U.S. patent application number 10/619253 was filed with the patent office on 2005-02-24 for antisense modulation of stearoyl-coa desaturase expression.
This patent application is currently assigned to Isis Pharmaceuticals, Inc.. Invention is credited to Crooke, Rosanne, Graham, Mark J..
Application Number | 20050043256 10/619253 |
Document ID | / |
Family ID | 34135466 |
Filed Date | 2005-02-24 |
United States Patent
Application |
20050043256 |
Kind Code |
A1 |
Crooke, Rosanne ; et
al. |
February 24, 2005 |
Antisense modulation of stearoyl-CoA desaturase expression
Abstract
Antisense compounds, compositions and methods are provided for
modulating the expression of stearoyl-CoA desaturase. The
compositions comprise antisense compounds, particularly antisense
oligonucleotides, targeted to nucleic acids encoding stearoyl-CoA
desaturase. Methods of using these compounds for modulation of
stearoyl-CoA desaturase expression and for treatment of diseases
associated with expression of stearoyl-CoA desaturase are
provided.
Inventors: |
Crooke, Rosanne; (Carlsbad,
CA) ; Graham, Mark J.; (San Clemente, CA) |
Correspondence
Address: |
LAW OFFICES OF JAMES E. WALTON, PLLC
1169 N. BURLESON BLVD.
SUITE 107-328
BURLESON
TX
76028
US
|
Assignee: |
Isis Pharmaceuticals, Inc.
Carlsbad
CA
|
Family ID: |
34135466 |
Appl. No.: |
10/619253 |
Filed: |
July 15, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10619253 |
Jul 15, 2003 |
|
|
|
09918187 |
Jul 30, 2001 |
|
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Current U.S.
Class: |
514/44A ;
536/23.1 |
Current CPC
Class: |
C12Y 114/19001 20130101;
A61P 43/00 20180101; C12N 2310/341 20130101; C12N 2310/3525
20130101; C12N 2310/321 20130101; C12N 2310/346 20130101; C12N
2310/11 20130101; C12N 2310/3341 20130101; C12N 9/0083 20130101;
A61P 3/10 20180101; C12N 2310/321 20130101; A61K 38/00 20130101;
Y02P 20/582 20151101; C12N 2310/315 20130101; C12N 15/1137
20130101; A61K 45/06 20130101 |
Class at
Publication: |
514/044 ;
536/023.1 |
International
Class: |
A61K 048/00; C07H
021/02 |
Claims
1. A compound 8 to 50 nucleobases in length targeted to a nucleic
acid molecule encoding human stearoyl-CoA desaturase, wherein the
compound specifically hybridizes with a nucleic acid molecule
encoding human stearoyl-CoA desaturase and inhibits the expression
of human stearoyl-CoA desaturase.
2. The compound according to claim 1, which is an antisense
oligonucleotide.
3. The compound according to claim 2, which hybridizes to a
sequence within a nucleic acid molecule encoding human stearoyl-CoA
desaturase SEQ ID NO: 3, provided that said sequence does not
include nucleotide sequences spanning 70 through nucleotide 91,
nucleotide 242 through nucleotide 262, or nucleotide 860 through
nucleotide 882 of SEQ ID NO: 3.
4. The compound according to claim 2, wherein the antisense
oligonucleotide comprises at least one modified internucleoside
linkage.
5. The compound according to claim 4, wherein the modified
internucleoside linkage is a phosphorothioate linkage.
6. The compound according to claim 2, wherein the antisense
oligonucleotide comprises at least one modified sugar moiety.
7. The compound according to claim 6, wherein the modified sugar
moiety is a 2'-O-methoxyethyl sugar moiety.
8. The compound according to claim 2, wherein the antisense
oligonucleotide comprises at least one modified nucleobase.
9. The compound according to claim 8, wherein the modified
nucleobase is a 5-methylcytosine.
10. The compound according to claim 2, wherein the antisense
oligonucleotide is a chimeric oligonucleotide.
11. The compound according to claim 2, wherein said antisense
oligonucleotide inhibits expression of said human stearoyl CoA
desaturase by at least 10% in a suitable assay.
12. The compound according to claim 2, wherein said antisense
oligonucleotide inhibits expression of said human stearoyl CoA
desaturase by at least 90% in a suitable assay.
13. The compound according to claim 1, wherein said compound
comprises a sequence selected from the group consisting of SEQ ID
NOS: 18, 19, 20, 23, 25, 26, 29, 30, 31, 33, 39, 43, 44, 83, 84,
85, 87, 88, 89, 91, 93, 94, 95, 97, 98, 100, 101, 102, 103, 105,
107, 108, 109, 110, 112, 113, 114, 115, 117, 118, 119, 120, 124,
126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,
139, 140, 141, 149, 150, 153, 154, 157, 158, 159, 160, 164, 165,
167, 168, 169, 188, 189, 197, 204, 207 and 210.
14. The compound according to claim 1, wherein said compound
comprises an antisense nucleic acid molecule that is specifically
hybridizable with a 5'-untranslated region (5' UTR) of stearoyl CoA
desaturase.
15. The compound according to claim 1, wherein said compound
comprises an antisense nucleic acid molecule that is specifically
hybridizable with a start codon region of stearoyl CoA
desaturase.
16. The compound according to claim 1, wherein said compound
comprises an antisense nucleic acid molecule that is specifically
hybridizable with a coding region of stearoyl CoA desaturase.
17. The compound according to claim 1, wherein said compound
comprises an antisense nucleic acid molecule that is specifically
hybridizable with a stop codon region of stearoyl CoA
desaturase.
18. The compound according to claim 1, wherein said compound
comprises an antisense nucleic acid molecule that is specifically
hybridizable with a 3'-untranslated region (3' UTR) of stearoyl CoA
desaturase.
19. A compound 8 to 50 nucleobases in length which specifically
hybridizes with at least an 8-nucleobase portion of an active site
on a nucleic acid molecule encoding human stearoyl-CoA
desaturase.
20. The compound according to claim 19, wherein said portion of
said active site falls outside of nucleotide sequences spanning 70
through nucleotide 91, nucleotide 242 through nucleotide 262, or
nucleotide 860 through nucleotide 882 of a nucleic acid molecule
encoding human stearoyl-CoA desaturase SEQ ID NO: 3.
21. A composition comprising the compound of claim 1 and a
pharmaceutically acceptable carrier or diluent.
22. The composition according to claim 21, further comprising a
colloidal dispersion system.
23. The composition according to claim 12, wherein the compound is
an antisense oligonucleotide.
24. A method of inhibiting the expression of human stearoyl-CoA
desaturase in cells or tissues comprising contacting the cells or
tissues with the compound of claim 1 so that expression of human
stearoyl-CoA desaturase is inhibited.
25. A method of treating a human having a disease or condition
associated with human stearoyl-CoA desaturase comprising
administering to the human a therapeutically or prophylactically
effective amount of the compound of claim 1 so that expression of
human stearoyl-CoA desaturase is inhibited.
26. The method according to claim 25, wherein the condition
involves abnormal lipid metabolism.
27. The method according to claim 25, wherein the condition
involves abnormal cholesterol metabolism.
28. The method according to claim 25, wherein the condition is
atherosclerosis.
29. The method according to claim 25, wherein the disease is
cardiovascular disease.
30. A method of screening for an antisense compound, the method
comprising the steps of: a. contacting a preferred target region of
a nucleic acid molecule encoding human stearoyl-CoA desaturase with
one or more candidate antisense compounds, said candidate antisense
compounds comprising at least an 8-nucleobase portion which is
complementary to said preferred target region, and b. selecting for
one or more candidate antisense compounds which inhibit the
expression of a nucleic acid molecule encoding human stearoyl-CoA
desaturase.
31. A method for improving liver function in an animal having
elevated liver enzymes comprising administering to said animal a
therapeutically or prophylactically effective amount of the
compound of claim 1 so that expression of stearoyl CoA desaturase
is inhibited and thereby lowers liver enzyme levels.
32. A method for treating an obese animal comprising administering
to said animal a therapeutically or prophylactically effective
amount of the compound of claim 1 so that expression of stearoyl
CoA desaturase is inhibited, thereby reducing said animal's weight
and appetite.
33. A duplexed antisense compound comprising: (a) a nucleobase
sequence 8 to 80 nucleobases in length targeted to a nucleic acid
molecule encoding stearoyl CoA desaturase with at least one natural
or modified nucleobase forming an overhang at a terminus of said
sequence; and (b) the complementary sequence of said sequence (a)
having optionally at least one natural or modified nucleobase
forming an overhang at a terminus of said complementary sequence;
wherein said sequences (a) and (b), when hybridized, have at least
one single-stranded overhang at at least one of terminus of said
hybridized duplex, and wherein said duplex when interacted with a
nucleic acid molecule encoding stearoyl CoA desaturase can modulate
the expression of said reductase.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of pending U.S.
patent application Ser. No. 09/918,187, filed Jul. 30, 2001.
BACKGROUND OF THE INVENTION
[0002] Saturated fatty acids are known to be the precursors of
unsaturated fatty acids in higher organisms. However, the control
mechanisms that govern the conversion of saturated fatty acids to
unsaturated fatty acids are not well understood. The relative
amounts of different fatty acids have effects on the physical
properties of membranes. Furthermore, regulation of unsaturated
fatty acids is important because they play a role in cellular
activity, metabolism and nuclear events that govern gene
transcription.
[0003] A critical committed step in the biosynthesis of
mono-unsaturated fatty acids is the introduction of the first
cis-double bond in the delta-9 position (between carbons 9 and 10).
This oxidative reaction is catalyzed by stearoyl-CoA desaturase
(SCD, also known as delta-9-desaturase) and involves cytochrome
b.sub.5, NADH (P)-cytochrome b.sub.5 reductase and molecular oxygen
(Ntambi, J. Lipid Res., 1999, 40, 1549-1558). Although the
insertion of the double bond occurs in several different
methylene-interrupted fatty acyl-CoA substrates, the preferred
substrates are palmitoyl- and stearoyl-CoA, which are converted to
palmitoleoyl- and oleoyl-CoA respectively (Ntambi, J. Lipid Res.,
1999, 40, 1549-1558).
[0004] It has been recognized that, regardless of diet, the major
storage fatty acids in human adipose tissue are oleic acid, an 18
carbon unsaturated fatty acid, and palmitoleic acid, a 16 carbon
unsaturated fatty acid (Ntambi, J. Lipid Res., 1999, 40,
1549-1558). During the de novo synthesis of fatty acids, the fatty
acid synthase enzyme stops at palmitoleic acid but the end product
of the pathway is usually oleic acid (Ntambi, J. Lipid Res., 1999,
40, 1549-1558).
[0005] The stearoyl-CoA desaturase gene was partially characterized
in 1994 via isolation of a 0.76 kb partial cDNA from human adipose
tissue (Li et al., Int. J. Cancer, 1994, 57, 348-352). Increased
levels of stearoyl-CoA desaturase mRNA were found in colonic and
esophageal carcinomas and in hepatocellular carcinoma (Li et al.,
Int. J. Cancer, 1994, 57, 348-352). The gene was fully
characterized in 1999 and it was found that alternative usage of
polyadenylation sites generates two transcripts of 3.9 and 5.2 kb
(Zhang et al., Biochem. J., 1999, 340, 255-264). Two loci for the
stearoyl-CoA desaturase gene were mapped to chromosomes 10 and 17
and it was determined that the chromosome 17 loci encodes a
transcriptionally inactive pseudogene (Ntambi, J. Lipid Res., 1999,
40, 1549-1558).
[0006] A nucleic acid molecule encoding the human stearoyl-CoA
desaturase and a nucleic acid molecule, which under suitable
conditions, specifically hybridizes to the nucleic acid molecule
encoding the human stearoyl-CoA desaturase, have been described
(Stenn et al., International patent publication WO 00/09754,
2000).
[0007] Stearoyl-CoA desaturase affects the ratio of stearate to
oleate, which in turn, affects cell membrane fluidity. Alterations
of this ratio have been implicated in various disease states
including cardiovascular disease, obesity, non-insulin-dependent
diabetes mellitus, skin disease, hypertension, neurological
diseases, immune disorders and cancer (Ntambi, J. Lipid Res., 1999,
40, 1549-1558). Stearoyl-CoA desaturase has been viewed as a
lipogenic enzyme not only for its key role in the biosynthesis of
monounsaturated fatty acids, but also for its pattern of regulation
by diet and insulin (Ntambi, J. Lipid Res., 1999, 40,
1549-1558).
[0008] The regulation of stearoyl-CoA desaturase is therefore of
considerable physiologic importance and its activity is sensitive
to dietary changes, hormonal imbalance, developmental processes,
temperature changes, metals, alcohol, peroxisomal proliferators and
phenolic compounds (Ntambi, J. Lipid Res., 1999, 40,
1549-1558).
[0009] Animal models have been very useful in investigations of the
regulation of stearoyl-CoA by polyunsaturated fatty acids (PUFAs).
For example, in adipose tissue of lean and obese Zucker rats, a 75%
decrease in stearoyl-CoA desaturase mRNA was observed when both
groups were fed a diet high in PUFAs relative to a control diet
(Jones et al., Am. J. Physiol., 1996, 271, E44-49).
[0010] Similar results have been obtained with tissue culture
systems. In the murine 3T3-L1 adipocyte cell line, arachidonic
linoleic, linolenic, and eicosapentanenoic acids decreased
stearoyl-CoA desaturase expression in a dose-dependent manner
(Sessler et al., J. Biol. Chem., 1996, 271, 29854-29858).
[0011] The molecular mechanisms by which PUFAs regulate
stearoyl-CoA desaturase gene expression in different tissues are
still poorly understood. The current understanding of the
regulatory mechanism involves binding of PUFAs to a putative
PUFA-binding protein, after which repression transcription occurs
via binding of the PUFA-binding protein to a cis-acting PUFA
response element of the stearoyl-CoA desaturase gene (SREBP)
(Ntambi, J. Lipid Res., 1999, 40, 1549-1558; Zhang et al., Biochem.
J., 2001, 357, 183-193).
[0012] Cholesterol has also been identified as a regulator of
stearoyl-CoA desaturase gene expression by a mechanism involving
repression of the maturation of the sterol regulatory element
binding protein (Bene et al., Biochem. Biophys. Res. Commun., 2001,
284, 1194-1198; Ntambi, J. Lipid Res., 1999, 40, 1549-1558).
[0013] Thiazolidinediones have been employed as regulators of
stearoyl-CoA desaturase activity in murine 3T3-L1 adipocytes (Kim
et al., J. Lipid Res., 2000, 41, 1310-1316), and in diabetic
rodents (Singh Ahuja et al., Mol. Pharmacol., 2001, 59,
765-773).
[0014] Compositions comprising a saponin in an amount effective to
inhibit stearoyl-CoA desaturase enzyme activity were described. The
saponin was derived from a source selected from the group
consisting of Quillaja saponaria, Panax trifolium, Panax
quinquefolium and Glycyrrhiza glabra (Chavali and Forse,
International patent publication No. WO 99/63979 1999).
[0015] An inhibitor of stearoyl-CoA desaturase was prepared in a
form suitable for oral, parenteral, rectal or dermal administration
for use in modifying the lipid structure of cell membranes. The
inhibitor was described as consisting of a saturated fatty acid
having from 12 to 28 carbon atoms in the alkyl chain, e.g. stearic
acid, or a pharmaceutically acceptable derivative thereof prepared
in a form suitable for parenteral, rectal or dermal administration
(Wood et al., European Patent No. EP 238198 1987). A stearoyl-CoA
desaturase antisense vector has been used to reduce expression
levels of stearoyl-CoA desaturase in chicken LMH hepatoma cells
(Diot et al., Arch. Biochem. Biophys., 2000, 380, 243-250).
[0016] To date, investigative strategies aimed at inhibiting
stearoyl-CoA desaturase function include the previously cited uses
of polyunsaturated fatty acids, saturated fatty acids,
thiazolidinediones, cholesterol, and an antisense vector. However,
these strategies are untested as therapeutic protocols.
Consequently, there remains a long felt need for additional agents
capable of effectively inhibiting stearoyl-CoA desaturase.
SUMMARY OF THE INVENTION
[0017] The present invention provides compositions and methods for
modulating the expression of stearoyl-CoA desaturase. Such
compositions and methods are shown to modulate the expression of
stearoyl-CoA desaturase, including inhibition of both isoforms of
stearoyl-CoA desaturase.
[0018] In particular, this invention relates to compounds,
particularly oligonucleotides, specifically hybridizable with
nucleic acids encoding stearoyl-CoA desaturase. Such compounds,
particularly antisense oligonucleotides, are targeted to a nucleic
acid encoding stearoyl-CoA desaturase, and modulate the expression
of stearoyl-CoA desaturase. Pharmaceutical and other compositions
comprising the compounds of the invention are also provided.
[0019] Further provided are methods of modulating the expression of
stearoyl-CoA desaturase in cells or tissues comprising contacting
the cells or tissues with one or more of the antisense compounds or
compositions of the invention. Further provided are methods of
treating an animal, particularly a human, suspected of having or
being prone to a disease or condition associated with expression of
stearoyl-CoA desaturase, by administering a therapeutically or
prophylactically effective amount of one or more of the antisense
compounds or compositions of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention employs oligomeric compounds,
particularly antisense oligonucleotides, for use in modulating the
function of nucleic acid molecules encoding stearoyl-CoA
desaturase, ultimately modulating the amount of stearoyl-CoA
desaturase produced. This is accomplished by providing antisense
compounds that specifically hybridize with one or more nucleic
acids encoding stearoyl-CoA desaturase.
[0021] Antisense technology is emerging as an effective means of
reducing the expression of specific gene products and is uniquely
useful in a number of therapeutic, diagnostic and research
applications involving modulation of stearoyl-CoA desaturase
expression.
[0022] As used herein, the terms "target nucleic acid" and "nucleic
acid encoding stearoyl-CoA desaturase" encompass DNA encoding
stearoyl-CoA desaturase, 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 that specifically hybridize to it is generally referred
to as "antisense". 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 stearoyl-CoA desaturase. In the context of the
present invention, "modulation" means either an increase
(stimulation) or a decrease (inhibition) in the expression of a
gene. In one embodiment of the present invention, inhibition is a
preferred form of modulation of gene expression and mRNA is a
preferred target.
[0023] For example, in one embodiment of the present invention, the
compounds of the present invention inhibit expression of
stearoyl-CoA desaturaseby at least 10% as measured in a suitable
assay, such as those described in the examples below. In another
embodiment, the compounds of the present invention inhibit
expression of stearoyl-CoA desaturase by at least 25%. In still
another embodiment of the invention, the compounds of the present
invention inhibit expression of stearoyl-CoA desaturase by at least
40%. In yet a further embodiment of this invention, the compounds
of the present invention inhibit expression of stearoyl-CoA
desaturase by at least 50%. In a further embodiment of this
invention, the compounds of the present invention inhibit
expression of stearoyl-CoA desaturase by at least 60%. In another
embodiment of this invention, the compounds of the present
invention inhibit expression of stearoyl-CoA desaturase by at least
70%. In still another embodiment of this invention, the compounds
of this invention inhibit expression of stearoyl-CoA desaturase by
at least 80%. In another embodiment of this invention, the
compounds of this invention inhibit expression of stearoyl-CoA
desaturase by at least 90% or higher. Exemplary compounds are
illustrated in Examples 15, and 17 to 24 below.
[0024] 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 as described herein begins with the
identification of a nucleic acid sequence encoding stearoyl-CoA
desaturase. This may be, for example, a cellular gene (or mRNA
transcribed from the gene). 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, results. In one
embodiment 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 stearoyl-CoA desaturase,
regardless of the sequence(s) of such codons.
[0025] 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.
[0026] 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 another
embodiment of a region of the nucleic acid sequence encoding
stearoyl-CoA desaturase which may be targeted effectively. Other
target regions of this invention include the 5' untranslated region
(5' UTR) of the nucleic acid sequence encoding stearoyl-CoA
desaturase, 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. Still another target region is the 3' untranslated region (3'
UTR) of the nucleic acid sequence encoding stearoyl-CoA desaturase,
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 of the nucleic acid sequence
encoding stearoyl-CoA desaturase may also be a preferred target
region.
[0027] 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, of the nucleic acid
sequence encoding stearoyl-CoA desaturase 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 of the nucleic acid sequence
encoding stearoyl-CoA desaturase, due to rearrangements or
deletions, are also preferred targets. In another embodiment of
this invention, introns of the nucleic acid sequence encoding
stearoyl-CoA desaturase can also be effective target regions for
antisense compounds targeted, for example, to DNA or pre-mRNA of
the nucleic acid sequence encoding stearoyl-CoA desaturase.
[0028] 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. See, e.g., Tables 1-5
below.
[0029] For example, Tables 1 and 2 illustrate antisense
oligonucleotides that hybridize to target regions of nucleotide 9
to 5100 of the nucleotide sequence of human stearoyl CoA desaturase
SEQ ID NO: 3. In one embodiment, for example, desirable
oligonucleotides target regions within nucleotides 13 to 71. In
another example, desirable oligonucleotides target regions within
nucleotides 178 to 247. In another example, desirable
oligonucleotides target regions within nucleotides 482 to 843. In
another example, desirable oligonucleotides target regions within
nucleotides 892 to 1064. In another example, desirable
oligonucleotides target regions within nucleotides 1303-1502. In
another example, desirable oligonucleotides target regions within
nucleotides 1597-2233. In another example, desirable
oligonucleotides target regions within nucleotides 2245-2589. In
another example, desirable oligonucleotides target regions within
nucleotides 2676-3278. In another example, desirable
oligonucleotides target regions within nucleotides 3342-3499. In
another example, desirable oligonucleotides target regions within
nucleotides 3655-3674. In another example, desirable
oligonucleotides target regions within nucleotides 3707-3790. In
another example, desirable oligonucleotides target regions within
nucleotides 3825-3853. In another example, desirable
oligonucleotides target regions within nucleotides 3911-4072. In
another example, desirable oligonucleotides target regions within
nucleotides 4132-4224. In another example, desirable
oligonucleotides target regions within nucleotides 4261-4398. In
another example, desirable oligonucleotides target regions within
nucleotides 4420-4554. In another example, desirable
oligonucleotides target regions within nucleotides 4645-4677. In
another example, desirable oligonucleotides target regions within
nucleotides 4834-4865. In another example, desirable
oligonucleotides target regions within nucleotides 4892-5100.
Oligonucletides that target any nucleotide sequence within SEQ ID
NO: 3, with the explicit exclusion of target regions between
nucleotides 70-91, 242-262 and 860-882, are included within this
invention.
[0030] As another example, Table 2 indicates illustrative
oligonucleotides that hybridize to target regions found within
nucleotides 505 to 14020 of the nucleotide sequence of human
stearoyl CoA desaturase SEQ ID NO: 81.
[0031] Tables 3-5 illustrate oligonucleotides that bind to target
regions within nucleotides 1 to 5366 of the nucleotide sequence of
mouse stearoyl CoA desaturase SEQ ID NO: 222.
[0032] 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 that 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 target
nucleic acid sequence (DNA or RNA) encoding stearoyl-CoA
desaturase.
[0033] 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 of the
stearoyl-CoA desaturase enzyme. There also must be a sufficient
degree of complementarity to avoid non-specific binding of the
antisense compound to non-stearoyl-CoA desaturase target sequences
under conditions in which specific binding is desired. Such
conditions include physiological conditions in the case of in vivo
assays or therapeutic treatment, and in the case of in vitro
assays, include conditions in which the assays are performed.
[0034] For example, in one embodiment, the antisense compounds of
the present invention comprise at least 80% sequence
complementarity to a target region within the target nucleic acid
of stearoyl-CoA desaturase to which they are targeted. In another
embodiment, the antisense compounds of the present invention
comprise at least 90% sequence complementarity to a target region
within the target nucleic acid of stearoyl-CoA desaturase to which
they are targeted. In still another embodiment of this invention,
the antisense compounds of the present invention comprise at least
95% sequence complementarity to a target region within the target
nucleic acid sequence of stearoyl-CoA desaturase to which they are
targeted. For example, an antisense compound in which 18 of 20
nucleobases of the antisense compound are complementary, and would
therefore specifically hybridize, to a target region would
represent 90 percent complementarity. Percent complementarity of an
antisense compound with a region of a target nucleic acid can be
determined routinely using basic local alignment search tools
(BLAST programs) (Altschul et al., J. Mol. Biol., 1990, 215,
403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
[0035] Antisense and other compounds of the invention that
hybridize to the target and inhibit expression of stearoyl-CoA
desaturase are identified as taught herein. In one embodiment of
this invention, the sequences of these compounds are hereinbelow
identified as preferred embodiments of the invention (see, e.g.,
Tables 1-5). The target sites to which these preferred sequences
are complementary are hereinbelow referred to as "active sites" and
are therefore preferred sites for targeting (see, e.g., Tables
1-5). Therefore another embodiment of the invention encompasses
compounds that hybridize to these active sites.
[0036] In one embodiment of this invention, the term "illustrative
target region" is defined as a nucleobase sequence of a target
region of stearoyl-CoA desaturase, to which an active antisense
compound is targeted. For example, an illustrative target region
may be at least 8 or at least 15 nucleobases in length. In still
another embodiment an illustrative target region is at least 25
nucleobases of the nucleic acid sequence or molecule encoding
stearoyl-CoA desaturase, to which an active antisense compound is
targeted. In still another embodiment an illustrative target region
is at 35 nucleobases. In yet another embodiment an illustrative
target region is at least 50 nucleobases of the nucleic acid
sequence or molecule encoding stearoyl-CoA desaturase, to which an
active antisense compound is targeted. In still another embodiment
an illustrative target region is at least 70 nucleobases. In
another embodiment an illustrative target region is at least 80
nucleobases or more. In still another embodiments, the illustrative
target regions consist of consecutive nucleobases of the lengths
identified above.
[0037] Exemplary additional target regions include DNA or RNA
sequences that comprise at least the 8 consecutive nucleobases from
the 5'-terminus of one of the illustrative target regions (the
remaining nucleobases being a consecutive stretch of the same DNA
or RNA beginning immediately upstream of the 5'-terminus of the
target region and continuing until the DNA or RNA contains about 8
to about 80 nucleobases). Similarly additional target regions are
represented by DNA or RNA sequences that comprise at least the 8
consecutive nucleobases from the 3'-terminus of one of the
illustrative target regions (the remaining nucleobases being a
consecutive stretch of the same DNA or RNA beginning immediately
downstream of the 3'-terminus of the target region and continuing
until the DNA or RNA contains about 8 to about 80 nucleobases).
[0038] Antisense compounds are commonly used as research reagents
and diagnostics. For example, 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. Antisense compounds are also used, for example,
to distinguish between functions of various members of a biological
pathway. Antisense modulation has, therefore, been harnessed for
research use.
[0039] For use in kits and diagnostics, the antisense compounds of
the present invention, either alone or in combination with other
antisense 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.
[0040] 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 that affect expression patterns.
[0041] 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 (reviewed in (To, Comb.
Chem. High Throughput Screen, 2000, 3, 235-41).
[0042] The specificity and sensitivity of antisense is 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,
particularly mammals, and including man. Antisense oligonucleotide
drugs, including ribozymes, 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.
[0043] 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.
[0044] 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. Antisense
compounds include ribozymes, external guide sequence (EGS)
oligonucleotides (oligozymes), and other short catalytic RNAs or
catalytic oligonucleotides that hybridize to the target nucleic
acid encoding stearoyl-CoA desaturase and modulate expression of
that enzyme.
[0045] The antisense compounds in accordance with this invention
preferably comprise from at least 8 nucleobases (i.e. about 8
linked nucleosides). Particularly preferred antisense compounds are
antisense oligonucleotides. In one embodiment, antisense compounds
of this invention are antisense oligonucleotides of at least about
15 nucleobases in length. In another embodiment, antisense
compounds of this invention comprise about 25 nucleobases in
length. In still another embodiment, antisense compounds of this
invention comprise about 35 nucleobases in length. In yet another
embodiment, antisense compounds of this invention comprise about 40
nucleobases in length. In still another embodiment, antisense
compounds of this invention comprise about 50 nucleobases in
length. In another embodiment, antisense compounds of this
invention comprise about 60 nucleobases in length. In still another
embodiment, antisense compounds of this invention comprise about 70
nucleobases in length. In yet another embodiment, antisense
compounds of this invention comprise about 80 nucleobases in
length.
