U.S. patent application number 10/394808 was filed with the patent office on 2004-09-23 for modulation of diacylglycerol acyltransferase 1 expression.
This patent application is currently assigned to Isis Pharmaceuticals Inc.. Invention is credited to Graham, Mark J., Monia, Brett P..
Application Number | 20040185559 10/394808 |
Document ID | / |
Family ID | 32988461 |
Filed Date | 2004-09-23 |
United States Patent
Application |
20040185559 |
Kind Code |
A1 |
Monia, Brett P. ; et
al. |
September 23, 2004 |
Modulation of diacylglycerol acyltransferase 1 expression
Abstract
Compounds, compositions and methods are provided for modulating
the expression of diacylglycerol acyltransferase 1. The
compositions comprise oligonucleotides, targeted to nucleic acid
encoding diacylglycerol acyltransferase 1. Methods of using these
compounds for modulation of diacylglycerol acyltransferase 1
expression and for diagnosis and treatment of disease associated
with expression of diacylglycerol acyltransferase 1 are
provided.
Inventors: |
Monia, Brett P.; (Encinitas,
CA) ; Graham, Mark J.; (San Clem, CA) |
Correspondence
Address: |
Mary E. Bak
Howson and Howson
Spring House Corporate Center
P.O. Box 457
Spring House
PA
19477
US
|
Assignee: |
Isis Pharmaceuticals Inc.
|
Family ID: |
32988461 |
Appl. No.: |
10/394808 |
Filed: |
March 21, 2003 |
Current U.S.
Class: |
435/375 ;
514/44A; 536/23.2 |
Current CPC
Class: |
C12N 2310/322 20130101;
C12Y 203/0102 20130101; C12N 15/1137 20130101; C12N 2310/315
20130101; A61K 38/00 20130101; C12N 2310/3341 20130101; C12N
2310/341 20130101; C12N 2310/346 20130101 |
Class at
Publication: |
435/375 ;
514/044; 536/023.2 |
International
Class: |
A61K 048/00; C07H
021/04 |
Claims
What is claimed is:
1. A compound 8 to 80 nucleobases in length targeted to a nucleic
acid molecule encoding diacylglycerol acyltransferase 1, wherein
said compound specifically hybridizes with said nucleic acid
molecule encoding diacylglycerol acyltransferase 1 (SEQ ID NO: 4)
and inhibits the expression of diacylglycerol acyltransferase
1.
2. The compound of claim 1 comprising 12 to 50 nucleobases in
length.
3. The compound of claim 2 comprising 15 to 30 nucleobases in
length.
4. The compound of claim 1 comprising an oligonucleotide.
5. The compound of claim 4 comprising an antisense
oligonucleotide.
6. The compound of claim 4 comprising a DNA oligonucleotide.
7. The compound of claim 4 comprising an RNA oligonucleotide.
8. The compound of claim 4 comprising a chimeric
oligonucleotide.
9. The compound of claim 4 wherein at least a portion of said
compound hybridizes with RNA to form an oligonucleotide-RNA
duplex.
10. The compound of claim 1 having at least 70% complementarity
with a nucleic acid molecule encoding diacylglycerol
acyltransferase 1 (SEQ ID NO: 4) said compound specifically
hybridizing to and inhibiting the expression of diacylglycerol
acyltransferase 1.
11. The compound of claim 1 having at least 80% complementarity
with a nucleic acid molecule encoding diacylglycerol
acyltransferase 1 (SEQ ID NO: 4) said compound specifically
hybridizing to and inhibiting the expression of diacylglycerol
acyltransferase 1.
12. The compound of claim 1 having at least 90% complementarity
with a nucleic acid molecule encoding diacylglycerol
acyltransferase 1 (SEQ ID NO: 4) said compound specifically
hybridizing to and inhibiting the expression of diacylglycerol
acyltransferase 1.
13. The compound of claim 1 having at least 95% complementarity
with a nucleic acid molecule encoding diacylglycerol
acyltransferase 1 (SEQ ID NO: 4) said compound specifically
hybridizing to and inhibiting the expression of diacylglycerol
acyltransferase 1.
14. The compound of claim 1 having at least one modified
internucleoside linkage, sugar moiety, or nucleobase.
15. The compound of claim 1 having at least one 2'-O-methoxyethyl
sugar moiety.
16. The compound of claim 1 having at least one phosphorothioate
internucleoside linkage.
17. The compound of claim 1 having at least one
5-methylcytosine.
18. A method of inhibiting the expression of diacylglycerol
acyltransferase 1 in cells or tissues comprising contacting said
cells or tissues with the compound of claim 1 so that expression of
diacylglycerol acyltransferase 1 is inhibited.
19. A method of screening for a modulator of diacylglycerol
acyltransferase 1, the method comprising the steps of: a.
contacting a preferred target segment of a nucleic acid molecule
encoding diacylglycerol acyltransferase 1 with one or more
candidate modulators of diacylglycerol acyltransferase 1, and b.
identifying one or more modulators of diacylglycerol
acyltransferase 1 expression which modulate the expression of
diacylglycerol acyltransferase 1.
20. The method of claim 19 wherein the modulator of diacylglycerol
acyltransferase 1 expression comprises an oligonucleotide, an
antisense oligonucleotide, a DNA oligonucleotide, an RNA
oligonucleotide, an RNA oligonucleotide having at least a portion
of said RNA oligonucleotide capable of hybridizing with RNA to form
an oligonucleotide-RNA duplex, or a chimeric oligonucleotide.
21. A diagnostic method for identifying a disease state comprising
identifying the presence of diacylglycerol acyltransferase 1 in a
sample using at least one of the primers comprising SEQ ID NOs 5 or
6, or the probe comprising SEQ ID NO: 7.
22. A kit or assay device comprising the compound of claim 1.
23. A method of treating an animal having a disease or condition
associated with diacylglycerol acyltransferase 1 comprising
administering to said animal a therapeutically or prophylactically
effective amount of the compound of claim 1 so that expression of
diacylglycerol acyltransferase 1 is inhibited.
24. The method of claim 23 wherein the condition involves abnormal
lipid metabolism.
25. The method of claim 23 wherein the condition involves abnormal
cholesterol metabolism.
26. The method of claim 23 wherein the condition is
atherosclerosis.
27. The method of claim 23 wherein the condition is an abnormal
metabolic condition.
28. The method of claim 27 wherein the abnormal metabolic condition
is hyperlipidemia.
29. The method of claim 23 wherein the disease is diabetes.
30. The method of claim 29 wherein the diabetes is Type 2
diabetes.
31. The method of claim 23 wherein the condition is obesity.
32. The method of claim 23 wherein the disease is cardiovascular
disease.
33. A method of modulating glucose levels in an animal comprising
administering to said animal the compound of claim 1.
34. The method of claim 33 wherein the animal is a human.
35. The method of claim 33 wherein the glucose levels are plasma
glucose levels.
36. The method of claim 33 wherein the glucose levels are serum
glucose levels.
37. The method of claim 33 wherein the animal is a diabetic
animal.
38. A method of preventing or delaying the onset of a disease or
condition associated with diacylglycerol acyltransferase 1 in an
animal comprising administering to said animal a therapeutically or
prophylactically effective amount of the compound of claim 1.
39. The method of claim 38 wherein the animal is a human.
40. The method of claim 38 wherein the condition is an abnormal
metabolic condition.
41. The method of claim 40 wherein the abnormal metabolic condition
is hyperlipidemia.
42. The method of claim 38 wherein the disease is diabetes.
43. The method of claim 42 wherein the diabetes is Type 2
diabetes.
44. The method of claim 38 wherein the condition is obesity.
45. A method of modulating cholesterol levels in an animal
comprising administering to said animal the compound of claim
1.
46. The method of claim 45 wherein the animal is a human.
47. The method of claim 45 wherein the cholesterol levels are
plasma cholesterol levels.
48. The method of claim 45 wherein the cholesterol levels are serum
cholesterol levels.
49. A method of lowering triglyceride levels in an animal
comprising administering to said animal the compound of claim
1.
50. The method of claim 49 wherein the animal is a human.
51. The method of claim 49 wherein the triglyceride levels are
plasma triglyceride levels.
52. The method of claim 49 wherein the triglyceride levels are
serum triglyceride levels.
Description
FIELD OF THE INVENTION
[0001] The present invention provides compositions and methods for
modulating the expression of diacylglycerol acyltransferase 1. In
particular, this invention relates to compounds, particularly
oligonucleotide compounds, which, in preferred embodiments,
hybridize with nucleic acid molecules encoding diacylglycerol
acyltransferase 1. Such compounds are shown herein to modulate the
expression of diacylglycerol acyltransferase 1.
BACKGROUND OF THE INVENTION
[0002] Triglycerides are one of the major energy storage molecules
in eukaryotes. The absorption of triglycerides (also called
triacylglycerols) from food is a very efficient process which
occurs by a series of steps wherein the dietary triacylglycerols
are hydrolyzed in the intestinal lumen and then resynthesized
within enterocytes. The resynthesis of triacylglycerols can occur
via the monoacylglycerol pathway which commences with
monoacylglycerol acyltransferase (MGAT) catalyzing the synthesis of
diacylglycerol from monoacylglycerol and fatty actyl-CoA. An
alternative synthesis of diacylglycerols is provided by the
glycerol-phosphate pathway which describes the coupling of two
molecules of fatty acyl-CoA to glycerol-3-phosphate. In either
case, diacylglycerol is then acylated with another molecule of
fatty acyl-CoA in a reaction catalyzed by one of two diacylglycerol
acyltransferase enzymes to form the triglyceride (Farese et al.,
Curr. Opin. Lipidol., 2000, 11, 229-234).
[0003] The reaction catalyzed by diacylglycerol acyltransferase 1
is the final and only committed step in, triglyceride synthesis. As
such, diacylglycerol acyltransferase 1 is involved in intestinal
fat absorption, lipoprotein assembly, regulating plasma
triglyceride concentrations, and fat storage in adipocytes.
Although identified in 1960, the genes encoding human and mouse
diacylglycerol acyltransferase 1 (also called DGAT1, acyl
CoA:diacylglycerol acyltransferase, acyl CoA:cholesterol
acyltransferase-related enzyme, ACAT related gene product, and
ARGP1) were not cloned until 1998 (Cases et al., Proc. Natl. Acad.
Sci. U.S.A., 1998, 95, 13018-13023; Oelkers et al., J. Biol. Chem.,
1998, 273, 26765-26771). Disclosed and claimed in U.S. Pat. No.
6,100,077 is an isolated nucleic acid encoding a human
diacylglycerol acyltransferase 1 (Sturley and Oelkers, 2000).
Diacylglycerol acyltransferase 1 is a microsomal membrane bound
enzyme and has 39% nucleotide identity to the related acyl
CoA:cholesterol acyltransferase (Oelkers et al., J. Biol. Chem.,
1998, 273, 26765-26771). A splice variant of diacylglycerol
acyltransferase 1 has also been cloned that contains a 77
nucleotide insert of unspliced intron with an in-frame stop codon,
resulting in a truncated form of diacylglycerol acyltransferase 1
that terminates at Arg-387 deleting 101 residues from the
C-terminus containing the putative active site (Cheng et al.,
Biochem. J., 2001, 359, 707-714).
[0004] Dysregulation of diacylglycerol acyltransferase 1 may play a
role in the development of obesity. Upon differentiation of mouse
3T3-L1 cells into mature adipocytes, a 90 fold increase in
diacylglycerol acyltransferase 1 protein levels is observed.
However, forced overexpression of diacylglycerol acyltransferase 1
in mature adipocytes results in only a 2 fold increase in
diacylglycerol acyltransferase 1 protein levels. This leads to an
increase in cellular triglyceride synthesis without a concomitant
increase in triglyceride lipolysis, leading to the suggestion that
manipulation of the steady state level of diacylglycerol
acyltransferase 1 may offer a potential means to treat obesity (Yu
et al., J. Biol. Chem., 2002, 277, 50876-50884).
[0005] Alterations in diacylglycerol acyltransferase 1 expression
may affect human body weight. In a random Turkish population, five
polymorphisms in the human diacylglycerol acyltransferase 1
promoter and 5' non-coding sequence have been identified. One
common variant, C79T, revealed reduced promoter activity for the
79T allele and is associated with a lower body mass index, higher
plasma cholesterol HDL levels, and lower diastolic blood pressure
in Turkish women (Ludwig et al., Clin. Genet., 2002, 62,
68-73).
[0006] Diacylglycerol acyltransferase 1 knockout mice exhibit
interesting phenotypes which indicate that inhibition of
diacylglycerol acyltransferase 1 may offer a strategy for treating
obesity and obesity-associated insulin resistance. Mice lacking
diacylglycerol acyltransferase 1 are viable and can still
synthesize triglycerides through other biological routes. However
the mice are lean and resistant to diet-induce obesity (Smith et
al., Nat. Genet., 2000, 25, 87-90), have decreased levels of tissue
triglycerides, and increased sensitivity to insulin and leptin
(Chen et al., J. Clin. Invest., 2002, 109, 1049-1055).
[0007] Currently, there are no known therapeutic agents which
effectively inhibit the synthesis of diacylglycerol acyltransferase
1 and to date, investigative strategies aimed at modulating
diacylglycerol acyltransferase 1 function have involved natural
occurring small molecule derivatives of roselipins and xanthohumols
isolated from Gliocladium roseum and Humulus lupulus, respectively
(Tabata et al., Phytochemistry, 1997, 46, 683-687; Tomoda et al.,
J. Antibiot. (Tokyo)., 1999, 52, 689-694).
[0008] Consequently, there remains a long felt need for additional
agents capable of effectively inhibiting diacylglycerol
acyltransferase 1 function.
[0009] Antisense technology is emerging as an effective means for
reducing the expression of specific gene products and may therefore
prove to be uniquely useful in a number of therapeutic, diagnostic,
and research applications for the modulation of diacylglycerol
acyltransferase 1 expression.
[0010] The present invention provides compositions and methods for
modulating diacylglycerol acyltransferase 1 expression.
SUMMARY OF THE INVENTION
[0011] The present invention is directed to compounds, especially
nucleic acid and nucleic acid-like oligomers, which are targeted to
a nucleic acid encoding diacylglycerol acyltransferase 1, and which
modulate the expression of diacylglycerol acyltransferase 1.
Pharmaceutical and other compositions comprising the compounds of
the invention are also provided. Further provided are methods of
screening for modulators of diacylglycerol acyltransferase 1 and
methods of modulating the expression of diacylglycerol
acyltransferase 1 in cells, tissues or animals comprising
contacting said cells, tissues or animals with one or more of the
compounds or compositions of the invention. Methods of treating an
animal, particularly a human, suspected of having or being prone to
a disease or condition associated with expression of diacylglycerol
acyltransferase 1 are also set forth herein. Such methods comprise
administering a therapeutically or prophylactically effective
amount of one or more of the compounds or compositions of the
invention to the person in need of treatment.
DETAILED DESCRIPTION OF THE INVENTION
[0012] A. Overview of the Invention
[0013] The present invention employs compounds, preferably
oligonucleotides and similar species for use in modulating the
function or effect of nucleic acid molecules encoding
diacylglycerol acyltransferase 1. This is accomplished by providing
oligonucleotides which specifically hybridize with one or more
nucleic acid molecules encoding diacylglycerol acyltransferase 1.
