U.S. patent application number 10/238442 was filed with the patent office on 2003-09-18 for antisense modulation of p38 mitogen activated protein kinase expression.
Invention is credited to Gaarde, William A., McKay, Robert, Monia, Brett P., Nero, Pamela.
Application Number | 20030176383 10/238442 |
Document ID | / |
Family ID | 23100663 |
Filed Date | 2003-09-18 |
United States Patent
Application |
20030176383 |
Kind Code |
A1 |
Monia, Brett P. ; et
al. |
September 18, 2003 |
Antisense modulation of p38 mitogen activated protein kinase
expression
Abstract
Compositions and methods for the treatment and diagnosis of
diseases or conditions amenable to treatment through modulation of
expression of a gene encoding a p38 mitogen-activated protein
kinase (p38 MAPK) are provided. Methods for the treatment and
diagnosis of diseases or conditions associated with aberrant
expression of one or more p38 MAPKs are also provided.
Inventors: |
Monia, Brett P.; (La Costa,
CA) ; Gaarde, William A.; (Carlsbad, CA) ;
Nero, Pamela; (San Diego, CA) ; McKay, Robert;
(San Diego, CA) |
Correspondence
Address: |
Licata & Tyrrell P.C.
66 E. Main Street
Marlton
NJ
08053
US
|
Family ID: |
23100663 |
Appl. No.: |
10/238442 |
Filed: |
September 9, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10238442 |
Sep 9, 2002 |
|
|
|
09640101 |
Aug 15, 2000 |
|
|
|
6448079 |
|
|
|
|
09640101 |
Aug 15, 2000 |
|
|
|
09286904 |
Apr 6, 1999 |
|
|
|
6140124 |
|
|
|
|
Current U.S.
Class: |
514/44A ;
435/455; 514/81; 536/23.2 |
Current CPC
Class: |
C12N 2310/3341 20130101;
C12N 2310/321 20130101; C12N 2310/346 20130101; A61P 43/00
20180101; A61P 29/00 20180101; C12Y 207/11024 20130101; C12N
2310/3525 20130101; A61K 38/00 20130101; C12N 2310/315 20130101;
A61P 37/06 20180101; Y02P 20/582 20151101; C12N 2310/341 20130101;
A61P 19/02 20180101; C12N 15/1137 20130101; C12N 2310/321
20130101 |
Class at
Publication: |
514/44 ; 514/81;
435/455; 536/23.2 |
International
Class: |
A61K 048/00; C07H
021/04; A61K 031/675; C12N 015/85 |
Claims
What is claimed is:
1. An antisense compound 8 to 30 nucleobases in length targeted to
the 5'-untranslated region, translational start site, translational
termination region or 3'-untranslated region of a nucleic acid
molecule encoding a p38 mitogen activated protein kinase, wherein
said antisense compound inhibits the expression of said p38 mitogen
activated protein kinase.
2. The antisense compound of claim 1 which is an antisense
oligonucleotide.
3. The antisense compound of claim 2 wherein the antisense
oligonucleotide comprises at least an 8-nucleobase portion of SEQ
ID NO: 17, 20, 38, 39, 41, 42, 43, 78, 94 or 95.
4. The antisense compound of claim 2 wherein the antisense
oligonucleotide comprises at least one modified internucleoside
linkage.
5. The antisense compound of claim 4 wherein the modified
internucleoside linkage is a phosphorothioate linkage.
6. The antisense compound of claim 2 wherein the antisense
oligonucleotide comprises at least one modified sugar moiety.
7. The antisense compound of claim 6 wherein the modified sugar
moiety is a 2'-O-methoxyethyl moiety.
8. The antisense compound of claim 2 wherein the antisense
oligonucleotide comprises at least one modified nucleobase.
9. The antisense compound of claim 8 wherein modified nucleobase is
a 5-methyl cytosine.
10. The antisense compound of claim 2 wherein the antisense
oligonucleotide is a chimeric oligonucleotide.
11. A pharmaceutical composition comprising the antisense compound
of claim 1 and a pharmaceutically acceptable carrier or
diluent.
12. The pharmaceutical composition of claim 11 further comprising a
colloidal dispersion system.
13. The pharmaceutical composition of claim 11 wherein the
antisense compound is an antisense oligonucleotide.
14. A method of inhibiting the expression of a p38 mitogen
activated protein kinase in cells or tissues comprising contacting
said cells or tissue with the antisense compound of claim 1 so that
expression of said p38 mitogen activated protein kinase is
inhibited.
15. A method of treating an animal having a disease or condition
associated with a p38 mitogen activated protein kinase comprising
administering to said animal a therapeutically or prophylactically
effective amount of the antisense compound of claim 1 so that
expression of said p38 mitogen-activated protein kinase is
inhibited.
16. The method of claim 15 wherein the disease or condition is an
inflammatory or autoimmune disease.
17. The method of claim 16 wherein said inflammatory or autoimmune
disease or condition is rheumatoid arthritis.
18. The method of claim 15 wherein said disease or condition is
heart disease.
19. An antisense compound 8 to 30 nucleobases in length targeted to
the coding region of a nucleic acid molecule encoding a p38
mitogen-activated protein kinase, wherein said antisense compound
inhibits the expression of said p38 mitogen-activated protein
kinase and comprises at least an 8-nucleobase portion of SEQ ID NO.
13, 26, 27, 28, 33, 35, 37, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 97, 98, 99, 100, 101, 102, 103, 104, 105 or
106.
20. The antisense compound of claim 19 which is an antisense
oligonucleotide.
21. The antisense compound of claim 20 wherein the antisense
oligonucleotide comprises at least one modified internucleoside
linkage.
22. The antisense compound of claim 21 wherein the modified
internucleoside linkage is a phosphorothioate linkage.
23. The antisense compound of claim 20 wherein the antisense
oligonucleotide comprises at least one modified sugar moiety.
24. The antisense compound of claim 23 wherein the modified sugar
moiety is a 2'-O-methoxyethyl moiety.
25. The antisense compound of claim 20 wherein the antisense
oligonucleotide comprises at least one modified nucleobase.
26. The antisense compound of claim 25 wherein modified nucleobase
is a 5-methyl cytosine.
27. The antisense compound of claim 20 wherein the antisense
oligonucleotide is a chimeric oligonucleotide.
28. A pharmaceutical composition comprising the antisense compound
of claim 19 and a pharmaceutically acceptable carrier or
diluent.
29. The pharmaceutical composition of claim 28 further comprising a
colloidal dispersion system.
30. The pharmaceutical composition of claim 28 wherein the
antisense compound is an antisense oligonucleotide.
31. A method of inhibiting the expression of p38 mitogen-activated
protein kinase in cells or tissues comprising contacting said cells
or tissue with the antisense compound of claim 19 so that
expression of p38 mitogen-activated protein kinase is
inhibited.
32. A method of treating an animal having a disease or condition
associated with a p38 mitogen activated protein kinase comprising
administering to said animal a therapeutically or prophylactically
effective amount of the antisense compound of claim 1 so that
expression of said p38 mitogen activated protein kinase is
inhibited.
33. The method of claim 32 wherein the disease or condition is an
inflammatory or autoimmune disease.
34. The method of claim 33 wherein said inflammatory or autoimmune
disease or condition is rheumatoid arthritis.
35. The method of claim 32 wherein said disease or condition is
heart disease.
36. An antisense compound 8 to 30 nucleobases in length targeted to
p38.alpha. mitogen activated protein kinase, wherein said antisense
compound inhibits the expression of said p38.alpha. mitogen
activated protein kinase and does not substantially inhibit the
expression of p38.beta. mitogen activated protein kinase.
37. An antisense compound 8 to 30 nucleobases in length targeted to
p38.beta. mitogen activated protein kinase, wherein said antisense
compound inhibits the expression of said p38.beta. mitogen
activated protein kinase and does not substantially inhibit the
expression of p38.alpha. mitogen activated protein kinase.
Description
INTRODUCTION
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/640,101 filed Aug. 15, 2000 which is a
continuation-in-part of U.S. patent application Ser. No.
09/286,904, filed Apr. 6, 1999.
FIELD OF THE INVENTION
[0002] This invention relates to compositions and methods for
modulating expression of p38 mitogen activated protein kinase
genes, a family of naturally present cellular genes involved in
signal transduction, and inflammatory and apoptotic responses. This
invention is also directed to methods for inhibiting inflammation
or apoptosis; these methods can be used diagnostically or
therapeutically. Furthermore, this invention is directed to
treatment of diseases or conditions associated with expression of
p38 mitogen activated protein kinase genes.
BACKGROUND OF THE INVENTION
[0003] Cellular responses to external factors, such as growth
factors, cytokines, and stress conditions, result in altered gene
expression. These signals are transmitted from the cell surface to
the nucleus by signal transduction pathways. Beginning with an
external factor binding to an appropriate receptor, a cascade of
signal transduction events is initiated. These responses are
mediated through activation of various enzymes and the subsequent
activation of specific transcription factors. These activated
transcription factors then modulate the expression of specific
genes.
[0004] The phosphorylation of enzymes plays a key role in the
transduction of extracellular signals into the cell. Mitogen
activated protein kinases (MAPKs), enzymes which effect such
phosphorylations are targets for the action of growth factors,
hormones, and other agents involved in cellular metabolism,
proliferation and differentiation (Cobb et al., J. Biol. Chem.,
1995, 270, 14843). Mitogen activated protein kinases were initially
discovered due to their ability to be tyrosine phosphorylated in
response to exposure to bacterial lipopolysaccharides or
hyperosmotic conditons (Han et al, Science, 1994, 265, 808). These
conditions activate inflammatory and apoptotic responses mediated
by MAPK. In general, MAP kinases are involved in a variety of
signal transduction pathways (sometimes overlapping and sometimes
parallel) that function to convey extracellular stimuli to
protooncogene products to modulate cellular proliferation and/or
differentiation (Seger et al., FASEB J., 1995, 9, 726; Cano et al.,
Trends Biochem. Sci., 1995, 20, 117).
[0005] One of the MAPK signal transduction pathways involves the
MAP kinases p38.alpha. and p38.beta._(also known as CSaids Binding
Proteins, CSBP). These MAP kinases are responsible for the
phosphorylation of ATF-2, MEFC2 and a variety of other cellular
effectors that may serve as substrates for p38 MAPK proteins
(Kummer et al, J. Biol. Chem., 1997, 272, 20490). Phosphorylation
of p38 MAPKs potentiates the ability of these factors to activate
transcription (Raingeaud et al, Mol. Cell Bio., 1996, 16, 1247; Han
et al, Nature, 1997, 386, 296). Among the genes activated by the
p38 MAPK signaling pathway is IL-6 (De Cesaris, P., et al., J.
Biol. Chem., 1998, 273, 7566-7571).
[0006] Besides p38.alpha. and p38.beta., other p38 MAPK family
members have been described, including p38.gamma. (Li et al,
Biochem. Biophys. Res. Commun., 1996, 228, 334), and p38.delta.
(Jiang et al, J. Biol. Chem., 1997, 272, 30122). The term "p38" as
used herein shall mean a member of the p38 MAPK family, including
but not limited to p38.alpha., p38.beta., p38.gamma. and
p38.delta., their isoforms (Kumar et al, Biochem. Biophys. Res.
