos- phorothioate oligonucleotides
having 2'-MOE wings and a deoxy gap TARGET SEQ SEQ ID TARGET % ID
ISIS # REGION NO SITE SEQUENCE INHIB NO 107769 5' UTR 3 1
cttagccccgaggcccgccc 0 17 107770 5' UTR 3 41 ctcggcccactgcgccgtct
58 18 107771 Start 3 74 catgacgggccagggcggct 60 19 Codon 107772
Coding 3 113 cccggacttgtcgatctgct 95 20 107773 Coding 3 154
ctggcttcatgtcggatatc 88 21 107774 Coding 3 178 ttggccactctacatgggaa
77 22 107775 Coding 3 223 ggactgacgtctctgtacct 75 23 107776 Coding
3 252 gatgtagtttaatccgacta 82 24 107777 Coding 3 280
ctagcgttgatatagtcatt 29 25 107778 Coding 3 324 gggtaagaatgtaactcctt
86 26 107779 Coding 3 352 tgaccgcatgtgttaggcaa 75 27 107780 Coding
3 381 ttttctgctcccacaccatc 30 28 107781 Coding 3 408
ctctgttgagcatgacgaca 78 29 107782 Coding 3 436 gcgcattttaacgaaccttt
83 30 107783 Coding 3 490 aaatttgtgtcttcaaagat 0 31 107784 Coding 3
519 tgatatcttcagagatcaat 57 32 107785 Coding 3 547
tctagctgtcgcactgtata 74 33 107786 Coding 3 575 agtttcttgggttgtaaggt
33 34 107787 Coding 3 604 gtggtatagtggaaatgtaa 51 35 107788 Coding
3 632 tgattcagggactccaaagt 55 36 107789 Coding 3 661
ttgaaaagaaagttcaagaa 17 37 107790 Coding 3 688 gggctgagtgaccctgactc
61 38 107791 Coding 3 716 gcagtgcaccacaacgggcc 81 39 107792 Coding
3 744 aggttccagacctgccgatg 81 40 107793 Coding 3 772
agcaggaggcaggtatcagc 2 41 107794 Coding 3 799 gaagaagggtctttcctctt
53 42 107795 Coding 3 826 tctaacagcactttcttgat 18 43 107796 Coding
3 853 atcaaccccatccgaaactt 0 44 107797 Coding 3 880
gagaagcgcagctggtcggc 82 45 107798 Coding 3 908 tttggcaccttcgatcacag
62 46 107799 Coding 3 952 agctccttccactgatcctg 70 47 107800 Coding
3 1024 tccaggattcgtttgggtgg 72 48 107801 Coding 3 1052
gaactccctgcatttcccat 68 49 107802 Coding 3 1079
ttccttcacccactggtgat 40 50 107803 Coding 3 1148
gtagggtgcggcatttaagg 0 51 107804 Coding 3 1176 cagtgtcttgactcatgctt
75 52 107805 Coding 3 1222 gcctgggcacctcgaagact 67 53 107806 Coding
3 1268 ctcgtccttctcgggcagtg 37 54 107807 Coding 3 1295
gggcttccagtaactcagtg 73 55 107808 Coding 3 1323
ccgtagccacgcacatgttg 80 56 107809 Coding 3 1351
tagcagaggtaagcgccggc 72 57 107810 Stop 3 1379 ctatgtgttgctgttgaaca
85 58 Codon 107811 3' UTR 3 1404 ggaggtggagtggaggaggg 51 59 107812
3' UTR 3 1433 ggctctgcgggcagaggcgg 81 60 107813 3' UTR 3 1460
ccgcggcatgcctgctagtc 84 61 107814 3' UTR 3 1489
tctctacgcggtccggcggc 84 62 107815 3' UTR 3 1533
aagatgggttttagtgcaga 65 63 107816 3' UTR 3 1634
gtactctctttcactctcct 69 64 107817 3' UTR 3 1662
ggccccttccctctgcgccg 59 65 107818 3' UTR 3 1707
ctccaggagggagccctggg 57 66 107819 3' UTR 3 1735
gggctgttggcgtgcgccgc 54 67 107820 3' UTR 3 1783
tttaaataaatatggagtgg 0 68 107821 3' UTR 3 1831 gttcaagaaaatgctagtgc
69 69 107822 3' UTR 3 1884 ttgataaagcccttgatgca 74 70 107823 3' UTR
3 1936 atggcaaagccttccattcc 26 71 107824 3' UTR 3 1973
gtcctccttcccagtactgg 60 72 107825 3' UTR 3 2011
ttacccacaatatcactaaa 39 73 107826 3' UTR 3 2045
attatatattatagcattgt 24 74 107827 3' UTR 3 2080
tcacatcatgtttcttatta 48 75 107828 3' UTR 3 2115
ataacagggaggagaataag 0 76 107829 3' UTR 3 2170 ttacatgcattctaatacac
21 77 107830 3' UTR 3 2223 gatcaaagtttctcatttca 81 78 107831 3' UTR
3 2274 ggtcatgcacaggcaggttg 82 79 107832 3' UTR 3 2309
caacaggcttaggaaccaca 65 80 107833 3' UTR 3 2344
aactgcaccctattgctgag 61 81 107834 3' UTR 3 2380
gtcatgccaggaattagcaa 0 82 107835 3' UTR 3 2413 acaggctgggcctcaccagg
58 83 107836 3' UTR 3 2443 tgagttacagcaagaccctg 44 84 107837 3' UTR
3 2473 gaatatggcttcccataccc 0 85 107838 3' UTR 3 2502
ccctaaatcatgtccagagc 87 86 107839 3' UTR 3 2558
gacttggaatggcggaggct 74 87 107840 3' UTR 3 2587
caaatcacggtctgctcaag 31 88 107841 3' UTR 3 2618
gaagtgtggtttccagcagg 56 89 107842 3' UTR 3 2648
cctaaaggaccgtcacccag 42 90 107843 3' UTR 3 2678
gtgaaccgggacagagacgg 25 91 107844 3' UTR 3 2724
gccccacagggtttgagggt 53 92 107845 3' UTR 3 2755
cctttgcaggaagagtcgtg 75 93 107846 3' UTR 3 2785
aaagccacttaatgtggagg 79 94 107847 3' UTR 3 2844
gtgaaaatgctggcaagaga 86 95 107848 3' UTR 3 2970
tcagaatgcttacagcctgg 61 96
[0228] As shown in Table 1, SEQ ID NOs 18, 19, 20, 21, 22, 23, 24,
26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 40, 42, 45, 46, 47, 48, 49,
50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
69, 70, 72, 73, 75, 78, 79, 80, 81, 83, 84, 86, 87, 89, 90, 92, 93,
94, 95, and 96 demonstrated at least 35% inhibition of human PTP1B
expression in this assay and are therefore preferred.
Example 16
Antisense Inhibition of Rat PTP1B Expression by Chimeric
Phosphorothioate Oligonucleotides Having 2'-MOE Wings and a Deoxy
Gap
[0229] In accordance with the present invention, a second series of
oligonucleotides were designed to target different regions of the
rat PTP1B RNA, using published sequences (GenBank accession number
M33962, incorporated herein as SEQ ID NO: 10). The oligonucleotides
are shown in Table 2. "Target site" indicates the first (5'-most)
nucleotide number on the particular target sequence to which the
oligonucleotide binds. All compounds in Table 2 are chimeric
oligonucleotides ("gapmers") 20 nucleotides in length, composed of
a central "gap" region consisting of ten 2'-deoxynucleotides, which
is flanked on both sides (5' and 3' directions) by five-nucleotide
"wings". The wings are composed of 2'-methoxyethyl (2'-MOE)
nucleotides. The internucleoside (backbone) linkages are
phosphoro-thioate (P.dbd.S) throughout the oligonucleotide. All
cytidine residues are 5-methylcytidines. The compounds were
analyzed for their effect on rat PTP1B mRNA levels by quantitative
real-time PCR as described in other examples herein. Data are
averages from two experiments. If present, "N.D." indicates "no
data". TABLE-US-00002 TABLE 2 Inhibition of rat PTP1B mRNA levels
by chimeric phosphor- othioate oligonucleotides having 2'-MOE wings
and a deoxy gap TARGET SEQ SEQ ID TARGET % ID ISIS # REGION NO SITE
SEQUENCE INHIB NO 111549 5' UTR 10 1 caacctccccagcagcggct 32 97
111550 5' UTR 10 33 tcgaggcccgtcgcccgcca 27 98 111551 5' UTR 10 73
cctcggccgtccgccgcgct 34 99 111552 Coding 10 132
tcgatctgctcgaattcctt 49 100 113669 Coding 10 164
cctggtaaatagccgcccag 36 101 113670 Coding 10 174
tgtcgaatatcctggtaaat 63 102 113671 Coding 10 184
actggcttcatgtcgaatat 58 103 113672 Coding 10 189
aagtcactggcttcatgtcg 40 104 111553 Coding 10 190
gaagtcactggcttcatgtc 27 105 113673 Coding 10 191
ggaagtcactggcttcatgt 54 106 113674 Coding 10 192
gggaagtcactggcttcatg 41 107 113675 Coding 10 193
tgggaagtcactggcttcat 56 108 113676 Coding 10 194
atgggaagtcactggcttca 31 109 113677 Coding 10 195
catgggaagtcactggcttc 59 110 413678 Coding 10 225
tttttgttcttaggaagttt 24 111 111554 Coding 10 228
cggtttttgttcttaggaag 45 112 111555 Coding 10 269
tccgactgtggtcaaaaggg 39 113 113679 Coding 10 273
ttaatccgactgtggtcaaa 45 114 113680 Coding 10 298
atagtcattatcttcctgat 49 115 111556 Coding 10 303
ttgatatagtcattatcttc 29 116 113681 Coding 10 330
gcttcctccatttttatcaa 67 117 111557 Coding 10 359
ggccctgggtgaggatatag 20 118 113682 Coding 10 399
cacaccatctcccagaagtg 29 119 111558 Coding 10 405
tgctcccacaccatctccca 48 120 113683 Coding 10 406
ctgctcccacaccatctccc 51 121 113684 Coding 10 407
tctgctcccacaccatctcc 37 122 113685 Coding 10 408
ttctgctcccacaccatctc 54 123 113686 Coding 10 417
cccctgctcttctgctccca 60 124 111559 Coding 10 438
atgcggttgagcatgaccac 15 125 113687 Coding 10 459
tttaacgagcctttctccat 33 126 113688 Coding 10 492
ttttcttctttctgtggcca 54 127 113689 Coding 10 502
gaccatctctttttcttctt 58 128 111560 Coding 10 540
tcagagatcagtgtcagctt 21 129 113690 Coding 10 550
cttgacatcttcagagatca 64 130 113691 Coding 10 558
taatatgacttgacatcttc 46 131 111561 Coding 10 579
aactccaactgccgtactgt 14 132 111562 Coding 10 611
tctctcgagcctcctgggta 38 133 113692 Coding 10 648
ccaaagtcaggccaggtggt 63 134 111563 Coding 10 654
gggactccaaagtcaggcca 31 135 113693 Coding 10 655
agggactccaaagtcaggcc 50 136 113694 Coding 10 656
cagggactccaaagtcaggc 45 137 113695 Coding 10 657
tcagggactccaaagtcagg 49 138 113696 Coding 10 663
ggtgactcagggactccaaa 34 139 111564 Coding 10 705
cctgactctcggactttgaa 53 140 113697 Coding 10 715
gctgagtgagcctgactctc 57 141 113698 Coding 10 726
ccgtgctctgggctgagtga 48 142 111565 Coding 10 774
aaggtccctgacctgccaat 28 143 111566 Coding 10 819
tctttcctcttgtccatcag 34 144 113699 Coding 10 820
gtctttcctcttgtccatca 41 145 113700 Coding 10 821
ggtctttcctcttgtccatc 66 146 113701 Coding 10 822
gggtctttcctcttgtccat 71 147 113702 Coding 10 852
aacagcactttcttgatgtc 39 148 111567 Coding 10 869
ggaacctgcgcatctccaac 0 149 111568 Coding 10 897
tggtcggccgtctggatgag 29 150 113703 Coding 10 909
gagaagcgcagttggtcggc 48 151 113704 Coding 10 915
aggtaggagaagcgcagttg 31 152 113705 Coding 10 918
gccaggtaggagaagcgcag 41 153 111569 Coding 10 919
agccaggtaggagaagcgca 56 154 113706 Coding 10 920
cagccaggtaggagaagcgc 58 155 113707 Coding 10 921
acagccaggtaggagaagcg 43 156 113708 Coding 10 922
cacagccaggtaggagaagc 49 157 113709 Coding 10 923
tcacagccaggtaggagaag 47 158 111570 Coding 10 924
atcacagccaggtaggagaa 51 159 113710 Coding 10 925
gatcacagccaggtaggaga 51 160 113711 Coding 10 926
cgatcacagccaggtaggag 63 161 113712 Coding 10 927
tcgatcacagccaggtagga 71 162 113713 Coding 10 932
caccctcgatcacagccagg 75 163 113714 Coding 10 978
tccttccactgatcctgcac 97 164 111571 Coding 10 979
ctccttccactgatcctgca 89 165 113715 Coding 10 980
gctccttccactgatcctgc 99 166 107799 Coding 10 981
agctccttccactgatcctg 99 167 113716 Coding 10 982
aagctccttccactgatcct 97 168 113717 Coding 10 983
aaagctccttccactgatcc 95 169 113718 Coding 10 984
gaaagctccttccactgatc 95 170 113719 Coding 10 985
ggaaagctccttccactgat 95 171 111572 Coding 10 986
gggaaagctccttccactga 89 172 113720 Coding 10 987
tgggaaagctccttccactg 97 173 113721 Coding 10 1036
tggccggggaggtgggggca 20 174 111573 Coding 10 1040
tgggtggccggggaggtggg 20 175 113722 Coding 10 1046
tgcgtttgggtggccgggga 18 176 111574 Coding 10 1073
tgcacttgccattgtgaggc 38 177 113723 Coding 10 1206
acttcagtgtcttgactcat 67 178 113724 Coding 10 1207
aacttcagtgtcttgactca 60 179 111575 Coding 10 1208
taacttcagtgtcttgactc 50 180 113725 Coding 10 1209
ctaacttcagtgtcttgact 53 181 111576 Coding 10 1255
gacagatgcctgagcacttt 32 182 106409 Coding 10 1333
gaccaggaagggcttccagt 32 183 113726 Coding 10 1334
tgaccaggaagggcttccag 39 184 111577 Coding 10 1335
ttgaccaggaagggcttcca 32 185 113727 Coding 10 1336
gttgaccaggaagggcttcc 41 186 113728 Coding 10 1342
gcacacgttgaccaggaagg 59 187 111578 Coding 10 1375
gaggtacgcgccagtcgcca 45 188 111579 Coding 10 1387
tacccggtaacagaggtacg 32 189 111580 Coding 10 1397
agtgaaaacatacccggtaa 30 190 111581 3' UTR 10 1456
caaatcctaacctgggcagt 31 191 111582 3' UTR 10 1519
ttccagttccaccacaggct 24 192 111583 3' UTR 10 1552
ccagtgcacagatgcccctc 47 193 111584 3' UTR 10 1609
acaggttaaggccctgagat 29 194 111585 3' UTR 10 1783
gcctagcatcttttgttttc 43 195 111586 3' UTR 10 1890
aagccagcaggaactttaca 36 196 111587 3' UTR 10 2002
gggacacctgagggaagcag 16 197 111588 3' UTR 10 2048
ggtcatctgcaagatggcgg 40 198 111589 3' UTR 10 2118
gccaacctctgatgaccctg 25 199 111590 3' UTR 10 2143
tggaagccccagctctaagc 25 200 111591 3' UTR 10 2165
tagtaatgactttccaatca 44 201 111592 3' UTR 10 2208
tgagtcttgctttacacctc 41 202 111593 3' UTR 10 2252
cctgcgcgcggagtgacttc 22 203 111594 3' UTR 10 2299
aggacgtcactgcagcagga 43 204 111595 3' UTR 10 2346
tcaggacaagtcttggcagt 32 205 111596 3' UTR 10 2405
gaggctgcacagtaagcgct 34 206 111597 3' UTR 10 2422
tcagccaaccagcatcagag 20 207 111598 3' UTR 10 2449
acccacagtgtccacctccc 30 208
111599 3' UTR 10 2502 agtgcgggctgtgctgctgg 30 209 111600 3' UTR 10
2553 cagctcgctctggcggcctc 8 210 111601 3' UTR 10 2608
aggaagggagctgcacgtcc 32 211 111602 3' UTR 10 2664
ccctcacgattgctcgtggg 24 212 111603 3' UTR 10 2756
cagtggagcggctcctctgg 18 213 111604 3' UTR 10 2830
caggctgacaccttacacgg 30 214 111605 3' UTR 10 2883
gtcctacctcaaccctagga 37 215 111606 3' UTR 10 2917
ctgccccagcaccagccaca 12 216 111607 3' UTR 10 2946
attgcttctaagaccctcag 33 217 111608 3' UTR 10 2978
ttacatgtcaccactgttgt 28 218 111609 3' UTR 10 3007
tacacatgtcatcagtagcc 37 219 111610 3' UTR 10 3080
ttttctaactcacagggaaa 30 220 111611 3' UTR 10 3153
gtgcccgccagtgagcaggc 23 221 111612 3' UTR 10 3206
cggcctcggcactggacagc 27 222 111613 3' UTR 10 3277
gtggaatgtctgagatccag 31 223 111614 3' UTR 10 3322
agggcgggcctgcttgccca 23 224 111615 3' UTR 10 3384
cggtcctggcctgctccaga 31 225 111616 3' UTR 10 3428
tacactgttcccaggagggt 42 226 111617 3' UTR 10 3471
tggtgccagcagcgctagca 10 227 111618 3' UTR 10 3516
cagtctcttcagcctcaaga 43 228 113729 3' UTR 10 3537
aagagtcatgagcaccatca 56 229 111619 3' UTR 10 3560
tgaaggtcaagttcccctca 40 230 111620 3' UTR 10 3622
ctggcaagaggcagactgga 30 231 111621 3' UTR 10 3666
ggctctgtgctggcttctct 52 232 111622 3' UTR 10 3711
gccatctcctcagcctgtgc 39 233 111623 3' UTR 10 3787
agcgcctgctctgaggcccc 16 234 111624 3' UTR 10 3854
tgctgagtaagtattgactt 35 235 111625 3' UTR 10 3927
ctatggccatttagagagag 36 236 113730 3' UTR 10 3936
tggtttattctatggccatt 59 237 111626 3' UTR 10 3994
cgctcctgcaaaggtgctat 11 238 111627 3' UTR 10 4053
gttggaaacggtgcagtcgg 39 239 111628 3' UTR 10 4095
atttattgttgcaactaatg 33 240
[0230] As shown in Table 2, SEQ ID NOs 97, 99, 100, 101, 102, 103,
104, 106, 107, 108, 109, 110, 112, 113, 114, 115, 117, 120, 121,
122, 123, 124, 126, 127, 128, 130, 131, 133, 134, 135, 136, 137,
138, 139, 140, 141, 142, 144, 145, 146, 147, 148, 151, 152, 153,
154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,
167, 168, 169, 170, 171, 172, 173, 177, 178, 179, 180, 181, 182,
183, 184, 185, 186, 187, 188, 189, 191, 193, 195, 196, 198, 201,
202, 204, 205, 206, 211, 215, 217, 219, 223, 225, 226, 228, 229,
230, 232, 233, 235, 236, 237, 239 and 240 demonstrated at least 30%
inhibition of rat PTP1B expression in this experiment and are
therefore preferred.
Example 17
Western Blot Analysis of PTP1B Protein Levels
[0231] Western blot analysis (immunoblot analysis) is carried out
using standard methods. Cells are harvested 16-20 h after
oligonucleotide treatment, washed once with PBS, suspended in
Laemmli buffer (100 .mu.L/well), boiled for 5 minutes and loaded on
a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and
transferred to membrane for western blotting. Appropriate primary
antibody directed to PTP1B is used, with a radiolabelled or
fluorescently labeled secondary antibody directed against the
primary antibody species. Bands are visualized using a
PHOSPHORIMAGER.TM. instrument (Molecular Dynamics, Sunnyvale
Calif.).
Example 18
Effects of Antisense Inhibition of PTP1B (ISIS 113715) on Blood
Glucose Levels
[0232] db/db mice are used as a model of Type 2 diabetes. These
mice are hyperglycemic, obese, hyperlipidemic, and insulin
resistant. The db/db phenotype is due to a mutation in the leptin
receptor on a C57BLKS background. However, a mutation in the leptin
gene on a different mouse background can produce obesity without
diabetes (ob/ob mice). Leptin is a hormone produced by fat that
regulates appetite and animals or humans with leptin deficiencies
become obese. Heterozygous db/wt mice (known as lean littermates)
do not display the hyperglycemia/hyperlipidemia or obesity
phenotype and are used as controls.
[0233] In accordance with the present invention, ISIS 113715
(GCTCCTTCCACTGATCCTGC, SEQ ID No: 166) was investigated in
experiments designed to address the role of PTP1B in glucose
metabolism and homeostasis. ISIS 113715 is completely complementary
to sequences in the coding region of the human, rat, and mouse
PTP1B nucleotide sequences incorporated herein as SEQ ID No: 3
(starting at nucleotide 951 of human PTP1B; Genbank Accession No.
M31724), SEQ ID No: 10 (starting at nucleotide 980 of rat PTP1B;
Genbank Accession No. M33962) and SEQ ID No: 241 (starting at
nucleotide 1570 of mouse PTP1B; Genbank Accession No. U24700). The
control used is ISIS 29848 (NNNNNNNNNNNNNNNNNNNN, SEQ ID No: 242)
where N is a mixture of A, G, T and C.
[0234] Male db/db mice and lean (heterozygous, i.e., db/wt)
littermates (age 9 weeks at time 0) were divided into matched
groups (n=6) with the same average blood glucose levels and treated
by intraperitoneal injection once a week with saline, ISIS 29848
(the control oligonucleotide) or ISIS 113715. db/db mice were
treated at a dose of 10, 25 or 50 mg/kg of ISIS 113715 or 50 mg/kg
of ISIS 29848 while lean littermates were treated at a dose of 50
or 100 mg/kg of ISIS 113715 or 100 mg/kg of ISIS 29848. Treatment
was continued for 4 weeks with blood glucose levels being measured
on day 0, 7, 14, 21 and 28.
[0235] By day 28 in db/db mice, blood glucose levels were reduced
at all doses from a starting level of 300 mg/dL to 225 mg/dL for
the 10 mg/kg dose, 175 mg/dL for the 25 mg/kg dose and 125 mg/dL
for the 50 mg/kg dose. These final levels are within normal range
for wild-type mice (170 mg/dL). The mismatch control and saline
treated levels levels were 320 mg/dL and 370 mg/dL at day 28,
respectively.
[0236] In lean littermates, blood glucose levels remained constant
throughout the study for all treatment groups (average 120 mg/dL).
These results indicate that treatment with ISIS 113715 reduces
blood glucose in db/db mice and that there is no hypoglycemia
induced in the db/db or the lean littermate mice as a result of the
oligonucleotide treatment.
[0237] In a similar experiment, ob/ob mice and their lean
littermates (heterozygous, i.e., ob/wt) were dosed twice a week at
50 mg/kg with ISIS 113715, ISIS 29848 or saline control and blood
glucose levels were measured at the end of day 7, 14 and 21.
Treatment of ob/ob mice with ISIS 113715 resulted in the largest
decrease in blood glucose over time going from 225 mg/dL at day 7
to 95 mg/dL at day 21. Ob/ob mice displayed an increase in plasma
glucose over time from 300 mg/dL to 325 mg/dL while treatment with
the control oligonucleotide reduced plasma glucose from an average
of 280 mg/dL to 130 mg/dL. In the lean littermates plasma glucose
levels remained unchanged in all treatment groups (average level
100 mg/dL).
Example 19
Effects of Antisense Inhibition of PTP1B (ISIS 113715) on mRNA
Expression in Liver
[0238] Male db/db mice and lean littermates (age 9 weeks at time 0)
were divided into matched groups (n=6) with the same average blood
glucose levels and treated by intraperitoneal injection once a week
with saline, ISIS 29848 (the control oligonucleotide) or ISIS
113715. db/db mice were treated at a dose of 10, 25 or 50 mg/kg of
ISIS 113715 or 50 mg/kg of ISIS 29848 while lean littermates were
treated at a dose of 50 or 100 mg/kg of ISIS 113715 or 100 mg/kg of
ISIS 29848. Treatment was continued for 4 weeks after which the
mice were sacrificed and tissues collected for mRNA analysis. RNA
values were normalized and are expressed as a percentage of saline
treated control.
[0239] ISIS 113715 successfully reduced PTP1B mRNA levels in the
livers of db/db mice at all doses examined (60% reduction of PTP 1
B mRNA), whereas the control oligonucleotide treated animals showed
no reduction in PTP1B mRNA, remaining at the level of the saline
treated control. Treatment of lean littermates with ISIS 113715
also reduced mRNA levels to 45% of control at the 50 mg/kg dose and
25% of control at the 100 mg/kg dose. The control oligonucleotide
(ISIS 29848) failed to show any reduction in mRNA levels.
Example 20
Effects of Antisense Inhibition of PTP1B (ISIS 113715) on Body
Weight
[0240] Male db/db mice and lean littermates (age 9 weeks at time 0)
were divided into matched groups (n=6) with the same average blood
glucose levels and treated by intraperitoneal injection once a week
with saline, ISIS 29848 (the control oligonucleotide) or ISIS
113715. db/db mice were treated at a dose of 10, 25 or 50 mg/kg of
ISIS 113715 or 50 mg/kg of ISIS 29848 while lean littermates were
treated at a dose of 50 or 100 mg/kg of ISIS 113715 or 100 mg/kg of
ISIS 29848. Treatment was continued for 4 weeks. At day 28 mice
were sacrificed and final body weights were measured.
[0241] Treatment of ob/ob mice with ISIS 113715 resulted in an
increase in body weight which was constant over the dose range with
animals gaining an average of 11.0 grams while saline treated
controls gained 5.5 grams. Animals treated with the control
oligonucleotide gained an average of 7.8 grams of body weight.
[0242] Lean littermate animals treated with 50 or 100 mg/kg of ISIS
113715 gained 3.8 grams of body weight compared to a gain of 3.0
grams for the saline controls.
[0243] In a similar experiment, ob/ob mice and their lean
littermates were dosed twice a week at 50 mg/kg with ISIS 113715,
ISIS 29848 or saline control and body weights were measured at the
end of day 7, 14 and 21.
[0244] Treatment of the ob/ob mice with ISIS 113715, ISIS 29848 or
saline control all resulted in a similar increase in body weight
across the 21-day timecourse. At the end of day 7 all ob/ob
treatment groups had an average weight of 42 grams. By day 21,
animals treated with ISIS 113715 had an average body weight of 48
grams, while those in the ISIS 29848 (control oligonucleotide) and
saline control group each had an average body weight of 52 grams.
All of the lean littermates had an average body weight of 25 grams
at the beginning of the timecourse and all lean littermate
treatment groups showed an increase in body weight, to 28 grams, by
day 21.
Example 21
Effects of Antisense Inhibition of PTP1B (ISIS 113715) on Plasma
Insulin Levels
[0245] Male db/db mice (age 9 weeks at time 0) were divided into
matched groups (n=6) with the same average blood glucose levels and
treated by intraperitoneal injection twice a week with saline, ISIS
29848 (the control oligonucleotide) or ISIS 113715 at a dose of 50
mg/kg. Treatment was continued for 3 weeks with plasma insulin
levels being measured on day 7, 14, and 21.
[0246] Mice treated with ISIS 113715 showed a decrease in plasma
insulin levels from 15 ng/mL at day 7 to 7.5 ng/mL on day 21.
Saline treated animals have plasma insulin levels of 37 ng/mL at
day 7, which dropped to 25 ng/mL on day 14, but rose again to 33
ng/mL by day 21. Mice treated with the control oligonucleotide also
showed a decrease in plasma insulin levels across the timecourse of
the study from 25 ng/mL at day 7 to 10 ng/mL on day 21. However,
ISIS 113715 was the most effective at reducing plasma insulin over
time.
Example 22
Antisense Inhibition of Human PTP1B Expression by Additional
Chimeric Phosphorothioate Oligonucleotides Having 2'-MOE Wings and
a Deoxy Gap
[0247] In accordance with the present invention, an additional
series of oligonucleotides were designed to target different
genomic regions of the human PTP1B RNA, using published sequences
(GenBank accession number M31724, incorporated herein as SEQ ID NO:
3), and concatenated genomic sequence derived from nucleotide
residues 1-31000 of Genbank accession number AL034429 followed by
nucleotide residues 1-45000 of Genbank accession number AL133230,
incorporated herein as SEQ ID NO: 243). The oligonucleotides are
shown in Table 3. "Target site" indicates the first (5'-most)
nucleotide number on the particular target sequence to which the
oligonucleotide binds. All compounds in Table 3 are chimeric
oligonucleotides ("gapmers") 20 nucleotides in length, composed of
a central "gap" region consisting of ten 2'-deoxynucleotides, which
is flanked on both sides (5' and 3' directions) by five-nucleotide
"wings". The wings are composed of 2'-methoxyethyl (2'-MOE)
nucleotides. The internucleoside (backbone) linkages are
phosphoro-thioate (P.dbd.S) throughout the oligonucleotide. All
cytidine residues are 5-methylcytidines. The compounds were
analyzed for their effect on RNA levels by quantitative real-time
PCR as described in other examples averages from two experiments.
If present, "N.D." indicates "no data". TABLE-US-00003 TABLE 3
Inhibition of human PTP1B mRNA levels by chimeric phosphorothioate
oligonucleotides hav- ing 2'-MOE wings and a deoxy gap TAR- SEQ
Isis # REGION GET TARGET SEQUENCE % ID 142020 5' UTR 3 6
GCGCTCTTAGCGCCGAGGC 61 244 142021 5' UTR 3 65 CCAGGGCGGCTGCTGCGC 56
245 142022 Start 3 80 CATCTCCATGACGGGCCAG 4 246 Codon G 142023
Start 3 85 TTTTCCATCTCCATGACGG 67 247 Codon G 142024 Start 3 90
ACTCCTTTTCCATCTCCAT 71 248 Codon G 142025 Exon 1 3 106
TTGTCGATCTGCTCGAACT 61 249 142026 Exon 1 3 109 GACTTGTCGATCTGCTCGA
66 250 142027 Exon 1 3 116 GCTCCCGGACTTGTCGATC 95 251 142028 Exon 1
3 119 CCAGCTCCCGGACTTGTCG 92 252 142029 Exon: 3 945
TCCACTGATCCTGCACGGA 44 253 Exon A Junc- tion 142030 Exon: 3 948
CCTTCCACTGATCCTGCAC 55 254 Exon G Junc- tion 142031 3' UTR 3 1453
TGCCTGCTAGTCGGGCGT 67 255 142032 3' UTR 3 1670 CGGGTGTAGGCCCCTTCCC
74 256 142033 3' UTR 3 1772 ATGGAGTGGAGAGTTGCT 63 257 142034 3' UTR
3 1893 TTGTACTTTTTGATAAAGC 61 258 142035 3' UTR 3 1962
CAGTACTGGTCTGACGCA 68 259 142036 3' UTR 3 2018 TCTCACGTTACCCACAATA
74 260 142037 3' UTR 3 2070 TTTCTTATTAAATACCCAC 61 261 142038 3'
UTR 3 2088 AAGTAATCTCACATCATGT 79 262 142039 3' UTR 3 2314
TTCAGCAACAGGCTTAGG 51 263 142040 3' UTR 3 2323 GACAATGACTTCAGCAAC
43 264 142041 3' UTR 3 2359 TGCCTATTCCTGGAAAACT 43 265 142042 3'
UTR 3 2395 GGAAGTCACTAGAGTGTC 14 266 142043 3' UTR 3 2418
CCAGGACAGGCTGGGCCT 67 267 142044 3' UTR 3 2426 CTGCTGTACCAGGACAGG
73 268 142045 3' UTR 3 2452 GGAATGTCTGAGTTACAG 74 269 142046 3' UTR
3 2566 AGAGTGTTGACTTGGAAT 43 270 142047 3' UTR 3 2574
GCTCAAGAAGAGTGTTGA 76 271 142048 3' UTR 3 2598 TGCCTCTCTTCCAAATCAC
43 272 142049 3' UTR 3 2800 TGTTTTTCATGTTAAAAAG 44 273 142050 3'
UTR 3 2895 TCCCACCACAGAATTTCTC 21 274 142051 3' UTR 3 2921
GCTCTGCAGGGTGACACCT 74 275 142052 3' UTR 3 3066 AGGAGGTTAAACCAGTAC
78 276 142053 3' UTR 3 3094 GGTGGAGAGCCAGCTGCT 59 277 142054 3' UTR
3 3153 TATTGGCTTAAGGCATATA 72 278 142055 3' UTR 3 3168
GACCTGATGAGTAAATATT 58 279 142084 5' UTR 243 859
TTCTTCATGTCAACCGGCA 11 280 142085 5' UTR 243 919 GCCCCGAGGCCCGCTGCA
83 281 142056 Intron 243 4206 TAGTGAACTATTGTTAGAA 70 282 1 142057
Intron 243 27032 TGCTAAGCGACTTCTAATC 72 283 1 142058 Intron 243
27203 CAGGATTCTAAGTTATTAA 32 284 1 142059 Intron 243 33720
TGGGGAGGATGGCTCTGG 21 285 1 142060 Intron 243 48065
TACAATACTATCTGTGACT 34 286 1 142061 Exon: 243 51931
GATACTTACAGGGACTGA 39 287 Intron CG Junc- tion 142086 Intron 243
52005 AACCCTGAGGCGAAAGGA 64 288 2 142062 Intron 243 54384
CCCCAGGTCACTAAAATTA 48 289 2 142063 Intron 243 55362
AAAGCAAAGGTGAGTTGG 56 290 2 142064 Intron 243 56093
GCTCAATTATTAAACCACT 64 291 3 142065 Intron 243 56717
AGTCCTCAAGAAGTCACTT 70 292 3 142066 Intron 243 61780
GAAAGCAGGGACTGCTGG 39 293 4 142067 Intron 243 64554
AAAACTGGGAGAGACAGC 71 294 4 142068 Intron 243 64869
ACATGGAAGCGATGGTCA 24 295 4 142069 Intron 243 67516
ATTGCTAGACTCACACTAG 68 296 5 142070 Intron 243 68052
GGCTGTGATCAAAAGGCA 51 297 5 142087 Intron 243 68481
CACTGGCTCTGGGCAACTT 70 298 5 142088 Intron 243 68563
GCTGGGCAGCCAGCCATA 71 299 5 142071 Intron 243 68648
AGTCCCCTCACCTCTTTTC 59 300 5 142072 Exon: 243 69107
CCTCCTTACCAGCAAGAGG 26 301 Intron C 142089 Intron 243 69198
TGTATTTTGGAAGAGGAG 53 302 6 142090 Intron 243 69220
ACAGACTAACACAGTGAG 53 303 6 142073 Intron 243 69264
CAAATTACCGAGTCTCAG 47 304 6 142074 Intron 243 69472
CATGAAAGGCTTGGTGCC 41 305 6 142075 Intron 243 70042
TTGGAAGATGAAATCTTTT 30 306 7 142076 Intron 243 70052
AGCCATGTACTTGGAAGA 69 307 7 142077 Intron 243 70661
CGAGCCCCTCATTCCAACA 42 308 8 142078 Intron 243 71005
CACCTCAGCGGACACCTCT 6 309 8 142079 Exon: 243 71938
GAAACATACCCTGTAGCA 52 310 Intron GA 142091 Intron 243 72131
CAGAGGGCTCCTTAAAAC 61 311 9 142092 Intron 243 72430
TTCGTAAAAGTTTGGGAT 34 312 9 142080 Intron 243 72453
CCCTCTTCTCCAAGGGAGT 73 313 9 142081 Intron 243 73158
GGAATGAAAGCAAACAGT 42 314 9 142082 Exon 243 75012
AAATGGTTTATTCCATGGC 66 315 10 142083 Exon 243 75215
AAAAATTTTATTGTTGCAG 48 316 10 142093 3' UTR 243 75095
CCGGTCATGCAGCCACGTA 85 317 142094 3' UTR 243 75165
GTTGGAAAACTGTACAGT 77 318 142095 3' UTR 243 75211
ATTTTATTGTTGCAGCTAA 46 319
[0248] As shown in Table 3, SEQ ID NOs, 244, 245, 247, 248, 249,
250, 251, 252, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263,
267, 268, 269, 271, 275, 276, 277, 278, 279, 281, 282, 283, 288,
290, 291, 292, 294, 296, 297, 298, 299, 300, 302, 303, 307, 310,
311, 313, 315, 317, and 318, demonstrated at least 50% inhibition
of human PTP1B expression in this assay and are therefore
preferred.