As used herein, the terms "target nucleic acid" and "nucleic acid
molecule encoding diacylglycerol acyltransferase 1" have been used
for convenience to encompass DNA encoding diacylglycerol
acyltransferase 1, RNA (including pre-mRNA and mRNA or portions
thereof) transcribed from such DNA, and also cDNA derived from such
RNA. The hybridization of a compound of this invention with its
target nucleic acid is generally referred to as "antisense".
Consequently, the preferred mechanism believed to be included in
the practice of some preferred embodiments of the invention is
referred to herein as "antisense inhibition." Such antisense
inhibition is typically based upon hydrogen bonding-based
hybridization of oligonucleotide strands or segments such that at
least one strand or segment is cleaved, degraded, or otherwise
rendered inoperable. In this regard, it is presently preferred to
target specific nucleic acid molecules and their functions for such
antisense inhibition.
[0014] The functions of DNA to be interfered with can include
replication and transcription. Replication and transcription, for
example, can be from an endogenous cellular template, a vector, a
plasmid construct or otherwise. The functions of RNA to be
interfered with can include functions such as translocation of the
RNA to a site of protein translation, translocation of the RNA to
sites within the cell which are distant from the site of RNA
synthesis, translation of protein from the RNA, splicing of the RNA
to yield one or more RNA species, and catalytic activity or complex
formation involving the RNA which may be engaged in or facilitated
by the RNA. One preferred result of such interference with target
nucleic acid function is modulation of the expression of
diacylglycerol acyltransferase 1. In the context of the present
invention, "modulation" and "modulation of expression" mean either
an increase (stimulation) or a decrease (inhibition) in the amount
or levels of a nucleic acid molecule encoding the gene, e.g., DNA
or RNA. Inhibition is often the preferred form of modulation of
expression and mRNA is often a preferred target nucleic acid.
[0015] In the context of this invention, "hybridization" means the
pairing of complementary strands of oligomeric compounds. In the
present invention, the preferred mechanism of pairing involves
hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed
Hoogsteen hydrogen bonding, between complementary nucleoside or
nucleotide bases (nucleobases) of the strands of oligomeric
compounds. For example, adenine and thymine are complementary
nucleobases which pair through the formation of hydrogen bonds.
Hybridization can occur under varying circumstances.
[0016] An antisense compound is specifically hybridizable when
binding of the compound to the target nucleic acid interferes with
the normal function of the target nucleic acid to cause a loss of
activity, and there is a sufficient degree of complementarity to
avoid non-specific binding of the antisense compound to non-target
nucleic acid sequences under conditions in which specific binding
is desired, i.e., under physiological conditions in the case of in
vivo assays or therapeutic treatment, and under conditions in which
assays are performed in the case of in vitro assays.
[0017] In the present invention the phrase "stringent hybridization
conditions" or "stringent conditions" refers to conditions under
which a compound of the invention will hybridize to its target
sequence, but to a minimal number of other sequences. Stringent
conditions are sequence-dependent and will be different in
different circumstances and in the context of this invention,
"stringent conditions" under which oligomeric compounds hybridize
to a target sequence are determined by the nature and composition
of the oligomeric compounds and the assays in which they are being
investigated.
[0018] "Complementary," as used herein, refers to the capacity for
precise pairing between two nucleobases of an oligomeric compound.
For example, if a nucleobase at a certain position of an
oligonucleotide (an oligomeric compound), is capable of hydrogen
bonding with a nucleobase at a certain position of a target nucleic
acid, said target nucleic acid being a DNA, RNA, or oligonucleotide
molecule, then the position of hydrogen bonding between the
oligonucleotide and the target nucleic acid is considered to be a
complementary position. The oligonucleotide and the further DNA,
RNA, or oligonucleotide molecule are complementary to each other
when a sufficient number of complementary positions in each
molecule are occupied by nucleobases which can hydrogen bond with
each other. Thus, "specifically hybridizable" and "complementary"
are terms which are used to indicate a sufficient degree of precise
pairing or complementarity over a sufficient number of nucleobases
such that stable and specific binding occurs between the
oligonucleotide and a target nucleic acid.
[0019] It is understood in the art that the sequence of an
antisense compound need not be 100% complementary to that of its
target nucleic acid to be specifically hybridizable. Moreover, an
oligonucleotide may hybridize over one or more segments such that
intervening or adjacent segments are not involved in the
hybridization event (e.g., a loop structure or hairpin structure).
It is preferred that the antisense compounds of the present
invention comprise at least 70% sequence complementarity to a
target region within the target nucleic acid, more preferably that
they comprise 90% sequence complementarity and even more preferably
comprise 95% sequence complementarity to the target region within
the target nucleic acid sequence to which they are targeted. For
example, an antisense compound in which 18 of 20 nucleobases of the
antisense compound are complementary to a target region, and would
therefore specifically hybridize, would represent 90 percent
complementarity. In this example, the remaining noncomplementary
nucleobases may be clustered or interspersed with complementary
nucleobases and need not be contiguous to each other or to
complementary nucleobases. As such, an antisense compound which is
18 nucleobases in length having 4 (four) noncomplementary
nucleobases which are flanked by two regions of complete
complementarity with the target nucleic acid would have 77.8%
overall complementarity with the target nucleic acid and would thus
fall within the scope of the present invention. Percent
complementarity of an antisense compound with a region of a target
nucleic acid can be determined routinely using BLAST programs
(basic local alignment search tools) and PowerBLAST programs known
in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410;
Zhang and Madden, Genome Res., 1997, 7, 649-656).
[0020] B. Compounds of the Invention
[0021] According to the present invention, compounds include
antisense oligomeric compounds, antisense oligonucleotides,
ribozymes, external guide sequence (EGS) oligonucleotides,
alternate splicers, primers, probes, and other oligomeric compounds
which hybridize to at least a portion of the target nucleic acid.
As such, these compounds may be introduced in the form of
single-stranded, double-stranded, circular or hairpin oligomeric
compounds and may contain structural elements such as internal or
terminal bulges or loops. Once introduced to a system, the
compounds of the invention may elicit the action of one or more
enzymes or structural proteins to effect modification of the target
nucleic acid. One non-limiting example of such an enzyme is RNAse
H, a cellular endonuclease which cleaves the RNA strand of an
RNA:DNA duplex. It is known in the art that single-stranded
antisense compounds which are "DNA-like" elicit RNAse H. Activation
of RNase H, therefore, results in cleavage of the RNA target,
thereby greatly enhancing the efficiency of
oligonucleotide-mediated inhibition of gene expression. Similar
roles have been postulated for other ribonucleases such as those in
the RNase III and ribonuclease L family of enzymes.
[0022] While the preferred form of antisense compound is a
single-stranded antisense oligonucleotide, in many species the
introduction of double-stranded structures, such as double-stranded
RNA (dsRNA) molecules, has been shown to induce potent and specific
antisense-mediated reduction of the function of a gene or its
associated gene products. This phenomenon occurs in both plants and
animals and is believed to have an evolutionary connection to viral
defense and transposon silencing.
[0023] The first evidence that dsRNA could lead to gene silencing
in animals came in 1995 from work in the nematode, Caenorhabditis
elegans (Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et
al. have shown that the primary interference effects of dsRNA are
posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA,
1998, 95, 15502-15507). The posttranscriptional antisense mechanism
defined in Caenorhabditis elegans resulting from exposure to
double-stranded RNA (dsRNA) has since been designated RNA
interference (RNAi). This term has been generalized to mean
antisense-mediated gene silencing involving the introduction of
dsRNA leading to the sequence-specific reduction of endogenous
targeted mRNA levels (Fire et al., Nature, 1998, 391, 806-811).
Recently, it has been shown that it is, in fact, the
single-stranded RNA oligomers of antisense polarity of the dsRNAs
which are the potent inducers of RNAi (Tijsterman et al., Science,
2002, 295, 694-697).
[0024] In the context of this invention, the term "oligomeric
compound" refers to a polymer or oligomer comprising a plurality of
monomeric units. 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, chimeras,
analogs and homologs 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 a target
nucleic acid and increased stability in the presence of
nucleases.
[0025] While oligonucleotides are a preferred form of the compounds
of this invention, the present invention comprehends other families
of compounds as well, including but not limited to oligonucleotide
analogs and mimetics such as those described herein.
[0026] The compounds in accordance with this invention preferably
comprise from about 8 to about 80 nucleobases (i.e. from about 8 to
about 80 linked nucleosides). One of ordinary skill in the art will
appreciate that-the invention embodies compounds of 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,
or 80 nucleobases in length.
[0027] In one preferred embodiment, the compounds of the invention
are 12 to 50 nucleobases in length. One having ordinary skill in
the art will appreciate that this embodies compounds of 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, or 50 nucleobases in length.
[0028] In another preferred embodiment, the compounds of the
invention are 15 to 30 nucleobases in length. One having ordinary
skill in the art will appreciate that this embodies compounds of
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30
nucleobases in length.
[0029] Particularly preferred compounds are oligonucleotides from
about 12 to about 50 nucleobases, even more preferably those
comprising from about 15 to about 30 nucleobases.
[0030] Antisense compounds 8-80 nucleobases in length comprising a
stretch of at least eight (8) consecutive nucleobases selected from
within the illustrative antisense compounds are considered to be
suitable antisense compounds as well.
[0031] Exemplary preferred antisense compounds include
oligonucleotide sequences that comprise at least the 8' consecutive
nucleobases from the 5'-terminus of one of the illustrative
preferred antisense compounds (the remaining nucleobases being a
consecutive stretch of the same oligonucleotide beginning
immediately upstream of the 5'-terminus of the antisense compound
which is specifically hybridizable to the target nucleic acid and
continuing until the oligonucleotide contains about 8 to about 80
nucleobases). Similarly preferred antisense compounds are
represented by oligonucleotide sequences that comprise at least the
8 consecutive nucleobases from the 3'-terminus of one of the
illustrative preferred antisense compounds (the remaining
nucleobases being a consecutive stretch of the same oligonucleotide
beginning immediately downstream of the 3'-terminus of the
antisense compound which is specifically hybridizable to the target
nucleic acid and continuing until the oligonucleotide contains
about 8 to about 80 nucleobases). One having skill in the art armed
with the preferred antisense compounds illustrated herein will be
able, without undue experimentation, to identify further preferred
antisense compounds.
[0032] C. Targets of the Invention
[0033] "Targeting" an antisense compound to a particular nucleic
acid molecule, in the context of this invention, can be a multistep
process. The process usually begins with the identification of a
target nucleic acid whose function is to be modulated. This target
nucleic acid may be, for example, a cellular gene (or mRNA
transcribed from the gene) whose expression is associated with a
particular disorder or disease state, or a nucleic acid molecule
from an infectious agent. In the present invention, the target
nucleic acid encodes diacylglycerol acyltransferase 1.
[0034] The targeting process usually also includes determination of
at least one target region, segment, or site within the target
nucleic acid for the antisense interaction to occur such that the
desired effect, e.g., modulation of expression, will result. Within
the context of the present invention, the term "region" is defined
as a portion of the target nucleic acid having at least one
identifiable structure, function, or characteristic. Within regions
of target nucleic acids are segments. "Segments" are defined as
smaller or sub-portions of regions within a target nucleic acid.
"Sites," as used in the present invention, are defined as positions
within a target nucleic acid.
[0035] 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 have 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 transcribed from a gene encoding
diacylglycerol acyltransferase 1, regardless of the sequence(s) of
such codons. 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).
[0036] 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. Consequently, the "start codon region" (or
"translation initiation codon region") and the "stop codon region"
(or "translation termination codon region") are all regions which
may be-targeted effectively with the antisense compounds of the
present invention.
[0037] The open reading frame (ORF) or "coding region," which is
known in the art to refer to the region between the translation
initiation codon and the translation termination codon, is also a
region which may be targeted effectively. Within the context of the
present invention, a preferred region is the intragenic region
encompassing the translation initiation or termination codon of the
open reading frame (ORF) of a gene.
[0038] Other target regions include the 5' untranslated region
(5'UTR), known in the art to refer to the portion of an mRNA in the
5' direction from the translation initiation codon, and thus
including nucleotides between the 5' cap site and the translation
initiation codon of an mRNA (or corresponding nucleotides on the
gene), and the 3' untranslated region (3'UTR), known in the art to
refer to the portion of an mRNA in the 3' direction from the
translation termination codon, and thus including nucleotides
between the translation termination codon and 3' end of an mRNA (or
corresponding nucleotides on the gene). The 5' cap site 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
site. It is also preferred to target the 5' cap region.
[0039] 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.
Targeting splice sites, i.e., intron-exon junctions or exon-intron
junctions, may also be particularly useful in situations where
aberrant splicing is implicated in disease, or where an
overproduction of a particular splice product is implicated in
disease. Aberrant fusion junctions due to rearrangements or
deletions are also preferred target sites. mRNA transcripts
produced via the process of splicing of two (or more) mRNAs from
different gene sources are known as "fusion transcripts". It is
also known that introns can be effectively targeted using antisense
compounds targeted to, for example, DNA or pre-mRNA.
[0040] It is also known in the art that alternative RNA transcripts
can be produced from the same genomic region of DNA. These
alternative transcripts are generally known as "variants". More
specifically, "pre-mRNA variants" are transcripts produced from the
same genomic DNA that differ from other transcripts produced from
the same genomic DNA in either their start or stop position and
contain both intronic and exonic sequence.
[0041] Upon excision of one or more exon or intron regions, or
portions thereof during splicing, pre-mRNA variants produce smaller
"mRNA variants". Consequently, mRNA variants are processed pre-mRNA
variants and each unique pre-mRNA variant must always produce a
unique mRNA variant as a result of splicing. These mRNA variants
are also known as "alternative splice variants". If no splicing of
the pre-mRNA variant occurs then the pre-mRNA variant is identical
to the mRNA variant.
[0042] It is also known in the art that variants can be produced
through the use of alternative signals to start or stop
transcription and that pre-mRNAs and mRNAs can possess more that
one start codon or stop codon. Variants that originate from a
pre-mRNA or mRNA that use alternative start codons are known as
"alternative start variants" of that pre-mRNA or mRNA. Those
transcripts that use an alternative stop codon are known as
"alternative stop variants" of that pre-mRNA or mRNA. One specific
type of alternative stop variant is the "polyA variant" in which
the multiple transcripts produced result from the alternative
selection of one of the "polyA stop signals" by the transcription
machinery, thereby producing transcripts that terminate at unique
polyA sites. Within the context of the invention, the types of
variants described herein are also preferred target nucleic
acids.
[0043] The locations on the target nucleic acid to which the
preferred antisense compounds hybridize are hereinbelow referred to
as "preferred target segments." As used herein the term "preferred
target segment" is defined as at least an 8-nucleobase portion of a
target region to which an active antisense compound is targeted.
While not wishing to be bound by theory, it is presently believed
that these target segments represent portions of the target nucleic
acid which are accessible for hybridization.