Commun., 1997, 235, 533) and other members of the p38 MAPK family
of proteins whether they function as p38 MAP kinases per se or
not.
[0007] Modulation of the expression of one or more p38 MAPKs is
desirable in order to interfere with inflammatory or apoptotic
responses associated with disease states and to modulate the
transcription of genes stimulated by ATF-2, MEFC2 and other p38
MAPK phosphorylation substrates.
[0008] Inhibitors of p38 MAPKs have been shown to have efficacy in
animal models of arthritis (Badger, A. M., et al., J. Pharmacol.
Exp. Ther., 1996, 279, 1453-1461) and angiogenesis (Jackson, J. R.,
et al., J. Pharmacol. Exp. Ther., 1998, 284, 687-692). MacKay, K.
and Mochy-Rosen, D. (J. Biol. Chem., 1999, 274, 6272-6279)
demonstrate that an inhibitor of p38 MAPKs prevents apoptosis
during ischemia in cardiac myocytes, suggesting that p38 MAPK
inhibitors can be used for treating ischemic heart disease. p38
MAPK also is required for T-cell HIV-1 replication (Cohen et al,
Mol. Med., 1997, 3, 339) and may be a useful target for AIDS
therapy. Other diseases believed to be amenable to treatment by
inhibitors of p38 MAPKs are disclosed in U.S. Pat. No. 5,559,137,
herein incorporated by reference.
[0009] Therapeutic agents designed to target p38 MAPKs include
small molecule inhibitors and antisense oligonucleotides. Small
molecule inhibitors based on pyridinylimidazole are described in
U.S. Pat. Nos. 5,670,527; 5,658,903; 5,656,644; 5,559,137;
5,593,992; and 5,593,991. WO 98/27098 and WO 99/00357 describe
additional small molecule inhibitors, one of which has entered
clinical trials. Other small molecule inhibitors are also
known.
[0010] Antisense therapy represents a potentially more specific
therapy for targeting p38 MAPKs and, in particular, specific p38
MAPK isoforms. Nagata, Y., et al. (Blood, 1998, 6, 1859-1869)
disclose an antisense phosphothioester oligonucleotide targeted to
the translational start site of mouse p38b (p38.beta.). Aoshiba,
K., et al. (J. Immunol., 1999, 162, 1692-1700) and Cohen, P. S., et
al. (Mol. Med., 1997, 3, 339-346) disclose a phosphorothioate
antisense oligonucleotide targeted to the coding regions of human
p38.alpha., human p38.beta. and rat p38.
[0011] There remains a long-felt need for improved compositions and
methods for modulating the expression of p38 MAP kinases.
SUMMARY OF THE INVENTION
[0012] The present invention provides antisense compounds which are
targeted to nucleic acids encoding a p38 MAPK and are capable of
modulating p38 MAPK expression. The present invention also provides
oligonucleotides targeted to nucleic acids encoding a p38 MAPK. The
present invention also comprises methods of modulating the
expression of a p38 MAPK, in cells and tissues, using the
oligonucleotides of the invention. Methods of inhibiting p38 MAPK
expression are provided; these methods are believed to be useful
both therapeutically and diagnostically. These methods are also
useful as tools, for example, for detecting and determining the
role of p38 MAPKs in various cell functions and physiological
processes and conditions and for diagnosing conditions associated
with expression of p38 MAPKs.
[0013] The present invention also comprises methods for diagnosing
and treating inflammatory diseases, particularly rheumatoid
arthritis. These methods are believed to be useful, for example, in
diagnosing p38 MAPK-associated disease progression. These methods
employ the oligonucleotides of the invention. These methods are
believed to be useful both therapeutically, including
prophylactically, and as clinical research and diagnostic
tools.
DETAILED DESCRIPTION OF THE INVENTION
[0014] p38 MAPKs play an important role in signal transduction in
response to cytokines, growth factors and other cellular stimuli.
Specific responses elicited by p38 include inflammatory and
apoptotic responses. Modulation of p38 may be useful in the
treatment of inflammatory diseases, such as rheumatoid
arthritis.
[0015] The present invention employs antisense compounds,
particularly oligonucleotides, for use in modulating the function
of nucleic acid molecules encoding a p38 MAPK, ultimately
modulating the amount of a p38 MAPK produced. This is accomplished
by providing oligonucleotides which specifically hybridize with
nucleic acids, preferably mRNA, encoding a p38 MAPK.
[0016] The antisense compounds may be used to modulate the function
of a particular p38 MAPK isoform, e.g. for research purposes to
determine the role of a particular isoform in a normal or disease
process, or to treat a disease or condition that may be associated
with a particular isoform. It may also be desirable to target
multiple p38 MAPK isoforms. In each case, antisense compounds can
be designed by taking advantage of sequence homology between the
various isoforms. If an antisense compound to a particular isoform
is desired, then the antisense compound is designed to a unique
region in the desired isoform's gene sequence. With such a
compound, it is desirable that this compound does not inhibit the
expression of other isoforms. Less desirable, but acceptable, are
compounds that do not "substantially" inhibit other isoforms. By
"substantially", it is intended that these compounds do not inhibit
the expression of other isoforms greater than 25%; more preferred
are compounds that do not inhibit other isoforms greater than 10%.
If an antisense compound is desired to target multiple p38
isoforms, then regions of significant homology between the isoforms
can be used.
[0017] This relationship between an antisense compound such as an
oligonucleotide and its complementary nucleic acid target, to which
it hybridizes, is commonly referred to as "antisense". "Targeting"
an oligonucleotide to a chosen nucleic acid target, in the context
of this invention, is a multistep process. The process usually
begins with identifying a nucleic acid sequence whose function is
to be modulated. This may be, as examples, a cellular gene (or mRNA
made from the gene) whose expression is associated with a
particular disease state, or a foreign nucleic acid from an
infectious agent. In the present invention, the target is a nucleic
acid encoding a p38 MAPK; in other words, a p38 MAPK gene or RNA
expressed from a p38 MAPK gene. p38 MAPK mRNA is presently the
preferred target. The targeting process also includes determination
of a site or sites within the nucleic acid sequence for the
antisense interaction to occur such that modulation of gene
expression will result.
[0018] In accordance with this invention, persons of ordinary skill
in the art will understand that messenger RNA includes not only the
information to encode a protein using the three letter genetic
code, but also associated ribonucleotides which form a region known
to such persons as the 5'-untranslated region, the 3'-untranslated
region, the 5' cap region and intron/exon junction ribonucleotides.
Thus, oligonucleotides may be formulated in accordance with this
invention which are targeted wholly or in part to these associated
ribonucleotides as well as to the informational ribonucleotides.
The oligonucleotide may therefore be specifically hybridizable with
a transcription initiation site region, a translation initiation
codon region, a 5' cap region, an intron/exon junction, coding
sequences, a translation termination codon region or sequences in
the 5'- or 3'-untranslated region. 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 (prokaryotes). It is also known in the art that
eukaryotic and prokaryotic genes may have two or more alternative
start codons, any one of which may be preferentially utilized for
translation initiation in a particular cell type or tissue, or
under a particular set of conditions. In the context of the
invention, "start codon" and "translation initiation codon" refer
to the codon or codons that are used in vivo to initiate
translation of an mRNA molecule transcribed from a gene encoding
p38, 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). 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. This region is a preferred target region.
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. This region is a preferred target region. The
open reading frame (ORF) or "coding region," which is known in the
art to refer to the region between the translation initiation codon
and the translation termination codon, is also a region which may
be targeted effectively. Other preferred 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). mRNA splice sites may also be preferred target regions, and
are particularly useful in situations where aberrant splicing is
implicated in disease, or where an overproduction of a particular
mRNA splice product is implicated in disease. Aberrant fusion
junctions due to rearrangements or deletions may also be preferred
targets.
[0019] Once the target site or sites have been identified,
oligonucleotides are chosen which are sufficiently complementary to
the target, i.e., hybridize sufficiently well and with sufficient
specificity, to give the desired modulation.
[0020] "Hybridization", in the context of this invention, means
hydrogen bonding, also known as Watson-Crick base pairing, between
complementary bases, usually on opposite nucleic acid strands or
two regions of a nucleic acid strand. Guanine and cytosine are
examples of complementary bases which are known to form three
hydrogen bonds between them. Adenine and thymine are examples of
complementary bases which form two hydrogen bonds between them.
[0021] "Specifically hybridizable" and "complementary" are terms
which are used to indicate a sufficient degree of complementarity
such that stable and specific binding occurs between the DNA or RNA
target and the oligonucleotide.
[0022] It is understood that an oligonucleotide need not be 100%
complementary to its target nucleic acid sequence to be
specifically hybridizable. An oligonucleotide is specifically
hybridizable when binding of the oligonucleotide to the target
interferes with the normal function of the target molecule to cause
a loss of utility, and there is a sufficient degree of
complementarity to avoid non-specific binding of the
oligonucleotide to non-target sequences under conditions in which
specific binding is desired, i.e., under physiological conditions
in the case of in vivo assays or therapeutic treatment and, in the
case of in vitro assays, under conditions in which the assays are
conducted.
[0023] Hybridization of antisense oligonucleotides with mRNA
interferes with one or more of the normal functions of mRNA. The
functions of mRNA to be interfered with include all vital functions
such as, for example, translocation of the RNA to the site of
protein translation, translation of protein from the RNA, splicing
of the RNA to yield one or more mRNA species, and catalytic
activity which may be engaged in by the RNA.
[0024] The overall effect of interference with mRNA function is
modulation of p38 MAPK expression. In the context of this invention
"modulation" means either inhibition or stimulation; i.e., either a
decrease or increase in expression. This modulation can be measured
in ways which are routine in the art, for example by Northern blot
assay of mRNA expression as taught in the examples of the instant
application or by Western blot or ELISA assay of protein
expression, or by an immunoprecipitation assay of protein
expression, as taught in the examples of the instant application.
Effects on cell proliferation or tumor cell growth can also be
measured, as taught in the examples of the instant application.
[0025] The oligonucleotides of this invention can be used in
diagnostics, therapeutics, prophylaxis, and as research reagents
and in kits. Since the oligonucleotides of this invention hybridize
to nucleic acids encoding a p38 MAPK, sandwich, calorimetric and
other assays can easily be constructed to exploit this fact.
Furthermore, since the oligonucleotides of this invention hybridize
specifically to nucleic acids encoding particular isoforms of p38
MAPK, such assays can be devised for screening of cells and tissues
for particular p38 MAPK isoforms. Such assays can be utilized for
diagnosis of diseases associated with various p38 MAPK isoforms.
Provision of means for detecting hybridization of oligonucleotide
with a p38 MAPK gene or mRNA can routinely be accomplished. Such
provision may include enzyme conjugation, radiolabelling or any
other suitable detection systems. Kits for detecting the presence
or absence of p38 MAPK may also be prepared.