Example 23
Antisense Inhibition of Human PTP1B Expression by Additional
Chimeric Phosphorothioate Oligonucleotides Having 2'-MOE Wings and
a Deoxy Gap
[0249] In accordance with the present invention, an additional
series of oligonucleotides were designed to target either the 3'UTR
or the 5'UTR of the human PTP1B RNA, using published sequences
(GenBank accession number M31724, incorporated herein as SEQ ID NO:
3) and concatenated genomic sequence derived from nucleotide
residues 1-31000 of Genbank accession number AL034429 followed by
nucleotide residues 1-45000 of Genbank accession number AL133230,
incorporated herein as SEQ ID NO: 243. The oligonucleotides are
shown in Table4. "Target site" indicates the first (5'-most)
nucleotide number on the particular target sequence to which the
oligonucleotide binds. All compounds in Table 3 are chimeric
oligonucleotides ("gapmers") 20 nucleotides in length, composed of
a central "gap" region consisting of ten 2'-deoxynucleotides, which
is flanked on both sides (5' and 3' directions) by five-nucleotide
"wings". The wings are composed of 2'-methoxyethyl (2'-MOE)
nucleotides. The internucleoside (backbone) linkages are
phosphoro-thioate (P.dbd.S) throughout the oligonucleotide. All
cytidine residues are 5-methylcytidines. The compounds were
analyzed for their effect on human PTP1B mRNA levels by
quantitative real-time PCR as described in other examples herein.
Data are averages from two experiments. If present, "N.D."
indicates "no data". TABLE-US-00004 TABLE 4 Inhibition of human
PTP1B mRNA levels by chimeric phos- phorothioate oligonucleotidies
having 2'-MOE wings and a deoxy gap TARGET SEQ ID TARGET % SEQ ID
ISIS # REGION NO. SITE SEQUENCE INHIB NO. 146879 5' UTR 3 50
CGCCTCCTTCTCGGCCCACT 29 320 146880 5' UTR 3 62 GGGCGGCTGCTGCGCCTGCT
34 321 146881 3' UTR 3 1601 GTGGATTTGGTACTCAAAGT 72 322 146882 3'
UTR 3 1610 AAATGGCTTGTGGATTTGGT 72 323 146883 3' UTR 3 1637
ATGGTACTCTCTTTCACTCT 61 324 146884 3' UTR 3 1643
GCCAGCATGGTACTCTCTTT 63 325 146885 3' UTR 3 1764
GAGAGTTGCTCCCTGCAGAT 62 326 146886 3' UTR 3 1770
GGAGTGGAGAGTTGCTCCCT 57 327 146887 3' UTR 3 1874
CCTTGATGCAAGGCTGACAT 65 328 146888 3' UTR 3 1879
AAAGCCCTTGATGCAAGGCT 59 329 146889 3' UTR 3 1915
AGTACTACCTGAGGATTTAT 46 330 146890 3' UTR 3 1925
TTCCATTCCCAGTACTACCT 41 331 146891 3' UTR 3 1938
CCATGGCAAAGCCTTCCATT 65 332 146892 3' UTR 3 1943
CAGGCCCATGGCAAAGCCTT 52 333 146893 3' UTR 3 1988
CAACTGCTTACAACCGTCCT 60 334 146894 3' UTR 3 2055
CCACGTGTTCATTATATATT 42 335 146895 3' UTR 3 2063
TTAAATACCCACGTGTTCAT 27 336 146896 3' UTR 3 2099
TAAGCGGGAGAAAGTAATG 47 337 146897 3' UTR 3 2118 CAGATAACAGGGAGGAGAA
31 338 146898 3' UTR 3 2133 GAGAACTAGATCTAGCAGA 0 339 146899 3' UTR
3 2140 AGTGATTGAGAACTAGATCT 62 340 146900 3' UTR 3 2184
GACACAAGAAGACCTTACA 49 341 146901 3' UTR 3 2212
CTCATTTCAAGCACATATTT 60 342 146902 3' UTR 3 2263
GGCAGGTTGGACTTGGACA 49 343 146903 3' UTR 3 2296
AACCACAGCCATGTAATGAT 43 344 146904 3' UTR 3 2332
TTGCTGAGCGACAATGACTT 42 345 146905 3' UTR 3 2350
CTGGAAAACTGCACCCTATT 31 346 146906 3' UTR 3 2409
GGTGGGCCTCACCAGGAAG 77 347 146907 3' UTR 3 2439
TTACAGCAAGACCCTGCTGT 28 348 146908 3' UTR 3 2457
ACCCTTGGAATGTCTGAGTT 65 349 146909 3' UTR 3 2464
TTCCCATACCCTTGGAATGT 62 350 146910 3' UTR 3 2471
ATATGGGTTCCCATACCCTT 47 351 146911 3' UTR 3 2477
GTGTGAATATGGCTTCCCAT 54 352 146912 3' UTR 3 2509
CCTGCTTCGGTAAATCATGT 65 353 146913 3' UTR 3 2514
GTGTCCCTGCTTCCCTAAAT 55 354 146914 3' UTR 3 2546
CGGAGGCTGATCCCAAAGG 55 355 146915 3' UTR 3 2602
CAGGTGCGTCTCTTCCAAAT 60 356 146916 3' UTR 3 2613
GTGGTTTCGAGCAGGTGCCT 63 357 146917 3' UTR 3 2628
GCTGTTTCAAGAAGTGTGGT 43 358 146918 3' UTR 3 2642
GGAGCGTGACCGAGGCTGTT 32 359 146919 3' UTR 3 2655
CAGGCTGCCTAAAGGACCG 60 360 146920 3' UTR 3 2732 ACCATCAGGCCCCACAGGG
58 361 146921 3' UTR 3 2759 GTTCCCTTTGCAGGAAGAGT 69 362 146922 3'
UTR 3 2772 GTGGAGGTCTTCAGTTCCCT 64 363 146923 3' UTR 3 2781
CCACTTAATGTGGAGGTCTT 54 364 146924 3' UTR 3 2814
AGCTACAGCTGCCGTGTTTT 51 365 146925 3' UTR 3 2862
CCACGAGAAAGGCAAAATG 50 366 146926 3' UTR 3 2885
GAATTTCTCTGTACTGGCTT 23 367 146927 3' UTR 3 2890
CCACAGAATTTCTCTGTACT 61 368 146928 3' UTR 3 2901
GAATGTTCCCACCACAGAAT 61 369 146929 3' UTR 3 2956
GCGTGGCACCTAAGCCTTAT 0 370 146930 3' UTR 3 2965
ATGCTTACAGCCTGGCACCT 55 371 146931 3' UTR 3 3008
CTACATACATATACAGGACT 65 372 146932 3' UTR 3 3042
TTTGAAATGCTACTATATAT 44 373 146933 3' UTR 3 3070
GGATAGGAGGTTAAACCAG 67 374 146934 3' UTR 3 3086
GCCAGCTGCTCTCCAAGGAT 42 375 146935 3' UTR 3 3121
CTACGTCTCTAACATAATGT 39 376 146936 3' UTR 3 3126
GCTCGCTACGTCTCTAACAT 68 377 146937 3' UTR 3 3143
AGGCATATAGCAGAGCAGC 61 378 146938 5' UTR 243 851
GTCAAGCGGCAGCCGGAAC 14 379 146942 5' UTR 243 891
CCTGCAGCTACCGCCGCCCT 69 380 146943 5' UTR 243 908
CGCTGCAATCCCCGACGCGT 87 381 146944 3' UTR 243 75050
ACCAAAACACCTTGCTTTTT 27 382 146945 3' UTR 243 75057
GTATTATACCAAAACACCTT 39 383 146946 3' UTR 243 75072
CACACACCTGAAAAGGTATT 42 384 146947 3' UTR 243 75097
ACCCGGTCATGCAGCCACGT 49 385 146948 3' UTR 243 75136
GTGAGGTCACAGAAGACCC 49 386 146949 3' UTR 243 75154
GTACAGTCTGACAGTTCTGT 40 387 146950 3' UTR 243 75172
ATGGCAAGTTGGAAAACTG 65 388 146951 3' UTR 243 75192
AATGCAAACCCATGATGAAT 43 389
[0250] As shown in Table 4, SEQ ID NOs, 322, 323, 324, 325, 326,
327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 340, 341, 342,
343, 344, 345, 347, 349, 350, 351, 352, 353, 354, 355, 356, 357,
358, 360, 361, 362, 363, 364, 365, 366, 368, 369, 371, 372, 373,
374, 375, 377, 378, 380, 381, 384, 385, 386, 387, 388, and 389
demonstrated at least 40% inhibition of human PTP1B expression in
this assay and are therefore preferred.
Example 24
Antisense Inhibition of PTP1B Expression (ISIS 113715) in Liver,
Muscle and Adipose Tissue of the Cynomolgus Monkey
[0251] In a further embodiment, male cynomolgus monkeys were
treated with ISIS 113715 (SEQ ID NO: 166) and levels of PTP1B mRNA
and protein were measured in muscle, adipose and liver tissue.
Serum samples were also measured for insulin levels.
[0252] Male cynomolgus monkeys were divided into two treatment
groups, control animals (n=4; saline treatment only) and treated
animals (n=8; treated with ISIS 113715). All animals had two
pre-dosing glucose tolerance tests (GTTs) performed to establish
insulin and glucose baseline values. Animals in the treatment group
were dosed subcutaneously on days 1, 8, and 15 with 3 mg/kg, 6
mg/kg and 12 mg/kg of ISIS 113715, respectively. Animals in the
control group were untreated. All animals had GTTs performed on
days 5, 13 and 19, four days post-dosing. Ten days after the last
dose of 12 mg/kg, all animals in the treatment group (ISIS 113715)
received a one-time dose of 6 mg/kg of ISIS 113715. Three days
later, all animals were sacrificed and tissues were taken for
analysis of PTP1B mRNA and protein levels. Levels of mRNA and
protein were normalized to those of the saline treated animals. Of
the tissue examined, PTP1B mRNA levels were reduced to the greatest
extent in the fat and liver, being reduced by 41% and 40%,
respectively. mRNA levels in muscle were reduced by 10%. Protein
levels were reduced by 60% in the liver and by 45% in the muscle
but were shown to increase by 10% in the fat.
[0253] Levels of the liver enzymes ALT and AST were measured weekly
and showed no change, indicating no ongoing toxic effects of the
oligonucleotide treatment.
[0254] The results of this study demonstrate a significant
reduction in liver PTP1B mRNA and protein upon treatment with ISIS
113715. Furthermore, there was no change seen in the fasting
insulin levels either between groups or between pre-treatment and
post-treatment of the same group. There was, however, a significant
lowering of insulin levels with no decrease in fasting glucose
levels in all groups suggesting that insulin efficiency
(sensitivity) was increased upon treatment with ISIS 113715.
Example 25
Effects of Antisense Inhibition of PTP1B (ISIS 113715) on mRNA
Expression in Fractionated Liver
[0255] Male db/db mice (age 9 weeks at time 0) were divided into
matched groups (n=6) with the same average blood glucose levels and
treated by intraperitoneal injection once a week with saline, ISIS
29848 (the control oligonucleotide) or ISIS 113715. db/db mice were
treated at a dose of 50 mg/kg of ISIS 113715 or 50 mg/kg of ISIS
29848 or 100 mg/kg of ISIS 29848. Treatment was continued for 3
weeks after which the mice were sacrificed and tissues were
collected for analysis. Liver tissue was removed and homogenized
whole or fractionated into hepatocytes and non-parenchymal (NP)
cell fractions by standard methods (Graham et al., J. Pharmacol.
Exp. Ther., 1998, 286, 447-458). During the study, plasma glucose
levels were measured as were PTP1B mRNA levels in both cell
fractions. RNA values were normalized and are expressed as a
percentage of saline treated control.
[0256] Treatment of db/db mice with ISIS 113715 caused a
significant reduction in plasma glucose levels (saline=500+/-25 vs.
treated=223+/-21 mg/dL; p=0.0001).
[0257] ISIS 113715 successfully reduced PTP 1B mRNA levels in both
hepatocytes and NP cell fractions, with an 80% reduction in
hepatocytes and a 30% reduction in the NP cell fraction. In
addition, PTP1B expression in the two cell fractions was found to
be dramatically different with a 5-8 fold greater level of
expression being found in the NP fraction. Thus, the inability of
ISIS 113715 to reduce PTP1B expression by no more than 60% in whole
liver as seen in previous experiments may result from a combination
of a relatively high expression of PTP1B in NP cells with a reduced
ability of ISIS 113715 to inhibit expression in this same cell
fraction. Consequently, distinct targeting of the compound to
hepatocytes, the key metabolic cell type in liver, results in a
much greater inhibition of PTP 1 B levels.
Example 26
Effects of Antisense Inhibition of PTP1B Expression (ISIS 113715)
in the Obese Insulin-Resistant Hyperinsulinemic Rhesus
Monkey-Improved Insulin Sensitivity
[0258] In a further embodiment, male obese insulin-resistant
hyperinsulinemic Rhesus monkeys were treated with ISIS 113715 (SEQ
ID NO: 166) and insulin sensitivity, glucose tolerance and PTP1B
mRNA and protein were measured. Serum samples were also measured
for insulin levels.
[0259] Male rhesus monkeys were divided into two treatment groups,
control animals (n=4; saline treatment only) and treated animals
(n=8; treated with ISIS 113715). All animals had two pre-dosing
glucose tolerance tests (GTTs) performed to establish insulin and
glucose baseline values. Animals in the treatment group were dosed
subcutaneously at a dose of 20 mg/kg (3 injections on alternate
days the first week followed by one injection per week for the next
two weeks). Fasted glucose/insulin levels and glucose tolerance
(IVGTTs) were measured as pharmacologic endpoints.
[0260] As compared to baseline values, a 50% reduction in fasting
insulin levels was observed (treated: 31.+-.9 vs. baseline: 67.+-.7
.mu.U/mL, p=0.0001), which was not accompanied by any change in
plasma glucose levels. Furthermore, a marked reduction in insulin
levels (AUC) was observed after IVGTTs (treated: 7295.+-.3178 vs.
baseline: 18968.+-.2113 .mu.U-min/mL, p=0.0002). Insulin
sensitivity was also significantly increased (glucose slope/insulin
AUC; 5-20 minutes).
[0261] Hypoglycemia was not observed, even in the 16 hour-fasted
animals. Levels of the liver enzymes ALT and AST were measured
weekly and showed no change, indicating no ongoing toxic effects of
the oligonucleotide treatment. Renal function tests were also
normal.
[0262] The results of this study are consistent with those seen in
previous rodent and monkey studies described herein which
demonstrate a significant lowering of insulin levels suggesting
that insulin efficiency (sensitivity) was increased upon treatment
with ISIS 113715.
Example 27
RNA Synthesis
[0263] In general, RNA synthesis chemistry is based on the
selective incorporation of various protecting groups at strategic
intermediary reactions. Although one of ordinary skill in the art
will understand the use of protecting groups in organic synthesis,
a useful class of protecting groups includes silyl ethers. In
particular bulky silyl ethers are used to protect the 5'-hydroxyl
in combination with an acid-labile orthoester protecting group on
the 2'-hydroxyl. This set of protecting groups is then used with
standard solid-phase synthesis technology. It is important to
lastly remove the acid labile orthoester protecting group after all
other synthetic steps. Moreover, the early use of the silyl
protecting groups during synthesis ensures facile removal when
desired, without undesired deprotection of 2'hydroxyl.
[0264] Following this procedure for the sequential protection of
the 5'-hydroxyl in combination with protection of the 2'-hydroxyl
by protecting groups that are differentially removed and are
differentially chemically labile, RNA oligonucleotides were
synthesized.
[0265] RNA oligonucleotides are synthesized in a stepwise fashion.
Each nucleotide is added sequentially (3'- to 5'-direction) to a
solid support-bound oligonucleotide. The first nucleoside at the
3'-end of the chain is covalently attached to a solid support. The
nucleotide precursor, a ribonucleoside phosphoramidite, and
activator are added, coupling the second base onto the 5'-end of
the first nucleoside. The support is washed and any unreacted
5'-hydroxyl groups are capped with acetic anhydride to yield
5'-acetyl moieties. The linkage is then oxidized to the more stable
and ultimately desired P(V) linkage. At the end of the nucleotide
addition cycle, the 5'-silyl group is cleaved with fluoride. The
cycle is repeated for each subsequent nucleotide.
[0266] Following synthesis, the methyl protecting groups on the
phosphates are cleaved in 30 minutes utilizing 1 M
disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate
(S.sub.2Na.sub.2) in DMF. The deprotection solution is washed from
the solid support-bound oligonucleotide using water. The support is
then treated with 40% methylamine in water for 10 minutes at
55.degree. C. This releases the RNA oligonucleotides into solution,
deprotects the exocyclic amines, and modifies the 2'-groups. The
oligonucleotides can be analyzed by anion exchange HPLC at this
stage.
[0267] The 2'-orthoester groups are the last protecting groups to
be removed. The ethylene glycol monoacetate orthoester protecting
group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is
one example of a useful orthoester protecting group, which has the
following important properties. It is stable to the conditions of
nucleoside phosphoramidite synthesis and oligonucleotide synthesis.
However, after oligonucleotide synthesis the oligonucleotide is
treated with methylamine, which not only cleaves the
oligonucleotide from the solid support but also removes the acetyl
groups from the orthoesters. The resulting 2-ethyl-hydroxyl
substituents on the orthoester are less electron withdrawing than
the acetylated precursor. As a result, the modified orthoester
becomes more labile to acid-catalyzed hydrolysis. Specifically, the
rate of cleavage is approximately 10 times faster after the acetyl
groups are removed. Therefore, this orthoester possesses sufficient
stability in order to be compatible with oligonucleotide synthesis.
Yet, when subsequently modified, it permits deprotection to be
carried out under relatively mild aqueous conditions compatible
with the final RNA oligonucleotide product.
[0268] Additionally, methods of RNA synthesis are well known in the
art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996;
Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821;
Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103,
3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett.,
1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990,
44, 639-641; Reddy, M. P., et al., Tetrahedron Lett., 1994, 25,
4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23,
2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23,
2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23,
2315-2331).
[0269] RNA antisense compounds (RNA oligonucleotides) of the
present invention can be synthesized by the methods herein or
purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once
synthesized, complementary RNA antisense compounds can then be
annealed by methods known in the art to form double stranded
(duplexed) antisense compounds. For example, duplexes can be formed
by combining 30 .mu.l of each of the complementary strands of RNA
oligonucleotides (50 .mu.M RNA oligonucleotide solution) and 15
.mu.l of 5.times. annealing buffer (100 mM potassium acetate, 30 mM
HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1
minute at 90.degree. C., then 1 hour at 37.degree. C. The resulting
duplexed antisense compounds can be used in kits, assays, screens,
or other methods to investigate the role of a target nucleic
acid.
Example 28
Design and Screening of Duplexed Antisense Compounds Targeting
PTP1B
[0270] In accordance with the present invention, a series of
nucleic acid duplexes comprising the antisense compounds of the
present invention and their complements can be designed to target
PTP1B. The nucleobase sequence of the antisense strand of the
duplex comprises at least a portion of an oligonucleotide in Table
1. The ends of the strands may be modified by the addition of one
or more natural or modified nucleobases to form an overhang. The
sense strand of the dsRNA is then designed and synthesized as the
complement of the antisense strand and may also contain
modifications or additions to either terminus. For example, in one
embodiment, both strands of the dsRNA duplex would be complementary
over the central nucleobases, each having overhangs at one or both
termini.
[0271] For example, a duplex comprising an antisense strand having
the sequence CGAGAGGCGGACGGGACCG (nucleotides 1-19 of SEQ ID NO:
414) and having a two-nucleobase overhang of deoxythymidine(dT)
would have the following structure: TABLE-US-00005
cgagaggcggacgggaccgTT Antisense (SEQ ID NO: 414)
||||||||||||||||||| Strand TTgctctccgcctgccctggc Comple- (SEQ ID
NO: 415) ment
The one or more nucleobases forming the single-stranded overhang(s)
may be dT as shown or may be another modified or unmodified
nucleobase.
[0272] A duplex comprising an antisense strand having the sequence
CGAGAGGCGGACGGGACCG (nucleotides 1-19 of SEQ ID NO: 414) may also
prepared with blunt ends (no single stranded overhang) as shown
(antisense strand below is nucleotides 1-19 of SEQ ID NO: 414;
complement below is nucleotides 1-19 of SEQ ID NO: 415):
TABLE-US-00006 cgagaggcggacgggaccg Antisense Strand
||||||||||||||||||| gctctccgcctgccctggc Complement
[0273] RNA strands of the duplex can be synthesized by methods
disclosed herein or purchased from Dharmacon Research Inc.,
(Lafayette, Colo.). Once synthesized, the complementary strands are
annealed. The single strands are aliquoted and diluted to a
concentration of 50 .mu.M. Once diluted, 30 .mu.L of each strand is
combined with 15 .mu.L of a 5.times. solution of annealing buffer.
The final concentration of said buffer is 100 mM potassium acetate,
30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final
volume is 75 .mu.L. This solution is incubated for 1 minute at
90.degree. C. and then centrifuged for 15 seconds. The tube is
allowed to sit for 1 hour at 37.degree. C. at which time the dsRNA
duplexes are used in experimentation. The final concentration of
the dsRNA duplex is 20 .mu.M. This solution can be stored frozen
(-20.degree. C.) and freeze-thawed up to 5 times.
[0274] Once prepared, the duplexed antisense compounds are
evaluated for their ability to modulate PTP1B expression. When
cells reached 80% confluency, they are treated with duplexed
antisense compounds of the invention. For cells grown in 96-well
plates, wells are washed once with 200 .mu.L OPTI-MEM.TM.-1
reduced-serum medium (Gibco BRL) and then treated with 130 .mu.L of
OPTI-MEM.TM.-1 medium containing 12 .mu.g/mL LIPOFECTIN.TM. reagent
(Gibco BRL) and the desired duplex antisense compound at a final
concentration of 200 nM. After 5 hours of treatment, the medium is
replaced with fresh medium. Cells are harvested 16 hours after
treatment, at which time RNA is isolated and target reduction
measured by RT-PCR.
[0275] A series of siRNA duplexes were prepared as described above
and were tested in A549 cells for their ability to reduce human
PTP1b expression. Also tested for comparison were several
single-stranded 2'-MOE gapped (chimeric) oligonucleotides as well
as two single-stranded controls (ISIS 116847, targeted to PTEN, and
ISIS 129700, a scrambled control) and two RNA duplex unrelated
controls (ISIS 271783:ISIS 297802 duplex and ISIS 263188:263189
duplex), which are targeted to PTEN. These compounds were tested at
two concentrations, 75 nM and 150 nM. The results are shown in
Table 5. TABLE-US-00007 TABLE 5 Inhibition of human PTP1B
expression by siRNA duplexes Isis No. Anti-sense Sense Target anti-
strand Strand site on % % sense: SEQ ID SEQ ID SEQ ID Inhib7 Inhib
Chem- sense Antisense strand sequence NO NO NO: 3 5 nM 150 nM
istry.sup.1 348290: UUCAUGUCGGAUAUCCUGGdTdA 390 403 148 53 70 R
348274 348291: UCACUGGCUUCAUGUCGGAdTdA 391 404 156 0 12 R 348275
348292: GGUAAGAAUGUAACUCCUUdTdG 392 405 322 8 9 R 348276 348293:
CUUCAGAGAUCAAUGUUAAdTdT 393 406 512 0 28 R 348277 348294:
UAGCUGUCGCACUGUAUAAdTdA 394 407 544 44 52 R 348278 348295:
CCAAUUCUAGCUGUCGCACdTdG 395 408 551 67 79 R 348279 342914:
UUGAUAAAGCCCUUGAUGCA 396 409 1884 66 80 R 342934 342916:
GGUCAUGCACAGGCAGGUUG 397 410 2274 55 72 R 342936 342908:
GAAGAAGGGUCUUUCCUCUU 398 411 799 61 77 R 342928 107772
CCCGGACTTGTCGATCTGCT 20 N/A 113 76 90 G 107804 CAGTGTCTTGACTCATGCTT
52 N/A 1176 85 95 G 107813 CCGCGGCATGCGTGCTAGTC 61 N/A 1460 71 88 G
107831 GGTCATGCACAGGGAGGTTG 79 N/A 2274 83 88 G 116847
CTGCTAGCCTCTGGATTTGA 399 N/A N/A 0 0 G 129700 TAGTGCGGACCTACCCACGA
400 N/A N/A 17 13 G 271783: GGGACGAACUGGUGUAAUGdTdT 401 412 N/A 0 4
R 297802 263188: CUUCUGGCAUCCGGUUUAGdTdT 402 413 N/A 28 9 R 263189
.sup.1Chemistry: "R" indicates an RNA duplex in which both strands
are 2' ribonucleic acid, except where 2' deoxynucleotides are
indicated by a "d" preceding the base. All linkages are P.dbd.S.
"G" indicates a single-stranded 2' MOE gapmer in which nucleotides
1-5 and 16-20 are 2' MOE nucleotides and nucleotides 6-15 are
2'-deoxy. All linkages are P.dbd.S and all cytosines are
5-methylcytosines.
[0276] As can be seen from the data in Table 5, both siRNA duplexes
and single stranded 2'MOE gapped oligonucleotides inhibit human
PTP1b target expression in a dose-dependent manner.