[0044] While the specific sequences of certain preferred target
segments are set forth herein, one of skill in the art will
recognize that these serve to illustrate and describe particular
embodiments within the scope of the present invention. Additional
preferred target segments may be identified by one having ordinary
skill.
[0045] Target segments 8-80 nucleobases in length comprising a
stretch of at least eight (8) consecutive nucleobases selected from
within the illustrative preferred target segments are considered to
be suitable for targeting as well.
[0046] Target segments can include DNA or RNA sequences that
comprise at least the 8 consecutive nucleobases from the
5'-terminus of one of the illustrative preferred target segments
(the remaining nucleobases being a consecutive stretch of the same
DNA or RNA beginning immediately upstream of the 5'-terminus of the
target segment and continuing until the DNA or RNA contains about 8
to about 80 nucleobases). Similarly preferred target segments are
represented by DNA or RNA sequences that comprise at least the 8
consecutive nucleobases from the 3'-terminus of one of the
illustrative preferred target segments (the remaining nucleobases
being a consecutive stretch of the same DNA or RNA beginning
immediately downstream of the 3'-terminus of the target segment and
continuing until the DNA or RNA contains about 8 to about 80
nucleobases). One having skill in the art armed with the preferred
target segments illustrated herein will be able, without undue
experimentation, to identify further preferred target segments.
[0047] Once one or more target regions, segments or sites have been
identified, antisense compounds are chosen which are sufficiently
complementary to the target, i.e., hybridize sufficiently well and
with sufficient specificity, to give the desired effect.
[0048] D. Screening and Target Validation
[0049] In a further embodiment, the "preferred target segments"
identified herein may be employed in a screen for additional
compounds that modulate the expression of diacylglycerol
acyltransferase 1. "Modulators" are those compounds that decrease
or increase the expression of a nucleic acid molecule encoding
diacylglycerol acyltransferase 1 and which comprise at least an
8-nucleobase portion which is complementary to a preferred target
segment. The screening method comprises the steps of contacting a
preferred target segment of a nucleic acid molecule encoding
diacylglycerol acyltransferase 1 with one or more candidate
modulators, and selecting for one or more candidate modulators
which decrease or increase the expression of a nucleic acid
molecule encoding diacylglycerol acyltransferase 1. Once it is
shown that the candidate modulator or modulators are capable of
modulating (e.g. either decreasing or increasing) the expression of
a nucleic acid molecule encoding diacylglycerol acyltransferase 1,
the modulator may then be employed in further investigative studies
of the function of diacylglycerol acyltransferase 1, or for use as
a research, diagnostic, or therapeutic agent in accordance with the
present invention.
[0050] The preferred target segments of the present invention may
be also be combined with their respective complementary antisense
compounds of the present invention to form stabilized
double-stranded (duplexed) oligonucleotides.
[0051] Such double stranded oligonucleotide moieties have been
shown in the art to modulate target expression and regulate
translation as well as RNA processsing via an antisense mechanism.
Moreover, the double-stranded moieties may be subject to chemical
modifications (Fire et al., Nature, 1998, 391, 806-811; Timmons and
Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263,
103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et
al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et
al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature,
2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200).
For example, such double-stranded moieties have been shown to
inhibit the target by the classical hybridization of antisense
strand of the duplex to the target, thereby triggering enzymatic
degradation of the target (Tijsterman et al., Science, 2002, 295,
694-697).
[0052] The compounds of the present invention can also be applied
in the areas of drug discovery and target validation. The present
invention comprehends the use of the compounds and preferred target
segments identified herein in drug discovery efforts to elucidate
relationships that exist between diacylglycerol acyltransferase 1
and a disease state, phenotype, or condition. These methods include
detecting or modulating diacylglycerol acyltransferase 1 comprising
contacting a sample, tissue, cell, or organism with the compounds
of the present invention, measuring the nucleic acid or protein
level of diacylglycerol acyltransferase 1 and/or a related
phenotypic or chemical endpoint at some time after treatment, and
optionally comparing the measured value to a non-treated sample or
sample treated with a further compound of the invention. These
methods can also be performed in parallel or in combination with
other experiments to determine the function of unknown genes for
the process of target validation or to determine the validity of a
particular gene product as a target for treatment or prevention of
a particular disease, condition, or phenotype.
[0053] E. Kits, Research Reagents, Diagnostics, and
Therapeutics
[0054] The compounds of the present invention can be utilized for
diagnostics, therapeutics, prophylaxis and as research reagents and
kits. Furthermore, antisense oligonucleotides, which are able to
inhibit gene expression with exquisite specificity, are often used
by those of ordinary skill to elucidate the function of particular
genes or to distinguish between functions of various members of a
biological pathway.
[0055] For use in kits and diagnostics, the compounds of the
present invention, either alone or in combination with other
compounds or therapeutics, can be used as tools in differential
and/or combinatorial analyses to elucidate expression patterns of a
portion or the entire complement of genes expressed within cells
and tissues.
[0056] As one nonlimiting example, expression patterns within cells
or tissues treated with one or more antisense compounds are
compared to control cells or tissues not treated with antisense
compounds and the patterns produced are analyzed for differential
levels of gene expression as they pertain, for example, to disease
association, signaling pathway, cellular localization, expression
level, size, structure or function of the genes examined. These
analyses can be performed on stimulated or unstimulated cells and
in the presence or absence of other compounds which affect
expression patterns.
[0057] Examples of methods of gene expression analysis known in the
art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett.,
2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE
(serial analysis of gene expression)(Madden, et al., Drug Discov.
Today, 2000, 5, 415-425), READS (restriction enzyme amplification
of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999,
303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et
al., Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 1976-81), protein
arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16;
Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed
sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000,
480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57),
subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.
Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,
203-208), subtractive cloning, differential display (DD) (Jurecic
and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative
genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl.,
1998, 31, 286-96), FISH (fluorescent in situ hybridization)
techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35,
1895-904) and mass spectrometry methods (To, Comb. Chem. High
Throughput Screen, 2000, 3, 235-41).
[0058] The compounds of the invention are useful for research and
diagnostics, because these compounds hybridize to nucleic acids
encoding diacylglycerol acyltransferase 1. For example,
oligonucleotides that are shown to hybridize with such efficiency
and under such conditions as disclosed herein as to be effective
diacylglycerol acyltransferase 1 inhibitors will also be effective
primers or probes under conditions favoring gene amplification or
detection, respectively. These primers and probes are useful in
methods requiring the specific detection of nucleic acid molecules
encoding diacylglycerol acyltransferase 1 and in the amplification
of said nucleic acid molecules for detection or for use in further
studies of diacylglycerol acyltransferase 1. Hybridization of the
antisense oligonucleotides, particularly the primers and probes, of
the invention with a nucleic acid encoding diacylglycerol
acyltransferase 1 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 diacylglycerol acyltransferase 1 in a sample may also be
prepared.
[0059] The specificity and sensitivity of antisense is also
harnessed by those of skill in the art for therapeutic uses.
Antisense compounds have been employed as therapeutic moieties in
the treatment of disease states in animals, including humans.
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
antisense compounds can be useful therapeutic modalities that can
be configured to be useful in treatment regimes for the treatment
of cells, tissues and animals, especially humans.
[0060] For therapeutics, an animal, preferably a human, suspected
of having a disease or disorder which can be treated by modulating
the expression of diacylglycerol acyltransferase 1 is treated by
administering antisense compounds in accordance with this
invention. For example, in one non-limiting embodiment, the methods
comprise the step of administering to the animal in need of
treatment, a therapeutically effective amount of a diacylglycerol
acyltransferase 1 inhibitor. The diacylglycerol acyltransferase 1
inhibitors of the present invention effectively inhibit the
activity of the diacylglycerol acyltransferase 1 protein or inhibit
the expression of the diacylglycerol acyltransferase 1 protein. In
one embodiment, the activity or expression of diacylglycerol
acyltransferase 1 in an animal is inhibited by about 10%.
Preferably, the activity or expression of diacylglycerol
acyltransferase 1 in an animal is inhibited by about 30%. More
preferably, the activity or expression of diacylglycerol
acyltransferase 1 in an animal is inhibited by 50% or more.
[0061] For example, the reduction of the expression of
diacylglycerol acyltransferase 1 may be measured in serum, adipose
tissue, liver or any other body fluid, tissue or organ of the
animal. Preferably, the cells contained within said fluids, tissues
or organs being analyzed contain a nucleic acid molecule encoding
diacylglycerol acyltransferase 1 protein and/or the diacylglycerol
acyltransferase 1 protein itself.
[0062] The compounds of the invention can be utilized in
pharmaceutical compositions by adding an effective amount of a
compound to a suitable pharmaceutically acceptable diluent or
carrier. Use of the compounds and methods of the invention may also
be useful prophylactically.
[0063] F. Modifications
[0064] 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 compound can be further joined to form a circular
compound, however, linear compounds are generally preferred. In
addition, linear compounds may have internal nucleobase
complementarity and may therefore fold in a manner as to produce a
fully or partially double-stranded compound. Within
oligonucleotides, 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.
[0065] Modified Internucleoside Linkages (Backbones)
[0066] 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.
[0067] Preferred modified oligonucleotide backbones containing a
phosphorus atom therein 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,
thionoalkylphosphotriesters, selenophosphates and boranophosphates
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 which 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] Modified Sugar and Internucleoside Linkages--Mimetics
[0072] 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 nucleobase
units are maintained for hybridization with an appropriate target
nucleic acid. One such 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.
[0073] 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.
[0074] Modified Sugars
[0075] 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)CH.sub.3].s- ub.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, polyalkylamino, substituted silyl, an RNA cleaving
group, a reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. A preferred
modification includes 2'-methoxyethoxy
(2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta,
1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred
modification includes 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples hereinbelow, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.3).sub.2, also described in
examples hereinbelow.
[0076] 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.
[0077] A further preferred modification of the sugar 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
methylene (--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 WO 98/39352 and WO 99/14226.
[0078] Natural and Modified Nucleobases
[0079] 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 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. and
are presently preferred base substitutions, even more particularly
when combined with 2'-O-methoxyethyl sugar modifications.
[0080] 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.
[0081] Conjugates
[0082] Another modification of the oligonucleotides of the
invention involves chemically linking to the oligonucleotide one or
more moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. These
moieties or conjugates 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 conjugate 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 uptake,
enhance resistance to degradation, and/or strengthen
sequence-specific hybridization with the target nucleic acid.
Groups that enhance the pharmacokinetic properties, in the context
of this invention, include groups that improve uptake,
distribution, metabolism or excretion of the compounds of the
present invention. Representative conjugate groups are disclosed in
International Patent Application PCT/US92/09196, filed Oct. 23,
1992, and U.S. Pat. No. 6,287,860, the entire disclosure of which
are incorporated herein by reference. Conjugate moieties include
but are not limited to lipid moieties such as a cholesterol moiety,
cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a
thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl
residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or
triethylammonium 1,2-di-O-hexadecyl-rac-glyc- ero-3-H-phosphonate,
a polyamine or a polyethylene glycol chain, or adamantane acetic
acid, a palmityl moiety, or an octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety. 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.
[0083] 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.
[0084] Chimeric Compounds
[0085] 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.
[0086] The present invention also includes antisense compounds
which are chimeric compounds. "Chimeric" antisense compounds or
"chimeras," in the context of this invention, are antisense
compounds, particularly oligonucleotides, which contain two or more
chemically distinct regions, each made up of at least one monomer
unit, i.e., a nucleotide in the case of an oligonucleotide
compound. These oligonucleotides typically contain at least one
region wherein the oligonucleotide is modified so as to confer upon
the oligonucleotide increased resistance to nuclease degradation,
increased cellular uptake, increased stability 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-mediated inhibition of gene
expression. The cleavage of RNA:RNA hybrids can, in like fashion,
be accomplished through the actions of endoribonucleases, such as
RNAseL which cleaves both cellular and viral RNA. Cleavage of the
RNA target can be routinely detected by gel electrophoresis and, if
necessary, associated nucleic acid hybridization techniques known
in the art.
[0087] 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.
[0088] G. Formulations
[0089] 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.
[0090] 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.
[0091] The term "prodrug" indicates a therapeutic agent that is
prepared in an inactive form that is converted to an active form
(i.e., drug) within the body or cells thereof by the action of
endogenous enzymes or other chemicals and/or conditions. In
particular, prodrug versions of the oligonucleotides of the
invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate]
derivatives according to the methods disclosed in WO 93/24510 to
Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S.
Pat. No. 5,770,713 to Imbach et al.
[0092] 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. For oligonucleotides,
preferred examples of pharmaceutically acceptable salts and their
uses are further described in U.S. Pat. No. 6,287,860, which is
incorporated herein in its entirety.
[0093] The present invention also includes pharmaceutical
compositions and formulations which include the antisense compounds
of the invention. The pharmaceutical compositions of the present
invention may be administered in a number of ways depending upon
whether local or systemic treatment is desired and upon the area to
be treated. Administration may be topical (including ophthalmic and
to mucous membranes including vaginal and rectal delivery),
pulmonary, e.g., by inhalation or insufflation of powders or
aerosols, including by nebulizer; intratracheal, intranasal,
epidermal and transdermal), oral or parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal or intramuscular injection or infusion; or
intracranial, e.g., intrathecal or intraventricular,
administration. Oligonucleotides with at least one
2'-O-methoxyethyl modification are believed to be particularly
useful for oral administration. 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.
[0094] 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.
[0095] 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 which increase the viscosity of the suspension
including, for example, sodium carboxymethylcellulose, sorbitol
and/or dextran. The suspension may also contain stabilizers.
[0096] Pharmaceutical compositions of the present invention
include, but are not limited to, solutions, emulsions, foams and
liposome-containing formulations. The pharmaceutical compositions
and formulations of the present invention may comprise one or more
penetration enhancers, carriers, excipients or other active or
inactive ingredients.
[0097] Emulsions are typically heterogenous systems of one liquid
dispersed in another in the form of droplets usually exceeding 0.1
.mu.m in diameter. 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. Microemulsions are included as an
embodiment of the present invention. Emulsions and their uses are
well known in the art and are further described in U.S. Pat. No.
6,287,860, which is incorporated herein in its entirety.
[0098] Formulations of the present invention include liposomal
formulations. As used in the present invention, the term "liposome"
means a vesicle composed of amphiphilic lipids arranged in a
spherical bilayer or bilayers. Liposomes are unilamellar or
multilamellar vesicles which have a membrane formed from a
lipophilic material and an aqueous interior that contains the
composition to be delivered. Cationic liposomes are positively
charged liposomes which are believed to interact with negatively
charged DNA molecules to form a stable complex. Liposomes that are
pH-sensitive or negatively-charged are believed to entrap DNA
rather than complex with it. Both cationic and noncationic
liposomes have been used to deliver DNA to cells.
[0099] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids that, when incorporated into liposomes,
result in enhanced circulation lifetimes relative to liposomes
lacking such specialized lipids. Examples of sterically stabilized
liposomes are those in which part of the vesicle-forming lipid
portion of the liposome comprises one or more glycolipids or is
derivatized with one or more hydrophilic polymers, such as a
polyethylene glycol (PEG) moiety. Liposomes and their uses are
further described in U.S. Pat. No. 6,287,860, which is incorporated
herein in its entirety.