[0026] The present invention is also suitable for diagnosing
abnormal inflammatory states in tissue or other samples from
patients suspected of having an inflammatory disease such as
rheumatoid arthritis. The ability of the oligonucleotides of the
present invention to inhibit inflammation may be employed to
diagnose such states. A number of assays may be formulated
employing the present invention, which assays will commonly
comprise contacting a tissue sample with an oligonucleotide of the
invention under conditions selected to permit detection and,
usually, quantitation of such inhibition. In the context of this
invention, to "contact" tissues or cells with an oligonucleotide or
oligonucleotides means to add the oligonucleotide(s), usually in a
liquid carrier, to a cell suspension or tissue sample, either in
vitro or ex vivo, or to administer the oligonucleotide(s) to cells
or tissues within an animal. Similarly, the present invention can
be used to distinguish p38 MAPK-associated diseases, from diseases
having other etiologies, in order that an efficacious treatment
regime can be designed.
[0027] The oligonucleotides of this invention may also be used for
research purposes. Thus, the specific hybridization exhibited by
the oligonucleotides may be used for assays, purifications,
cellular product preparations and in other methodologies which may
be appreciated by persons of ordinary skill in the art.
[0028] In the context of this invention, the term "oligonucleotide"
refers to an oligomer or polymer of ribonucleic acid or
deoxyribonucleic acid. This term includes oligonucleotides composed
of naturally-occurring nucleobases, sugars and covalent intersugar
(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 binding to target and increased
stability in the presence of nucleases.
[0029] The antisense compounds in accordance with this invention
preferably comprise from about 5 to about 50 nucleobases.
Particularly preferred are antisense oligonucleotides comprising
from about 8 to about 30 nucleobases (i.e. from about 8 to about 30
linked nucleosides). Preferred embodiments comprise at least an
8-nucleobase portion of a sequence of an antisense compound which
inhibits the expression of a p38 mitogen activated kinase. As is
known in the art, a nucleoside is a base-sugar combination. The
base portion of the nucleoside is normally a heterocyclic base. The
two most common classes of such heterocyclic bases are the purines
and the pyrimidines. Nucleotides are nucleosides that further
include a phosphate group covalently linked to the sugar portion of
the nucleoside. For those nucleosides that include a pentofuranosyl
sugar, the phosphate group can be linked to either the 2=, 3= or
5=hydroxyl moiety of the sugar. In forming oligonucleotides, the
phosphate groups covalently link adjacent nucleosides to one
another to form a linear polymeric compound. In turn the respective
ends of this linear polymeric structure can be further joined to
form a circular structure, however, open linear structures are
generally preferred. Within the oligonucleotide structure, the
phosphate groups are commonly referred to as forming the
internucleoside backbone of the oligonucleotide. The normal linkage
or backbone of RNA and DNA is a 3=to 5=phosphodiester linkage.
[0030] Specific examples of some preferred modified
oligonucleotides envisioned for this invention include those
containing phosphorothioates, phosphotriesters, methyl
phosphonates, short chain alkyl or cycloalkyl intersugar linkages
or short chain heteroatomic or heterocyclic intersugar linkages.
Most preferred are oligonucleotides with phosphorothioates (usually
abbreviated in the art as P.dbd.S) and those with
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
backbones, wherein the native phosphodiester (usually abbreviated
in the art as P.dbd.O) backbone is represented as
O--P--O--CH.sub.2). Also preferred are oligonucleotides having
morpholino backbone structures (Summerton and Weller, U.S. Pat. No.
5,034,506). Further preferred are oligonucleotides with
NR--C(*)--CH.sub.2--CH.sub.2, CH.sub.2--NR--C(*)--CH.sub.2,
CH.sub.2--CH.sub.2--NR--C(*), C(*)--NR--CH.sub.2--CH.sub.2 and
CH.sub.2--C(*)--NR--CH.sub.2 backbones, wherein "*" represents O or
S (known as amide backbones; DeMesmaeker et al., WO 92/20823,
published Nov. 26, 1992). In other preferred embodiments, such as
the peptide nucleic acid (PNA) backbone, the phosphodiester
backbone of the oligonucleotide is replaced with a polyamide
backbone, the nucleobases being bound directly or indirectly to the
aza nitrogen atoms of the polyamide backbone (Nielsen et al.,
Science, 254, 1497 (1991); U.S. Pat. No. 5,539,082). Other
preferred modified oligonucleotides may contain one or more
substituted sugar moieties comprising one of the following at the
2' position: OH, SH, SCH.sub.3, F, OCN, OCH.sub.3OCH.sub.3,
OCH.sub.3O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nNH.sub.2 or
O(CH.sub.2).sub.nCH.sub.3 where n is from 1 to about 10; C.sub.1 to
C.sub.10 lower alkyl, alkoxyalkoxy, substituted lower alkyl,
alkaryl or aralkyl; Cl; Br; CN; CF.sub.3; OCF.sub.3; O-, S-, or
N-alkyl; O--, S--, or N-alkenyl; 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'-O-methoxyethyl [which can be written as
2'-O--CH.sub.2CH.sub.2OCH.sub.- 3, and is also known in the art as
2'-O-(2-methoxyethyl) or 2'-methoxyethoxy] [Martin et al., Helv.
Chim. Acta, 78, 486 (1995)]. Other preferred modifications include
2'-methoxy (2'-O--CH.sub.3), 2'-propoxy
(2'-OCH.sub.2CH.sub.2CH.sub.3), 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2'-fluoro (2'-F). 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. Similar
modifications may also be made at other positions on the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide and the 5' position of the 5' terminal
nucleotide. Oligonucleotides may also have sugar mimetics such as
cyclobutyls in place of the pentofuranosyl group.
[0031] The oligonucleotides of the invention may additionally or
alternatively include nucleobase modifications or substitutions. As
used herein, "unmodified" or "natural" nucleobases include adenine
(A), guanine (G), thymine (T), cytosine (C) and uracil (U).
Modified nucleobases include nucleobases found only infrequently or
transiently in natural nucleic acids, e.g., hypoxanthine,
6-methyladenine and 5-methylcytosine, as well as synthetic
nucleobases, e.g., 5-bromouracil, 5-hydroxymethyluracil,
8-azaguanine, 7-deazaguanine, N.sup.6(6-aminohexyl)adenine and 2,
6-diaminopurine [Kornberg, A., DNA Replication, 1974, W. H. Freeman
& Co., San Francisco, 1974, pp. 75-77; Gebeyehu, G., et al.,
Nucleic Acids Res., 15, 4513 (1987)]. 5-methylcytosine (5-me-C) is
presently a preferred nucleobase, particularly in combination with
2'-O-methoxyethyl modifications.
[0032] Another preferred additional or alternative modification of
the oligonucleotides of the invention involves chemically linking
to the oligonucleotide one or more lipophilic moieties which
enhance the cellular uptake of the oligonucleotide. Such lipophilic
moieties may be linked to an oligonucleotide at several different
positions on the oligonucleotide. Some preferred positions include
the 3' position of the sugar of the 3' terminal nucleotide, the 5'
position of the sugar of the 5' terminal nucleotide, and the 2'
position of the sugar of any nucleotide. The N.sup.6 position of a
purine nucleobase may also be utilized to link a lipophilic moiety
to an oligonucleotide of the invention (Gebeyehu, G., et al.,
Nucleic Acids Res., 1987, 15, 4513). Such lipophilic moieties
include but are not limited to a cholesteryl moiety [Letsinger et
al., Proc. Natl. Acad. Sci. USA, 86, 6553 (1989)], cholic acid
[Manoharan et al., Bioorg. Med. Chem. Let., 4, 1053 (1994)], a
thioether, e.g., hexyl-S-tritylthiol [Manoharan et al., Ann. N.Y.
Acad. Sci., 660, 306 (1992); Manoharan et al., Bioorg. Med. Chem.
Let., 3, 2765 (1993)], a thiocholesterol [Oberhauser et al., Nucl.
Acids Res., 20, 533 (1992)], an aliphatic chain, e.g., dodecandiol
or undecyl residues [Saison-Behmoaras et al., EMBO J., 10, 111
(1991); Kabanov et al., FEBS Lett., 259, 327 (1990); Svinarchuk et
al., Biochimie., 75, 49(1993)], a phospholipid, e.g.,
di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate [Manoharan et al.,
Tetrahedron Lett., 36, 3651 (1995); Shea et al., Nucl. Acids Res.,
18, 3777 (1990)], a polyamine or a polyethylene glycol chain
[Manoharan et al., Nucleosides & Nucleotides, 14, 969 (1995)],
or adamantane acetic acid [Manoharan et al., Tetrahedron Lett., 36,
3651 (1995)], a palmityl moiety [Mishra et al., Biochim. Biophys.
Acta, 1264, 229 (1995)], or an octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety [Crooke et al., J.
Pharmacol. Exp. Ther., 277, 923 (1996)]. Oligonucleotides
comprising lipophilic moieties, and methods for preparing such
oligonucleotides, as disclosed in U.S. Pat. No. 5,138,045, No.
5,218,105 and No. 5,459,255, the contents of which are hereby
incorporated by reference.
[0033] The present invention also includes oligonucleotides which
are chimeric oligonucleotides. "Chimeric" oligonucleotides or
"chimeras," in the context of this invention, are oligonucleotides
which contain two or more chemically distinct regions, each made up
of at least one nucleotide. These oligonucleotides typically
contain at least one region wherein the oligonucleotide is modified
so as to confer upon the oligonucleotide increased resistance to
nuclease degradation, increased cellular uptake, and/or increased
binding affinity for the target nucleic acid. An additional region
of the oligonucleotide may serve as a substrate for enzymes capable
of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H
is a cellular endonuclease which cleaves the RNA strand of an
RNA:DNA duplex. Activation of RNase H, therefore, results in
cleavage of the RNA target, thereby greatly enhancing the
efficiency of antisense inhibition of gene expression. Cleavage of
the RNA target can be routinely detected by gel electrophoresis
and, if necessary, associated nucleic acid hybridization techniques
known in the art. This RNAse H-mediated cleavage of the RNA target
is distinct from the use of ribozymes to cleave nucleic acids.
Ribozymes are not comprehended by the present invention.
[0034] Examples of chimeric oligonucleotides include but are not
limited to "gapmers," in which three distinct regions are present,
normally with a central region flanked by two regions which are
chemically equivalent to each other but distinct from the gap. A
preferred example of a gapmer is an oligonucleotide in which a
central portion (the "gap") of the oligonucleotide serves as a
substrate for RNase H and is preferably composed of
2'-deoxynucleotides, while the flanking portions (the 5' and 3'
"wings") are modified to have greater affinity for the target RNA
molecule but are unable to support nuclease activity (e.g.,
2'-fluoro- or 2'-O-methoxyethyl-substituted). Other chimeras
include "wingmers," also known in the art as "hemimers," that is,
oligonucleotides with two distinct regions. In a preferred example
of a wingmer, the 5' portion of the oligonucleotide serves as a
substrate for RNase H and is preferably composed of
2'-deoxynucleotides, whereas the 3' portion is modified in such a
fashion so as to have greater affinity for the target RNA molecule
but is unable to support nuclease activity (e.g., 2'-fluoro- or
2'-O-methoxyethyl-substituted), or vice-versa. In one embodiment,
the oligonucleotides of the present invention contain a
2'-O-methoxyethyl (2'-O--CH.sub.2CH.sub.2OCH.sub.3) modification on
the sugar moiety of at least one nucleotide. This modification has
been shown to increase both affinity of the oligonucleotide for its
target and nuclease resistance of the oligonucleotide. According to
the invention, one, a plurality, or all of the nucleotide subunits
of the oligonucleotides of the invention may bear a
2'-O-methoxyethyl (--O--CH.sub.2CH.sub.2OCH.sub.3) modification.