Sequence CWU 1
1
415 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1
tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence Antisense
Oligonucleotide 2 atgcattctg cccccaagga 20 3 3247 DNA Homo sapiens
CDS (91)...(1398) 3 gggcgggcct cggggctaag agcgcgacgc ctagagcggc
agacggcgca gtgggccgag 60 aaggaggcgc agcagccgcc ctggcccgtc atg gag
atg gaa aag gag ttc gag 114 Met Glu Met Glu Lys Glu Phe Glu 1 5 cag
atc gac aag tcc ggg agc tgg gcg gcc att tac cag gat atc cga 162 Gln
Ile Asp Lys Ser Gly Ser Trp Ala Ala Ile Tyr Gln Asp Ile Arg 10 15
20 cat gaa gcc agt gac ttc cca tgt aga gtg gcc aag ctt cct aag aac
210 His Glu Ala Ser Asp Phe Pro Cys Arg Val Ala Lys Leu Pro Lys Asn
25 30 35 40 aaa aac cga aat agg tac aga gac gtc agt ccc ttt gac cat
agt cgg 258 Lys Asn Arg Asn Arg Tyr Arg Asp Val Ser Pro Phe Asp His
Ser Arg 45 50 55 att aaa cta cat caa gaa gat aat gac tat atc aac
gct agt ttg ata 306 Ile Lys Leu His Gln Glu Asp Asn Asp Tyr Ile Asn
Ala Ser Leu Ile 60 65 70 aaa atg gaa gaa gcc caa agg agt tac att
ctt acc cag ggc cct ttg 354 Lys Met Glu Glu Ala Gln Arg Ser Tyr Ile
Leu Thr Gln Gly Pro Leu 75 80 85 cct aac aca tgc ggt cac ttt tgg
gag atg gtg tgg gag cag aaa agc 402 Pro Asn Thr Cys Gly His Phe Trp
Glu Met Val Trp Glu Gln Lys Ser 90 95 100 agg ggt gtc gtc atg ctc
aac aga gtg atg gag aaa ggt tcg tta aaa 450 Arg Gly Val Val Met Leu
Asn Arg Val Met Glu Lys Gly Ser Leu Lys 105 110 115 120 tgc gca caa
tac tgg cca caa aaa gaa gaa aaa gag atg atc ttt gaa 498 Cys Ala Gln
Tyr Trp Pro Gln Lys Glu Glu Lys Glu Met Ile Phe Glu 125 130 135 gac
aca aat ttg aaa tta aca ttg atc tct gaa gat atc aag tca tat 546 Asp
Thr Asn Leu Lys Leu Thr Leu Ile Ser Glu Asp Ile Lys Ser Tyr 140 145
150 tat aca gtg cga cag cta gaa ttg gaa aac ctt aca acc caa gaa act
594 Tyr Thr Val Arg Gln Leu Glu Leu Glu Asn Leu Thr Thr Gln Glu Thr
155 160 165 cga gag atc tta cat ttc cac tat acc aca tgg cct gac ttt
gga gtc 642 Arg Glu Ile Leu His Phe His Tyr Thr Thr Trp Pro Asp Phe
Gly Val 170 175 180 cct gaa tca cca gcc tca ttc ttg aac ttt ctt ttc
aaa gtc cga gag 690 Pro Glu Ser Pro Ala Ser Phe Leu Asn Phe Leu Phe
Lys Val Arg Glu 185 190 195 200 tca ggg tca ctc agc ccg gag cac ggg
ccc gtt gtg gtg cac tgc agt 738 Ser Gly Ser Leu Ser Pro Glu His Gly
Pro Val Val Val His Cys Ser 205 210 215 gca ggc atc ggc agg tct gga
acc ttc tgt ctg gct gat acc tgc ctc 786 Ala Gly Ile Gly Arg Ser Gly
Thr Phe Cys Leu Ala Asp Thr Cys Leu 220 225 230 ctg ctg atg gac aag
agg aaa gac cct tct tcc gtt gat atc aag aaa 834 Leu Leu Met Asp Lys
Arg Lys Asp Pro Ser Ser Val Asp Ile Lys Lys 235 240 245 gtg ctg tta
gaa atg agg aag ttt cgg atg ggg ttg atc cag aca gcc 882 Val Leu Leu
Glu Met Arg Lys Phe Arg Met Gly Leu Ile Gln Thr Ala 250 255 260 gac
cag ctg cgc ttc tcc tac ctg gct gtg atc gaa ggt gcc aaa ttc 930 Asp
Gln Leu Arg Phe Ser Tyr Leu Ala Val Ile Glu Gly Ala Lys Phe 265 270
275 280 atc atg ggg gac tct tcc gtg cag gat cag tgg aag gag ctt tcc
cac 978 Ile Met Gly Asp Ser Ser Val Gln Asp Gln Trp Lys Glu Leu Ser
His 285 290 295 gag gac ctg gag ccc cca ccc gag cat atc ccc cca cct
ccc cgg cca 1026 Glu Asp Leu Glu Pro Pro Pro Glu His Ile Pro Pro
Pro Pro Arg Pro 300 305 310 ccc aaa cga atc ctg gag cca cac aat ggg
aaa tgc agg gag ttc ttc 1074 Pro Lys Arg Ile Leu Glu Pro His Asn
Gly Lys Cys Arg Glu Phe Phe 315 320 325 cca aat cac cag tgg gtg aag
gaa gag acc cag gag gat aaa gac tgc 1122 Pro Asn His Gln Trp Val
Lys Glu Glu Thr Gln Glu Asp Lys Asp Cys 330 335 340 ccc atc aag gaa
gaa aaa gga agc ccc tta aat gcc gca ccc tac ggc 1170 Pro Ile Lys
Glu Glu Lys Gly Ser Pro Leu Asn Ala Ala Pro Tyr Gly 345 350 355 360
atc gaa agc atg agt caa gac act gaa gtt aga agt cgg gtc gtg ggg
1218 Ile Glu Ser Met Ser Gln Asp Thr Glu Val Arg Ser Arg Val Val
Gly 365 370 375 gga agt ctt cga ggt gcc cag gct gcc tcc cca gcc aaa
ggg gag ccg 1266 Gly Ser Leu Arg Gly Ala Gln Ala Ala Ser Pro Ala
Lys Gly Glu Pro 380 385 390 tca ctg ccc gag aag gac gag gac cat gca
ctg agt tac tgg aag ccc 1314 Ser Leu Pro Glu Lys Asp Glu Asp His
Ala Leu Ser Tyr Trp Lys Pro 395 400 405 ttc ctg gtc aac atg tgc gtg
gct acg gtc ctc acg gcc ggc gct tac 1362 Phe Leu Val Asn Met Cys
Val Ala Thr Val Leu Thr Ala Gly Ala Tyr 410 415 420 ctc tgc tac agg
ttc ctg ttc aac agc aac aca tag cctgaccctc 1408 Leu Cys Tyr Arg Phe
Leu Phe Asn Ser Asn Thr 425 430 435 ctccactcca cctccaccca
ctgtccgcct ctgcccgcag agcccacgcc cgactagcag 1468 gcatgccgcg
gtaggtaagg gccgccggac cgcgtagaga gccgggcccc ggacggacgt 1528
tggttctgca ctaaaaccca tcttccccgg atgtgtgtct cacccctcat ccttttactt
1588 tttgcccctt ccactttgag taccaaatcc acaagccatt ttttgaggag
agtgaaagag 1648 agtaccatgc tggcggcgca gagggaaggg gcctacaccc
gtcttggggc tcgccccacc 1708 cagggctccc tcctggagca tcccaggcgg
cgcacgccaa cagccccccc cttgaatctg 1768 cagggagcaa ctctccactc
catatttatt taaacaattt tttccccaaa ggcatccata 1828 gtgcactagc
attttcttga accaataatg tattaaaatt ttttgatgtc agccttgcat 1888
caagggcttt atcaaaaagt acaataataa atcctcaggt agtactggga atggaaggct
1948 ttgccatggg cctgctgcgt cagaccagta ctgggaagga ggacggttgt
aagcagttgt 2008 tatttagtga tattgtgggt aacgtgagaa gatagaacaa
tgctataata tataatgaac 2068 acgtgggtat ttaataagaa acatgatgtg
agattacttt gtcccgctta ttctcctccc 2128 tgttatctgc tagatctagt
tctcaatcac tgctcccccg tgtgtattag aatgcatgta 2188 aggtcttctt
gtgtcctgat gaaaaatatg tgcttgaaat gagaaacttt gatctctgct 2248
tactaatgtg ccccatgtcc aagtccaacc tgcctgtgca tgacctgatc attacatggc
2308 tgtggttcct aagcctgttg ctgaagtcat tgtcgctcag caatagggtg
cagttttcca 2368 ggaataggca tttgctaatt cctggcatga cactctagtg
acttcctggt gaggcccagc 2428 ctgtcctggt acagcagggt cttgctgtaa
ctcagacatt ccaagggtat gggaagccat 2488 attcacacct cacgctctgg
acatgattta gggaagcagg gacacccccc gccccccacc 2548 tttgggatca
gcctccgcca ttccaagtca acactcttct tgagcagacc gtgatttgga 2608
agagaggcac ctgctggaaa ccacacttct tgaaacagcc tgggtgacgg tcctttaggc
2668 agcctgccgc cgtctctgtc ccggttcacc ttgccgagag aggcgcgtct
gccccaccct 2728 caaaccctgt ggggcctgat ggtgctcacg actcttcctg
caaagggaac tgaagacctc 2788 cacattaagt ggctttttaa catgaaaaac
acggcagctg tagctcccga gctactctct 2848 tgccagcatt ttcacatttt
gcctttctcg tggtagaagc cagtacagag aaattctgtg 2908 gtgggaacat
tcgaggtgtc accctgcaga gctatggtga ggtgtggata aggcttaggt 2968
gccaggctgt aagcattctg agctggcttg ttgtttttaa gtcctgtata tgtatgtagt
3028 agtttgggtg tgtatatata gtagcatttc aaaatggacg tactggttta
acctcctatc 3088 cttggagagc agctggctct ccaccttgtt acacattatg
ttagagaggt agcgagctgc 3148 tctgctatat gccttaagcc aatatttact
catcaggtca ttatttttta caatggccat 3208 ggaataaacc atttttacaa
aaataaaaac aaaaaaagc 3247 4 21 DNA Artificial Sequence PCR Primer 4
ggagttcgag cagatcgaca a 21 5 21 DNA Artificial Sequence PCR Primer
5 ggccactcta catgggaagt c 21 6 24 DNA Artificial Sequence PCR Probe
6 agctgggcgg ccatttacca ggat 24 7 19 DNA Artificial Sequence PCR
Primer 7 gaaggtgaag gtcggagtc 19 8 20 DNA Artificial Sequence PCR
Primer 8 gaagatggtg atgggatttc 20 9 20 DNA Artificial Sequence PCR
Probe 9 caagcttccc gttctcagcc 20 10 4127 DNA Rattus norvegicus CDS
(120)...(1418) 10 agccgctgct ggggaggttg gggctgaggt ggtggcgggc
gacgggcctc gagacgcgga 60 gcgacgcggc ctagcgcggc ggacggccga
gggaactcgg gcagtcgtcc cgtcccgcc 119 atg gaa atg gag aag gaa ttc gag
cag atc gat aag gct ggg aac tgg 167 Met Glu Met Glu Lys Glu Phe Glu
Gln Ile Asp Lys Ala Gly Asn Trp 1 5 10 15 gcg gct att tac cag gat
att cga cat gaa gcc agt gac ttc cca tgc 215 Ala Ala Ile Tyr Gln Asp
Ile Arg His Glu Ala Ser Asp Phe Pro Cys 20 25 30 aga ata gcg aaa
ctt cct aag aac aaa aac cgg aac agg tac cga gat 263 Arg Ile Ala Lys
Leu Pro Lys Asn Lys Asn Arg Asn Arg Tyr Arg Asp 35 40 45 gtc agc
cct ttt gac cac agt cgg att aaa ttg cat cag gaa gat aat 311 Val Ser
Pro Phe Asp His Ser Arg Ile Lys Leu His Gln Glu Asp Asn 50 55 60
gac tat atc aat gcc agc ttg ata aaa atg gag gaa gcc cag agg agc 359
Asp Tyr Ile Asn Ala Ser Leu Ile Lys Met Glu Glu Ala Gln Arg Ser 65
70 75 80 tat atc ctc acc cag ggc cct tta cca aac acg tgc ggg cac
ttc tgg 407 Tyr Ile Leu Thr Gln Gly Pro Leu Pro Asn Thr Cys Gly His
Phe Trp 85 90 95 gag atg gtg tgg gag cag aag agc agg ggc gtg gtc
atg ctc aac cgc 455 Glu Met Val Trp Glu Gln Lys Ser Arg Gly Val Val
Met Leu Asn Arg 100 105 110 atc atg gag aaa ggc tcg tta aaa tgt gcc
cag tat tgg cca cag aaa 503 Ile Met Glu Lys Gly Ser Leu Lys Cys Ala
Gln Tyr Trp Pro Gln Lys 115 120 125 gaa gaa aaa gag atg gtc ttc gat
gac acc aat ttg aag ctg aca ctg 551 Glu Glu Lys Glu Met Val Phe Asp
Asp Thr Asn Leu Lys Leu Thr Leu 130 135 140 atc tct gaa gat gtc aag
tca tat tac aca gta cgg cag ttg gag ttg 599 Ile Ser Glu Asp Val Lys
Ser Tyr Tyr Thr Val Arg Gln Leu Glu Leu 145 150 155 160 gag aac ctg
gct acc cag gag gct cga gag atc ctg cat ttc cac tac 647 Glu Asn Leu
Ala Thr Gln Glu Ala Arg Glu Ile Leu His Phe His Tyr 165 170 175 acc
acc tgg cct gac ttt gga gtc cct gag tca cct gcc tct ttc ctc 695 Thr
Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala Ser Phe Leu 180 185
190 aat ttc cta ttc aaa gtc cga gag tca ggc tca ctc agc cca gag cac
743 Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu Ser Pro Glu His
195 200 205 ggc ccc att gtg gtc cac tgc agt gct ggc att ggc agg tca
ggg acc 791 Gly Pro Ile Val Val His Cys Ser Ala Gly Ile Gly Arg Ser
Gly Thr 210 215 220 ttc tgc ctg gct gac acc tgc ctc tta ctg atg gac
aag agg aaa gac 839 Phe Cys Leu Ala Asp Thr Cys Leu Leu Leu Met Asp
Lys Arg Lys Asp 225 230 235 240 ccg tcc tct gtg gac atc aag aaa gtg
ctg ttg gag atg cgc agg ttc 887 Pro Ser Ser Val Asp Ile Lys Lys Val
Leu Leu Glu Met Arg Arg Phe 245 250 255 cgc atg ggg ctc atc cag acg
gcc gac caa ctg cgc ttc tcc tac ctg 935 Arg Met Gly Leu Ile Gln Thr
Ala Asp Gln Leu Arg Phe Ser Tyr Leu 260 265 270 gct gtg atc gag ggt
gca aag ttc atc atg ggc gac tcg tca gtg cag 983 Ala Val Ile Glu Gly
Ala Lys Phe Ile Met Gly Asp Ser Ser Val Gln 275 280 285 gat cag tgg
aag gag ctt tcc cat gaa gac ctg gag cct ccc cct gag 1031 Asp Gln
Trp Lys Glu Leu Ser His Glu Asp Leu Glu Pro Pro Pro Glu 290 295 300
\ cac gtg ccc cca cct ccc cgg cca ccc aaa cgc aca ttg gag cct cac
1079 His Val Pro Pro Pro Pro Arg Pro Pro Lys Arg Thr Leu Glu Pro
His 305 310 315 320 \ aat ggc aag tgc aag gag ctc ttc tcc aac cac
cag tgg gtg agc gag 1127 Asn Gly Lys Cys Lys Glu Leu Phe Ser Asn
His Gln Trp Val Ser Glu 325 330 335 \ gag agc tgt gag gat gag gac
atc ctg gcc aga gag gaa agc aga gcc 1175 Glu Ser Cys Glu Asp Glu
Asp Ile Leu Ala Arg Glu Glu Ser Arg Ala 340 345 350 \ ccc tca att
gct gtg cac agc atg agc agt atg agt caa gac act gaa 1223 Pro Ser
Ile Ala Val His Ser Met Ser Ser Met Ser Gln Asp Thr Glu 355 360 365
\ gtt agg aaa cgg atg gtg ggt gga ggt ctt caa agt gct cag gca tct
1271 Val Arg Lys Arg Met Val Gly Gly Gly Leu Gln Ser Ala Gln Ala
Ser 370 375 380 \ gtc ccc act gag gaa gag ctg tcc cca acc gag gag
gaa caa aag gca 1319 Val Pro Thr Glu Glu Glu Leu Ser Pro Thr Glu
Glu Glu Gln Lys Ala 385 390 395 400 \ cac agg cca gtt cac tgg aag
ccc ttc ctg gtc aac gtg tgc atg gcc 1367 His Arg Pro Val His Trp
Lys Pro Phe Leu Val Asn Val Cys Met Ala 405 410 415 \ acg gcc ctg
gcg act ggc gcg tac ctc tgt tac cgg gta tgt ttt cac 1415 Thr Ala
Leu Ala Thr Gly Ala Tyr Leu Cys Tyr Arg Val Cys Phe His 420 425 430
\ tga cagactgctg tgaggcatga gcgtggtggg cgctgccact gcccaggtta 1468
ggatttggtc tgcggcgtct aacctggtgt agaagaaaca acagcttaca agcctgtggt
1528 ggaactggaa gggccagccc caggaggggc atctgtgcac tgggctttga
aggagcccct 1588 ggtcccaaga acagagtcta atctcagggc cttaacctgt
tcaggagaag tagaggaaat 1648 gccaaatact cttcttgctc tcacctcact
cctccccttt ctctggttcg tttgtttttg 1708 gaaaaaaaaa aaaaagaatt
acaacacatt gttgttttta acatttataa aggcaggttt 1768 ttgttatttt
tagagaaaac aaaagatgct aggcactggt gagattctct tgtgcccttt 1828
ggcatgtgat cagattcacg atttacgttt atttccgggg gagggtccca cctgtcagga
1888 ctgtaaagtt cctgctggct tggtcagccc ccccaccccc ccaccccgag
cttgcaggtg 1948 ccctgctgtg aggagagcag cagcagaggc tgcccctgga
cagaagccca gctctgcttc 2008 cctcaggtgt ccctgcgttt ccatcctcct
tctttgtgac cgccatcttg cagatgaccc 2068 agtcctcagc accccacccc
tgcagatggg tttctccgag ggcctgcctc agggtcatca 2128 gaggttggct
gccagcttag agctggggct tccatttgat tggaaagtca ttactattct 2188
atgtagaagc cactccactg aggtgtaaag caagactcat aaaggaggag ccttggtgtc
2248 atggaagtca ctccgcgcgc aggacctgta acaacctctg aaacactcag
tcctgctgca 2308 gtgacgtcct tgaaggcatc agacagatga tttgcagact
gccaagactt gtcctgagcc 2368 gtgattttta gagtctggac tcatgaaaca
ccgccgagcg cttactgtgc agcctctgat 2428 gctggttggc tgaggctgcg
gggaggtgga cactgtgggt gcatccagtg cagttgcttt 2488 tgtgcagttg
ggtccagcag cacagcccgc actccagcct cagctgcagg ccacagtggc 2548
catggaggcc gccagagcga gctggggtgg atgcttgttc acttggagca gccttcccag
2608 gacgtgcagc tcccttcctg ctttgtcctt ctgcttcctt ccctggagta
gcaagcccac 2668 gagcaatcgt gaggggtgtg agggagctgc agaggcatca
gagtggcctg cagcggcgtg 2728 aggccccttc ccctccgaca cccccctcca
gaggagccgc tccactgtta tttattcact 2788 ttgcccacag acacccctga
gtgagcacac cctgaaactg accgtgtaag gtgtcagcct 2848 gcacccagga
ccgtcaggtg cagcaccggg tcagtcctag ggttgaggta ggactgacac 2908
agccactgtg tggctggtgc tggggcaggg gcaggagctg agggtcttag aagcaatctt
2968 caggaacaga caacagtggt gacatgtaaa gtccctgtgg ctactgatga
catgtgtagg 3028 atgaaggctg gcctttctcc catgactttc tagatcccgt
tccccgtctg ctttccctgt 3088 gagttagaaa acacacaggc tcctgtcctg
gtggtgccgt gtgcttgaca tgggaaactt 3148 agatgcctgc tcactggcgg
gcacctcggc atcgccacca ctcagagtga gagcagtgct 3208 gtccagtgcc
gaggccgcct gactcccggc aggactcttc aggctctggc ctgccccagc 3268
acaccccgct ggatctcaga cattccacac ccacacctca ttccctggac acttgggcaa
3328 gcaggcccgc ccttccacct ctggggtcag cccctccatt ccgagttcac
actgctctgg 3388 agcaggccag gaccggaagc aaggcagctg gtgaggagca
ccctcctggg aacagtgtag 3448 gtgacagtcc tgagagtcag cttgctagcg
ctgctggcac cagtcacctt gctcagaagt 3508 gtgtggctct tgaggctgaa
gagactgatg atggtgctca tgactcttct gtgaggggaa 3568 cttgaccttc
acattgggtg gcttttttta aaataagcga aggcagctgg aactccagtc 3628
tgcctcttgc cagcacttca cattttgcct ttcacccaga gaagccagca cagagccact
3688 ggggaaggcg atggccttgc ctgcacaggc tgaggagatg gctcagccgg
cgtccaggct 3748 gtgtctggag cagggggtgc acagcagcct cacaggtggg
ggcctcagag caggcgctgc 3808 cctgtcccct gccccgctgg aggcagcaaa
gctgctgcat gccttaagtc aatacttact 3868 cagcagggcg ctctcgttct
ctctctctct ctctctctct ctctctctct ctctctctct 3928 ctctctaaat
ggccatagaa taaaccattt tacaaaaata aaagccaaca acaaagtgct 3988
ctggaatagc acctttgcag gagcgggggg tgtctcaggg tcttctgtga cctcaccgaa
4048 ctgtccgact gcaccgtttc caacttgtgt ctcactaatg ggtctgcatt
agttgcaaca 4108 ataaatgttt ttaaagaac 4127 11 21 DNA Artificial
Sequence PCR Primer 11 cgagggtgca aagttcatca t 21 12 21 DNA
Artificial Sequence PCR Primer 12 ccaggtcttc atgggaaagc t 21 13 26
DNA Artificial Sequence PCR Probe 13 cgactcgtca gtgcaggatc agtgga
26 14 23 DNA Artificial Sequence PCR Primer 14 tgttctagag
acagccgcat ctt 23 15 21 DNA Artificial Sequence PCR Primer 15
caccgacctt caccatcttg t 21 16 24 DNA Artificial Sequence PCR Probe
16 ttgtgcagtg ccagcctcgt ctca 24 17 20 DNA Artificial Sequence
Antisense Oligonucleotide 17 cttagccccg aggcccgccc
20 18 20 DNA Artificial Sequence Antisense Oligonucleotide 18
ctcggcccac tgcgccgtct 20 19 20 DNA Artificial Sequence Antisense
Oligonucleotide 19 catgacgggc cagggcggct 20 20 20 DNA Artificial
Sequence Antisense Oligonucleotide 20 cccggacttg tcgatctgct 20 21
20 DNA Artificial Sequence Antisense Oligonucleotide 21 ctggcttcat
gtcggatatc 20 22 20 DNA Artificial Sequence Antisense
Oligonucleotide 22 ttggccactc tacatgggaa 20 23 20 DNA Artificial
Sequence Antisense Oligonucleotide 23 ggactgacgt ctctgtacct 20 24
20 DNA Artificial Sequence Antisense Oligonucleotide 24 gatgtagttt
aatccgacta 20 25 20 DNA Artificial Sequence Antisense
Oligonucleotide 25 ctagcgttga tatagtcatt 20 26 20 DNA Artificial
Sequence Antisense Oligonucleotide 26 gggtaagaat gtaactcctt 20 27
20 DNA Artificial Sequence Antisense Oligonucleotide 27 tgaccgcatg
tgttaggcaa 20 28 20 DNA Artificial Sequence Antisense
Oligonucleotide 28 ttttctgctc ccacaccatc 20 29 20 DNA Artificial
Sequence Antisense Oligonucleotide 29 ctctgttgag catgacgaca 20 30
20 DNA Artificial Sequence Antisense Oligonucleotide 30 gcgcatttta
acgaaccttt 20 31 20 DNA Artificial Sequence Antisense
Oligonucleotide 31 aaatttgtgt cttcaaagat 20 32 20 DNA Artificial
Sequence Antisense Oligonucleotide 32 tgatatcttc agagatcaat 20 33
20 DNA Artificial Sequence Antisense Oligonucleotide 33 tctagctgtc
gcactgtata 20 34 20 DNA Artificial Sequence Antisense
Oligonucleotide 34 agtttcttgg gttgtaaggt 20 35 20 DNA Artificial
Sequence Antisense Oligonucleotide 35 gtggtatagt ggaaatgtaa 20 36
20 DNA Artificial Sequence Antisense Oligonucleotide 36 tgattcaggg
actccaaagt 20 37 20 DNA Artificial Sequence Antisense
Oligonucleotide 37 ttgaaaagaa agttcaagaa 20 38 20 DNA Artificial
Sequence Antisense Oligonucleotide 38 gggctgagtg accctgactc 20 39
20 DNA Artificial Sequence Antisense Oligonucleotide 39 gcagtgcacc
acaacgggcc 20 40 20 DNA Artificial Sequence Antisense
Oligonucleotide 40 aggttccaga cctgccgatg 20 41 20 DNA Artificial
Sequence Antisense Oligonucleotide 41 agcaggaggc aggtatcagc 20 42
20 DNA Artificial Sequence Antisense Oligonucleotide 42 gaagaagggt
ctttcctctt 20 43 20 DNA Artificial Sequence Antisense
Oligonucleotide 43 tctaacagca ctttcttgat 20 44 20 DNA Artificial
Sequence Antisense Oligonucleotide 44 atcaacccca tccgaaactt 20 45
20 DNA Artificial Sequence Antisense Oligonucleotide 45 gagaagcgca
gctggtcggc 20 46 20 DNA Artificial Sequence Antisense
Oligonucleotide 46 tttggcacct tcgatcacag 20 47 20 DNA Artificial
Sequence Antisense Oligonucleotide 47 agctccttcc actgatcctg 20 48
20 DNA Artificial Sequence Antisense Oligonucleotide 48 tccaggattc
gtttgggtgg 20 49 20 DNA Artificial Sequence Antisense
Oligonucleotide 49 gaactccctg catttcccat 20 50 20 DNA Artificial
Sequence Antisense Oligonucleotide 50 ttccttcacc cactggtgat 20 51
20 DNA Artificial Sequence Antisense Oligonucleotide 51 gtagggtgcg
gcatttaagg 20 52 20 DNA Artificial Sequence Antisense
Oligonucleotide 52 cagtgtcttg actcatgctt 20 53 20 DNA Artificial
Sequence Antisense Oligonucleotide 53 gcctgggcac ctcgaagact 20 54
20 DNA Artificial Sequence Antisense Oligonucleotide 54 ctcgtccttc
tcgggcagtg 20 55 20 DNA Artificial Sequence Antisense
Oligonucleotide 55 gggcttccag taactcagtg 20 56 20 DNA Artificial
Sequence Antisense Oligonucleotide 56 ccgtagccac gcacatgttg 20 57
20 DNA Artificial Sequence Antisense Oligonucleotide 57 tagcagaggt
aagcgccggc 20 58 20 DNA Artificial Sequence Antisense
Oligonucleotide 58 ctatgtgttg ctgttgaaca 20 59 20 DNA Artificial
Sequence Antisense Oligonucleotide 59 ggaggtggag tggaggaggg 20 60
20 DNA Artificial Sequence Antisense Oligonucleotide 60 ggctctgcgg
gcagaggcgg 20 61 20 DNA Artificial Sequence Antisense
Oligonucleotide 61 ccgcggcatg cctgctagtc 20 62 20 DNA Artificial
Sequence Antisense Oligonucleotide 62 tctctacgcg gtccggcggc 20 63
20 DNA Artificial Sequence Antisense Oligonucleotide 63 aagatgggtt
ttagtgcaga 20 64 20 DNA Artificial Sequence Antisense
Oligonucleotide 64 gtactctctt tcactctcct 20 65 20 DNA Artificial
Sequence Antisense Oligonucleotide 65 ggccccttcc ctctgcgccg 20 66
20 DNA Artificial Sequence Antisense Oligonucleotide 66 ctccaggagg
gagccctggg 20 67 20 DNA Artificial Sequence Antisense
Oligonucleotide 67 gggctgttgg cgtgcgccgc 20 68 20 DNA Artificial
Sequence Antisense Oligonucleotide 68 tttaaataaa tatggagtgg 20 69
20 DNA Artificial Sequence Antisense Oligonucleotide 69 gttcaagaaa
atgctagtgc 20 70 20 DNA Artificial Sequence Antisense
Oligonucleotide 70 ttgataaagc ccttgatgca 20 71 20 DNA Artificial
Sequence Antisense Oligonucleotide 71 atggcaaagc cttccattcc 20 72
20 DNA Artificial Sequence Antisense Oligonucleotide 72 gtcctccttc
ccagtactgg 20 73 20 DNA Artificial Sequence Antisense
Oligonucleotide 73 ttacccacaa tatcactaaa 20 74 20 DNA Artificial
Sequence Antisense Oligonucleotide 74 attatatatt atagcattgt 20 75
20 DNA Artificial Sequence Antisense Oligonucleotide 75 tcacatcatg
tttcttatta 20 76 20 DNA Artificial Sequence Antisense
Oligonucleotide 76 ataacaggga ggagaataag 20 77 20 DNA Artificial
Sequence Antisense Oligonucleotide 77 ttacatgcat tctaatacac 20 78
20 DNA Artificial Sequence Antisense Oligonucleotide 78 gatcaaagtt
tctcatttca 20 79 20 DNA Artificial Sequence Antisense
Oligonucleotide 79 ggtcatgcac aggcaggttg 20 80 20 DNA Artificial
Sequence Antisense Oligonucleotide 80 caacaggctt aggaaccaca 20 81
20 DNA Artificial Sequence Antisense Oligonucleotide 81 aactgcaccc
tattgctgag 20 82 20 DNA Artificial Sequence Antisense
Oligonucleotide 82 gtcatgccag gaattagcaa 20 83 20 DNA Artificial
Sequence Antisense Oligonucleotide 83 acaggctggg cctcaccagg 20 84
20 DNA Artificial Sequence Antisense Oligonucleotide 84 tgagttacag
caagaccctg 20 85 20 DNA Artificial Sequence Antisense
Oligonucleotide 85 gaatatggct tcccataccc 20 86 20 DNA Artificial
Sequence Antisense Oligonucleotide 86 ccctaaatca tgtccagagc 20 87
20 DNA Artificial Sequence Antisense Oligonucleotide 87 gacttggaat
ggcggaggct 20 88 20 DNA Artificial Sequence Antisense
Oligonucleotide 88 caaatcacgg tctgctcaag 20 89 20 DNA Artificial
Sequence Antisense Oligonucleotide 89 gaagtgtggt ttccagcagg 20 90
20 DNA Artificial Sequence Antisense Oligonucleotide 90 cctaaaggac
cgtcacccag 20 91 20 DNA Artificial Sequence Antisense
Oligonucleotide 91 gtgaaccggg acagagacgg 20 92 20 DNA Artificial
Sequence Antisense Oligonucleotide 92 gccccacagg gtttgagggt 20 93
20 DNA Artificial Sequence Antisense Oligonucleotide 93 cctttgcagg
aagagtcgtg 20 94 20 DNA Artificial Sequence Antisense
Oligonucleotide 94 aaagccactt aatgtggagg 20 95 20 DNA Artificial
Sequence Antisense Oligonucleotide 95 gtgaaaatgc tggcaagaga 20 96
20 DNA Artificial Sequence Antisense Oligonucleotide 96 tcagaatgct
tacagcctgg 20 97 20 DNA Artificial Sequence Antisense
Oligonucleotide 97 caacctcccc agcagcggct 20 98 20 DNA Artificial
Sequence Antisense Oligonucleotide 98 tcgaggcccg tcgcccgcca 20 99
20 DNA Artificial Sequence Antisense Oligonucleotide 99 cctcggccgt
ccgccgcgct 20 100 20 DNA Artificial Sequence Antisense
Oligonucleotide 100 tcgatctgct cgaattcctt 20 101 20 DNA Artificial
Sequence Antisense Oligonucleotide 101 cctggtaaat agccgcccag 20 102
20 DNA Artificial Sequence Antisense Oligonucleotide 102 tgtcgaatat
cctggtaaat 20 103 20 DNA Artificial Sequence Antisense
Oligonucleotide 103 actggcttca tgtcgaatat 20 104 20 DNA Artificial
Sequence Antisense Oligonucleotide 104 aagtcactgg cttcatgtcg 20 105
20 DNA Artificial Sequence Antisense Oligonucleotide 105 gaagtcactg
gcttcatgtc 20 106 20 DNA Artificial Sequence Antisense
Oligonucleotide 106 ggaagtcact ggcttcatgt 20 107 20 DNA Artificial
Sequence Antisense Oligonucleotide 107 gggaagtcac tggcttcatg 20 108
20 DNA Artificial Sequence Antisense Oligonucleotide 108 tgggaagtca
ctggcttcat 20 109 20 DNA Artificial Sequence Antisense
Oligonucleotide 109 atgggaagtc actggcttca 20 110 20 DNA Artificial
Sequence Antisense Oligonucleotide 110 catgggaagt cactggcttc 20 111
20 DNA Artificial Sequence Antisense Oligonucleotide 111 tttttgttct
taggaagttt 20 112 20 DNA Artificial Sequence Antisense
Oligonucleotide 112 cggtttttgt tcttaggaag 20 113 20 DNA Artificial
Sequence Antisense Oligonucleotide 113 tccgactgtg gtcaaaaggg 20 114
20 DNA Artificial Sequence Antisense Oligonucleotide 114 ttaatccgac
tgtggtcaaa 20 115 20 DNA Artificial Sequence Antisense
Oligonucleotide 115 atagtcatta tcttcctgat 20 116 20 DNA Artificial
Sequence Antisense Oligonucleotide 116 ttgatatagt cattatcttc 20 117
20 DNA Artificial Sequence Antisense Oligonucleotide 117 gcttcctcca
tttttatcaa 20 118 20 DNA Artificial Sequence Antisense
Oligonucleotide 118 ggccctgggt gaggatatag 20 119 20 DNA Artificial
Sequence Antisense Oligonucleotide 119 cacaccatct cccagaagtg 20 120
20 DNA Artificial Sequence Antisense Oligonucleotide 120 tgctcccaca
ccatctccca 20 121 20 DNA Artificial Sequence Antisense
Oligonucleotide 121 ctgctcccac accatctccc 20 122 20 DNA Artificial
Sequence Antisense Oligonucleotide 122 tctgctccca caccatctcc 20 123
20 DNA Artificial Sequence Antisense Oligonucleotide 123 ttctgctccc
acaccatctc 20 124 20 DNA Artificial Sequence Antisense
Oligonucleotide 124 cccctgctct tctgctccca 20 125 20 DNA Artificial
Sequence Antisense Oligonucleotide 125 atgcggttga gcatgaccac 20 126
20 DNA Artificial Sequence Antisense Oligonucleotide 126 tttaacgagc
ctttctccat 20 127 20 DNA Artificial Sequence Antisense
Oligonucleotide 127 ttttcttctt tctgtggcca 20 128 20 DNA Artificial
Sequence Antisense Oligonucleotide 128 gaccatctct ttttcttctt 20 129
20 DNA Artificial Sequence Antisense Oligonucleotide 129 tcagagatca