[0100] The pharmaceutical formulations and compositions of the
present invention may also include surfactants. The use of
surfactants in drug products, formulations and in emulsions is well
known in the art. Surfactants and their uses are further described
in U.S. Pat. No. 6,287,860, which is incorporated herein in its
entirety.
[0101] In one embodiment, the present invention employs various
penetration enhancers to effect the efficient delivery of nucleic
acids, particularly oligonucleotides. In addition to aiding the
diffusion of non-lipophilic drugs across cell membranes,
penetration enhancers also enhance the permeability of lipophilic
drugs. 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.
Penetration enhancers and their uses are further described in U.S.
Pat. No. 6,287,860, which is incorporated herein in its
entirety.
[0102] One of skill in the art will recognize that formulations are
routinely designed according to their intended use, i.e. route of
administration.
[0103] Preferred formulations for topical administration 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).
[0104] For topical or other administration, 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,
pharmaceutically acceptable salts thereof, and their uses are
further described in U.S. Pat. No. 6,287,860, which is incorporated
herein in its entirety. 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.
[0105] 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 and fatty acids and their uses are further described in
U.S. Pat. No. 6,287,860, which is incorporated herein in its
entirety. 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 and their uses are further
described in U.S. Pat. No. 6,287,860, which is incorporated herein
in its entirety. Oral formulations for oligonucleotides and their
preparation are described in detail in U.S. application Ser. Nos.
09/108,673 (filed Jul. 1, 1998), 09/315,298 (filed May 20, 1999)
and 10/071,822, filed Feb. 8, 2002, each of which is incorporated
herein by reference in their entirety.
[0106] 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.
[0107] Certain embodiments of the invention provide pharmaceutical
compositions containing one or more oligomeric compounds and one or
more other chemotherapeutic agents which function by a
non-antisense mechanism. Examples of such chemotherapeutic agents
include but are not limited to cancer chemotherapeutic drugs such
as 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-hydroxyperoxycyclophosphoramide, 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). 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. Combinations of
antisense compounds and other non-antisense drugs are also within
the scope of this invention. Two or more combined compounds may be
used together or sequentially.
[0108] 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. Alternatively, compositions of the invention may contain
two or more antisense compounds targeted to different regions of
the same nucleic acid target. Numerous examples of antisense
compounds are known in the art. Two or more combined compounds may
be used together or sequentially.
[0109] H. Dosing
[0110] The formulation of therapeutic compositions and their
subsequent administration (dosing) 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 ug to 100 g per kg of body
weight, once or more daily, to once every 20 years.
[0111] 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
Synthesis of Nucleoside Phosphoramidites
[0112] The following compounds, including amidites and their
intermediates were prepared as described in U.S. Pat. No. 6,426,220
and published PCT WO 02/36743; 5'-O-Dimethoxytrityl-thymidine
intermediate for 5-methyl dC amidite,
5'-O-Dimethoxytrityl-2'-deoxy-5-methylcytidine intermediate for
5-methyl-dC amidite,
5'-O-Dimethoxytrityl-2'-deoxy-N4-benzoyl-5-methylcyt- idine
penultimate intermediate for 5-methyl dC amidite,
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-deoxy-N.sup.4-benzoyl-5-methylcy-
tidin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite
(5-methyl dC amidite), 2'-Fluorodeoxyadenosine,
2'-Fluorodeoxyguanosine, 2'-Fluorouridine, 2'-Fluorodeoxycytidine,
2'-O-(2-Methoxyethyl) modified amidites,
2'-O-(2-methoxyethyl)-5-methyluridine intermediate,
5'-O-DMT-2'-O-(2-methoxyethyl)-5-methyluridine penultimate
intermediate,
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-5-methyluridi-
n-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T
amidite),
5'-O-Dimethoxytrityl-2'-O-(2-methoxyethyl)-5-methylcytidine
intermediate,
5'-O-dimethoxytrityl-2'-O-(2-methoxyethyl)-N.sup.4-benzoyl-5-methyl-cytid-
ine penultimate intermediate,
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(-
2-methoxyethyl)-N.sup.4-benzoyl-5-methylcytidin-3'-O-yl]-2-cyanoethyl-N,N--
diisopropylphosphoramidite (MOE 5-Me-C amidite),
[5'-O-(4,4'-Dimethoxytrip-
henylmethyl)-2'-O-(2-methoxyethyl)-N.sup.4-benzoyladenosin-3'-O-yl]-2-cyan-
oethyl-N,N-diisopropylphosphoramidite (MOE A amdite),
[5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.sup.4-isobu-
tyrylguanosin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite
(MOE G amidite), 2'-O-(Aminooxyethyl) nucleoside amidites and
2'-O-(dimethylaminooxyethyl) nucleoside amidites,
2'-(Dimethylaminooxyeth- oxy) nucleoside amidites,
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-- 5-methyluridine,
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-meth-
yluridine,
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methylu-
ridine,
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-me-
thyluridine, 5'-O-tert-Butyldiphenylsilyl-2'-O-[N,N
dimethylaminooxyethyl]-5-methyluridine,
2'-O-(dimethylaminooxyethyl)-5-me- thyluridine,
5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine,
5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoe-
thyl)-N,N-diisopropylphosphoramidite], 2'-(Aminooxyethoxy)
nucleoside amidites,
N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(-
4,4'-dimethoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphora-
midite], 2'-dimethylaminoethoxyethoxy (2'-DMAEOE) nucleoside
amidites, 2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl
uridine,
5'-O-dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl
uridine and
5'-O-Dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)-ethyl-
)]-5-methyl
uridine-3'-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.
Example 2
Oligonucleotide and Oligonucleoside Synthesis
[0113] 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.
[0114] Oligonucleotides: Unsubstituted and substituted
phosphodiester (P.dbd.O) oligonucleotides are synthesized on an
automated DNA synthesizer (Applied Biosystems model 394) using
standard phosphoramidite chemistry with oxidation by iodine.
[0115] Phosphorothioates (P.dbd.S) are synthesized similar to
phosphodiester oligonucleotides with the following exceptions:
thiation was effected by utilizing a 10% w/v solution of
3,H-1,2-benzodithiole-3-o- ne 1,1-dioxide in acetonitrile for the
oxidation of the phosphite linkages. The thiation reaction step
time was increased to 180 sec and preceded by the normal capping
step. After cleavage from the CPG column and deblocking in
concentrated ammonium hydroxide at 55.degree. C. (12-16 hr), the
oligonucleotides were recovered by precipitating with >3 volumes
of ethanol from a 1 M NH.sub.4OAc solution. Phosphinate
oligonucleotides are prepared as described in U.S. Pat. No.
5,508,270, herein incorporated by reference.
[0116] Alkyl phosphonate oligonucleotides are prepared as described
in U.S. Pat. No. 4,469,863, herein incorporated by reference.
[0117] 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.
[0118] 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.
[0119] Alkylphosphonothioate oligonucleotides are prepared as
described in published PCT applications PCT/US94/00902 and
PCT/US93/06976 (published as WO 94/17093 and WO 94/02499,
respectively), herein incorporated by reference.
[0120] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are
prepared as described in U.S. Pat. No. 5,476,925, herein
incorporated by reference.
[0121] Phosphotriester oligonucleotides are prepared as described
in U.S. Pat. No. 5,023,243, herein incorporated by reference.
[0122] Borano phosphate oligonucleotides are prepared as described
in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated
by reference.
[0123] Oligonucleosides: Methylenemethylimino linked
oligonucleosides, also identified as MMI linked oligonucleosides,
methylenedimethylhydrazo linked oligonucleosides, also identified
as MDH linked oligonucleosides, and methylenecarbonylamino linked
oligonucleosides, also identified as amide-3 linked
oligonucleosides, and methyleneaminocarbonyl linked
oligonucleosides, also identified as amide-4 linked
oligonucleosides, as well as mixed backbone compounds having, for
instance, alternating MMI and P.dbd.O or P.dbd.S linkages are
prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023,
5,489,677, 5,602,240 and 5,610,289, all of which are herein
incorporated by reference.
[0124] 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.
[0125] Ethylene oxide linked oligonucleosides are prepared as
described in U.S. Pat. No. 5,223,618, herein incorporated by
reference.
Example 3
RNA Synthesis
[0126] 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.
[0127] 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.
[0128] 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.
[0129] 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.
[0130] 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-ethylhydroxyl 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.
[0131] 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).
[0132] 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 4
Synthesis of Chimeric Oligonucleotides
[0133] 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".
[0134] [2'-O--Me]-[2'-deoxy]-[2'-O--Me] Chimeric Phosphorothioate
Oligonucleotides
[0135] Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate
and 2'-deoxy phosphorothioate oligonucleotide segments are
synthesized using an Applied Biosystems automated DNA synthesizer
Model 394, 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
incorporating coupling steps with increased reaction times for the
5'-dimethoxytrityl-2'-O-methyl-3'-O- -phosphoramidite. The fully
protected oligonucleotide is cleaved from the support and
deprotected in concentrated ammonia (NH.sub.4OH) for 12-16 hr at
55.degree. C. The deprotected oligo is then recovered by an
appropriate method (precipitation, column chromatography, volume
reduced in vacuo and analyzed spetrophotometrically for yield and
for purity by capillary electrophoresis and by mass
spectrometry.
[0136] [2'-O-(2-Methoxyethyl)]-[2'-deoxy]-[2'-O-(Methoxyethyl)]
Chimeric Phosphorothioate Oligonucleotides
[0137] [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.
[0138] [2'-O-(2-Methoxyethyl)Phosphodiester]-[2'-deoxy
Phosphorothioate]-[2'-O-(2-Methoxyethyl) Phosphodiester] Chimeric
Oligonucleotides
[0139] [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,
oxidation 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.
[0140] 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 5
Design and Screening of Duplexed Antisense Compounds Targeting
Diacylglycerol Acyltransferase 1
[0141] 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
diacylglycerol acyltransferase 1. 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.
[0142] 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:
1 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
[0143] 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 uM. Once diluted, 30 uL of each strand is
combined with 15 uL of a 5.times. solution of annealing buffer. The
final concentration of said buffer is 100 mM potassium acetate, 30
mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume
is 75 uL. 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 uM. This solution can be stored frozen (-20.degree. C.) and
freeze-thawed up to 5 times.
[0144] Once prepared, the duplexed antisense compounds are
evaluated for their ability to modulate diacylglycerol
acyltransferase 1 expression.
[0145] 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 .mu.L of
OPTI-MEM-1 containing 12 .mu.g/mL LIPOFECTIN (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 6
Oligonucleotide Isolation
[0146] After cleavage from the controlled pore glass solid support
and deblocking in concentrated ammonium hydroxide at 55.degree. C.
for 12-16 hours, the oligonucleotides or oligonucleosides are
recovered by precipitation out of 1 M NH.sub.4OAc with >3
volumes of ethanol. Synthesized oligonucleotides were analyzed by
electrospray mass spectroscopy (molecular weight determination) and
by capillary gel electrophoresis and judged to be at least 70% full
length material. The relative amounts of phosphorothioate and
phosphodiester linkages obtained in the synthesis was determined by
the ratio of correct molecular weight relative to the -16 amu
product (+/-32+/-48). 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
[0147] Oligonucleotides were synthesized via solid phase P(III)
phosphoramidite chemistry on an automated synthesizer capable of
assembling 96 sequences simultaneously in a 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-cyanoethyl-diiso-propyl 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 standard or patented methods.
They are utilized as base protected beta-cyanoethyldiisopropyl
phosphoramidites.
[0148] 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
[0149] 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
[0150] 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 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.
[0151] T-24 Cells:
[0152] 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 (Invitrogen Corporation, Carlsbad, Calif.)
supplemented with 10% fetal calf serum (Invitrogen Corporation,
Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin
100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.).
Cells were routinely passaged by trypsinization and dilution when
they reached 90% confluence. Cells were seeded into 96-well plates
(Falcon-Primaria #353872) at a density of 7000 cells/well for use
in RT-PCR analysis.
[0153] 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.
[0154] A549 Cells:
[0155] 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 (Invitrogen
Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf
serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100
units per mL, and streptomycin 100 micrograms per mL (Invitrogen
Corporation, Carlsbad, Calif.). Cells were routinely passaged by
trypsinization and dilution when they reached 90% confluence.
[0156] NHDF Cells:
[0157] 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.
[0158] HEK Cells:
[0159] 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.
[0160] HepG2 Cells:
[0161] 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.
[0162] 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.
[0163] b.END Cells:
[0164] 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 supplemented
with 10% fetal bovine serum (Gibco/Life Technologies, Gaithersburg,
Md.). Cells were routinely passaged by trypsinization and dilution
when they reached 90% confluence. Cells were seeded into 24-well
plates (Falcon-Primaria #3047) at a density of 40,000 cells/well
for use in RT-PCR analysis.
[0165] 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.
[0166] Treatment with Antisense Compounds:
[0167] When cells reached 65-75% confluency, they were treated with
oligonucleotide. For cells grown in 24-well plates, wells were
washed once with 400 .mu.L Eagle's DMEM medium and then treated
with 100 .mu.L of Eagle's DMEM containing 3.75 .mu.g/mL
LIPOFECTIN.TM. (Invitrogen Corporation, Carlsbad, Calif.) and the
desired concentration of oligonucleotide. Cells are treated and
data are obtained in triplicate. After 4-7 hours of treatment at
37.degree. C., the medium was replaced with Eagle's DMEM
supplemented with 10% fetal bovine serum. Cells were harvested
20-24 hours after oligonucleotide treatment.
[0168] 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 selected from either ISIS 13920
(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human
H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is
targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are
2'-O-methoxyethyl gapmers (2'-O-methoxyethyls shown in bold) with a
phosphorothioate backbone. For mouse or rat cells the positive
control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID
NO: 3, 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-H-ras (for ISIS
13920), JNK2 (for ISIS 18078) 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
c-H-ras, JNK2 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. The concentrations of antisense oligonucleotides used
herein are from 50 nM to 300 nM.
Example 10
Analysis of Oligonucleotide Inhibition of Diacylglycerol
Acyltransferase 1 Expression
[0169] Antisense modulation of diacylglycerol acyltransferase 1
expression can be assayed in a variety of ways known in the art.
For example, diacylglycerol acyltransferase 1 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. The preferred
method of RNA analysis of the present invention is the use of total
cellular RNA as described in other examples herein. Methods of RNA
isolation are well known in the art. Northern blot analysis is also
routine in the art. Real-time quantitative (PCR) can be
conveniently accomplished using the commercially available ABI
PRISM.TM. 7600, 7700, or 7900 Sequence Detection System, available
from PE-Applied Biosystems, Foster City, Calif. and used according
to manufacturer's instructions.
[0170] Protein levels of diacylglycerol acyltransferase 1 can be
quantitated in a variety of ways well known in the art, such as
immunoprecipitation, Western blot analysis (immunoblotting),
enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated
cell sorting (FACS). Antibodies directed to diacylglycerol
acyltransferase 1 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 monoclonal
or polyclonal antibody generation methods well known in the
art.
Example 11
Design of Phenotypic Assays and in Vivo Studies for the Use of
Diacylglycerol Acyltransferase 1 Inhibitors
[0171] Phenotypic Assays
[0172] Once diacylglycerol acyltransferase 1 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.