Oligonucleotides comprising a plurality of nucleotide subunits
having a 2'-O-methoxyethyl modification can have such a
modification on any of the nucleotide subunits within the
oligonucleotide, and may be chimeric oligonucleotides. Aside from
or in addition to 2'-O-methoxyethyl modifications, oligonucleotides
containing other modifications which enhance antisense efficacy,
potency or target affinity are also preferred. Chimeric
oligonucleotides comprising one or more such modifications are
presently preferred. Through use of such modifications, active
oligonucleotides have been identified which are shorter than
conventional "first generation" oligonucleotides active against
p38. Oligonucleotides in accordance with this invention are from 5
to 50 nucleotides in length. In the context of this invention it is
understood that this encompasses non-naturally occurring oligomers
as hereinbefore described, having from 5 to 50 monomers.
[0035] The oligonucleotides 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 Applied Biosystems. Any other
means for such synthesis may also be employed; the actual synthesis
of the oligonucleotides is well within the talents of the
routineer. It is well known to use similar techniques to prepare
oligonucleotides such as the phosphorothioates and 2'-alkoxy or
2'-alkoxyalkoxy derivatives, including 2'-O-methoxyethyl
oligonucleotides [Martin, P., Helv. Chim. Acta, 78, 486 (1995)]. It
is also well known to use similar techniques and commercially
available modified amidites and controlled-pore glass (CPG)
products such as biotin, fluorescein, acridine or psoralen-modified
amidites and/or CPG (available from Glen Research, Sterling Va.) to
synthesize fluorescently labeled, biotinylated or other conjugated
oligonucleotides.
[0036] The antisense compounds of the present invention include
bioequivalent compounds, including pharmaceutically acceptable
salts and prodrugs. This is intended to 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 pharmaceutically
acceptable salts of the nucleic acids of the invention and prodrugs
of such nucleic acids.
[0037] Pharmaceutically acceptable "salts" are physiologically and
pharmaceutically acceptable salts of the nucleic acids of the
invention: i.e., salts that retain the desired biological activity
of the parent compound and do not impart undesired toxicological
effects thereto [see, for example, Berge et al., "Pharmaceutical
Salts," J. of Pharma Sci., 66:1 (1977)].
[0038] For oligonucleotides, examples of pharmaceutically
acceptable salts include but are not limited to (a) salts formed
with cations such as sodium, potassium, ammonium, magnesium,
calcium, polyamines such as spermine and spermidine, etc.; (b) acid
addition salts formed with inorganic acids, for example
hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric
acid, nitric acid and the like; (c) salts formed with organic acids
such as, for example, acetic acid, oxalic acid, tartaric acid,
succinic acid, maleic acid, fumaric acid, gluconic acid, citric
acid, malic acid, ascorbic acid, benzoic acid, tannic acid,
palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic
acid, methanesulfonic acid, p-toluenesulfonic acid,
naphthalenedisulfonic acid, polygalacturonic acid, and the like;
and (d) salts formed from elemental anions such as chlorine,
bromine, and iodine.
[0039] The oligonucleotides of the invention may additionally or
alternatively be prepared to be delivered in a "prodrug" form. 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.
[0040] For therapeutic or prophylactic treatment, oligonucleotides
are administered in accordance with this invention. Oligonucleotide
compounds of the invention may be formulated in a pharmaceutical
composition, which may include pharmaceutically acceptable
carriers, thickeners, diluents, buffers, preservatives, surface
active agents, neutral or cationic lipids, lipid complexes,
liposomes, penetration enhancers, carrier compounds and other
pharmaceutically acceptable carriers or excipients and the like in
addition to the oligonucleotide. Such compositions and formulations
are comprehended by the present invention.
[0041] Pharmaceutical compositions comprising the oligonucleotides
of the present invention may include penetration enhancers in order
to enhance the alimentary delivery of the oligonucleotides.
Penetration enhancers may be classified as belonging to one of five
broad categories, i.e., fatty acids, bile salts, chelating agents,
surfactants and non-surfactants (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, 8:91-192; Muranishi,
Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7:1).
One or more penetration enhancers from one or more of these broad
categories may be included.
[0042] The compositions of the present invention may additionally
contain other adjunct components conventionally found in
pharmaceutical compositions, at their art-established usage levels.
Thus, for example, the compositions may contain additional
compatible pharmaceutically-active materials such as, e.g.,
antipruritics, astringents, local anesthetics or anti-inflammatory
agents, or may contain additional materials useful in physically
formulating various dosage forms of the composition of present
invention, such as dyes, flavoring agents, preservatives,
antioxidants, opacifiers, thickening agents and stabilizers.
However, such materials, when added, should not unduly interfere
with the biological activities of the components of the
compositions of the invention.
[0043] Regardless of the method by which the oligonucleotides of
the invention are introduced into a patient, colloidal dispersion
systems may be used as delivery vehicles to enhance the in vivo
stability of the oligonucleotides and/or to target the
oligonucleotides to a particular organ, tissue or cell type.
Colloidal dispersion systems include, but are not limited to,
macromolecule complexes, nanocapsules, microspheres, beads and
lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles, liposomes and lipid:oligonucleotide complexes of
uncharacterized structure. A preferred colloidal dispersion system
is a plurality of liposomes. Liposomes are microscopic spheres
having an aqueous core surrounded by one or more outer layers made
up of lipids arranged in a bilayer configuration [see, generally,
Chonn et al., Current Op. Biotech., 6, 698 (1995)].
[0044] 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, vaginal,
rectal, intranasal, epidermal and transdermal), oral or parenteral.
Parenteral administration includes intravenous drip, subcutaneous,
intraperitoneal or intramuscular injection, pulmonary
administration, e.g., by inhalation or insufflation, 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.
[0045] 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.
[0046] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets or tablets. Thickeners, flavoring agents,
diluents, emulsifiers, dispersing aids or binders may be
desirable.
[0047] Compositions for parenteral administration may include
sterile aqueous solutions which may also contain buffers, diluents
and other suitable additives. In some cases it may be more
effective to treat a patient with an oligonucleotide of the
invention in conjunction with other traditional therapeutic
modalities in order to increase the efficacy of a treatment
regimen. In the context of the invention, the term "treatment
regimen" is meant to encompass therapeutic, palliative and
prophylactic modalities. For example, a patient may be treated with
conventional chemotherapeutic agents, particularly those used for
tumor and cancer treatment. Examples of such chemotherapeutic
agents include but are not limited to daunorubicin, daunomycin,
dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,
bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,
bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,
mithramycin, prednisone, hydroxyprogesterone, testosterone,
tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,
pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,
methylcyclohexylnitrosurea, nitrogen mustards, melphalan,
cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),
5-azacytidine, hydroxyurea, deoxycoformycin,
4-hydroxyperoxycyclophosphor- amide, 5-fluorouracil (5-FU),
5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX),
colchicine,taxol, vincristine, vinblastine, etoposide,
trimetrexate, teniposide, cisplatin and diethylstilbestrol (DES).
See, generally, The Merck Manual of Diagnosis and Therapy, 15th
Ed., pp. 1206-1228, Berkow et al., eds., Rahay, N.J., 1987). 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).
[0048] The formulation of therapeutic compositions and their
subsequent administration is believed to be within the skill of
those in the art. Dosing is dependent on severity and
responsiveness of the disease state to be treated, with the course
of treatment lasting from several days to several months, or until
a cure is effected or a diminution of the disease state is
achieved. Optimal dosing schedules can be calculated from
measurements of drug accumulation in the body of the patient.
Persons of ordinary skill can easily determine optimum dosages,
dosing methodologies and repetition rates. Optimum dosages may vary
depending on the relative potency of individual oligonucleotides,
and can generally be estimated based on EC.sub.50s found to be
effective in in vitro and in vivo animal models. In general, dosage
is from 0.01 .mu.g to 100 g per kg of body weight, and may be given
once or more daily, weekly, monthly or yearly, or even once every 2
to 20 years. Persons of ordinary skill in the art can easily
estimate repetition rates for dosing based on measured residence
times and concentrations of the drug in bodily fluids or tissues.
Following successful treatment, it may be desirable to have the
patient undergo maintenance therapy to prevent the recurrence of
the disease state, wherein the oligonucleotide is administered in
maintenance doses, ranging from 0.01 .mu.g to 100 g per kg of body
weight, once or more daily, to once every 20 years.
[0049] Thus, in the context of this invention, by "therapeutically
effective amount" is meant the amount of the compound which is
required to have a therapeutic effect on the treated mammal. This
amount, which will be apparent to the skilled artisan, will depend
upon the type of mammal, the age and weight of the mammal, the type
of disease to be treated, perhaps even the gender of the mammal,
and other factors which are routinely taken into consideration when
treating a mammal with a disease. A therapeutic effect is assessed
in the mammal by measuring the effect of the compound on the
disease state in the animal. For example, if the disease to be
treated is cancer, therapeutic effects are assessed by measuring
the rate of growth or the size of the tumor, or by measuring the
production of compounds such as cytokines, production of which is
an indication of the progress or regression of the tumor.
[0050] The following examples illustrate the present invention and
are not intended to limit the same.
EXAMPLES
Example 1
Synthesis of Oligonucleotides
[0051] Unmodified oligodeoxynucleotides are synthesized on an
automated DNA synthesizer (Applied Biosystems model 380B) using
standard phosphoramidite chemistry with oxidation by iodine.
.beta.-cyanoethyldiisopropyl-phosphoramidites were purchased from
Applied Biosystems (Foster City, Calif.). For phosphorothioate
oligonucleotides, the standard oxidation bottle was replaced by a
0.2 M solution of .sup.3H-1,2-benzodithiole-3-one 1,1-dioxide in
acetonitrile for the stepwise thiation of the phosphite linkages.
The thiation cycle wait step was increased to 68 seconds and was
followed by the capping step.
[0052] 2'-methoxy oligonucleotides are synthesized using 2'-methoxy
.beta.-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham,
Mass.) and the standard cycle for unmodified oligonucleotides,
except the wait step after pulse delivery of tetrazole and base was
increased to 360 seconds. Other 2'-alkoxy oligonucleotides were
synthesized by a modification of this method, using appropriate
2'-modified amidites such as those available from Glen Research,
Inc., Sterling, Va.