gtgtcagctt 20 130 20 DNA Artificial Sequence Antisense
Oligonucleotide 130 cttgacatct tcagagatca 20 131 20 DNA Artificial
Sequence Antisense Oligonucleotide 131 taatatgact tgacatcttc 20 132
20 DNA Artificial Sequence Antisense Oligonucleotide 132 aactccaact
gccgtactgt 20 133 20 DNA Artificial Sequence Antisense
Oligonucleotide 133 tctctcgagc ctcctgggta 20 134 20 DNA Artificial
Sequence Antisense Oligonucleotide 134 ccaaagtcag gccaggtggt 20 135
20 DNA Artificial Sequence Antisense Oligonucleotide 135 gggactccaa
agtcaggcca 20 136 20 DNA Artificial Sequence Antisense
Oligonucleotide 136 agggactcca aagtcaggcc 20 137 20 DNA Artificial
Sequence Antisense Oligonucleotide 137 cagggactcc aaagtcaggc 20 138
20 DNA Artificial Sequence Antisense Oligonucleotide 138 tcagggactc
caaagtcagg 20 139 20 DNA Artificial Sequence Antisense
Oligonucleotide 139 ggtgactcag ggactccaaa 20 140 20 DNA Artificial
Sequence Antisense Oligonucleotide 140 cctgactctc ggactttgaa 20 141
20 DNA Artificial Sequence Antisense Oligonucleotide 141 gctgagtgag
cctgactctc 20 142 20 DNA Artificial Sequence Antisense
Oligonucleotide 142 ccgtgctctg ggctgagtga 20 143 20 DNA Artificial
Sequence Antisense
Oligonucleotide 143 aaggtccctg acctgccaat 20 144 20 DNA Artificial
Sequence Antisense Oligonucleotide 144 tctttcctct tgtccatcag 20 145
20 DNA Artificial Sequence Antisense Oligonucleotide 145 gtctttcctc
ttgtccatca 20 146 20 DNA Artificial Sequence Antisense
Oligonucleotide 146 ggtctttcct cttgtccatc 20 147 20 DNA Artificial
Sequence Antisense Oligonucleotide 147 gggtctttcc tcttgtccat 20 148
20 DNA Artificial Sequence Antisense Oligonucleotide 148 aacagcactt
tcttgatgtc 20 149 20 DNA Artificial Sequence Antisense
Oligonucleotide 149 ggaacctgcg catctccaac 20 150 20 DNA Artificial
Sequence Antisense Oligonucleotide 150 tggtcggccg tctggatgag 20 151
20 DNA Artificial Sequence Antisense Oligonucleotide 151 gagaagcgca
gttggtcggc 20 152 20 DNA Artificial Sequence Antisense
Oligonucleotide 152 aggtaggaga agcgcagttg 20 153 20 DNA Artificial
Sequence Antisense Oligonucleotide 153 gccaggtagg agaagcgcag 20 154
20 DNA Artificial Sequence Antisense Oligonucleotide 154 agccaggtag
gagaagcgca 20 155 20 DNA Artificial Sequence Antisense
Oligonucleotide 155 cagccaggta ggagaagcgc 20 156 20 DNA Artificial
Sequence Antisense Oligonucleotide 156 acagccaggt aggagaagcg 20 157
20 DNA Artificial Sequence Antisense Oligonucleotide 157 cacagccagg
taggagaagc 20 158 20 DNA Artificial Sequence Antisense
Oligonucleotide 158 tcacagccag gtaggagaag 20 159 20 DNA Artificial
Sequence Antisense Oligonucleotide 159 atcacagcca ggtaggagaa 20 160
20 DNA Artificial Sequence Antisense Oligonucleotide 160 gatcacagcc
aggtaggaga 20 161 20 DNA Artificial Sequence Antisense
Oligonucleotide 161 cgatcacagc caggtaggag 20 162 20 DNA Artificial
Sequence Antisense Oligonucleotide 162 tcgatcacag ccaggtagga 20 163
20 DNA Artificial Sequence Antisense Oligonucleotide 163 caccctcgat
cacagccagg 20 164 20 DNA Artificial Sequence Antisense
Oligonucleotide 164 tccttccact gatcctgcac 20 165 20 DNA Artificial
Sequence Antisense Oligonucleotide 165 ctccttccac tgatcctgca 20 166
20 DNA Artificial Sequence Antisense Oligonucleotide 166 gctccttcca
ctgatcctgc 20 167 20 DNA Artificial Sequence Antisense
Oligonucleotide 167 agctccttcc actgatcctg 20 168 20 DNA Artificial
Sequence Antisense Oligonucleotide 168 aagctccttc cactgatcct 20 169
20 DNA Artificial Sequence Antisense Oligonucleotide 169 aaagctcctt
ccactgatcc 20 170 20 DNA Artificial Sequence Antisense
Oligonucleotide 170 gaaagctcct tccactgatc 20 171 20 DNA Artificial
Sequence Antisense Oligonucleotide 171 ggaaagctcc ttccactgat 20 172
20 DNA Artificial Sequence Antisense Oligonucleotide 172 gggaaagctc
cttccactga 20 173 20 DNA Artificial Sequence Antisense
Oligonucleotide 173 tgggaaagct ccttccactg 20 174 20 DNA Artificial
Sequence Antisense Oligonucleotide 174 tggccgggga ggtgggggca 20 175
20 DNA Artificial Sequence Antisense Oligonucleotide 175 tgggtggccg
gggaggtggg 20 176 20 DNA Artificial Sequence Antisense
Oligonucleotide 176 tgcgtttggg tggccgggga 20 177 20 DNA Artificial
Sequence Antisense Oligonucleotide 177 tgcacttgcc attgtgaggc 20 178
20 DNA Artificial Sequence Antisense Oligonucleotide 178 acttcagtgt
cttgactcat 20 179 20 DNA Artificial Sequence Antisense
Oligonucleotide 179 aacttcagtg tcttgactca 20 180 20 DNA Artificial
Sequence Antisense Oligonucleotide 180 taacttcagt gtcttgactc 20 181
20 DNA Artificial Sequence Antisense Oligonucleotide 181 ctaacttcag
tgtcttgact 20 182 20 DNA Artificial Sequence Antisense
Oligonucleotide 182 gacagatgcc tgagcacttt 20 183 20 DNA Artificial
Sequence Antisense Oligonucleotide 183 gaccaggaag ggcttccagt 20 184
20 DNA Artificial Sequence Antisense Oligonucleotide 184 tgaccaggaa
gggcttccag 20 185 20 DNA Artificial Sequence Antisense
Oligonucleotide 185 ttgaccagga agggcttcca 20 186 20 DNA Artificial
Sequence Antisense Oligonucleotide 186 gttgaccagg aagggcttcc 20 187
20 DNA Artificial Sequence Antisense Oligonucleotide 187 gcacacgttg
accaggaagg 20 188 20 DNA Artificial Sequence Antisense
Oligonucleotide 188 gaggtacgcg ccagtcgcca 20 189 20 DNA Artificial
Sequence Antisense Oligonucleotide 189 tacccggtaa cagaggtacg 20 190
20 DNA Artificial Sequence Antisense Oligonucleotide 190 agtgaaaaca
tacccggtaa 20 191 20 DNA Artificial Sequence Antisense
Oligonucleotide 191 caaatcctaa cctgggcagt 20 192 20 DNA Artificial
Sequence Antisense Oligonucleotide 192 ttccagttcc accacaggct 20 193
20 DNA Artificial Sequence Antisense Oligonucleotide 193 ccagtgcaca
gatgcccctc 20 194 20 DNA Artificial Sequence Antisense
Oligonucleotide 194 acaggttaag gccctgagat 20 195 20 DNA Artificial
Sequence Antisense Oligonucleotide 195 gcctagcatc ttttgttttc 20 196
20 DNA Artificial Sequence Antisense Oligonucleotide 196 aagccagcag
gaactttaca 20 197 20 DNA Artificial Sequence Antisense
Oligonucleotide 197 gggacacctg agggaagcag 20 198 20 DNA Artificial
Sequence Antisense Oligonucleotide 198 ggtcatctgc aagatggcgg 20 199
20 DNA Artificial Sequence Antisense Oligonucleotide 199 gccaacctct
gatgaccctg 20 200 20 DNA Artificial Sequence Antisense
Oligonucleotide 200 tggaagcccc agctctaagc 20 201 20 DNA Artificial
Sequence Antisense Oligonucleotide 201 tagtaatgac tttccaatca 20 202
20 DNA Artificial Sequence Antisense Oligonucleotide 202 tgagtcttgc
tttacacctc 20 203 20 DNA Artificial Sequence Antisense
Oligonucleotide 203 cctgcgcgcg gagtgacttc 20 204 20 DNA Artificial
Sequence Antisense Oligonucleotide 204 aggacgtcac tgcagcagga 20 205
20 DNA Artificial Sequence Antisense Oligonucleotide 205 tcaggacaag
tcttggcagt 20 206 20 DNA Artificial Sequence Antisense
Oligonucleotide 206 gaggctgcac agtaagcgct 20 207 20 DNA Artificial
Sequence Antisense Oligonucleotide 207 tcagccaacc agcatcagag 20 208
20 DNA Artificial Sequence Antisense Oligonucleotide 208 acccacagtg
tccacctccc 20 209 20 DNA Artificial Sequence Antisense
Oligonucleotide 209 agtgcgggct gtgctgctgg 20 210 20 DNA Artificial
Sequence Antisense Oligonucleotide 210 cagctcgctc tggcggcctc 20 211
20 DNA Artificial Sequence Antisense Oligonucleotide 211 aggaagggag
ctgcacgtcc 20 212 20 DNA Artificial Sequence Antisense
Oligonucleotide 212 ccctcacgat tgctcgtggg 20 213 20 DNA Artificial
Sequence Antisense Oligonucleotide 213 cagtggagcg gctcctctgg 20 214
20 DNA Artificial Sequence Antisense Oligonucleotide 214 caggctgaca
ccttacacgg 20 215 20 DNA Artificial Sequence Antisense
Oligonucleotide 215 gtcctacctc aaccctagga 20 216 20 DNA Artificial
Sequence Antisense Oligonucleotide 216 ctgccccagc accagccaca 20 217
20 DNA Artificial Sequence Antisense Oligonucleotide 217 attgcttcta
agaccctcag 20 218 20 DNA Artificial Sequence Antisense
Oligonucleotide 218 ttacatgtca ccactgttgt 20 219 20 DNA Artificial
Sequence Antisense Oligonucleotide 219 tacacatgtc atcagtagcc 20 220
20 DNA Artificial Sequence Antisense Oligonucleotide 220 ttttctaact
cacagggaaa 20 221 20 DNA Artificial Sequence Antisense
Oligonucleotide 221 gtgcccgcca gtgagcaggc 20 222 20 DNA Artificial
Sequence Antisense Oligonucleotide 222 cggcctcggc actggacagc 20 223
20 DNA Artificial Sequence Antisense Oligonucleotide 223 gtggaatgtc
tgagatccag 20 224 20 DNA Artificial Sequence Antisense
Oligonucleotide 224 agggcgggcc tgcttgccca 20 225 20 DNA Artificial
Sequence Antisense Oligonucleotide 225 cggtcctggc ctgctccaga 20 226
20 DNA Artificial Sequence Antisense Oligonucleotide 226 tacactgttc
ccaggagggt 20 227 20 DNA Artificial Sequence Antisense
Oligonucleotide 227 tggtgccagc agcgctagca 20 228 20 DNA Artificial
Sequence Antisense Oligonucleotide 228 cagtctcttc agcctcaaga 20 229
20 DNA Artificial Sequence Antisense Oligonucleotide 229 aagagtcatg
agcaccatca 20 230 20 DNA Artificial Sequence Antisense
Oligonucleotide 230 tgaaggtcaa gttcccctca 20 231 20 DNA Artificial
Sequence Antisense Oligonucleotide 231 ctggcaagag gcagactgga 20 232
20 DNA Artificial Sequence Antisense Oligonucleotide 232 ggctctgtgc
tggcttctct 20 233 20 DNA Artificial Sequence Antisense
Oligonucleotide 233 gccatctcct cagcctgtgc 20 234 20 DNA Artificial
Sequence Antisense Oligonucleotide 234 agcgcctgct ctgaggcccc 20 235
20 DNA Artificial Sequence Antisense Oligonucleotide 235 tgctgagtaa
gtattgactt 20 236 20 DNA Artificial Sequence Antisense
Oligonucleotide 236 ctatggccat ttagagagag 20 237 20 DNA Artificial
Sequence Antisense Oligonucleotide 237 tggtttattc tatggccatt 20 238
20 DNA Artificial Sequence Antisense Oligonucleotide 238 cgctcctgca
aaggtgctat 20 239 20 DNA Artificial Sequence Antisense
Oligonucleotide 239 gttggaaacg gtgcagtcgg 20 240 20 DNA Artificial
Sequence Antisense Oligonucleotide 240 atttattgtt gcaactaatg 20 241
2346 DNA Mus musculus CDS (710)...(2008) 241 gaattcggga tccttttgca
cattcctagt tagcagtgca tactcatcag actggagatg 60 tttaatgaca
tcagggaacc aaacggacaa cccatagtac ccgaagacag ggtgaaccag 120
acaatcgtaa gcttgatggt gttttccctg actgggtagt tgaagcatct catgaatgtc
180 agccaaattc cgtacagttc ggtgcggatc cgaacgaaac acctcctgta
ccaggttccc 240 gtgtcgctct caatttcaat cagctcatct atttgtttgg
gagtcttgat tttatttacc 300 gtgaagacct tctctggctg gccccgggct
ctcatgttgg tgtcatgaat taacttcaga 360 atcatccagg cttcatcatg
ttttcccacc tccagcaaga accgagggct ttctggcatg 420 aaggtgagag
ccaccacaga ggagacgcat gggagcgcac agacgatgac gaagacgcgc 480
cacgtgtgga actggtaggc tgaacccatg ctgaagctcc acccgtagtg gggaatgatg
540 gcccaggcat ggcggaggct agatgccgcc aatcatccag aacatgcaga
agccgctgct 600 ggggagcttg gggctgcggt ggtggcgggt gacgggcttc
gggacgcgga gcgacgcggc 660 ctagcgcggc ggacggccgt gggaactcgg
gcagccgacc cgtcccgcc atg gag atg 718 Met Glu Met 1 gag aag gag ttc
gag gag atc gac aag gct ggg aac tgg gcg gct att 766 Glu Lys Glu Phe
Glu Glu Ile Asp Lys Ala Gly Asn Trp Ala Ala Ile 5 10 15 tac cag gac
att cga cat gaa gcc agc gac ttc cca tgc aaa gtc gcg 814 Tyr Gln Asp
Ile Arg His Glu Ala Ser Asp Phe Pro Cys Lys Val Ala 20 25 30 35 aag
ctt cct aag aac aaa aac cgg aac agg tac cga gat gtc agc cct 862 Lys
Leu Pro Lys Asn Lys Asn Arg Asn Arg Tyr Arg Asp Val Ser Pro 40 45
50 ttt gac cac agt cgg att aaa ttg cac cag gaa gat aat gac tat atc
910 Phe Asp His Ser Arg Ile Lys Leu His Gln Glu Asp Asn Asp Tyr Ile
55 60 65 aat gcc agc ttg ata aaa atg gaa gaa gcc cag agg agc tat
att ctc 958 Asn Ala Ser Leu Ile Lys Met Glu Glu Ala Gln Arg Ser Tyr
Ile Leu 70 75 80 acc cag ggc cct tta cca aac aca tgt ggg cac ttc
tgg gag atg gtg 1006 Thr Gln Gly Pro Leu Pro Asn Thr Cys Gly His
Phe Trp Glu Met Val 85 90 95 tgg gag cag aag agc agg ggc gtg gtc
atg ctc aac cgc atc atg gag 1054 Trp Glu Gln Lys Ser Arg Gly Val
Val Met Leu Asn Arg Ile Met Glu 100 105 110 115 aaa ggc tcg tta aaa
tgt gcc cag tat tgg cca cag caa gaa gaa aag 1102 Lys Gly Ser Leu
Lys Cys Ala Gln Tyr Trp Pro Gln Gln Glu Glu Lys 120 125 130 gag atg
gtc ttt gat gac aca ggt ttg aag ttg aca cta atc tct gaa 1150 Glu
Met Val Phe Asp Asp Thr Gly Leu Lys Leu Thr Leu Ile Ser Glu 135 140
145 gat gtc aag tca tat tac aca gta cga cag ttg gag ttg gaa aac ctg
1198 Asp Val Lys Ser Tyr Tyr Thr Val Arg Gln Leu Glu Leu Glu Asn
Leu 150 155 160 act acc aag gag act cga gag atc ctg cat ttc cac tac
acc aca tgg 1246 Thr Thr Lys Glu Thr Arg Glu Ile Leu His Phe His
Tyr Thr Thr Trp 165 170 175 cct gac ttt gga gtc ccc gag tca ccg gct
tct ttc ctc aat ttc ctt 1294 Pro Asp Phe Gly Val Pro Glu Ser Pro
Ala Ser Phe Leu Asn Phe Leu 180 185 190 195 ttc aaa gtc cga gag tca
ggc tca ctc agc ctg gag cat ggc ccc att 1342 Phe Lys Val Arg Glu
Ser Gly Ser Leu Ser Leu Glu His Gly Pro Ile 200 205 210 gtg gtc cac
tgc agc gcc ggc atc ggg agg tca ggg acc ttc tgt ctg 1390 Val Val
His Cys Ser Ala Gly Ile Gly Arg Ser Gly Thr Phe Cys Leu 215 220 225
gct gac acc tgc ctc tta ctg atg gac aag agg aaa gac cca tct tcc
1438 Ala Asp Thr Cys Leu Leu Leu Met Asp Lys Arg Lys Asp Pro Ser
Ser 230 235 240 gtg gac atc aag aaa gta ctg ctg gag atg cgc agg ttc
cgc atg ggg 1486 Val Asp Ile Lys Lys Val Leu Leu Glu Met Arg Arg
Phe Arg Met
Gly 245 250 255 ctc atc cag act gcc gac cag ctg cgc ttc tcc tac ctg
gct gtc atc 1534 Leu Ile Gln Thr Ala Asp Gln Leu Arg Phe Ser Tyr
Leu Ala Val Ile 260 265 270 275 gag ggc gcc aag ttc atc atg ggc gac
tcg tca gtg cag gat cag tgg 1582 Glu Gly Ala Lys Phe Ile Met Gly
Asp Ser Ser Val Gln Asp Gln Trp 280 285 290 aag gag ctc tcc cgg gag
gat cta gac ctt cca ccc gag cac gtg ccc 1630 Lys Glu Leu Ser Arg
Glu Asp Leu Asp Leu Pro Pro Glu His Val Pro 295 300 305 cca cct ccc
cgg cca ccc aaa cgc aca ctg gag cct cac aac ggg aag 1678 Pro Pro
Pro Arg Pro Pro Lys Arg Thr Leu Glu Pro His Asn Gly Lys 310 315 320
tgc aag gag ctc ttc tcc agc cac cag tgg gtg agc gag gag acc tgt
1726 Cys Lys Glu Leu Phe Ser Ser His Gln Trp Val Ser Glu Glu Thr
Cys 325 330 335 ggg gat gaa gac agc ctg gcc aga gag gaa ggc aga gcc
cag tca agt 1774 Gly Asp Glu Asp Ser Leu Ala Arg Glu Glu Gly Arg
Ala Gln Ser Ser 340 345 350 355 gcc atg cac agc gtg agc agc atg agt
cca gac act gaa gtt agg aga 1822 Ala Met His Ser Val Ser Ser Met
Ser Pro Asp Thr Glu Val Arg Arg 360 365 370 cgg atg gtg ggt gga ggt
ctt caa agt gct cag gcg tct gtc ccc acc 1870 Arg Met Val Gly Gly
Gly Leu Gln Ser Ala Gln Ala Ser Val Pro Thr 375 380 385 gag gaa gag
ctg tcc tcc act gag gag gaa cac aag gca cat tgg cca 1918 Glu Glu
Glu Leu Ser Ser Thr Glu Glu Glu His Lys Ala His Trp Pro 390 395 400
agt cac tgg aag ccc ttc ctg gtc aat gtg tgc atg gcc acg ctc ctg
1966 Ser His Trp Lys Pro Phe Leu Val Asn Val Cys Met Ala Thr Leu
Leu 405 410 415 gcc acc ggc gcg tac ttg tgc tac cgg gtg tgt ttt cac
tga 2008 Ala Thr Gly Ala Tyr Leu Cys Tyr Arg Val Cys Phe His * 420
425 430 cagactggga ggcactgcca ctgcccagct taggatgcgg tctgcggcgt
ctgacctggt 2068 gtagagggaa caacaactcg caagcctgct ctggaactgg
aagggcctgc cccaggaggg 2128 tattagtgca ctgggctttg aaggagcccc
tggtcccacg aacagagtct aatctcaggg 2188 ccttaacctg ttcaggagaa
gtagaggaaa tgccaaatac tcttcttgct ctcacctcac 2248 tcctcccctt
tctctgattc atttgttttt ggaaaaaaaa aaaaaaagaa ttacaacaca 2308
ttgttgtttt taacatttat aaaggcaggc ccgaattc 2346 242 20 DNA
Artificial Sequence unsure (1)..(20) Antisense Oligonucleotide 242
nnnnnnnnnn nnnnnnnnnn 20 243 75899 DNA Homo sapiens 243 gatcttcctg
cctcagcctc cccagcagct gggccccacc acaccggcta attttttaac 60
ttttagtagt gacgaggtct gattctgtta cccaggctgg tctggaactc ctggcctcaa
120 gacatccgcc tgcctctgcc tcccaaagtg ctgggattac agatgtaagc
caccgcgcct 180 gggctcctat gatttttatt taacataatg caccatggaa
tttgtgctct gcttagttca 240 gtctgagcag gagttccttg atacttcggg
aaacactgaa aatcattcca tccccatcca 300 ttcattcctg cagcacccaa
gtggaaattc tgcgtttcag acagggacac tacccttaga 360 gagcagtggg
cttccccagc agcgtagtga aacatgatac tcctgagttt catgaaaaaa 420
gggcagacat ctggccagag ctgggaggca ggaaatagag cacggtgccc tcctcccata
480 ctccagcttg gattactgag gctggggccc aggccctgca ggaaaggagg
tgcatgacta 540 ctttaaggcc actcactctg tgactcaacg ggccgggtcg
gggctggaac tcaatgccct 600 cccgggcctg gagagcccac gcgccgtggg
cggggctccc ggggtcgcct aggcaacagg 660 cgcgcgccgc gcccgagccc
agagccccaa agcggaggag ggaacgcgcg ctattagata 720 tctcgcggtg
ctggggccac ttcccctagc accgcccccg gctcctcccc gcggaagtgc 780
ttgtcgaaat tctcgatcgc tgattggtcc ttctgcttca ggggcggagc ccctggcagg
840 cgtgatgcgt agttccggct gccggttgac atgaagaagc agcagcggct
agggcggcgg 900 tagctgcagg ggtcggggat tgcagcgggc ctcggggcta
agagcgcgac gcggcctaga 960 gcggcagacg gcgcagtggg ccgagaagga
ggcgcagcag ccgccctggc ccgtcatgga 1020 gatggaaaag gagttcgagc
agatcgacaa gtccgggagc tgggcggcca tttaccaggt 1080 gcgggagcgc
cccggagcgt ggcgggccct tcgcttaggc cgcttgaaca tcccctcaga 1140
cctccaggcc ccagactccc tctgggtctt gccctctgcc tcgctcctac tgcttgagga
1200 ttcgatggga cagcgacgca ctgcgtcccc ccaccctttg tccccggggc
gggcgtgttt 1260 ctcgccgcag cgtcggagcc cccttcgatc ccccacctcc
cttctgttct ccagctcggg 1320 tgatctctca agccggggga ccgccggtct
gtgctctcaa cgcgaatccc tcgcaccccg 1380 accccgcccc ctgcctgtcc
actctttgtc ccctggggtg atttagcacc cccactattt 1440 ccttttctgg
agtggaccac ctcagactct cttcctttgt ctccctgggg gaaaaggtta 1500
ctccccccgt ccctccttca catttccttt cccctagtct cagtgtgcgt cgagtcccag
1560 agatgacagt cccctttccc ctttctgttc attcatttat tggataggag
ttggcaagct 1620 tattctgtgc taggcaccgc ttaggcattg gaggtggtgt
ttgctaatca ggacaggcaa 1680 gatcctagcc ttagtggggc ctagagtcga
atagggcaat caaacacaaa agcaaataat 1740 ttcagatagt gacaggtgct
gtgaagagaa cgacttccta acggggtaca gggtgactgc 1800 atagaaggcc
ggctgtctta gagaagggga tcagggaagg cctgtcaaag gaggagacat 1860
ttgctttgtg agctgaacca agaggagcag aaagccgtga gaatatgggg ctaaagaacc
1920 ttctagccag gaggcctgcg gtacccactc cattggggcc atgatattat
tctttcaggc 1980 agggactcag gaaggttaac gttttaaccc tctctaaaat
agcatctttc ctcaatgagc 2040 agcttagtct ttggtcgtgg cagagatgac
cttgtcttag gagtcatctc cttgtgtgtt 2100 aaaaagttag gaaaggaggg
tttctcatat atctataaaa caagtagtta aaaacacaaa 2160 gagctcttcc
tttcacaagc agctgaataa gatacatact cccaattaaa tgtcattgcg 2220
ggggttgtta agattaacta aaaccacact tgcacagtat cttaaataag cgatatacag
2280 aatagagaga ttttgttact tgtgtaaagg gagacagcag atgattctgt
tttcagctta 2340 taggctcaaa aggcaaattg tgagatccat cagctgtagt
attaaaatct attttgagct 2400 ccgcttagaa aggaaaaaag gtttaagcag
ttctttggta tgcttgacta acaaaagcct 2460 ttttttttgg cagccttgat
tttcatgtgg atttacatca agcttatttg acaggattct 2520 ttttatttgg
actgtagtgt gtatattagt ttctgctaga ctaatatttc taaccactgt 2580
aatctatata ctaataagta tgattgatca gtatataaaa tttgtatgcc atatctggtc
2640 tctgaattag ctgaatgaat tccataaggg actttgagac tgtgtagaca
aattttctgc 2700 atcagtttaa tgcagtagag tctaaaatgt ctttaaatga
aaattgttgg tctgaagtgt 2760 tggagttgat tatgatacac cccatcacag
tggaagcatt gtggagagaa gtcttttcca 2820 ctgaaattga ctgagttgac
aacaagaaat acgtattgta acttagttct tagttgaatt 2880 ttatttctta
caattttaag ccagagtggg ttgacctgtc acccaagcat ggttaaaatt 2940
gtattcagca tgcaactagc atggagtgtg tcagtcttca attcatttcc ttcattgttc
3000 ttaagttttt ctgccacaat taaaccccac aagttagtca aggtgttgag
attttcactg 3060 cttcttaatg gattgccaca ttccctgagg tagtttcttt
tggtcttaga gaattgtcag 3120 ggccagcttt tctcacctcc actgtatgga
tatttttctt ttctaagatc ttgaaatcag 3180 aagcttttct cctaagtgta
aaagtagctc tttgtcatac aactgtagcg ttttctgaaa 3240 cagagttcag
atgaccttga gtctaaagtg gctaactttc caaggtgtgt atcgctttac 3300
caaaaccatt atttttcaag gattcaaaga atgtgtttac aattgataga aaatggaagt
3360 ttaaaaaaat taatacttta tagcatgttg aaatgagggc agccttatac
aaagtcatac 3420 tttgagcttg cctagcctat tgtgatcaga gaataatgta
atttttgctt acaacttggt 3480 aagcaggtca gttattctaa cttattttct
gattagaaca aaaagatgta aaaacttgaa 3540 aactattggg aaaagaacaa
agagtgaaga ggacttttga gtgctgagga atgtggcagc 3600 ttggaaaaca
aactttttag gcagagattc tttgctaggt cagtttgata aagtgagcat 3660
aaccgtattt ttaatcttta atgctaatga atagcataga tgctaataag catctaggtc
3720 tataaaaagt cagctttgat agtgtatata gatggcttta aacattgttt
tctagcattt 3780 aaacactttc aaatcatccg gttgcttgat tgggcctagc
tgtctaagag gagagaatga 3840 gcccagatga ggaaaagaga ttgattttac
tgagctagaa tgagaggaga gagggttgag 3900 tgaatgaaaa gaatagctca
tgtgctcccc tccatctgta gtttaagagg ggttgggtcc 3960 ggtgttttgc
ttgttttctc gtctgtaaat tctttgattc tctgacacca ctcactatat 4020
ttcattgtga atgatttgat tgtttcagat aaaggggact gcaataatac cttgtgacat
4080 gaaggcaaga tttattcatg ttagaggcag gctttgtaaa atgggccact
cttccaattg 4140 acatttgttt ttatagctgt tttcattatg aaatacaatc
taatgcctga ctaggttaaa 4200 accatgttgt aacaatagtt cactaaaatt
ccttactgat atacagctta tgttgttata 4260 ttccaaaaag atgaatatta
aaatttgcca ataatgttta tttaaatact attttcttca 4320 gaggaaaaaa
aactatttta tgcaaaggag aaagatctat acactatgac tcacttcact 4380
taaaaaaaaa aagactaacg gaaatgacat ggagagactg ggaagttcta gtcatcttga
4440 gtgacccatt agatctaaat gttcttgttt agccctggtt tgagtgaact
aaatttaggt 4500 gtctgatcag tactttggaa atggtgtaaa tgcctttgta
attgtctgga ctgatattag 4560 attaactggg agcacaagta gaaatagtga
aggaaagaac tttttgctat tgttatttga 4620 catcactggc atatttatag
gaatactttg gtgtttttgg aagtaagtaa accaaccagt 4680 ggttctaaaa
agtcagctgg gggataatgg taatgccgct gtttcttagc tgcaagttat 4740
ctgccgttac ttctcctcca ttttgcattt tatcttgaat agctcctcaa aacctattaa
4800 aatacctggt attgaataat gtaattgaat gtgtactgaa tttcacagtg
gaaatgaata 4860 agaaatttcc tgtggaggtt ttttgactta gctactgaaa
taacggcctt ttgttgtgtg 4920 attctttccc ttttctcttt gttaaagaaa
actgtcttgt gatcttgtag attacagaat 4980 ccttttggca atttctgttc
ctagcactgc tttttctttc tttctttctt ttaaatagaa 5040 atggggtttt
gctgtgttgc ccaggttggt cttgaactcc tggcttcaag cgatcctccc 5100
accttggcct cctgaagttg ggattgcagg cgtgagcagg tactttttct gaggcctgcc
5160 tgagcctata tatattttgc acaatttggc attcctccct acagtgttta
tgctgatttg 5220 tttctggtaa caactaatac tggcaaatcg gctgggcatg
ttactttatg ctgcccatat 5280 tcaggaaaat tggaattcta gctgggtcat
tgttcccaga tgatgtagtt tggcaccagc 5340 cattccatgt tcacattttg
agtatccagg agggctgggg actttggagt agttggtgat 5400 tccctctgcc
acatttcact ggttggtcac tatggcatcc tttccaccac actagtagtc 5460
taggttctca gatgttgctt atgagcctgc aatggtttct agtttcacac tgcagaaatg
5520 agtgaagccg gttacccgtt aatatggtcc catcatcact agagtaattc
attgttctaa 5580 aaccagatct gagtctctca ctcctctgca actacttctg
attctttcat aacacttgta 5640 aagtccaaac tcctctttag catggcagcc
agcttccagt ccttccctcc tatgtggctt 5700 ccattctagc cagacaagaa
agggcagcgt tctccaaact catcctcgcc cttcattcct 5760 ctataccatt
gctgagcact ttgttgagga tgcctctccc gttcaatcta gcttgcatct 5820
tccagctcga atgtgtgctt ccttgcacca gagttttgtt ccgtcacctg tgtgttttca
5880 tacaagctgg cacatatctc ttctaaagcc ctgctgtcat tgtagctgcg
tctttacaaa 5940 catttttttt ttaaattttt ataaagtcaa ggtctcacta
tattgcccag gctggtctca 6000 aactcctggg ctcaagtgat cctcctgcct
tggcctccca gagtgctggg attataggta 6060 tgagacactg tgcccagctg
tagctgctac tttatatccc aggtctatct ccaatggagc 6120 ccaagcttcc
tgaggccacc tgttgtatct ttctcattca tcttgaagtc ctctgctcct 6180
ggcacagagt aggtacctaa caagagttgg gattgaattg atggtcagta ctttgctagc
6240 ctgatggtat aaagatgtac aaaacatgtt cctggctccc actctagggg
ggcaatgatg 6300 gaaacaaata gattagccca cattagtacc aatagtagag
gtcactctgg gagaaggccc 6360 ccaccacatt ttgagtcatg gcctaatgag
gtaatttagt attgcctgct gcagtggctt 6420 tggaagaaag gctggcattc
ttagccagta gaagctgata ccactgattt gtttcacaga 6480 agctttaaat
ataacaataa atttgtgctt ggcctacggt gaactttaca ggcaacttgg 6540
aggtaatatg tttgtctctc taagaattgt tgaattcctc ttccctcatc cctcctgact
6600 ggttctcaca agcctagcgg gcctttgcat gtggttggtt cataaaatac
tttttgattt 6660 tgggatataa aatatagttc tccataaaat aacgactgtt
accaagtctt tgattttttt 6720 tttcaaacta taaatggtaa tgacattctt
tggcctttga tcagaccacc cttaggggca 6780 agagagtagt ttcatgtttt
gctttttcta gtgtcccctg tgtctgggta tagttgcagt 6840 ctcagctgtc
atactaacag tgctgagtga gtcccttact ttctttgggt tttggtttct 6900
cccttgtaaa aatgatcctg gactaactga tcattaagtt caggtcaagt aataaaaatc
6960 cttaatgtac tcacaaatac aatttaatgt tcctgaataa tccttgtaaa
aactgcagca 7020 gttactcagt tttgtaaggt gtggttgggt actattaggc
tcaaaagttt ataggagctt 7080 tgtgagtata gttaacaact caaaagaatg
gggtgttttt tcccgagggg catgaaatgt 7140 ttttgataaa tagagttcat
ttgacttggt aatgtggaaa atgagtagcc ctgacacgta 7200 cgctatgctt
ttgcagtttt tctctcaagt agcaattggg tggcttttcc tgtaaaagat 7260
agaggaactg attcttgaga atttacgaaa gcttcaaccc taactaggta tgcaaagaat
7320 agttgccctt tatgttgtaa ttttaggaag aaacctacat ctggtctaag
tttcatttga 7380 ataatatgat agtttacaca tctgccatat ttgagaagaa
agtacctaag tctccagcat 7440 tttagaaata atgctttact ttgtgtagaa
atggtcttta gagtttaata gctgctgccc 7500 tctccttttt caaagcagct
tgacataatc atgagtatct tgctgacagc ttgtaaattt 7560 tgattgtatg
aaaactgaaa ataagaccat ttcacatgga agattccctc ctgccctgaa 7620
acagccaaag aaaactgtag ccatcaaatc tattgatctc tgggctttgg tacaagtcac
7680 actactacaa ataaaataat accaagtact tataaatgat tttcagtcct
tttaaagttt 7740 atttttttaa tatttttttt gagatggggt cttgctgtgt
cgtccaggct ggagtgcagt 7800 ggcacaatct tggctcactg caacctccac