[0046] In other embodiments, exemplary antisense compounds include
DNA or RNA sequences that comprise at least the 8 consecutive
nucleobases from the 5'-terminus of one of the illustrative
antisense compounds (the remaining nucleobases being a consecutive
stretch of the same DNA or RNA beginning immediately upstream of
the 5'-terminus of the antisense compound which is specifically
hybridizable to the target nucleic acid and continuing until the
DNA or RNA contains about 8 to about 80 nucleobases). Similarly, in
another embodiment, such antisense compounds include at least 12
consecutive nucleobases from the 5'-terminus of one of the
illustrative antisense compounds. In yet another embodiment, the
antisense compound includes at least 25 consecutive nucleobases
from the 5'-terminus of one of the illustrative antisense
compounds. In a further embodiment, the antisense compound includes
at least 30 consecutive nucleobases from the 5'-terminus of one of
the illustrative antisense compounds. In yet another embodiment,
the antisense compound includes at least 50 consecutive nucleobases
from the 5'-terminus of one of the illustrative antisense
compounds. In still another embodiment, the antisense compound
includes at least 60 or more consecutive nucleobases from the
5'-terminus of one of the illustrative antisense compounds.
[0047] Similarly in another embodiment antisense compounds are
represented by DNA or RNA sequences that comprise at least the 8
consecutive nucleobases from the 3'-terminus of one of the
illustrative antisense compounds (the remaining nucleobases being a
consecutive stretch of the same DNA or RNA beginning immediately
downstream of the 3'-terminus of the antisense compound which is
specifically hybridizable to the target nucleic acid and continuing
until the DNA or RNA contains about 8 to about 80 nucleobases). In
another embodiment, such antisense compounds include at least 12
consecutive nucleobases from the 3'-terminus of one of the
illustrative antisense compounds. In yet another embodiment, the
antisense compound includes at least 25 consecutive nucleobases
from the 3'-terminus of one of the illustrative antisense
compounds. In a further embodiment, the antisense compound includes
at least 30 consecutive nucleobases from the 3'-terminus of one of
the illustrative antisense compounds. In yet another embodiment,
the antisense compound includes at least 50 consecutive nucleobases
from the 3'-terminus of one of the illustrative antisense
compounds. In still another embodiment, the antisense compound
includes at least 60 or more consecutive nucleobases from the
3'-terminus of one of the illustrative antisense compounds. One
having skill in the art, once armed with the antisense compounds
illustrated, and other teachings herein will be able, without undue
experimentation, to identify further antisense compounds of this
invention.
[0048] Specific sequences of particular exemplary target regions of
stearoyl-CoA desaturase and representative antisense and other
compounds of the invention, which hybridize to the target, and
inhibit expression of the target, are identified below are set
forth below in Tables 1-5. One of skill in the art will recognize
that these serve to illustrate and describe particular embodiments
within the scope of the present invention. Once armed with the
teachings of the illustrative target regions described herein may
without undue experimentation identify further target regions, as
described above. In addition, one having ordinary skill in the art
using the teachings contained herein will also be able to identify
additional compounds, including oligonucleotide probes and primers,
that specifically hybridize to these illustrative target regions
using techniques available to the ordinary practitioner in the
art.
[0049] 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.
[0050] 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.
[0051] Preferred modified oligonucleotide backbones include, for
example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates, 5'-alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriest- ers,
selenophosphates and borano-phosphates having normal 3'-5'
linkages, 2'-5' linked analogs of these, and those having inverted
polarity wherein one or more internucleotide linkages is a 3' to
3', 5' to 5' or 2' to 2' linkage. Preferred oligonucleotides having
inverted polarity comprise a single 3' to 3' linkage at the 3'-most
internucleotide linkage, i.e. a single inverted nucleoside residue
that may be abasic (the nucleobase is missing or has a hydroxyl
group in place thereof). Various salts, mixed salts and free acid
forms are also included.
[0052] 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; 5,194,599; 5,565,555;
5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are
commonly owned with this application, and each of which is herein
incorporated by reference.
[0053] 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; riboacetyl 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.
[0054] 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; 5,792,608;
5,646,269 and 5,677,439, certain of which are commonly owned with
this application, and each of which is herein incorporated by
reference.
[0055] 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.
[0056] 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.
[0057] 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.su- b.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, alkenyl, alkynyl, 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, poly-alkylamino, 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. 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'-dimethylamino-ethoxyethoxy (also known in the art as
2'-O-dimethylamino-ethoxyethyl 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.
[0058] A further preferred modification includes Locked Nucleic
Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or
4' carbon atom of the sugar ring thereby forming a bicyclic sugar
moiety. The linkage is preferably a methelyne (--CH.sub.2--).sub.n
group bridging the 2' oxygen atom and the 4' carbon atom wherein n
is 1 or 2. LNAs and preparation thereof are described in
International patent publication Nos. WO 98/39352 and WO
99/14226.
[0059] Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2'-allyl
(2'-CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl
(2'-O--CH.sub.2--CH.dbd.CH.sub- .2) and 2'-fluoro (2'-F). The
2'-modification may be in the arabino (up) position or ribo (down)
position. A preferred 2'-arabino modification is 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; 5,792,747;
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.
[0060] 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
(--C.ident.C--CH.sub.3) uracil and cytosine and other alkynyl
derivatives of pyrimidine bases, 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, 2-F-adenine, 2-amino-adenine, 8-azaguanine and
8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine
and 3-deazaadenine. Further modified nucleobases include tricyclic
pyrimidines such as phenoxazine
cytidine(1H-pyrimido[5,4-b][1,4]benzoxazi- n-2(3H)-one),
phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin--
2(3H)-one), G-clamps such as a substituted phenoxazine cytidine
(e.g.
9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole
cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).
Modified nucleobases may also include those in which the purine or
pyrimidine base is replaced with other heterocycles, for example
7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
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.degree. 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.
[0061] 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; 5,645,985; 5,830,653;
5,763,588; 6,005,096; 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.
[0062] Another modification of the oligonucleotides of the
invention involves chemically linking to the oligonucleotide one or
more moieties or conjugates that enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. The
compounds of the invention can include conjugate groups covalently
bound to functional groups such as primary or secondary hydroxyl
groups. Conjugate groups of the invention include intercalators,
reporter molecules, polyamines, polyamides, polyethylene glycols,
polyethers, groups that enhance the pharmacodynamic properties of
oligomers, and groups that enhance the pharmacokinetic properties
of oligomers. Typical conjugates groups include cholesterols,
lipids, phospholipids, biotin, phenazine, folate, phenanthridine,
anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and
dyes. Groups that enhance the pharmacodynamic properties, in the
context of this invention, include groups that improve oligomer
uptake, enhance oligomer resistance to degradation, and/or
strengthen sequence-specific hybridization with RNA. Groups that
enhance the pharmacokinetic properties, in the context of this
invention, include groups that improve oligomer uptake,
distribution, metabolism or excretion. Representative conjugate
groups are disclosed in International Patent Publication No.
WO93/07883 (Application PCT/US92/09196, filed Oct. 23, 1992) the
entire disclosure of which is incorporated herein by reference.
Conjugate 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-S-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. Oligonucleotides of the
invention may also be conjugated to active drug substances, for
example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen,
fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,
dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,
folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,
indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an
antidiabetic, an antibacterial or an antibiotic.
Oligonucleotide-drug conjugates and their preparation are described
in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15,
1999), which is incorporated herein by reference in its
entirety.
[0063] 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.
[0064] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within an oligonucleotide.
The present invention also includes antisense compounds that are
chimeric compounds. "Chimeric" antisense compounds or "chimeras,"
in the context of this invention, are antisense 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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 International
Patent Publication Nos. WO 93/24510 to Gosselin et al., published
Dec. 9, 1993 or WO 94/26764, and U.S. Pat. No. 5,770,713 to Imbach
et al.
[0071] 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.
[0072] 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'-dibenzylethylene-diamine, 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 the 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-methylbenzenesulfonic 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.
[0073] 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.
[0074] 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 that can be treated by
modulating the expression of stearoyl-CoA desaturase is treated by
administering antisense compounds in accordance with this
invention. Among such diseases or disorder are included, for
example, cardiovascular disease, obesity, non-insulin-dependent
diabetes mellitus, skin disease, hypertension, neurological
diseases, immune disorders and cancer.
[0075] 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 to prevent such
diseases or disorders, e.g., to prevent or delay infection, undue
weight gain, inflammation or tumor formation, for example.
[0076] The antisense compounds of the invention are useful for
research and diagnostics, because these compounds hybridize to
nucleic acids encoding stearoyl-CoA desaturase, 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 stearoyl-CoA desaturase 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 stearoyl-CoA
desaturase in a sample may also be prepared.
[0077] The present invention also includes pharmaceutical
compositions and formulations that 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.
[0078] 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. Preferred
topical formulations include those in which the oligonucleotides of
the invention are in admixture with a topical delivery agent such
as lipids, liposomes, fatty acids, fatty acid esters, steroids,
chelating agents and surfactants. Preferred lipids and liposomes
include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,
dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl
choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and
cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and
dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of the
invention may be encapsulated within liposomes or may form
complexes thereto, in particular to cationic liposomes.
Alternatively, oligonucleotides may be complexed to lipids, in
particular to cationic lipids. Preferred fatty acids and esters
include but are not limited arachidonic acid, oleic acid,
eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic
acid, palmitic acid, stearic acid, linoleic acid, linolenic acid,
dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or
a C.sub.1-10 alkyl ester (e.g. isopropylmyristate IPM),
monoglyceride, diglyceride or pharmaceutically acceptable salt
thereof. Topical formulations are described in detail in U.S.
patent application Ser. No. 09/315,298 filed on May 20, 1999, which
is incorporated herein by reference in its entirety.
[0079] Compositions and formulations for oral administration
include powders or granules, microparticulates, nanoparticulates,
suspensions or solutions in water or non-aqueous media, capsules,
gel capsules, sachets, tablets or minitablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable. Preferred oral formulations are those in which
oligonucleotides of the invention are administered in conjunction
with one or more penetration enhancers, surfactants and chelators.
Preferred surfactants include fatty acids and/or esters or salts
thereof, bile acids and/or salts thereof. Preferred bile
acids/salts include chenodeoxycholic acid (CDCA) and
ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic
acid, deoxycholic acid, glucholic acid, glycholic acid,
glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid,
sodium tauro-24,25-dihydro-fusid- ate, sodium
glycodihydrofusidate,. Preferred fatty acids include arachidonic
acid, undecanoic acid, oleic acid, lauric acid, caprylic acid,
capric acid, myristic acid, palmitic acid, stearic acid, linoleic
acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin,
glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an
acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or
a pharmaceutically acceptable salt thereof (e.g. sodium). Also
preferred are combinations of penetration enhancers, for example,
fatty acids/salts in combination with bile acids/salts. A
particularly preferred combination is the sodium salt of lauric
acid, capric acid and UDCA. Further penetration enhancers include
polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
Oligonucleotides of the invention may be delivered orally in
granular form including sprayed dried particles, or complexed to
form micro or nanoparticles. Oligonucleotide complexing agents
include poly-amino acids; polyimines; polyacrylates;
polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates;
cationized gelatins, albumins, starches, acrylates,
polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates;
DEAE-derivatized polyimines, pollulans, celluloses and starches.
Particularly preferred complexing agents include chitosan,
N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,
polyspermines, protamine, polyvinylpyridine,
polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.
p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),
poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),
poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,
DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,
polyhexylacrylate, poly(D,L-lactic acid),
poly(DL-lactic-co-glycolic acid (PLGA), alginate, and
polyethyleneglycol (PEG). Oral formulations for oligonucleotides
and their preparation are described in detail in U.S. Published
patent application No. 2003/0040497 (Feb. 27, 2003) and its parent
applications; U.S. Published patent application No. 2003/0027780
(Feb. 6, 2003) and its parent applications; and U.S. patent
application Ser. No. 09/082,624 (filed May 21, 1998), each of which
is incorporated herein by reference in their entirety.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] The compositions of the present invention may be formulated
into any of many possible dosage forms such as, but not limited to,
tablets, capsules, gel 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 that increase the viscosity of the suspension including,
for example, sodium carboxymethylcellulose, sorbitol and/or
dextran. The suspension may also contain stabilizers.
[0084] 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.
[0085] Emulsions
[0086] 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 .mu.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.
[0087] 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,
cited above).
[0088] 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, cited
above). 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, cited above).
[0089] 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.
[0090] 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, cited above).
[0091] 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.
[0092] 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.
[0093] The application of emulsion formulations via dermatological,
oral and parenteral routes and methods for their manufacture has
been reviewed in the literature (Idson, cited above). 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, cited above; Idson, cited
above). 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.
[0094] 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, cited above).
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).
[0095] 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, cited above; Block, cited above). Compared to conventional
emulsions, microemulsions offer the advantage of solubilizing
water-insoluble drugs in a formulation of thermodynamically stable
droplets that are formed spontaneously.
[0096] 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 (MO750), decaglycerol sequioleate (SO750),
decaglycerol decaoleate (DAO750), 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 (C8-C12) mono, di, and tri-glycerides,
polyoxyethylated glyceryl fatty acid esters, fatty alcohols,
polyglycolized glycerides, saturated polyglycolized C8-C10
glycerides, vegetable oils and silicone oil.
[0097] 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., 1994 cited above; 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.
[0098] 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.
[0099] Liposomes
[0100] 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.
[0101] 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.
[0102] 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 that is highly
deformable and able to pass through such fine pores.
[0103] 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, cited above).
Important considerations in the preparation of liposome
formulations are the lipid surface charge, vesicle size and the
aqueous volume of the liposomes.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] Liposomes fall into two broad classes. Cationic liposomes
are positively charged liposomes that 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).
[0108] Liposomes that 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).
[0109] One major type of liposomal composition includes
phospholipids other than naturally-derived phosphatidylcholine.
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
phosphatidylethanolamine (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.
[0110] 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).
[0111] 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 Novasome.TM. I
(glyceryl dilaurate/cholesterol/po- lyoxyethylene-10-stearyl ether)
and Novasome.TM. 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).
[0112] Liposomes also include "sterically stabilized" liposomes.
This latter term, 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).
[0113] 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 International Patent
Publication No. 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
International Patent Publication No. WO 97/13499 (Lim et al.).
[0114] 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 phosphatidylethanolamine
(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 International Patent Publication No. 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 International Patent Publication No. WO
91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in
International Patent Publication No. WO 94/20073 (Zalipsky et al.)
Liposomes comprising PEG-modified ceramide lipids are described in
International Patent Publication No. 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.
[0115] A limited number of liposomes comprising nucleic acids are
known in the art. International Patent Publication No. 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. Patent No. 5,665,710 to Rahman et al. describes
certain methods of encapsulating oligodeoxynucleotides in
liposomes. International Patent Publication No. WO 97/04787 to Love
et al. discloses liposomes comprising antisense oligonucleotides
targeted to the raf gene.
[0116] 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 that are so highly deformable that they are easily able to
penetrate through pores that 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.
[0117] 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, cited above).
[0118] 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.
[0119] 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.
[0120] 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.
[0121] 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.
[0122] The use of surfactants in drug products, formulations and in
emulsions has been reviewed (Rieger, cited above).
[0123] Penetration Enhancers
[0124] In one embodiment, the present invention employs various
penetration enhancers to effect 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.
[0125] 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.
[0126] Surfactants: 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., 1991, cited above); and perfluorochemical emulsions,
such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40,
252).
[0127] Fatty acids: 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., , 1991, p.92, cited above; Muranishi, Critical Reviews
in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et
al., J. Pharm. Pharmacol., 1992, 44, 651-654).
[0128] Bile salts: 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 taurodeoxycholate), chenodeoxycholic
acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA),
sodium tauro-24,25-dihydro-fusidate (STDHF), sodium
glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee
et al., 1991, page 92, cited above; 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).
[0129] Chelating Agents: 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 ethylenediamine tetraacetate (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.,
1991, page 92, cited above; Muranishi, 1990, cited above; Buur et
al., J. Control Rel., 1990, 14, 43-51).
[0130] Non-chelating non-surfactants: 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, 1990,
cited above). This class of penetration enhancers include, for
example, unsaturated cyclic ureas, 1-alkyl- and
1-alkenylazacycloalkanone derivatives (Lee et al., 1991, page 92,
cited above); and non-steroidal anti-inflammatory agents such as
diclofenac sodium, indomethacin and phenylbutazone (Yamashita et
al., J. Pharm. Pharmacol., 1987, 39, 621-626).
[0131] 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., International Patent
Publication No. WO 97/30731), are also known to enhance the
cellular uptake of oligonucleotides.
[0132] 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.
[0133] Carriers
[0134] 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).
[0135] 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 that 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 that 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.
[0140] Other Components
[0141] 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.
[0142] Aqueous suspensions may contain substances that increase the
viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
[0143] 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 daunorubicin, daunomycin,
dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,
bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,
bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,
mithramycin, prednisone, hydroxyprogesterone, testosterone,
tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,
pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,
methylcyclohexylnitrosurea, nitrogen mustards, melphalan,
cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-azacytidine, hydroxyurea, deoxycoformycin,
4-hydroxyperoxycyclophosphor- amide, 5-fluorouracil (5-FU),
5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine,
taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate,
irinotecan, topotecan, gemcitabine, teniposide, cisplatin and
diethylstilbestrol (DES). See, generally, The Merck Manual of
Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al.,
eds., Rahway, N.J. When used with the compounds of the invention,
such chemotherapeutic agents may be used individually (e.g., 5-FU
and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide
for a period of time followed by MTX and oligonucleotide), or in
combination with one or more other such chemotherapeutic agents
(e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and
oligonucleotide). 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.
[0144] 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.
[0145] 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 ug 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 g to 100 g per kg of body
weight, once or more daily, to once every 20 years.
[0146] 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
[0147] 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.
[0148] 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
[0149] 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
[0150] The synthesis of 2'-deoxy-2'-fluoroguanosine was
accomplished using tetraisopropyldisiloxanyl (TPDS) protected
9-beta-D-arabinofuranosylguani- ne as starting material, and
conversion to the intermediate
diisobutyryl-arabinofuranosylguanosine. 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'-phosphoramidi- tes.
2'-Fluorouridine
[0151] Synthesis of 2'-deoxy-2'-fluorouridine was accomplished by
the modification of a literature procedure in which
2,2'-anhydro-1-beta-D-ara- binofuranosyluracil was treated with 70%
hydrogen fluoride-pyridine. Standard procedures were used to obtain
the 5'-DMT and 5'-DMT-3' phosphoramidites.
2'-Fluorodeoxycytidine
[0152] 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
[0153] 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]
[0154] 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 mL). 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
[0155] 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 155-160.degree. 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
[0156] 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
[0157] 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.degree. 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
[0158] A first solution was prepared by dissolving
3'-O-acetyl-2'-O-methox-
yethyl-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 0-10.degree. C., 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
[0159] A solution of
3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5--
methyl-4-triazoleuridine (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 liter 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
[0160] 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
[0161]
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-(isopropyl)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)nu- cleoside amidites
2'-(Dimethylaminooxyethoxy)nucleoside amidites
[0162] 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
[0163] 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
[0164] 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
[0165]
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-butyldiphenylsilyl-5-methyluridine
as white foam (21.819 g, 86%).
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methylurid-
ine
[0166]
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridi-
ne (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 stirred for 1 h.
Solvent was removed under vacuum; residue chromatographed to get
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluri-
dine as white foam (1.95 g, 78%).
5'-O-tert-Butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methylurid-
ine
[0167]
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-met-
hyluridine (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.degree. 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-dimethylaminooxyethyll-5-methyluri-
dine as a white foam (14.6 g, 80%).
2'-O-(dimethylaminooxyethyl)-5-methyluridine
[0168] 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-butyldiphenylsil-
yl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (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
[0169] 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]
[0170] 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.sub.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-dim-
ethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoethyl)-N,N-diisopropylphos-
phoramidite] as a foam (1.04 g, 74.9%).
2'-(Aminooxyethoxy)nucleoside amidites
[0171] 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]
[0172] 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. (McGee et al., International Patent
Publication No. WO 94/02501). Standard protection procedures should
afford 2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)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-hydroxyethyl)-5'-O-(4,4'-dim-
ethoxytrityl)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-diphen-
ylcarbamoyl-2'-O-([2-phthalmidoxy]ethyl)-5'-O-(4,4'-dimethoxytrityl)guanos-
ine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].
2'-dimethylaminoethoxyethoxy (2'-DMAEOE) nucleoside amidites
[0173] 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
[0174] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol)
is slowly added to a solution of borane in tetra-hydrofuran (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
[0175] To 0.5 g (1.3 mmol) of
2'-O-[2(2-N,N-dimethylamino-ethoxy)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.
[0176] 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-dimethylaminoethoxy)-ethyl)]-5-methyl
uridine-3'-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite
[0177] Diisopropylaminotetrazolide (0.6 g) and
2-cyanoethoxy-N,N-diisoprop- yl 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
[0178] Unsubstituted and substituted phosphodiester (P=O)
oligonucleotides are synthesized on an automated DNA synthesizer
(Applied Biosystems model 380B) using standard phosphoramidite
chemistry with oxidation by iodine.
[0179] 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.degree. C. (18 h), the oligonucleotides were purified by
precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl
solution.
[0180] Phosphinate oligonucleotides are prepared as described in
U.S. Pat. No. 5,508,270, herein incorporated by reference.
[0181] Alkyl phosphonate oligonucleotides are prepared as described
in U.S. Pat. No. 4,469,863, herein incorporated by reference.
[0182] 3'-Deoxy-3'-methylene phosphonate oligonucleotides are
prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050,
herein incorporated by reference.
[0183] Phosphoramidite 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.
[0184] Alkylphosphonothioate oligonucleotides are prepared as
described in published International Patent Publication Nos. WO
94/17093 and WO 94/02499, herein incorporated by reference.
[0185] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are
prepared as described in U.S. Pat. No. 5,476,925, herein
incorporated by reference.
[0186] Phosphotriester oligonucleotides are prepared as described
in U.S. Pat. No. 5,023,243, herein incorporated by reference.
[0187] 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
[0188] Methylenemethylimino linked oligonucleosides, also
identified as MMI linked oligonucleosides,
methylenedimethyl-hydrazo 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.
[0189] 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.
[0190] Ethylene oxide linked oligonucleosides are prepared as
described in U.S. Pat. No. 5,223,618, herein incorporated by
reference.
Example 4
PNA Synthesis
[0191] 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
[0192] 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
[0193] 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
[0194]
[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
[0195] (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.
[0196] 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
[0197] 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
[0198] 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.
[0199] 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
[0200] 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) or, for individually prepared samples, on a
commercial CE apparatus (e.g., Beckman P/ACE.TM. 5000, ABI 270).
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
[0201] 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 seven 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.
[0202] T-24 cells:
[0203] 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 5A 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.
[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.
[0205] A549 Cells:
[0206] 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.
[0207] NHDF Cells:
[0208] 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.
[0209] HEK Cells:
[0210] 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.
[0211] HepG2 Cells:
[0212] The human hepatoblastoma cell line HepG2 was obtained from
the American Type Culture Collection (Manassas, Va.). HepG2 cells
were routinely cultured in Eagle's MEM supplemented with 10% fetal
calf serum, non-essential amino acids, and 1 mM sodium pyruvate
(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.
[0213] For Northern blotting or other analyses, cells may be seeded
onto 100 mm or other standard tissue culture plates and treated
similarly, using appropriate volumes of medium and
oligonucleotide.
[0214] AML12 Cells:
[0215] AML12 (alpha mouse liver 12) cell line was established from
hepatocytes from a mouse (CD1 strain, line MT42) transgenic for
human TGF alpha. Cells are cultured in a 1:1 mixture of Dulbecco's
modified Eagle's medium and Ham's F12 medium with 0.005 mg/ml
insulin, 0.005 mg/ml transferrin, 5 ng/ml selenium, and 40 ng/ml
dexamethasone, and 90%; 10% fetal bovine serum. For subculturing,
spent medium is removed; and fresh media of 0.25% trypsin, 0.03%
EDTA solution is added. Fresh trypsin solution (1 to 2 ml) is added
and the culture is left to sit at room temperature until the cells
detach.
[0216] 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.
[0217] For Northern blotting or other analyses, cells may be seeded
onto 100 mm or other standard tissue culture plates and treated
similarly, using appropriate volumes of medium and
oligonucleotide.
[0218] b.END Cells:
[0219] The mouse brain endothelial cell line b.END was obtained
from Dr. Werner Risau at the Max Plank Instititute (Bad Nauheim,
Germany). b.END cells were routinely cultured in DMEM, high glucose
(Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%
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 3000 cells/well for use in
RT-PCR analysis.
[0220] Primary Mouse Hepatocytes:
[0221] Primary mouse hepatocytes were prepared from CD-1 mice
purchased from Charles River Labs (Wilmington, Mass.) and were
routinely cultured in Hepatoyte Attachment Media (Gibco)
supplemented with 10% Fetal Bovine Serum (Gibco/Life Technologies,
Gaithersburg, Md.), 250 nM dexamethasone (Sigma), and 10 nM bovine
insulin (Sigma). Cells were seeded into 96-well plates
(Falcon-Primaria #3872) at a density of 10000 cells/well for use in
RT-PCR analysis.
[0222] For Northern blotting or other analyses, cells are plated
onto 100 mm or other standard tissue culture plates coated with rat
tail collagen (200 .mu.g/mL) (Becton Dickinson) and treated
similarly using appropriate volumes of medium and
oligonucleotide.
[0223] Treatment with Antisense Compounds:
[0224] 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.
[0225] 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 Stearoyl-CoA Desaturase
Expression
[0226] Antisense modulation of stearoyl-CoA desaturase expression
can be assayed in a variety of ways known in the art. For example,
stearoyl-CoA desaturase 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.
[0227] Protein levels of stearoyl-CoA desaturase 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 stearoyl-CoA desaturase 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.
[0228] 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
[0229] 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, 60 .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. 55 .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.
[0230] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
Example 12
Total RNA Isolation
[0231] Total RNA 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. 100 .mu.L Buffer RLT was
added to each well and the plate vigorously agitated for 20
seconds. 100 .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. 1 mL of Buffer RW1 was added to each well of the RNEASY
96.TM. plate and the vacuum again applied for 15 seconds. 1 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.
[0232] The repetitive pipetting and elution steps may be automated
using a QIAGEN Bio-Robot 9604 (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 stearoyl-CoA Desaturase mRNA
Levels
[0233] Quantitation of stearoyl-CoA desaturase 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.
[0234] 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
multiplexable. Other methods of PCR are also known in the art.
[0235] 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 total RNA 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).
[0236] Gene target quantities obtained by real time RT-PCR are
normalized using either the expression level of GAPDH, a gene whose
expression is constant, or by quantifying total RNA using
RiboGreen.TM. reagent (Molecular Probes, Inc. Eugene, OR). GAPDH
expression is quantified by real time RT-PCR, by being run
simultaneously with the target, multiplexing, or separately. Total
RNA is quantified using RiboGreen.TM. RNA quantification reagent
from Molecular Probes. Methods of RNA quantification by
RiboGreen.TM. reagent are taught in Jones, L. J., et al, Analytical
Biochemistry, 1998, 265, 368-374.
[0237] In this assay, 175 .mu.L of RiboGreen.TM. working reagent
(RiboGreen.TM. reagent diluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA,
pH 7.5) is pipetted into a 96-well plate containing 25 uL purified,
cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied
Biosystems) with excitation at 480 nm and emission at 520 nm.
[0238] Probes and primers to human stearoyl-CoA desaturase were
designed to hybridize to a human stearoyl-CoA desaturase sequence,
using published sequence information (GenBank accession number
AF097514, incorporated herein as SEQ ID NO:3). For human
stearoyl-CoA desaturase the PCR primers were: forward primer:
GATCCCGGCATCCGAGA (SEQ ID NO: 4) reverse primer:
GGTATAGGAGCTAGAGATATCGTCCTG (SEQ ID NO: 5) and the PCR probe was:
FAM-CCAAGATGCCGGCCCACTTGC-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:
[0239] forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7)
[0240] 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.
Example 14
Northern Blot Analysis of Stearoyl-CoA Desaturase mRNA Levels
[0241] 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 apparatus (Stratagene, Inc, La Jolla, Calif.)
and then robed using QUICKHYB.TM. hybridization solution
(Stratagene, La Jolla, Calif.) using manufacturer's recommendations
for stringent conditions.
[0242] To detect human stearoyl-CoA desaturase, a human
stearoyl-CoA desaturase specific probe was prepared by PCR using
the forward primer GATCCCGGCATCCGAGA (SEQ ID NO: 4) and the reverse
primer GGTATAGGAGCTAGAGATATCGTCCTG (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.).