[0053] 2'-fluoro oligonucleotides are synthesized as described in
Kawasaki et al., J. Med. Chem., 36, 831 (1993). Briefly, the
protected nucleoside N.sup.6-benzoyl-2'-deoxy-2'-fluoroadenosine is
synthesized utilizing commercially available
9-.beta.-D-arabinofuranosyladenine as starting material and by
modifying literature procedures whereby the 2'-a-fluoro atom is
introduced by a S.sub.N2-displacement of a 2'-.beta.-O-trifyl
group. Thus N.sup.6-benzoyl-9-.beta.-D-arabinofuranosyladenine is
selectively protected in moderate yield as the
3',5'-ditetrahydropyranyl (THP) intermediate. Deprotection of the
THP and N.sup.6-benzoyl groups is accomplished using standard
methodologies and standard methods are used to obtain the
5'-dimethoxytrityl-(DMT) and 5'-DMT-3'-phosphoramidite
intermediates.
[0054] The synthesis of 2'-deoxy-2'-fluoroguanosine is accomplished
using tetraisopropyldisiloxanyl (TPDS) protected
9-.beta.-D-arabinofuranosylgua- nine as starting material, and
conversion to the intermediate
diisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS
group is followed by protection of the hydroxyl group with THP to
give diisobutyryl di-THP protected arabinofuranosylguanine.
Selective O-deacylation and triflation is followed by treatment of
the crude product with fluoride, then deprotection of the THP
groups. Standard methodologies are used to obtain the 5'-DMT- and
5'-DMT-3'-phosphoramidit- es.
[0055] Synthesis of 2'-deoxy-2'-fluorouridine is accomplished by
the modification of a known procedure in which
2,2'-anhydro-1-.beta.-D-arabin- ofuranosyluracii is treated with
70% hydrogen fluoride-pyridine. Standard procedures are used to
obtain the 5'-DMT and 5'-DMT-3'phosphoramidites.
[0056] 2'-deoxy-2'-fluorocytidine is synthesized via amination of
2'-deoxy-2'-fluorouridine, followed by selective protection to give
N.sup.4-benzoyl-2'-deoxy-2'-fluorocytidine. Standard procedures are
used to obtain the 5'-DMT and 5'-DMT-3'phosphoramidites.
[0057] 2'-(2-methoxyethyl)-modified amidites were synthesized
according to Martin, P., Helv. Chim. Acta, 78,486 (1995). For ease
of synthesis, the last nucleotide was a deoxynucleotide.
2'-O--CH.sub.2CH.sub.2OCH.sub.3cyt- osines may be 5-methyl
cytosines.
[0058] Synthesis of 5-Methyl Cytosine Monomers:
[0059]
2,2'-Anhydro[1-(.beta.-D-arabinofuranosyl)-5-methyluridine]:
[0060] 5-Methyluridine (ribosylthymine, commercially available
through Yamasa, Choshi, Japan) (72.0 g, 0.279 M),
diphenyl-carbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g,
0.024 M) were added to DMF (300 mL). The mixture was heated to
reflux, with stirring, allowing the evolved carbon dioxide gas to
be released in a controlled manner. After 1 hour, the slightly
darkened solution was concentrated under reduced pressure. The
resulting syrup was poured into diethylether (2.5 L), with
stirring. The product formed a gum. The ether was decanted and the
residue was dissolved in a minimum amount of methanol (ca. 400 mL).
The solution was poured into fresh ether (2.5 L) to yield a stiff
gum. The ether was decanted and the gum was dried in a vacuum oven
(60EC at 1 mm Hg for 24 hours) to give a solid which was crushed to
a light tan powder (57 g, 85% crude yield). The material was used
as is for further reactions.
[0061] 2'-O-Methoxyethyl-5-methyluridine:
[0062] 2,2'-Anhydro-5-methyluridine (195 g, 0.81 M),
tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol
(1.2 L) were added to a 2 L stainless steel pressure vessel and
placed in a pre-heated oil bath at 160EC. After heating for 48
hours at 155-160EC, the vessel was opened and the solution
evaporated to dryness and triturated with MeOH (200 mL). The
residue was suspended in hot acetone (1 L). The insoluble salts
were filtered, washed with acetone (150 mL) and the filtrate
evaporated. The residue (280 g) was dissolved in CH.sub.3CN (600
mL) and evaporated. A silica gel column (3 kg) was packed in
CH.sub.2Cl.sub.2/acetone/MeOH (20:5:3) containing 0.5% Et.sub.3NH.
The residue was dissolved in CH.sub.2Cl.sub.2 (250 mL) and adsorbed
onto silica (150 g) prior to loading onto the column. The product
was eluted with the packing solvent to give 160 g (63%) of
product.
[0063] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine:
[0064] 2'-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was
co-evaporated with pyridine (250 mL) and the dried residue
dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl
chloride (94.3 g, 0.278 M) was added and the mixture stirred at
room temperature for one hour. A second aliquot of dimethoxytrityl
chloride (94.3 g, 0.278 M) was added and the reaction stirred for
an additional one hour. Methanol (170 mL) was then added to stop
the reaction. HPLC showed the presence of approximately 70%
product. The solvent was evaporated and triturated with CH.sub.3CN
(200 mL). The residue was dissolved in CHCl.sub.3 (1.5 L) and
extracted with 2.times.500 mL of saturated NaHCO.sub.3 and
2.times.500 mL of saturated NaCl. The organic phase was dried over
Na.sub.2SO.sub.4, filtered and evaporated. 275 g of residue was
obtained. The residue was purified on a 3.5 kg silica gel column,
packed and eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5%
Et.sub.3NH. The pure fractions were evaporated to give 164 g of
product. Approximately 20 g additional was obtained from the impure
fractions to give a total yield of 183 g (57%).
[0065]
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine:
[0066] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (106
g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from
562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38
mL, 0.258 M) were combined and stirred at room temperature for 24
hours. The reaction was monitored by tlc by first quenching the tlc
sample with the addition of MeOH. Upon completion of the reaction,
as judged by tlc, MeOH (50 mL) was added and the mixture evaporated
at 35EC. The residue was dissolved in CHCl.sub.3 (800 mL) and
extracted with 2.times.200 mL of saturated sodium bicarbonate and
2.times.200 mL of saturated NaCl. The water layers were back
extracted with 200 mL of CHCl.sub.3. The combined organics were
dried with sodium sulfate and evaporated to give 122 g of residue
(approx. 90% product). The residue was purified on a 3.5 kg silica
gel column and eluted using EtOAc/Hexane (4:1). Pure product
fractions were evaporated to yield 96 g (84%).
[0067]
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triaz-
oleuridine:
[0068] A first solution was prepared by dissolving
3'-O-acetyl-2'-O-methox-
yethyl-5'-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in
CH.sub.3CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M)
was added to a solution of triazole (90 g, 1.3 M) in CH.sub.3CN (1
L), cooled to -5EC and stirred for 0.5 h using an overhead stirrer.
POCl.sub.3 was added dropwise, over a 30 minute period, to the
stirred solution maintained at 0-10EC, and the resulting mixture
stirred for an additional 2 hours. The first solution was added
dropwise, over a 45 minute period, to the later solution. The
resulting reaction mixture was stored overnight in a cold room.
Salts were filtered from the reaction mixture and the solution was
evaporated. The residue was dissolved in EtOAc (1 L) and the
insoluble solids were removed by filtration. The filtrate was
washed with 1.times.300 mL of NaHCO.sub.3 and 2.times.300 mL of
saturated NaCl, dried over sodium sulfate and evaporated. The
residue was triturated with EtOAc to give the title compound.
[0069] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine:
[0070] A solution of
3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5--
methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and
NH.sub.4OH (30 mL) was stirred at room temperature for 2 hours. The
dioxane solution was evaporated and the residue azeotroped with
MeOH (2.times.200 mL). The residue was dissolved in MeOH (300 mL)
and transferred to a 2 liter stainless steel pressure vessel. MeOH
(400 mL) saturated with NH.sub.3 gas was added and the vessel
heated to 100EC for 2 hours (tic showed complete conversion). The
vessel contents were evaporated to dryness and the residue was
dissolved in EtOAc (500 mL) and washed once with saturated NaCl
(200 mL). The organics were dried over sodium sulfate and the
solvent was evaporated to give 85 g (95%) of the title
compound.
[0071]
N.sup.4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcyti-
dine:
[0072] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (85
g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride
(37.2 g, 0.165 M) was added with stirring. After stirring for 3
hours, tic showed the reaction to be approximately 95% complete.
The solvent was evaporated and the residue azeotroped with MeOH
(200 mL). The residue was dissolved in CHCl.sub.3 (700 mL) and
extracted with saturated NaHCO.sub.3 (2.times.300 mL) and saturated
NaCl (2.times.300 mL), dried over MgSO.sub.4 and evaporated to give
a residue (96 g). The residue was chromatographed on a 1.5 kg
silica column using EtOAc/Hexane (1:1) containing 0.5% Et.sub.3NH
as the eluting solvent. The pure product fractions were evaporated
to give 90 g (90%) of the title compound.
[0073]
N'-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine--
3'-amidite:
[0074]
N.sup.4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcyti-
dine (74 g, 0.10 M) was dissolved in CH.sub.2Cl.sub.2 (1 L).
Tetrazole diisopropylamine (7.1 g) and
2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) were
added with stirring, under a nitrogen atmosphere. The resulting
mixture was stirred for 20 hours at room temperature (tic showed
the reaction to be 95% complete). The reaction mixture was
extracted with saturated NaHCO.sub.3 (1.times.300 mL) and saturated
NaCl (3.times.300 mL). The aqueous washes were back-extracted with
CH.sub.2Cl.sub.2 (300 mL), and the extracts were combined, dried
over MgSO.sub.4 and concentrated. The residue obtained was
chromatographed on a 1.5 kg silica column using
EtOAc.backslash.Hexane (3:1) as the eluting solvent. The pure
fractions were combined to give 90.6 g (87%) of the title
compound.
[0075] 5-methyl-2'-deoxycytidine (5-me-C) containing
oligonucleotides were synthesized according to published methods
[Sanghvi et al., Nucl. Acids Res., 21, 3197 (1993)] using
commercially available phosphoramidites (Glen Research, Sterling
Va. or ChemGenes, Needham Mass.).
[0076] 2=-O-(dimethylaminooxyethyl) Nucleoside Amidites
[0077] 2'-(Dimethylaminooxyethoxy) nucleoside amidites [also known
in the art as 2'-O-(dimethylaminooxyethyl) nucleoside amidites] are
prepared as described in the following paragraphs. Adenosine,
cytidine and guanosine nucleoside amidites are prepared similarly
to the thymidine (5-methyluridine) except the exocyclic amines are
protected with a benzoyl moiety in the case of adenosine and
cytidine and with isobutyryl in the case of guanosine.
[0078]
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-5-methyluridine
[0079] O.sup.2-2'-anhydro-5-methyluridine (Pro. Bio. Sint., Varese,
Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013
eq, 0.0054 mmol) are dissolved in dry pyridine (500 ml) at ambient
temperature under an argon atmosphere and with mechanical stirring.
tert-Butyldiphenylchlor- osilane (125.8 g, 119.0 mL, 1.1 eq, 0.458
mmol) is added in one portion. The reaction is stirred for 16 h at
ambient temperature. TLC (Rf 0.22, ethyl acetate) indicates a
complete reaction. The solution is concentrated under reduced
pressure to a thick oil. This is partitioned between
dichloromethane (1 L) and saturated sodium bicarbonate (2.times.1
L) and brine (1 L). The organic layer is dried over sodium sulfate
and concentrated under reduced pressure to a thick oil. The oil is
dissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600
mL) and the solution is cooled to -10.degree. C. The resulting
crystalline product is collected by filtration, washed with ethyl
ether (3.times.200 mL) and dried (40.degree. C., 1 mm Hg, 24 h) to
149 g (74.8%) of white solid. TLC and NMR are used to check
consistency with pure product.