ctcctgggct caagtgatcc tcccacctca 7860 ggctcccaag tagctgagac
tacaggcatg tgccatcacg cccagctaat ttttgtattt 7920 ttttggagta
gagatgggat tttgctgtgt tgcccaggct ggtcttgaac tcctgggctt 7980
aagccatctg tctgcctcag gctcccaaag tgttgggatt acaggtgtga gccactgtgc
8040 ccggcccagc ccttttttta agagaaaaac gtatgacatc gttcgattta
ctgagtgctt 8100 atggttttac taaggcagta aggttttatg gataccctat
ggtaattaga tagaattagt 8160 gctctgaagt cagctctgta atatggactc
agagtaaaca tggcaaaggg acacttaagg 8220 tctgcatttt ctctgggaaa
taaacgtatt ctttactact ctgaatctag tgctgggaaa 8280 ttctaaatcc
ttcttgagga ttaaccactt gaagtaaagt tttgggtccc aagtaggctt 8340
gtgtccctgt ctccttctct ttacttttca gatgtttctt cctagagact gaggtatatt
8400 ttacttttac agatgaagaa ggaagcctcg gctgtgtttg tggcttttgt
gggtgagcaa 8460 catcacttgc aaagataaga tgagcatagc aaaactaggc
tttcaaaata atttttaaaa 8520 atttcttagt gattagaaaa ggaaaactct
tcccttgtct ctgttaagaa acgtttttcg 8580 acttttttcc tttcttaatg
gatcttttat tggcacttct cttccttttg cagaatctta 8640 cttaaaagtc
actacgttac attacagcaa acagcttagc taatttttat ccagatgggc 8700
cccggttaca ggattgtaca ctattgcgaa tttcttacag gaaagtgaac atcaagtaat
8760 tattccaaat agagttctct taagaacgtg agttacttaa aaatgtctaa
ggatgaagtc 8820 acttctgaat ataacttcac tcaagagaac aaataagcaa
actgcattta gcataacatg 8880 gtaaattagc tttaactctc cttgatgttt
gaacatttgt cgctgttaac tactgtttca 8940 cttttcaaat agtcagggct
tagtttgctt ctgtaaggat aaagggaaaa tacgccttca 9000 ctgagtcata
aatatttttg tggctaactt ttgcacagag aaaagaggcc tctaagaagg 9060
tacccagtga attttttttt cggggcaggg agagaatatg tcattttttg gtttgttgtt
9120 gttgttgtca ttgttttgct ttgttgtttt tactctgaac tgaactgtat
cttgacagca 9180 cttttgaatt aagagcatta ctcttattgt tctctactac
ctggacgcca cctccctgtt 9240 gccatagtgt taaggatcat gctccgaggt
ggggtgaggc agaatggggc caagatcaga 9300 aagttacatt aagctacatc
aggtttatac aagcataaaa ccaaattttt ggagcagtcc 9360 ccagaataca
acctggttta gccacaccta aaggttgctc ttgaatattc cttgagaatc 9420
cacatcccta gaatgctggg tttcaatggg ccctttatgt acctatcatg gtgtcatttc
9480 tgagcatttc taaatattcc ttcatgtctt actgacagtt tttcttgaat
aaatcttagg 9540 aatattagtg ccattatcag tattttgttt ggtctgttca
caccacaaat aactacccag 9600 gtctgctact tgcccctatt tctctacctg
ctaatgaaaa tgcttttgaa agtttgagta 9660 acagtattgg agtgtgcaca
gtggtattgg taggttctgt actcatcctt aaccacttgt 9720 tttcatcctt
tgtgagcttg aagtttctcc aaaaaattta tcacaaaact tatcagacat 9780
agttaataca ctcagagaga gaatcactga aaaagtagat gtagtttaac aaacccagtg
9840 cctttttttt acccatgaat acatatttgt caactaaacc tcattttgca
acttgttcca 9900 ctactcgaat ggtaacaaac ttttggtttc ccaatagatt
tggaagatgt tgcttttgaa 9960 agtaggaaat agatggcttt agaagatgga
agaatatttt gtttgaagtg ggagcgtggt 10020 atgtccttag ctgtctgtga
aatgcagctg aagatgggtg tgggccttca tctgcatttc 10080 ccatcttcag
tttgaggagg tagttaccct tctaaccact taagaactgc atggtacatg 10140
ctgttttatt tacagggcaa aactgtgctc ccgtagtttc cctggtgctt gccttcacgt
10200 taacacagtg tcatcgtttg gcagtgttta tgtgccaggg tccatgttag
aaggaggaaa 10260 ggtatagcga agttaaaggg tgcagttggc ctcccacctt
tagttttgta agtgccttta 10320 aagtttgatt tttgtaggtt gatcataagg
aagtgataag tatgttaggt tatttgtggt 10380 ttgagctaat tttagtctct
ttttacagct tgctttgtat cctttgccat taaaacatgc 10440 tttctagaaa
gacaactttt gaatgtagga cacagtctat attctatact tggctacatt 10500
tcaaaaaata ttttctcagt actttggaag ttggacagtt ggaagcatag tgacagtatt
10560 taaaaatctt tgattccggc cgggcatggt ggctcacgcc tgtaatccca
gcactttggg 10620 aggccgaggt gggtggatca cttgaggtcc ggagttcagg
accagcctga ccaacatggt 10680 gaaaccctgt ctctactaaa aatacaaaat
tagccgagcg tggtggtaca tgcctgtaat 10740 cccagctact caggaggctg
aggcaggaga atcgcttgaa tctgggaggc ggaggttgca 10800 ttgagccgag
atcataccat tgcactacag cctgggggac aagagtgaaa ctctgtctca 10860
aaaaaaaaaa aaaaattaag tgatttcttt gctttgtgac acttctactt ttccagcaag
10920 taaattatat tctttcatac aggtatgaaa ttcttgttcc aagctagtgg
ttaaaaaggc 10980 acagttgata ttagaggatt tgtaaaagat tatgaccacg
cctgcaatgt actgaagcaa 11040 ggctttgctg ggctgtgtat aggaaacctt
ccccagcctg tgcccttgct tgatagaaca 11100 ttttgctcct aagggtaggt
gcctgtatct gtctccagta ctggttagtt tcacacagaa 11160 cagttgtgtt
tcagagcttt agtctcaagc tgccctgctc ccctgaagca gccaccctga 11220
gcatgtgcac tcacaggagg ggacatgtga ggtcatggaa gaagacgact caggaagaag
11280 aagacttggg tttgggttct gactctgcct ttgactgttg tgggattttg
aggagttgca 11340 tacaggatct gtaaaatgta gtcattagac tagactagac
agccatatag cattacctag 11400 atgtaacttt ctacaaagac atggtcacag
gagaagacca gagggtgggg tgatctttct 11460 ggaaaaattg gggcttcatg
ccttactcat gctagatatg gtagcattat atggctgtgc 11520 ctgatccccc
taatctaaaa gtgggacaga actttaaaat ttcatattaa ctcaaattaa 11580
aacttgaaaa aaacccatta tttccttaaa aataataaaa tgccctgtgg gggcataagt
11640 cacattatat tttaaaattc ctgaatgcca catggatgaa tgtagttcct
tttgaaattc 11700 ttcttttgtc taaagaggaa tgttggattt tgtaattgga
ctaaaaaatc ttccatttga 11760 gagagaaaca gtctgctgca tgttctaccc
ttgttcagga taaaacccac taatagctaa 11820 catttattga attctgtgtt
gtgcctcagg cactgtgcaa agtcctttac atgcaatgct 11880 gtttattata
tactgtcaat tggtctataa cagcaggaaa tgtttcagga ggacaatgag 11940
gtcccagacc ctcagtcttc tcctgtgtcc tggattcagc ttcacaatag cactatggca
12000 gtgtggccac tgcttcagct tccacataca tggctgtgaa gagagacagg
ggattgtgct 12060 aagcctcccc gatttattag gacataggag gagagagttt
gtagtttttg acctttgcct 12120 agttttctaa cctctttcct agatgtcaca
aattggccac ccacagtcat attttgcttg 12180 cttcacgcaa tgctttttaa
aaaagagaag agtttaattt gtgccattgt ttataaatga 12240 atcaggagaa
atgacatgca actctggatt ctggcctctc ttgaaaaatc tgaaaatcac 12300
accgtctgag cttacactgg cagtggtctg ctggactgag ggacacaact ccttttggat
12360 gtacatgtgt gcgttgcaga gtttaccaca gtcccacagt gggtcacact
gtccttgtcg 12420 gtgtacacta cctagcactt gagtttgcaa cccctacccc
aagctgagtt ttctcgtcaa 12480 gcttgatgtt aatgttatgt gatgcttggc
cttgtaggta tttggtatat tatcgttaga 12540 taaaattgaa gcaaagggct
aaagggttgg tggcctgagg gagtgccctt gacagtaaag 12600 tctaggataa
aatcattggc caggtactcc ttcccttccc gcccttcctc ttttctcttt 12660
atcctcagcc tccttctgct attttgagga agttagaagc caccaccatt ttttcccacc
12720 tcaggcaact gagtgtggct gtatttctgt cccatgttca gttatttcca
ggaactattt
12780 ttgatgacca acttgaagtt acattgggtg ggcctaatgg gggctgataa
aagaatgagg 12840 tgaccaaata tgcttgcact gagacggcta cgaagtaagg
tttttaatga cttgctttgt 12900 gacttggtca ggagtgatac catttgtcat
gtgtccaact tcatgactaa atggttgctc 12960 taccttatcc tcatagctat
aataaaataa aataaataca tacattgcag ggaggaatgt 13020 atcttgttaa
aggtctctcc cttttagcaa caaaagtaca tattatgttg tagaacatgc 13080
tttttctttg atccttcttg aacacctatt actctataga ggtatgttgt gtatggcaaa
13140 ttagaacaag caatagataa ggatgattct ttaccattat aacccagtca
aggtctttgt 13200 cctaagtttt gtacctttct ccagagggaa aggtatttgt
atttatttat ttatttttga 13260 ggcagagttt tgctcttgtt gcccaggctg
gggtgcaatg gcacgatctc agctcactgt 13320 aacatccgcc tcccgagttc
aagtgattct cctgcctcag cctcccgagt agctgggatt 13380 acaggtgcct
gccacgatgc ccggctaatt tttttttttt tttttgtatt tttagtagag 13440
atggggtttc atcatgttgg ccaggctggt cttgaactcc tgacctcagg tgatccatcc
13500 acctcggcct cccaaagtgt tgggattaca ggcatcagcc actgcctccg
gccaggtatt 13560 tgtattttta gtctctatgc cttaccgtct cagatcagga
ggatttggtg atttatcgaa 13620 tgtgggggaa ggggaagaag aggaaacggg
aggaatgttc cagattaggg aaatagctag 13680 atggaagatg cagcccctca
tcaaggtggg gacacaggaa aaggaacgtg tgcaaagaag 13740 atggtgatct
ggttgtgacc atgttgttag aggacgtcca gggaagcatc tggtaggtgg 13800
tggggtgttt aaatatagaa cattcggaga atgctccgaa gcttcagaga acccttccca
13860 aaaggacaaa accagctcag tgttttagca ctccgggatc atatggcatg
acagcatggc 13920 tgctttatac ttttttgtgt atgtgaaatt aaaaccaacc
actcaggacc aatttctctg 13980 aagctttttg tcaatctttc atttgctttt
ctcgtctaga ttgtaagctc cttgcagcca 14040 gtgtctgttg attcagtcat
tcaaaaaata atacatgaac agctactagg taccaggctc 14100 tgtgctgggc
agttgggata tgtggtgagg aagacaaact tggtccctgc ccttaggaag 14160
ttcagtagtc cagcagacaa agtggctgaa taaagataat ctcagttcac agtgataaga
14220 gctcttacag gcctaggctc caggtgctgt ggggatgctc aggaaaaggt
atctaattgg 14280 gattgggagc aggcaaaaca aataaaggat agtgtataaa
ggtaatatct agttgaagtt 14340 ctgaagggca aggaggagtg agcctgtata
ttctctgagt ctctccctaa tctgggattg 14400 acttcttgtc cgtctctgtt
catattaagt gtcacctagg cttgaaaggg tgagatcata 14460 tttcacttcc
ttcctctttg gtcttaacct ttctctgcta ccccctcaca caatgcatat 14520
gcattattct cttattgtat atatttttcc tctcttcctt ttcatgtttc ctctgccatt
14580 acttttaacc tcgactgcca tatggcctct aaacgcttcc agaagggtag
cctagtggag 14640 gttattccat catggccttg agctcatgcg accagatagt
gaaggcatct gtgtaggtgt 14700 cttctccagg agggtgatat ttgtttcatt
gtaaattttg tagccctaga acaccaacaa 14760 cagtgcacag taattagtag
gcaggcagta caggattcat tgaagtgaag tgataacttt 14820 tatccaagta
tgtatgcaga taatctttga tttgtacaaa aaaaattata ttttaatatg 14880
taaagatttt ttaaaagaat cttcaagttt tagccttccc actaggaata tattgaaaac
14940 atgtgcctag ttcactgact tgcagctgcc actatgagaa taaaggtctc
atttagttgt 15000 tgtgaatttt aagggatatt ttcaatgatg ttggctggtt
tatcccatta tgtggtcttt 15060 tttttttttt tttttttttt ttgaggtgga
gtctcgctct gtcacccagg ctggagtgca 15120 gtggcgcaat ctcgactcac
tgcaacctcc gcctcccggg ttcaagcgat tctgctgtct 15180 cagcctccta
agtagctggg attacaggcg cctgccacta cgcccagcta atttttggta 15240
tttttggtag agaagggttt caccatgttg gtcaggctgg tctcgaactc ctgacctcat
15300 gatccactca cttcagcctc ccaaagtgct gggattacag gcgtgagcca
ccatgcccag 15360 cctatgtgct cttattagca attctcagta cacagatagc
tttgagtgat tctttcaagt 15420 caagtacctt attaaaaaac tcaagtgtac
tgataattat cttactttta aatggctaag 15480 tgataagact gaatttttag
gtactgtaac acttcagatt acagattctg atatttttat 15540 ggttatttat
atttatttat ttttgagatg gagttttgct cttgctgcct aggctggagt 15600
gcaatggcac gatctcggct cactgcaacc tccgcctccc aggttcaagc gattctcctg
15660 cctcagcctc ctgagtagct gggattacag tcacccgcca ctacagccgg
ctaatttttg 15720 ttatttttaa tagagacaat gtttcaccat gttggccagg
gtggtctcgc acttctgacc 15780 tctggcgatc cgcccgcctc ggcctcccaa
agtgctggga ttacaggcgt gagccaccgc 15840 acctggcctg gttacttaaa
tttaaataca aaaattatgt tgattaattc tgaatgattt 15900 cctgattgct
ccccgtttac cattcacaca tttattaaat tcttcgcttg ccatatagaa 15960
gcagtctctc tgccatatat gccatataga taacagaact agctgtctgc aaaccactga
16020 aattgtgaaa acatctcccc ttttttcctg tttctaattc tagctatgag
gattatatac 16080 agaagtagtc ctggatttga tttttttttt tttttgatga
ttgttttttg atagttgttg 16140 actacaaatc atttaaacgt ctgaaagggg
aaaggttttc cttaaaaatg gatgacaaag 16200 gagaataaaa aggtattttg
actatttttt tgaatgatga gttttttttt tctctttctt 16260 gttttctttt
ggagtcattt atgtgtcact gagtggatac catggaacat gtggcagaag 16320
tagatatatg gggtaaaaga accatagttc ataagctcct tgacagaatc actgaagtgt
16380 agccgttata tggccactgt cgcaggggga ggcagcagtt ttgaagaagg
ggatgagtaa 16440 taatgagtga taaaaaggca tcctggatag aagaccaaac
tctgcagaag accccagttt 16500 gattatgctt ttgttttctg atttgcggag
gagagtgaaa atgcctgagg ggtgcggggg 16560 agcacatagg gtgtatgtgt
gtgtgtgtgc gcgtgcagat tctctctttc actgtatgta 16620 tttgtatgca
tgtatgtatc ttaggactta agctttctag tcaataaatt gccatagtgg 16680
ggaattgctt aattgcttgc cttctgttgt tgtatttaat ttaattttat ttttaatgat
16740 ttttttggtg gggtacaggg tcttaactat gttgtccagg ctggtcttga
actcctaaac 16800 tcaagtgatc ctcccgcctc gggctcccaa aatgctggga
ttacaggtgt gagccaccat 16860 gcccagctta gttgtatttt aaatgggcct
gtttgcagca ttccctactc cccttagttt 16920 acctggctca caacctgtct
ttccatatca aggcttctgt cacccctggc ccatgtcagt 16980 gcatttgggc
agcccaccca gcatcatcac ctcatgtccc agggaacttc ctgttcctct 17040
cttccagcta tttccttccc tggcagttga gatagtctct acctttgacc tactgttaag
17100 ctcagacctt ctgctctcta gttacagcct ctgtgctgcc agattccctc
gctcagttgc 17160 tttctctagt ttgggttttc tcctttattc agatttccag
ctgtttctct cctcccccca 17220 ccgcagcctc ctcacttccc tccttatgca
tctgagactg tggtcagtca ctttagatgc 17280 tgcctctcca ctgtacttgt
gtccatcttc ttacctacca cctctagccc tggagcaggc 17340 tcttcccctg
tctttgtctt cctgggccca ggctcctaag cgctgctgga aaaaaaatcc 17400
cccagtattg agcccctaga aatccagtct ttaatcccaa atctgtctcc cccagcatct
17460 ggccatcaga tctaaagctt acctgccatc ctttccacct catttctctc
acaggggaaa 17520 aggagccttt gctcctagag tctgcgctcc tgaccccttc
ccatctcacc tgttcaaggc 17580 atcttgcaat aaggggttgg tgactctcga
ggaatggatc ccaggccctc cctattatca 17640 tcttatgtat gccagttcaa
cgttctcagc ttcctccagc cgagacggcc cctccagcca 17700 ctgctttata
ctctccttct ctggttgaaa tttttgaagt aaataggtca ctctgcccat 17760
cgttcatctt ccagtcactc tgtgtgttta tcttccaggg aagtgaggct ctatgctacc
17820 aagccactga aataattttt ttttttttcc agactgagtc ttgctctgtc
acccaggctg 17880 gagtgcagtg ccgcagtctt ggctcactgc aacctctgcc
tcccggcttc aggcgattct 17940 cctgccccag cctcctgagt agctgggatt
acaggtgcct gtcatcacgc ctggctaatt 18000 ttttgtattt ttggtagaga
tggggcttca ccatgttggc caggcttgtt ggcatgttga 18060 ccatgttggc
caggctagcc tcaagtgatc cacccgtcag cctcccaaag tgctgagatt 18120
acaggtgtga gccaccgcac ctggcctgaa ataattcttg acaagatctg cttccttgtt
18180 actaatacag tggatatttt gcatcctaat tttaatgcag ttcagtgtgg
tagacctgta 18240 tttgcatatt gaatattccc ttccctgttt taataactct
attttttcct tttcttttat 18300 atctcctgct tctctagcta gtcctagacc
ttactcatcg gtgtcttctc tgtttgttcc 18360 tcaacttgag gagttcctac
agggtttacc caatctgctg ctttcattta gcccttttgt 18420 tctttttgag
ccatctcatt cactcaccca ggatgtagca tcggcccttg aattcagtgt 18480
gcacacatac actgtgcact atgggacagc cttcagaggc actttgttcc tgaaattgtg
18540 gtggtctttg cctctcatgg agccttgcat atgctgtttc ctctgcctgg
aatatcctac 18600 cttttactta actgattctc gttcttcttt ccagtcacat
tttgtacatt tcttctggga 18660 agctttctct gatttcccct ttccacaggt
ccaagttaac tgccttgtct aggtcctccc 18720 atggccctct gaaggcctcc
tttcatagca ccatgtctga gtatactgta ataacacgca 18780 ttgctctgta
atagcctgtt tacttaccta ttgccaagta atctatcaag tcttataaag 18840
ggcggggctg cttttgttct agtcatttgt atctcttagt acccaatata gtgtttggca
18900 tatagaaaat acccaacaag gccagtcgca gtggctcata cctgtaatcc
gagcactttg 18960 gtaggctgag gtgggcggat cacttgaggt caggagtttg
agaccagcct ggccaacatg 19020 gtgaaaccct gtctctacta aaaatacaaa
aattagccag gcgtggtggc gggtgcctgt 19080 agtcccagct acttgggagg
ctgaggcagg agaatcactt gaactgggga ggtggaggtt 19140 gcagtgagct
gagatcactc cactgcactc cagcctgggt gacagagtga gactccatct 19200
taaaaaaaaa aaagactcca tcttaaaaaa aaaaaaaaag aaaaaagaaa gaaaataccc
19260 aataagtagt tcctgaatga atagatgaga atgctgttta gaaggttcat
gaattggaaa 19320 ccgtgattgc tagggaggct ttgagttgat ggtattgtgt
tgaaccatgt gttacccagg 19380 atcaatttag attttacact ttgttttctc
tgttcctttt tatagtaatt ttctgtatgt 19440 ggtgttttcc ccccatgaga
ttgtatacca tttctcagcg agaactgtgt gtaatgcttg 19500 gtggctccct
catggtgcct tgcatggaat tggacttcgt ttcagtggat ctgatcccag 19560
ttatgttaat gctcgatgga gctaagtctt atctcgaagc agtccatgtc ttcatcagct
19620 ggccctgcct ccatgccctg cacagaccat gccactctgg agaggtagtt
tccctgtggc 19680 ttattagtct tatgttccag tgtgctggcc aagtatgaga
gacatcagtg gtatgagaga 19740 gtctctctca ttcaaacttc gtaggttttg
tagctgggac tgaccagtgc tgacaggaaa 19800 tagaggcatt tattaaaagc
cagagatttt tcaagttgca ggaagcaaag ctcttgttag 19860 ctatgatttt
gtggtgggtt tggtagtcca atataaaagt aaaaactgga tgacaatggg 19920
aggagcatgc ttgggtctcc aaagttagat catttttcct aagtaatttg tctttaaact
19980 tttactggtt tggaatttcc tgagattttg atcttgccag aaagtttata
gcaaaagttc 20040 tgagcagatg acacttttgc gtctgaaacc aaatcattgt
ttttgttttt aacttttttc 20100 ttaatatatt atccttagtt cagccctgaa
gattattctg ttatttgtgg atctcaactt 20160 tccccccatc tcctggatct
ttgtgaaatg aatggtatta attgaataga gaaggaagat 20220 ataaacataa
acttagtcaa aaacttgttc ttgactaggc aagttgggct ttatagcttt 20280
gagctgatga catgtctatt cttgtgaaaa agggattttt agtgttggtt tggcttcttg
20340 ttatatttga tttattatta ttatcattat cattattttt gagacagagt
cttgctctgt 20400 cgcccaggct ggagtgcagt ggctcaatct cggctcagtg
caacctccgc ctcccaggtt 20460 caagcgattc tcgtgcctca gcctctggag
tagctgggat tacaggcggg tgccactaca 20520 cctggctaat atttgtattt
ttagtagaga caggtttcac catgttggct aggctggtct 20580 tgaactcctg
acctcaggtg atccacctgc cttggcctcc caaagtgctg ggattacagg 20640
ccttagccac tgtgcctggc tgattttttt tttttttttt tttttaggtt tgttttaact
20700 ggaactttac gtgaatgtaa ttgaatttag aataaaagca cttaatttca
cagtgtgcag 20760 tgaactttct gttacttatt ttaacagtaa aaccccttgc
agtaaatgac ttggagcaaa 20820 gattgctttt ttaaaaaatg ttttaatttg
tttttctttt cttgagatgg agtcttgctc 20880 tgtcaccagg ctggagtatg
gtggcgcgat cttggctcac tgcagcctcc ccgcctccta 20940 ggttcaagcg
aatctcctgc ctcagcctcc tgagtagctg ggactacagg cacatgccac 21000
catgcccagc taatttttgt atttttagta gagacagggt ttcaccatgt tggtcaggat
21060 ggtcttgatc tcttgacccc gtgatccacc ctcctcggcc tcccaaagtg
ctgggattac 21120 aactgctggg attacaagtg ctgggattac aagcgtgagc
caccacgcct ggccaatttt 21180 tttttttttt ttctttttga gacagagttt
cactctgtca cccaggctgg agtgcagtgt 21240 cacagtcaaa actcactggc
agccttaacc tcctgggctc gaatgatcct cctgcctcag 21300 cctcccaagt
aactgagact acaggcatgt accactgtgc ccagctaatt gtttttttat 21360
tttttatttt ttgtagggac agggtctcgc tattttgccc aggctagtct acaactcttg
21420 ggctcaagca gtcctcctgc cttgacctcc caaaatgttg ggattacagg
gacaagccac 21480 tgcacctggc caaggattgt tttttaagtg aactgagacc
cagccttatt agtggtccca 21540 gagcagacct gggacctgaa gggaaccctt
ttcttctggt ccagcgtctt tcctctgatg 21600 ggctactttc ctggagcctt
tgattgcctg tcatcagagt aactgagttt gaacagagta 21660 ggtagttcct
ctccagacca ccacactcac cagctttcat tctgcttctc tcgtttagac 21720
tgtggttctg aatcctcagt tctatttact gagtgttttt aaacataaaa atgcctttta
21780 atgagattga aggccagagg tgggacagtt gaggacaaag tagaaataaa
accttcaagg 21840 cggggttgtt ggtgggagtc tttttttgtt tgtttgtttt
ttgagactga gtctcgctct 21900 gtcacccagg ctggagtgca gtggcacaat
ctcagctcac tgcaacctcc gcctcccgag 21960 ttcaagctat tctcctgcct
cagcctcctt agtagctggg atttcaggct cccgccacca 22020 tgcccagcta
atttttgtat ttttggtaga aacggggttt caccatgttg gccaggctgg 22080
tctcaaactc ctgacctcag gtgatctgtc tgcctcagcc tcccaaagtg ctgggattac
22140 aggcgtgagc cactgtgcct ggcagggagt cttatagaag ctgtcgtgga
caatgtggga 22200 agtagtgagc ctttgtattc cagtatgctg ggctccactg
tgcttgctct ggcccccggt 22260 cgctctctgt gtgttattga gtccccatcc
acggccatac tcttcgtcct gcttctctcc 22320 ttaccatcct ctccccgcta
gtggtaccac ggctaccact agcaattact gacatgtggg 22380 atcttagggc
tacttcccta taaggctgca gggcatgtgg tgttggctac gcgcatggta 22440
accatggtag ccctgtggtt ctccacatgt gcgccttgtg acctgggatt ggctgcagac
22500 tagtaataaa ctgcgtcttc tggtatggaa tctgtctgta gttgtacttt
ctacctctgt 22560 atttaagggg agatctgtaa cctaccaatg ccagttgaag
aggatggatg atagagatgt 22620 taacaaacag ctgaaaaact aactacaatg
gcctgcaaaa tagaacagca ggtttttgtg 22680 gcaaaacttt gtgtccatga
gtttgttttt taaatatcct catataatct gttttaaatc 22740 gagaggcttt
gggtaaaagc catggctagt cttacatgtc atggagtacc tagcttgtga 22800
ggttcacagt ttattattta cagagtgtcc ccttaaatct tctttgggtc ggttcagcga
22860 atgttgctca gatggacttt tttggctgac atagagtcaa aatggtaatc
aagcatgaaa 22920 gtacagacag tccttaacgc acaaatgtgt catgcttgaa
aagttggaaa gttggttctc 22980 tggagctctg attgtattgt cctgtagaat
ccgtgttgtg aatggtggtt aaatcccaaa 23040 tgagtccgta gaacctatat
aatctgcaat atacctgcag tattccaatt aatatgtaat 23100 tcccccatag
aactatgtta atgatttgta tgtatggtat ttaatattat acataataat 23160
gattgtatga ataaaaaaca ttctgggctc catgtggatg atggggtgtg tgtgtgtgtc
23220 tgtctatgtg tgggtgggtg tgtgttcata gatccctttt cctgcaatcc
tggcactgga 23280 attggtttta tcatttccaa ttaagtttca ttcccatgaa
ttttggagta cagactgggt 23340 ccaggtatgc agggcataga ttagagccct
gagaaatagg attaggctgg aattgctggg 23400 ttggagatca gtagcttcca
ggaacacttt ttgggcctgg ctgtcttcat tatccccttt 23460 tgttttctcc
tggggtctgc aggtattgcc ctgttttgtt cctctaatat cacttttttt 23520
ttttttctgc ttttgaccag ggtttttgcc tctggtctac aactgaatat cctatcagac
23580 tctcctgatt ttgaaataaa tatatagttt ttttgaggtg ttctagcgaa
tttctaaatc 23640 taaatgttgt ggcagagtta ttacatacta attttgctat
gagaggttgt agaatcccag 23700 atgactaatc ttgtaaacca tacacgcatt
tccatctaat tctccattgt atatcatgtt 23760 gcagaaaata acagcctcta
gagtttacat tgcctccttt gactatattt cttatttaag 23820 attagttttc
agataagacc ttttcatggc agtacataac tgtacagagg gcttccaact 23880
tgtcttggga gctctcatct ctgggagaca tcacattacc cactgccccc tgccccccgc
23940 ccccagcctg gatgcactca gcctgtaccc catttctgtc ctcagccaaa
cactgctgaa 24000 atgcaagagc tttcaattgc tagccagtga agatgcagac
taagggattt ccatgtagaa 24060 gcccgctctt ttcagctggc tcgtcgagag
ctggaggccc cttgcttgtt cacatgaggc 24120 tttttgtccc tgacttggtg
gctgctgttt cacttctcag cagaaaggga cacccttgcc 24180 cccccccaga
aaggaagatt tgatgtacca cttccgaaag gttcagtcgg gcatcactgt 24240
aaccaagaag ataggtcagg tgaggctgga ggtggaacag ggctgctcgc tagaactcca
24300 gattgttcca caagtgcctt ctggcagaga atgatggaag cttccgtgat
ttttttttct 24360 ccttaatagt tatgagcaca gaagaggagc agattgtctg
gctatagaag ctgtcttatt 24420 ttttattttt gtttttgaga tggagtcttt
ctctcttgcc caggctaaag tgcaatggcg 24480 cgatctcggc tcactgcaac
ctccgcctcc cgagttcaag cgattctcct gcctcagcct 24540 cctgagtagc
tgggaattac aggcatgcgc caccatgcca gactgatttt tgtattagag 24600
acagggtttc accatgttgg tcagtctggt ttcgaactcc tgacctcaag atctgcccac
24660 ctcagcctcc caaagtgttg ggattacagg tgttagccac tgcacccggc
cgaagctgtc 24720 atattaaata gcactttctg cttttagcaa atttaatcca
aatgagactt tagattttct 24780 tgctctgact taccagcagt tccttgaaac
acatttaatt atttttgcca gaaaatcact 24840 caagcactta cgccattttt
ttaccgtgaa aatatgctgc attattttaa aatatattag 24900 aagtcagtaa
ccataagatt ttatatgttt tctaatgtat tctgtaagct ttctgctgct 24960
tttgtttgga aggtgtattt tgtaacgtag aggactgctt tatctgcttg taagcttgat
25020 ttttgttttt actgtaattt ttttttcttt tgctgtattg agaaatacat
tgagtaatta 25080 taaagtcagt ggcatgttta taagttaata tttgtatcta
ttccttagtt actctaactc 25140 aaaacctaaa gtaatcttca actctaattt
actctgacat ccagttgact gccaagtcct 25200 ccaacttaat ccttatcctt
ttttttttaa agagatgcag tcttgctttg tcacccaggc 25260 tggagtgcag
tggtgcaatc atagcttact gtaacctcaa attcctgggc tcaaatgatc 25320
ctcccacgtc agcctctgga gtagctgggg ctacaggctc ttgctaccat gcccagctaa
25380 ctttttattt ttatttttta tagagacaga gtctcactgt tgctcaggct
ggccttgaac 25440 tcctgccttc aggcggaact cctgccttca ggcggtcctc
ctgcattggc ctcccaaagt 25500 gctggaatta caggcccaat tttattcttg
ggatgtatgt ctgaaactct ttccttcact 25560 tccttcccaa gccttagttc
aggcccttct catctgtggt cttcaaagtc gccttcagct 25620 ggttcaggtc
cttcctttct gctgtatctt tcatgggagg acatgttatg tatcactgtc 25680
ctacttgaaa acttccattc cccattgatg agggtgttac ctccagattc ctaacacagg
25740 tgctgaaggc atgcctggat aaaggcactc ccttgatctc ctggccaggt
ccccgtacac 25800 ctgcagcgca tgctccacat tctgtcttta ctgatgctgt
gtcttctgcc tgcggagcca 25860 cccaccattc tattcacagc ccctgcctca
gcggagcacg tgcctccctc ttcctacact 25920 gagctgtcct ttctattgaa
tcccctcttt tttgtagtat gggaaatatt ttattatgaa 25980 tactcttttc
tctgttgcct ccgtgaccac gttaactttg ccctaattcg ccttaggact 26040
ccatctgctt aggggaaagt taggatttgg ttacagaaag caagctgcta gaaagaacag
26100 tgtttagctt ctgacaggca aaataggatt ttgcaacatg cttttccttt
ttaatgctta 26160 gacattttat atgaattaat atttttattt ggttgcttat
acattacttt ctttttagct 26220 agaatgtgaa ccctatagga acatggggat
tgcctttcac atctttgtat cctcagtacc 26280 taatgttcag tcaccctgtg
gtcttgtgtc gtatatacat ttagccttcc ttaattaaac 26340 catatgtact
ggtccccgtc ccccaccccc aaatagagag aaagaaattc cttgaatact 26400
acattgccag tatcaaacca caccttgata tcctctgggg aaagggaggt atcagttgaa
26460 aagagaaaag aggttaaaat ctaggcatta aaatgtgtaa ggcttagatg
ctggcaattt 26520 aaggtatgtt ttcctgaggt taattttgat tgtgtgcaaa
ttttacctca tatctaactg 26580 taggatttag tcaccacata agatgggata
cctccataaa tccttcagaa atgtttgtga 26640 aattaaataa agccttattg
aagactcagc tcttgagagt catctaccta cctaacagtt 26700 attcttgaac
agaagagtct tacttttccc tataaggcag tgtgatagcc atctgtatat 26760
tcatataatt tatgttggcg cttacttcat ttaaaaatgt attccgtgaa tgcagttgcc
26820 aggcggtgtg ctgatcagaa acgtgtacca atggcctctt ttataattat
aagaggaaga 26880 ccaacctgaa acagtcacac aaatgattaa ttttaattgt
ggaggagtgc tgggaaagaa 26940 aaataaaaga tgcaatgcaa gtgtttacaa
aggagctttg agcttgtttg aagtggtcct 27000 tgggcactta agcaaggctt
aaagaatgat gtgattagaa gtggcttagc aattctaaag 27060 aacacaggga
aggcgtgtgg ccagaacatt ggtccctaga gcacatcgcc tcctgacata 27120
ccatttcctt aagttaatgt tttaccacta tacataggcc ctcccctttg tttacccaga
27180 tttttttaat tttaaggatg tttttaataa cttagaatcc tgtaatttgt
tgaacagtcc 27240 tgtattccct ttacttatat tccttgagat tttataaaat
attttttaca tgtcccaagt 27300 cttgattata tctttttacc tcttgttaag
aaatacttac ttttctattt ttatgctata 27360 tttcatgttt actgtagaaa
acaaaaaaag taaaattttt ctttattcct atcactgcag 27420 cttataagca
ctctaaacat tttgatctat attttgccaa tcatatattt tagttaaaat 27480
tgttgttgac ataattgtag attcctgtgc agttgaaaga aataatacag agctgagcgc
27540 ggtggctcac gcctgtaatc ccagcacttt gggaggccga ggcaggcaga
tcatgaggtc 27600 aggagtttga gaccagactg gccaacatgg cgaaaccctg
tctctactaa aaatacaaaa 27660 attagctggg tgtggtggcg ggcacctgta
atcccagcta gttgggaggc tgaggcagga 27720 gaatcgtttg aactccggag
gcagaggttg cagtgagccg agatggtacc attccactcc 27780 agcctgggca
acaagagcaa gactgcatct caaaaataat aataataata ataaataaac
27840 tttaaaaata aaacagagag atcccatgtg cgctttgcct agttccccca
tccactgccc 27900 ataacatttt gcagaactgc agtacagtat cacaaccaca
atactgacat tgatacagtc 27960 tgctcatctt attcatattt ccccagtgtt