[0243] Hybridized membranes were visualized and quantitated using a
PHOSPHORIMAGER.TM. apparatus 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 Stearoyl-CoA Desaturase Expression by
Chimeric Phosphorothioate Oligonucleotides having 2'-MOE Wings and
a Deoxy Gap
[0244] In accordance with the present invention, a series of
oligonucleotides was designed to target different regions of the
human stearoyl-CoA desaturase RNA, using published sequence
(GenBank accession number AF097514, 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 stearoyl-CoA
desaturase mRNA levels in HepG2 cells by quantitative real-time PCR
as described in other example herein. Data are averages from two
experiments. If present, "N.D." indicates "no data".
1TABLE 1 Inhibition of human stearoyl-CoA desaturase mRNA levels by
chimeric phosphorothioate oligonucleotides having 2'-MOE wings and
a deoxy gap TARGET TARGET SEQ ID ISIS # REGION SEQ ID NO SITE
SEQUENCE % INHIB NO 147899 5'UTR 3 9 GTCCGGTATTTCCTCAGCCC N.D. 10
147900 5'UTR 3 72 CCGCGGTGCGTGGAGGTCCC N.D. 11 147901 5'UTR 3 121
TACGCGCTGAGCCGCGGCGC N.D. 12 147902 5'UTR 3 141
GCGGTTTCGAAGCCCGCCGG N.D. 13 147903 Coding 3 311
CCTCCATTCTGCAGGACCCT N.D. 14 147904 Coding 3 471
TCCCAAGTGTAGCAGAGACA N.D. 15 147905 Coding 3 571
CTCCTGCTGTTATGCCCAGG N.D. 16 147906 Coding 3 691
CACGGTGGTCACGAGCCCAT N.D. 17 147907 Coding 3 771
CAGCCAACCCACGTGAGAGA 22 18 147908 Coding 3 824 GACAAGTCTAGCGTACTCCC
49 19 147909 Coding 3 1011 GTTCACCAGCCAGGTGGCAT 10 20 147910 Coding
3 1111 TGTGGAAGCCCTCACCCACA 0 21 147911 Coding 3 1171
AGTTGATGTGCCAGCGGTAC 6 22 147912 Stop 3 1307 GGACCCCAAACTCAGCCACT
25 23 Codon 147913 3'UTR 3 1581 TGCCTGGGAGGCAATAAGGG 8 24 147914
3'UTR 3 1861 ATACATGCTAACTCTCTCCC 10 25 147915 3'UTR 3 1941
AAGTCCTCATTAGGTAGGCA 37 26 147916 3'UTR 3 2241 TGTAATGAGCAGCTCATGGA
0 27 147917 3'UTR 3 2616 TCAGTAACCTTCTCAAGCCC 0 28 147918 3'UTR 3
2980 GGAGCTGCCTGGACAGCAAG 16 29 147919 3'UTR 3 3011
TCAGTGACCCTGAGCATTCT 30 30 147920 3'UTR 3 3231 TGGCTGGCCCACTGGCTCAA
11 31 147921 3'UTR 3 3291 GCATGCCCTCTGGTTCTGAC 7 32 147922 3'UTR 3
3471 GCTTTGCAGTTCACCCTGAC 23 33 147923 3'UTR 3 3502
GTGGTATCTCAAATCCCAGG 0 34 147924 3'UTR 3 3791 TAGTCCAGGCTAACCCCTGT
0 35 147925 3'UTR 3 3851 GTGATCTTCCCTTAGATCCT 0 36 147926 3'UTR 3
4101 CTCAGCAGACACACTCCCAC 3 37 147927 3'UTR 3 4226
GCTAAGTTGTCAGCACACCC 0 38 147928 3'UTR 3 4406 AAGTTTCCAGAATGAAGCCC
25 39 147929 3'UTR 3 4571 AGAGAATACACCCAAGATAC 0 40 147930 3'UTR 3
4708 TAGTTAAGTGACTTGCCCAG 0 41 147931 3'UTR 3 4771
GCCCTTTGAGGTAGGTCAGT 4 42 147932 3'UTR 3 4921 CCATATAGACTAATGACAGC
10 43 147933 3'UTR 3 5021 CTGTATGTTTCCGTGGCAAT 11 44 168231 5'UTR 3
101 CTTGCACGCTAGCTGGTTGT N.D. 45 168232 Coding 3 331
GCATCGTCTCCAACTTATCT N.D. 46 168233 Coding 3 451
TAAGGATGATGTTTCTCCAG N.D. 47 168234 Coding 3 526
CCCAAAGCCAGGTGTAGAAC N.D. 48 168235 Coding 3 601
TGTAAGAGCGGTGGCTCCAC N.D. 49 168236 Coding 3 661
CATTCTGGAATGCCATTGTG N.D. 50 168237 Coding 3 731
TTATGAGGATCAGCATGTGT N.D. 51 168238 Coding 3 861
CCTCTGGAACATCACCAGTT N.D. 52 168239 Coding 3 901
GGATGAAGCACATCAGCAGC N.D. 53 168240 Coding 3 936
TTCACCCCAGAAATACCAGG N.D. 54 168241 Coding 3 1082
GAAACCAGGATATTCTCCCG N.D. 55 168242 Coding 3 1151
TCACTGGCAGAGTAGTCATA N.D. 56 168243 Coding 3 1261
TAATCCTGGCCAAGATGGCG N.D. 57 168244 3'UTR 3 1401
TCATCATCTTTAGCATCCTG N.D. 58 168245 3'UTR 3 1601
GCAAAGACTGACCAGCTGCT N.D. 59 168246 3'UTR 3 1748
GACTACCCAGAAGATTCTGT N.D. 60 168247 3'UTR 3 1881
CTTCCCTCATCCTTACATTC N.D. 61 168248 3'UTR 3 1985
CCCGAGCCAGGAGAGAAAGG N.D. 62 168249 3'UTR 3 2102
CTTCCCCAGCAGAGACCACT N.D. 63 168250 3'UTR 3 2281
CCAATATCCTGAAGATGGCA N.D. 64 168251 3'UTR 3 2481
CCCAACTAATTCCTCCTCTC N.D. 65 168252 3'UTR 3 2541
TATAGATCCTGTCCCTCAGC N.D. 66 168253 3'UTR 3 2631
CTCCCAATAACTCACTCAGT N.D. 67 168254 3'UTR 3 2826
AAGAGATTCCTAACCCTGCC N.D. 68 168255 3'UTR 3 2941
CACACAAAGGAGGCTGCCTG N.D. 69 168256 3'UTR 3 3051
AAGTGGCAGCTAGCTCTACT N.D. 70 168257 3'UTR 3 3321
CACCCTCACCAAGTAAGCAG N.D. 71 168258 3'UTR 3 3401
TGCTTCTTCCCAGTGAGAAC N.D. 72 168259 3'UTR 3 3941
ATCAAGCAGGCATCTGATGA N.D. 73 168260 3'UTR 3 4052
CCCTCAGCCTGAGGTGCCAT N.D. 74 168261 3'UTR 3 4357
ATAATCCTCCACTCAGGCCC N.D. 75 168262 3'UTR 3 4431
CACTTAAGAAAAGCAGCCCT N.D. 76 168263 3'UTR 3 4681
CAGCAAGTCAGTGGCACAGT N.D. 77 168264 3'UTR 3 4971
GGCTAGTTATCCACCGCTTC N.D. 78 168265 3'UTR 3 5044
CCCAATCACAGAAAAGGCAT N.D. 79 168266 3'UTR 3 5081
AACTACTATATCCCACATAA N.D. 80
[0245] As shown in Table 1, SEQ ID NOs 18, 19, 20, 23, 25, 26, 29,
30, 31, 33, 39, 43 and 44 demonstrated at least 10% inhibition of
human stearoyl-CoA desaturase expression in this assay. The target
sites to which these preferred sequences are complementary are
herein referred to as "active sites" and are therefore preferred
sites for targeting by compounds of the present invention.
Example 16
Western Blot Analysis of Stearoyl-CoA Desaturase Protein Levels
[0246] 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 stearoyl-CoA desaturase is used, with a
radiolabelled or fluorescently labeled secondary antibody directed
against the primary antibody species. Bands are visualized using a
PHOSPHORIMAGER.TM. apparatus(Molecular Dynamics, Sunnyvale
Calif.).
Example 17
Antisense Inhibition of Human Stearoyl-CoA Desaturase Expression by
Chimeric Phosphorothioate Oligonucleotides Having 2'-MOE Wings and
a Deoxy Gap
[0247] In accordance with the present invention, a series of
oligonucleotides was designed to target different regions of the
human stearoyl-CoA desaturase RNA, using published sequence
(GenBank accession number AF097514, incorporated herein as SEQ ID
NO: 3 and nucleotides 7371062 to 7389569 of the nucleotide sequence
with the GenBank accession number NT.sub.--030059.7, incorporated
herein as SEQ ID NO: 81). 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 phosphorothioate (P.dbd.S)
throughout the oligonucleotide. All cytidine residues are
5-methylcytidines. The compounds were analyzed for their effect on
human stearoyl-CoA desaturase mRNA levels in HepG2 cells by
quantitative real-time PCR as described in other examples herein.
The positive control oligonucleotide is ISIS 18078
(GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 82), a 2'-O-methoxyl gapmer
(2'-O-methoxyethyls shown in bold) with a rothioate backbone, which
is targeted to human Jun-N-terminal kinase-2 (JNK2). Data are
averages from two experiments and are shown in Table 2. If present,
"N.D." indicates "no data".
2TABLE 2 Inhibition of human stearoyl-CoA desaturase mRNA levels by
chimeric phosphorothioate oligonucleotides having 2'-MOE wings and
a deoxy gap Seq Control ISIS Target Target % ID SEQ ID # Region Seq
ID Site SEQUENCE Inhib No NO 300870 5'UTR 3 13 ccgtgtccggtatttcctca
53 83 82 300871 5'UTR 3 25 ggcaacgggtgaccgtgtcc 75 84 82 300872
5'UTR 3 42 atttaaaggctagagctggc 60 85 82 300873 5'UTR 3 52
cgagccgggaatttaaaggc 40 86 82 300874 5'UTR 3 178
gaggctccggagcggagttc 63 87 82 300875 5'UTR 3 215
ttggctctcggatgccggga 69 88 82 300876 Start Codon 3 228
gtgggccggcatcttggctc 54 89 82 300877 Coding 3 239
tcctgcagcaagtgggccgg 88 90 82 300878 Coding 3 253
agctagagatatcgtcctgc 82 91 82 300879 Coding 3 482
ccatacagggctcccaagtg 42 92 82 300880 Coding 3 513
gtagaacttgcaggtaggaa 57 93 82 300881 Coding 3 566
gctgttatgcccagggcact 93 94 82 300882 Coding 3 667
agacatcattctggaatgcc 76 95 82 300883 Coding 3 709
ctgaaaacttgtggtgggca 42 96 82 300884 Coding 3 715
gtgtttctgaaaacttgtgg 60 97 82 300885 Coding 3 821
aagtctagcgtactcccctt 69 98 82 300886 Coding 3 873
tttgtagtacctcctctgga 36 99 82 300887 Coding 3 1045
cataaggacgatatccgaag 52 100 82 300888 Stop Codon 3 1303
cccaaactcagccactcttg 50 101 82 300889 3'UTR 3 1347
aaacctctgcctggctggtt 87 102 82 300890 3'UTR 3 1381
gtagcattattcagtagtta 58 103 82 300891 3'UTR 3 1419
tactggaatgggttaacatc 42 104 82 300892 3'UTR 3 1484
tcagcttagcatcataaagg 71 105 82 300893 3'UTR 3 1597
agactgaccagctgcttgcc 45 106 82 300894 3'UTR 3 1613
gctggacactgagcaaagac 65 107 82 300895 3'UTR 3 1620
tttggaagctggacactgag 51 108 82 300896 3'UTR 3 1668
tctggagcaaagaccattcg 91 109 82 300897 3'UTR 3 1704
cttcaaagctcacaacagct 69 110 82 300898 3'UTR 3 1711
ccacctacttcaaagctcac 68 111 82 300899 3'UTR 3 1716
tcaagccacctacttcaaag 45 112 82 300900 3'UTR 3 1723
ctctagctcaagccacctac 57 113 82 300901 3'UTR 3 1814
tgtgtcaaatgaagttgctt 66 114 82 300902 3'UTR 3 1842
cccgacaatttacctgcttt 75 115 82 300903 3'UTR 3 1869
ttacattcatacatgctaac 22 116 82 300904 3'UTR 3 1915
tgtctgatcatggcgagagg 90 117 82 300905 3'UTR 3 1969
aaggaagcatgctatgtggt 87 118 82 300906 3'UTR 3 1976
ggagagaaaggaagcatgct 81 119 82 300907 3'UTR 3 2040
aactatatgttgcggcattg 59 120 82 300908 3'UTR 3 2781
tagatgttaacagagacccc 15 121 82 300909 3'UTR 3 2839
aatcagggtagtgaagagat 27 122 82 300910 3'UTR 3 2859
gggtagagccaggaatcaag 32 123 82 300911 3'UTR 3 3020
agcagtggttcagtgaccct 83 124 82 300912 3'UTR 3 3035
tactttcaaaagagaagcag 27 125 82 300913 3'UTR 3 3056
cgtgaaagtggcagctagct 81 126 82 300914 3'UTR 3 3122
ccttgtcttgagccatcagt 77 127 82 300915 3'UTR 3 3132
ggtttgccagccttgtcttg 78 128 82 300916 3'UTR 3 3222
cactggctcaacatgagcgc 71 129 82 300917 3'UTR 3 3238
tgctctgtggctggcccact 93 130 82 300918 3'UTR 3 3252
aataaaccctcttttgctct 52 131 82 300919 3'UTR 3 3259
gactgaaaataaaccctctt 54 132 82 300920 3'UTR 3 3342
agagcactgactcaggcggg 81 133 82 300921 3'UTR 3 3357
ttgcactgccagctgagagc 88 134 82 300922 3'UTR 3 3371
tacttctacaagcattgcac 83 135 82 300923 3'UTR 3 3383
actgtttcctcctacttcta 63 136 82 300924 3'UTR 3 3409
cttgcccttgcttcttccca 70 137 82 300925 3'UTR 3 3432
tttcgaggtgaggcacttgg 60 138 82 300926 3'UTR 3 3480
tcagccaaagctttgcagtt 77 139 82 300927 3'UTR 3 3655
ttctgctttgatgactgagc 86 140 82 300928 3'UTR 3 2052
atcctcggcctcaactatat 58 141 82 300929 3'UTR 3 2136
tccttgttattaaagaaaaa 35 142 82 300930 3'UTR 3 2146
ctaagaaatctccttgttat 28 143 82 300931 3'UTR 3 2162
cttcttgatatatgaactaa 11 144 82 300932 3'UTR 3 2171
acttcaagacttcttgatat 45 145 82 300933 3'UTR 3 2214
aaattccatgagctgctgtt 27 146 82 300934 3'UTR 3 2245
gaactgtaatgagcagctca 36 147 82 300935 3'UTR 3 2272
tgaagatggcagagcagaaa 27 148 82 300936 3'UTR 3 2321
tggaaatgccacagccatct 70 149 82 300937 3'UTR 3 2361
cgacttcacctccttaaatc 56 150 82 300938 3'UTR 3 2397
gcaatgtatatatgtatata 31 151 82 300939 3'UTR 3 2506
ccagcagtggagaggaaatt 27 152 82 300940 3'UTR 3 2525
cagcctctccatctcatgtc 61 153 82 300941 3'UTR 3 2570
ctatgtgaagttcgctctta 66 154 82 300942 3'UTR 3 2589
cgtgttctcagatcccttcc 0 155 82 300943 3'UTR 3 2676
aactaattaatgaatggacc 36 156 82 300944 3'UTR 3 2700
ttactcatttcaaggagaaa 54 157 82 300945 3'UTR 3 2715
gaagccttctagtttttact 55 158 82 300946 3'UTR 3 2726
cactgtggagagaagccttc 71 159 82 300947 3'UTR 3 2732
gcacaacactgtggagagaa 58 160 82 300948 3'UTR 3 3679
aatcttaatagagcaaagcc 0 161 82 300949 3'UTR 3 3707
gactgagtgtttggtagtgt 26 162 82 300950 3'UTR 3 3771
agcctctacgcaattaacac 38 163 82 300951 3'UTR 3 3825
ctgaggtgaatagctcaaaa 51 164 82 300952 3'UTR 3 3834
ccttttctactgaggtgaat 65 165 82 300953 3'UTR 3 3911
tagaaataccagcagacatt 37 166 82 300954 3'UTR 3 3993
gcacacgattacaataggaa 62 167 82 300955 3'UTR 3 3999
tccatggcacacgattacaa 64 168 82 300956 3'UTR 3 4004
tcagatccatggcacacgat 54 169 82 300957 3'UTR 3 4041
aggtgccatccagccttatg 12 170 82 300958 3'UTR 3 4053
gccctcagcctgaggtgcca 21 171 82 300959 3'UTR 3 4132
agctttagaatcttgaaaat 25 172 82 300960 3'UTR 3 4150
aatgtgtcacttgaattgag 26 173 82 300961 3'UTR 3 4193
ctgttagaaatccggactct 33 174 82 300962 3'UTR 3 4205
ccaaagcagggactgttaga 41 175 82 300963 3'UTR 3 4261
caacactgtgattagaaaag 20 176 82 300964 3'UTR 3 4321
cttcagtagggtctcaggtg 43 177 82 300965 3'UTR 3 4331
ctaccagccacttcagtagg 37 178 82 300966 3'UTR 3 4347
actcaggcccctttttctac 34 179 82 300967 3'UTR 3 4364
gatactgataatcctccact 18 180 82 300968 3'UTR 3 4379
aatcctgcaaatcgtgatac 34 181 82 300969 3'UTR 3 4420
agcagccctaacaaaagttt 34 182 82 300970 3'UTR 3 4535
aaattttccattttaaatgc 23 183 82 300971 3'UTR 3 4578
cacttacagagaatacaccc 38 184 82 300972 3'UTR 3 4584
gagctacacttacagagaat 26 185 82 300973 3'UTR 3 4628
aacatggccacctcgctttt 16 186 82 300974 3'UTR 3 4645
gccttaaccaccagcataac 40 187 82 300975 3'UTR 3 4653
aggccctggccttaaccacc 60 188 82 300976 3'UTR 3 4658
tggagaggccctggccttaa 55 189 82 300977 3'UTR 3 4786
tcatgcctcaaaactgccct 34 190 82 300978 3'UTR 3 4800
ctaaaaagcattagtcatgc 0 191 82 300979 3'UTR 3 4834
agaattcctgtgctgaagga 13 192 82 300980 3'UTR 3 4841
ggtcttgagaattcctgtgc 47 193 82 300981 3'UTR 3 4846
actcaggtcttgagaattcc 43 194 82 300982 3'UTR 3 4868
ggacattcctattataaaaa 9 195 82 300983 3'UTR 3 4892
acacggacgtatcaagttca 41 196 82 300984 3'UTR 3 5024
cctctgtatgtttccgtggc 67 197 82 300985 exon 81 505
tgcgaggagttgactggcgc 39 198 82 300986 exon 81 512
ggcaaagtgcgaggagttga 45 199 82 300987 exon: intron 81 799
ggaaactcacatcgtcctgc 33 200 82 300988 exon: intron 81 1614
ggctgcttaccccaaagcca 29 201 82 300989 intron 81 2854
ctcagttgcatttcactgta 45 202 82 300990 intron 81 3557
gtgggaagagaagatgtcca 4 203 82 300991 intron 81 5287
gccttctctaaggttttaag 50 204 82 300992 intron: exon 81 5633
tagaatacccctgccaggag 34 205 82 300993 exon: intron 81 5764
aacttcttacctggaatgcc 18 206 82 300994 intron 81 7232
ccttgcaaaagagctcatac 56 207 82 300995 exon: intron 81 7900
cttcactcacctcctctgga 0 208 82 300996 intron 81 8630
tttgcactgtctctccccac 13 209 82 300997 intron 81 8878
tcagtggtttcttacacttg 77 210 82 300998 intron: exon 81 9799
tttgtagtacctacattgac 4 211 82 300999 exon: intron 81 10032
gctgacttacccacagctcc 10 212 82 301000 intron 81 10163
tactgccccctaattttata 0 213 82 301001 intron 81 12377
ccatttgcgatacaggaaac 27 214 82 301002 intron: exon 81 14001
aagccctcacctgaaacaaa 22 215 82
[0248] As shown in Table 2, SEQ ID NOs 83, 84, 85, 87, 88, 89, 90,
91, 93, 94, 95, 97, 98, 100, 101, 102, 103, 105, 107, 108, 109,
110, 111, 113, 114, 115, 117, 118, 119, 120, 124, 126, 127, 128,
129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,
149, 150, 153, 154, 157, 158, 159, 160, 164, 165, 167, 168, 169,
188, 189, 197, 204, 207 and 210 demonstrated at least 50%
inhibition of human stearoyl-CoA desaturase expression in this
assay. Preferred antisense oligonucleotide sequences are SEQ ID NOs
94, 130, 140 and 134. The target sites to which these preferred
sequences are complementary are herein referred to as "active
sites" and are therefore preferred sites for targeting by compounds
of the present invention.
Example 18
Antisense Inhibition of Mouse Stearoyl-CoA Desaturase Expression by
Chimeric Phosphorothioate Oligonucleotides having 2'-MOE Wings and
a Deoxy Gap
[0249] In accordance with the present invention, a series of
oligonucleotides was designed to target different regions of the
mouse stearoyl-CoA desaturase RNA, using published sequence
(GenBank Accession number M21280.1, incorporated herein as SEQ ID
NO: 216; GenBank Accession number M21281.1, incorporated herein as
SEQ ID NO: 217; GenBank Accession number M21282.1, incorporated
herein as SEQ ID NO: 218; GenBank Accession number M21283.1,
incorporated herein as SEQ ID NO: 219; GenBank Accession number
M21284.1, incorporated herein as SEQ ID NO: 220; GenBank Accession
number M21285.1, incorporated herein as SEQ ID NO: 221; the
concatenation of SEQ ID NOs 216, 217, 218, 219, 220 and 221,
incorporated herein as SEQ ID NO: 222). 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
phosphorothioate (P.dbd.S) throughout the oligonucleotide. All
cytidine residues are 5-methylcytidines.
3TABLE 3 Chimeric phosphorothioate oligonucleotides hav- ing 2'-MOE
wings and a deoxy gap targeted to mouse stearoyl-CoA Target SEQ
ISIS SEQ ID Target ID # Region NO Site Sequence NO 180548 5'UTR 222
9 agatctcttggagcatgtgg 223 180549 5'UTR 222 71 cttctctcgttcatttccgg
224 180550 5'UTR 222 126 cttctttcatttcaggacgg 225 180551 5'UTR 222
161 tccctcctcatcctgatagg 226 180552 5'UTR 222 191
cctccagacgtactccagct 227 180553 5'UTR 222 211 aggaccatgagaatgatgtt
228 180554 5'UTR 222 231 acaggcctcccaagtgcagc 229 180555 5'UTR 222
250 ggaaccagtatgatcccgta 230 180557 5'UTR 222 291
agtagaaaatcccgaagagg 231 180558 5'UTR 222 321 cggctgtgatgcccagagcg
232 180559 5'UTR 222 341 gctccagaggcgatgagccc 233 180560 5'UTR 222
361 cgagccttgtaagttctgtg 234 180561 5'UTR 222 391
gcaatgattaggaagatccg 235 180562 5'UTR 222 421 tacacgtcattttggaacgc
236 180563 5'UTR 222 441 ggtgatctcgggcccagtcg 237 180564 5'UTR 222
471 cgtgtgtttctgagaacttg 238 180565 5'UTR 222 591
cggctttcaggtcagacatg 239 180566 5'UTR 222 611 ctggaacatcaccagcttct
240 180567 5'UTR 222 648 agcacatcagcaggaggccg 241 180568 5'UTR 222
651 tgaagcacatcagcaggagg 242 180569 5'UTR 222 691
gtctcgccccagcagtacca 243 180570 5'UTR 222 741 gcaccagagtgtatcgcaag
244 180571 5'UTR 222 761 caccagccaggtggcgttga 245 180572 5'UTR 222
781 tagagatgcgcggcactgtt 246 180573 5'UTR 222 812
ttgaatgttcttgtcgtagg 247 180574 Start 222 855 cctcgcccacggcacccagg
248 Codon 180575 Coding 222 869 gtagttgtggaagccctcgc 249 180576
Coding 222 881 gaaggtgtggtggtagttgt 250 180577 Coding 222 911
gcggtactcactggcagagt 251 180578 Coding 222 929 ggtgaagttgatgtgccagc
252 180579 Coding 222 1011 tcctggctaagacagtagcc 253 180580 Coding
222 1031 cccgtctccagttctcttaa 254 180581 Coding 222 1039
ttgtgactcccgtctccagt 255 180582 Coding 222 1049
tcagctactcttgtgactcc 256
[0250] In a further embodiment of the present invention, a series
of oligonucleotides was designed to target different regions of the
mouse stearoyl-CoA desaturase RNA, using published sequences
(GenBank Accession number M21280.1, incorporated herein as SEQ ID
NO: 216; GenBank Accession number M21281.1, incorporated herein as
SEQ ID NO: 217; GenBank Accession number M21282.1, incorporated
herein as SEQ ID NO: 218; GenBank Accession number M21283.1,
incorporated herein as SEQ ID NO: 219; GenBank Accession number
M21284.1, incorporated herein as SEQ ID NO: 220; GenBank Accession
number M21285.1, incorporated herein as SEQ ID NO: 221; the
concatenation of SEQ ID NOs 216, 217, 218, 219, 220 and 221,
incorporated herein as SEQ ID NO: 222). The oligonucleotides are
shown in Table 4. "Target site" indicates the first (5'-most)
nucleotide number on the particular target sequence to which the
oligonucleotide binds. All compounds in Table 4 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.
[0251] Probes and primers to mouse stearoyl-CoA desaturase were
designed to hybridize to a mouse stearoyl-CoA desaturase sequence,
using published sequence information (SEQ ID NO: 222). For mouse
stearoyl-CoA desaturase the PCR primers were:
[0252] forward primer: ACACCAGAGACATGGGCAAGT (SEQ ID NO: 257)
[0253] reverse primer: CATCACACACTGGCTTCAGGAA (SEQ ID NO: 258) and
the PCR probe was: FAM-CTGAAGTGAGGTCCATTAG-TAMRA (SEQ ID NO: 259)
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 mouse GAPDH the PCR primers
were:
[0254] forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO:260)
[0255] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:261) and the
PCR probe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3' (SEQ ID No:
262) where JOE is the fluorescent reporter dye and TAMRA is the
quencher dye.
[0256] The compounds were analyzed for their effect on mouse
stearoyl-CoA desaturase mRNA levels in b.END cells by quantitative
real-time PCR as described in other examples herein. The positive
control oligonucleotide is ISIS 18078 (GTGCGCGCGAGCCCGAAATC, SEQ ID
NO: 82), a 2'-O-methoxyl gapmer (2'-O-methoxyethyls shown in bold)
with a phosphorothioate backbone, which is targeted to human
Jun-N-terminal kinase-2 (JNK2). Data are averages from two
experiments and are shown in Table 4. If present, "N.D." indicates
"no data".