[0173] 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 diacylglycerol
acyltransferase 1 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.).
[0174] 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 diacylglycerol acyltransferase 1 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.
[0175] 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.
[0176] 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
diacylglycerol acyltransferase 1 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.
[0177] In Vivo Studies
[0178] The individual subjects of the in vivo studies described
herein are warm-blooded vertebrate animals, which includes
humans.
[0179] 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 diacylglycerol acyltransferase 1
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 diacylglycerol acyltransferase 1 inhibitor
or a placebo. Using this randomization approach, each volunteer has
the same chance of being given either the new treatment or the
placebo.
[0180] Volunteers receive either the diacylglycerol acyltransferase
1 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 diacylglycerol
acyltransferase 1 or diacylglycerol acyltransferase 1 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.
[0181] 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.
[0182] 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 diacylglycerol
acyltransferase 1 inhibitor treatment. In general, the volunteers
treated with placebo have little or no response to treatment,
whereas the volunteers treated with the diacylglycerol
acyltransferase 1 inhibitor show positive trends in their disease
state or condition index at the conclusion of the study.
Example 12
RNA Isolation
[0183] Poly(A)+ mRNA Isolation
[0184] Poly(A)+ mRNA was isolated according to Miura et al., (Clin.
Chem., 1996, 42, 1758-1764). Other methods for poly(A)+mRNA
isolation are routine in the art. 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.
[0185] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
[0186] Total RNA Isolation
[0187] 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. 150 .mu.L Buffer RLT was
added to each well and the plate vigorously agitated for 20
seconds. 150 .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 1
minute. 500 .mu.L of Buffer RW1 was added to each well of the
RNEASY 96.TM. plate and incubated for 15 minutes and the vacuum was
again applied for 1 minute. An additional 500 .mu.L of Buffer RW1
was added to each well of the RNEASY 96.TM. plate and the vacuum
was applied for 2 minutes. 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 90 seconds. The Buffer RPE wash was then repeated and the
vacuum was applied for an additional 3 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 140 .mu.L of RNAse free
water into each well, incubating 1 minute, and then applying the
vacuum for 3 minutes.
[0188] 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 Diacylglycerol
Acyltransferase 1 mRNA Levels
[0189] Quantitation of diacylglycerol acyltransferase 1 mRNA levels
was accomplished by real-time quantitative PCR using the ABI
PRISM.TM. 7600, 7700, or 7900 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., FAM or JOE,
obtained from either PE-Applied Biosystems, Foster City, Calif.,
Operon Technologies Inc., Alameda, Calif. or Integrated DNA
Technologies Inc., Coralville, Iowa) is attached to the 5' end of
the probe and a quencher dye (e.g., TAMRA, obtained from either
PE-Applied Biosystems, Foster City, Calif., Operon Technologies
Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,
Coralville, Iowa) 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. 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.
[0190] 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.
[0191] PCR reagents were obtained from Invitrogen Corporation,
(Carlsbad, Calif.). RT-PCR reactions were carried out by adding 30
.mu.L PCR cocktail (2.5.times. PCR buffer minus MgCl.sub.2, 6.6 mM
MgCl.sub.2, 375 .mu.M each of DATP, dCTP, dCTP and dGTP, 375 nM
each of forward primer and reverse primer, 125 nM of probe, 4 Units
RNAse inhibitor, 1.25 Units PLATINUM.RTM. Taq, 5 Units MuLV reverse
transcriptase, and 2.5.times. ROX dye) to 96-well plates containing
20 .mu.L total RNA solution (20-200 ng). 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
PLATINUM.RTM. Taq, 45 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).
[0192] 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. (Molecular Probes, Inc. Eugene, Oreg.). 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
(Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA
quantification by RiboGreen.TM. are taught in Jones, L. J., et al,
(Analytical Biochemistry, 1998, 265, 368-374).
[0193] In this assay, 180 .mu.L of RiboGreen.TM. working reagent
(RiboGreen.TM. reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA,
pH 7.5) is pipetted into a 96-well plate containing 20 .mu.L
purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE
Applied Biosystems) with excitation at 485 nm and emission at 530
nm.
[0194] Probes and primers to human diacylglycerol acyltransferase 1
were designed to hybridize to a human diacylglycerol
acyltransferase 1 sequence, using published sequence information
(GenBank accession number NM.sub.--012079.2, incorporated herein as
SEQ ID NO:4). For human diacylglycerol acyltransferase 1 the PCR
primers were: forward primer: TCCCCGCATCCGGAA (SEQ ID NO: 5)
reverse primer: CTGGGTGAAGAACAGCATCTCA (SEQ ID NO: 6) and the PCR
probe was: FAM-CGCTTTCTGCTGCGACGGATCC-TAMRA (SEQ ID NO: 7) where
FAM is the fluorescent dye and TAMRA is the quencher dye. For human
GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC(SEQ
ID NO:8) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the
PCR probe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC- TAMRA 3' (SEQ ID NO:
10) where JOE is the fluorescent reporter dye and TAMRA is the
quencher dye.
[0195] Probes and primers to mouse diacylglycerol acyltransferase 1
were designed to hybridize to a mouse diacylglycerol
acyltransferase 1 sequence, using published sequence information
(GenBank accession number AF078752.1, incorporated herein as SEQ ID
NO:11). For mouse diacylglycerol acyltransferase 1 the PCR primers
were:
[0196] forward primer: GTTCCGCCTCTGGGCATT (SEQ ID NO:12)
[0197] reverse primer: GAATCGGCCCACAATCCA (SEQ ID NO: 13) and
the
[0198] PCR probe was: FAM-CAGCCATGATGGCTCAGGTCCCACT-TAMRA (SEQ ID
NO: 14) where FAM is the fluorescent reporter dye and TAMRA is the
quencher dye. For mouse GAPDH the PCR primers were:
[0199] forward primer: GGCAAATTCAACGGCACAGT(SEQ ID NO:15)
[0200] reverse primer: GGGTCTCGCTCCTGGAAGAT(SEQ ID NO:16) and
the
[0201] PCR probe was: 5' JOE-AAGGCCGAGAATGGGAAGCTTGTCATC- TAMRA 3'
(SEQ ID NO: 17) where JOE is the fluorescent reporter dye and TAMRA
is the quencher dye.
Example 14
Northern Blot Analysis of Diacylglycerol Acyltransferase 1 mRNA
Levels
[0202] Eighteen hours after antisense treatment, cell monolayers
were washed twice with cold PBS and lysed in 1 mL RNAZOL.TM.
(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 (Stratagene, Inc, La Jolla, Calif.) and then
probed using QUICKHYB.TM. hybridization solution (Stratagene, La
Jolla, Calif.) using manufacturer's recommendations for stringent
conditions.
[0203] To detect human diacylglycerol acyltransferase 1, a human
diacylglycerol acyltransferase 1 specific probe was prepared by PCR
using the forward primer TCCCCGCATCCGGAA (SEQ ID NO: 5) and the
reverse primer CTGGGTGAAGAACAGCATCTCA (SEQ ID NO: 6). 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.).
[0204] To detect mouse diacylglycerol acyltransferase 1, a mouse
diacylglycerol acyltransferase 1 specific probe was prepared by PCR
using the forward primer GTTCCGCCTCTGGGCATT (SEQ ID NO: 12) and the
reverse primer GAATCGGCCCACAATCCA (SEQ ID NO: 13). To normalize for
variations in loading and transfer efficiency membranes were
stripped and probed for mouse glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).
[0205] Hybridized membranes were visualized and quantitated using a
PHOSPHORIMAGER.TM. 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 Diacylglycerol Acyltransferase 1
Expression by Chimeric Phosphorothioate Oligonucleotides Having
2'-MOE Wings and a Deoxy Gap
[0206] In accordance with the present invention, a series of
antisense compounds were designed to target different regions of
the human diacylglycerol acyltransferase 1 RNA, using published
sequences (GenBank accession number NM.sub.--012079.2, incorporated
herein as SEQ ID NO: 4). The compounds are shown in Table 1.
"Target site" indicates the first (5'-most) nucleotide number on
the particular target sequence to which the compound 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 diacylglycerol acyltransferase 1 mRNA levels by quantitative
real-time PCR as described in other examples herein. Data are
averages from three experiments in which HepG2 cells were treated
with the antisense oligonucleotides of the present invention. The
positive control for each datapoint is identified in the table by
sequence ID number. If present, "N.D." indicates "no data".
2TABLE 1 Inhibition of human diacylglycerol acyltransferase 1 mRNA
levels by chimeric phosphorothioate oligonucleotides having 2'-MOE
wings and a deoxy gap TARGET CONTROL SEQ ID TARGET % SEQ ID SEQ ID
ISIS # REGION NO SITE SEQUENCE INHIB NO NO 191617 5' UTR 4 1
gccgcctctctcgtccattc 57 18 1 191619 5' UTR 4 21
gagccgctaactaatggacg 37 19 1 191621 5' UTR 4 41
acaacggctgcgttgctccg 30 20 1 191623 5' UTR 4 71
ccgcccgcgtcaggcccgtc 40 21 1 191625 5' UTR 4 91
gcctcaccagcgcgttcaac 20 22 1 191627 5' UTR 4 120
ccctgccggccgccgtagcc 24 23 1 191629 5' UTR 4 151
ctccgggccctagacaacgg 45 24 1 191631 5' UTR 4 181
gttcgtagcgcccgaggcgc 53 25 1 191633 5' UTR 4 211
cccggccgcagccaagcgtg 44 26 1 191635 Start 4 231
gcccatggcctcagcccgca 77 27 1 Codon 191637 Coding 4 281
tggctcgagggccgcgaccc 58 28 1 191639 Coding 4 301
ccgcaggcccgccgccgccg 49 29 1 191641 Coding 4 321
ccgcacctcttcttccgccg 40 30 1 191643 Coding 4 401
acgccggcgtctccgtcctt 92 31 1 191645 Coding 4 421
gctcccagtggccgctgccc 60 32 1 191647 Coding 4 441
ctgcaggcgatggcacctca 85 33 1 191649 Coding 4 491
aggatgccacggtagttgct 62 34 1 191651 Coding 4 511
gcatcaccacacaccagttc 37 35 1 191653 Coding 4 561
gccatacttgatgaggttct 48 36 1 191655 Coding 4 651
gacattggccgcaataacca 47 37 1 191657 Coding 4 681
cttctcaacctggaatgcag 29 38 1 191659 Coding 4 721
gcagtcccgcctgctccgtc 50 39 1 191661 Coding 4 741
caggttggctacgtgcagca 31 40 1 191663 Coding 4 781
ccagtaagaccacagccgct 62 41 1 191665 Coding 4 831
ggtgtgcgccatcagcgcca 59 42 1 191667 Coding 4 931
cagcactgctggccttcttc 52 43 1 191669 Coding 4 1021
tgagctcgtagcacaaggtg 43 44 1 191671 Coding 4 1121
cactgctggatcagccccac 20 45 1 191673 Coding 4 1181
atgcgtgagtagtccatgtc 59 46 1 191675 Coding 4 1231
tgagccagatgaggtgattg 62 47 1 191677 Coding 4 1281
gagctcagccacggcattca 76 48 1 191679 Coding 4 1351
tctgccagaagtaggtgaca 30 49 1 191681 Coding 4 1611
gatgagcgacagccacacag 21 50 1 191683 Coding 4 1671
ctcatagttgagcacgtagt 73 51 1 191685 3' UTR 4 1721
cagtgagaagccaggccctc 68 52 1 191687 3' UTR 4 1781
ccatccccagcactcgaggc 68 53 1 191689 3' UTR 4 1801
aggatgctgtgcagccaggc 73 54 1 191691 3' UTR 4 1851
ggtgcaggacagagccccat 72 55 1 191693 3' UTR 4 1881
gtgtctggcctgctgtcgcc 71 56 1 191695 3' UTR 4 1901
ctcccagctggcatcagact 76 57 1
[0207] As shown in Table 1, SEQ ID NOs 18, 24, 25, 26, 27, 28, 29,
31, 32, 33, 34, 36, 37, 39, 41, 42, 43, 44, 46, 47, 48, 51, 52, 53,
54, 55, 56 and 57 demonstrated at least 43% inhibition of human
diacylglycerol acyltransferase 1 expression in this assay and are
therefore preferred. More preferred are SEQ ID NOs 31, 33, 27, and
57. 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. These preferred target segments are shown in
Table 3. The sequences represent the reverse complement of the
preferred antisense compounds shown in Table 1. "Target site"
indicates the first (5'-most) nucleotide number on the particular
target nucleic acid to which the oligonucleotide binds. Also shown
in Table 3 is the species in which each of the preferred target
segments was found.
Example 16
Antisense Inhibition of Mouse Diacylglycerol Acyltransferase 1
Expression by Chimeric Phosphorothioate Oligonucleotides Having
2'-MOE Wings and a Deoxy Gap
[0208] In accordance with the present invention, a second series of
antisense compounds were designed to target different regions of
the mouse diacylglycerol acyltransferase 1 RNA, using published
sequences (GenBank accession number AF078752.1, incorporated herein
as SEQ ID NO: 11). The compounds are shown in Table 2. "Target
site" indicates the first (5'-most) nucleotide number on the
particular target nucleic acid to which the compound 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
mouse diacylglycerol acyltransferase 1 mRNA levels by quantitative
real-time PCR a described in other examples herein. Data are
averages from three experiments in which b.END cells were treated
with the antisense oligonucleotides of the present invention. If
present, "N.D." indicates "no data".