[0080]
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine
[0081] In a 2 L stainless steel, unstirred pressure reactor is
added borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the
fume hood and with manual stirring, ethylene glycol (350 mL,
excess) is added cautiously at first until the evolution of
hydrogen gas subsided.
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-5-methyluridine
(149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) are
added with manual stirring. The reactor is sealed and heated in an
oil bath until an internal temperature of 160.degree. C. is reached
and then maintained for 16 h (pressure <100 psig). The reaction
vessel is cooled to ambient and opened. TLC (Rf 0.67 for desired
product and Rf 0.82 for ara-T side product, ethyl acetate)
indicates % conversion to the product. In order to avoid additional
side product formation, the reaction is stopped, concentrated under
reduced pressure (10 to 1 mm Hg) in a warm water bath
(40-100.degree. C.) with the more extreme conditions used to remove
the ethylene glycol. [Alternatively, once the low boiling solvent
is gone, the remaining solution can be partitioned between ethyl
acetate and water. The product will be in the organic phase.] The
residue is purified by column chromatography (2 kg silica gel,
ethyl acetate-hexanes gradient 1:1 to 4:1). The appropriate
fractions are combined, stripped and dried to product as a white
crisp foam (84 g, 50%), contaminated starting material (17.4 g) and
pure reusable starting material 20 g. TLC and NMR are used to
determine consistency with pure product.
[0082]
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridi-
ne
[0083]
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine
(20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g,
44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was
then dried over P.sub.2O.sub.5 under high vacuum for two days at
40.degree. C. The reaction mixture was flushed with argon and dry
THF (369.8 mL, Aldrich, sure seal bottle) was added to get a clear
solution. Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added
dropwise to the reaction mixture. The rate of addition is
maintained such that resulting deep red coloration is just
discharged before adding the next drop. After the addition was
complete, the reaction was stirred for 4 hrs. By that time TLC
showed the completion of the reaction (ethylacetate:hexane, 60:40).
The solvent was evaporated in vacuum. Residue obtained was placed
on a flash column and eluted with ethyl acetate:hexane (60:40), to
get
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine
as white foam (21.819, 86%).
[0084]
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-met-
hyluridine
[0085]
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridi-
ne (3.1 g, 4.5 mmol) is dissolved in dry CH.sub.2Cl.sub.2 (4.5 mL)
and methylhydrazine (300 mL, 4.64 mmol) is added dropwise at
-10.degree. C. to 0.degree. C. After 1 hr the mixture is filtered,
the filtrate is washed with ice cold CH.sub.2Cl.sub.2 and the
combined organic phase is washed with water, brine and dried over
anhydrous Na.sub.2SO.sub.4. The solution is concentrated to get
2'-O-(aminooxyethyl) thymidine, which is then dissolved in MeOH
(67.5 mL). To this formaldehyde (20% aqueous solution, w/w, 1.1
eg.) is added and the mixture for 1 hr. Solvent is removed under
vacuum; residue chromatographed to get
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)
ethyl]-5-methyluridine as white foam.
[0086]
5'-O-tert-Butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-met-
hyluridine
[0087]
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-met-
hyluridine (1.77 g, 3.12 mmol) is dissolved in a solution of 1M
pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium
cyanoborohydride (0.39 g, 6.13 mmol) is added to this solution at
10.degree. C. under inert atmosphere. The reaction mixture is
stirred for 10 minutes at 10.degree. C. After that the reaction
vessel is removed from the ice bath and stirred at room temperature
for 2 hr, the reaction monitored by TLC (5% MeOH in
CH.sub.2Cl.sub.2). Aqueous NaHCO.sub.3 solution (5%, 10 mL) is
added and extracted with ethyl acetate (2.times.20 mL). Ethyl
acetate phase is dried over anhydrous Na.sub.2SO.sub.4, evaporated
to dryness. Residue is dissolved in a solution of 1M PPTS in MeOH
(30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) is added and
the reaction mixture is stirred at room temperature for 10 minutes.
Reaction mixture cooled to 10.degree. C. in an ice bath, sodium
cyanoborohydride (0.39 g, 6.13 mmol) is added and reaction mixture
stirred at 10.degree. C. for 10 minutes. After 10 minutes, the
reaction mixture is removed from the ice bath and stirred at room
temperature for 2 hrs. To the reaction mixture 5% NaHCO.sub.3 (25
mL) solution is added and extracted with ethyl acetate (2.times.25
mL). Ethyl acetate layer is dried over anhydrous Na.sub.2SO.sub.4
and evaporated to dryness. The residue obtained is purified by
flash column chromatography and eluted with 5% MeOH in CH.sub.2Cl 2
to get 5'-O-tert-butyldiphenylsilyl-2'-O-[N,N-dimethylaminoo-
xyethyl]-5-methyluridine as a white foam (14.6 g).
[0088] 2'-O-(dimethylaminooxyethyl)-5-methyluridine
[0089] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) is
dissolved in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept
over KOH). This mixture of triethylamine-2HF is then added to
5'-O-tert-butyldiphenylsily-
l-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4
mmol) and stirred at room temperature for 24 hrs. Reaction is
monitored by TLC (5% MeOH in CH.sub.2Cl.sub.2). Solvent is removed
under vacuum and the residue placed on a flash column and eluted
with 10% MeOH in CH.sub.2Cl.sub.2 to get
2'-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg).
[0090] 5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine
[0091] 2'-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17
mmol) is dried over P.sub.2O.sub.5 under high vacuum overnight at
40.degree. C. It is then co-evaporated with anhydrous pyridine (20
mL). The residue obtained is dissolved in pyridine (11 mL) under
argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol),
4,4'-dimethoxytrityl chloride (880 mg, 2.60 mmol) is added to the
mixture and the reaction mixture is stirred at room temperature
until all of the starting material disappeared. Pyridine is removed
under vacuum and the residue chromatographed and eluted with 10%
MeOH in CH.sub.2Cl.sub.2 (containing a few drops of pyridine) to
get 5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-m- ethyluridine (1.13
g).
[0092]
5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-F(2--
cyanoethyl)-N,N-diisopropylphosphoramiditel
[0093] 5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine (1.08
g, 1.67 mmol) is co-evaporated with toluene (20 mL). To the residue
N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) is added and
dried over P.sub.2O.sub.5 under high vacuum overnight at 40.degree.
C. Then the reaction mixture is dissolved in anhydrous acetonitrile
(8.4 mL) and 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphoramidite
(2.12 mL, 6.08 mmol) is added. The reaction mixture is stirred at
ambient temperature for 4 hrs under inert atmosphere. The progress
of the reaction was monitored by TLC (hexane:ethyl acetate 1:1).
The solvent is evaporated, then the residue is dissolved in ethyl
acetate (70 mL) and washed with 5% aqueous NaHCO.sub.3 (40 mL).
Ethyl acetate layer is dried over anhydrous Na.sub.2SO.sub.4 and
concentrated. Residue obtained is chromatographed (ethyl acetate as
eluent) to get 5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyeth-
yl)-5-methyluridine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]
as a foam (1.04 g).
[0094] 2'-(Aminooxyethoxy) Nucleoside Amidites
[0095] 2'-(Aminooxyethoxy) nucleoside amidites [also known in the
art as 2'-O-(aminooxyethyl) nucleoside amidites] are prepared as
described in the following paragraphs. Adenosine, cytidine and
thymidine nucleoside amidites are prepared similarly.
[0096]
N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-
-dimethoxytrityl)guanosine-3'-F(2-cyanoethyl)-N,N-diisopropylphosphoramidi-
tel
[0097] The 2'-O-aminooxyethyl guanosine analog may be obtained by
selective 2'-O-alkylation of diaminopurine riboside. Multigram
quantities of diaminopurine riboside may be purchased from Schering
AG (Berlin) to provide 2'-O-(2-ethylacetyl) diaminopurine riboside
along with aminor amount of the 3'-O-isomer. 2'-O-(2-ethylacetyl)
diaminopurine riboside may be resolved and converted to
2'-O-(2-ethylacetyl)guanosine by treatment with adenosine
deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO
94/02501 A1 940203.) Standard protection procedures should afford
2'-O-(2-ethylacetyl) -5'-O-(4,4'-dimethoxytrityl)guanosine and
2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'--
dimethoxy trityl)guanosine which may be reduced to provide
2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dime-
thoxytrityl) guanosine. As before the hydroxyl group may be
displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the
protected nucleoside may phosphitylated as usual to yield
2-N-isobutyryl-6-O-diphen-
ylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine-3'-[-
(2-cyanoethyl)-N,N-diisopropylphosphoramidite].
[0098] Oligonucleotides having methylene (methylimino) (MMI)
backbones are synthesized according to U.S. Pat. No. 5,378,825,
which is coassigned to the assignee of the present invention and is
incorporated herein in its entirety. For ease of synthesis, various
nucleoside dimers containing MMI linkages were synthesized and
incorporated into oligonucleotides. Other nitrogen-containing
backbones are synthesized according to WO 92/20823 which is also
coassigned to the assignee of the present invention and
incorporated herein in its entirety.
[0099] Oligonucleotides having amide backbones are synthesized
according to De Mesmaeker et al., Acc. Chem. Res., 28, 366 (1995).
The amide moiety is readily accessible by simple and well-known
synthetic methods and is compatible with the conditions required
for solid phase synthesis of oligonucleotides.
[0100] Oligonucleotides with morpholino backbones are synthesized
according to U.S. Pat. No. 5,034,506 (Summerton and Weller).
[0101] Peptide-nucleic acid (PNA) oligomers are synthesized
according to P. E. Nielsen et al., Science, 254, 1497 (1991).
[0102] After cleavage from the controlled pore glass column
(Applied Biosystems) and deblocking in concentrated ammonium
hydroxide at 55EC for 18 hours, the oligonucleotides are purified
by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.
Synthesized oligonucleotides were analyzed by polyacrylamide gel
electrophoresis on denaturing gels and judged to be at least 85%
full length material. The relative amounts of phosphorothioate and
phosphodiester linkages obtained in synthesis were periodically
checked by .sup.31p nuclear magnetic resonance spectroscopy, and
for some studies oligonucleotides were purified by HPLC, as
described by Chiang et al., J. Biol. Chem., 266, 18162 (1991).
Results obtained with HPLC-purified material were similar to those
obtained with non-HPLC purified material.
Example 2
Human p38.alpha. Oligonucleotide Sequences
[0103] Antisense oligonucleotides were designed to target human
p38.alpha.. Target sequence data are from the p38 MAPK cDNA
sequence; Genbank accession number L35253, provided herein as SEQ
ID NO: 1. Oligonucleotides was synthesized as chimeric
oligonucleotides ("gapmers") 20 nucleotides in length, composed of
a central "gap" region consisting of eight 2'-deoxynucleotides,
which is flanked on both sides (5' and 3' directions) by
six-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
2'-MOE cytosines were 5-methyl-cytosines. These oligonucleotide
sequences are shown in Table 1.