4TABLE 4 Inhibition of mouse stearoyl-CoA desaturase mRNA levels by
chimeric phosphorothioate oligonucleotides having 2'-MOE wings and
a deoxy gap Target Control Isis SEQ ID Target % SEQ ID SEQ ID #
Region NO Site Sequence Inhib NO NO 185154 exon: intron 216 876
ggaagctcacctcttggagc 48 263 82 185155 exon: intron 217 269
ctgctcaccgaagagggcag 0 264 82 185156 intron: exon 218 1
gtagtagaaaatccctgcaa 6 265 82 185157 exon: intron 219 202
tcccttacctcctctggaac 0 266 82 185158 exon: intron 220 228
tgacttacccacggcaccca 41 267 82 185159 intron: exon 221 1
gtggaagccctcgcctgcaa 83 268 82 185160 5'UTR 222 68
ctggctaccgccactcacaa 0 269 82 185161 5'UTR 222 142
aagcctaggactttggtctg 51 270 82 185162 5'UTR 222 148
gtgtgcaagcctaggacttt 0 271 82 185163 5'UTR 222 156
taggaattgtgtgcaagcct 0 272 82 185164 5'UTR 222 275
atctgctgttccctctgcct 0 273 82 185165 5'UTR 222 445
tccagagtagaccttggagg 15 274 82 185166 5'UTR 222 571
ctagccaaggaagccaggcg 12 275 82 185167 5'UTR 222 581
gcagagatagctagccaagg 2 276 82 185168 5'UTR 222 612
ttttatcggctgccagcaaa 41 277 82 185169 5'UTR 222 644
ggatgaccgtgttcagtatt 41 278 82 185170 5'UTR 222 697
tggctgtgcacagatctcct 82 279 82 185171 5'UTR 222 708
tcagcccggtctggctgtgc 56 280 82 185172 5'UTR 222 748
gcgcttggaaacctgccctc 59 281 82 185173 5'UTR 222 768
tgtaggcgagtggcggaact 43 282 82 185174 5'UTR 222 795
gtggacttcggttccggagc 31 283 82 185175 5'UTR 222 830
ttgctcgcctcactttccca 79 284 82 185176 5'UTR 222 854
gtgggccggcatgatgatag 67 285 82 185177 5'UTR 222 877
gaactggagatctcttggag 51 286 82 185178 Coding 222 1150
tagaaaatcccgaagagggc 0 287 82 185179 Coding 222 1160
ggtcatgtagtagaaaatcc 10 288 82 185180 Coding 222 1165
gcgctggtcatgtagtagaa 40 289 82 185181 Coding 222 1676
ggattgaatgttcttgtcgt 81 290 82 185182 Coding 222 1681
tcccgggattgaatgttctt 46 291 82 185183 Coding 222 1688
gatattctcccgggattgaa 39 292 82 185184 Coding 222 1858
gtagccttagaaactttctt 52 293 82 185185 Stop Codon 222 1918
cccaaagctcagctactctt 65 294 82 185186 3'UTR 222 1934
aacaggaactcagaagccca 90 295 82 185187 3'UTR 222 1967
cagaatattaaatctctgcc 48 296 82 185188 3'UTR 222 1984
agttgttagttaatcaacag 0 297 82 185189 3'UTR 222 2159
aattgtatatgcatttatca 62 298 82 185190 3'UTR 222 2208
ctgtatagaatgttcaaatt 2 299 82 185191 3'UTR 222 2236
acagcatgttccttggcttt 51 300 82 185192 3'UTR 222 2246
tagcatcaaaacagcatgtt 46 301 82 185193 3'UTR 222 2259
accatgctcaccctagcatc 45 302 82 185194 3'UTR 222 2408
aaggatcagtatttcagaaa 39 303 82 185195 3'UTR 222 2552
tctctcgagacaatctactt 67 304 82 185196 3'UTR 222 2821
cttcagttaccaaaagctaa 37 305 82 185197 3'UTR 222 2887
aaatgtcagctgtttagtta 0 306 82 185198 3'UTR 222 3002
ggcaacccaggcaacacctc 39 307 82 185199 3'UTR 222 3017
gccacgaaagaaactggcaa 23 308 82 185200 3'UTR 222 3102
atgttccccaagggcttcat 86 309 82 185201 3'UTR 222 3112
tccctggcagatgttcccca 76 310 82 185202 3'UTR 222 3427
ctggctctgcttcctgaagc 48 311 82 185203 3'UTR 222 3569
gctgagctgttaactcacaa 71 312 82 185204 3'UTR 222 3640
cacacaccgagacagatcaa 79 313 82 185205 3'UTR 222 3828
caggaagcagaccctcttcc 42 314 82 185206 3'UTR 222 3958
aatactgatgtgatgttttc 65 315 82 185207 3'UTR 222 3968
atggttctaaaatactgatg 36 316 82 185208 3'UTR 222 4046
actgagtgtttggcacctta 79 317 82 185209 3'UTR 222 4066
ggctctgattctacaagtga 61 318 82 185210 3'UTR 222 4116
tcaacaaaacagctcagagc 83 319 82 185211 3'UTR 222 4127
gattttctacttcaacaaaa 56 320 82 185212 3'UTR 222 4333
cttaaagacaccaggacctc 56 321 82 185213 3'UTR 222 4387
catctggaaactgttataaa 45 322 82 185214 3'UTR 222 4466
ctaagggaaggagtgagact 42 323 82 185215 3'UTR 222 4608
ttacttcccaccaaatttga 59 324 82 185216 3'UTR 222 4652
tgacaatgataacgaggacg 81 325 82 185217 3'UTR 222 4760
cagatggtggctttgctaac 0 326 82 185218 3'UTR 222 4825
ttgttacaagagaaaggata 68 327 82 185219 3'UTR 222 4884
tcagatacttagcccaggag 74 328 82 185220 3'UTR 222 4902
tgttgagatgtgagactgtc 50 329 82 185221 3'UTR 222 5010
cacctcagaactgcccttga 73 330 82 185222 3'UTR 222 5018
gctctaatcacctcagaact 84 331 82 185223 3'UTR 222 5074
ggagtctgtatgaatacctc 64 332 82 185224 3'UTR 222 5132
tctctgggaagagcaatgta 58 333 82 185225 3'UTR 222 5170
gtaggtagtcttgcactttg 36 334 82 185226 3'UTR 222 5211
aggaagggaaaggtttcctg 38 335 82 185227 3'UTR 222 5268
tacacttgggtcacaaataa 49 336 82 185228 3'UTR 222 5280
aatcatccaaattacacttg 50 337 82 185229 3'UTR 222 5303
cttcaagagttgatattaat 60 338 82 185230 3'UTR 222 5329
atacaatctcaatcagtaca 76 339 82 185231 3'UTR 222 5347
cacttttattaggaacaaat 0 340 82
[0257] As shown in Table 4, SEQ ID NOs 263, 267, 268, 270, 277,
278, 279, 280, 281, 282, 284, 285, 286, 289, 290, 291, 293, 294,
295, 296, 298, 300, 301, 302, 304, 309, 310, 311, 312, 313, 314,
315, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 328, 329,
330, 331, 332, 333, 336, 337, 338, 339 demonstrated at least 40%
inhibition of stearoyl-CoA desaturase in this experiment and are
therefore preferred. More preferred are SEQ ID NOs 295, 331 and
268. The target regions to which these preferred sequences are
complementary are herein referred to as "preferred target segments"
and are therefore preferred for targeting by compounds of the
present invention.
[0258] In a further embodiment of the present invention, a series
of oligonucleotides was designed to target different regions of the
mouse stearoyl-CoA desaturase RNA, using published sequences
(GenBank Accession number M21280.1, incorporated herein as SEQ ID
NO: 216; GenBank Accession number M21281.1, incorporated herein as
SEQ ID NO: 217; GenBank Accession number M21282.1, incorporated
herein as SEQ ID NO: 218; GenBank Accession number M21283.1,
incorporated herein as SEQ ID NO: 219; GenBank Accession number
M21284.1, incorporated herein as SEQ ID NO: 220; GenBank Accession
number M21285.1, incorporated herein as SEQ ID NO: 221; the
concatenation of SEQ ID NOs 216, 217, 218, 219, 220 and 221,
incorporated herein as SEQ ID NO: 222). The oligonucleotides are
shown in Table 5. "Target site" indicates the first (5'-most)
nucleotide number on the particular target sequence to which the
oligonucleotide binds. All compounds in Table 5 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.
[0259] Probes and primers to mouse stearoyl-CoA desaturase were
designed to hybridize to a mouse stearoyl-CoA desaturase sequence,
using published sequence information (SEQ ID NO: 222). For mouse
stearoyl-CoA desaturase the PCR primers were:
[0260] forward primer: TTCCGCCACTCGCCTACA (SEQ ID NO: 341)
[0261] reverse primer: CTTTCCCAGTGCTGAGATCGA (SEQ ID NO: 342) and
the PCR probe was: FAM-CAACGGGCTCCGGAACCGAA-TAMRA (SEQ ID NO: 343)
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 mouse GAPDH the PCR primers
were:
[0262] forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO:261)
[0263] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:262) and the
PCR probe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3' (SEQ ID NO:
263) where JOE is the fluorescent reporter dye and TAMRA is the
quencher dye.
[0264] The compounds were analyzed for their effect on mouse
stearoyl-CoA desaturase mRNA levels in primary mouse hepatocytes by
quantitative real-time PCR as described in other examples herein.
The positive control oligonucleotide is ISIS 18078
(GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 82), a 2'-O-methoxyl gapmer
(2'-O-methoxyethyls shown in bold) with a phosphorothioate
backbone, which is targeted to human Jun-N-terminal kinase-2
(JNK2). Data are averages from two experiments and are shown in
Table 5. If present, "N.D." indicates "no data".
5TABLE 5 Inhibition of mouse stearoyl-CoA desaturase mRNA levels by
chimeric phosphorothioate oligonucleotides having 2'-MOE wings and
a deoxy gap Target Control Isis SEQ ID Target % SEQ SEQ ID # Region
NO Site Sequence Inhib ID NO NO 180556 5'UTR 222 261
gcttgcaggagggaaccagt 40 344 82 244459 5'UTR 222 138
ctaggactttggtctggcgc 6 345 82 244461 5'UTR 222 280
gcgcaatctgctgttccctc 0 346 82 244464 5'UTR 222 401
agggcgcgctgctccaaccc 0 347 82 244467 5'UTR 222 462
aagaaagccaagtagattcc 0 348 82 244470 5'UTR 222 692
gtgcacagatctcctgggct 35 349 82 244472 5'UTR 222 736
ctgccctcctgactctcggg 54 350 82 244476 Coding 222 878
agaactggagatctcttgga 71 351 82 244479 Coding 222 1020
cctcctcatcctgataggtg 16 352 82 244481 Coding 222 1045
acgtactccagcttgggcgg 50 353 82 244484 Coding 222 1057
atgttcctccagacgtactc 33 354 82 244487 Coding 222 1062
gaatgatgttcctccagacg 43 355 82 244490 Coding 222 1068
ccatgagaatgatgttcctc 50 356 82 244493 Coding 222 1098
tcccgtacaggcctcccaag 54 357 82 244495 Coding 222 1128
tgtagagcttgcaggaggga 10 358 82 244498 Coding 222 1264
gccatggtgttggcaatgat 41 359 82 244501 Coding 222 1324
tctgagaacttgtggtgggc 18 360 82 244504 Coding 222 1329
gtgtttctgagaacttgtgg 50 361 82 244507 Coding 222 1334
ggcgtgtgtttctgagaact 63 362 82 244510 Coding 222 1347
aattgtgagggtcggcgtgt 11 363 82 244514 Coding 222 1357
ccacggcgggaattgtgagg 47 364 82 244517 Coding 222 1363
aagaagccacggcgggaatt 16 365 82 244520 Coding 222 1387
agcagccaacccacgtgaga 51 366 82 244523 Coding 222 1395
tgcgcacaagcagccaaccc 67 367 82 244526 Coding 222 1400
gtgtttgcgcacaagcagcc 52 368 82 244528 Coding 222 1408
acagccgggtgtttgcgcac 63 369 82 244532 Coding 222 1413
ctttgacagccgggtgtttg 3 370 82 244535 Coding 222 1418
cttctctttgacagccgggt 50 371 82 244538 Coding 222 1423
ccgcccttctctttgacagc 64 372 82 244541 Coding 222 1435
atgtccagttttccgccctt 55 373 82 244542 Coding 222 1440
cagacatgtccagttttccg 55 374 82 244546 Coding 222 1445
caggtcagacatgtccagtt 48 375 82 244549 Coding 222 1450
gctttcaggtcagacatgtc 49 376 82 244553 Coding 222 1455
tctcggctttcaggtcagac 50 377 82 244554 Coding 222 1460
cagcttctcggctttcaggt 37 378 82 244557 Coding 222 1465
atcaccagcttctcggcttt 20 379 82 244560 Coding 222 1470
ggaacatcaccagcttctcg 42 380 82 244565 Coding 222 1477
ctcctctggaacatcaccag 17 381 82 244567 Coding 222 1486
ttgtagtacctcctctggaa 0 382 82 244569 Coding 222 1516
aggatgaagcacatcagcag 29 383 82 244572 Coding 222 1525
agcgtgggcaggatgaagca 45 384 82 244577 Coding 222 1538
gtaccagggcaccagcgtgg 37 385 82 244578 Coding 222 1543
cagcagtaccagggcaccag 25 386 82 244581 Coding 222 1548
cgccccagcagtaccagggc 13 387 82 244585 Coding 222 1583
gaaggtgctaacgaacaggc 18 388 82 244589 Coding 222 1627
ctgttcaccagccaggtggc 37 389 82 244591 Coding 222 1633
gcggcactgttcaccagcca 3 390 82 244595 Coding 222 1693
accaggatattctcccggga 62 391 82 244598 Coding 222 1732
tggtagttgtggaagccctc 54 392 82 244599 Coding 222 1768
tactcactggcagagtagtc 19 393 82 244602 Coding 222 1773
agcggtactcactggcagag 31 394 82 244607 Coding 222 1778
gtgccagcggtactcactgg 5 395 82 244609 Coding 222 1783
ttgatgtgccagcggtactc 29 396 82 244613 Coding 222 1792
gtggtgaagttgatgtgcca 40 397 82 244615 Coding 222 1798
aagaacgtggtgaagttgat 12 398 82 244619 Coding 222 1860
cagtagccttagaaactttc 75 399 82 244620 Coding 222 1885
ccagttctcttaatcctggc 63 400 82 244623 Coding 222 1891
ccgtctccagttctcttaat 37 401 82 244626 3'UTR 222 2006
taacaccccgatagcaatat 59 402 82 244630 3'UTR 222 2365
gagggtggacagacacaggc 4 403 82 244633 3'UTR 222 2445
cttgaagctaggaacaagga 68 404 82 244636 3'UTR 222 2647
tatggctacctctctctctc 82 405 82 244639 3'UTR 222 2920
ttttcatagtttcacaccat 47 406 82 244643 3'UTR 222 2970
tattttctaagtgaaatagt 5 407 82 244644 3'UTR 222 3243
taggcagcactaggcaggct 33 408 82 244647 3'UTR 222 3373
aggaacaggcctggacagca 36 409 82 244650 3'UTR 222 4168
gagggctataggtcagtaga 34 410 82 244655 3'UTR 222 4329
aagacaccaggacctcaatg 18 411 82 244656 3'UTR 222 4532
ccaatgtactgatgactctc 62 412 82 244660 3'UTR 222 4737
tcacaccacctcactggagc 62 413 82 244663 3'UTR 222 4987
agtaggtcagtattaataac 35 414 82 244667 3'UTR 222 5220
atctcattcaggaagggaaa 0 415 82 244668 3'UTR 222 5272
aaattacacttgggtcacaa 57 416 82 244673 3'UTR 222 5326
caatctcaatcagtacaagt 37 417 82
[0265] As shown in Table 5, SEQ ID NOs 344, 350, 351, 353, 355,
356, 357, 359, 361, 362, 364, 366, 367, 368, 369, 371, 372, 373,
374, 375, 376, 377, 380, 384, 391, 392, 397, 399, 400, 402, 404,
405, 406, 412, 413, 416 exhibited at least 40% inhibition of
stearoyl-CoA desaturase in this experiment. A more preferred
sequence is SEQ ID NO: 373. The target regions to which these
preferred sequences are complementary are herein referred to as
"preferred target segments" are therefore preferred for targeting
by compounds of the present invention.
Example 19
Effects of Antisense Inhibition of Mouse Stearoyl-CoA Desaturase
Expression in Mice: mRNA Levels in Liver and Fat Tissue
[0266] Ob/ob mice harbor a mutation in the leptin gene. The leptin
mutation on a C57B/16 background yields a db/db phenotype,
characterized by, hyperglycemia, obesity, hyperlipidemia, and
insulin resistance. However, a mutation in the leptin gene on a
different mouse background can produce obesity without diabetes,
and these mice are referred to as ob/ob mice. Leptin is a hormone
that regulates appetite. Leptin deficiency results in obesity in
animals and humans.
[0267] In accordance with the present invention, ISIS 185222 (SEQ
ID NO: 332) was further investigated for its ability to reduce
target levels in liver and fat tissue in ob/ob mice maintained on a
high-fat (11% kcal from fat) or low-fat (2% from fat) diet.
[0268] ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, SEQ ID NO: 418) is a
scrambled control oligonucleotide. ISIS 141923 is a chimeric
oligonucleotide ("gapmer") 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.
[0269] Eight-week old male ob/ob mice were dosed twice weekly by
intraperitoneal injection with saline or 25 mg/kg of ISIS 185222 or
ISIS 141923. Mice were maintained on a low-fat or high-fat diet. At
the end of the ten-week investigation period, mice were sacrificed
and evaluated for stearoyl-CoA desaturase and stearoyl-CoA
desaturase-2 mRNA levels in liver and fat tissue. Inhibition of
mRNA expression was determined by quantitative real-time PCR as
described in other examples herein. The data are the averages of
mRNA levels from nine mice per group and are presented in Table
6.
6TABLE 6 Antisense inhibition of stearoyl-CoA desaturase in mouse
liver and fat tissue Percent Inhibition Saline ISIS ISIS control
185222 141923 mRNA Diet Liver Fat Liver Fat Liver Fat stearoyl-CoA
High-fat 0 0 93 96 29 28 desaturase Low-fat 0 0 94 98 0 0
stearoyl-CoA High-fat 0 0 37 40 52 5 desaturase-2 Low-fat 0 0 37 0
0 0
[0270] The data demonstrate that the oligonucleotide of the present
invention can inhibit the expression of stearoyl-CoA desaturase in
vivo, in both liver and fat tissues. The data also suggest that
antisense inhibition of stearoyl-CoA desaturase can reduce
expression of stearoyl-CoA desaturase-2.
Example 20
Effects of Antisense Inhibition of Stearoyl-CoA Desaturase in a
Mouse Model of Obesity: Organ Weights and Levels of Serum
Cholesterol, Triglyceride and Liver Enzymes
[0271] In accordance with the present invention, further
investigation of the effects antisense inhibition of stearoyl-CoA
desaturase was conducted in ob/ob mice. The saline-treated and
antisense oligonucleotide-treated ob/ob mice described in Example
19 were also evaluated for body organ weight, levels of serum
cholesterol and triglyceride and levels of liver enzymes ALT and
AST at the end of the ten-week investigation period. Increased
levels of ALT and AST are indicative of impaired liver function.
Blood samples were collected and evaluated for cholesterol,
triglyceride, ALT and AST levels. White adipose tissue (WAT),
spleen and liver were individually weighed. Data are expressed as
percent change relative to the saline control for the respective
diet. The data represent the average of nine mice per treatment
group and are presented in Table 7.
7TABLE 7 Effects of antisense inhibtion of stearoyl-CoA desaturase
on cholesterol, triglyceride, ALT, AST and organ weight Percent
Change Liver Organ Enzymes Weight ALT AST CHOL TRIG Liver Spleen
WAT ISIS High-fat -76 -72 -11 3 -8 19 2 185222 Low-fat -60 -55 15
29 -29 -19 -4 ISIS High-fat -42 -39 -1 -10 -4 20 9 141923 Low-fat
45 34 50 70 31 -41 27
[0272] The data demonstrate that, concomitant with reducing target
mRNA expression (shown in Example 19), the oligonucleotide of the
present invention lowers the levels of the liver enzyme ALT and AST
in animals maintained on either a high-fat or low-fat diet, which
is indicative of improved liver function. Histologically, mice
treated with ISIS 185222 and maintained on a low-fat diet exhibit
lowered hepatic fatty degeneration.
Example 21
Effects of Antisense Inhibition of Stearoyl-CoA Desaturase in a
Mouse Model of Obesity: Plasma Glucose and Insulin, Body Weight,
Food Consumption and Oxygen Consumption
[0273] In accordance with the present invention, the ob/ob mice
described in Example 19 were further evaluated to assess the
effects of antisense inhibition of stearoyl-CoA desaturase.
[0274] Mice were evaluated for plasma glucose and oxygen
consumption following three weeks of treatment. The glucose
evaluation was conducted following an overnight fast. The oxygen
consumption was determined by measuring metabolic rate (MR) and
respiratory quotient (RER) during both light and dark cycles.
Plasma glucose and insulin (both in non-fasting mice), food
consumption, oxygen consumption and total body weight were measured
throughout the ten-week treatment period. Shown in Table 8 are
plasma insulin (non-fasting) following five weeks of treatment,
food consumption following six weeks of treatment and plasma
glucose (non-fasting) and total body weight following seven weeks
of treatment. The data are the averages of measurements from seven
to nine mice and are expressed as percent change relative to saline
control for the respective diet. The data are presented in Table
8.
8TABLE 8 Effects of antisense inhibtion of stearoyl-CoA desaturase
on body weight, food consumption, insulin, glucose and oxygen
consumption Percent Change Weight Oxygen consumption Total Food
Insulin Glucose MR RER Body Consumed Fed Fed Fast Dark Light Dark
Light ISIS High-fat -3 -10 -3 2 -3 3 0 0 2 185222 Low-fat -60 -55
15 29 -29 4 11 -6 0 ISIS High-fat -42 -39 -1 -10 -4 -5 -5 3 0
141923 Low-fat 45 34 50 70 31 6 5 2 0
[0275] The data suggest that body weight and food consumption are
lowered by treatment of ob/ob mice with the oligonucleotide of the
present invention. Comparison of blood glucose, insulin and oxygen
consumption in mice fed the same diet does not reveal any
significant changes between saline-treated and antisense
oligonucleotide-treated mice.
Example 22
RNA Synthesis
[0276] 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.
[0277] 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.
[0278] 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.
[0279] Following synthesis, the methyl protecting groups on the
phosphates are cleaved in 30 minutes utilizing 1 M
disodium-2-carbamoyl-2-cyanoethyl- ene-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.
[0280] 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
and yet, when subsequently modified, permits deprotection to be
carried out under relatively mild aqueous conditions compatible
with the final RNA oligonucleotide product.
[0281] 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., Tetrahedrom 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).
[0282] 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 uM 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 23
Design and Screening of Duplexed Antisense Compounds Targeting
Stearoyl-CoA Desaturase
[0283] 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
stearoyl-CoA desaturase. 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.
[0284] For example, a duplex comprising an antisense strand having
the sequence CGAGAGGCGGACGGGACCG and having a two-nucleobase
overhang of deoxythymidine(dT) would have the following
structure:
9 cgagaggcggacgggaccgTT Antisense Strand
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline. TTgctctccgcctgccctggc
Complement
[0285] 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 the 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.
[0286] Once prepared, the duplexed antisense compounds are
evaluated for their ability to modulate stearoyl-CoA desaturase
expression.
[0287] 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-1
reduced-serum medium (Gibco BRL) and then treated with 130 .L of
OPTI-MEM-1 medium containing 12 .mu.g/mL LIPOFECTIN 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.
Example 24
Design of Phenotypic Assays and in vivo Studies for the use of
Stearoyl-CoA Desaturase Inhibitors Phenotypic Assays
[0288] Once stearoyl-CoA desaturase inhibitors have been identified
by the methods disclosed herein, the compounds are further
investigated in one or more phenotypic assays, each having
measurable endpoints predictive of efficacy in the treatment of a
particular disease state or condition. Phenotypic assays, kits and
reagents for their use are well known to those skilled in the art
and are herein used to investigate the role and/or association of
stearoyl-CoA desaturase in health and disease. Representative
phenotypic assays, which can be purchased from any one of several
commercial vendors, include those for determining cell viability,
cytotoxicity, proliferation or cell survival (Molecular Probes,
Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays
including enzymatic assays (Panvera, LLC, Madison, Wis.; BD
Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San
Diego, Calif.), cell regulation, signal transduction, inflammation,
oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor,
Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.),
angiogenesis assays, tube formation assays, cytokine and hormone
assays and metabolic assays (Chemicon International Inc., Temecula,
Calif.; Amersham Biosciences, Piscataway, N.J.).
[0289] In one non-limiting example, cells determined to be
appropriate for a particular phenotypic assay (i.e., MCF-7 cells
selected for breast cancer studies; adipocytes for obesity studies)
are treated with stearoyl-CoA desaturase inhibitors identified from
the in vitro studies as well as control compounds at optimal
concentrations which are determined by the methods described above.
At the end of the treatment period, treated and untreated cells are
analyzed by one or more methods specific for the assay to determine
phenotypic outcomes and endpoints.
[0290] Phenotypic endpoints include changes in cell morphology over
time or treatment dose as well as changes in levels of cellular
components such as proteins, lipids, nucleic acids, hormones,
saccharides or metals. Measurements of cellular status, which
include pH, stage of the cell cycle, intake or excretion of
biological indicators by the cell, are also endpoints of
interest.
[0291] Analysis of the geneotype of the cell (measurement of the
expression of one or more of the genes of the cell) after treatment
is also used as an indicator of the efficacy or potency of the
stearoyl-CoA desaturase inhibitors. Hallmark genes, or those genes
suspected to be associated with a specific disease state,
condition, or phenotype, are measured in both treated and untreated
cells.
In vivo Studies
[0292] The individual subjects of the in vivo studies described
herein are warm-blooded vertebrate animals, which includes
humans.
[0293] The clinical trial is subjected to rigorous controls to
ensure that individuals are not unnecessarily put at risk and that
they are fully informed about their role in the study. To account
for the psychological effects of receiving treatments, volunteers
are randomly given placebo or stearoyl-CoA desaturase inhibitor.
Furthermore, to prevent the doctors from being biased in
treatments, they are not informed as to whether the medication they
are administering is a stearoyl-CoA desaturase inhibitor or a
placebo. Using this randomization approach, each volunteer has the
same chance of being given either the new treatment or the
placebo.
[0294] Volunteers receive either the stearoyl-CoA desaturase
inhibitor or placebo for eight week period with biological
parameters associated with the indicated disease state or condition
being measured at the beginning (baseline measurements before any
treatment), end (after the final treatment), and at regular
intervals during the study period. Such measurements include the
levels of nucleic acid molecules encoding stearoyl-CoA desaturase
or stearoyl-CoA desaturase protein levels in body fluids, tissues
or organs compared to pre-treatment levels. Other measurements
include, but are not limited to, indices of the disease state or
condition being treated, body weight, blood pressure, serum titers
of pharmacologic indicators of disease or toxicity as well as ADME
(absorption, distribution, metabolism and excretion)
measurements.
[0295] Information recorded for each patient includes age (years),
gender, height (cm), family history of disease state or condition
(yes/no), motivation rating (some/moderate/great) and number and
type of previous treatment regimens for the indicated disease or
condition.
[0296] Volunteers taking part in this study are healthy adults (age
18 to 65 years) and roughly an equal number of males and females
participate in the study. Volunteers with certain characteristics
are equally distributed for placebo and stearoyl-CoA desaturase
inhibitor treatment. In general, the volunteers treated with
placebo have little or no response to treatment, whereas the
volunteers treated with the stearoyl-CoA desaturase inhibitor show
positive trends in their disease state or condition index at the
conclusion of the study.