actcgtatcc acgtgtgtat gcattgtgtt 28020 ttcaatactc ttttattata
aagctgtttt taatgtgatt caattctagg ttgttttgtt 28080 ctgccctcaa
aaagcattcc ctctcctaat catatctccg tcataccctt gtatgttttc 28140
tttaaacctg ttttaagaaa gcagctacct gtaagagaaa tgagattgaa aacagaattg
28200 ccaatctgct tgtactttat aagcctgttg attgtttaga tacggtttag
ccagtttata 28260 gttaccctgg gtgctgaaag gtatgctgga tgatacctaa
ccaacagaga accattgaat 28320 gccgttcaaa atggactgaa gcatcagcaa
tgtctgaaaa aggcctgaca gtaatgtaca 28380 tgtcaaatgg cccgtaattt
aagcagagta gagtaagtag aagaataaac atggggaaag 28440 ttccagcaac
agaggaggct ttgagctttt gctcttcatc ttgagtggat gttgttctca 28500
ggtggtaata ggccatcgag ctttctccac tggctgcctc tctggggaac aaataaccga
28560 aaagatactc agcaccctgg ttggtacata ggtggtcagt tgatttatac
ttcctggttt 28620 tcagtgttgc ttgaattttc taaatggaaa cacagtacct
ttataatcag aaaacaatcc 28680 cgagttttga tttgagggtg ttgtaaaaag
ttaaaaaaaa aaaaacagaa atgtgaaaag 28740 gaagttgtgt tagagtattt
ggagttgaga aagcatgaaa aggacagaag agaagctggt 28800 tgtcaggttg
catggggtag ctacaagcac actgaccaga aagtcagctg gaaaaaaaat 28860
gtagaaacag gagataaaac ggccaagggg ctatacaagc aaacagcaag gacctgagaa
28920 gaaaaactag ttaggtgtga ctgtcagagt gatgtgtaca gtgtgatcct
ttctgtgtaa 28980 aaacaagcag taagaattcg ctgtttacgt ttgcgtgtgt
ttggagaaga gtggggaaga 29040 gtaggcactg ccagactgtg aacactggtt
aggttattgt tatatctttg tattatatac 29100 actggacatg ttatttgtat
aatatgagaa gaaattttat aaatcattaa atcttttggc 29160 atttaggaac
atttgtgttt tctaatagtt gcttctatac tattatcttt attatatgcc 29220
cttcatcttc tcagtgtttg gctgttgttg tgattccctt ttgtgagcag tgttgaagtt
29280 agctaatatt catttcttct cccttctttc accctcctcc agagtctgat
ttgaagtatt 29340 cctagctgct acctataaaa gcaataagca agattgtttt
acttttcaca aactcgtcct 29400 gttctgtgcc tctgcctcgg acatagctgt
agtatagagt gttgtctccc ttacatcctt 29460 ctatcttaga cctactagta
aatattaatg ctcactctaa gttcttctca attctttttt 29520 tttttttttt
tttttttttt gagaaagagt ttcgctcttg ttgcccaggc tggagtgcaa 29580
cggcacgatt tcggctcacc gcaacctcca ccttctgggt ttaagcgact ctcctgcctc
29640 agcctcctga gtagctggga ttacagtcac gtgccaccac ccctggcaaa
ttttgtattt 29700 ttagtagaga caaggtttct tccatgttgg ccaggctggt
ctcaaactcc cgacctcagg 29760 tgatccacct gcctcagcct tccaaagtgc
tgggattcca ggcgtgagcc accgcgccca 29820 gcctcttctc tcaattcttc
ctgaagctct ttctgcacta gattcctcag gaagggcttg 29880 tgggaacaat
cttctgtgaa tcaacagtac atattcataa tagtttgtca gcagcctatt 29940
attttaaggc catttggtct gtatataaaa atgtttggat cacattttct ttctttaagg
30000 taaatatgtt attctgttgt cttctggtat aaagcattgc tgtaaatgtt
tgacagtcta 30060 attatctttt gcttataagt gacttagggt tttttgtcta
tgtgcccaaa ggattttttc 30120 cctctttctc tctttttttt tttttttttt
tttttttaaa cagacaggat ctcaccctgt 30180 tgcccaggct ttagtgcagt
gaggcagtca gagcttactg aagttttgaa ctcctgggct 30240 tgaggaacaa
aggatttttt taacctttta attcaaagtc tcatcattta tgcaaccatg 30300
tcttggtgtt ggctgttttg ggttgttctc cctcaaaaat ccatgtgctc tttcaatatg
30360 tagttttaaa tctttttttt ttaatttcag gaaaatcttg aattagagtt
ttccgttttt 30420 cgtctggtac attgcttggg tttccttctt caggaactca
gcctgttatg tgtatgtttg 30480 atcttctttg cctgtcgtct gtttctttca
cttcctctca cttttttaaa cttcatttat 30540 taaaaaaaaa tttttttttc
gagacagagt ttcgctcttg ttgcccaggc tggagtgcaa 30600 tggcgtgatc
tcggctcact gcaacctccg cctcccaggt tcaagtgatt ctcctgcctc 30660
agtctcccaa gtagctggga ttacaggcat gcgccaccac gcccagctaa ttttttgtat
30720 ttttagtaga gacagggttt ctccatgttg gtcaggctgg tcttgaactc
ctgacctcgt 30780 gatctgcccg cctcagcctc ccaaagtgct gggattacag
gcgtgagcca ctgtgcccag 30840 ccttattaaa aattttaaaa acatacattt
aaacttaaca gaaaaattat gagagagaag 30900 ggggtggtgc caggcttttt
taaacaacca gctcttacat gaactcatag agtgataact 30960 cattaccatg
aggacggcat caagccgttc atgaaggatc tggccccgtg acccagacac 31020
ctcctactag gtccattttt aacattgggg atcacatttc aacgtgagat ttggaggggg
31080 caaaactaca aaccatgtca ctcagggatt ggaggagcaa gtaccaccta
tactttggac 31140 tcaggtagaa aggcaaaata tccaggaaat aagctgctac
cgtccagggt tcagcagagg 31200 tgcccatcag cctgccaagt actcaagagt
ccagcctcta gggagctaat catcatggtg 31260 agctcttcga ggcacaggga
gctgggaaga cagtgcttgc cacccctgcc tgaatagtgt 31320 ttgcacagag
agttctgttg tgtcttgatt gggtcctcct gccactggga atgctgtgga 31380
ttatactagg tctctatctg gcttgtttca gggctccatg tgaaaacctt cttgatatcc
31440 tagccatcca cctgctcagt ccctagtttg caaggaggct gtggggagcc
tagattctgt 31500 gtcagataga atgtactaca ttccgtctca ggaatgtacc
acatcagaaa acagtgcgac 31560 ctgcaggaga agtagaggtg aagaggcaca
ttcttccgag aaatgtttct ctcaacaccc 31620 agcattccct ggatatcagc
aggaaattac tcactgctag aaaatgcccc atgagccttc 31680 tgttaaggag
gtcaagggag agaacagaga aagttctcaa agttgacttg gtcactggta 31740
ctttcttatg cggttcttat tttgtttgcc atcgtcatca tcatgctatg tctattttct
31800 caatccaaat ccactgcttt caccttggtt ctttctgacc ggtttggcac
actcattcag 31860 taaatcctta tggagagccc aatgtctgca taattgtgct
gtgctgatga ccaagctaga 31920 cctacgagtg tcggctcctt tgagatgtac
gggacagctc ttctgtcatc tcttctggga 31980 agcctctcca ggcttggtga
acagtggcaa gatgtttaac agttgtacat gtgtcccatg 32040 ttcctttcta
agagcctggg caaaccagac ccggtcgcag gtcatcgtag tatggcgtga 32100
gcttcctctc tcctttctga ccttttgtgt gatggcaaga acctgcagag tgacacaagc
32160 agcaggcttc tgaggttgct ctagcctcag aatggccgtc ccttctccac
cctggccctc 32220 attgctgagg tttcctttga agcaacagtg ccggaacaga
ctaggggaag cagcttggac 32280 atagctgtat gatttattac cacccattga
ggccaaccaa agtcggcaag gagaggtagc 32340 aggtcagtgg tgcctggaag
cttcctcttt cctttgcacc agatgtgact gctctgcaat 32400 tactcctaaa
tttgctactc tcgtttttac tagccaacct tgatgttttt cccttcttcc 32460
tgtagaatag acttcccctc tgatcagtac tttctactca acactatttg tggccacagt
32520 gggaactcat tgaggacagg gaccatgaca ttactacctg acccatcaac
acttggcata 32580 acttgaaatg caaggacaaa aattggctgc aagtacaatg
tggtcttcac tctgaaggtg 32640 atccttaaaa cttggctttg gcatcatatt
gccttaatat acctagggga ttgggtaaaa 32700 ccagttactt taaaagagtt
ttacaattct ggccttctag ctatcttgtc ttcttaaaca 32760 agagcacaag
atgaatgtat cttagtgaaa ttttatatgg tttgctttga gtaatcttgc 32820
gaagattgat ttttagcaca gtaggaaaga cacattctaa tagtgatttt tttccccgag
32880 tttatgtact gctgttgcat gaaaatctga ctagatttaa tgttcctaaa
gttctttgtt 32940 catcctgatt tttgcaggtc ctagggaaag ctttgttttc
ctcttaacct aacttagatg 33000 ttgtcatttc atgagctttg gaggaagagt
gtatagccaa ttgtgtaatg tctttaaagg 33060 atattatctc tgcaatagtt
gtttataagg cctaagttat tcatgtaata atagtggccc 33120 cggatctgtt
tctagcaata ggtatatgga ttttggttcc tatatagttg tagttgtggc 33180
tttgagatat tgagcaagcc cttttaagaa aggatttggc atccctcagc cttcaaaagc
33240 ttctcaaaat tgatcatatg ttattagcaa aggtttactg cctgcttcca
ttgtatagac 33300 aatttatttt ttatgtattc cgttctaaga aggcagatga
ccaaaagatc ttgcatctgt 33360 tgcccaaggc ttgtgactag agaggaaaga
gataagaata cttttttaaa atcccatttt 33420 actaaatatg ttgaggaagt
ggtaagatat attaatttgt tgagattttt ctgttatgcc 33480 tattatatga
aataggtact ctgaacatgg cttcttaatt aaatatattt gataaaatac 33540
aacttgcttc ccctggagtt tagaagtcag ataactgcca tggagagcta tgctttcttt
33600 gttttaaaga tctgcttatg aacatgataa acaggaacaa tttaatgttt
tcaatatttt 33660 cttgtatttt actgcaagtt tatacacaac ataaatatgg
gggaaggggg aaatgtttat 33720 accagagcca tcctgcccat tctttcctta
cagaaggaca aaggagcagt atttatttta 33780 actacaaaaa tactattgta
ggttttaaaa attccgtata ttttgatatc ttgtgttcct 33840 cttgaccttt
aatttgctaa atagttgcaa agaatgaagg taacctgcat catcttctta 33900
aaaaccaact ctatctaatt ataatagttt gtctatctct gaaaaatagt gatgtgttca
33960 ttctgaaatc agaactaccg gatgcagctg cattttgtta ctatttgaat
ttcgggagag 34020 ggaggaggat gcagcctttc gagctgctga aatacacaaa
cacaaagaag acaccaagca 34080 tagtagaact gtgttaagct gaccaagcca
gaagaagcac ctattctcag catagtatga 34140 gacgtaaagg caatataatg
ggcatagttg aagatggtag aaggaaaata gactctgatg 34200 gtttaatgtt
aaatgctttt tttaaaaaag tggtattcca atatcgaaga agaagacttt 34260
ctacttttag aagcaataaa ggaaattgca gaggaaaggg tcaataggtt ggaatacata
34320 aaaattaaaa acttttaaac tttttttttt tgagacagag tctcactctg
tcacccaggc 34380 tggagtgcaa tggtgcaatc tcggctcgct acaacctccg
cttcctgagt tcaagcaatt 34440 ctcctgcctc agcctcccga gtagctggga
ttacaggcat gggccaccac tcctggctaa 34500 tatttgtatt tttagtagag
acagggtttc accatgttgt ccaggctgat ctcaaactcc 34560 tgacctcgtg
atccgcctgc ctcggcctcc caaagtgctg ggattacagg catgagccac 34620
cgcgcctgga ctaaattgtt tcagtattaa ttttttttaa aacaagatct tactgttgcc
34680 caggctgaag tacagtggcc caatcatggc taactgcagc cttgacttct
gggcctcaag 34740 ggatcctccc acctcagcgt cccgagtagc tgggaccaca
gacatgtacc accacaccca 34800 gctacttgtt ttatttttat ttttgtagag
atgaggtttc accatgttgc ccaggctggt 34860 ctcgaactcc tgggcccaag
caatcctcct cccttggcct cccaaagtgc tggtattaca 34920 ggtgtaagcc
attgcgccct gcctgatttt ttaaatgtgc aaacagataa gttggaaaag 34980
tgatttccaa taaagataaa gagttgatgg ttttaaaata cgtaaagagc ttatatgaat
35040 gagaaaaaca ctaacattcc aaaagattag aaggcaaagg acagaaagaa
acaaatcact 35100 atgtctggga agggacatga aggagcaggt tcccactggg
ccagcggggc tcaaacccac 35160 tggggacgtc cgagagactg caagggccat
gccttcacat tgccgtacct gagaagcaag 35220 gagctggggt atttatctct
ttcacacttt gggaggctga ggtgggcgga tcacctgagg 35280 tcaggagttc
gagactagcc tggccaacac agtgaaaccc cgtctctact aaaactagaa 35340
ataattagct gggtgtggtg gcacacacct gtaatcccag ctacttggaa ggctgaggca
35400 tgagaattgc ttgagcccag gaggtagagg ctgcagtgag cataaattgc
accactgcac 35460 tccagcctgg gtgaaactct gtctcaaaaa gtaataataa
tcatgataaa taaaataaca 35520 ttagattgtt agcagaagta gccacaggtt
tctcccacct ctctgcaagt tgctgagtgt 35580 gattcccatc aagaggtaca
atgtcttttt atttttattt tatttatttt atttatattg 35640 cctatgttgt
ctaggctggt tccaaactcc tgagctcaag tgatccttct acgtcagccc 35700
cccaaagtgt tgggattaca ggcatcagcc actgcacctg gcccagatac tttttcttga
35760 gtaggaattt cgagtcaccc tgaacattgc atgccttcgt agtggggaag
acaataggaa 35820 accacaggct gtaggctaaa atgggttgtg tttcttgtaa
cgtcatgaca aggcataacc 35880 catcttggca tagtaaatag taagcactca
ctgaactgat gattttaaat ctttgctgtt 35940 tattcagcaa tatcctaaat
tagcgctatg ttagtggagt tgcatctccc tcatggatta 36000 gtctgaaaaa
gatgagaaat ctgtatgtag accaagttat ccttaaactg ctcataatgt 36060
atgatgcacg tggttttacg tgtacagcct ggtaccattg ttcttaggca catttcagtg
36120 ccagaactct taatacccag gaagaagcaa aaagaaagat ggaggtgcag
ctagaggttg 36180 tggcctttga acgattcatt ctgccttaat aagagtggtc
tggctgagct cggtggctca 36240 cacctgtaat cccagcactt tgggaggcca
aggcaggcag atcgcttgag cccaggagtt 36300 caagaccagc ccaggcagca
tagcgagacc ccccctcccc ccgtctctac aaaaaaatag 36360 aaacaatgag
ccaggcatgg tggaacgtag tgcgtggtgc ctgtagtctc agctacccag 36420
ttggctgagg tgggaggatc acctgagccc tagaagtcga ggcttcagtg agcccttatt
36480 gtgccactgc actccactct aggtgacaga gcgagacagg tcctgtctcg
aaaagaaaga 36540 agaagaatta aaaaaagtga ttagatccct tgtgtttggg
acacttgttg gcagcaggga 36600 tggtagcgtt tatgagggtt gcatgtaaca
tcgcctagct cagacatctg tttgactgtc 36660 ttcccccctg aagcgcaggc
tctgtgaggg caggtctttt gtctttcttg ttaatcttca 36720 tatgcttagt
gcttgccaca tagttgatgc tcagtcgata tttggatgaa ttgaagggat 36780
taatgcattg aatctgaacc ttgctttctt aatgcatatg gggagttctt tggaaagcca
36840 cacagaggag cttggttgcc tgcttcctct cttccccaga ttgtcttttt
attgttgtgg 36900 cttcactgaa gcactctcac ttcaaataat tttgggcatt
ggtcgtattt tattctttgt 36960 tccttcttca tccttacccc tcagatggta
tgtagaaaag tacactacat ctagaaagta 37020 ctttataaac tcatttggtt
gataataata catatgcctt ttccttggtc ctggtagcag 37080 aatcttgtgc
cactcttgga atacaaacga aattcttaac caaagccagt ttcattttga 37140
tgttctattt tcctcccatt cacactccaa attgtgcacc aaagtatcat cctagttttg
37200 tgaggatggt tctccatact tcagggtagg agtatcatgt ggattcctat
gatacctttc 37260 tccctgggac catggagggc agcagctggt gattgatagt
ctgattcccg gtgaggaaag 37320 ctgtgagcct tccacttgca gatgtctgcc
aactacatgt gtccttagtc aactgtacca 37380 ctgtcctccg gcaaacagca
gaagcccagg gcctgaagtt cttaagctgt cattatggaa 37440 agcagaaggt
aaacaaaaca gaagtgaaag tagatttaat tttttagact gttctcttac 37500
aggaatggtt ttgtggttct cagcatttta aaaaaaatag tggttccaat atgttttatt
37560 gacatcaatt actgtaagtc tgattcattt tctgcctatt gatttctacc
caaggtgaaa 37620 ttcatgacat ttaacagaaa gcataagtga ttttttaaaa
gcagacacta ttagggacgg 37680 taaaaataag atttaaagtc gggacacttg
aaaaagcaat ttttatacct ttggtaacga 37740 ttctattctg attctttgta
taaataatat aaacaaaggc tctagaagct tactataatg 37800 aagttggtgt
gctgtttcta aattctggtt taaggcccaa attcatttta tctgcattaa 37860
cttttttttt tttgagagtc tcgctctgtc acccaggcta gagtgcaatg gtatgatctc
37920 ggctcactgc aacctctgcc tcccgggttc aagcgattct cctgcctcag
cctcccgagt 37980 agctgggatt ataggtgtgc gccaccacgc ccggctaatt
tttgtatttt tagtagagac 38040 ggggtttcac tatgctggtc aggctggtct
caaactcctg accttgtgat ccgcctgcct 38100 cggcctccca aagtgctggg
attacaggcg tgagccactg cacccggccg tgttaaaatt 38160 tttcagtggt
agaccactat gtcaatatgt tgctttcact gacaacagta ttttcttaaa 38220
gataggatac cccatttcta gatgaatctc attctagctg gaaaataatt tttcagttct
38280 gaaactacat caggcctcag ggaatcaaaa ctagctatta gccacacaca
tataaagtgg 38340 ctttgcttta taaacgattt agggtcacca tcaatgacaa
tggtcccttt ttattgtatt 38400 tttaagagtt tcttatctta aatggctgca
taactgtaga gttttaaaaa aattaagtaa 38460 atgaccatgt taatgctcta
ttaagcttcc aaacaatatt gtaatttact ttgaagattt 38520 ttttttattc
tcaacatcct gcagcttgac cgtttgcctc cgtgtctcag tgctgcttat 38580
tttgaggtgt ggactggagt ccatctgtcc cccttgcctc tgaactgctc cgttttgtgt
38640 ttcgtaattc ttcatgctgc atcctgggcg catttctctg tagtagcttt
caatttgctc 38700 atgctttgac tgggcttagt ctagcgttta tcctatctct
taaggttttt taaaaaattt 38760 tcatgattat tcatttattt ccaggatttc
tcatttcttc agtcacatct ccttgttctg 38820 gttttacttc ttcctgtttt
tattcataac atctttttta tacacgattc cttcatgtat 38880 ttctaatctt
aagtatattt aattgcttat ttgattcttt ttttttttta ttgagacagg 38940
gtcttactct gccaccaggc cggagtgcag tgacatagtc atagctcact gcagcctcaa
39000 ctacttggac tcaagcgacc ttcccacctc agcctcccag gtagctagga
atacaggtgt 39060 gagagccgcc acacccagct gatttgtctt actatgttgc
ccaggctggt cttgaattcc 39120 tgggctcatg tgatctgccc ttcttggcct
cctgaagtgc tgagattata ggtgtgaacc 39180 actgcacctg gccaagtatg
tttatttatt tattctaatt tgagagggag tctcgctctg 39240 tcgtgcccag
gctgtagtgc agtggcacaa tcccagctca ctgcaacctc tgcctcctgg 39300
gttcatgcga ttctcttgcc tcagcctcct gagtacctgg ggttacagtt gcgtgccacc
39360 acacctagct aatttttgtg tttttagtac aggcggggtt ttaccctgtt
ggccaggctg 39420 gtcttgaact tgtgacctga agtgatccgc ccgccttggc
ctcccaaagt gctgggatta 39480 caggcatgag ccaccacgct tggcccaagt
atgtttattt ttaaagtccc caacaagcta 39540 tacaataaat tgcatatgga
atggattttt gttctagttg atttgttggt tatcatttgt 39600 agaactaact
agttgtcttc tgtgtttgat accttgcttc taggtcattt tgagttggga 39660
gccttttgtt ttgtttttat tctcatgctg tttttgagcc tagctgtgcc tttatggttt
39720 tctctaaatt taattgacca ttgttttata tttggagcag tgggtgtaca
tcagagtgtg 39780 aaagcagccc caccctctcc accagaaggt ctccatgcca
gtttcacgaa gcatttttca 39840 tgccctcatt cctgccctta tcccttgatt
tgtggggagt ttgtaaagca gttgattgtt 39900 ttttttccac gtagttttcc
aagtgcacat aattgttctg ttagtgactt gtagctccat 39960 tatctattaa
ccttgcccca gaccactgta caagcggacc caacgcttcc tccagctgtg 40020
gcagggacag ttacttggta tcctgctgcc ttttcaatgc tgaccagttt tgccccttcc
40080 tcccctcaac ccctgtcttt cattcaacta tcaccaaacc aaaagattct
ggtttgcttt 40140 ttagtatgtg ttcttattca gtacatagtc attttaaaat
ttaaaccaaa acagacttgg 40200 tactgattag cttaatttta agctttttct
ttattattaa acagtgtagt ttatcttagc 40260 atttcatatt aagtatatga
tttatttcat attgcttata tgaatgtaca cataaatata 40320 ataaaaatat
tttcctaagg tttttgtagt aaattatatc gtttcattaa ctttcatata 40380
tagcattgct tttgacctgg aagacattga acctctgatg atttgtatat tcctcggagt
40440 atactttgtt acatagaaat tttctcattt ataatgagat ttgtgattaa
caaaatttgt 40500 tcaacatgca ttactttgaa gatctggttt ctaaaatttt
atgctagtta ccccaccccc 40560 ccttctatat atatctccct attcagcgac
tactgcaaga gttccaggaa atgtacactg 40620 tgtgttcact tactgcattt
taaatcattg cctttactat atttctgcat ttcccttcaa 40680 tctagctctg
tctgtacatt tctgaaagcc agtagcttcc ctgaagaacc aggtaacaac 40740
ccgaacaatc aaattagata accatttgta gaatggaggt tccgggagat cttagaagat
40800 gtgatgggtg ctaagggact ttgtagttcc ctgaagttcc agtgagtaaa
aggtaccctt 40860 ggaatttttt attccttcag acttttaaaa cagagatcac
tttcaaaaat tactctttct 40920 gctttgaatc catgttttag taactatttt
gacactgttt ggtcagaagg ctgtgtgggt 40980 caactgcaaa taaataaaat
aaatgtgatt tcagtaattt ccattttgta acaagtaatt 41040 gagaaaatag
gattggatca gatatttgct tatacacatt ccctttcagg agcacttctg 41100
ttctataaag aatgttggta tattgttaag gacacttcaa gctttgggaa cctttgaagt
41160 atccattgat tcagttaaca aaattatgtt gagtgcctac cctgggcctg
ggcctgtgtt 41220 aggaggggac actaagatga gagtccaaag cacttcttct
cagactcctg gctgctaatg 41280 ggttgctgcc tctacttctt cacttagcag
atagctttaa aatgagtaat gcattttacc 41340 atggagcccg taagagacat
tcacccagtt gtggaccgag gagaagggtg ttaaacccag 41400 attgtgatgt
ttcacttgat gaagtgctta atataaacat ggaaatattt ccgcaaggat 41460
aaactggctt ttatgcctgt gtgttttcag gagaaataga aatctctaat caaatattgc
41520 cagcttttca cccaagtttg actttttgcc taattgagtt tgggaggtgt
ctgaataatg 41580 gataatgagc tttcctgaat aaatataaaa attaattaac
tccaggctct aattcattct 41640 gttaccagag ttttgtaagc atgttacccc
tttgtgttca ttgggagatc atctgttacc 41700 ttcttaaatg agtggggaag
gatgggaaat gaggaagagc tataaaaact attcaggtga 41760 agaaggtttc
tgcccctcct tgcccctttt aaaatctcca gctcagcaga tgctttgttt 41820
aaacttgatc aagtgcttgt gaatcttcct agcctagcta aatcataact ttggaaggac
41880 ttgctttttt ctctcatgac aatggtttac cacagaaatg attcagatca
ctttgtgtgc 41940 ctgatgccta tgtaaaatga tacagtgaaa tggaaaccat
ttacctgtaa gctttgggca 42000 cacccaagcc tgcttcagga gcacatgatc
aggcgtgcac tctgggagag ccgtacacat 42060 ttgacatcta tgatgtgtgg
cgttttattc tatcacattt ctgaaatcta cactaagaga 42120 aaggaggctc
ttaaaaaacc actgaggtgt ggactggggg aaggagagat ccgtaaagaa 42180
cctgtttgtt acctgttgat actatttccc attggtaaaa tttctaattt agtgtgatcc
42240 agccctgaaa tgctgaggca cacactgaat gactcctgac atctttagtg
tttttgttca 42300 ggggactctt ctgggaatct gtttcatggc aagtttatta
ttcccttttg gtttggctca 42360 tcagtttacc cagcagtcat cttaatcggt
tttaaaggct tttattttat tttgttttct 42420 ctgtggaaat tttacacatt
cagtagatta gaagtagtta tttaatcttt ggttagcata 42480 ataaaagatc
ttctagggac attttttgct tgcagtggaa ggctagttaa atgtgttcat 42540
tagtcatgaa tctgcttttt ctatagctgt tggaaacgta gctcccctgt gatacagttg
42600 tagaatacag aaatctcgtt ttgctgttac ggtacggtag tctacttact
ttcttccaaa 42660 ccattaatgt tatagttacc tttaattgcg taggtcctat
cacccctcaa ttttaagact 42720 ctaagcctgg cattttatct tacaaaatga
aatataaaga cttgtactca gagtatgtgt 42780 gtgttttcca tataccattc
taaagtagag aaagatgagg gattcgccag aaactgattt 42840 ctaataaatt
atccagaaac tgaccccttc tcacctcttc tgttactgtc actgtggttt
42900 cagccacagc atcctttgct gcattgttac cttagtttcc tgactgtatc
cttccttaca 42960 ccattgatcc ctgcaatccc atctgcgcgt agcagccaga
agggatccac ttactgctgt 43020 gatcagaaat cctcagccag gtgcagtggc
tcatgcctgt aatctcagca ctatgggagg 43080 ctgagactgg agaattattt
gagcccagga gtttgagacc agcctcaaac tgggtaatat 43140 aatgagacct
catctctaca aacaggaaaa aaaaaatttt tttttttttt ttaactagcc 43200
aggtatagtg ctaatatacc tgttctggga tccagcatgc tctccctgac ctgcagcttc
43260 atctccacca ctttgcccct cactcccacc acaatggctt tcttctcttc
ctcagacatg 43320 ccgtgcgtcc tcctacctgg aatattcccc tccaaacatt
cccatggctc actccctcac 43380 cttcatcaga tctctgttcc agtgtcactt
ttactggaag gtcttttgtg accatcctac 43440 ttattataaa aaaataatct
gcccaacctt ctccttttat ttcctctact tgatttttca 43500 atttagtact
tatcagctga catatatttt gtctctctgt ctctctctgt ctctcataga 43560
aggtaaattc tataaaggaa ggaattttta tgtttggttc tttgctgtag ctccaatatt
43620 caaaacagtg cctgacacac agtaggccct ttatatttgt tgaataaatg
ttgacactct 43680 gatatctaat ttttgtctgg tgactaatac gaaaactata
gagtgataat aaaagcatta 43740 ccttagtaga ctggaaaggg atgagcgcta
ggatgaactt tctgcctggc gatcttgctg 43800 aatttaggag gcagattggg
gttcaaagga ggctgaaatg gctaggattt gcagagcagg 43860 gtactaagga
tgagcaggct atgacagaaa gaactccaga aatctgcaaa gggatcacct 43920
tgagtctggc tggatacagt gtacactttg tagggtgtct cttcatgagc ttggataaag
43980 aacaactgtt ggggagtgga taattcccag cactcattca agcttgcatc
ggccagaacg 44040 gagagagaca gacctctgta atacgtagga tatttggtag
aaacattcaa ccgaaaacca 44100 tcagatatgc aaaaagtaat aataataagt
aaacaatgtg atgcatagct agaagaaaaa 44160 tcagacatta gaagcaagcc
cagaaatgac agatgataaa ttagcagata aggacattaa 44220 aacagctatt
ataaataact tagcagattt aaagaaaaac aacataatga ggataatgga 44280
agaaaaacaa ccgaatacca tttctaaaga agaaaaatac aatatctgaa atgagaattt
44340 agctggatag gattaatagt ttaggcactg cagaagaaaa aaacagcatc
tatatgagaa 44400 tatacccaag ggaagtacag agaggaaaaa aatgtggatt
ggggggtgcc tcagtgacat 44460 atggaacaat attaaacaag tctgccccca
aaatacttga aggaataagg ttcaagtttt 44520 ttccaggttt aatgaaaact
ataagcctac agattcaagc atttcaacaa accttcagca 44580 aaataaacaa
aaccacagta ggcctggcac actgtctcat gcctgcaatc ccagcacttt 44640
gggagcctga gtcaggagga ttgcttgaga tctgcttggg caacatagcc agaccctgtc
44700 tctacaaaaa ataaaatgaa ataaattagc tggatgtgga ggtccacacc
tgtaactcta 44760 gctagcctgg aggctaagaa gggaggattg cctgagccca
gtagttcaag gctggagtga 44820 gctaggactg catcactgca ctccagccta
ggcaacagca agaccacatc tctctctctc 44880 tctctctctc tctctcaaaa
ggcagtgaaa taacgactta tttggggaaa aaataaaggc 44940 agagaatttg
ttgccagcag actagcataa aaaaaaggaa gtccttgaaa cagaagagaa 45000
atgataaaag atggaaattt ggatatatac taaagaatga ggattgctaa aagtgacata
45060 catagataaa tatgaaatat atttttattt taaaatttat ttaaagcaaa
aataaaaata 45120 catcatattt ataacataga aataaaaaat gtatgataat
agcataaagg ataagtggac 45180 aaatgctgtt gtcgtatttt tggtaaaatg
cactattatt tgaaagtaga ccatcgtgaa 45240 ttcgatgcat attgtaaacc
aaatagaaca ctaaaaaatg aaaataaaga gatatggcta 45300 atgtgccaat
ggtggagata agatagatgc aaaaaaagaa aaacattcaa aagaaggcag 45360
agacagagga aaaaaggacc aaagatcaaa tgagtcaaat agaaagcagc taaactagca
45420 atatggcaga tttaaatcta gccatgtcaa tagttatatt aaatgtaaat
gttctaaata 45480 cctgaattaa aggatgaaga ttgtcagatt agattgaaaa
agcatgaccc aactacatgc 45540 tgtctgtaag aaattagaaa aagaacaaat
taaatccaaa gtaagaagaa aggaaataga 45600 gtagaagtta gtgaagtata
aaacaaagag caaagaaaat caattaaatg aaaagctggt 45660 tctttgtaaa
gatcagtaaa attgataaat ttctagctaa actggccaag aaaaaagaaa 45720
agacatacaa attaacagta tcaggaagaa aaacagagaa ttcaaaggag tgtaatgcaa
45780 actttatgct agtaaatgca ataagttaga tggtatggaa aaaaatgtga
acaatacaaa 45840 gcagactgtg gttgcctttg gtggcagtag cggggtggga
gtggaaggtt gaattgactg 45900 gaaccagaag cacaagtgaa ctttttgggg
tgatggaaat gttttgtatc ttggttgcat 45960 tgatagttaa atggttgtag
acattgctta aaactcactg aacacttaag tgggtatgtt 46020 ttattatttg
taaaatatac ctcaaaagca gttttaaaaa tgtattcaag tacatactta 46080
agatctttgc attttactct gagtatacct taattttaaa atctgttttt taaaaagtat
46140 tatgtagata ccttttattt tcccaatgtc tttattaaat gacatctcca
cgttttgctt 46200 cttacctcta tttttttttt tttatttctc tgtctctcag
gcatgcacac acacacacca 46260 aaaaaagtac atatgcataa tccttttggc
tgaataaaat cagttgcaac tgttatttcg 46320 gcccttattt gctccgggta
aatattcgtt agctgagtgg tttatctgta tcagatattt 46380 cttacatctt
catccagtca caccagctgg actgaccaga ttgtttttca cttcaagggc 46440
agaatttgta ctcactgctg aatgcttcca aatgatacgt agaataacaa atttaagact
46500 tagattttta ctttttcagg tctttttttt tttttctgtg ctgtatagca
tttccctgaa 46560 agcttaatct catctgtaag tgatgcagtg gatgtgttac
tattggatta atttatttac 46620 tcttaggtag gtttgtaatc tgtcatcatg
ctgttgtttt tttgtgtggg tttgtttttg 46680 gttttgagac agggtctcac
tctgctgccc aggctggaga ggctagagtg cagtgatgtg 46740 tttatgggtc
actgcagatt caatctcctg ggctcaagtg atcttcctgc ctcaacccct 46800
tgtgtagatg gaagcacagg tgcacgccac cacacccggc tattttttta aatgtattgt
46860 agagacgagg catcattttt ttgcccaagg ctgatcttga actcctgggc
tcaaacaatc 46920 ctcccacctc ggctcccaaa gtgctgggat tacagatgtg
aaccaccact cgagctccat 46980 cattctgtta ttagttgttc tctagtatga
gtcaaaaact cttacctgcc cttttacagt 47040 tttataaata agtaagcaga
atagcagaat gtggacattt tttaaatcca aattgaatat 47100 gcacatgact
caaggagtca aatagtaccg taatcggttt atgataaaat ccagtggttt 47160
ggctgggtgt cgtggctcac acttgtaatc ccagcacctt gggaggctga ggcaggtgga
47220 tcacctgaag tcaggagttt gagaccagtc tgacctacat ggtgaaacta
ctaaaataca 47280 aaattagctg ggcatggtgg tgcatgcctg taatcccagc
tacttgggag gctgaggcag 47340 gagaattgct tcaacccggg aggcagaggt
tgtggtgagc cgatatcgca ttatttcaga 47400 acaattttcc acaagatcag
tgagtgctgt ccaatagaca tataatacaa cccacataca 47460 tgactttaca
ttttcttgta gccatagtag aaaaggtcaa aagaagcaga tgaaattaat 47520
agcctgggca acaagagcaa aaccccatct tttaaaaaat aaaataaaat atggtggttt
47580 gctgtcccca cctcagacca tttctctggt ctttctcatt gaccaccact
cccaatcttt 47640 gttctgctga ttgattacag cttgtatata tctccatatt
tctaagcaaa atgtttatct 47700 tttttaaatt tataaattct ttttattatt
tttcagagac agggtcttaa ctctgtcgcc 47760 caggctggag tacagtggca
ccatcgtagc tcactgtagc ctcgaactcc tgggctcaag 47820 cagtcttcct
gcctccgcct ctcaggtagc tgagactacg ctacaggcac ataccaccat 47880
gcccagctca aaatgtttat cttttgatac attattcgag accattatta aggtggatga
47940 tttagttttc ttaaacagcc atcccctttc ttttcctccc ctctgcttca
ccgcccccat 48000 tttcccaatg ttttaccttt tggttaaatc agtactcatt
gtttacatta tttgcctctg 48060 cacatagtca cagatagtat tgtactgtac
tgtactgtgt ttctttttta aacattattt 48120 ctgttgttaa taattgactt