3TABLE 2 Inhibition of mouse diacylglycerol acyltransferase 1 mRNA
levels by chimeric phosphorothioate oligonucleotides having 2'-MOE
wings and a deoxy gap TARGET SEQ ID TARGET % SEQ ID ISIS # REGION
NO SITE SEQUENCE INHIB NO 191723 5' UTR 11 1 ctacttatttccattcatcc 2
58 191724 5' UTR 11 21 tatcctaagtatgcctaatt 0 59 191725 5' UTR 11
31 gcttgagccctatcctaagt 0 60 191726 5' UTR 11 61
ctcgtcgcggcccaatcttc 21 61 191727 Start 11 81 cccatggcttcggcccgcac
48 62 Codon 191729 Coding 11 191 cagccgcgtctcgcacctcg 74 63 191730
Coding 11 232 cggagccggcgcgtcacccc 63 64 191731 Coding 11 281
ccacgctggtccgcccgtct 67 65 191732 Coding 11 301
cagatcccagtagccgtcgc 59 66 191733 Coding 11 321
tcttgcagacgatggcacct 49 67 191734 Coding 11 371
tcaggataccacgataattg 48 68 191735 Coding 11 391
cagcatcaccacacaccaat 52 69 191736 Coding 11 411
aaccttgcattactcaggat 62 70 191737 Coding 11 451
atccaccaggatgccatact 29 71 191738 Coding 11 471
agagacaccacctggatagg 42 72 191740 Coding 11 601
cagcagccccatctgctctg 63 73 191741 Coding 11 621
gccaggttaaccacatgtag 58 74 191742 Coding 11 661
aaccagtaaggccacagctg 16 75 191743 Coding 11 681
cccactggagtgatagactc 42 76 191744 Coding 11 711
atggagtatgatgccagagc 53 77 191745 Coding 11 771
acccttcgctggcggcacca 68 78 191746 Coding 11 841
tggatagctcacagcttgct 56 79 191747 Coding 11 861
tctcggtaggtcaggttgtc 32 80 191748 Coding 11 961
ctcaagaactcgtcgtagca 60 81 191749 Coding 11 1001
gttggatcagccccacttga 37 82 191750 Coding 11 1061
gtgaatagtccatatccttg 48 83 191751 Coding 11 1081
taagagacgctcaatgatcc 18 84 191752 Coding 11 1161
tctgccacagcattgagaca 50 85 191753 Coding 11 1201
ccaatctctgtagaactcgc 55 86 191754 Coding 11 1221
gtgacagactcagcattcca 56 87 191755 Coding 11 1271
gtctgatgcaccacttgtgc 72 88 191756 Coding 11 1301
tgccatgtctgagcataggc 70 89 191757 Coding 11 1331
atactcctgtcctggccacc 65 90 191759 Coding 11 1471
attgccatagttcccttgga 68 91 191760 Coding 11 1491
agtgtcacccacacagctgc 66 92 191761 Coding 11 1511
ccaccggttgcccaatgatg 71 93 191762 Coding 11 1531
gtggacatacatgagcacag 62 94 191763 Coding 11 1551
tagttgagcacgtagtagtc 40 95 191764 Stop 11 1586 ctttggcagtagctcatacc
37 96 Codon 191765 3' UTR 11 1621 tccagaactccaggcccagg 59 97
[0209] As shown in Table 2, SEQ ID NOs 62, 63, 64, 65, 66, 67, 68,
69, 70, 73, 74, 77, 78, 79, 81, 83, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94 and 97 demonstrated at least 43% inhibition of mouse
diacylglycerol acyltransferase 1 expression in this experiment and
are therefore preferred. More preferred are SEQ ID Nos: 63, 88, 91,
and 93. 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. These preferred target segments of the mRNA are
shown in Table 3 as the appropriate RNA sequence, where thymine (T)
has been replaced with uracil (U) to reflect correct representation
of an RNA sequence. The sequences represent the reverse complement
of the preferred antisense compounds shown in Tables 1 and 2.
"Target site" indicates the first (5'-most) nucleotide number on
the particular target nucleic acid to which the oligonucleotide
binds. Also shown in Table 3 is the species in which each of the
preferred target segments was found.
4TABLE 3 Sequence and position of preferred target segments
identified in diacylglycerol acyltransferase 1. TARGET SITE SEQ ID
TARGET REV COMP SEQ ID ID NO SITE SEQUENCE OF SEQ ID ACTIVE IN NO
108088 4 1 gaauggacgagagaggcggc 18 H. sapiens 98 108094 4 151
ccguugucuagggcccggag 24 H. sapiens 99 108095 4 181
gcgccucgggcgcuacgaac 25 H. sapiens 100 108096 4 211
cacgcuuggcugcggccggg 26 H. sapiens 101 108097 4 231
ugcgggcugaggccaugggc 27 H. sapiens 102 108098 4 281
gggucqcggcccucgagcca 28 H. sapiens 103 108099 4 301
cggcggcggcgggccugcgg 29 H. sapiens 104 108101 4 401
aaggacggagacgccggcgu 31 H. sapiens 105 108102 4 421
gggcagcggccacugggagc 32 H. sapiens 106 108103 4 441
ugaggugccaucgccugcag 33 H. sapiens 107 108104 4 491
agcaacuaccguggcauccu 34 H. sapiens 108 108106 4 561
agaaccucaucaaguauggc 36 H. sapiens 109 108107 4 651
ugguuauugcggccaauguc 37 H. sapiens 110 108109 4 721
gacggagcaggcgggacugc 39 H. sapiens 111 108111 4 781
agcggcuguggucuuacugg 41 H. sapiens 112 108112 4 831
uggcgcugauggcgcacacc 42 H. sapiens 113 108113 4 931
gaagaaggccagcagugcug 43 H. sapiens 114 108114 4 1021
caccuugugcuacgagcuca 44 H. sapiens 115 108116 4 1181
gacauggacuacucacgcau 46 H. sapiens 116 108117 4 1231
caaucaccucaucuggcuca 47 H. sapiens 117 108118 4 1281
ugaaugccguggcugagcuc 48 H. sapiens 118 108121 4 1671
acuacgugcucaacuaugag 51 H. sapiens 119 108122 4 1721
gagggccuggcuucucacug 52 H. sapiens 120 108123 4 1781
gccucgagugcuggggaugg 53 H. sapiens 121 108124 4 1801
gccuggcugcacagcauccu 54 H. sapiens 122 108125 4 1851
auggggcucuguccugcacc 55 H. sapiens 123 108126 4 1881
ggcgacagcaggccagacac 56 H. sapiens 124 108127 4 1901
agucugaugccagcugggag 57 H. sapiens 125 108139 11 81
gugcgggccgaagccauggg 62 M. musculus 126 108141 11 191
cgaggugcgagacgcggcug 63 M. musculus 127 108142 11 232
ggggugacgcgccggcuccg 64 M. musculus 128 108143 11 281
agacgggcggaccagcgugg 65 M. musculus 129 108144 11 301
gcgacggcuacugggaucug 66 M. musculus 130 108145 11 321
aggugccaucgucugcaaga 67 M. musculus 131 108146 11 371
caauuaucgugguauccuga 68 M. musculus 132 108147 11 391
auugguguguggugaugcug 69 M. musculus 133 108148 11 411
auccugaguaaugcaagguu 70 M. musculus 134 108152 11 601
cagagcagauggggcugcug 73 M. musculus 135 108153 11 621
cuacaugugguuaaccuggc 74 M. musculus 136 108156 11 711
gcucuggcaucauacuccau 77 M. musculus 137 108157 11 771
uggugccgccagcgaagggu 78 M. musculus 138 108158 11 841
agcaagcugugagcuaucca 79 M. musculus 139 108160 11 961
ugcuacgacgaguucuugag 81 M. musculus 140 108162 11 1061
caaggauauggacuauucac 83 M. musculus 141 108164 11 1161
ugucucaaugcuguggcaga 85 M. musculus 142 108165 11 1201
gcgaguucuacagagauugg 86 M. musculus 143 108166 11 1221
uggaaugcugagucugucac 87 M. musculus 144 108167 11 1271
gcacaaguggugcaucagac 88 M. musculus 145 108168 11 1301
gccuaugcucagacauggca 89 M. musculus 146 108169 11 1331
gguggccaggacaggaguau 90 M. musculus 147 108171 11 1471
uccaagggaacuauggcaau 91 M. musculus 148 108172 11 1491
gcagcugugugggugacacu 92 M. musculus 149 108173 11 1511
caucauugggcaaccggugg 93 M. musculus 150 108174 11 1531
cugugcucauguauguccac 94 M. musculus 151 108177 11 1621
ccugggccuggaguucugga 97 M. musculus 152
[0210] As these "preferred target segments" have been found by
experimentation to be open to, and accessible for, hybridization
with the antisense compounds of the present invention, one of skill
in the art will recognize or be able to ascertain, using no more
than routine experimentation, further embodiments of the invention
that encompass other compounds that specifically hybridize to these
preferred target segments and consequently inhibit the expression
of diacylglycerol acyltransferase 1.
[0211] According to the present invention, antisense compounds
include antisense oligomeric compounds, antisense oligonucleotides,
ribozymes, external guide sequence (EGS) oligonucleotides,
alternate splicers, primers, probes, and other short oligomeric
compounds which hybridize to at least a portion of the target
nucleic acid.
Example 17
Western Blot Analysis of Diacylglycerol Acyltransferase 1 Protein
Levels
[0212] 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 ul/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 diacylglycerol acyltransferase 1 is used, with
a radiolabeled or fluorescently labeled secondary antibody directed
against the primary antibody species. Bands are visualized using a
PHOSPHORIMAGER.TM. (Molecular Dynamics, Sunnyvale, Calif.).
Example 18
Antisense Inhibition of Diacylglycerol Acyltransferase 1
Expression--Dose Response in HepG2 Cells
[0213] In accordance with the present invention, 6 oligonucleotides
targeted to diacylglycerol acyltransferase 1, ISIS 191729 (SEQ ID
NO: 63), ISIS 191731 (SEQ ID NO: 65), ISIS 191755 (SEQ ID NO: 88),
ISIS 191756 (SEQ ID NO: 89),
[0214] ISIS 191759 (SEQ ID NO: 91), and ISIS 191761 (SEQ ID NO:
93), were further investigated in a dose response study.
[0215] In the dose-response experiment, with mRNA levels as the
endpoint, HepG2 cells were treated with ISIS 191729, ISIS 191731,
ISIS 191755, ISIS 191756, ISIS 191759, or ISIS 191761 at doses of
1, 5, 10, 25, 50, and 100 nM oligonucleotide. Data were obtained by
real-time quantitative PCR as described in other examples herein
and are averaged from two experiments with mRNA levels in the
treatment groups being normalized to an untreated control group.
The data are shown in Table 4.
5TABLE 4 Inhibition of diacylglycerol acyltransferase 1 mRNA levels
by chimeric phosphorothioate oligonucleotides having 2'-MOE wings
and a deoxy gap - Dose Response Dose (nM) 1 5 10 25 50 100 ISIS NO.
% Inhibition 191729 26 62 78 80 83 83 191731 27 58 57 58 82 85
191755 41 59 72 75 83 79 191756 13 39 59 65 81 75 191759 26 44 74
80 82 86 191761 23 63 71 80 85 87
[0216] From these data, it is evident that all of the
oligonucleotides presented in Table 4 are capable of reducing
diacylglycerol acyltransferase 1 mRNA levels in a dose-dependent
manner.
Example 19
Effects of Antisense Inhibition of Diacylglycerol Acyltransferase 1
(ISIS 191729 and ISIS 191755) on Serum Glucose Levels--In Vivo
Studies
[0217] C57BL/6 ob/ob mice, a strain reported to be susceptible to
hyperlipidemia-induced atherosclerotic plaque formation were used
in the following studies to evaluate diacylglycerol acyltransferase
1 antisense oligonucleotides as potential agents to lower serum
glucose levels.
[0218] Male C57BL/6 mice (n=8) receiving a Purina 5015 diet were
evaluated over the course of 4 weeks for the effects of ISIS 191729
(SEQ ID No: 63) and ISIS 191755 (SEQ ID NO: 88) on serum glucose
levels. Control animals received saline treatment. Mice were dosed
intraperitoneally twice a week with 25 mg/kg ISIS 191729, ISIS
191755, or saline for 4 weeks.
[0219] Both antisense oligonucleotides were able to reduce serum
glucose levels relative to the saline-treated animals. Before any
tratment was started (week 0), the measured glucose levels for each
group of animals were 357, 368, and 346 mg/dL for the groups which
would be treated with saline, ISIS 191729, and ISIS 191755,
respectively. After two weeks, serum glucose levels were 300 and
278 mg/dL for ISIS 191729 and ISIS 191755, respectively, compared
to 360 mg/dL for saline control. After four weeks of treatment, the
serum glucose levels were further reduced to 224 and 188 mg/dL for
ISIS 191729 and ISIS 191755, respectively, compared to 313 mg/dL
for saline control.
[0220] These data indicate that ISIS 191729 and ISIS 191755 are
able to significantly reduce serum glucose levels in vivo. ISIS
191755 also caused no change in food intake or body weight, but
reduced epididymal fat pad weight by 12%. (See Table 5 for a
summary of in vivo data).
Example 20
Effects of Antisense Inhibition of Diacylglycerol Acyltransferase 1
(ISIS 191729 and ISIS 191755) on Diacylglycerol Acyltransferase 1
mRNA Levels in C57BL/6 Mice
[0221] Male C57BL/6 mice (n=8) receiving a Purina 5015 diet were
evaluated after 4 weeks of treatment for the effects of ISIS 191729
(SEQ ID No: 63) and ISIS 191755 (SEQ ID NO: 88) to lower the level
of diacylglycerol acyltransferase 1 mRNA levels in white adipose
tissue and liver. Control animals received saline treatment. Mice
were dosed intraperitoneally twice a week with 25 mg/kg ISIS
191729, ISIS 191755, or saline for 4 weeks.
[0222] The diacylglycerol acyltransferase 1 mRNA levels in white
adipose tissue of the mice dosed with ISIS 191729 were 29% that of
the saline treated mice, and those dosed with ISIS 191755 were 16%
that of the saline treated mice. The diacylglycerol acyltransferase
1 mRNA levels in liver of the mice dosed with ISIS 191729 were 8%
that of the saline treated mice, and those dosed with ISIS 191755
were 4% that of the saline treated mice.
[0223] It has been reported in the art that diacylglycerol
acyltransferase 1 knockout mice demonstrate enhanced resistance to
diet-induced obesity and this was not coupled with changes in
energy expenditure or plasma glucose levels in ob/ob mice due to a
compensatory upregulation of diacylglycerol acyltransferase 2
expression in white adipose tissue. The results of studies
described herein using antisense compounds to transiently modulate
diacylglycerol acyltransferase 1 mRNA levles are in contrast to
those seen in the diacylglycerol acyltransferase 1 knockout
studies. The results shown herein indicate that antisense
oligonucleotides ISIS 191729 and ISIS 191755 are able to reduce
diacylglycerol acyltransferase 1 mRNA levels, reduce serum glucose
levels, and reduce fat pad weight while not affecting food intake
and total body weight. (See Table 5 for a summary of in vivo
data).