Sequence CWU 1
1
418 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1
tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence Antisense
Oligonucleotide 2 atgcattctg cccccaagga 20 3 5221 DNA Homo sapiens
CDS (236)...(1315) 3 ataaaagggg gctgaggaaa taccggacac ggtcacccgt
tgccagctct agcctttaaa 60 ttcccggctc ggggacctcc acgcaccgcg
gctagcgccg acaaccagct agcgtgcaag 120 gcgccgcggc tcagcgcgta
ccggcgggct tcgaaaccgc agtcctccgg cgaccccgaa 180 ctccgctccg
gagcctcagc cccctggaaa gtgatcccgg catccgagag ccaag atg 238 Met 1 ccg
gcc cac ttg ctg cag gac gat atc tct agc tcc tat acc acc acc 286 Pro
Ala His Leu Leu Gln Asp Asp Ile Ser Ser Ser Tyr Thr Thr Thr 5 10 15
acc acc att aca gcg cct ccc tcc agg gtc ctg cag aat gga gga gat 334
Thr Thr Ile Thr Ala Pro Pro Ser Arg Val Leu Gln Asn Gly Gly Asp 20
25 30 aag ttg gag acg atg ccc ctc tac ttg gaa gac gac att cgc cct
gat 382 Lys Leu Glu Thr Met Pro Leu Tyr Leu Glu Asp Asp Ile Arg Pro
Asp 35 40 45 ata aaa gat gat ata tat gac ccc acc tac aag gat aag
gaa ggc cca 430 Ile Lys Asp Asp Ile Tyr Asp Pro Thr Tyr Lys Asp Lys
Glu Gly Pro 50 55 60 65 agc ccc aag gtt gaa tat gtc tgg aga aac atc
atc ctt atg tct ctg 478 Ser Pro Lys Val Glu Tyr Val Trp Arg Asn Ile
Ile Leu Met Ser Leu 70 75 80 cta cac ttg gga gcc ctg tat ggg atc
act ttg att cct acc tgc aag 526 Leu His Leu Gly Ala Leu Tyr Gly Ile
Thr Leu Ile Pro Thr Cys Lys 85 90 95 ttc tac acc tgg ctt tgg ggg
gta ttc tac tat ttt gtc agt gcc ctg 574 Phe Tyr Thr Trp Leu Trp Gly
Val Phe Tyr Tyr Phe Val Ser Ala Leu 100 105 110 ggc ata aca gca gga
gct cat cgt ctg tgg agc cac cgc tct tac aaa 622 Gly Ile Thr Ala Gly
Ala His Arg Leu Trp Ser His Arg Ser Tyr Lys 115 120 125 gct cgg ctg
ccc cta cgg ctc ttt ctg atc att gcc aac aca atg gca 670 Ala Arg Leu
Pro Leu Arg Leu Phe Leu Ile Ile Ala Asn Thr Met Ala 130 135 140 145
ttc cag aat gat gtc tat gaa tgg gct cgt gac cac cgt gcc cac cac 718
Phe Gln Asn Asp Val Tyr Glu Trp Ala Arg Asp His Arg Ala His His 150
155 160 aag ttt tca gaa aca cat gct gat cct cat aat tcc cga cgt ggc
ttt 766 Lys Phe Ser Glu Thr His Ala Asp Pro His Asn Ser Arg Arg Gly
Phe 165 170 175 ttc ttc tct cac gtg ggt tgg ctg ctt gtg cgc aaa cac
cca gct gtc 814 Phe Phe Ser His Val Gly Trp Leu Leu Val Arg Lys His
Pro Ala Val 180 185 190 aaa gag aag ggg agt acg cta gac ttg tct gac
cta gaa gct gag aaa 862 Lys Glu Lys Gly Ser Thr Leu Asp Leu Ser Asp
Leu Glu Ala Glu Lys 195 200 205 ctg gtg atg ttc cag agg agg tac tac
aaa cct ggc ttg ctg ctg atg 910 Leu Val Met Phe Gln Arg Arg Tyr Tyr
Lys Pro Gly Leu Leu Leu Met 210 215 220 225 tgc ttc atc ctg ccc acg
ctt gtg ccc tgg tat ttc tgg ggt gaa act 958 Cys Phe Ile Leu Pro Thr
Leu Val Pro Trp Tyr Phe Trp Gly Glu Thr 230 235 240 ttt caa aac agt
gtg ttc gtt gcc act ttc ttg cga tat gct gtg gtg 1006 Phe Gln Asn
Ser Val Phe Val Ala Thr Phe Leu Arg Tyr Ala Val Val 245 250 255 ctt
aat gcc acc tgg ctg gtg aac agt gct gcc cac ctc ttc gga tat 1054
Leu Asn Ala Thr Trp Leu Val Asn Ser Ala Ala His Leu Phe Gly Tyr 260
265 270 cgt cct tat gac aag aac att agc ccc cgg gag aat atc ctg gtt
tca 1102 Arg Pro Tyr Asp Lys Asn Ile Ser Pro Arg Glu Asn Ile Leu
Val Ser 275 280 285 ctt gga gct gtg ggt gag ggc ttc cac aac tac cac
cac tcc ttt ccc 1150 Leu Gly Ala Val Gly Glu Gly Phe His Asn Tyr
His His Ser Phe Pro 290 295 300 305 tat gac tac tct gcc agt gag tac
cgc tgg cac atc aac ttc acc aca 1198 Tyr Asp Tyr Ser Ala Ser Glu
Tyr Arg Trp His Ile Asn Phe Thr Thr 310 315 320 ttc ttc att gat tgc
atg gcc gcc ctc ggt ctg gcc tat gac cgg aag 1246 Phe Phe Ile Asp
Cys Met Ala Ala Leu Gly Leu Ala Tyr Asp Arg Lys 325 330 335 aaa gtc
tcc aag gcc gcc atc ttg gcc agg att aaa aga acc gga gat 1294 Lys
Val Ser Lys Ala Ala Ile Leu Ala Arg Ile Lys Arg Thr Gly Asp 340 345
350 gga aac tac aag agt ggc tga g tttggggtc cctcaggttt cctttttcaa
1345 Gly Asn Tyr Lys Ser Gly * 355 aaaccagcca ggcagaggtt ttaatgtctg
tttattaact actgaataat gctaccagga 1405 tgctaaagat gatgatgtta
acccattcca gtacagtatt cttttaaaat tcaaaagtat 1465 tgaaagccaa
caactctgcc tttatgatgc taagctgata ttatttcttc tcttatcctc 1525
tctctcttct aggcccattg tcctcctttt cactttattg ctatcgccct cctttccctt
1585 attgcctccc aggcaagcag ctggtcagtc tttgctcagt gtccagcttc
caaagcctag 1645 acaacctttc tgtagcctaa aacgaatggt ctttgctcca
gataactctc tttccttgag 1705 ctgttgtgag ctttgaagta ggtggcttga
gctagagata aaacagaatc ttctgggtag 1765 tcccctgttg attatcttca
gcccaggctt ttgctagatg gaatggaaaa gcaacttcat 1825 ttgacacaaa
gcttctaaag caggtaaatt gtcgggggag agagttagca tgtatgaatg 1885
taaggatgag ggaagcgaag caagaggaac ctctcgccat gatcagacat acagctgcct
1945 acctaatgag gacttcaagc cccaccacat agcatgcttc ctttctctcc
tggctcgggg 2005 taaaaagtgg ctgcggtgtt tggcaatgct aattcaatgc
cgcaacatat agttgaggcc 2065 gaggataaag aaaagacatt ttaagtttgt
agtaaaagtg gtctctgctg gggaagggtt 2125 ttcttttctt tttttcttta
ataacaagga gatttcttag ttcatatatc aagaagtctt 2185 gaagttgggt
gtttccagaa ttggtaaaaa cagcagctca tggaattttg agtattccat 2245
gagctgctca ttacagttct ttcctctttc tgctctgcca tcttcaggat attggttctt
2305 cccctcatag taataagatg gctgtggcat ttccaaacat ccaaaaaaag
ggaaggattt 2365 aaggaggtga agtcgggtca aaaataaaat atatatacat
atatacattg cttagaacgt 2425 taaactatta gagtatttcc cttccaaaga
gggatgtttg gaaaaaactc tgaaggagag 2485 gaggaattag ttgggatgcc
aatttcctct ccactgctgg acatgagatg gagaggctga 2545 gggacaggat
ctataggcag cttctaagag cgaacttcac ataggaaggg atctgagaac 2605
acgttgccag gggcttgaga aggttactga gtgagttatt gggagtctta ataaaataaa
2665 ctagatatta ggtccattca ttaattagtt ccagtttctc cttgaaatga
gtaaaaacta 2725 gaaggcttct ctccacagtg ttgtgcccct tcactcattt
ttttttgagg agaagggggt 2785 ctctgttaac atctagccta aagtatacaa
ctgcctgggg ggcagggtta ggaatctctt 2845 cactaccctg attcttgatt
cctggctcta ccctgtctgt cccttttctt tgaccagatc 2905 tttctcttcc
ctgaacgttt tcttctttcc ctggacaggc agcctccttt gtgtgtattc 2965
agaggcagtg atgacttgct gtccaggcag ctccctcctg cacacagaat gctcagggtc
3025 actgaaccac tgcttctctt ttgaaagtag agctagctgc cactttcacg
tggcctccgc 3085 agtgtctcca cctacacccc tgtgctcccc tgccacactg
atggctcaag acaaggctgg 3145 caaaccctcc cagaaacatc tctggcccag
aaagcctctc tctccctccc tctctcatga 3205 ggcacagcca agccaagcgc
tcatgttgag ccagtgggcc agccacagag caaaagaggg 3265 tttattttca
gtcccctctc tctgggtcag aaccagaggg catgctgaat gccccctgct 3325
tacttggtga gggtgccccg cctgagtcag tgctctcagc tggcagtgca atgcttgtag
3385 aagtaggagg aaacagttct cactgggaag aagcaagggc aagaacccaa
gtgcctcacc 3445 tcgaaaggag gccctgttcc ctggagtcag ggtgaactgc
aaagctttgg ctgagacctg 3505 ggatttgaga taccacaaac cctgctgaac
acagtgtctg ttcagcaaac taaccagcat 3565 tccctacagc ctagggcaga
caatagtata gaagtctgga aaaaaacaaa aacagaattt 3625 gagaaccttg
gaccactcct gtccctgtag ctcagtcatc aaagcagaag tctggctttg 3685
ctctattaag attggaaatg tacactacca aacactcagt ccactgttga gccccagtgc
3745 tggaagggag gaaggccttt cttctgtgtt aattgcgtag aggctacagg
ggttagcctg 3805 gactaaaggc atccttgtct tttgagctat tcacctcagt
agaaaaggat ctaagggaag 3865 atcactgtag tttagttctg ttgacctgtg
cacctacccc ttggaaatgt ctgctggtat 3925 ttctaattcc acaggtcatc
agatgcctgc ttgataatat ataaacaata aaaacaactt 3985 tcacttcttc
ctattgtaat cgtgtgccat ggatctgatc tgtaccatga ccctacataa 4045
ggctggatgg cacctcaggc tgagggcccc aatgtatgtg tggctgtggg tgtgggtggg
4105 agtgtgtctg ctgagtaagg aacacgattt tcaagattct aaagctcaat
tcaagtgaca 4165 cattaatgat aaactcagat ctgatcaaga gtccggattt
ctaacagtcc ctgctttggg 4225 gggtgtgctg acaacttagc tcaggtgcct
tacatctttt ctaatcacag tgttgcatat 4285 gagcctgccc tcactccctc
tgcagaatcc ctttgcacct gagaccctac tgaagtggct 4345 ggtagaaaaa
ggggcctgag tggaggatta tcagtatcac gatttgcagg attcccttct 4405
gggcttcatt ctggaaactt ttgttagggc tgcttttctt aagtgcccac atttgatgga
4465 gggtggaaat aatttgaatg tatttgattt ataagttttt tttttttttt
gggttaaaag 4525 atggttgtag catttaaaat ggaaaatttt ctccttggtt
tgctagtatc ttgggtgtat 4585 tctctgtaag tgtagctcaa ataggtcatc
atgaaaggtt aaaaaagcga ggtggccatg 4645 ttatgctggt ggttaaggcc
agggcctctc caaccactgt gccactgact tgctgtgtga 4705 ccctgggcaa
gtcacttaac tataaggtgc ctcagttttc cttctgttaa aatggggata 4765
ataatactga cctacctcaa agggcagttt tgaggcatga ctaatgcttt ttagaaagca
4825 ttttgggatc cttcagcaca ggaattctca agacctgagt attttttata
ataggaatgt 4885 ccaccatgaa cttgatacgt ccgtgtgtcc cagatgctgt
cattagtcta tatggttctc 4945 caagaaactg aatgaatcca ttggagaagc
ggtggataac tagccagaca aaatttgaga 5005 atacataaac aacgcattgc
cacggaaaca tacagaggat gccttttctg tgattgggtg 5065 ggattttttc
cctttttatg tgggatatag tagttacttg tgacaaaaat aattttggaa 5125
taatttctat taatatcaac tctgaagcta attgtactaa tctgagattg tgtttgttca
5185 taataaaagt gaagtgaatc taaaaaaaaa aaaaaa 5221 4 17 DNA
Artificial Sequence PCR Primer 4 gatcccggca tccgaga 17 5 27 DNA
Artificial Sequence PCR Primer 5 ggtataggag ctagagatat cgtcctg 27 6
21 DNA Artificial Sequence PCR Probe 6 ccaagatgcc ggcccacttg c 21 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
20 DNA Artificial Sequence Antisense Oligonucleotide 10 gtccggtatt
tcctcagccc 20 11 20 DNA Artificial Sequence Antisense
Oligonucleotide 11 ccgcggtgcg tggaggtccc 20 12 20 DNA Artificial
Sequence Antisense Oligonucleotide 12 tacgcgctga gccgcggcgc 20 13
20 DNA Artificial Sequence Antisense Oligonucleotide 13 gcggtttcga
agcccgccgg 20 14 20 DNA Artificial Sequence Antisense
Oligonucleotide 14 cctccattct gcaggaccct 20 15 20 DNA Artificial
Sequence Antisense Oligonucleotide 15 tcccaagtgt agcagagaca 20 16
20 DNA Artificial Sequence Antisense Oligonucleotide 16 ctcctgctgt
tatgcccagg 20 17 20 DNA Artificial Sequence Antisense
Oligonucleotide 17 cacggtggtc acgagcccat 20 18 20 DNA Artificial
Sequence Antisense Oligonucleotide 18 cagccaaccc acgtgagaga 20 19
20 DNA Artificial Sequence Antisense Oligonucleotide 19 gacaagtcta
gcgtactccc 20 20 20 DNA Artificial Sequence Antisense
Oligonucleotide 20 gttcaccagc caggtggcat 20 21 20 DNA Artificial
Sequence Antisense Oligonucleotide 21 tgtggaagcc ctcacccaca 20 22
20 DNA Artificial Sequence Antisense Oligonucleotide 22 agttgatgtg
ccagcggtac 20 23 20 DNA Artificial Sequence Antisense
Oligonucleotide 23 ggaccccaaa ctcagccact 20 24 20 DNA Artificial
Sequence Antisense Oligonucleotide 24 tgcctgggag gcaataaggg 20 25
20 DNA Artificial Sequence Antisense Oligonucleotide 25 atacatgcta
actctctccc 20 26 20 DNA Artificial Sequence Antisense
Oligonucleotide 26 aagtcctcat taggtaggca 20 27 20 DNA Artificial
Sequence Antisense Oligonucleotide 27 tgtaatgagc agctcatgga 20 28
20 DNA Artificial Sequence Antisense Oligonucleotide 28 tcagtaacct
tctcaagccc 20 29 20 DNA Artificial Sequence Antisense
Oligonucleotide 29 ggagctgcct ggacagcaag 20 30 20 DNA Artificial
Sequence Antisense Oligonucleotide 30 tcagtgaccc tgagcattct 20 31
20 DNA Artificial Sequence Antisense Oligonucleotide 31 tggctggccc
actggctcaa 20 32 20 DNA Artificial Sequence Antisense
Oligonucleotide 32 gcatgccctc tggttctgac 20 33 20 DNA Artificial
Sequence Antisense Oligonucleotide 33 gctttgcagt tcaccctgac 20 34
20 DNA Artificial Sequence Antisense Oligonucleotide 34 gtggtatctc
aaatcccagg 20 35 20 DNA Artificial Sequence Antisense
Oligonucleotide 35 tagtccaggc taacccctgt 20 36 20 DNA Artificial
Sequence Antisense Oligonucleotide 36 gtgatcttcc cttagatcct 20 37
20 DNA Artificial Sequence Antisense Oligonucleotide 37 ctcagcagac
acactcccac 20 38 20 DNA Artificial Sequence Antisense
Oligonucleotide 38 gctaagttgt cagcacaccc 20 39 20 DNA Artificial
Sequence Antisense Oligonucleotide 39 aagtttccag aatgaagccc 20 40
20 DNA Artificial Sequence Antisense Oligonucleotide 40 agagaataca
cccaagatac 20 41 20 DNA Artificial Sequence Antisense
Oligonucleotide 41 tagttaagtg acttgcccag 20 42 20 DNA Artificial
Sequence Antisense Oligonucleotide 42 gccctttgag gtaggtcagt 20 43
20 DNA Artificial Sequence Antisense Oligonucleotide 43 ccatatagac
taatgacagc 20 44 20 DNA Artificial Sequence Antisense
Oligonucleotide 44 ctgtatgttt ccgtggcaat 20 45 20 DNA Artificial
Sequence Antisense Oligonucleotide 45 cttgcacgct agctggttgt 20 46
20 DNA Artificial Sequence Antisense Oligonucleotide 46 gcatcgtctc
caacttatct 20 47 20 DNA Artificial Sequence Antisense
Oligonucleotide 47 taaggatgat gtttctccag 20 48 20 DNA Artificial
Sequence Antisense Oligonucleotide 48 cccaaagcca ggtgtagaac 20 49
20 DNA Artificial Sequence Antisense Oligonucleotide 49 tgtaagagcg
gtggctccac 20 50 20 DNA Artificial Sequence Antisense
Oligonucleotide 50 cattctggaa tgccattgtg 20 51 20 DNA Artificial
Sequence Antisense Oligonucleotide 51 ttatgaggat cagcatgtgt 20 52
20 DNA Artificial Sequence Antisense Oligonucleotide 52 cctctggaac
atcaccagtt 20 53 20 DNA Artificial Sequence Antisense
Oligonucleotide 53 ggatgaagca catcagcagc 20 54 20 DNA Artificial
Sequence Antisense Oligonucleotide 54 ttcaccccag aaataccagg 20 55
20 DNA Artificial Sequence Antisense Oligonucleotide 55 gaaaccagga
tattctcccg 20 56 20 DNA Artificial Sequence Antisense
Oligonucleotide 56 tcactggcag agtagtcata 20 57 20 DNA Artificial
Sequence Antisense Oligonucleotide 57 taatcctggc caagatggcg 20 58
20 DNA Artificial Sequence Antisense Oligonucleotide 58 tcatcatctt
tagcatcctg 20 59 20 DNA Artificial Sequence Antisense
Oligonucleotide 59 gcaaagactg accagctgct 20 60 20 DNA Artificial
Sequence Antisense Oligonucleotide 60 gactacccag aagattctgt 20 61
20 DNA Artificial Sequence Antisense Oligonucleotide 61 cttccctcat
ccttacattc 20 62 20 DNA Artificial Sequence Antisense
Oligonucleotide 62 cccgagccag gagagaaagg 20 63 20 DNA Artificial
Sequence Antisense
Oligonucleotide 63 cttccccagc agagaccact 20 64 20 DNA Artificial
Sequence Antisense Oligonucleotide 64 ccaatatcct gaagatggca 20 65
20 DNA Artificial Sequence Antisense Oligonucleotide 65 cccaactaat
tcctcctctc 20 66 20 DNA Artificial Sequence Antisense
Oligonucleotide 66 tatagatcct gtccctcagc 20 67 20 DNA Artificial
Sequence Antisense Oligonucleotide 67 ctcccaataa ctcactcagt 20 68
20 DNA Artificial Sequence Antisense Oligonucleotide 68 aagagattcc
taaccctgcc 20 69 20 DNA Artificial Sequence Antisense
Oligonucleotide 69 cacacaaagg aggctgcctg 20 70 20 DNA Artificial
Sequence Antisense Oligonucleotide 70 aagtggcagc tagctctact 20 71
20 DNA Artificial Sequence Antisense Oligonucleotide 71 caccctcacc
aagtaagcag 20 72 20 DNA Artificial Sequence Antisense
Oligonucleotide 72 tgcttcttcc cagtgagaac 20 73 20 DNA Artificial
Sequence Antisense Oligonucleotide 73 atcaagcagg catctgatga 20 74
20 DNA Artificial Sequence Antisense Oligonucleotide 74 ccctcagcct
gaggtgccat 20 75 20 DNA Artificial Sequence Antisense
Oligonucleotide 75 ataatcctcc actcaggccc 20 76 20 DNA Artificial
Sequence Antisense Oligonucleotide 76 cacttaagaa aagcagccct 20 77
20 DNA Artificial Sequence Antisense Oligonucleotide 77 cagcaagtca
gtggcacagt 20 78 20 DNA Artificial Sequence Antisense
Oligonucleotide 78 ggctagttat ccaccgcttc 20 79 20 DNA Artificial
Sequence Antisense Oligonucleotide 79 cccaatcaca gaaaaggcat 20 80
20 DNA Artificial Sequence Antisense Oligonucleotide 80 aactactata
tcccacataa 20 81 18508 DNA H. sapiens 81 gagatgttag tggtgggcgc
cccccgaggg ttcaccactg tttcctgaga aacttcccca 60 gtgcccaccc
acccgttctc cgtgtgcccg agggccggtc ctgggctagg ctccgcgccc 120
cagccccaaa ccgggtcccc agccccttcc agagagaaag ctcccgacgc gggatgccgg
180 gcagaggccc agcggcgggt ggaagagaag ctgagaagga gaaacagagg
ggagggggag 240 cgaggagctg gcggcagagg gaacagcaga ttgcgccgag
ccaatggcaa cggcaggacg 300 aggtggcacc aaattccctt cggccaatga
cgagccggag tttacagaag cctcattagc 360 atttccccag aggcaggggc
aggggcagag gccgggtggt gtggtgtcgg tgtcggcagc 420 atccccggcg
ccctgctgcg gtcgccgcga gcctcggcct ctgtctcctc cccctcccgc 480
ccttacctcc acgcgggacc gcccgcgcca gtcaactcct cgcactttgc ccctgcttgg
540 cagcggataa aagggggctg aggaaatacc ggacacggtc acccgttgcc
agctctagcc 600 tttaaattcc cggctcgggg acctccacgc accgcggcta
gcgccgacaa ccagctagcg 660 tgcaaggcgc cgcggctcag cgcgtaccgg
cgggcttcga aaccgcagtc ctccggcgac 720 cccgaactcc gctccggagc
ctcagccccc tggaaagtga tcccggcatc cgagagccaa 780 gatgccggcc
cacttgctgc aggacgatgt gagtttccca gcctggcccc gtaccgccgg 840
gtcgcaggcg cgggctgggc ttccagggga cgggttggtg gcagaagaga ggggagagct
900 ccgcggagga cttggtcatc tttttcgagt tgtgctgcct tccgtgagtt
gggaatgtgg 960 attgtaattt ggggacttga gtctccaact ttagtttctt
aagctttaaa gaaaaatccg 1020 gtcgtgctgg tgcttttatg aattatgcgg
gttttccttt gtcttcgtgg ggatgtgagt 1080 gctttacttc tccttcctac
tgccgcctcc cgataggttt cgcgcccctc gtccccctcg 1140 ccctccgccc
ctaatgtatc tgtacagttt cagggaactt ttctccgttg cgtctcggat 1200
acaccctacc ctcagtgaac tacggcgctg cggaagggtc cgtactgtcc acccttcccc
1260 cagcgtgatt agagagcgga gtggccccag ctgcctccac gtgtctcttc
tcctgactct 1320 cctcttcctc ccccttccag atctctagct cctataccac
caccaccacc attacagcgc 1380 ctccctccag ggtcctgcag aatggaggag
ataagttgga gacgatgccc ctctacttgg 1440 aagacgacat tcgccctgat
ataaaagatg atatatatga ccccacctac aaggataagg 1500 aaggcccaag
ccccaaggtt gaatatgtct ggagaaacat catccttatg tctctgctac 1560
acttgggagc cctgtatggg atcactttga ttcctacctg caagttctac acctggcttt
1620 ggggtaagca gcctccctgt cctcctgacc tagtcctcca ggtactcact
gctcttttaa 1680 taaggtagga tcttacagag gacaccagcc cctcccagcc
tcccggttgg ggtttttctc 1740 aggcatttct ctttggttgc ttcaggccta
gtgggctggg aagaacctgg gggtactgag 1800 atgcaggact gtcaatgctg
ggggttgaga gctttggaac cttgtgcttg tgggctttga 1860 agtgtgctgt
gctggagagt cagctttccc tgaaagagct tttctgtttg agtcatttgc 1920
ttggctgcct gtcccacccc tgccagccac aaaagaatca aacccctcta cctgggaggc
1980 atgggaacat ggtactaaac cattggcttt ctagggtttg tgttgagcaa
tcataggcta 2040 gcacaaggga aactactcag ggagagagtt tattggaaag
ggaggagaga gctcattgga 2100 aagagaggaa aagagaggtt gttatccaca
aaactatttt gacctgaccc cttggccaga 2160 ccaggcttct tcgttacaga
ggaagatgaa gctcccgtgc aacccaagtc acacaggtgt 2220 gtgttgctct
gttactctac attccagggt gctgcacata ccagtcaaca aaatgctctt 2280
agaatgaaat caggctgaag atatgtttca tgttccatcc cccgtccagc ctgcttcagg
2340 cccccctact gatgccatga agtgccaaga attctgcacc ttccaacacc
agtcctaccc 2400 atccctcctt gccagggaca tcgaggccag gccagaaaag
agagcccggt cttgaggctg 2460 ctgtaatgag ggagacagtt cccagtttgt
ccccgacctg cccttagctc agctctttca 2520 gggacctgtt ggtgtggcct
gatggcatgg ccctgccccc tggatacaca catgcgcttt 2580 gcaggctttt
tcacctctgt cactatgagc ctaagttggg aagggaaagc agggagctct 2640
gcagatccag gggccctgca ctgctactct ctaaggggtg gtgagccagg acttctggtg
2700 tgggaaactt tctgacagct cgctcttgaa gggtagttat gcagaagggc
tggtaggcca 2760 agttttagct gtttccctca ggacctctta ggtcttctga
agatggtgag atctggttca 2820 ttgagctttt gtttccagaa atcctccagg
tgctacagtg aaatgcaact gagaagagcc 2880 cctttgtcag gatctcagtt
tttctaggta ccacctgtgg gcaacagtac ttttttagga 2940 tggggaaggt
agcattcctt gggttacata tgtgtcacag ccgcagagac agaacaggcc 3000
aggtgcagat cagttcatct gcataccagg aagagctctt gtatttgtga ggggagggcg
3060 acatctgttg tctagcccct ctgaggatcc agcagaactt cagttgcagc
tgagggatga 3120 ggagcaggga tagcattccc caggcagtct gtggatgcct
ctctccaccc tttctcccca 3180 agaggatagt cttcacaagt agttttctct
cctgcaagct ccttccagcc tcagcacccc 3240 ctgtaggttt cctgaagccc
agagtttgac ccgattgcct ctgcttggtt atcctcagtt 3300 gctactgcaa
ggagcagaat tccatcccaa agtgaaggct gtgcctgcta catttttttg 3360
gtgacctgcc catgggtgtg tgctgtgccc ttgggcagat aaatgtggct cacagagact
3420 agatggtaac ttattttact gggccctttg gtggatggct tagctgaaag
aatgaccatg 3480 tcgatgtggg gatatcagaa ctcctctgtc ttgagaaaca
aggagggtga ggggaggagg 3540 gtcagaggtc tcaacttgga catcttctct
tcccaccttc agaatttagg agcttcacct 3600 ttcttgtttt aatccctatg
gacaatgtta aattagcaac tggtcgtgat tccagcattt 3660 cttatgactg
agcacttttg agagccttaa atctctttca ttttggagtc tcccaaagaa 3720
gcccttagga gaatgcctct ggcaacactg agagccagag gcaaaaccat tcaagtctct
3780 ttcttaaggc cagagcaatg agatgttgct taaccatcaa tctggacatt
ttcctcccct 3840 ggggaggaga gaaggaataa atctccaact gatttaagcc
cttctatttg ataggcttag 3900 aggtgtttct agaaaaggca ccagagtgcc
tgagttcttt ccccaatttc cctgttaata 3960 taggatctta tatacgttct
gagtcaacac tgccctaggg tcttgcagca aataacaacc 4020 ctagaagctc
ccttgctcat tagagagagc tctggctcaa ctgatggttt cccgagtaag 4080
atgcaggtag atactgtatc tggggagatg tcagtcacca cttggcttga gggcagatca
4140 cctggggcct cagtccccac tctgagggaa tgtggagaga gctcttgttg
gggctgactg 4200 gctctgtggc tctcctggag atcttatagg aggaatagaa
gagaagaaag gaggtagggt 4260 tggggggaaa aggaggttgt ttaggaaaca
gctatcaatg gctgcaaaag aacacaggac 4320 aatttgttac aatgtgtggt
gtctccaact gcaactaagt tctgtggcca ctgaggatct 4380 attgtttcta
gctgtttccc taggaatgaa acattgttaa gagtttctat caaggccaca 4440
gcttctgcct gctagagcta ctgaacagag gaagatatgg ggacgcccag cagcccactt
4500 cccagttaga gattatatct ggcacctcct gagcctgcag gcctccagga