[0104] The human Jurkat T-cell line (American Type Culture
Collection, Manassas, Va.) was maintained in RPMI 1640 growth media
supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan,
Utah). HUVEC cells (Clonetics, San Diego, Calif.) were cultivated
in endothelial basal media supplemented with 10% FBS (Hyclone,
Logan, Utah).
[0105] Jurkat cells were grown to approximately 75% confluency and
resuspended in culture media at a density of 1.times.10.sup.7
cells/ml. A total of 3.6.times.10.sup.6 cells were employed for
each treatment by combining 360 .mu.l of cell suspension with
oligonucleotide at the indicated concentrations to reach a final
volume of 400 .mu.l. Cells were then transferred to an
electroporation cuvette and electroporated using an Electrocell
Manipulator 600 instrument (Biotechnologies and Experimental
Research, Inc.) employing 150 V, 1000 .mu.F, at 13 .OMEGA..
Electroporated cells were then transferred to conical tubes
containing 5 ml of culture media, mixed by inversion, and plated
onto 10 cm culture dishes.
[0106] HUVEC cells were allowed to reach 75% confluency prior to
use. The cells were washed twice with warm (37.degree. C.)
OPTIMEM.TM. (Life Technologies). The cells were incubated in the
presence of the appropriate culture medium, without the growth
factors added, and the oligonucleotide formulated in LIPOFECTIN7
(Life Technologies), a 1:1 (w/w) liposome formulation of the
cationic lipid N-[1-(2,3-dioleyloxy)pro-
pyl]-n,n,n-trimethylammonium chloride (DOTMA), and dioleoyl
phosphotidylethanolamine (DOPE) in membrane filtered water. HUVEC
cells were treated with 100 nM oligonucleotide in 10 .mu.g/ml
LIPOFECTIN7. Treatment was for four hours.
[0107] Total mRNA was isolated using the RNEASY7 Mini Kit (Qiagen,
Valencia, Calif.; similar kits from other manufacturers may also be
used), separated on a 1% agarose gel, transferred to HYBOND.TM.-N+
membrane (Amersham Pharmacia Biotech, Piscataway, N.J.), a
positively charged nylon membrane, and probed. p38 MAPK probes were
made using the Prime-A-Gene7 kit (Promega Corporation, Madison,
Wis.), a random primer labeling kit, using mouse p38.alpha. or
p38.beta. cDNA as a template. A glyceraldehyde 3-phosphate
dehydrogenase (G3PDH) probe was purchased from Clontech (Palo Alto,
Calif.), Catalog Number 9805-1. The fragments were purified from
low-melting temperature agarose, as described in Maniatis, T., et
al., Molecular Cloning: A Laboratory Manual, 1989. The G3PDH probe
was labeled with REDIVUE.TM. .sup.32P-dCTP (Amersham Pharmacia
Biotech, Piscataway, N.J.) and Strip-EZ labelling kit (Ambion,
Austin, Tex.). mRNA was quantitated by a Phospholmager (Molecular
Dynamics, Sunnyvale, Calif.).
1TABLE 1 Nucleotide Sequences of Human p38.alpha. Chimeric (deoxy
gapped) Phosphorothicate Oligonucleotides TARGET GENE SEQ
NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE.sup.1 ID CO- TARGET NO 5'
-> 3') NO ORDINATES.sup.2 REGION 16486 AAGACCGGGCCCGGAATTCC 3
0001-0020 5'-UTR 16487 GTGGAGGCCAGTCCCCGGGA 4 0044-0063 5'-UTR
16488 TGGCAGCAAAGTGCTGCTGG 5 0087-0106 5'-UTR 16489
CAGAGAGCCTCCTGGGAGGG 6 0136-0155 5'-UTR 16490 TGTGCCGAATCTCGGCCTCT
7 0160-0179 5'-UTR 16491 GGTCTCGGGCGACCTCTCCT 8 0201-0220 5'-UTR
16492 CAGCCGCGGGACCAGCGGCG 9 0250-0269 5'-UTR 16493
CATTTTCCAGCGGCAGCCGC 10 0278-0297 AUG 16494 TCCTGAGACATTTTCCAGCG 11
0286-0305 AUG 16495 CTGCCGGTAGAACGTGGGCC 12 0308-0327 coding 16496
GTAAGCTTCTGACATTTCAC 13 0643-0662 coding 16497 TTTAGGTCCCTGTGAATTAT
14 0798-0817 coding 16498 ATGTTCTTCCAGTCAACAGC 15 0939-0958 coding
16499 TAAGGAGGTCCCTGCTTTCA 16 1189-1208 coding 16500
AACCAGGTGCTCAGGACTCC 17 1368-1387 stop 16501 GAAGTGGGATCAACAGAACA
18 1390-1409 3'-UTR 16502 TGAAAAGGCCTTCCCCTCAC 19 1413-1432 3'-UTR
16503 AGGCACTTGAATAATATTTG 20 1444-1463 3'-UTR 16504
CTTCCACCATGGAGGAAATC 21 1475-1494 3'-UTR 16505 ACACATGCACACACACTAAC
22 1520-1539 3'-UTR .sup.1 Emboldened residues,
2'-methoxyethoxy-residues (others are 2'-deoxy-) including "C"
residues, 5-methyl-cytosines; all linkages are phosphorothioate
linkages. .sup.2 Co-ordinates from Genbank Accession No. L35253,
locus name "HUMMAPKNS", SEQ ID NO. 1.
[0108] For an initial screen of human p38.alpha. antisense
oligonucleotides, Jurkat cells were electroporated with 10 .mu.M
oligonucleotide. mRNA was measured by Northern blot. Results are
shown in Table 2. Oligonucleotides 16496 (SEQ ID NO. 13), 16500
(SEQ ID NO. 17) and 16503 (SEQ ID NO. 20) gave 35% or greater
inhibition of p38.alpha. mRNA.
2TABLE 2 Inhibition of Human p38.alpha. mRNA expression in Jurkat
Cells by Chimeric (deoxy gapped) Phosphoro- thioate
Oligonucleotides SEQ GENE ISIS ID TARGET % mRNA % mRNA No: NO:
REGION EXPRESSION INHIBITION control -- 100% 0% 16486 3 5'-UTR 212%
-- 16487 4 5'-UTR 171% -- 16488 5 5'-UTR 157% -- 16489 6 5'-UTR
149% -- 16490 7 5'-UTR 152% -- 16491 8 5'-UTR 148% -- 16492 9
5'-UTR 125% -- 16493 10 AUG 101% -- 16494 11 AUG 72% 28% 16495 12
coding 72% 28% 16496 13 coding 61% 39% 16497 14 coding 104% --
16498 15 coding 88% 12% 16499 16 coding 74% 26% 16500 17 stop 63%
37% 16501 18 3'-UTR 77% 23% 16502 19 3'-UTR 79% 21% 16503 20 3'-UTR
65% 35% 16504 21 3'-UTR 72% 28% 16505 22 3'-UTR 93% 7%
[0109] The most active human p38.alpha. oligonucleotides were
chosen for dose response studies. Oligonucleotide 16490 (SEQ ID NO.
7) which showed no inhibition in the initial screen was included as
a negative control. Jurkat cells were grown and treated as
described above except the concentration of oligonucleotide was
varied as indicated in Table 3. Results are shown in Table 3. Each
of the active oligonucleotides showed a dose response effect with
IC.sub.50s around 10 nM. Maximum inhibition was approximately 70%
with 16500 (SEQ ID NO. 17). The most active oligonucleotides were
also tested for their ability to inhibit p38.beta.. None of these
oligonucleotides significantly reduced p38.beta. mRNA
expression.
3TABLE 3 Dose Response of p38.alpha. mRNA in Jurkat cells to human
p38.alpha. Chimeric (deoxy gapped) Phosphorothioate
Oligonucleotides SEQ ID ASO Gene % mRNA % mRNA ISIS # NO: Target
Dose Expression Inhibition control -- -- -- 100% 0% 16496 13 coding
2.5 nM 94% 6% " " " 5 nM 74% 26% " " " 10 nM 47% 53% " " " 20 nM
41% 59% 16500 17 stop 2.5 nM 82% 18% " " " 5 nM 71% 29% " " " 10 nM
49% 51% " " " 20 nM 31% 69% 16503 20 3'-UTR 2.5 nM 74% 26% " " " 5
nM 61% 39% " " " 10 nM 53% 47% " " " 20 nM 41% 59% 16490 7 5'-UTR
2.5 nM 112% -- " " " 5 nM 109% -- " " " 10 nM 104% -- " " " 20 nM
97% 3%
Example 3
Human p38.beta. Oligonucleotide Sequences
[0110] Antisense oligonucleotides were designed to target human
p38.beta.. Target sequence data are from the p38.beta. MAPK cDNA
sequence; Genbank accession number U53442, provided herein as SEQ
ID NO: 23. Oligonucleotides was synthesized as 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
2'-MOE cytosines were 5-methyl-cytosines. These oligonucleotide
sequences are shown in Table 4.
4TABLE 4 Nucleotide Sequences of Human p38.beta. Phosphorothioate
Oligonucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE
SEQUENCE ID CO- TARGET NO. (5' -> 3') NO: ORDINATES.sup.2 REGION
17891 CGACATGTCCGGAGCAGAAT 25 0006-0025 AUG 17892
TTCAGCTCCTGCCGGTAGAA 26 0041-0060 coding 17893 TGCGGCACCTCCCACACGGT
27 0065-0084 coding 17894 CCGAACAGACGGAGCCGTAT 28 0121-0140 coding
17895 GTGCTTCAGGTGCTTGAGCA 29 0240-0259 coding 17896
GCGTGAAGACGTCCAGAAGC 30 0274-0293 coding 17897 ACTTGACCATGTTGTTCAGG
31 0355-0374 coding 17898 AACGTGCTCGTCAAGTGCCA 32 0405-0424 coding
17899 ATCCTGAGCTCACAGTCCTC 33 0521-0540 coding 17900
ACTGTTTGGTTGTAATGCAT 34 0635-0654 coding 17901 ATGATCCGCTTCAGCTGGTC
35 0731-0750 coding 17902 GCCAGTGCCTCAGCTGCACT 36 0935-0954 coding
17903 AACGCTCTCATCATATGGCT 37 1005-1024 coding 17904
CAGCACCTCACTGCTCAATC 38 1126-1145 stop 17905 TCTGTGACCATAGGAGTGTG
39 1228-1247 3'-UTR 17906 ACACATCTTTGTGCATGCAT 40 1294-1313 3'-UTR
17907 CCTACACATCGCAAGCACAT 41 1318-1337 3'-UTR 17908
TCCAGCCTGAGCACCTCTAA 42 1581-1600 3'-UTR 17909 AGTGCACCCTCATCCACACG
43 1753-1772 3'-UTR 17910 CTTGCCAGATATGGCTGCTG 44 1836-1855 3'-UTR
.sup.1Emboldened residues, 2'-methoxyethoxy-residues (others are
2'-deoxy-) including "C" residues, 5-methyl-cytosines; all linkages
are phosphorothioate linkages. .sup.2Co-ordinates from Genbank
Accession No. U53442, locus name "HSU53442", SEQ ID NO. 23.