6TABLE 5 Effects of ISIS 191729 or ISIS 191755 treatment on serum
glucose levels and diacylglycerol acyltransferase 1 mRNA levels in
C57BL/6 mice. Treated with Biological Marker ISIS ISIS Measured
saline 191729 191755 week Glucose 0 357 368 346 mg/dL 2 360 300 278
4 313 224 188 tissue mRNA Liver 100 8 4 % of control White adipose
100 29 16
[0224]
Sequence CWU 1
1
152 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1
tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence Antisense
Oligonucleotide 2 gtgcgcgcga gcccgaaatc 20 3 20 DNA Artificial
Sequence Antisense Oligonucleotide 3 atgcattctg cccccaagga 20 4
1976 DNA H. sapiens CDS (245)...(1711) 4 gaatggacga gagaggcggc
cgtccattag ttagcggctc cggagcaacg cagccgttgt 60 ccttgaggcc
gacgggcctg acgcgggcgg gttgaacgcg ctggtgaggc ggtcacccgg 120
gctacggcgg ccggcagggg gcagtggcgg ccgttgtcta gggcccggag gtggggccgc
180 gcgcctcggg cgctacgaac ccggcaggcc cacgcttggc tgcggccggg
tgcgggctga 240 ggcc atg ggc gac cgc ggc agc tcc cgg cgc cgg agg aca
ggg tcg cgg 289 Met Gly Asp Arg Gly Ser Ser Arg Arg Arg Arg Thr Gly
Ser Arg 1 5 10 15 ccc tcg agc cac ggc ggc ggc ggg cct gcg gcg gcg
gaa gaa gag gtg 337 Pro Ser Ser His Gly Gly Gly Gly Pro Ala Ala Ala
Glu Glu Glu Val 20 25 30 cgg gac gcc gct gcg ggc ccc gac gtg gga
gcc gcg ggg gac gcg cca 385 Arg Asp Ala Ala Ala Gly Pro Asp Val Gly
Ala Ala Gly Asp Ala Pro 35 40 45 gcc ccg gcc ccc aac aag gac gga
gac gcc ggc gtg ggc agc ggc cac 433 Ala Pro Ala Pro Asn Lys Asp Gly
Asp Ala Gly Val Gly Ser Gly His 50 55 60 tgg gag ctg agg tgc cat
cgc ctg cag gat tct tta ttc agc tct gac 481 Trp Glu Leu Arg Cys His
Arg Leu Gln Asp Ser Leu Phe Ser Ser Asp 65 70 75 agt ggc ttc agc
aac tac cgt ggc atc ctg aac tgg tgt gtg gtg atg 529 Ser Gly Phe Ser
Asn Tyr Arg Gly Ile Leu Asn Trp Cys Val Val Met 80 85 90 95 ctg atc
ttg agc aat gcc cgg tta ttt ctg gag aac ctc atc aag tat 577 Leu Ile
Leu Ser Asn Ala Arg Leu Phe Leu Glu Asn Leu Ile Lys Tyr 100 105 110
ggc atc ctg gtg gac ccc atc cag gtg gtt tct ctg ttc ctg aag gat 625
Gly Ile Leu Val Asp Pro Ile Gln Val Val Ser Leu Phe Leu Lys Asp 115
120 125 ccc cat agc tgg ccc gcc cca tgc ctg gtt att gcg gcc aat gtc
ttt 673 Pro His Ser Trp Pro Ala Pro Cys Leu Val Ile Ala Ala Asn Val
Phe 130 135 140 gct gtg gct gca ttc cag gtt gag aag cgc ctg gcg gtg
ggt gcc ctg 721 Ala Val Ala Ala Phe Gln Val Glu Lys Arg Leu Ala Val
Gly Ala Leu 145 150 155 acg gag cag gcg gga ctg ctg ctg cac gta gcc
aac ctg gcc acc att 769 Thr Glu Gln Ala Gly Leu Leu Leu His Val Ala
Asn Leu Ala Thr Ile 160 165 170 175 ctg tgt ttc cca gcg gct gtg gtc
tta ctg gtt gag tct atc act cca 817 Leu Cys Phe Pro Ala Ala Val Val
Leu Leu Val Glu Ser Ile Thr Pro 180 185 190 gtg ggc tcc ctg ctg gcg
ctg atg gcg cac acc atc ctc ttc ctc aag 865 Val Gly Ser Leu Leu Ala
Leu Met Ala His Thr Ile Leu Phe Leu Lys 195 200 205 ctc ttc tcc tac
cgc gac gtc aac tca tgg tgc cgc agg gcc agg gcc 913 Leu Phe Ser Tyr
Arg Asp Val Asn Ser Trp Cys Arg Arg Ala Arg Ala 210 215 220 aag gct
gcc tct gca ggg aag aag gcc agc agt gct gct gcc ccg cac 961 Lys Ala
Ala Ser Ala Gly Lys Lys Ala Ser Ser Ala Ala Ala Pro His 225 230 235
acc gtg agc tac ccg gac aat ctg acc tac cgc gat ctc tac tac ttc
1009 Thr Val Ser Tyr Pro Asp Asn Leu Thr Tyr Arg Asp Leu Tyr Tyr
Phe 240 245 250 255 ctc ttc gcc ccc acc ttg tgc tac gag ctc aac ttt
ccc cgc tct ccc 1057 Leu Phe Ala Pro Thr Leu Cys Tyr Glu Leu Asn
Phe Pro Arg Ser Pro 260 265 270 cgc atc cgg aag cgc ttt ctg ctg cga
cgg atc ctt gag atg ctg ttc 1105 Arg Ile Arg Lys Arg Phe Leu Leu
Arg Arg Ile Leu Glu Met Leu Phe 275 280 285 ttc acc cag ctc cag gtg
ggg ctg atc cag cag tgg atg gtc ccc acc 1153 Phe Thr Gln Leu Gln
Val Gly Leu Ile Gln Gln Trp Met Val Pro Thr 290 295 300 atc cag aac
tcc atg aag ccc ttc aag gac atg gac tac tca cgc atc 1201 Ile Gln
Asn Ser Met Lys Pro Phe Lys Asp Met Asp Tyr Ser Arg Ile 305 310 315
atc gag cgc ctc ctg aag ctg gcg gtc ccc aat cac ctc atc tgg ctc
1249 Ile Glu Arg Leu Leu Lys Leu Ala Val Pro Asn His Leu Ile Trp
Leu 320 325 330 335 atc ttc ttc tac tgg ctc ttc cac tcc tgc ctg aat
gcc gtg gct gag 1297 Ile Phe Phe Tyr Trp Leu Phe His Ser Cys Leu
Asn Ala Val Ala Glu 340 345 350 ctc atg cag ttt gga gac cgg gag ttc
tac cgg gac tgg tgg aac tcc 1345 Leu Met Gln Phe Gly Asp Arg Glu
Phe Tyr Arg Asp Trp Trp Asn Ser 355 360 365 gag tct gtc acc tac ttc
tgg cag aac tgg aac atc cct gtg cac aag 1393 Glu Ser Val Thr Tyr
Phe Trp Gln Asn Trp Asn Ile Pro Val His Lys 370 375 380 tgg tgc atc
aga cac ttc tac aag ccc atg ctt cga cgg ggc agc agc 1441 Trp Cys
Ile Arg His Phe Tyr Lys Pro Met Leu Arg Arg Gly Ser Ser 385 390 395
aag tgg atg gcc agg aca ggg gtg ttc ctg gcc tcg gct ttc ttc cac
1489 Lys Trp Met Ala Arg Thr Gly Val Phe Leu Ala Ser Ala Phe Phe
His 400 405 410 415 gag tac ctg gtg agc gtc cct ctg cga atg ttc cgc
ctc tgg gct ttc 1537 Glu Tyr Leu Val Ser Val Pro Leu Arg Met Phe
Arg Leu Trp Ala Phe 420 425 430 acg ggc atg atg gct cag atc cca ctg
gcc tgg ttc gtg ggc cgc ttt 1585 Thr Gly Met Met Ala Gln Ile Pro
Leu Ala Trp Phe Val Gly Arg Phe 435 440 445 ttc cag ggc aac tat ggc
aac gca gct gtg tgg ctg tcg ctc atc atc 1633 Phe Gln Gly Asn Tyr
Gly Asn Ala Ala Val Trp Leu Ser Leu Ile Ile 450 455 460 gga cag cca
ata gcc gtc ctc atg tac gtc cac gac tac tac gtg ctc 1681 Gly Gln
Pro Ile Ala Val Leu Met Tyr Val His Asp Tyr Tyr Val Leu 465 470 475
aac tat gag gcc cca gcg gca gag gcc tga gctgcacctg agggcctggc 1731
Asn Tyr Glu Ala Pro Ala Ala Glu Ala 480 485 ttctcactgc cacctcaaac
ccgctgccag agcccacctc tcctcctagg cctcgagtgc 1791 tggggatggg
cctggctgca cagcatcctc ctctggtccc agggaggcct ctctgcccta 1851
tggggctctg tcctgcaccc ctcagggatg gcgacagcag gccagacaca gtctgatgcc
1911 agctgggagt cttgctgacc ctgccccggg tccgagggtg tcaataaagt
gctgtccagt 1971 gggag 1976 5 15 DNA Artificial Sequence PCR Primer
5 tccccgcatc cggaa 15 6 22 DNA Artificial Sequence PCR Primer 6
ctgggtgaag aacagcatct ca 22 7 22 DNA Artificial Sequence PCR Probe
7 cgctttctgc tgcgacggat cc 22 8 19 DNA Artificial Sequence PCR
Primer 8 gaaggtgaag gtcggagtc 19 9 20 DNA Artificial Sequence PCR
Primer 9 gaagatggtg atgggatttc 20 10 20 DNA Artificial Sequence PCR
Probe 10 caagcttccc gttctcagcc 20 11 1650 DNA M. musculus CDS
(96)...(1592) 11 ggatgaatgg aaataagtag aattaggcat acttaggata
gggctcaagc cgcggcccgt 60 gaagattggg ccgcgacgag gtgcgggccg aagcc atg
ggc gac cgc gga ggc 113 Met Gly Asp Arg Gly Gly 1 5 gcg gga agc tct
cgg cgt cgg agg acc ggc tcg cgg gtt tcc gtc cag 161 Ala Gly Ser Ser
Arg Arg Arg Arg Thr Gly Ser Arg Val Ser Val Gln 10 15 20 ggt ggt
agt ggg ccc aag gta gaa gag gac gag gtg cga gac gcg gct 209 Gly Gly
Ser Gly Pro Lys Val Glu Glu Asp Glu Val Arg Asp Ala Ala 25 30 35
gtg agc ccc gac ttg ggc gcc ggg ggt gac gcg ccg gct ccg gct ccg 257
Val Ser Pro Asp Leu Gly Ala Gly Gly Asp Ala Pro Ala Pro Ala Pro 40
45 50 gct cca gcc cat acc cgg gac aaa gac ggg cgg acc agc gtg ggc
gac 305 Ala Pro Ala His Thr Arg Asp Lys Asp Gly Arg Thr Ser Val Gly
Asp 55 60 65 70 ggc tac tgg gat ctg agg tgc cat cgt ctg caa gat tct
ttg ttc agc 353 Gly Tyr Trp Asp Leu Arg Cys His Arg Leu Gln Asp Ser
Leu Phe Ser 75 80 85 tca gac agt ggt ttc agc aat tat cgt ggt atc
ctg aat tgg tgt gtg 401 Ser Asp Ser Gly Phe Ser Asn Tyr Arg Gly Ile
Leu Asn Trp Cys Val 90 95 100 gtg atg ctg atc ctg agt aat gca agg
tta ttt tta gag aac ctt atc 449 Val Met Leu Ile Leu Ser Asn Ala Arg
Leu Phe Leu Glu Asn Leu Ile 105 110 115 aag tat ggc atc ctg gtg gat
cct atc cag gtg gtg tct ctg ttt ttg 497 Lys Tyr Gly Ile Leu Val Asp
Pro Ile Gln Val Val Ser Leu Phe Leu 120 125 130 aag gac ccc tac agc
tgg cct gcc cca tgc gtg att att gca tcc aat 545 Lys Asp Pro Tyr Ser
Trp Pro Ala Pro Cys Val Ile Ile Ala Ser Asn 135 140 145 150 att ttt
gtt gtg gct gca ttt cag att gag aag cgc ctg gca gtg ggt 593 Ile Phe
Val Val Ala Ala Phe Gln Ile Glu Lys Arg Leu Ala Val Gly 155 160 165
gcc ctg aca gag cag atg ggg ctg ctg cta cat gtg gtt aac ctg gcc 641
Ala Leu Thr Glu Gln Met Gly Leu Leu Leu His Val Val Asn Leu Ala 170
175 180 aca atc att tgc ttc cca gca gct gtg gcc tta ctg gtt gag tct
atc 689 Thr Ile Ile Cys Phe Pro Ala Ala Val Ala Leu Leu Val Glu Ser
Ile 185 190 195 act cca gtg ggt tcc gtg ttt gct ctg gca tca tac tcc
atc atg ttc 737 Thr Pro Val Gly Ser Val Phe Ala Leu Ala Ser Tyr Ser
Ile Met Phe 200 205 210 ctc aag ctt tat tcc tac cgg gat gtc aac ctg
tgg tgc cgc cag cga 785 Leu Lys Leu Tyr Ser Tyr Arg Asp Val Asn Leu
Trp Cys Arg Gln Arg 215 220 225 230 agg gtc aag gcc aaa gct gtc tct
aca ggg aag aag gtc agt ggg gct 833 Arg Val Lys Ala Lys Ala Val Ser
Thr Gly Lys Lys Val Ser Gly Ala 235 240 245 gct gcc cag caa gct gtg
agc tat cca gac aac ctg acc tac cga gat 881 Ala Ala Gln Gln Ala Val
Ser Tyr Pro Asp Asn Leu Thr Tyr Arg Asp 250 255 260 ctc tat tac ttc
atc ttt gct cct act ttg tgt tat gaa ctc aac ttt 929 Leu Tyr Tyr Phe
Ile Phe Ala Pro Thr Leu Cys Tyr Glu Leu Asn Phe 265 270 275 cct cgg
tcc ccc cga ata cga aag cgc ttt ctg cta cga cga gtt ctt 977 Pro Arg
Ser Pro Arg Ile Arg Lys Arg Phe Leu Leu Arg Arg Val Leu 280 285 290
gag atg ctc ttt ttt acc cag ctt caa gtg ggg ctg atc caa cag tgg
1025 Glu Met Leu Phe Phe Thr Gln Leu Gln Val Gly Leu Ile Gln Gln
Trp 295 300 305 310 atg gtc cct act atc cag aac tcc atg aag ccc ttc
aag gat atg gac 1073 Met Val Pro Thr Ile Gln Asn Ser Met Lys Pro
Phe Lys Asp Met Asp 315 320 325 tat tca cgg atc att gag cgt ctc tta
aag ctg gcg gtc ccc aac cat 1121 Tyr Ser Arg Ile Ile Glu Arg Leu
Leu Lys Leu Ala Val Pro Asn His 330 335 340 ctg atc tgg ctt atc ttc
ttc tat tgg ttt ttc cac tcc tgt ctc aat 1169 Leu Ile Trp Leu Ile
Phe Phe Tyr Trp Phe Phe His Ser Cys Leu Asn 345 350 355 gct gtg gca
gag ctt ctg cag ttt gga gac cgc gag ttc tac aga gat 1217 Ala Val
Ala Glu Leu Leu Gln Phe Gly Asp Arg Glu Phe Tyr Arg Asp 360 365 370
tgg tgg aat gct gag tct gtc acc tac ttt tgg cag aac tgg aat atc
1265 Trp Trp Asn Ala Glu Ser Val Thr Tyr Phe Trp Gln Asn Trp Asn
Ile 375 380 385 390 ccc gtg cac aag tgg tgc atc aga cac ttc tac aag
cct atg ctc aga 1313 Pro Val His Lys Trp Cys Ile Arg His Phe Tyr
Lys Pro Met Leu Arg 395 400 405 cat ggc agc agc aaa tgg gtg gcc agg
aca gga gta ttt ttg acc tca 1361 His Gly Ser Ser Lys Trp Val Ala
Arg Thr Gly Val Phe Leu Thr Ser 410 415 420 gcc ttc ttc cat gag tac
cta gtg agc gtt ccc ctg cgg atg ttc cgc 1409 Ala Phe Phe His Glu
Tyr Leu Val Ser Val Pro Leu Arg Met Phe Arg 425 430 435 ctc tgg gca
ttc aca gcc atg atg gct cag gtc cca ctg gcc tgg att 1457 Leu Trp
Ala Phe Thr Ala Met Met Ala Gln Val Pro Leu Ala Trp Ile 440 445 450
gtg ggc cga ttc ttc caa ggg aac tat ggc aat gca gct gtg tgg gtg