aggtgaggga 4560 agacaatggt ggggtgcttc actgacagct tgaagaatat
cccaccattg tctagagagc 4620 gttgcccggt gagagtatgg gctgacaggg
atgtggcaaa ggcgagacag aggagttgtg 4680 catgtatctt gggggaggtg
gatggtatag ctggaacgtg aaatctttgg taaagcttaa 4740 gacactgtac
aaattggatt tatgcacagg gctaattttt ccctgatttg gccacactga 4800
ctgcttgaat atttaaatgc ttttttgtac cagttgataa atggccatag tctgaatgcc
4860 ctaagagtcc cctacaacta agggcttctc aaattggtca gtgcccagat
tgtactggct 4920 gttattttat tttttgagat aaggtcttgc tgtgttgata
aggcttgagt gcagtggcgc 4980 gatcttggct caccgcagcc tcgacctcct
gagctcaaga gatcctccca cctcaacctc 5040 ttgagtagct gagaccacag
gcaggtgcca ccatgccaag ctaattttta aaattatctg 5100 tagaggcaag
gtctccctat gttatcccag ctggtcttga actcctgggc tcaaggggat 5160
cttcctgcct tggcctccca aaatgctggg atgacaaagt ggttcatact acacctggcc
5220 ctctgctgtt attttaataa caccctggct tatgggttct gagctctgca
ggagtagttt 5280 gtggccctta aaaccttaga gaaggcctag atagaggtga
aaggagatag ctaggccctg 5340 ggagaatgcc tttaagatga agaatgagtg
gtaagagcac tattctctct cctgcctttc 5400 tgactcctta gttcctggga
ttctttagct tatctttttt tcctgggttg agaggttggg 5460 gggtgatatt
tttcaagtgg taaaatctaa gggatgtggt tatccctaga gttcatggta 5520
aagccagttc tcacccaaag cctgacgaag acagtttcta gcatccagag agtgtctctg
5580 gcatcctttc ccagatggaa ctcacactga ttggtgactc ccccactgtc
ttctcctggc 5640 aggggtattc tactattttg tcagtgccct gggcataaca
gcaggagctc atcgtctgtg 5700 gagccaccgc tcttacaaag ctcggctgcc
cctacggctc tttctgatca ttgccaacac 5760 aatggcattc caggtaagaa
gttgtctctg ctcagctgtt tgtcctccac actattaatg 5820 atccggggac
agaaaggagg gatcagcacc agagaggagc cacacctgac agccatttca 5880
ctttcctctc tcctgtagtc acctcaagtt ccagttcagt ccttaagtcc ataaagcatg
5940 aagagacttc tgagtcttgg aaaagggaac tggaagataa ttggaaaata
ctcctgatgt 6000 gtaggaatat ttttgatcct aaggtccctg tgttgtcaca
caatctggcc gttgtggctc 6060 ttcatcataa ggggctttgg cacataagcc
agagactgac cttagattcc tgggcagaca 6120 ctggacaata aattcactat
ttaaggtaaa tatcttaggg aggcagagct gggaggatca 6180 ctggagccca
ggagtttgaa gctgcagtga gccatgatat caccactgca ctccagcctg 6240
ggtgacaaga ccccaacttt aaaaaaaaaa tttcaaaagt gaataactta ggactccacc
6300 acagtgggat tgaagttgat gttcccagac tcgtgaactc ttattttgag
ataatgagag 6360 caaacacttt tattgcactg actatgagcc tggcactatt
ctaagcattt gatataaagt 6420 cctcacaaaa atcttaggaa ataggtacta
ttatccccat tttacagatg aggaaaccaa 6480 gttacagaga gattagatag
accagcccaa cattgtggcc ctttcttggg ctgccatggt 6540 ggctaagtaa
aacattgagg tttgttaagg caaaaaacaa acctgggcac ggtggctcac 6600
gcctgtaatc tcagaacttt gggaggccga ggtgggcaga tcacaaggtc aggagatcaa
6660 gatcatcctg gctaacacag tgaaactctg tctctactaa aaatacaaaa
aattagccag 6720 gcgtggtggc gggcacctgt agtcccagct acgtgggagg
ctgaggcagg agaatggtgt 6780 gaacctggga ggcagagctt gcagtgagct
gagatcgcgc cactgcactc cagcctgggc 6840 aacagcaaga ctccatctca
aaaaaaaaaa aaaaaaagtt tatagcaaag acagaatgaa 6900 gggaaatggg
gaaagggaat gcatcatagt cattaagctg tagcaatggg caaatgatag 6960
catgtggcgt ttggcttagc ttggagcaag aggaagaaag gaaaccaact tagtggtggc
7020 aatatcccaa gaacttgcca catttgcatg atcatctctg ccatagcagc
ttataccttg 7080 aaggcttcca aagttgtcct gtggagcaaa aggaaggaag
agagatattg gtacattctt 7140 taagggatgg aaaaagtcat gaagaagccc
agaggtcgtt tgaaaatgca gtcatgatca 7200 cgagttgcat gcctggcctt
gttattgggt tgtatgagct cttttgcaag gcaccagaat 7260 ggtgcaccct
gcagctgcag cttatctact gattgagacc ctaggacaca aggctgcctg 7320
cctcatgttc cccatgccta gggattaggt accccatgag gatcttttcc aacattcctt
7380 gcttaaagaa ttgcaatgtt ctcacttctt gaaactctct gagctctgta
tgatttacct 7440 ccgttccacc caccatataa ctttcaagaa acagcagttc
tattgctatg gtcctgggac 7500 tttaagttgc ttttttctac ttaagcttca
gtggcaagtt gggagaagaa gggaggcaac 7560 tccatgactc ctttggagcc
cagattcctg ggtattttgt gaggttgggc tgagcgcctt 7620 gggctcttga
tacctgtcca ttgggattct cctaataggg tgtctatcct caagccttac 7680
attcctcttc tctctctccc cagaatgatg tctatgaatg ggctcgtgac caccgtgccc
7740 accacaagtt ttcagaaaca catgctgatc ctcataattc ccgacgtggc
tttttcttct 7800 ctcacgtggg ttggctgctt gtgcgcaaac acccagctgt
caaagagaag gggagtacgc 7860 tagacttgtc tgacctagaa gctgagaaac
tggtgatgtt ccagaggagg tgagtgaagc 7920 cctgatggag gtggggatat
ggccctggca cctggtcatt agggacccca ttttttctcc 7980 tgagactttc
aaaatataag ctgagaaatt tgctgggttt gcatgttcac aatcttaatt 8040
taaaatccca atttttaaca tcccacgggc ccgtagccat agactattgc tccatttctt
8100 tctctctgac tatcttaatt aaacccatta cattcaagag atgtttattg
tcctaggaca 8160 gtcatagatt caaagatgat tatagcctag ttgcctaggt
ttgtttgttt gtttttgtgt 8220 ttgtgtttca acagtctttc tctcttgccc
aggctggagt gcagtggcac aatcatggct 8280 cactgcagcc ttgacttccc
aggctcaagc aatccttcta cctcaacctc ctgagtatct 8340 gggactacag
gcacacaccg ccatgcctgg ctaatttttt gtggggacaa ggtctcactc 8400
actatattgc ccaggccggt agcttagttc ttaccttcaa aaagtttgta gcctatcggg
8460 gtggagagat aagccaagta tccagataac catggcataa ggcagaatat
tttctgtact 8520 atgagaggta caaaggggag ggagattgct caatgggcaa
caccaaggaa gtgatatgaa 8580 ataaatagtg ttggaatcca ccaacggata
gaaattttta caactatgtg tggggagaga 8640 cagtgcaaac agaagaaaca
gaatgagcta aaacacgaag catgttccag caatagagtc 8700 cttttgcttg
aagtataggg tatgggaaga agtaagactg gagagactaa tgccattctt 8760
gtcgagtcct aaaagcagac ttaggactta attcaataag caataggaag ccattacatc
8820 ttttgaactg caatgtggca tagttacgga cgtgctttag gaaggctgct
tttagaacaa 8880 gtgtaagaaa ccactgagcc aaagtgagag gtagggacat
aagttaggta atgaggaccc 8940 ctgctagcga agcagtggca gaaatggaga
aaagagttgg gtgcagggaa tgtcagtgat 9000 gtaaaagtca aagacttgac
tgctgaagga atgtagggaa tcagtgccct tggaatgtca 9060 atggcctggt
ctacattgag aatgaagact gagaaagggc ttcctgaggg acagagagct 9120
gcaggtgatc aaggacactc aatgggtctc tgagggaaaa gaagaccaaa gaattaggga
9180 gtagctagca gaaaatggag gcatgacact aaacacagac tgaaaaagag
tgctgattag 9240 aaagagaaag gagcccaaag gcagatggga aaaccagcca
aggatggaga gacgtctgtt 9300 cattagtttg tagtttggac ctcacctatc
ttaccaatgt ggtattatgc tctagtaaaa 9360 agtcagcgat ggccgggcat
ggtggctcat gcccgtaatc ccagcacttt gggaggctga 9420 ggcgggagga
ttgctggaaa gttcaggcat tcgagaccag cctgggcaac atagtgagac 9480
ctcatctcta caaaaaatta aaaactaaat gggcacgatg gttcatgcct gtggtcccag
9540 ctactcagga ggctggggtg ggaggatctc ttggcccagg agttcaaggc
tgtggtgaac 9600 taaggtcacg ccactgcact ccagccttgg caacagagtg
agaccctgtc aaaaaaaaca 9660 aacaaaaata aataaaacaa tgaacttaga
gtcagacaag caattcaaca tggaagaaag 9720 acagcccatc ccctcccaat
tagtgtggaa gatccatgta ggtgtggagt ccccctccat 9780 tgacctggtg
tctggtctgt caatgtaggt actacaaacc tggcttgctg atgatgtgct 9840
tcatcctgcc cacgcttgtg ccctggtatt tctggggtga aacttttcaa aacagtgtgt
9900 tcgttgccac tttcttgcga tatgctgtgg tgcttaatgc cacctggctg
gtgaacagtg 9960 ctgcccacct cttcggatat cgtccttatg acaagaacat
tagcccccgg gagaatatcc 10020 tggtttcact tggagctgtg ggtaagtcag
ctgtccaagt aagactacat ccagtggtct 10080 gctgattagg ggattaggct
aggagccaga aaaactagat aaatctgttt tttatggcta 10140 ctttgtatct
cagtttttcc actataaaat tagggggcag tatactggaa aacgcttttg 10200
agagtcaggc aacatgtttt atgtaaaaat gaaaggataa gaaacaaaac acaaaaaaac
10260 actgattttg attccaggtt ctaaaacaat ttccaaatgc catgtatgct
ccgggcccgg 10320 cggctcatgc ctgtaatccc agcactttgg gaggctgagg
cgggcaggtc acctgaggtc 10380 aggagtttga gaccagtctg gccaacacgg
tgaaaccctg tttctattaa aaagcaaaac 10440 aaacaaacaa aaaagaaaac
caaatgccat acatgatgag caccttagag ttttcctttc 10500 tttcatcaac
tctgggctgg actgcagtct tgcttgaggc aaggaatgca taaatagaac 10560
aatgggatca tctgagtggc cagtaagcct ggtgttttat gaacttcaag tatgcacaca
10620 aagtattttt tatctttcca ctctactcag atatgccttg ctttaagagt
gtgccgtgcc 10680 ttactgtttg gtgatgccat tatgaagggc atcaaaataa
ctgctggtgg ccctttacta 10740 ccacctaccc tcttgtctcc tcttgtcctc
tatttttctc tcttctactt ctattctggg 10800 ctaggaacat cccttccccc
aacatgcctt caggaatctc ccaataaagc agtgtgatca 10860 caagttcctg
ctcaattctc taatgttgat cttatctttt ctcttttctt tcctttcatt 10920
ttctttcttt tcttttcttt tcctttcttt tctaatgaga cagggtctca ccatggtgcc
10980 caggctggtc ttgaaccctg ggctgaagtg atcctcctgc ctcagcctcc
caaagtatct 11040 acattttttt ctctgtccct ctttccaact gaatatagac
tttcaccaag gccctgaatg 11100 aattttcctt aaatagatct ggcgacctct
tctttccagt aattggtgct attggtcatt 11160 caataatatc tagacaacca
cactactcca cacatttagg caggtcattg cctaacactc 11220 attttctttt
tctctttaaa aatcttcctt tatattctca accttaacca tctttattat 11280
cttttaaatt gttgttgaga cagtctcact ctgttgccca ggtttcagtg cagtggtgtg
11340 atcacagctc actgcagcta tgacctcctg ggctcaagcg atcctcgggg
ttcagcctcc 11400 caagtaactg ggattacagg tgcatgccac catgcttggc
taatttttct atttttttgt 11460 agagacatgg ttttgccatg ttgcacaggc
tggtctcgaa ctcctgagct caagtgatct 11520 tcctgccttg gcctcccaaa
gtgctggaat tataatagcc gtgagccact gcgcctggcc 11580 tactatgttt
attaaaagga tttattgcct gtaatcccag cactttggga ggccgaggcg 11640
ggtggatcat gaggtcagga gatcgagacc atcctggcta acaaggtgaa accccgtctc
11700 tactaaaaat acaaaaaaat tagccaggcg cagtggcggg cgcctgtagt
cccagctact 11760 tgggaggctg aggcaggaga atggcgtgaa cccgggaagc
ggagcttgca gtgagccgag 11820 attccgccac tgcagtccgc agtccggcct
gggcgacaga gcgagactcc gtctcaaaaa 11880 aaaaaaaaaa aaaagattta
tttgtctagg cgtggtggct cacacctgta atcccagcac 11940 tttgggaagc
caaagtgggt ggttcacttg aggtcaggag ttagagatca gccaggccaa 12000
tatggtgaac ctctgtctct acttaaaaaa aaaaaaaaaa aaaagtacaa aaaacttagc
12060 caggcatggt ggcacgtgcc tgtagtccca gctactcagg aggctgaggc
agaagaatcg 12120 cttgaacccg ggaggcagag gttgcaatga gccgagattg
tgccactgca ctccagcctg 12180 ggtgacagag actccatctc aaaaatatat
aataaaaata aaagcatttt ttttctctct 12240 ttttaacttt cacatatctc
ttttcaggca ccttcttacc attgtgccta ttcttacttt 12300 aacccatgat
taaaataaat catatacact gtataaatct gagattatca taggaatgga 12360
gtttctggca tgagatgttt cctgtatcgc aaatggatct ataatgacct tccccacctc
12420 cagcctctgg gtggccatga gttcaaagtg gctgccaata tctgacctgt
tgttgttatc 12480 attcactcct ccttgcctgc tgctttcctc ccttatcacc
tcaccctttg ttctcctcca 12540 gctctgtttc ctgccaccct aatctttttg
tttcttgaat tacctccccc actgtcacat 12600 gctcatcttc tctgccaaat
taaccttctc ccttgagcct ttcttgggct gtctcttgct 12660 gccccagttg
caaagtcctg tcttctttct acccgttgac cctcttcttt ttttttcctc 12720
ccttgtctct gtgtgcatct gattccattt taaatctggt aaccaaaggc ctggctagtg
12780 cttacacaca gcccagctgc aaaaccatta atggacatta ataatcctca
gtacctttat 12840 tcctggcatt ctacccccct cctccccagt tcacactgca
gatcatcagg tgtcacagag 12900 agaggacata ccttgaaatg
ccctagatga tgtcatttac tttgcaggac ttccttgcct 12960 tgcttctgat
taatgtcatg actggtctgt ctgagggtac tgttatctac aaagagccaa 13020
atattagctc ttagtagcta ttctttatcc atgcctgatt agggtcagta ttatttttgg
13080 ctgtggttca gaaagaagag tcctgccaag cgttggcaaa ctctctatct
gtcgagtttc 13140 caaagcttta cacgttagag aaattgctgt gaatccagaa
tttgtttgtt ttcctccctc 13200 cagcaaagtg aaatgttcat cccaagagtc
ctcaaaatct cagaggttac agggtatttt 13260 tcttcctcag agagcttctg
ttttatcagc acctccccca caccagggtc aaagctcaaa 13320 aaagttggag
cagcccctgg gaactgcagt ggctgaggac attccagccc ctgggctggc 13380
ctttcttctg atctttggct gcagggccca ctcttttgga acctcccacc cctagaggtg
13440 gttccagtgt ggtggggaaa ggtgtgcttc tttactcatt tttttaagag
tcatagccag 13500 agtgcttcat tctgcaagga cgtgcacatg cacatgcaca
cagagccttg agggcagggc 13560 caagagtgaa tttggaattt tccaacctga
tacccattcc caaaagtagg agcttctctc 13620 tagtcatttt atcctctgag
aaactgtcag ttctcctccc acaaggctcc cagacagcca 13680 cgggtgacca
gggtctccaa tcactcctta agatgccttt gactggctgg gcgcagtgac 13740
tcatgactgt aatcctagca ctttgggtgg tcaacgtggg agggttgctt gcaacatggc
13800 aagaccccgt ctctacaaaa aaagtaaaat aataaaagta aagatgcctc
tgaggggatc 13860 tgtttggttc atattaaaag agactgaatt catccattca
acaatagcga gtttctccca 13920 ggtgtgaggt accctgctag ctaactggtg
tgcacaaatc aagaaaacct caatgcaccg 13980 tcactccata acttctcgct
tttgtttcag gtgagggctt ccacaactac caccactcct 14040 ttccctatga
ctactctgcc agtgagtacc gctggcacat caacttcacc acattcttca 14100
ttgattgcat ggccgccctc ggtctggcct atgaccggaa gaaagtctcc aaggccgcca
14160 tcttggccag gattaaaaga accggagatg gaaactacaa gagtggctga
gtttggggtc 14220 cctcaggttc ctttttcaaa aaccagccag gcagaggttt
taatgtctgt ttattaacta 14280 ctgaataatg ctaccaggat gctaaagatg
atgatgttaa cccattccag tacagtattc 14340 ttttaaaatt caaaagtatt
gaaagccaac aactctgcct ttatgatgct aagctgatat 14400 tatttcttct
cttatcctct ctctcttcta ggcccattgt cctccttttc actttattgc 14460
tatcgccctc ctttccctta ttgcctccca ggcaagcagc tggtcagtct ttgctcagtg
14520 tccagcttcc aaagcctaga caacctttct gtagcctaaa acgaatggtc
tttgctccag 14580 ataactctct ttccttgagc tgttgtgagc tttgaagtag
gtggcttgag ctagagataa 14640 aacagaatct tctgggtagt cccctgttga
ttatcttcag cccaggcttt tgctagatgg 14700 aatggaaaag caacttcatt
tgacacaaag cttctaaagc aggtaaattg tcgggggaga 14760 gagttagcat
gtatgaatgt aaggatgagg gaagcgaagc aagaggaacc tctcgccatg 14820
atcagacata cagctgccta cctaatgagg acttcaagcc ccaccacata gcatgcttcc
14880 tttctctcct ggctcggggt aaaaagtggc tgcggtgttt ggcaatgcta
attcaatgcc 14940 gcaacatata gttgaggccg aggataaaga aaagacattt
taagtttgta gtaaaagtgg 15000 tctctgctgg ggaagggttt tcttttcttt
ttttctttaa taacaaggag atttcttagt 15060 tcatatatca agaagtcttg
aagttgggtg tttccagaat tggtaaaaac agcagctcat 15120 agaattttga
gtattccatg agctgctcat tacagttctt tcctctttct gctctgccat 15180
cttcaggata ttggttcttc ccctcatagt aataagatgg ctgtggcatt tccaaacatc
15240 caaaaaaagg gaaggattta aggaggtgaa gtcgggtcaa aaataaaata
tatatacata 15300 tatacattgc ttagaacgtt aaactattag agtatttccc
ttccaaagag ggatgtttgg 15360 aaaaaactct gaaggagagg aggaattagt
tgggatgcca atttcctctc cactgctgga 15420 catgagatgg agaggctgag
ggacaggatc tataggcagc ttctaagagc gaacttcaca 15480 taggaaggga
tctgagaaca cgttgccagg ggcttgagaa ggttactgag tgagttattg 15540
ggagtcttaa taaaataaac tagatattag gtccattcat taattagttc cagtttctcc
15600 ttgaaatgag taaaaactag aaggcttctc tccacagtgt tgtgcccctt
cactcatttt 15660 tttttgagga gaagggggtc tctgttaaca tctagcctaa
agtatacaac tgcctggggg 15720 gcagggttag gaatctcttc actaccctga
ttcttgattc ctggctctac cctgtctgtc 15780 ccttttcttt gaccagatct
ttctcttccc tgaacgtttt cttctttccc tggacaggca 15840 gcctcctttg
tgtgtattca gaggcagtga tgacttgctg tccaggcagc tccctcctgc 15900
acacagaatg ctcagggtca ctgaaccact gcttctcttt tgaaagtaga gctagctgcc
15960 actttcacgt ggcctccgca gtgtctccac ctacacccct gtgctcccct
gccacactga 16020 tggctcaaga caaggctggc aaaccctccc agaaacatct
ctggcccaga aagcctctct 16080 ctccctccct ctctcatgag gcacagccaa
gccaagcgct catgttgagc cagtgggcca 16140 gccacagagc aaaagagggt
ttattttcag tcccctctct ctgggtcaga accagagggc 16200 atgctgaatg
ccccctgctt acttggtgag ggtgccccgc ctgagtcagt gctctcagct 16260
ggcagtgcaa tgcttgtaga agtaggagga aacagttctc actgggaaga agcaagggca
16320 agaacccaag tgcctcacct cgaaaggagg ccctgttccc tggagtcagg
gtgaactgca 16380 aagctttggc tgagacctgg gatttgagat accacaaacc
ctgctgaaca cagtgtctgt 16440 tcagcaaact aaccagcatt ccctacagcc
tagggcagac aatagtatag aagtctggaa 16500 aaaaacaaaa acagaatttg
agaaccttgg accactcctg tccctgtagc tcagtcatca 16560 aagcagaagt
ctggctttgc tctattaaga ttggaaatgt acactaccaa acactcagtc 16620
cactgttgag ccccagtgct ggaagggagg aaggcctttc ttctgtgtta attgcgtaga
16680 ggctacaggg gttagcctgg actaaaggca tccttgtctt ttgagctatt
cacctcagta 16740 gaaaaggatc taagggaaga tcactgtagt ttagttctgt
tgacctgtgc acctacccct 16800 tggaaatgtc tgctggtatt tctaattcca
caggtcatca gatgcctgct tgataatata 16860 taaacaataa aaacaacttt
cacttcttcc tattgtaatc gtgtgccatg gatctgatct 16920 gtaccatgac
cctacataag gctggatggc acctcaggct gagggcccca atgtatgtgt 16980
ggctgtgggt gtgggtggga gtgtgtctgc tgagtaagga acacgatttt caagattcta
17040 aagctcaatt caagtgacac attaatgata aactcagatc tgatcaagag
tccggatttc 17100 taacagtcct tgctttgggg ggtgtgctga caacttagct
caggtgcctt acatcttttc 17160 taatcacagt gttgcatatg agcctgccct
cactccctct gcagaatccc tttgcacctg 17220 agaccctact gaagtggctg
gtagaaaaag gggcctgagt ggaggattat cagtatcacg 17280 atttgcagga
ttcccttctg ggcttcattc tggaaacttt tgttagggct gcttttctta 17340
agtgcccaca tttgatggag ggtggaaata atttgaatgt atttgattta taagtttttt
17400 tttttttttt gggttaaaag atggttgtag catttaaaat ggaaaatttt
ctccttggtt 17460 tgctagtatc ttgggtgtat tctctgtaag tgtagctcaa
ataggtcatc atgaaaggtt 17520 aaaaaagcga ggtggccatg ttatgctggt
ggttaaggcc agggcctctc caaccactgt 17580 gccactgact tgctgtgtga
ccctgggcaa gtcacttaac tataaggtgc ctcagttttc 17640 cttctgttaa
aatggggata ataatactga cctacctcaa agggcagttt tgaggcatga 17700
ctaatgcttt ttagaaagca ttttgggatc cttcagcaca ggaattctca agacctgagt
17760 attttttata ataggaatgt ccaccatgaa cttgatacgt ccgtgtgtcc
cagatgctgt 17820 cattagtcta tatggttctc caagaaactg aatgaatcca
ttggagaagc ggtggataac 17880 tagccagaca aaatttgaga atacataaac
aacgcattgc cacggaaaca tacagaggat 17940 gccttttctg tgattgggtg
ggattttttc cctttttatg tgggatatag tagttacttg 18000 tgacaagaat
aattttggaa taatttctat taatatcaac tctgaagcta attgtactaa 18060
tctgagattg tgtttgttca taataaaagt gaagtgaatc tgattgcact gggtctggga
18120 gtttcttttg gctgtgattc aaagtcttgg gatttgtctc tggctcatat
ctatgtctgt 18180 acctttaaga gtataaaaga agtagaagtt acaatggttg
actcataccc cattaacctg 18240 ccctgctgtc ctaaaggtaa ctttaggtta
aactctggga cgcaggaagc caggagtctg 18300 ctgccattaa atcaaacaca
ttaacaccag ctggcaactt ggccctgggg aagtgccagg 18360 gttctcgggt
gtgtcacgtg gtcggtcaca tagacctaag ataaagacac tgggagagga 18420
aaagaccagc aggagaagcg ctccctggga aagcagtttt ttgctcttgc ccaggctgga
18480 gtgcaatggt gtgatctggg ctcactgc 18508 82 20 DNA Artificial
Sequence Antisense Oligonucleotide 82 gtgcgcgcga gcccgaaatc 20 83
20 DNA Artificial Sequence Antisense Oligonucleotide 83 ccgtgtccgg
tatttcctca 20 84 20 DNA Artificial Sequence Antisense
Oligonucleotide 84 ggcaacgggt gaccgtgtcc 20 85 20 DNA Artificial
Sequence Antisense Oligonucleotide 85 atttaaaggc tagagctggc 20 86
20 DNA Artificial Sequence Antisense Oligonucleotide 86 cgagccggga
atttaaaggc 20 87 20 DNA Artificial Sequence Antisense
Oligonucleotide 87 gaggctccgg agcggagttc 20 88 20 DNA Artificial
Sequence Antisense Oligonucleotide 88 ttggctctcg gatgccggga 20 89
20 DNA Artificial Sequence Antisense Oligonucleotide 89 gtgggccggc
atcttggctc 20 90 20 DNA Artificial Sequence Antisense
Oligonucleotide 90 tcctgcagca agtgggccgg 20 91 20 DNA Artificial
Sequence Antisense Oligonucleotide 91 agctagagat atcgtcctgc 20 92
20 DNA Artificial Sequence Antisense Oligonucleotide 92 ccatacaggg
ctcccaagtg 20 93 20 DNA Artificial Sequence Antisense
Oligonucleotide 93 gtagaacttg caggtaggaa 20 94 20 DNA Artificial
Sequence Antisense Oligonucleotide 94 gctgttatgc ccagggcact 20 95
20 DNA Artificial Sequence Antisense Oligonucleotide 95 agacatcatt
ctggaatgcc 20 96 20 DNA Artificial Sequence Antisense
Oligonucleotide 96 ctgaaaactt gtggtgggca 20 97 20 DNA Artificial
Sequence Antisense Oligonucleotide 97 gtgtttctga aaacttgtgg 20 98
20 DNA Artificial Sequence Antisense Oligonucleotide 98 aagtctagcg
tactcccctt 20 99 20 DNA Artificial Sequence Antisense
Oligonucleotide 99 tttgtagtac ctcctctgga 20 100 20 DNA Artificial
Sequence Antisense Oligonucleotide 100 cataaggacg atatccgaag 20 101
20 DNA Artificial Sequence Antisense Oligonucleotide 101 cccaaactca
gccactcttg 20 102 20 DNA Artificial Sequence Antisense
Oligonucleotide 102 aaacctctgc ctggctggtt 20 103 20 DNA Artificial
Sequence Antisense Oligonucleotide 103 gtagcattat tcagtagtta 20 104
20 DNA Artificial Sequence Antisense Oligonucleotide 104 tactggaatg
ggttaacatc 20 105 20 DNA Artificial Sequence Antisense
Oligonucleotide 105 tcagcttagc atcataaagg 20 106 20 DNA Artificial
Sequence Antisense Oligonucleotide 106 agactgacca gctgcttgcc 20 107
20 DNA Artificial Sequence Antisense Oligonucleotide 107 gctggacact
gagcaaagac 20 108 20 DNA Artificial Sequence Antisense
Oligonucleotide 108 tttggaagct ggacactgag 20 109 20 DNA Artificial
Sequence Antisense Oligonucleotide 109 tctggagcaa agaccattcg 20 110
20 DNA Artificial Sequence Antisense Oligonucleotide 110 cttcaaagct
cacaacagct 20 111 20 DNA Artificial Sequence Antisense
Oligonucleotide 111 ccacctactt caaagctcac 20 112 20 DNA Artificial
Sequence Antisense Oligonucleotide 112 tcaagccacc tacttcaaag 20 113
20 DNA Artificial Sequence Antisense Oligonucleotide 113 ctctagctca
agccacctac 20 114 20 DNA Artificial Sequence Antisense
Oligonucleotide 114 tgtgtcaaat gaagttgctt 20 115 20 DNA Artificial
Sequence Antisense Oligonucleotide 115 cccgacaatt tacctgcttt 20 116
20 DNA Artificial Sequence Antisense Oligonucleotide 116 ttacattcat
acatgctaac 20 117 20 DNA Artificial Sequence Antisense
Oligonucleotide 117 tgtctgatca tggcgagagg 20 118 20 DNA Artificial
Sequence Antisense Oligonucleotide 118 aaggaagcat gctatgtggt 20 119
20 DNA Artificial Sequence Antisense Oligonucleotide 119 ggagagaaag
gaagcatgct 20 120 20 DNA Artificial Sequence Antisense