tttaattttt ttcctatttt gttttttaaa gagatggggt 48180 cttactatat
tgcccaggct agagttcagt ggctcttcgc gggcatgatc ccactgctga 48240
tcagtacagg aatttccacc tgctccattt ccaacctgga ccagttcacc ccttcttagg
48300 caacctggtg gtcccccatt cccgggaggt cagcatattg atgccaaact
tagtgcggac 48360 acccgatcgg cataacgcat gcagcccagg actcctgggc
tcaagcagtc ctcccgggct 48420 caagcagtcc tcccacctaa gcctcccgcg
tagctgagac tacagacact tgccaccaca 48480 ccaggttaat ttttgtgttt
tttgtagagg tggggttttg ccatgttgtc cagactcatc 48540 tcaaacttct
cagctcaagt gagcctcctg cctcagcttc ccaagtagct gggattatag 48600
acgcatgcca ccacacccca tgataattgc cttttttttt aatttgcata attttctttg
48660 tagcttttgc taatgttccc atatcttctt atagccttac agaatgattt
tccacaagat 48720 cagtgagtgc tgtccagtag acatataata caacccacat
acatgatttt accttttttt 48780 gtagccatag taaaaaaggt caaaagaagc
agatgaaatt aatagtatct tttacttaac 48840 ccagttcatt caaaatgtta
tttcaataaa tggtcaatat ttaaaatact tgagatattt 48900 tgcttttatt
tatttctttt gttactaagt cttcaaaatc caatgtgtat tttacactta 48960
cagaacatct ctttttagac tggccacatg tagctcaggg ttactgtatt ggacagagtg
49020 gtttcagttt caagtttttc cttggagaca tcctacttga aatttccatt
ctccatgtat 49080 ctgggtggtt ggtctataga cttgccactc acagctgtca
tcttgagact ttctttgctt 49140 ttcttctcta ttggatattc agtttcctgg
atttcaggtc ttctcatttt cctctagtag 49200 ttttgttagg tcatggttgg
tatggcatgg ttgggatagc gtgttcacac agctatctcg 49260 tgagtcatac
tcctccaatc cagcctgctc gcttcccgtg tctgtcatgt agttgtcacc 49320
ctgctatctc tccctccagt ttttgcagaa atttcctttg tcttcactct tggtcttcct
49380 ctcccatccc ccatgtatcc tatatctttc tctttcttgg tttatttcat
cactcaggtg 49440 gaaaagatgc tccagtggat tactgggaaa agggggagca
tggatgataa aggtattgag 49500 accttacacg tcagggaatt tttttttttt
tttttttttg agacggagtt ttgctcttgt 49560 ccaggttgga gtgcagtggc
gccaactcgg ctcactgcaa cctccacctc ctgggttcaa 49620 gtgattctcc
tgcctcagcc tcctgagtag ctgggattac aggtgcccgc caccacgccc 49680
agctaatttt ttgtattttt aatagagacg aggtttcact gtgttggcca ggctggtctt
49740 gaactcctac ttcaggcaat ccacccacct cggaatgttt ttattgtccc
ttctcatttc 49800 atgactgctg ggctaggtat agaattccag aatcattgtt
cttagaatct cgaaggcatt 49860 gcttcattgc tggccagctt tcagtgttct
tgcaaagtct gaagctgtgc taatcacctc 49920 atcctttgaa agtgaactgt
tttttcttcc cagaaactta cagaacattc tctttgtccg 49980 cagaattctg
ggattgcaat tactgtgcct tagaatgggt ctgtttttat cattatgaag 50040
agtactggat gggtcgggag gttttcttga attacttctt gatgttttct ttccttgtat
50100 ttttttgttt gctaattttc tatttttttt tcttggttta ctttcttggg
cagggggatt 50160 tcttctactt atatttgatt cttcagttga gcttgtcatt
tttgctatct tgtttttaag 50220 tttcgagaga catctttgtt ttatataaca
ttctgttctt aatacataga tgcaagatct 50280 tttctttctg agtatattaa
tatgtatttg aaatctttct attctctgca gtttgtttcc 50340 cccaagggtt
tttttttttt tctggttttt gttttttgtt tttatgttag agactttcct 50400
gttatatctg gtcatcagtg gtacctgcat gtggtggaga gtaggggctt attggagtat
50460 gagaaccttg agcaggtgta aggagcctgt caacactgcg ctggcctcag
ggcctctagg 50520 gaggctgcca gttgtgcatt ctgaggatac cttttggttg
tgccttttgt ctggtcagat 50580 tatctagaga tgctctgcct cctacctgga
ggagaagggt ctagctgcca gcggtgtgag 50640 tgtctcttgg ggaaaaggac
tcgagttcct ggtgtttggc ttgtgtatgg ccgcttaccc 50700 catttttggt
ggagcgctca catcttccac tgtgccaaca gtcttgctgc agttcataga 50760
ccttctggtt tacatttttc cagaaagtat gtctttagat ttctgcagaa gtctgaggag
50820 catggaagga gcttggggaa tgagatggca atccaggtct tcccagatgg
ctctaccttt 50880 atcccctgca gggaatccca ctcctccttc ctgactggga
gcacagccag agccttggga 50940 ggaatctgga gtggaaatct cgggcggtct
ggctttctta ctgttcactt gtaattttgc 51000 tttctcacaa ctgccaacca
ctaatcagcc tgatttccag cttccagaat tctattgctg 51060 ttgtctgctc
tcctattccc accgtagggg atggggctgt cttttttttt ttttttaatt 51120
ttggtaaaac atacaaaaca taaagtgttc cattttagcc atttttaggt acacagttca
51180 gtggcagtaa gtacattcac gttgtgtgta tttgtttttt tagtaataaa
caatataaaa 51240 ttttttaagt aataaaacac aaataaaaga ttgtttaatg
tgattatcgt ggaattttag 51300 gtgtgatcag gagccatggt gtagtcttct
gttgaaacag ggtgatagga tttgtttacc 51360 acctcctagg aaagcagttg
gatagtttgt tggcataaaa gtacatttta tctattttta 51420 ataatcgtag
ctttatagaa attgcagttg gaactcccag gcctggcatt caaggctctc 51480
tgagatctgg gctacccacc catgtcctcc agccgtctgt cgcacctcct actgcccact
51540 cactgttcct ggcatgagat gtgatctcca gcccccatgc ctttgctgtg
cagggtgttc 51600 cagagtgaat tgtccctcct gtctgtctct ctgccctctt
cctcgtcttt ccatcttcct 51660 gccccacatc actgcctcct acccaaggcc
tgtgctcatt cctcctcggt tttcccccat 51720 ggcctggtac atacctctga
attatcacct tgcatttccc atattgcccg gctctctttg 51780 atgtctgttt
ctttgctggg tcttcctcag tgtctgacgg tcagttaaat gtctttattc 51840
ttttttgtag gatatccgac atgaagccag tgacttccca tgtagagtgg ccaagcttcc
51900 taagaacaaa aaccgaaata ggtacagaga cgtcagtccc tgtaagtatc
cacgtggccg 51960 gtaccagtct tgctcttcct ttgctgcagg cctttttagt
caagactcct ttcgcctcag 52020 ggtttagtat aataataaat caatgtagca
gaggtttatg acgcgattgt ttcctatagt 52080 aaaggcatta gagacttata
gtaatagctc atttttccac cattatagaa gggctcaggt 52140 ttcagtttct
ggaaaattca gtgaagttca aagcactttt cttaagcttt gactgttttt 52200
gtgatgaatc attttcctac cagctgaagc agagtatagc aggcataata aaaccttttc
52260 tggatgactc agcagcagcg tcattagggc atgagcactg tgttccgctg
taatgaagcc 52320 ccgcacaggc attcggggtg ggcactgtcg tcccctgcgc
tgaatatgca aggcagctct 52380 gtctggagtc cccaccgcct ccacccccgc
caacctcatc atttttctcc ctctttcctg 52440 ctgttagttc ttcctaggat
tgtcagtgtg cctgctggcc tgtggcagcc ctgtccgcct 52500 tctgagtgat
tggctgtcag tctgccggta gctgaaaagt aaataactta acatgttaga 52560
atttgcataa agtaaggaaa actggagctg agtacaggac ttgaactgcg ccatctcctc
52620 taggccacag aggccttttt gacccccttc caggtcttta gacattgtca
ggcagtgagg 52680 ggtcgtagct gccagtgtct ccatggtagc gtgctctgcc
agggatgcag aagattctcc 52740 agtcattcct ccagtgggca cttcctgcag
gtcctgtgcc catggctggg agtggtggct 52800 gtcattgttc tctgccagaa
gggttagcag tgcatcctga cctgacttat gtggcgccca 52860 gattcctgga
aggggtctaa aaatggacct agacttggtg tagaacgtgt gcctcttggc 52920
ctgccaccat ggttccctgc ctggttttgt gtgtcagctc tgccgcttaa gaactgagtg
52980 gcttcgggca agttgttctc tctcatagga gtgtgtgaag atgaagcaac
ataagctgct 53040 tagcccagcg cccagtacct cacgcagaca taagtgctca
gtaaatgttg tctgtggtgg 53100 ggatggttgt caccaacatc tgaagtgcac
ttctaggtca tcaggtgaca tgattggcgc 53160 caacacatgg tactcttgat
ttagcacatc tcagctgagg cacctcattg atatttgttt 53220 aaaaacaaaa
acaaaaaacc ttggtgattc tgctgtgaag tcctggccag aaacctccag 53280
accgctgatc aacacgcaac agaaccatca ccgttcacct ctttgacatg gtgccaggat
53340 accctggatc tctagctttt gctatagttg ctctaattag ggaataatct
tgtctttaat 53400 attcctttgc tacatttttt aacatttctt atctaaatgg
ttttatgaat cagttttaca 53460 gagaaaaaaa accagtattt aaaatattct
tccaggggct ggtccaagta cagtagtgtt 53520 tacaactatg tgatcacaac
cagttacaga tttctttgtt ccttctccat ccccactgct 53580 ttacttgact
agccaaaaaa aaaaaaaaaa aaagttattc cagggaaaca attctccaac 53640
tttttcactc ccaatctcac tcctcttatc ttcctcccgt actcctatcc tcctcccgta
53700 ctcctatcct cctcccctac tcctatcctc cagtagaaac agtcatttgc
tgtgaaggtt 53760 atgggggaga atgagtcaag gtagaaggtc acctgctgcc
cagctcacag tgctgctggt 53820 gatgacagca gtccacagtt acaggcactt
gctgaacgag gggctctgta tacacctcag 53880 ctcattgact cttcccacaa
ccctcttgtc acctaccatt tagcaaatga aaaaaccaag 53940 gctctgaggt
gagttgtttg cccagagtca cccagtgctg tttgaaccca ctcacataac 54000
caaccaatac cattatgtaa tttttgaggt cttttatctc tgtgatccac ttaaaaatta
54060 tccaagtatc tttatttgta ctaagcctcc ataatgagaa acagtgttcc
agatggtggc 54120 tagttttcaa agacatctct ctttggaatt cttctttaga
acaaaaagcc ccagaccact 54180 tatccccatt catatcccct ttggacctag
ggagaaggta ctatttatag gtgatcacct 54240 gagtttattg tcccttgtgc
tgtgccagaa ataaaggtcc ccacctgctc ttattagctc 54300 tactaacagg
ataaggaaag tggccctcag agagctactg cttttgtgac aaacaaatga 54360
tacaagaaaa aaaaagtggc tttttaattt tagtgacctg gggcaggact tccaaatgaa
54420 agtttatttc taaaaactaa aaggtaaatt taatatactt tcagtgtttg
ggcttaaatt 54480 ctctttcaag tgtctttgtg atatgctctg aattttaaaa
atttagaatc attgaagttc 54540 attatacttg aactttaaaa aaaaaaaaca
aaaacctcgt ataaaggtca aggtatgact 54600 tcatgctgct gtgtacttag
gtcatttaat cttcaaacca ctggatagag gttaggttga 54660 agttcgatct
taaatcctac ctactgtagc tcattgtacc agcaacagct gtagggacta 54720
ggtggaattc atggtgggtt ttgttccctt ttaaagattg aagccaccat attttctgcc
54780 ctctaaaagt ttatgtcagc caggcatggg tggctcacac ttgtaatccc
agcactttgg 54840 ggaggctgag gtgggtggat cacttgaggc caggagttcg
agaccagcct ggccaacatg 54900 gtgaaacccc atctctacta aaaatagaaa
aattaggtga gcatggtggc ctgcgcctgt 54960 aatcccagct actcgggagg
ctgaggcagg agaaacattt gaatccggga gatggaggct 55020 gcagtgagct
gagaacatgc cactgcactc cagcctgggt gacagagtga gactcttgac 55080
tcaaaaaaaa aagttatgca tcagagaaca gatcctttga tgccctcctc tgccctgaaa
55140 ggtttttggg ggagagtaat aagtatcaca acaagatatg acctgagaac
agatttccca 55200 gataggacat gatccatgtt ttaatatggc ttactgctgt
tgcttcatag tgtgaagctt 55260 cagacacttc tgaaaaccct ttcagaaaat
cccagtcgcc ccatactgat gactaatctc 55320 aactaaaaca gggcttcagc
cagtgtgaat gccactaatg ccaccaactc acctttgctt 55380 ttctgtaggg
tgtgcacctg tatgtacaca ttcagctttt ccgggattaa cctctgagtt 55440
ctggtttgtc tttcagttga ccatagtcgg attaaactac atcaagaaga taatgactat
55500 atcaacgcta gtttgataaa aatggaagaa gcccaaagga gttacattct
tacccaggta 55560 agcagattgt ctgaattttc tatttaatgt caatttaaga
gtttgagagt gctgttatcc 55620 acacctcaaa taaaatctgc cacatccttt
agaaggtcag gatttcagca taccaaaaag 55680 cagcaaggaa gggggaaaaa
tcatccttca aaggttcagt ttggttataa ggaacgctaa 55740 tcttttctgg
gaagcataag atgacattgc tggaaatgag agcttataga aaacaacatt 55800
aaaatgccag agttgcctgt gtggtctgtt ggcagagaca gcagagccat ggctggagga
55860 gggtctgtac ctgtgttgct tccagaagta tttgtcgtag agcacttgtg
atggcaaatc 55920 taagaacgtt agcagtagac caggaatctc tgtccagagc
cattcagagt agctcagcat 55980 ggttctcatt ctttggccag aagaaaggca
tcattggatc atgtgaacaa gcatgaaaaa 56040 tgacttaaaa tttctgttgg
cttttggcat ctttatggaa acaaaatcct gaaagtggtt 56100 taataattga
gcctcttgta aaacactcag tggcatgtga ccaaaagggt atctgggaaa 56160
gaggataaaa agagtttctt tttaattaat cttctcaagt cttaacttgt tacctgtaag
56220 ttggtctaaa aagactgggt ttcttatttt gtttttcatc ataatttttg
tttctcattc 56280 catgtcagct ttcagtctta tatggcttta ggccacaggg
cgattttgaa catttgtaat 56340 tttgcttaat aattaggaaa ttaaaattct
ggggaagaca gaatgctcta tgaagaaagg 56400 ctgctttgag caaggagcta
ggtcagggcg cgttcaactg aggcctttct tcactgcctt 56460 tttgtcttgt
cccagttcct ccccatttat gactaaaatc agcccagatg cttctcgtca 56520
tctgggatgc agagcatcag cccagctgtg ttcagtccta tggggccatt gagtaagttc
56580 ttggtgcatg gatacagggc aggcctttac caggccctga gcccctggtc
ctcccagcac 56640 ctctggggta tttaggggag gctgatgggg gagggggttg
ataaggcggg agatgtctgg 56700 ggatgaggtt gaggcaaaag tgacttcttg
aggactttgc tttttggaga agtcaaattt 56760 cctacttctt gatttcagcc
cttcaactct ggtatggagt caggaagccc tttaaatacc 56820 tgttgtcggg
tgtatcatgt caagtgttgc attagcaaat gaccatgtat ccttgtgcta 56880
ctgtcctgcc taccccgcat cctagcgctt ccttgggaca tgagaagctc tgtctggttt
56940 gtgaggtggc actggggatg ttgagaaact gtttacacag tttccctttg
ccctggggat 57000 ttactaaagg agtcgaggca gcctgacccc aaagcatcac
ccctggacac tatgaccgaa 57060 acatttcccc agtgcccaaa ccaagaacac
ccttcccatt tttttttcag tggtgttcat 57120 tatgtaataa tacaagtctc
tcttctcatt ttttaaaagt cagaagtaca gaagagcaga 57180 gaataatgtc
caaggggccc tccttcacct cccccgtgca gtgtcagcta agtgtggtgc 57240
gtgtccttgc agatcttagg ggattgtgat ccttcagacc attctaaact ggggtggtgc
57300 tgggagttag ggaaggcatg aagggagtag tggagagctg cagtgactgg
ggtcttcatg 57360 ccagggtgga gaatgcaagg cccaggtggc cagccatgtg
ccacgggatt tctggctgcc 57420 aagagctgtt tatctgttca ctggggaggg
aagagttaaa tgtggtctgc ttttctccga 57480 gtcccttcag cacagggagt
gctgacttgt cttgttcagg tagtaagttc aagatgagct 57540 caggaaagaa
agtgagagga cactgagggc tagtggttga gccaagtgtg atgggactta 57600
aagggagaag atttaaagaa taaggagctt atgggccggg gacggtggct tacgcctgta
57660 atcccagcac tttgggaggc tgaagcaggt ggatcacttg ggtcaggagt
tcgaggccag 57720 cctggccaac atggtgaaac cccgtctcta ctaaaaatac
agaaattagc tgggtgtggt 57780 agtgtgcacc tgtaatccca gctacttggg
aggctgagac aggagaatcg cttgagccca 57840 ggaggcagag gttgcagtga
gccaggattg cgcccctgta ctccagcctg ggtgatggag 57900 cgagactctg
cctcaaaaaa aattaaaaaa aaataaagag gttaggtgaa aatagatgag
57960 aatggaaacc atgagaagaa gtgatgctgg ccaaggacat gacaggttct
gatgtggagg 58020 tgataggcaa tgtctcttcc agccactgct aataattgag
acaaactcaa ggcattcata 58080 ccctgtgtcc agtaaacatc tgtgcccatt
gccaggtgag ctggattgaa atgggccagc 58140 tgctcagcag acaccctcat
gccccagtga ctctgttccc cttgggccac ctcattgacc 58200 atttatgttt
ctacatctcc taagtttgtt gggccaagga tggaggctgt ctgccgtcag 58260
ggtcctcatt gctgatggta ggaatagttg ctgatgtttc attggatgtt gctgtattct
58320 agggactgtg ctaagtactt tatagaaatg aacatacttc attttcacag
ttttatgaat 58380 agggactatt attagtcaag taagcgatgg ggaaactggg
gcagggagcg atgaagtgac 58440 ttgcgcaagg tcacaagatg atgtgattgg
aaccaagaga agtgttgtgg ttggccacgc 58500 ccccacactg cctctcatct
gcaccaagga gttttgtccc atagcccaag ggccttgggg 58560 acgaatctca
gtggaggccc ttagcgggcc tgcctgagcc agaaagcaga atcggcattt 58620
ttctgtcctt ggttggccca gccctgaact gagatgcgga aatcgccttt cgctgcctgg
58680 tagaaaatgg agctgcagtt actgaccacc aggcagagag aggtgggtcc
ctgtcccagc 58740 ctcagccacc actctgccta agctgtgggg actgagggcg
ctgtcgttag ctgactgcag 58800 aaggtgagca cacgctgtag catgttatgt
ttcagatgtc acatgttgtg ttattgtgtc 58860 tttgcagggc cctttgccta
acacatgcgg tcacttttgg gagatggtgt gggagcagaa 58920 aagcaggggt
gtcgtcatgc tcaacagagt gatggagaaa ggttcggtaa gtctcggctt 58980
catttgctgt gtatgtgatc atgcatacca ctccatatag ttaccatttt cgtccagatt
59040 tttaaattat ttttcttgcc tttgtatttc ctttacgtag tatttttatt
taaaaaaatt 59100 aaaacagcag catataaatg catgttggtt gtcaaccagt
taatgaagtg aataaaaggg 59160 aggaggcgga agaactgcac ggacctcttc
gcccccgcct tctcctgtgt ggtgcgtgtg 59220 gcgctccgcc cacctgtgct
gcctgtgcgg ctctcatcac agtgtggagt tgtgtgtgga 59280 gttatggaga
cctgctttta tcttgaaaag caagttctta gtgcatcttc atggtgtctg 59340
attttttggc tggtgagagt gtggctacct ctgcggagct gtgggagcgg ctgactagat
59400 gagatttgcc tccattcagt acctagactc ttgccctgcc acacctcttc
ggagtgagca 59460 ttgacttcag gatgtgtgtc attctaagtt cctgcaactt
ttcaaacacc cctcgggcta 59520 gcgtgtggct gcacggtgtc catttgtgca
ggccaccact cctcttgcat ctgggtctag 59580 ccacctctcc ttcttgactt
accatagttc attttgtacc atgctttcag aatgagcttt 59640 ctcaaatcca
agtctcacca cggttcttcc cagctgaaaa cccttgtgcg gttccctttg 59700
cctcacagga taatacatgg tgtggcttac ggaaccctgc aggtctggcc ctaggcccct
59760 ggacacagac ctctcaccac tcttggaact ttagccagga caaagttttc
tgtttttagt 59820 ttcttaccat gttctctggg ccgaggagtc ccagtgccca
cgttcatccc acttgcaggc 59880 acccctggac ggctgccccc agctccccaa
ctgcctgcat tctcccctgc cctcctcact 59940 ctgttggaat agctgagaat
agccgatttc tgggcagccg gcctcctgtg tagactgtcc 60000 tgtgtagact
gtcctgtgta gattgtctgt gtagactgtc ctgtgtagat tgtctgtgta 60060
gactgtcctg tgtagactgt ccatgtaaac tgtcctgtgt agattgtctg tgtagactgt
60120 cctgtgtaga ctgtcctgtg tagattgtct gtgtagactg tctctgtaga
ccgtcgtgta 60180 tagactgtcc tgtgtagact gtctctgtag accgtcgtgt
atagactgtc ctgtgtagac 60240 tgtctctgta gaccgtcctg tgtagattgt
ctgtgtagac catcctgtgt agaccatccc 60300 atttagacca tctgcctgtg
caggcgcagg ccagtgttca gcagggccac aggctcctcg 60360 gcctccctgc
cctcgctgct ccccaacact gccaaccctg ctgcggggtc caggaggaga 60420
tgggctgagg atcgtggaga ccagcaggag cgtgtggccc aggagcaggg aactgggtgt
60480 ccttgggcct tgccaggtcc aggctcagct aggacacggc tctcacagct
gtcctggttg 60540 cctccggcca cagaagaagg tgagggctcc agagaggcca
cctttccaaa aaaagcacag 60600 tcatggccct agaatgtaaa aaatccaagt
gttaagaagg aacacatcaa aggaaacttc 60660 agcagtgaaa acttgaagca
ttaaccacga agcctctgcc tccaccacac acaaagaaac 60720 ggctttagtt
actcgcagaa agtcttcctc ttaggacagc gcgtgtttaa aatcataggg 60780
gtttggtttg ttttgttttg gggttggggt tttttggggg ttttttaccc ttgcctactt
60840 tttaaaaaat gaaagtgttt atttgcccaa caataacaga cagggagctt
gcctaagtgt 60900 tctgttgatg atataatgta tcttgtctta gaaaaaaact
ttttcagtga aaggtggttt 60960 ttaaattttt tcttccctcc ttagtagctt
gattagtaaa atgtgaagtt acaaatgtga 61020 agcaaacccc cacccttcac
cactagtcag caattttgag taaagaaaca aagcatcagg 61080 tgctcacagc
acacactgtc ttagagggaa ggggaagcct ggtggcctgt ggaagccttc 61140
agcatagctc catctgcagg cttctgaccc tcagcactac tgacacttgg gctggatcat
61200 tgtctgctag ggatccgggc agggagtggc tgtgctgggc gctgtaggaa
gtttagcagc 61260 atctctggcc tctatccacc agatgccagt agcaccccct
ccccagatgt ggcagtcaga 61320 tgtgtttctg tctagactcc agactttgtc
caacgtcccc tggtaggcca aattgccccc 61380 ggttgagaac caccgctcta
gatggtattg agggttggga attttaaatc aagacattta 61440 ttcagaaatt
accagatata gtagcatttg cttcttattt atttctttgt tgctaagtgt 61500
ttggcaaaac ctctttgctg tgagcacaag gtttgcttta gcaattgttg tcacattaca
61560 gcaaggagtg gtgtccagcg ctgtagttat gtatttgagc agtgtccagt
gctgtagtta 61620 tgtgttccag cctcaccagg ccctgtgctt cattgtctcc
cactcaagac tgaccacaaa 61680 tggcccacag atccactgtg acaacctttc
cctttgggtt actgtggtgg catcgagaac 61740 atggctggtt ggctttgctg
tagtttactg tgataactgt gccagcagtc cctgctttcc 61800 tttgttaagt
atcccattcc actggaggat tacttgggcg tgcagattgg catgaaaagc 61860
aatgtatggt ttgagattgt taaagtttct ttgggatcaa cattttcaat tctgtatcag
61920 cattatccct cccagagggc tggctgggag aaatcatgag aagttacagt
atcttatttg 61980 ctcagctaat ctaattataa atgatccaca cagcttgtgg
taaaaccagc ttttggggag 62040 ttttcattta atgcatactt gtcttctgat
ttccttcctt caccaaatag tgtaggatgc 62100 tccctcttat ttttggcaaa
catgcctgtt atcttttggg accctgggct tcctggaaac 62160 cagttatgca
gaagatgatt gtgtgtgtta gactggggtc atccagatgg ctagagttct 62220
cactggttct gtttaaggat tgactttaga cacctcagtg taggctgcac catggcgtaa
62280 gggttgggat tgttgtttag aagggggaag taagcaaggt gagtttaatt
ggccattgca 62340 gaatctcacc cgtatctccc tcctgaaatc ctcactaaag
ctgccgtttg ctttcaggtg 62400 ctttcatgca caagacactg cattttgtat
cacagggtcc atataattca tttttctctc 62460 gtacttagtt ctctgtgtta
agaattactt acttagttct ctgtgttaat aatttttggc 62520 gaaaccaaat
tacccgtcac agggttactg tagatgtctt tcataggttt tccaaacacc 62580
acttgcccac ttgtttggga aggccccaag gactgtttaa catctgcctt catggtggaa
62640 acagcaacta tgagagatgc tagcatgttg gcactgccat gttcctctgg
taccagccca 62700 agataggact caatttgagg cctggtgaag tactgtgttc
taataaaaat ccatctactt 62760 ttcatggccg tatatatcaa tgtaataggg
taactggaaa tgtgatcttg tgccttttaa 62820 aaattttgtg tgtttaaaac
aaaaatttct attggaaatg acagagcata gcttgttgct 62880 gtagacacct
gagagtcctt aaaaataaat attgggttat tgacacttag ttgcatgaca 62940
gaattcctca cttgtacagt tccaaagtct tagtctttac ccagattaca gagggttatt
63000 aagcattagg tttggttttg aaagtgagtg cttgctgtct ggaggtgagc
tttaagactc 63060 gtctgccctg cttatgagat gaggaagggt ggcctcttcc
tcctgcattt ctgttcttcg 63120 cttccttctc tgtctgctca ctctgtggaa
tgcccacccc agcacgggtg gggtggaacc 63180 tgtcagatca gtctcttgtt
tctggggtct tgaggcatta taagatctag ttgttagaag 63240 tgtgggatta
attcatcttt tcacattctt ctaagttcct gcttttagct gccacaccca 63300
ctttggctaa gtgggggtct tgccatgtaa ttagcgcctc catgccaagt ggcagaattg
63360 cttcaatggt gacagattgt ccccattcaa gagttcactt ttggcaactc
atcattgatc 63420 caggaaggtg acatggatga aactggctaa gacttcagac
aggcttgtgt ccagactctt 63480 gagaaagctc tgttggcttc tggtctggca
ctgtgaagtt tgctgtgatg ctggcaccac 63540 aacctggtgt ttcctaattt
gtttctccca cattttgctt tggttttgtc ttttgggcag 63600 cttccagctc
cagtagagca ggaccaatag gcatttgtgg ttctatattc accctcctca 63660
cgtgcttcct ggctcctcat tgcccccaga tgatgccaca ggtccctggg cctgctgcca
63720 gtcgtctgtg atctgggcct ctgctggccc cttctccagc tgctcttttc
agcctcttat 63780 ttgcagtcac tgcctaggaa atcctagtca tccttcaaaa
cctgcctctt gcacagagct 63840 ttctctgatc tctcttttct gtaaccttgg
ctgacctgaa acatttccct cttctgaatt 63900 cctgctgcat gtccgtagca
tttccccctc agccctcccc catagtccac cttgtcactg 63960 ctgggcacag
cagtgtcttc tgacagacag ctggccctga agtggttccc ttcacccaca 64020
ccatcctttg ccccagagga ggtattgagt gggtcagtgc acgtgaactg ccagtgtcat
64080 ttgccaaaga gctgttgaca cacgctgaca tttcttttgc tgaaaatcat
aagggctttg 64140 agcttccctc tgtccaggca catggtcagg ctgacccggt
agctctgccc ctgctgacct 64200 gccatttttg tccacaacag ttatccatga
gcagaaacat ttgtgtaact gaggcagaaa 64260 cttagttcaa gtaaaatgtc
actaaattcg agtcagtttt tgtcttagac cctaaatgaa 64320 accaaatttt
cataaatttt cttgttttaa agaaaaattt aatgagctac atttaaactg 64380
agaacatcag atagtgtctg agattatcaa aatagaacat caaaagtatt tttctgaatg
64440 aactgaacca aaccagaatg aaagggcaag ccctggggag cctgtctcca
agccttctct 64500 gaaagggagt ctgtatttgg tgataactgc tcagcctctc
caaagggcct cacctgctgt 64560 ctctcccagt tttattttta attgcctgtg
agttttctgt gcagggtaag gcacctacat 64620 tctatgccag cagcctgatc
aggtcctggg taatgtttga aatggctaca cagaggagtt 64680 tcaaagcctt
ttgttcaatc tggcttcacc tcgtagacgg tgagaaagcg tcagagccct 64740
gcaggatccc gttgccacgt ttgaccgggg agccgatggg tttggaagtc tgagccctgt
64800 ctgcacaacc tgccccggtc agcagcttcg tgcccccacc cccatctccc
catgaggcag 64860 gcatctgtgc tgaccatggc ttccatgttc agaaaccccc
aggcctttga gttatcatga 64920 agcttgtggg atgtgctcca agcctcctgc
catagaaaaa ctgccatatt gctcacaata 64980 attcactatt atttgtttcc
ccagttaaaa tgcgcacaat actggccaca aaaagaagaa 65040 aaagagatga
tctttgaaga cacaaatttg aaattaacat tgatctctga agatatcaag 65100
tcatattata cagtgcgaca gctagaattg gaaaacctta cagtgagtat agcacacact
65160 tcagcacttc aggcggctac tggttcacat gcctcttcct ttatcccttg
ggtgatatta 65220 cctaatgtca gtgttcctgg cttttgtata ccccgagcaa
gatgtggttt gggcactgtg 65280 gtgagcggag cttacttgtg tacctaccaa
gtgcccaggg agggtggagg ccacagtgct 65340 ctctctgacc tttaacaaca
gttaacacca gttcttaggg aaaggagagt ttcttaccca 65400 aaagactggt
tcctgcttgt gcagctgcag agggactgga gcggcagcct gcaagtccca 65460
gtgaagcatg ctgccttctt tgtggtcctc agtcttcgag tctgaagaga gggaagaagg
65520 ggtatagggg ctcactccag tttcatagct agtgaaagtt ttctgggcca
ggtcttgggt 65580 ttttttgttg tgggaagagt ttataacacc agctacttgc
ttggtaaaag ttggtcttgg 65640 aacatggcaa ggcattgtgg caagcagcac
tgccgctgaa cgcgctgctc ctggggcttt 65700 ggaataattc ccctggatcc
gtaacttggg ggtgttcatg tcattctggg gaacagtgga 65760 gggagtgcgc
ggcagcacct gggggcacca gtgaagagtg gccagccacc aacctctaga 65820
acctaactgg ggtcgaatcc tggccccacc ttactagctc atcacagtgt ctccgtttcc
65880 tcttctgtca aactcaggtt ttgcgagggt tctgggaggt cctatacggg
aagggttagc 65940 agttaccatg ggtgtgtagc acgggcttta tctgaaggga
aggtggagcc gtagggagac 66000 catgtggagt ggggctccag ggctgtgtgg
gtggggaggg atctgcttct gggttacccc 66060 atgcctcccc ttctcaagta
ctacttttta atcatcatgg ctcctgccat tcatttcata 66120 gttgatgtaa
gccaggtgcg gtggctcacg tctttaatcc cagcacttgg ggaggctgag 66180
gccaggagga tcactcgagg ccaggagttc aagaccagct tgggcaacat agtgagaccc
66240 ccgtctctac aaaaaaacaa aaacagttag tcagacatcg tggtgctccc
ctatagtcca 66300 gctactcagg aggctgaggc aggaggattg cttgtgcccg
ggagttcaag gctgcagtga 66360 gctatgcttg caccactgca ctctagcctg
ggtgacagag caagaccctg tctcaaaaat 66420 aaataaataa aaaaaatagt
agaagtaaga tctagaatgt agcacaggtt accaggacgt 66480 aggcaagggg
ttcgggctgc ctggctcttg aggatggtag cagtgcagct gatgtgagtg 66540
ctttctgccc tctggtggtg accgcgccgg agtcaccagc cctgccatag ccctgatggg
66600 gcagagggtt ctgagtacgg tggatggagg tgctttctgg aagattctca
ggagtaacat 66660 gggcagtgtg ttggaatgtg ctagaggatt tatgcagtag
ccttttaaaa gaatgctttt 66720 tagcatttgc aagcctgaca ttaagagtga
cttctgggaa actatttgct tgttgaggga 66780 aactgaattt caacagagca
gaagagctgt gcgctttttg cttggcagag tgaatacagc 66840 cagctcagag
gttttgatgt taggatctgt ttgctccaac agactttgtt tttaaaaggc 66900
ttttctcagc catagctgtc tgttctagca caaggctgga atgagttcct tgtgaaagag
66960 gtgagcaggt gtgagggagg gtgtcagtgg gcggtaaccc acaccttcaa
ggattaaagg 67020 aaaacttgca tttggcatgc ttgcttctta ttcaatttta
aaatacattt taacggccgg 67080 gcacggtggc taacacctgt aatcccagca
ctttgagggg ctgaggtggg tggttcacga 67140 ggccaggggt tcaagaccag
cctggccaag atggtgaaac cccatctcta ctaaaaatac 67200 aaaaaaaaaa
aaaattagcc gggcgtggtg gcgggcacct gtaatcccag ctactcggga 67260
ggctgaggca gagaattgct tgaacccagg aggcggaggt tgcattgagc cgagatcatg
67320 ccactgcatt ccagcctggg cggcagagca agactctgtc tcaaaataat
aataataatt 67380 ttttaaaaat acattttaag tccttttctt ccccacctgc
ctccacccac caaatagaag 67440 aggtatttct tcttctttaa tgtcattaag
gttatatgga taccattttc tagagaggaa 67500 agaatgatgg aattgcctag
tgtgagtcta gcaattatcc taacatacac aaatttctcc 67560 ttgttctgtg
ccaagatact gtatttaata tttaatgaac attaaatatt atttactagt 67620
gtatttaatg gctgaggcag ggttaaatat gtattatttt catcccagca gagttggggg
67680 aggtcctagt aactatgcca tgagctctgt gagggtgagg tggtgtcttt
gcccccgcct 67740 ccctggcaca gtgactggca catgattggc atagtgtgga
cattcgtcaa gtgaaggaag 67800 gcatcatgag cagatctctg gcctgaatcc
ttctgccatc agctgctcgc caggtggccc 67860 tggcactggg ccacagggaa
actctccagg ctggtatggt tcctgtctgt ggctgtcttc 67920 ccgggcccat
gttaggagac tttcacttcc agagcccttt ccctctcagg gccttgctta 67980
ccaagtgact ggttcccatt tactaggagc tcttaggtca ttgaagatgt tgcgtactcc
68040 ccccagtgag ggctgccttt tgatcacagc cgccagaagc ctcaaggaag
gagcagagct 68100 ggaaacagac gccaggccat tgcttctgtt cctctggggc
agacccagcc acggaagaga 68160 cattctggga caagggctgg ggtccacctt
tcaaacgtgt ctgcagcagg ctctcagcat 68220 ggactctctg cctccaaaca
tccacctcct catcggaaaa tggatgggag tgcctgcctg 68280 gagcagctgg