1505 Val Gly Arg Phe Phe Gln Gly Asn Tyr Gly Asn Ala Ala Val Trp
Val 455 460 465 470 aca ctc atc att ggg caa ccg gtg gct gtg ctc atg
tat gtc cac gac 1553 Thr Leu Ile Ile Gly Gln Pro Val Ala Val Leu
Met Tyr Val His Asp 475 480 485 tac tac gtg ctc aac tac gat gcc cca
gtg ggg gta tga gctactgcca 1602 Tyr Tyr Val Leu Asn Tyr Asp Ala Pro
Val Gly Val 490 495 aaggccagcc ctccctaacc tgggcctgga gttctggagg
ggttcctg 1650 12 18 DNA Artificial Sequence PCR Primer 12
gttccgcctc tgggcatt 18 13 18 DNA Artificial Sequence PCR Primer 13
gaatcggccc acaatcca 18 14 25 DNA Artificial Sequence PCR Probe 14
cagccatgat ggctcaggtc ccact 25 15 20 DNA Artificial Sequence PCR
Primer 15 ggcaaattca acggcacagt 20 16 20 DNA Artificial Sequence
PCR Primer 16 gggtctcgct cctggaagat 20 17 27 DNA Artificial
Sequence PCR Probe 17 aaggccgaga atgggaagct tgtcatc 27 18 20 DNA
Artificial Sequence Antisense Oligonucleotide 18 gccgcctctc
tcgtccattc 20 19 20 DNA Artificial Sequence Antisense
Oligonucleotide 19 gagccgctaa ctaatggacg 20 20 20 DNA Artificial
Sequence Antisense Oligonucleotide 20 acaacggctg cgttgctccg 20 21
20 DNA Artificial Sequence Antisense Oligonucleotide 21 ccgcccgcgt
caggcccgtc 20 22 20 DNA Artificial Sequence Antisense
Oligonucleotide 22 gcctcaccag cgcgttcaac 20 23 20 DNA Artificial
Sequence Antisense Oligonucleotide 23 ccctgccggc cgccgtagcc 20 24
20 DNA Artificial Sequence Antisense Oligonucleotide 24 ctccgggccc
tagacaacgg 20 25 20 DNA Artificial Sequence Antisense
Oligonucleotide 25 gttcgtagcg cccgaggcgc 20 26 20 DNA Artificial
Sequence Antisense Oligonucleotide 26 cccggccgca gccaagcgtg 20 27
20 DNA Artificial Sequence Antisense Oligonucleotide 27 gcccatggcc
tcagcccgca 20 28 20 DNA Artificial Sequence Antisense
Oligonucleotide 28 tggctcgagg gccgcgaccc 20 29 20 DNA Artificial
Sequence Antisense Oligonucleotide 29 ccgcaggccc gccgccgccg 20 30
20 DNA Artificial Sequence Antisense Oligonucleotide 30 ccgcacctct
tcttccgccg 20 31 20 DNA Artificial Sequence Antisense
Oligonucleotide 31 acgccggcgt ctccgtcctt 20 32 20 DNA Artificial
Sequence Antisense Oligonucleotide 32 gctcccagtg gccgctgccc 20 33
20 DNA Artificial Sequence Antisense Oligonucleotide 33 ctgcaggcga
tggcacctca 20 34 20 DNA Artificial Sequence Antisense
Oligonucleotide 34 aggatgccac ggtagttgct 20 35 20 DNA Artificial
Sequence Antisense Oligonucleotide 35 gcatcaccac acaccagttc 20 36
20 DNA Artificial Sequence Antisense Oligonucleotide 36 gccatacttg
atgaggttct 20 37 20 DNA Artificial Sequence Antisense
Oligonucleotide 37 gacattggcc gcaataacca 20 38 20 DNA Artificial
Sequence Antisense Oligonucleotide 38 cttctcaacc tggaatgcag 20 39
20 DNA Artificial Sequence Antisense Oligonucleotide 39 gcagtcccgc
ctgctccgtc 20 40 20 DNA Artificial Sequence Antisense
Oligonucleotide 40 caggttggct acgtgcagca 20 41 20 DNA Artificial
Sequence Antisense Oligonucleotide 41 ccagtaagac cacagccgct 20 42
20 DNA Artificial Sequence Antisense
Oligonucleotide 42 ggtgtgcgcc atcagcgcca 20 43 20 DNA Artificial
Sequence Antisense Oligonucleotide 43 cagcactgct ggccttcttc 20 44
20 DNA Artificial Sequence Antisense Oligonucleotide 44 tgagctcgta
gcacaaggtg 20 45 20 DNA Artificial Sequence Antisense
Oligonucleotide 45 cactgctgga tcagccccac 20 46 20 DNA Artificial
Sequence Antisense Oligonucleotide 46 atgcgtgagt agtccatgtc 20 47
20 DNA Artificial Sequence Antisense Oligonucleotide 47 tgagccagat
gaggtgattg 20 48 20 DNA Artificial Sequence Antisense
Oligonucleotide 48 gagctcagcc acggcattca 20 49 20 DNA Artificial
Sequence Antisense Oligonucleotide 49 tctgccagaa gtaggtgaca 20 50
20 DNA Artificial Sequence Antisense Oligonucleotide 50 gatgagcgac
agccacacag 20 51 20 DNA Artificial Sequence Antisense
Oligonucleotide 51 ctcatagttg agcacgtagt 20 52 20 DNA Artificial
Sequence Antisense Oligonucleotide 52 cagtgagaag ccaggccctc 20 53
20 DNA Artificial Sequence Antisense Oligonucleotide 53 ccatccccag
cactcgaggc 20 54 20 DNA Artificial Sequence Antisense
Oligonucleotide 54 aggatgctgt gcagccaggc 20 55 20 DNA Artificial
Sequence Antisense Oligonucleotide 55 ggtgcaggac agagccccat 20 56
20 DNA Artificial Sequence Antisense Oligonucleotide 56 gtgtctggcc
tgctgtcgcc 20 57 20 DNA Artificial Sequence Antisense
Oligonucleotide 57 ctcccagctg gcatcagact 20 58 20 DNA Artificial
Sequence Antisense Oligonucleotide 58 ctacttattt ccattcatcc 20 59
20 DNA Artificial Sequence Antisense Oligonucleotide 59 tatcctaagt
atgcctaatt 20 60 20 DNA Artificial Sequence Antisense
Oligonucleotide 60 gcttgagccc tatcctaagt 20 61 20 DNA Artificial
Sequence Antisense Oligonucleotide 61 ctcgtcgcgg cccaatcttc 20 62
20 DNA Artificial Sequence Antisense Oligonucleotide 62 cccatggctt
cggcccgcac 20 63 20 DNA Artificial Sequence Antisense
Oligonucleotide 63 cagccgcgtc tcgcacctcg 20 64 20 DNA Artificial
Sequence Antisense Oligonucleotide 64 cggagccggc gcgtcacccc 20 65
20 DNA Artificial Sequence Antisense Oligonucleotide 65 ccacgctggt
ccgcccgtct 20 66 20 DNA Artificial Sequence Antisense
Oligonucleotide 66 cagatcccag tagccgtcgc 20 67 20 DNA Artificial
Sequence Antisense Oligonucleotide 67 tcttgcagac gatggcacct 20 68
20 DNA Artificial Sequence Antisense Oligonucleotide 68 tcaggatacc
acgataattg 20 69 20 DNA Artificial Sequence Antisense
Oligonucleotide 69 cagcatcacc acacaccaat 20 70 20 DNA Artificial
Sequence Antisense Oligonucleotide 70 aaccttgcat tactcaggat 20 71
20 DNA Artificial Sequence Antisense Oligonucleotide 71 atccaccagg
atgccatact 20 72 20 DNA Artificial Sequence Antisense
Oligonucleotide 72 agagacacca cctggatagg 20 73 20 DNA Artificial
Sequence Antisense Oligonucleotide 73 cagcagcccc atctgctctg 20 74
20 DNA Artificial Sequence Antisense Oligonucleotide 74 gccaggttaa
ccacatgtag 20 75 20 DNA Artificial Sequence Antisense
Oligonucleotide 75 aaccagtaag gccacagctg 20 76 20 DNA Artificial
Sequence Antisense Oligonucleotide 76 cccactggag tgatagactc 20 77
20 DNA Artificial Sequence Antisense Oligonucleotide 77 atggagtatg
atgccagagc 20 78 20 DNA Artificial Sequence Antisense
Oligonucleotide 78 acccttcgct ggcggcacca 20 79 20 DNA Artificial
Sequence Antisense Oligonucleotide 79 tggatagctc acagcttgct 20 80
20 DNA Artificial Sequence Antisense Oligonucleotide 80 tctcggtagg
tcaggttgtc 20 81 20 DNA Artificial Sequence Antisense
Oligonucleotide 81 ctcaagaact cgtcgtagca 20 82 20 DNA Artificial
Sequence Antisense Oligonucleotide 82 gttggatcag ccccacttga 20 83
20 DNA Artificial Sequence Antisense Oligonucleotide 83 gtgaatagtc
catatccttg 20 84 20 DNA Artificial Sequence Antisense
Oligonucleotide 84 taagagacgc tcaatgatcc 20 85 20 DNA Artificial
Sequence Antisense Oligonucleotide 85 tctgccacag cattgagaca 20 86
20 DNA Artificial Sequence Antisense Oligonucleotide 86 ccaatctctg
tagaactcgc 20 87 20 DNA Artificial Sequence Antisense
Oligonucleotide 87 gtgacagact cagcattcca 20 88 20 DNA Artificial
Sequence Antisense Oligonucleotide 88 gtctgatgca ccacttgtgc 20 89
20 DNA Artificial Sequence Antisense Oligonucleotide 89 tgccatgtct
gagcataggc 20 90 20 DNA Artificial Sequence Antisense
Oligonucleotide 90 atactcctgt cctggccacc 20 91 20 DNA Artificial
Sequence Antisense Oligonucleotide 91 attgccatag ttcccttgga 20 92
20 DNA Artificial Sequence Antisense Oligonucleotide 92 agtgtcaccc
acacagctgc 20 93 20 DNA Artificial Sequence Antisense
Oligonucleotide 93 ccaccggttg cccaatgatg 20 94 20 DNA Artificial
Sequence Antisense Oligonucleotide 94 gtggacatac atgagcacag 20 95
20 DNA Artificial Sequence Antisense Oligonucleotide 95 tagttgagca
cgtagtagtc 20 96 20 DNA Artificial Sequence Antisense
Oligonucleotide 96 ctttggcagt agctcatacc 20 97 20 DNA Artificial
Sequence Antisense Oligonucleotide 97 tccagaactc caggcccagg 20 98
20 RNA H. sapiens 98 gaauggacga gagaggcggc 20 99 20 RNA H. sapiens
99 ccguugucua gggcccggag 20 100 20 RNA H. sapiens 100 gcgccucggg
cgcuacgaac 20 101 20 RNA H. sapiens 101 cacgcuuggc ugcggccggg 20
102 20 RNA H. sapiens 102 ugcgggcuga ggccaugggc 20 103 20 RNA H.
sapiens 103 gggucgcggc ccucgagcca 20 104 20 RNA H. sapiens 104
cggcggcggc gggccugcgg 20 105 20 RNA H. sapiens 105 aaggacggag
acgccggcgu 20 106 20 RNA H. sapiens 106 gggcagcggc cacugggagc 20
107 20 RNA H. sapiens 107 ugaggugcca ucgccugcag 20 108 20 RNA H.
sapiens 108 agcaacuacc guggcauccu 20 109 20 RNA H. sapiens 109
agaaccucau caaguauggc 20 110 20 RNA H. sapiens 110 ugguuauugc
ggccaauguc 20 111 20 RNA H. sapiens 111 gacggagcag gcgggacugc 20
112 20 RNA H. sapiens 112 agcggcugug gucuuacugg 20 113 20 RNA H.
sapiens 113 uggcgcugau ggcgcacacc 20 114 20 RNA H. sapiens 114
gaagaaggcc agcagugcug 20 115 20 RNA H. sapiens 115 caccuugugc
uacgagcuca 20 116 20 RNA H. sapiens 116 gacauggacu acucacgcau 20
117 20 RNA H. sapiens 117 caaucaccuc aucuggcuca 20 118 20 RNA H.
sapiens 118 ugaaugccgu ggcugagcuc 20 119 20 RNA H. sapiens 119
acuacgugcu caacuaugag 20 120 20 RNA H. sapiens 120 gagggccugg
cuucucacug 20 121 20 RNA H. sapiens 121 gccucgagug cuggggaugg 20
122 20 RNA H. sapiens 122 gccuggcugc acagcauccu 20 123 20 RNA H.
sapiens 123 auggggcucu guccugcacc 20 124 20 RNA H. sapiens 124
ggcgacagca ggccagacac 20 125 20 RNA H. sapiens 125 agucugaugc
cagcugggag 20 126 20 RNA M. musculus 126 gugcgggccg aagccauggg 20
127 20 RNA M. musculus 127 cgaggugcga gacgcggcug 20 128 20 RNA M.
musculus 128 ggggugacgc gccggcuccg 20 129 20 RNA M. musculus 129
agacgggcgg accagcgugg 20 130 20 RNA M. musculus 130 gcgacggcua
cugggaucug 20 131 20 RNA M. musculus 131 aggugccauc gucugcaaga 20
132 20 RNA M. musculus 132 caauuaucgu gguauccuga 20 133 20 RNA M.
musculus 133 auuggugugu ggugaugcug 20 134 20 RNA M. musculus 134
auccugagua augcaagguu 20 135 20 RNA M. musculus 135 cagagcagau
ggggcugcug 20 136 20 RNA M. musculus 136 cuacaugugg uuaaccuggc 20
137 20 RNA M. musculus 137 gcucuggcau cauacuccau 20 138 20 RNA M.
musculus 138 uggugccgcc agcgaagggu 20 139 20 RNA M. musculus 139
agcaagcugu gagcuaucca 20 140 20 RNA M. musculus 140 ugcuacgacg
aguucuugag 20 141 20 RNA M. musculus 141 caaggauaug gacuauucac 20
142 20 RNA M. musculus 142 ugucucaaug cuguggcaga 20 143 20 RNA M.
musculus 143 gcgaguucua cagagauugg 20 144 20 RNA M. musculus 144
uggaaugcug agucugucac 20 145 20 RNA M. musculus 145 gcacaagugg
ugcaucagac 20 146 20 RNA M. musculus 146 gccuaugcuc agacauggca 20
147 20 RNA M. musculus 147 gguggccagg acaggaguau 20 148 20 RNA M.
musculus 148 uccaagggaa cuauggcaau 20 149 20 RNA M. musculus 149
gcagcugugu gggugacacu 20 150 20 RNA M. musculus 150 caucauuggg
caaccggugg 20 151 20 RNA M. musculus 151 cugugcucau guauguccac 20
152 20 RNA M. musculus 152 ccugggccug gaguucugga 20
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