Oligonucleotide 120 aactatatgt tgcggcattg 20 121 20 DNA Artificial
Sequence Antisense Oligonucleotide 121 tagatgttaa cagagacccc 20 122
20 DNA Artificial Sequence Antisense Oligonucleotide 122 aatcagggta
gtgaagagat 20 123 20 DNA Artificial Sequence Antisense
Oligonucleotide 123 gggtagagcc aggaatcaag 20 124 20 DNA Artificial
Sequence Antisense Oligonucleotide 124 agcagtggtt cagtgaccct 20 125
20 DNA Artificial Sequence Antisense Oligonucleotide 125 tactttcaaa
agagaagcag 20 126 20 DNA Artificial Sequence Antisense
Oligonucleotide 126 cgtgaaagtg gcagctagct 20 127 20 DNA Artificial
Sequence Antisense Oligonucleotide 127 ccttgtcttg agccatcagt 20 128
20 DNA Artificial Sequence Antisense Oligonucleotide 128 ggtttgccag
ccttgtcttg 20 129 20 DNA Artificial Sequence Antisense
Oligonucleotide 129 cactggctca acatgagcgc 20 130 20 DNA Artificial
Sequence Antisense Oligonucleotide 130 tgctctgtgg ctggcccact 20 131
20 DNA Artificial Sequence Antisense Oligonucleotide 131 aataaaccct
cttttgctct 20 132 20 DNA Artificial Sequence Antisense
Oligonucleotide 132 gactgaaaat aaaccctctt 20 133 20 DNA Artificial
Sequence Antisense Oligonucleotide 133 agagcactga ctcaggcggg 20 134
20 DNA Artificial Sequence Antisense Oligonucleotide 134 ttgcactgcc
agctgagagc 20 135 20 DNA Artificial Sequence Antisense
Oligonucleotide 135 tacttctaca agcattgcac 20 136 20 DNA Artificial
Sequence Antisense Oligonucleotide 136 actgtttcct cctacttcta 20 137
20 DNA Artificial Sequence Antisense Oligonucleotide 137 cttgcccttg
cttcttccca 20 138 20 DNA Artificial Sequence Antisense
Oligonucleotide 138 tttcgaggtg aggcacttgg 20 139 20 DNA Artificial
Sequence Antisense Oligonucleotide 139 tcagccaaag ctttgcagtt 20 140
20 DNA Artificial Sequence Antisense Oligonucleotide 140 ttctgctttg
atgactgagc 20 141 20 DNA Artificial Sequence Antisense
Oligonucleotide 141 atcctcggcc tcaactatat 20 142 20 DNA Artificial
Sequence Antisense Oligonucleotide 142 tccttgttat taaagaaaaa 20 143
20 DNA Artificial Sequence Antisense Oligonucleotide 143 ctaagaaatc
tccttgttat 20 144 20 DNA Artificial Sequence Antisense
Oligonucleotide 144 cttcttgata tatgaactaa 20 145 20 DNA Artificial
Sequence Antisense Oligonucleotide 145 acttcaagac ttcttgatat 20 146
20 DNA Artificial Sequence Antisense Oligonucleotide 146 aaattccatg
agctgctgtt 20 147 20 DNA Artificial Sequence Antisense
Oligonucleotide 147 gaactgtaat gagcagctca 20 148 20 DNA Artificial
Sequence Antisense Oligonucleotide 148 tgaagatggc agagcagaaa 20 149
20 DNA Artificial Sequence Antisense Oligonucleotide 149 tggaaatgcc
acagccatct 20 150 20 DNA Artificial Sequence Antisense
Oligonucleotide 150 cgacttcacc tccttaaatc 20 151 20 DNA Artificial
Sequence Antisense Oligonucleotide 151 gcaatgtata tatgtatata 20 152
20 DNA Artificial Sequence Antisense Oligonucleotide 152 ccagcagtgg
agaggaaatt 20 153 20 DNA Artificial Sequence Antisense
Oligonucleotide 153 cagcctctcc atctcatgtc 20 154 20 DNA Artificial
Sequence Antisense Oligonucleotide 154 ctatgtgaag ttcgctctta 20 155
20 DNA Artificial Sequence Antisense Oligonucleotide 155 cgtgttctca
gatcccttcc 20 156 20 DNA Artificial Sequence Antisense
Oligonucleotide 156 aactaattaa tgaatggacc 20 157 20 DNA Artificial
Sequence Antisense Oligonucleotide 157 ttactcattt caaggagaaa 20 158
20 DNA Artificial Sequence Antisense Oligonucleotide 158 gaagccttct
agtttttact 20 159 20 DNA Artificial Sequence Antisense
Oligonucleotide 159 cactgtggag agaagccttc 20 160 20 DNA Artificial
Sequence Antisense Oligonucleotide 160 gcacaacact gtggagagaa
20 161 20 DNA Artificial Sequence Antisense Oligonucleotide 161
aatcttaata gagcaaagcc 20 162 20 DNA Artificial Sequence Antisense
Oligonucleotide 162 gactgagtgt ttggtagtgt 20 163 20 DNA Artificial
Sequence Antisense Oligonucleotide 163 agcctctacg caattaacac 20 164
20 DNA Artificial Sequence Antisense Oligonucleotide 164 ctgaggtgaa
tagctcaaaa 20 165 20 DNA Artificial Sequence Antisense
Oligonucleotide 165 ccttttctac tgaggtgaat 20 166 20 DNA Artificial
Sequence Antisense Oligonucleotide 166 tagaaatacc agcagacatt 20 167
20 DNA Artificial Sequence Antisense Oligonucleotide 167 gcacacgatt
acaataggaa 20 168 20 DNA Artificial Sequence Antisense
Oligonucleotide 168 tccatggcac acgattacaa 20 169 20 DNA Artificial
Sequence Antisense Oligonucleotide 169 tcagatccat ggcacacgat 20 170
20 DNA Artificial Sequence Antisense Oligonucleotide 170 aggtgccatc
cagccttatg 20 171 20 DNA Artificial Sequence Antisense
Oligonucleotide 171 gccctcagcc tgaggtgcca 20 172 20 DNA Artificial
Sequence Antisense Oligonucleotide 172 agctttagaa tcttgaaaat 20 173
20 DNA Artificial Sequence Antisense Oligonucleotide 173 aatgtgtcac
ttgaattgag 20 174 20 DNA Artificial Sequence Antisense
Oligonucleotide 174 ctgttagaaa tccggactct 20 175 20 DNA Artificial
Sequence Antisense Oligonucleotide 175 ccaaagcagg gactgttaga 20 176
20 DNA Artificial Sequence Antisense Oligonucleotide 176 caacactgtg
attagaaaag 20 177 20 DNA Artificial Sequence Antisense
Oligonucleotide 177 cttcagtagg gtctcaggtg 20 178 20 DNA Artificial
Sequence Antisense Oligonucleotide 178 ctaccagcca cttcagtagg 20 179
20 DNA Artificial Sequence Antisense Oligonucleotide 179 actcaggccc
ctttttctac 20 180 20 DNA Artificial Sequence Antisense
Oligonucleotide 180 gatactgata atcctccact 20 181 20 DNA Artificial
Sequence Antisense Oligonucleotide 181 aatcctgcaa atcgtgatac 20 182
20 DNA Artificial Sequence Antisense Oligonucleotide 182 agcagcccta
acaaaagttt 20 183 20 DNA Artificial Sequence Antisense
Oligonucleotide 183 aaattttcca ttttaaatgc 20 184 20 DNA Artificial
Sequence Antisense Oligonucleotide 184 cacttacaga gaatacaccc 20 185
20 DNA Artificial Sequence Antisense Oligonucleotide 185 gagctacact
tacagagaat 20 186 20 DNA Artificial Sequence Antisense
Oligonucleotide 186 aacatggcca cctcgctttt 20 187 20 DNA Artificial
Sequence Antisense Oligonucleotide 187 gccttaacca ccagcataac 20 188
20 DNA Artificial Sequence Antisense Oligonucleotide 188 aggccctggc
cttaaccacc 20 189 20 DNA Artificial Sequence Antisense
Oligonucleotide 189 tggagaggcc ctggccttaa 20 190 20 DNA Artificial
Sequence Antisense Oligonucleotide 190 tcatgcctca aaactgccct 20 191
20 DNA Artificial Sequence Antisense Oligonucleotide 191 ctaaaaagca
ttagtcatgc 20 192 20 DNA Artificial Sequence Antisense
Oligonucleotide 192 agaattcctg tgctgaagga 20 193 20 DNA Artificial
Sequence Antisense Oligonucleotide 193 ggtcttgaga attcctgtgc 20 194
20 DNA Artificial Sequence Antisense Oligonucleotide 194 actcaggtct
tgagaattcc 20 195 20 DNA Artificial Sequence Antisense
Oligonucleotide 195 ggacattcct attataaaaa 20 196 20 DNA Artificial
Sequence Antisense Oligonucleotide 196 acacggacgt atcaagttca 20 197
20 DNA Artificial Sequence Antisense Oligonucleotide 197 cctctgtatg
tttccgtggc 20 198 20 DNA Artificial Sequence Antisense
Oligonucleotide 198 tgcgaggagt tgactggcgc 20 199 20 DNA Artificial
Sequence Antisense Oligonucleotide 199 ggcaaagtgc gaggagttga 20 200
20 DNA Artificial Sequence Antisense Oligonucleotide 200 ggaaactcac
atcgtcctgc 20 201 20 DNA Artificial Sequence Antisense
Oligonucleotide 201 ggctgcttac cccaaagcca 20 202 20 DNA Artificial
Sequence Antisense Oligonucleotide 202 ctcagttgca tttcactgta 20 203
20 DNA Artificial Sequence Antisense Oligonucleotide 203 gtgggaagag
aagatgtcca 20 204 20 DNA Artificial Sequence Antisense
Oligonucleotide 204 gccttctcta aggttttaag 20 205 20 DNA Artificial
Sequence Antisense Oligonucleotide 205 tagaataccc ctgccaggag 20 206
20 DNA Artificial Sequence Antisense Oligonucleotide 206 aacttcttac
ctggaatgcc 20 207 20 DNA Artificial Sequence Antisense
Oligonucleotide 207 ccttgcaaaa gagctcatac 20 208 20 DNA Artificial
Sequence Antisense Oligonucleotide 208 cttcactcac ctcctctgga 20 209
20 DNA Artificial Sequence Antisense Oligonucleotide 209 tttgcactgt
ctctccccac 20 210 20 DNA Artificial Sequence Antisense
Oligonucleotide 210 tcagtggttt cttacacttg 20 211 20 DNA Artificial
Sequence Antisense Oligonucleotide 211 tttgtagtac ctacattgac 20 212
20 DNA Artificial Sequence Antisense Oligonucleotide 212 gctgacttac
ccacagctcc 20 213 20 DNA Artificial Sequence Antisense
Oligonucleotide 213 tactgccccc taattttata 20 214 20 DNA Artificial
Sequence Antisense Oligonucleotide 214 ccatttgcga tacaggaaac 20 215
20 DNA Artificial Sequence Antisense Oligonucleotide 215 aagccctcac
ctgaaacaaa 20 216 912 DNA M. musculus 216 tcccagtctc ccggggtttc
tctttgctgg tgcctggaag tgggggtaga tgtgaagtta 60 gaccgagttg
tgagtggcgg tagccagtgt cttcctcact tctttcgatg cgatttcccc 120
agtgaaccat ttgctaagcg ccagaccaaa gtcctaggct tgcacacaat tcctacttgg
180 aatcacgtta tcctgctctt aaagaaaagt cacccatcag cccacagcaa
agaggataag 240 gagaaaaaga ggggaggaga gacggagaag ctagaggcag
agggaacagc agattgcgcc 300 tagccaatgg aaaaggcagg acaaggtggc
accaaattct ctttggccaa tgacaagacg 360 ggcttcacag gaggcacatt
agcatttatc cccaggcagg gggttggagc agcgcgccct 420 gttgatgcct
tcagcatccc ggcgcctcca aggtctactc tggaatctac ttggctttct 480
ttcccgttct tggtcccgcc ctctctctct ccctccctcc ctccctccct tcctccctcc
540 ctccctccct ccctccctcc ctcacctcca cgcctggctt ccttggctag
ctatctctgc 600 gctctttacc ctttgctggc agccgataaa agggggctga
ggaaatactg aacacggtca 660 tcccatcgcc tgctctaccc tttaaaatcc
cagcccagga gatctgtgca cagccagacc 720 gggctgaaca cccatcccga
gagtcaggag ggcaggtttc caagcgcagt tccgccactc 780 gcctacacca
acgggctccg gaaccgaagt ccacgctcga tctcagcact gggaaagtga 840
ggcgagcaac tgactatcat catgccggcc cacatgctcc aagaggtgag cttccagaag
900 cggccctcgc tc 912 217 288 DNA M. musculus 217 tgcagatct
ccagttctta cacgaccacc accaccatca ctgcacctcc ctccggaaat 60
gaacgagaga aggtgaagac agtgcccctc cacctggaag aagacatccg tcctgaaatg
120 aaagaagata ttcacgaccc cacctatcag gatgaggagg gacccccgcc
caagctggag 180 tacgtctgga ggaacatcat tctcatggtc ctgctgcact
tgggaggcct gtacgggatc 240 atactggttc cctcctgcaa gctctacact
gccctcttcg gtgagcag 288 218 144 DNA M. musculus 218 ttgcagggat
tttctactac atgaccagcg ctctgggcat cacagccggg gctcatcgcc 60
tctggagcca cagaacttac aaggctcggc tgcccctgcg gatcttccta atcattgcca
120 acaccatggc gttccagtaa gaag 144 219 221 DNA M. musculus 219
ttccagaaat gacgtgtacg actgggcccg agatcaccgc gcccaccaca agttctcaga
60 aacacacgcc gaccctcaca attcccgccg tggcttcttc ttctctcacg
tgggttggct 120 gcttgtgcgc aaacacccgg ctgtcaaaga gaagggcgga
aaactggaca tgtctgacct 180 gaaagccgag aagctggtga tgttccagag
gaggtaaggg a 221 220 247 DNA M. musculus 220 atgtaggtac tacaagcccg
gcctcctgct gatgtgcttc atcctgccca cgctggtgcc 60 ctggtactgc
tggggcgaga cttttgtaaa cagcctgttc gttagcacct tcttgcgata 120
cactctggtg ctcaacgcca cctggctggt gaacagtgcc gcgcatctct atggatatcg
180 cccctacgac aagaacattc aatcccggga gaatatcctg gtttccctgg
gtgccgtggg 240 taagtca 247 221 3660 DNA M. musculus 221 ttgcaggcga
gggcttccac aactaccacc acaccttccc cttcgactac tctgccagtg 60
agtaccgctg gcacatcaac ttcaccacgt tcttcatcga ctgcatggct gccctgggcc
120 tggcttacga ccggaagaaa gtttctaagg ctactgtctt agccaggatt
aagagaactg 180 gagacgggag tcacaagagt agctgagctt tgggcttctg
agttcctgtt tcaaacgttt 240 tctggcagag atttaatatt ctgttgatta
actaacaact ggatattgct atcggggtgt 300 taatgatgca tttaacctat
tccggtacag tattcttata aaatgagaaa gctttgatca 360 cgttttgagg
taataaatat tttatttagc taggattaac catgccacaa gacattatat 420
atttctaagc acacatgata aatgcatata caattttgca caacagcttt aaataataac
480 aataaatttg aacattctat acagagagga tcaaagccaa ggaacatgct
gttttgatgc 540 tagggtgagc atggtgctca gtccctgttt gtttgcatgg
tgtccagctt tgtttcttct 600 ctgtcatcac caccttcagg caaatagttg
accaaccact ggcctgtgtc tgtccaccct 660 ccaaagccca ggccaccttt
ctgttttctg aaatactgat ccttcctcct gaatacatcc 720 ctccttgttc
ctagcttcaa gactgctgcc tcaaataggg atagagcaag tccccgctgc 780
aggttgtgct agatgggatg gagaaattat cttcatttga tacagagcaa gtagattgtc
840 tcgagagaaa agttagcatg cgtggtatga tttgtaagta aagatggaag
agagagagag 900 agagagagag agagagagag agagagagag agaggtagcc
atatctaaca gcctacttac 960 caaagacccc aggcctctct gcttggcatg
cctcctttct gtccatcctc tgaaccccag 1020 agattagtga gatttgaata
attaaatcat tttcagagtg aagggggtta atgcagggtc 1080 tgtgctaggg
gagggtttta gcttttggta actgaagatt ttttcatgga aaaagtcttc 1140
gtgttcaatg tgcctagaac tgataactaa acagctgaca tttgtcgggg acagatatgg
1200 tgtgaaacta tgaaaatata agcaaaatct tcacttggaa catgaaacta
tttcacttag 1260 aaaataatcg aaggacccga ggtgttgcct gggttgccag
tttctttcgt ggctgggcag 1320 gaactagtga ggttgagggg cagtgtctgt
aagtagctgc taagaggtgc atttccagat 1380 gaagcccttg gggaacatct
gccagggatc cgcatggtgt tggctccatc cattgcttta 1440 gtttcctcct
tggattgtgt agaaacttgg cttcccatgg ttttgaacct tccatgcctt 1500
ctttgctttg tggccaccca gcctgcctag tgctgcctag gaagctctta cccacctgat
1560 ttcttctgac atttctttct ttggcctttt tttctttctc cggacatgca
gctagttgcc 1620 tgagtgtatc aagagcaccc aggacttgct gctgtccagg
cctgttcctc ccccagtatc 1680 cgtgggtgtg gaagagctgt gtagcttcag
gaagcagagc caggtgccac ctttctgtgg 1740 cttccagatc ctccctacct
ccaactcatg tgcctctgtc acagtgattt caggaaagct 1800 tggtagaccc
tctagcaaca tctcggttca gaaagtctct ctggtttgtg agttaacagc 1860
tcagctaagt gctgttttgt ctcagtgagt taaccactga atgcgagggt tggttgttga
1920 tctgtctcgg tgtgtgtcgg agtagacagc atatgcactt ctccctgtgc
gctttgcaag 1980 gtaatgtggc tttggctgat ccatgcaggc aggtagtggt
acagtgctgc tgaaaggaag 2040 aagttcccca ttttatctgt taaaacacca
gagacatggg caagtgctaa tggacctcac 2100 ttcaggaaga gggtctgctt
cctgaagcca gtgtgtgatg aaaagtgact gagacctgat 2160 atctaaggtg
agacctgata cctaacactc tgtcacacag tccagggcca acagtgctat 2220
aggaaagtct agaagaaaac atcacatcag tattttagaa ccatcaacca tctcttgtcc
2280 ctatagccca atccagaggc ctggttttta gaactggctg tgtaaggtgc
caaacactca 2340 gttcacttgt agaatcagag ccttttttcc cccctatgtt
aattgaacac gcgctctgag 2400 ctgttttgtt gaagtagaaa atctcataga
aaaatcactg tagatctact gacctatagc 2460 cctctggaaa tgcctttgag
atggttttac ttttctaggt catagatgcc tgattataaa 2520 gatgaacaat
aaaatcagct ttctttcttt ctcttctgat cttattcccc agatctgatt 2580
caggccatgt tccaaagcaa ggctacattg aggtcctggt gtctttaagt aaaggacatc
2640 tttcagatcc tctcaaagaa ggatttataa cagtttccag atgaatgtac
taatagcttt 2700 gggtgcctta tctctttcct aatctgtagt gcctgtgagc
tcagtctcac tccttccctt 2760 agcccggaga ccccttagat cgagtgggaa
tagtcaagag gctggctgga gagtcatcag 2820 tacattggtt tgcagaaatc
ttttacaggc tacattttgg aatttttttt tttttagtaa 2880 gtgatcaaat
ttggtgggaa gtaattcgag tgtattcgat tgtattgtcg tcctcgttat 2940
cattgtcaaa catgttatag acggcagttg gcactggggc tgctaatctc tgggtgtagt
3000 ctctgaaact gtagctccag tgaggtggtg tgaaaggtta gcaaagccac
catctgctgg 3060 tgctccagcc aaggtgcctc ttagccactg aattgctatg
ttatcctttc tcttgtaaca 3120 aacccacccc agagataaag cctttaatca
acccaagaaa ctcctgggct aagtatctga 3180 cagtctcaca tctcaacagt
gtgaattaag tgtccatagc atcagctcag gaggacactc 3240 tgggagagtg
ctgacaaaaa agggttatta atactgacct actacttcaa gggcagttct 3300
gaggtgatta gagctttttt taaaaaccaa gtatttgggg atcctcagca gaggtattca
3360 tacagactcc caaagaacta tatatgttcc tgagaccatc gtttagtcta
cattgctctt 3420 cccagagact gacagatatg accagtcaaa gtgcaagact
acctacccac tgccatgaaa 3480 accattgcag gaaacctttc ccttcctgaa
tgagattttt tttttccctt tttatgtggg 3540 gtaattattt gtgacccaag
tgtaatttgg atgatttcca ttaatatcaa ctcttgaagc 3600 ctacttgtac
tgattgagat tgtatttgtt cctaataaaa gtggatctgg ttgtactgtc 3660 222
5383 DNA M. musculus CDS (862)...(1929) Antisense Oligonucleotide
222 tcccagtctc ccggggtttc tctttgctgg tgcctggaag tgggggtaga
tgtgaagtta 60 gaccgagttg tgagtggcgg tagccagtgt cttcctcact
tctttcgatg cgatttcccc 120 agtgaaccat ttgctaagcg ccagaccaaa
gtcctaggct tgcacacaat tcctacttgg 180 aatcacgtta tcctgctctt
aaagaaaagt cacccatcag cccacagcaa agaggataag 240 gagaaaaaga
ggggaggaga gacggagaag ctagaggcag agggaacagc agattgcgcc 300
tagccaatgg aaaaggcagg acaaggtggc accaaattct ctttggccaa tgacaagacg
360 ggcttcacag gaggcacatt agcatttatc cccaggcagg gggttggagc
agcgcgccct 420 gttgatgcct tcagcatccc ggcgcctcca aggtctactc
tggaatctac ttggctttct 480 ttcccgttct tggtcccgcc ctctctctct
ccctccctcc ctccctccct tcctccctcc 540 ctccctccct ccctccctcc
ctcacctcca cgcctggctt ccttggctag ctatctctgc 600 gctctttacc
ctttgctggc agccgataaa agggggctga ggaaatactg aacacggtca 660
tcccatcgcc tgctctaccc tttaaaatcc cagcccagga gatctgtgca cagccagacc
720 gggctgaaca cccatcccga gagtcaggag ggcaggtttc caagcgcagt
tccgccactc 780 gcctacacca acgggctccg gaaccgaagt ccacgctcga
tctcagcact gggaaagtga 840 ggcgagcaac tgactatcat c atg ccg gcc cac
atg ctc caa gag atc tcc 891 Met Pro Ala His Met Leu Gln Glu Ile Ser
1 5 10 agt tct tac acg acc acc acc acc atc act gca cct ccc tcc gga
aat 939 Ser Ser Tyr Thr Thr Thr Thr Thr Ile Thr Ala Pro Pro Ser Gly
Asn 15 20 25 gaa cga gag aag gtg aag aca gtg ccc ctc cac ctg gaa
gaa gac atc 987 Glu Arg Glu Lys Val Lys Thr Val Pro Leu His Leu Glu
Glu Asp Ile 30 35 40 cgt cct gaa atg aaa gaa gat att cac gac ccc
acc tat cag gat gag 1035 Arg Pro Glu Met Lys Glu Asp Ile His Asp
Pro Thr Tyr Gln Asp Glu 45 50 55 gag gga ccc ccg ccc aag ctg gag
tac gtc tgg agg aac atc att ctc 1083 Glu Gly Pro Pro Pro Lys Leu
Glu Tyr Val Trp Arg Asn Ile Ile Leu 60 65 70 atg gtc ctg ctg cac
ttg gga ggc ctg tac ggg atc ata ctg gtt ccc 1131 Met Val Leu Leu
His Leu Gly Gly Leu Tyr Gly Ile Ile Leu Val Pro 75 80 85 90 tcc tgc
aag ctc tac act gcc ctc ttc ggg att ttc tac tac atg acc 1179 Ser
Cys Lys Leu Tyr Thr Ala Leu Phe Gly Ile Phe Tyr Tyr Met Thr 95 100
105 agc gct ctg ggc atc aca gcc ggg gct cat cgc ctc tgg agc cac aga
1227 Ser Ala Leu Gly Ile Thr Ala Gly Ala His Arg Leu Trp Ser His
Arg 110 115 120 act tac aag gct cgg ctg ccc ctg cgg atc ttc cta atc
att gcc aac 1275 Thr Tyr Lys Ala Arg Leu Pro Leu Arg Ile Phe Leu
Ile Ile Ala Asn 125 130 135 acc atg gcg ttc caa aat gac gtg tac gac
tgg gcc cga gat cac cgc 1323 Thr Met Ala Phe Gln Asn Asp Val Tyr
Asp Trp Ala Arg Asp His Arg 140 145 150 gcc cac cac aag ttc tca gaa
aca cac gcc gac cct cac aat tcc cgc 1371 Ala His His Lys Phe Ser
Glu Thr His Ala Asp Pro His Asn Ser Arg 155 160 165 170 cgt
ggc ttc ttc ttc tct cac gtg ggt tgg ctg ctt gtg cgc aaa cac 1419
Arg Gly Phe Phe Phe Ser His Val Gly Trp Leu Leu Val Arg Lys His 175
180 185 ccg gct gtc aaa gag aag ggc gga aaa ctg gac atg tct gac ctg
aaa 1467 Pro Ala Val Lys Glu Lys Gly Gly Lys Leu Asp Met Ser Asp
Leu Lys 190 195 200 gcc gag aag ctg gtg atg ttc cag agg agg tac tac
aag ccc ggc ctc 1515 Ala Glu Lys Leu Val Met Phe Gln Arg Arg Tyr
Tyr Lys Pro Gly Leu 205 210 215 ctg ctg atg tgc ttc atc ctg ccc acg
ctg gtg ccc tgg tac tgc tgg 1563 Leu Leu Met Cys Phe Ile Leu Pro
Thr Leu Val Pro Trp Tyr Cys Trp 220 225 230 ggc gag act ttt gta aac
agc ctg ttc gtt agc acc ttc ttg cga tac 1611 Gly Glu Thr Phe Val
Asn Ser Leu Phe Val Ser Thr Phe Leu Arg Tyr 235 240 245 250 act ctg
gtg ctc aac gcc acc tgg ctg gtg aac agt gcc gcg cat ctc 1659 Thr
Leu Val Leu Asn Ala Thr Trp Leu Val Asn Ser Ala Ala His Leu 255 260
265 tat gga tat cgc ccc tac gac aag aac att caa tcc cgg gag aat atc
1707 Tyr Gly Tyr Arg Pro Tyr Asp Lys Asn Ile Gln Ser Arg Glu Asn
Ile 270 275 280 ctg gtt tcc ctg ggt gcc gtg ggc gag ggc ttc cac aac
tac cac cac 1755 Leu Val Ser Leu Gly Ala Val Gly Glu Gly Phe His
Asn Tyr His His 285 290 295 acc ttc ccc ttc gac tac tct gcc agt gag
tac cgc tgg cac atc aac 1803 Thr Phe Pro Phe Asp Tyr Ser Ala Ser
Glu Tyr Arg Trp His Ile Asn 300 305 310 ttc acc acg ttc ttc atc gac
tgc atg gct gcc ctg ggc ctg gct tac 1851 Phe Thr Thr Phe Phe Ile
Asp Cys Met Ala Ala Leu Gly Leu Ala Tyr 315 320 325 330 gac cgg aag
aaa gtt tct aag gct act gtc tta gcc agg att aag aga 1899 Asp Arg
Lys Lys Val Ser Lys Ala Thr Val Leu Ala Arg Ile Lys Arg 335 340 345
act gga gac ggg agt cac aag agt agc tga gctttgggct tctgagttcc 1949
Thr Gly Asp Gly Ser His Lys Ser Ser * 350 355 tgtttcaaac gttttctggc
agagatttaa tattctgttg attaactaac aactggatat 2009 tgctatcggg
gtgttaatga tgcatttaac ctattccggt acagtattct tataaaatga 2069
gaaagctttg atcacgtttt gaggtaataa atattttatt tagctaggat taaccatgcc
2129 acaagacatt atatatttct aagcacacat gataaatgca tatacaattt
tgcacaacag 2189 ctttaaataa taacaataaa tttgaacatt ctatacagag
aggatcaaag ccaaggaaca 2249 tgctgttttg atgctagggt gagcatggtg
ctcagtccct gtttgtttgc atggtgtcca 2309 gctttgtttc ttctctgtca
tcaccacctt caggcaaata gttgaccaac cactggcctg 2369 tgtctgtcca
ccctccaaag cccaggccac ctttctgttt tctgaaatac tgatccttcc 2429
tcctgaatac atccctcctt gttcctagct tcaagactgc tgcctcaaat agggatagag
2489 caagtccccg ctgcaggttg tgctagatgg gatggagaaa ttatcttcat
ttgatacaga 2549 gcaagtagat tgtctcgaga gaaaagttag catgcgtggt
atgatttgta agtaaagatg 2609 gaagagagag agagagagag agagagagag