tgggagagcg cagcgccagc acgtaggaca cactcggttc atgggctgat 68340
gccgttcgca ttgactgcct cttcagctgg gtgttgagcc acaccttgga gtcaccagtc
68400 tttggagacc aagtctgcta cttttttctc taaagtgaca atcctctgaa
acctccagat 68460 catcttgaag cccccgtctg aaagttgccc agagccagtg
cctcacctgc tgttccttgt 68520 tcactttttc acgggaggcc ttgcagggct
ttatgacaag attttatggg tggctgccca 68580 gcatcattgt gactcgtgag
acagagagaa accagttgta accatgtaga cagtggaagt 68640 gatagggaga
aaagaggtga ggggactctt caatccgaag ggaaatgaag tctaagcagg 68700
cgcaccctgc aggttcagtg tcaagcccag ggcctggccc cagggtgtgg tatttgttga
68760 ctgggtgtgt ggaccctggg agaaagtctg agaatgaatg ttcctcttag
aggtagagag 68820 tggaaggtga ctctgtgtgt acttggaatt agtgatttct
gtacagatga ttcttttaga 68880 atcatcatga gtatttttct ctttcagacc
caagaaactc gagagatctt acatttccac 68940 tataccacat ggcctgactt
tggagtccct gaatcaccag cctcattctt gaactttctt 69000 ttcaaagtcc
gagagtcagg gtcactcagc ccggagcacg ggcccgttgt ggtgcactgc 69060
agtgcaggca tcggcaggtc tggaaccttc tgtctggctg atacctgcct cttgctggta
69120 aggaggccct cgcgggtgcc ctggggagct cctctacctg ctctgctgtg
atgttttttc 69180 ctaagtagaa actgaagcgc tcctcttcca aaatacagag
actcactgtg ttagtctgtt 69240 tttgcgttac taataaaggc gtacctgaga
ctcggtaatt tgtaaagaaa agaggtttaa 69300 ctggctcccg gttctgcagg
ctgtacaagc atggcaccag catctgctcg gctcctgggg 69360 aggcctcagg
gagcttccag tcatggtgga aggtgaaggg gagcaggagc aagagatggg 69420
ggaggtccca gactcttaac cagctctctt gtgaatgcat tgcctcaggg agggcaccaa
69480 gcctttcatg agggacctgt ccccctgacc cagacacctc ccacccagcc
ccacctccaa 69540 cactagggat cacatttcag catgagattg ggaggggaca
gacatctaac ggtgttatta 69600 acgttgccct tgagaattgg acctggctga
cttatatctc ctctctggct ttcagatgga 69660 caagaggaaa gacccttctt
ccgttgatat caagaaagtg ctgttagaaa tgaggaagtt 69720 tcggatgggg
ctgatccaga cagccgacca gctgcgcttc tcctacctgg ctgtgatcga 69780
aggtgccaaa ttcatcatgg gggactcttc cgtgcaggtc agcattgcct ttgtttgaat
69840 ccaggtgtga ccattttaac ttttttgtct ttgaaggagg ctgtcagttg
taaaagttca 69900 aacaccgtct ggtgtcaggg gaaatagcta cccttcatgt
ttaaaatagc tagaaagttg 69960 tcaaaatgtt caccatgttg cactttgtgc
ctttgaagtg ctcacataga gagcattgat 70020 aggaagacga gactttattt
tcaaaagatt tcatcttcca agtacatggc tgcagccctg 70080 agaggccgag
agcccctcgc caagccgtca cctctgctca tgcaaaggga tttcctgaca 70140
aaccagccga agtgaacact aataggactt cctcttgctg ctctttcaag gatcagtgga
70200 aggagctttc ccacgaggac ctggagcccc cacccgagca tatcccccca
cctccccggc 70260 cacccaaacg aatcctggag ccacacaatg ggaaatgcag
ggagttcttc ccaaatcacc 70320 agtgggtgaa ggaagagacc caggaggata
aagactgccc catcaaggaa gaaaaaggaa 70380 gccccttaaa tgccgcaccc
tacggcatcg aaaggtaata tgattgggtc ccagcttgtt 70440 ggggtgaggg
gaaatgactt tctgttctag aaacacacgc tggtactgaa accctgtgga 70500
tgcagcctcc tgttggcaag cagcgcttcc gcatccttgg ggaacagggc gcgtggacca
70560 cagccactcc actcctggct gctggaggtc cggtattggg cacagggtgg
ccgcaggaca 70620 tgagccactt ctgtgggctt ctagtgccac cttgtggtgc
ttgttggaat gaggggctcg 70680 gagccaccga gtagggtttt tctgcccccc
ctgacgacag cgccctcccc caggtttccg 70740 gacagtcctg aaatgtgatg
tccaggcttg agtgccctca gtccccacag tggtcctttg 70800 gggaatgtaa
ccttttttat gtggtcttga ttaaatccca ttttacttcc ttgcaggtta 70860
acaaccatta ttgagtacct attgatatgt gtggtgtact gagttaacta gaacatgtcc
70920 cctggtctgt gttctagacc atcttgctgg gaaaaaggca gacccaaagc
atattttggt 70980 gggggcccat ggacagtgat gtgatagagg tgtccgctga
ggtggtcagg gaaggctgct 71040 tgcagtaggt ggccgtgcac ggaaagtttg
cagaatgagc aggtgttagt tccagctgga 71100 gatgactgcc ggctgtgccc
ttggtacctg ctttctggag ggaagtttta agacgtgtgc 71160 atacttgacc
cagcagttgt atacatggag aaatttactt tgcagcaact ctcaaaacaa 71220
gcgtgtaaag atgtgtatag gtagttgtgt ttgttgtggc attgtttgta gtagtgaaaa
71280 attagagaca ggccaatgat ataaccaggg acctgatcaa ttatgttctc
tcccggtgtt 71340 gggatattct gtagctctta aagaatgaga tctgggtgta
ctgatgtggc cagacattgc 71400 aattgcagta catgagaagg caaatcatac
agtagtgtgt acaccagtga gtcctccagc 71460 cagataaatc ctcacagtga
ccagtcgccc aggcaccttg tgaaccctac cctgggtgtg 71520 ggtgctatct
gaagtacctg ggggaggggg tgacaagtgg acttcaggct gatgtgggcc 71580
ctggcctggc cctccctcca agcagagggg gctggctcgc tggaaggtta acatcatcca
71640 actctgtcta cacgtggctt gttttttcct agaattcctg ccacaatagc
agcatccttg 71700 ccattcattt tctccaaagt gagtaaccca tctctgccct
ctgattcctc agcatgagtc 71760 aagacactga agttagaagt cgggtcgtgg
ggggaagtct tcgaggtgcc caggctgcct 71820 ccccagccaa aggggagccg
tcactgcccg agaaggacga ggaccatgca ctgagttact 71880 ggaagccctt
cctggtcaac atgtgcgtgg ctacggtcct cacggccggc gcttacctct 71940
gctacagggt atgtttccac tgacagacgc gctggcgaga tgctcgtgtg cagagagcac
72000 tggccgctag cccgatggta ggattcagtt ctgtggtgca tctgagccag
tctcagaaga 72060 aacagatcaa aggtttttaa agtctggaac tgtggaaggg
ctaacaagag aattaaggat 72120 cgatgcactg gggttttaag gagccctctg
gtcccaagaa tataagagtc taatctcagg 72180 gccttaacct attcaggagt
aagtagagaa aatgccaaat acgtctgttt ctctctctct 72240 tttttttttt
attcctttgt ttttggaaaa aaatagagtt acaacacatt gttgttttta 72300
acctttataa aaagcagctt tttgttattt ctggaacaaa aaaaaacaaa gtaggcactt
72360 atgaaacttt ctcataccct taggtgatgt aatcagccat ataatttata
tttgatttcc 72420 cagggaagga atcccaaact tttacgaatg taaactccct
tggagaagag ggttaggacg 72480 ctgttgcgct caagcccccc tcagctgtgt
gcacactgag ccaggacagg gtctttgagc 72540 tttcccacta taagaagaac
agcaacaaaa ggccgtctag aaaaacagaa cctgcctctg 72600 cttctgctca
gggtgtcccc gctgggtttc cattgtcctt tctccattgc tccctcctgt 72660
gacagccatc ttgctcatgt accagccctc atcaccccat ccccataaat gggtgtcctc
72720 gaggcctctg cctgggggtc agaggtcacc acagggtggc cattggcatg
tcaacccgct 72780 gttaattcag agaagtgggc tccacctcat tgggagaagt
gccatttcag cagaaattca 72840 cacgttagac gtgtgttgct gttaagtaag
gggaagagag aggactagcc tcagagctct 72900 ggccatggaa atgacctcct
aagacttttt cgtggtttta aatattttac ctctttccag 72960 gtggcatctg
agtacatcag atggttttgc aaaatgcaaa caattttttc cttggggatg
73020 atttttgggg agagggggct actgtaaaaa ataaaaccaa aacccccttt
gctccctcgg 73080 aggttgaagt tgccgggggg tgtggccggg gtcatgcatg
aggcgacagc tctgcaggtg 73140 cgggtctggg ctcatctgaa ctgtttggtt
tcattccagt tcctgttcaa cagcaacaca 73200 tagcctgacc ctcctccact
ccacctccac ccactgtccg cctctgcccg cagagcccac 73260 gcccgactag
caggcatgcc gcggtaggta agggccgccg gaccgcgtag agagccgggc 73320
cccggacgga cgttggttct gcactaaaac ccatcttccc cggatgtgtg tctcacccct
73380 catcctttta ctttttgccc cttccacttt gagtaccaaa tccacaagcc
attttttgag 73440 gagagtgaaa gagagtacca tgctggcggc gcagagggaa
ggggcctaca cccgtcttgg 73500 ggctcgcccc acccagggct ccctcctgga
gcatcccagg cgggcggcac gccaacagcc 73560 ccccccttga atctgcaggg
agcaactctc cactccatat ttatttaaac aattttttcc 73620 ccaaaggcat
ccatagtgca ctagcatttt cttgaaccaa taatgtatta aaattttttg 73680
atgtcagcct tgcatcaagg gctttatcaa aaagtacaat aataaatcct caggtagtac
73740 tgggaatgga aggctttgcc atgggcctgc tgcgtcagac cagtactggg
aaggaggacg 73800 gttgtaagca gttgttattt agtgatattg tgggtaacgt
gagaagatag aacaatgcta 73860 taatatataa tgaacacgtg ggtatttaat
aagaaacatg atgtgagatt actttgtccc 73920 gcttattctc ctccctgtta
tctgctagat ctagttctca atcactgctc ccccgtgtgt 73980 attagaatgc
atgtaaggtc ttcttgtgtc ctgatgaaaa atatgtgctt gaaatgagaa 74040
actttgatct ctgcttacta atgtgcccca tgtccaagtc caacctgcct gtgcatgacc
74100 tgatcattac atggctgtgg ttcctaagcc tgttgctgaa gtcattgtcg
ctcagcaata 74160 gggtgcagtt ttccaggaat aggcatttgc ctaattcctg
gcatgacact ctagtgactt 74220 cctggtgagg cccagcctgt cctggtacag
cagggtcttg ctgtaactca gacattccaa 74280 gggtatggga agccatattc
acacctcacg ctctggacat gatttaggga agcagggaca 74340 ccccccgccc
cccacctttg ggatcagcct ccgccattcc aagtcaacac tcttcttgag 74400
cagaccgtga tttggaagag aggcacctgc tggaaaccac acttcttgaa acagcctggg
74460 tgacggtcct ttaggcagcc tgccgccgtc tctgtcccgg ttcaccttgc
cgagagaggc 74520 gcgtctgccc caccctcaaa ccctgtgggg cctgatggtg
ctcacgactc ttcctgcaaa 74580 gggaactgaa gacctccaca ttaagtggct
ttttaacatg aaaaacacgg cagctgtagc 74640 tcccgagcta ctctcttgcc
agcattttca cattttgcct ttctcgtggt agaagccagt 74700 acagagaaat
tctgtggtgg gaacattcga ggtgtcaccc tgcagagcta tggtgaggtg 74760
tggataaggc ttaggtgcca ggctgtaagc attctgagct gggcttgttg tttttaagtc
74820 ctgtatatgt atgtagtagt ttgggtgtgt atatatagta gcatttcaaa
atggacgtac 74880 tggtttaacc tcctatcctt ggagagcagc tggctctcca
ccttgttaca cattatgtta 74940 gagaggtagc gagctgctct gctatatgcc
ttaagccaat atttactcat caggtcatta 75000 ttttttacaa tggccatgga
ataaaccatt tttacaaaaa taaaaacaaa aaaagcaagg 75060 tgttttggta
taataccttt tcaggtgtgt gtggatacgt ggctgcatga ccgggtgggt 75120
gggggggagt gtctcagggt cttctgtgac ctcacagaac tgtcagactg tacagttttc
75180 caacttgcca tattcatgat gggtttgcat tttagctgca acaataaaat
ttttttctaa 75240 agaacatgaa tttggggtgc ttcccatttt tttctttgct
taatagagct aaaccaggat 75300 gagtaactcc tgtttctttc tatccctgct
gatgtgaaac agatgttgtc aatcagctgg 75360 ggttagagtt ttccacttct
aagaattaac ctcagcatcc ctgcattgcc agcaccctca 75420 ggctggagcg
ctttccttga ctgtgagctt gttgaacacc ttaggcctca gcccatttcc 75480
ttcccaaatt gacgctttgc ctgtgtaggg ccctcagata acttaacaaa cttaccagtg
75540 ttgtttgaag aacagtgttt tgagttgtaa tctcaaaacc atatccctta
cccaattacc 75600 tgtaagacac aatggttacc acatctcagt acgtaaagtc
cacttgatat agaattgact 75660 tagaaataag acagattagt atagtttttc
atttgtgtac aaaattaaac aatgtaaatt 75720 ccccccaaag tgattttttt
gactttttga agtaattttg gacttgcaaa atgttgccaa 75780 aatagtacga
agagttcccc agtaccctcg aagtttcctc gactgtttca aagctggctg 75840
caggcccagg ctcatgagac tgggaagagg acaggctgtg gtcatgtgga cccacaggg
75899 244 20 DNA Artificial Sequence Antisense Oligonucleotide 244
gcgctcttag ccccgaggcc 20 245 20 DNA Artificial Sequence Antisense
Oligonucleotide 245 ccagggcggc tgctgcgcct 20 246 20 DNA Artificial
Sequence Antisense Oligonucleotide 246 catctccatg acgggccagg 20 247
20 DNA Artificial Sequence Antisense Oligonucleotide 247 ttttccatct
ccatgacggg 20 248 20 DNA Artificial Sequence Antisense
Oligonucleotide 248 actccttttc catctccatg 20 249 20 DNA Artificial
Sequence Antisense Oligonucleotide 249 ttgtcgatct gctcgaactc 20 250
20 DNA Artificial Sequence Antisense Oligonucleotide 250 gacttgtcga
tctgctcgaa 20 251 20 DNA Artificial Sequence Antisense
Oligonucleotide 251 gctcccggac ttgtcgatct 20 252 20 DNA Artificial
Sequence Antisense Oligonucleotide 252 ccagctcccg gacttgtcga 20 253
20 DNA Artificial Sequence Antisense Oligonucleotide 253 tccactgatc
ctgcacggaa 20 254 20 DNA Artificial Sequence Antisense
Oligonucleotide 254 ccttccactg atcctgcacg 20 255 20 DNA Artificial
Sequence Antisense Oligonucleotide 255 atgcctgcta gtcgggcgtg 20 256
20 DNA Artificial Sequence Antisense Oligonucleotide 256 cgggtgtagg
ccccttccct 20 257 20 DNA Artificial Sequence Antisense
Oligonucleotide 257 atggagtgga gagttgctcc 20 258 20 DNA Artificial
Sequence Antisense Oligonucleotide 258 ttgtactttt tgataaagcc 20 259
20 DNA Artificial Sequence Antisense Oligonucleotide 259 cagtactggt
ctgacgcagc 20 260 20 DNA Artificial Sequence Antisense
Oligonucleotide 260 tctcacgtta cccacaatat 20 261 20 DNA Artificial
Sequence Antisense Oligonucleotide 261 tttcttatta aatacccacg 20 262
20 DNA Artificial Sequence Antisense Oligonucleotide 262 aagtaatctc
acatcatgtt 20 263 20 DNA Artificial Sequence Antisense
Oligonucleotide 263 ttcagcaaca ggcttaggaa 20 264 20 DNA Artificial
Sequence Antisense Oligonucleotide 264 gacaatgact tcagcaacag 20 265
20 DNA Artificial Sequence Antisense Oligonucleotide 265 tgcctattcc
tggaaaactg 20 266 20 DNA Artificial Sequence Antisense
Oligonucleotide 266 ggaagtcact agagtgtcat 20 267 20 DNA Artificial
Sequence Antisense Oligonucleotide 267 ccaggacagg ctgggcctca 20 268
20 DNA Artificial Sequence Antisense Oligonucleotide 268 ctgctgtacc
aggacaggct 20 269 20 DNA Artificial Sequence Antisense
Oligonucleotide 269 tggaatgtct gagttacagc 20 270 20 DNA Artificial
Sequence Antisense Oligonucleotide 270 agagtgttga cttggaatgg 20 271
20 DNA Artificial Sequence Antisense Oligonucleotide 271 gctcaagaag
agtgttgact 20 272 20 DNA Artificial Sequence Antisense
Oligonucleotide 272 tgcctctctt ccaaatcacg 20 273 20 DNA Artificial
Sequence Antisense Oligonucleotide 273 tgtttttcat gttaaaaagc 20 274
20 DNA Artificial Sequence Antisense Oligonucleotide 274 tcccaccaca
gaatttctct 20 275 20 DNA Artificial Sequence Antisense
Oligonucleotide 275 gctctgcagg gtgacacctc 20 276 20 DNA Artificial
Sequence Antisense Oligonucleotide 276 aggaggttaa accagtacgt 20 277
20 DNA Artificial Sequence Antisense Oligonucleotide 277 ggtggagagc
cagctgctct 20 278 20 DNA Artificial Sequence Antisense
Oligonucleotide 278 tattggctta aggcatatag 20 279 20 DNA Artificial
Sequence Antisense Oligonucleotide 279 gacctgatga gtaaatattg 20 280
20 DNA Artificial Sequence Antisense Oligonucleotide 280 ttcttcatgt
caaccggcag 20 281 20 DNA Artificial Sequence Antisense
Oligonucleotide 281 gccccgaggc ccgctgcaat 20 282 20 DNA Artificial
Sequence Antisense Oligonucleotide 282 tagtgaacta ttgttacaac 20 283
20 DNA Artificial Sequence Antisense Oligonucleotide 283 tgctaagcca
cttctaatca 20 284 20 DNA Artificial Sequence Antisense
Oligonucleotide 284 caggattcta agttattaaa 20 285 20 DNA Artificial
Sequence Antisense Oligonucleotide 285 tgggcaggat ggctctggta 20 286
20 DNA Artificial Sequence Antisense Oligonucleotide 286 tacaatacta
tctgtgacta 20 287 20 DNA Artificial Sequence Antisense
Oligonucleotide 287 gatacttaca gggactgacg 20 288 20 DNA Artificial
Sequence Antisense Oligonucleotide 288 aaccctgagg cgaaaggagt 20 289
20 DNA Artificial Sequence Antisense Oligonucleotide 289 ccccaggtca
ctaaaattaa 20 290 20 DNA Artificial Sequence Antisense
Oligonucleotide 290 aaagcaaagg tgagttggtg 20 291 20 DNA Artificial
Sequence Antisense Oligonucleotide 291 gctcaattat taaaccactt 20 292
20 DNA Artificial Sequence Antisense Oligonucleotide 292 agtcctcaag
aagtcacttt 20 293 20 DNA Artificial Sequence Antisense
Oligonucleotide 293 gaaagcaggg actgctggca 20 294 20 DNA Artificial
Sequence Antisense Oligonucleotide 294 aaaactggga gagacagcag 20 295
20 DNA Artificial Sequence Antisense Oligonucleotide 295 acatggaagc
catggtcagc 20 296 20 DNA Artificial Sequence Antisense
Oligonucleotide 296 attgctagac tcacactagg 20 297 20 DNA Artificial
Sequence Antisense Oligonucleotide 297 ggctgtgatc aaaaggcagc 20 298
20 DNA Artificial Sequence Antisense Oligonucleotide 298 cactggctct
gggcaacttt 20 299 20 DNA Artificial Sequence Antisense
Oligonucleotide 299 gctgggcagc cacccataaa 20 300 20 DNA Artificial
Sequence Antisense Oligonucleotide 300 agtcccctca cctcttttct 20 301
20 DNA Artificial Sequence Antisense Oligonucleotide 301 cctccttacc
agcaagaggc 20 302 20 DNA Artificial Sequence Antisense
Oligonucleotide 302 tgtattttgg aagaggagcg 20 303 20 DNA Artificial
Sequence Antisense Oligonucleotide 303 acagactaac acagtgagtc 20 304
20 DNA Artificial Sequence Antisense Oligonucleotide 304 acaaattacc
gagtctcagg 20 305 20 DNA Artificial Sequence Antisense
Oligonucleotide 305 tcatgaaagg cttggtgccc 20 306 20 DNA Artificial
Sequence Antisense Oligonucleotide 306 ttggaagatg aaatcttttg 20 307
20 DNA Artificial Sequence Antisense Oligonucleotide 307 agccatgtac
ttggaagatg 20 308 20 DNA Artificial Sequence Antisense
Oligonucleotide 308 cgagcccctc attccaacaa 20 309 20 DNA Artificial
Sequence Antisense Oligonucleotide 309 cacctcagcg gacacctcta 20 310
20 DNA Artificial Sequence Antisense Oligonucleotide 310 gaaacatacc
ctgtagcaga 20 311 20 DNA Artificial Sequence Antisense
Oligonucleotide 311 cagagggctc cttaaaaccc 20 312 20 DNA Artificial
Sequence Antisense Oligonucleotide 312 attcgtaaaa gtttgggatt 20 313
20 DNA Artificial Sequence Antisense Oligonucleotide 313 ccctcttctc
caagggagtt 20 314 20 DNA Artificial Sequence Antisense
Oligonucleotide 314 ggaatgaaac caaacagttc 20 315 20 DNA Artificial
Sequence Antisense Oligonucleotide 315 aaatggttta ttccatggcc 20 316
20 DNA Artificial Sequence Antisense Oligonucleotide 316 aaaaatttta
ttgttgcagc 20 317 20 DNA Artificial Sequence Antisense
Oligonucleotide 317 ccggtcatgc agccacgtat 20 318 20 DNA Artificial
Sequence Antisense Oligonucleotide 318 gttggaaaac tgtacagtct 20 319
20 DNA Artificial Sequence Antisense Oligonucleotide 319 attttattgt
tgcagctaaa 20 320 20 DNA Artificial Sequence Antisense
Oligonucleotide 320 cgcctccttc tcggcccact 20 321 20 DNA Artificial
Sequence Antisense Oligonucleotide 321 gggcggctgc tgcgcctcct 20 322
20 DNA Artificial Sequence Antisense Oligonucleotide 322 gtggatttgg
tactcaaagt 20 323 20 DNA Artificial Sequence Antisense
Oligonucleotide 323 aaatggcttg tggatttggt 20 324 20 DNA Artificial
Sequence Antisense Oligonucleotide 324 atggtactct ctttcactct 20 325
20 DNA Artificial Sequence Antisense Oligonucleotide 325 gccagcatgg
tactctcttt 20 326 20 DNA Artificial Sequence Antisense
Oligonucleotide 326 gagagttgct ccctgcagat 20 327 20 DNA Artificial
Sequence Antisense Oligonucleotide 327 ggagtggaga gttgctccct 20 328
20 DNA Artificial Sequence Antisense Oligonucleotide 328 ccttgatgca
aggctgacat 20 329 20 DNA Artificial Sequence Antisense
Oligonucleotide 329 aaagcccttg atgcaaggct 20 330 20 DNA Artificial
Sequence Antisense Oligonucleotide 330 agtactacct gaggatttat 20 331
20 DNA Artificial Sequence Antisense Oligonucleotide 331 ttccattccc
agtactacct 20 332 20 DNA Artificial Sequence Antisense
Oligonucleotide 332 ccatggcaaa gccttccatt 20 333 20 DNA Artificial
Sequence Antisense Oligonucleotide 333 caggcccatg gcaaagcctt 20 334
20 DNA Artificial Sequence Antisense Oligonucleotide 334 caactgctta
caaccgtcct 20 335 20 DNA Artificial Sequence Antisense
Oligonucleotide 335 ccacgtgttc attatatatt 20 336 20 DNA Artificial
Sequence Antisense Oligonucleotide 336 ttaaataccc acgtgttcat 20 337
20 DNA Artificial Sequence Antisense Oligonucleotide 337 taagcgggac
aaagtaatct 20 338 20 DNA Artificial Sequence Antisense
Oligonucleotide 338 cagataacag ggaggagaat 20 339 20 DNA Artificial
Sequence Antisense Oligonucleotide 339 gagaactaga tctagcagat 20 340
20 DNA Artificial Sequence Antisense Oligonucleotide 340 agtgattgag
aactagatct 20 341 20 DNA Artificial Sequence Antisense
Oligonucleotide 341 gacacaagaa gaccttacat 20 342 20 DNA Artificial
Sequence Antisense Oligonucleotide 342 ctcatttcaa gcacatattt 20 343
20 DNA Artificial Sequence Antisense Oligonucleotide 343 ggcaggttgg
acttggacat 20 344 20 DNA Artificial Sequence Antisense
Oligonucleotide 344 aaccacagcc atgtaatgat 20 345 20 DNA Artificial
Sequence Antisense Oligonucleotide 345 ttgctgagcg acaatgactt
20 346 20 DNA Artificial Sequence Antisense Oligonucleotide 346
ctggaaaact gcaccctatt 20 347 20 DNA Artificial Sequence Antisense
Oligonucleotide 347 gctgggcctc accaggaagt 20 348 20 DNA Artificial
Sequence Antisense Oligonucleotide 348 ttacagcaag accctgctgt 20 349
20 DNA Artificial Sequence Antisense Oligonucleotide 349 acccttggaa
tgtctgagtt 20 350 20 DNA Artificial Sequence Antisense
Oligonucleotide 350 ttcccatacc cttggaatgt 20 351 20 DNA Artificial
Sequence Antisense Oligonucleotide 351 atatggcttc ccataccctt 20 352
20 DNA Artificial Sequence Antisense Oligonucleotide 352 gtgtgaatat
ggcttcccat 20 353 20 DNA Artificial Sequence Antisense
Oligonucleotide 353 cctgcttccc taaatcatgt 20 354 20 DNA Artificial
Sequence Antisense Oligonucleotide 354 gtgtccctgc ttccctaaat 20 355
20 DNA Artificial Sequence Antisense Oligonucleotide 355 cggaggctga
tcccaaaggt 20 356 20 DNA Artificial Sequence Antisense
Oligonucleotide 356 caggtgcctc tcttccaaat 20 357 20 DNA Artificial
Sequence Antisense Oligonucleotide 357 gtggtttcca gcaggtgcct 20 358
20 DNA Artificial Sequence Antisense Oligonucleotide 358 gctgtttcaa
gaagtgtggt 20 359 20 DNA Artificial Sequence Antisense
Oligonucleotide 359 ggaccgtcac ccaggctgtt 20 360 20 DNA Artificial
Sequence Antisense Oligonucleotide 360 caggctgcct aaaggaccgt 20 361
20 DNA Artificial Sequence Antisense Oligonucleotide 361 accatcaggc
cccacagggt 20 362 20 DNA Artificial Sequence Antisense
Oligonucleotide 362 gttccctttg caggaagagt 20 363 20 DNA Artificial
Sequence Antisense Oligonucleotide 363 gtggaggtct tcagttccct 20 364
20 DNA Artificial Sequence Antisense Oligonucleotide 364 ccacttaatg
tggaggtctt 20 365 20 DNA Artificial Sequence Antisense
Oligonucleotide 365 agctacagct gccgtgtttt 20 366 20 DNA Artificial
Sequence Antisense Oligonucleotide 366 ccacgagaaa ggcaaaatgt 20 367
20 DNA Artificial Sequence Antisense Oligonucleotide 367 gaatttctct
gtactggctt 20 368 20 DNA Artificial Sequence Antisense
Oligonucleotide 368 ccacagaatt tctctgtact 20 369 20 DNA Artificial
Sequence Antisense Oligonucleotide 369 gaatgttccc accacagaat 20 370
20 DNA Artificial Sequence Antisense Oligonucleotide 370 gcctggcacc
taagccttat 20 371 20 DNA Artificial Sequence Antisense
Oligonucleotide 371 atgcttacag cctggcacct 20 372 20 DNA Artificial
Sequence Antisense Oligonucleotide 372 ctacatacat atacaggact 20 373
20 DNA Artificial Sequence Antisense Oligonucleotide 373 tttgaaatgc
tactatatat 20 374 20 DNA Artificial Sequence Antisense
Oligonucleotide 374 ggataggagg ttaaaccagt 20 375 20 DNA Artificial
Sequence Antisense Oligonucleotide 375 gccagctgct ctccaaggat 20 376
20 DNA Artificial Sequence Antisense Oligonucleotide 376 ctacctctct
aacataatgt 20 377 20 DNA Artificial Sequence Antisense
Oligonucleotide 377 gctcgctacc tctctaacat 20 378 20 DNA Artificial
Sequence Antisense Oligonucleotide 378 aggcatatag cagagcagct 20 379
20 DNA Artificial Sequence Antisense Oligonucleotide 379 gtcaaccggc
agccggaact 20 380 20 DNA Artificial Sequence Antisense
Oligonucleotide 380 cctgcagcta ccgccgccct 20 381 20 DNA Artificial
Sequence Antisense Oligonucleotide 381 cgctgcaatc cccgacccct 20 382
20 DNA Artificial Sequence Antisense Oligonucleotide 382 accaaaacac
cttgcttttt 20 383 20 DNA Artificial Sequence Antisense
Oligonucleotide 383 gtattatacc aaaacacctt 20 384 20 DNA Artificial
Sequence Antisense Oligonucleotide 384 cacacacctg aaaaggtatt 20 385
20 DNA Artificial Sequence Antisense Oligonucleotide 385 acccggtcat
gcagccacgt 20 386 20 DNA Artificial Sequence Antisense
Oligonucleotide 386 gtgaggtcac agaagaccct 20 387 20 DNA Artificial
Sequence Antisense Oligonucleotide 387 gtacagtctg acagttctgt 20 388
20 DNA Artificial Sequence Antisense Oligonucleotide 388 atggcaagtt
ggaaaactgt 20 389 20 DNA Artificial Sequence Antisense
Oligonucleotide 389 aatgcaaacc catcatgaat 20 390 21 DNA Artificial
Sequence Antisense strand of siRNA duplex 390 uucaugucgg auauccuggt
a 21 391 21 DNA Artificial Sequence Antisense strand of siRNA
duplex 391 ucacuggcuu caugucggat a 21 392 21 DNA Artificial
Sequence Antisense strand of siRNA duplex 392 gguaagaaug uaacuccuut
g 21 393 21 DNA Artificial Sequence Antisense strand of siRNA
duplex 393 cuucagagau caauguuaat t 21 394 21 DNA Artificial
Sequence Antisense strand of siRNA duplex 394 uagcugucgc acuguauaat
a 21 395 21 DNA Artificial Sequence Antisense strand of siRNA
duplex 395 ccaauucuag cugucgcact g 21 396 20 RNA Artificial
Sequence Antisense strand of siRNA duplex 396 uugauaaagc ccuugaugca
20 397 20 RNA Artificial Sequence Antisense strand of siRNA duplex
397 ggucaugcac aggcagguug 20 398 20 RNA Artificial Sequence
Antisense strand of siRNA duplex 398 gaagaagggu cuuuccucuu 20 399
20 DNA Artificial Sequence Antisense Oligonucleotide 399 ctgctagcct
ctggatttga 20 400 20 DNA Artificial Sequence Antisense
Oligonucleotide 400 tagtgcggac ctacccacga 20 401 21 DNA Artificial
Sequence Antisense strand of siRNA duplex 401 gggacgaacu gguguaaugt
t 21 402 21 DNA Artificial Sequence Antisense strand of siRNA
duplex 402 cuucuggcau ccgguuuagt t 21 403 21 DNA Artificial
Sequence Sense strand of siRNA duplex 403 ccaggauauc cgacaugaag c
21 404 21 DNA Artificial Sequence Sense strand of siRNA duplex 404
uccgacauga agccagugac t 21 405 21 DNA Artificial Sequence Sense
strand of siRNA duplex 405 aaggaguuac auucuuaccc a 21 406 21 DNA
Artificial Sequence Sense strand of siRNA duplex 406 uuaacauuga
ucucugaaga t 21 407 21 DNA Artificial Sequence Sense strand of
siRNA duplex 407 uuauacagug cgacagcuag a 21 408 21 DNA Artificial
Sequence Sense strand of siRNA duplex 408 gugcgacagc uagaauugga a
21 409 20 RNA Artificial Sequence Sense strand of siRNA duplex 409
ugcaucaagg gcuuuaucaa 20 410 20 RNA Artificial Sequence Sense
strand of siRNA duplex 410 caaccugccu gugcaugacc 20 411 20 RNA
Artificial Sequence Sense strand of siRNA duplex 411 ucucuugcca
gcauuuucac 20 412 21 DNA Artificial Sequence Sense strand of siRNA
duplex 412 cauuacacca guucguccct t 21 413 21 DNA Artificial
Sequence Sense strand of siRNA duplex 413 cuaaaccgga ugccagaagt t
21 414 21 DNA Artificial Sequence Antisense strand of siRNA duplex
414 cgagaggcgg acgggaccgt t 21 415 21 DNA Artificial Sequence Sense
strand of siRNA duplex 415 cggtcccgtc cgcctctcgt t 21
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