agagagagag agagagaggt agccatatct 2669 aacagcctac ttaccaaaga
ccccaggcct ctctgcttgg catgcctcct ttctgtccat 2729 cctctgaacc
ccagagatta gtgagatttg aataattaaa tcattttcag agtgaagggg 2789
gttaatgcag ggtctgtgct aggggagggt tttagctttt ggtaactgaa gattttttca
2849 tggaaaaagt cttcgtgttc aatgtgccta gaactgataa ctaaacagct
gacatttgtc 2909 ggggacagat atggtgtgaa actatgaaaa tataagcaaa
atcttcactt ggaacatgaa 2969 actatttcac ttagaaaata atcgaaggac
ccgaggtgtt gcctgggttg ccagtttctt 3029 tcgtggctgg gcaggaacta
gtgaggttga ggggcagtgt ctgtaagtag ctgctaagag 3089 gtgcatttcc
agatgaagcc cttggggaac atctgccagg gatccgcatg gtgttggctc 3149
catccattgc tttagtttcc tccttggatt gtgtagaaac ttggcttccc atggttttga
3209 accttccatg ccttctttgc tttgtggcca cccagcctgc ctagtgctgc
ctaggaagct 3269 cttacccacc tgatttcttc tgacatttct ttctttggcc
tttttttctt tctccggaca 3329 tgcagctagt tgcctgagtg tatcaagagc
acccaggact tgctgctgtc caggcctgtt 3389 cctcccccag tatccgtggg
tgtggaagag ctgtgtagct tcaggaagca gagccaggtg 3449 ccacctttct
gtggcttcca gatcctccct acctccaact catgtgcctc tgtcacagtg 3509
atttcaggaa agcttggtag accctctagc aacatctcgg ttcagaaagt ctctctggtt
3569 tgtgagttaa cagctcagct aagtgctgtt ttgtctcagt gagttaacca
ctgaatgcga 3629 gggttggttg ttgatctgtc tcggtgtgtg tcggagtaga
cagcatatgc acttctccct 3689 gtgcgctttg caaggtaatg tggctttggc
tgatccatgc aggcaggtag tggtacagtg 3749 ctgctgaaag gaagaagttc
cccattttat ctgttaaaac accagagaca tgggcaagtg 3809 ctaatggacc
tcacttcagg aagagggtct gcttcctgaa gccagtgtgt gatgaaaagt 3869
gactgagacc tgatatctaa ggtgagacct gatacctaac actctgtcac acagtccagg
3929 gccaacagtg ctataggaaa gtctagaaga aaacatcaca tcagtatttt
agaaccatca 3989 accatctctt gtccctatag cccaatccag aggcctggtt
tttagaactg gctgtgtaag 4049 gtgccaaaca ctcagttcac ttgtagaatc
agagcctttt ttccccccta tgttaattga 4109 acacgcgctc tgagctgttt
tgttgaagta gaaaatctca tagaaaaatc actgtagatc 4169 tactgaccta
tagccctctg gaaatgcctt tgagatggtt ttacttttct aggtcataga 4229
tgcctgatta taaagatgaa caataaaatc agctttcttt ctttctcttc tgatcttatt
4289 ccccagatct gattcaggcc atgttccaaa gcaaggctac attgaggtcc
tggtgtcttt 4349 aagtaaagga catctttcag atcctctcaa agaaggattt
ataacagttt ccagatgaat 4409 gtactaatag ctttgggtgc cttatctctt
tcctaatctg tagtgcctgt gagctcagtc 4469 tcactccttc ccttagcccg
gagacccctt agatcgagtg ggaatagtca agaggctggc 4529 tggagagtca
tcagtacatt ggtttgcaga aatcttttac aggctacatt ttggaatttt 4589
ttttttttta gtaagtgatc aaatttggtg ggaagtaatt cgagtgtatt cgattgtatt
4649 gtcgtcctcg ttatcattgt caaacatgtt atagacggca gttggcactg
gggctgctaa 4709 tctctgggtg tagtctctga aactgtagct ccagtgaggt
ggtgtgaaag gttagcaaag 4769 ccaccatctg ctggtgctcc agccaaggtg
cctcttagcc actgaattgc tatgttatcc 4829 tttctcttgt aacaaaccca
ccccagagat aaagccttta atcaacccaa gaaactcctg 4889 ggctaagtat
ctgacagtct cacatctcaa cagtgtgaat taagtgtcca tagcatcagc 4949
tcaggaggac actctgggag agtgctgaca aaaaagggtt attaatactg acctactact
5009 tcaagggcag ttctgaggtg attagagctt tttttaaaaa ccaagtattt
ggggatcctc 5069 agcagaggta ttcatacaga ctcccaaaga actatatatg
ttcctgagac catcgtttag 5129 tctacattgc tcttcccaga gactgacaga
tatgaccagt caaagtgcaa gactacctac 5189 ccactgccat gaaaaccatt
gcaggaaacc tttcccttcc tgaatgagat tttttttttc 5249 cctttttatg
tggggtaatt atttgtgacc caagtgtaat ttggatgatt tccattaata 5309
tcaactcttg aagcctactt gtactgattg agattgtatt tgttcctaat aaaagtggat
5369 ctggttgtac tgtc 5383 223 20 DNA Artificial Sequence Antisense
Oligonucleotide 223 agatctcttg gagcatgtgg 20 224 20 DNA Artificial
Sequence Antisense Oligonucleotide 224 cttctctcgt tcatttccgg 20 225
20 DNA Artificial Sequence Antisense Oligonucleotide 225 cttctttcat
ttcaggacgg 20 226 20 DNA Artificial Sequence Antisense
Oligonucleotide 226 tccctcctca tcctgatagg 20 227 20 DNA Artificial
Sequence Antisense Oligonucleotide 227 cctccagacg tactccagct 20 228
20 DNA Artificial Sequence Antisense Oligonucleotide 228 aggaccatga
gaatgatgtt 20 229 20 DNA Artificial Sequence Antisense
Oligonucleotide 229 acaggcctcc caagtgcagc 20 230 20 DNA Artificial
Sequence Antisense Oligonucleotide 230 ggaaccagta tgatcccgta 20 231
20 DNA Artificial Sequence Antisense Oligonucleotide 231 agtagaaaat
cccgaagagg 20 232 20 DNA Artificial Sequence Antisense
Oligonucleotide 232 cggctgtgat gcccagagcg 20 233 20 DNA Artificial
Sequence Antisense Oligonucleotide 233 gctccagagg cgatgagccc 20 234
20 DNA Artificial Sequence Antisense Oligonucleotide 234 cgagccttgt
aagttctgtg 20 235 20 DNA Artificial Sequence Antisense
Oligonucleotide 235 gcaatgatta ggaagatccg 20 236 20 DNA Artificial
Sequence Antisense Oligonucleotide 236 tacacgtcat tttggaacgc 20 237
20 DNA Artificial Sequence Antisense Oligonucleotide 237 ggtgatctcg
ggcccagtcg 20 238 20 DNA Artificial Sequence Antisense
Oligonucleotide 238 cgtgtgtttc tgagaacttg 20 239 20 DNA Artificial
Sequence Antisense Oligonucleotide 239 cggctttcag gtcagacatg 20 240
20 DNA Artificial Sequence Antisense Oligonucleotide 240 ctggaacatc
accagcttct 20 241 20 DNA Artificial Sequence Antisense
Oligonucleotide 241 agcacatcag caggaggccg 20 242 20 DNA Artificial
Sequence Antisense Oligonucleotide 242 tgaagcacat cagcaggagg 20 243
20 DNA Artificial Sequence Antisense Oligonucleotide 243 gtctcgcccc
agcagtacca 20 244 20 DNA Artificial Sequence Antisense
Oligonucleotide 244 gcaccagagt gtatcgcaag 20 245 20 DNA Artificial
Sequence Antisense Oligonucleotide 245 caccagccag gtggcgttga 20 246
20 DNA Artificial Sequence Antisense Oligonucleotide 246 tagagatgcg
cggcactgtt 20 247 20 DNA Artificial Sequence Antisense
Oligonucleotide 247 ttgaatgttc ttgtcgtagg 20 248 20 DNA Artificial
Sequence Antisense Oligonucleotide 248 cctcgcccac ggcacccagg 20 249
20 DNA Artificial Sequence Antisense Oligonucleotide 249 gtagttgtgg
aagccctcgc 20 250 20 DNA Artificial Sequence Antisense
Oligonucleotide 250 gaaggtgtgg tggtagttgt 20 251 20 DNA Artificial
Sequence Antisense Oligonucleotide 251 gcggtactca ctggcagagt 20 252
20 DNA Artificial Sequence Antisense Oligonucleotide 252 ggtgaagttg
atgtgccagc 20 253 20 DNA Artificial Sequence Antisense
Oligonucleotide 253 tcctggctaa gacagtagcc 20 254 20 DNA Artificial
Sequence Antisense Oligonucleotide 254 cccgtctcca gttctcttaa 20 255
20 DNA Artificial Sequence Antisense Oligonucleotide 255 ttgtgactcc
cgtctccagt 20 256 20 DNA Artificial Sequence Antisense
Oligonucleotide 256 tcagctactc ttgtgactcc 20 257 21 DNA Artificial
Sequence PCR Primer 257 acaccagaga catgggcaag t 21 258 22 DNA
Artificial Sequence PCR Primer 258 catcacacac tggcttcagg aa 22 259
19 DNA Artificial Sequence PCR Probe 259 ctgaagtgag gtccattag 19
260 19 DNA Artificial Sequence PCR Primer 260 gaaggtgaag gtcggagtc
19 261 20 DNA Artificial Sequence PCR Primer 261 gaagatggtg
atgggatttc 20 262 20 DNA Artificial Sequence PCR Probe 262
caagcttccc gttctcagcc 20 263 20 DNA Artificial Sequence Antisense
Oligonucleotide 263 ggaagctcac ctcttggagc 20 264 20 DNA Artificial
Sequence Antisense Oligonucleotide 264 ctgctcaccg aagagggcag 20 265
20 DNA Artificial Sequence Antisense Oligonucleotide 265 gtagtagaaa
atccctgcaa 20 266 20 DNA Artificial Sequence Antisense
Oligonucleotide 266 tcccttacct cctctggaac 20 267 20 DNA Artificial
Sequence Antisense Oligonucleotide 267 tgacttaccc acggcaccca 20 268
20 DNA Artificial Sequence Antisense Oligonucleotide 268 gtggaagccc
tcgcctgcaa 20 269 20 DNA Artificial Sequence Antisense
Oligonucleotide 269 ctggctaccg ccactcacaa 20 270 20 DNA Artificial
Sequence Antisense Oligonucleotide 270 aagcctagga ctttggtctg 20 271
20 DNA Artificial Sequence Antisense Oligonucleotide 271 gtgtgcaagc
ctaggacttt 20 272 20 DNA Artificial Sequence Antisense
Oligonucleotide 272 taggaattgt gtgcaagcct 20 273 20 DNA Artificial
Sequence Antisense Oligonucleotide 273 atctgctgtt ccctctgcct 20 274
20 DNA Artificial Sequence Antisense Oligonucleotide 274 tccagagtag
accttggagg 20 275 20 DNA Artificial Sequence Antisense
Oligonucleotide 275 ctagccaagg aagccaggcg 20 276 20 DNA Artificial
Sequence Antisense Oligonucleotide 276 gcagagatag ctagccaagg 20 277
20 DNA Artificial Sequence Antisense Oligonucleotide 277 ttttatcggc
tgccagcaaa 20 278 20 DNA Artificial Sequence Antisense
Oligonucleotide 278 ggatgaccgt gttcagtatt 20 279 20 DNA Artificial
Sequence Antisense Oligonucleotide 279 tggctgtgca cagatctcct 20 280
20 DNA Artificial Sequence Antisense Oligonucleotide 280 tcagcccggt
ctggctgtgc 20 281 20 DNA Artificial Sequence Antisense
Oligonucleotide 281 gcgcttggaa acctgccctc 20 282 20 DNA Artificial
Sequence Antisense Oligonucleotide 282 tgtaggcgag tggcggaact 20 283
20 DNA Artificial Sequence Antisense Oligonucleotide 283 gtggacttcg
gttccggagc 20 284 20 DNA Artificial Sequence Antisense
Oligonucleotide 284 ttgctcgcct cactttccca 20 285 20 DNA Artificial
Sequence Antisense Oligonucleotide 285 gtgggccggc atgatgatag 20 286
20 DNA Artificial Sequence Antisense Oligonucleotide 286 gaactggaga
tctcttggag 20 287 20 DNA Artificial Sequence Antisense
Oligonucleotide 287 tagaaaatcc cgaagagggc 20 288 20 DNA Artificial
Sequence Antisense Oligonucleotide 288 ggtcatgtag tagaaaatcc 20 289
20 DNA Artificial Sequence Antisense Oligonucleotide 289 gcgctggtca
tgtagtagaa 20 290 20 DNA Artificial Sequence Antisense
Oligonucleotide 290 ggattgaatg ttcttgtcgt 20 291 20 DNA Artificial
Sequence Antisense Oligonucleotide 291 tcccgggatt gaatgttctt 20 292
20 DNA Artificial Sequence Antisense Oligonucleotide 292 gatattctcc
cgggattgaa 20 293 20 DNA Artificial Sequence Antisense
Oligonucleotide 293 gtagccttag aaactttctt 20 294 20 DNA Artificial
Sequence Antisense Oligonucleotide 294 cccaaagctc agctactctt 20 295
20 DNA Artificial Sequence Antisense Oligonucleotide 295 aacaggaact
cagaagccca 20 296 20 DNA Artificial Sequence Antisense
Oligonucleotide 296 cagaatatta aatctctgcc 20 297 20 DNA Artificial
Sequence Antisense Oligonucleotide 297 agttgttagt taatcaacag 20 298
20 DNA Artificial Sequence Antisense Oligonucleotide 298 aattgtatat
gcatttatca 20 299 20 DNA Artificial Sequence Antisense
Oligonucleotide 299 ctgtatagaa tgttcaaatt 20 300 20 DNA Artificial
Sequence Antisense Oligonucleotide 300 acagcatgtt ccttggcttt 20 301
20 DNA Artificial Sequence Antisense Oligonucleotide 301 tagcatcaaa
acagcatgtt 20 302 20 DNA Artificial Sequence Antisense
Oligonucleotide 302 accatgctca ccctagcatc 20 303 20 DNA Artificial
Sequence Antisense Oligonucleotide 303 aaggatcagt atttcagaaa 20 304
20 DNA Artificial Sequence Antisense Oligonucleotide 304 tctctcgaga
caatctactt 20 305 20 DNA Artificial Sequence Antisense
Oligonucleotide 305 cttcagttac caaaagctaa 20 306 20 DNA Artificial
Sequence Antisense Oligonucleotide 306 aaatgtcagc tgtttagtta 20 307
20 DNA Artificial Sequence Antisense Oligonucleotide 307 ggcaacccag
gcaacacctc 20 308 20 DNA Artificial Sequence Antisense
Oligonucleotide 308 gccacgaaag aaactggcaa 20 309 20 DNA Artificial
Sequence Antisense Oligonucleotide 309 atgttcccca agggcttcat 20 310
20 DNA Artificial Sequence Antisense Oligonucleotide 310 tccctggcag
atgttcccca 20 311 20 DNA Artificial Sequence Antisense
Oligonucleotide 311 ctggctctgc ttcctgaagc 20 312 20 DNA Artificial
Sequence Antisense Oligonucleotide 312 gctgagctgt taactcacaa 20 313
20 DNA Artificial Sequence Antisense Oligonucleotide 313 cacacaccga
gacagatcaa 20 314 20 DNA Artificial Sequence Antisense
Oligonucleotide 314 caggaagcag accctcttcc 20 315 20 DNA Artificial
Sequence Antisense Oligonucleotide 315 aatactgatg tgatgttttc 20 316
20 DNA Artificial Sequence Antisense Oligonucleotide 316 atggttctaa
aatactgatg 20 317 20 DNA Artificial Sequence Antisense
Oligonucleotide 317 actgagtgtt tggcacctta 20 318 20 DNA Artificial
Sequence Antisense Oligonucleotide 318 ggctctgatt ctacaagtga 20 319
20 DNA Artificial Sequence Antisense Oligonucleotide 319 tcaacaaaac
agctcagagc 20 320 20 DNA Artificial Sequence Antisense
Oligonucleotide 320 gattttctac ttcaacaaaa 20 321 20 DNA Artificial
Sequence Antisense Oligonucleotide 321 cttaaagaca ccaggacctc 20 322
20 DNA Artificial Sequence Antisense Oligonucleotide 322 catctggaaa
ctgttataaa 20 323 20 DNA Artificial Sequence Antisense
Oligonucleotide 323 ctaagggaag gagtgagact 20 324 20 DNA Artificial
Sequence Antisense Oligonucleotide 324 ttacttccca ccaaatttga 20 325
20 DNA Artificial Sequence Antisense Oligonucleotide 325 tgacaatgat
aacgaggacg 20 326 20 DNA Artificial Sequence Antisense
Oligonucleotide 326 cagatggtgg ctttgctaac 20 327 20 DNA Artificial
Sequence Antisense Oligonucleotide 327 ttgttacaag agaaaggata 20 328
20 DNA Artificial Sequence Antisense Oligonucleotide 328 tcagatactt
agcccaggag 20 329 20 DNA Artificial Sequence Antisense
Oligonucleotide 329 tgttgagatg tgagactgtc 20 330 20 DNA Artificial
Sequence Antisense Oligonucleotide 330 cacctcagaa ctgcccttga 20 331
20 DNA Artificial Sequence Antisense Oligonucleotide 331 gctctaatca
cctcagaact 20 332 20 DNA Artificial Sequence Antisense
Oligonucleotide 332 ggagtctgta tgaatacctc 20 333 20 DNA Artificial
Sequence Antisense Oligonucleotide 333 tctctgggaa gagcaatgta 20 334
20 DNA Artificial Sequence Antisense Oligonucleotide 334 gtaggtagtc
ttgcactttg 20 335 20 DNA Artificial Sequence Antisense
Oligonucleotide 335 aggaagggaa aggtttcctg 20 336 20 DNA Artificial
Sequence Antisense Oligonucleotide 336 tacacttggg tcacaaataa 20 337
20 DNA Artificial Sequence Antisense Oligonucleotide 337 aatcatccaa
attacacttg 20 338 20 DNA Artificial Sequence Antisense
Oligonucleotide 338 cttcaagagt tgatattaat 20 339 20 DNA Artificial
Sequence Antisense Oligonucleotide 339 atacaatctc aatcagtaca 20 340
20 DNA Artificial Sequence Antisense Oligonucleotide 340 cacttttatt
aggaacaaat 20 341 18 DNA Artificial Sequence PCR Primer 341
ttccgccact cgcctaca 18 342 21 DNA Artificial Sequence PCR Primer
342 ctttcccagt gctgagatcg a 21 343 20 DNA Artificial Sequence PCR
Probe 343 caacgggctc cggaaccgaa 20 344 20 DNA Artificial Sequence
Antisense Oligonucleotide 344 gcttgcagga gggaaccagt 20 345 20 DNA
Artificial Sequence Antisense Oligonucleotide 345 ctaggacttt
ggtctggcgc 20 346 20 DNA Artificial Sequence Antisense
Oligonucleotide 346 gcgcaatctg ctgttccctc 20 347 20 DNA Artificial
Sequence Antisense Oligonucleotide 347 agggcgcgct gctccaaccc 20 348
20 DNA Artificial Sequence Antisense Oligonucleotide 348 aagaaagcca
agtagattcc 20 349 20 DNA Artificial Sequence Antisense
Oligonucleotide 349 gtgcacagat ctcctgggct 20 350 20 DNA Artificial
Sequence Antisense Oligonucleotide 350 ctgccctcct gactctcggg 20 351
20 DNA Artificial Sequence Antisense Oligonucleotide 351 agaactggag
atctcttgga 20 352 20 DNA Artificial Sequence Antisense
Oligonucleotide 352 cctcctcatc ctgataggtg 20 353 20 DNA Artificial
Sequence Antisense Oligonucleotide 353 acgtactcca gcttgggcgg 20 354
20 DNA Artificial Sequence Antisense Oligonucleotide 354 atgttcctcc
agacgtactc 20 355 20 DNA Artificial Sequence Antisense
Oligonucleotide 355 gaatgatgtt cctccagacg 20 356 20 DNA Artificial
Sequence Antisense Oligonucleotide 356 ccatgagaat gatgttcctc 20 357
20 DNA Artificial Sequence Antisense Oligonucleotide 357 tcccgtacag
gcctcccaag 20 358 20 DNA Artificial Sequence Antisense
Oligonucleotide 358 tgtagagctt gcaggaggga 20 359 20 DNA Artificial
Sequence Antisense Oligonucleotide 359 gccatggtgt tggcaatgat 20 360
20 DNA Artificial Sequence Antisense Oligonucleotide 360 tctgagaact
tgtggtgggc 20 361 20 DNA Artificial Sequence Antisense
Oligonucleotide 361 gtgtttctga gaacttgtgg 20 362 20 DNA Artificial
Sequence Antisense Oligonucleotide 362 ggcgtgtgtt tctgagaact 20 363
20 DNA Artificial Sequence Antisense Oligonucleotide 363 aattgtgagg
gtcggcgtgt 20 364 20 DNA Artificial Sequence Antisense
Oligonucleotide 364 ccacggcggg aattgtgagg 20 365 20 DNA Artificial
Sequence Antisense Oligonucleotide 365 aagaagccac ggcgggaatt 20 366
20 DNA Artificial Sequence Antisense Oligonucleotide 366 agcagccaac
ccacgtgaga 20 367 20 DNA Artificial Sequence Antisense
Oligonucleotide 367 tgcgcacaag cagccaaccc 20 368 20 DNA Artificial
Sequence Antisense Oligonucleotide 368 gtgtttgcgc acaagcagcc 20 369
20 DNA Artificial Sequence Antisense Oligonucleotide 369 acagccgggt
gtttgcgcac 20 370 20 DNA Artificial Sequence Antisense
Oligonucleotide 370 ctttgacagc cgggtgtttg 20 371 20 DNA Artificial
Sequence Antisense Oligonucleotide 371 cttctctttg acagccgggt 20 372
20 DNA Artificial Sequence Antisense Oligonucleotide 372 ccgcccttct
ctttgacagc 20 373 20 DNA Artificial Sequence Antisense
Oligonucleotide 373 atgtccagtt ttccgccctt 20 374 20 DNA Artificial
Sequence Antisense Oligonucleotide 374 cagacatgtc cagttttccg 20 375
20 DNA Artificial Sequence Antisense Oligonucleotide 375 caggtcagac
atgtccagtt 20 376 20 DNA Artificial Sequence Antisense
Oligonucleotide 376 gctttcaggt cagacatgtc 20 377 20 DNA Artificial
Sequence Antisense Oligonucleotide 377 tctcggcttt caggtcagac 20 378
20 DNA Artificial Sequence Antisense Oligonucleotide 378 cagcttctcg
gctttcaggt 20 379 20 DNA Artificial Sequence Antisense
Oligonucleotide 379 atcaccagct tctcggcttt 20 380 20 DNA Artificial
Sequence Antisense Oligonucleotide 380 ggaacatcac cagcttctcg 20 381
20 DNA Artificial Sequence Antisense Oligonucleotide 381 ctcctctgga
acatcaccag 20 382 20 DNA Artificial Sequence Antisense
Oligonucleotide 382 ttgtagtacc tcctctggaa 20 383 20 DNA Artificial
Sequence Antisense Oligonucleotide 383 aggatgaagc acatcagcag 20 384
20 DNA Artificial Sequence Antisense Oligonucleotide 384 agcgtgggca
ggatgaagca 20 385 20 DNA Artificial Sequence Antisense
Oligonucleotide 385 gtaccagggc accagcgtgg 20 386 20 DNA Artificial
Sequence Antisense Oligonucleotide 386 cagcagtacc agggcaccag 20 387
20 DNA Artificial Sequence Antisense Oligonucleotide 387 cgccccagca
gtaccagggc 20 388 20 DNA Artificial Sequence Antisense
Oligonucleotide 388 gaaggtgcta acgaacaggc 20 389 20 DNA Artificial
Sequence Antisense Oligonucleotide 389 ctgttcacca gccaggtggc 20 390
20 DNA Artificial Sequence Antisense Oligonucleotide 390 gcggcactgt
tcaccagcca 20 391 20 DNA Artificial Sequence Antisense
Oligonucleotide 391 accaggatat tctcccggga 20 392 20 DNA Artificial
Sequence Antisense Oligonucleotide 392 tggtagttgt ggaagccctc 20 393
20 DNA Artificial Sequence Antisense Oligonucleotide 393 tactcactgg
cagagtagtc 20 394 20 DNA Artificial Sequence Antisense
Oligonucleotide 394 agcggtactc actggcagag 20 395 20 DNA Artificial
Sequence Antisense Oligonucleotide 395 gtgccagcgg tactcactgg 20 396
20 DNA Artificial Sequence Antisense Oligonucleotide 396 ttgatgtgcc
agcggtactc 20 397 20 DNA Artificial Sequence Antisense
Oligonucleotide 397 gtggtgaagt tgatgtgcca 20 398 20 DNA Artificial
Sequence Antisense Oligonucleotide 398 aagaacgtgg tgaagttgat 20 399
20 DNA Artificial Sequence Antisense Oligonucleotide 399 cagtagcctt
agaaactttc 20 400 20 DNA Artificial Sequence Antisense
Oligonucleotide 400 ccagttctct taatcctggc 20 401 20 DNA Artificial
Sequence Antisense Oligonucleotide 401 ccgtctccag ttctcttaat 20 402
20 DNA Artificial Sequence Antisense Oligonucleotide 402 taacaccccg
atagcaatat 20 403 20 DNA Artificial Sequence Antisense
Oligonucleotide 403 gagggtggac agacacaggc 20 404 20 DNA Artificial
Sequence Antisense Oligonucleotide 404 cttgaagcta ggaacaagga 20 405
20 DNA Artificial Sequence Antisense Oligonucleotide 405 tatggctacc
tctctctctc 20 406 20 DNA Artificial Sequence Antisense
Oligonucleotide 406 ttttcatagt ttcacaccat 20 407 20 DNA Artificial
Sequence Antisense Oligonucleotide 407 tattttctaa gtgaaatagt 20 408
20 DNA Artificial Sequence Antisense Oligonucleotide 408 taggcagcac
taggcaggct 20 409 20 DNA Artificial Sequence Antisense
Oligonucleotide 409 aggaacaggc ctggacagca 20 410 20 DNA Artificial
Sequence Antisense Oligonucleotide 410 gagggctata ggtcagtaga 20 411
20 DNA Artificial Sequence Antisense Oligonucleotide 411 aagacaccag
gacctcaatg 20 412 20 DNA Artificial Sequence Antisense
Oligonucleotide 412 ccaatgtact gatgactctc 20 413 20 DNA Artificial
Sequence Antisense Oligonucleotide 413 tcacaccacc tcactggagc 20 414
20 DNA Artificial Sequence Antisense Oligonucleotide 414 agtaggtcag
tattaataac 20 415 20 DNA Artificial Sequence Antisense
Oligonucleotide 415 atctcattca ggaagggaaa 20 416 20 DNA Artificial
Sequence Antisense Oligonucleotide 416 aaattacact tgggtcacaa 20 417
20 DNA Artificial Sequence Antisense Oligonucleotide 417 caatctcaat
cagtacaagt 20 418 20 DNA Artificial Sequence Antisense
Oligonucleotide 418 ccttccctga aggttcctcc 20 ISPH-0590 PATENT
* * * * *