[0111] For an initial screen of human p38.beta. antisense
oligonucleotides, HUVEC cells were cultured and treated as
described in Example 2. mRNA was measured by Northern blot as
described in Example 2. Results are shown in Table 5. Every
oligonucleotide tested gave at least 50% inhibition.
Oligonucleotides 17892 (SEQ ID NO. 26), 17893 (SEQ ID NO. 27),
17894 (SEQ ID NO. 28), 17899 (SEQ ID NO. 33), 17901 (SEQ ID NO.
35), 17903 (SEQ ID NO. 37), 17904 (SEQ ID NO. 38), 17905 (SEQ ID
NO. 39), 17907 (SEQ ID NO. 41), 17908 (SEQ ID NO. 42), and 17909
(SEQ ID NO. 43) gave greater than approximately 85% inhibition and
are preferred.
5TABLE 5 Inhibition of Human p38.beta. mRNA expression in Huvec
Cells by Chimeric (deoxy gapped) Phosphorothioate Oligonucleotides
SEQ GENE ID TARGET % mRNA % mRNA ISIS No: NO: REGION EXPRESSION
INHIBITION control -- -- 100% 0% 17891 25 AUG 22% 78% 17892 26
coding 10% 90% 17893 27 coding 4% 96% 17894 28 coding 13% 87% 17895
29 coding 25% 75% 17896 30 coding 24% 76% 17897 31 coding 25% 75%
17898 32 coding 49% 51% 17899 33 coding 5% 95% 17900 34 coding 40%
60% 17901 35 coding 15% 85% 17902 36 coding 49% 51% 17903 37 coding
11% 89% 17904 38 stop 9% 91% 17905 39 3'-UTR 14% 86% 17906 40
3'-UTR 22% 78% 17907 41 3'-UTR 8% 92% 17908 42 3'-UTR 17% 83% 17909
43 3'-UTR 13% 87% 17910 44 3'-UTR 26% 74%
[0112] Oligonucleotides 17893 (SEQ ID NO. 27), 17899 (SEQ ID NO.
33), 17904 (SEQ ID NO. 38), and 17907 (SEQ ID NO. 41) were chosen
for dose response studies. HUVEC cells were cultured and treated as
described in Example 2 except that the oligonucleotide
concentration was varied as shown in Table 6. The Lipofectin7/Oligo
ratio was maintained at 3 .mu.g Lipofectin7/100 nM oligo, per ml.
mRNA was measured by Northern blot as described in Example 2.
[0113] Results are shown in Table 6. Each oligonucleotide tested
had an IC.sub.50 of less than 10 nM. The effect of these
oligonucleotides on human p38.alpha. was also determined. Only
oligonucleotide 17893 (SEQ ID NO. 27) showed an effect on
p38.alpha. mRNA expression. The IC.sub.50 of this oligonucleotide
was approximately 4 fold higher for p38.alpha. compared to
p38.beta..
6TABLE 6 Dose Response of p38.beta. in Huvec cells to human
p38.beta. Chimeric (deoxy gapped) Phosphorothioate Oligonucleotides
SEQ ID ASO Gene % mRNA % mRNA ISIS # NO: Target Dose Expression
Inhibition control -- -- -- 100% 0% 17893 27 coding 10 nM 37% 63% "
" " 25 nM 18% 82% " " " 50 nM 16% 84% " " " 100 nM 19% 81% 17899 33
coding 10 nM 37% 63% " " " 25 nM 23% 77% " " " 50 nM 18% 82% " " "
100 nM 21% 79% 17904 38 stop 10 nM 31% 69% " " " 25 nM 21% 79% " "
" 50 nM 17% 83% " " " 100 nM 19% 81% 17907 41 3'-UTR 10 nM 37% 63%
" " " 25 nM 22% 78% " " " 50 nM 18% 72% " " " 100 nM 18% 72%
Example 4
Rat p38.alpha. Oligonucleotide Sequences
[0114] Antisense oligonucleotides were designed to target rat
p38.alpha.. Target sequence data are from the p38 MAPK cDNA
sequence; Genbank accession number U73142, provided herein as SEQ
ID NO: 45. Oligonucleotides was synthesized as 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 in the wings
are phosphodiester (P.dbd.O). Internucleoside linkages in the
central gap are phosphorothioate (P.dbd.S). All 2'-MOE cytosines
and 2'-OH cytosines were 5-methyl-cytosines. These oligonucleotide
sequences are shown in Table 7.
[0115] bEND.3, a mouse endothelial cell line (gift of Dr. Werner
Risau; see Montesano et al., Cell, 1990, 62, 435, and Stepkowski et
al., J. Immunol., 1994, 153, 5336) were grown in high-glucose DMEM
(Life Technologies, Gaithersburg, Md.) medium containing 10% fetal
bovine serum (FBS) and 1% Penicillin/Streptomycinin. Cells were
plated at approximately 2.times.10.sup.5 cells per 100 mm dish.
Within 48 hours of plating, the cells were washed with
phosphate-buffered saline (Life Technologies). Then, Opti-MEM7
medium containing 3 .mu.g/mL LIPOFECTIN.sup.7 and an appropriate
amount of oligonucleotide were added to the cells. As a control,
cells were treated with LIPOFECTIN.sup.7 without oligonucleotide
under the same conditions and for the same times as the
oligonucleotide-treated samples.
[0116] After 4 hours at 37.degree. C., the medium was replaced with
high glucose DMEM medium containing 10% FBS and 1%
Penicillin/Streptomycinin. The cells were typically allowed to
recover overnight (about 18 to 24 hours) before RNA and/or protein
assays were performed as described in Example 2. The p38.alpha.,
p38.beta. and G3PDH probes used were identical to those described
in Example 2.
7TABLE 7 Nucleotide Sequences of Rat p38.alpha. Phosphorothioate
Oligonucleotides TARGET GENE NUCLEOTIDE GENE ISIS NUCLEOTIDE
SEQUENCE.sup.1 CO- TARGET NO. (5' -> 3') SEQ ID NO
ORDINATES.sup.2 REGION 21844
CoToGoCoGsAsCsAsTsTsTsTsCsCsAsGoCoGoGoC 47 0001-0020 AUG 21845
GoGoToAoAsGsCsTsTsCsTsGsAsCsAsCoToToCoA 48 0361-0380 coding 21846
GoGoCoCoAsGsAsGsAsCsTsGsAsAsTsGoTo- AoGoT 49 0781-0800 coding 21871
CoAoToCoAsTsCsAsGsGsGsTsCs- GsTsGoGoToAoC 50 0941-0960 coding 21872
GoGoCoAoCsAsAsAsGsCsTsAsAsTsGsAoCoToToC 51 1041-1060 coding 21873
AoGoGoToGsCsTsCsAsGsGsAsCsTsCsCoAoToToT 52 1081-1100 stop 21874
GoGoAoToGsGsAsCsAsGsAsAsCsAsGsAoAoGoCoA 53 1101-1120 3'-UTR 21875
GoAoGoCoAsGsGsCsAsGsAsCsTsGsCsCoAoAoGoG 54 1321-1340 3'-UTR 21876
AoGoGoCoTsAsGsAsGsCsCsCsAsGsGsAoGo- CoCoA 55 1561-1580 3'-UTR 21877
GoAoGoCoCsTsGsTsGsCsCsTsGs- GsCsAoCoToGoG 56 1861-1880 3'-UTR 21878
ToGoCoAoCsCsAsCsAsAsGsCsAsCsCsToGoGoAoG 57 2081-2100 3'-UTR 21879
GoGoCoToAsCsCsAsTsGsAsGsTsGsAsGoAoAoGoA 58 2221-2240 3'-UTR 21880
GoToCoCoCsTsGsCsAsCsTsGsAsTsAsGoAoGoAoA 59 2701-2720 3'-UTR 21881
ToCoToToCsCsAsAsTsGsGsAsGsAsAsAoCoToGoG 60 3001-3020 3'-UTR .sup.1
Emboldened residues, 2'-methoxyethoxy-residues (others are
2'-deoxy-); 2'-MOE cytosines and 2'-deoxy cytosine residues are
5-methyl-cytosines; "s" linkages are phosphorothioate linkages; "o"
linkages are phosphodiester linkages. .sup.2 Co-ordinates from
Genbank Accession No. U73142, locus name "RNU73142", SEQ ID NO.
45.
[0117] Rat p38.alpha. antisense oligonucleotides were screened in
bEND.3 cells for inhibition of p38.alpha. and p38.beta. mRNA
expression. The concentration of oligonucleotide used was 100 nM.
Results are shown in Table 8. Oligonucleotides 21844 (SEQ ID NO.
47), 21845 (SEQ ID NO. 48), 21872 (SEQ ID NO. 51), 21873 (SEQ ID
NO. 52), 21875 (SEQ ID NO. 54), and 21876 (SEQ ID NO. 55) showed
greater than approximately 70% inhibition of p38.alpha. mRNA with
minimal effects on p38.beta. mRNA levels. Oligonucleotide 21871
(SEQ ID NO. 50) inhibited both p38.alpha. and p38.beta. levels
greater than 70%.
8TABLE 8 Inhibition of Mouse p38 mRNA expression in bEND.3 Cells by
Chimeric (deoxy gapped) Mixed Backbone p38.alpha. Antisense
Oligonucleotides SEQ GENE % p38.alpha. ID TARGET mRNA % p38.beta.
mRNA ISIS No: NO: REGION INHIBITION INHIBITION control -- -- 0% 0%
21844 47 AUG 81% 20% 21845 48 coding 75% 25% 21871 50 coding 90%
71% 21872 51 coding 87% 23% 21873 52 stop 90% 3% 21874 53 3'-UTR
38% 21% 21875 54 3'-UTR 77% -- 21876 55 3'-UTR 69% -- 21877 56
3'-UTR 55% 13% 21878 57 3'-UTR 25% 10% 21879 58 3'-UTR -- -- 21881
60 3'-UTR -- --
[0118] Several of the most active oligonucleotides were selected
for dose response studies. bEND.3 cells were cultured and treated
as described above, except that the concentration of
oligonucleotide was varied as noted in Table 9. Results are shown
in Table 9.
9TABLE 9 Dose Response of bEND.3 cells to rat p38.beta. Chimeric
(deoxy gapped) Phosphorothioate Oligonucleotides % p38.alpha. %
p38.beta. SEQ ID ASO Gene mRNA mRNA ISIS # NO: Target Dose
Inhibition Inhibition control -- -- -- 100% 0% 21844 47 AUG 1 nM --
-- " " " 5 nM -- -- " " " 25 nM 36% 8% " " " 100 nM 80% 5% 21871 50
coding 1 nM 1% -- " " " 5 nM 23% 4% " " " 25 nM 34% 24% " " " 100
nM 89% 56% 21872 51 stop 1 nM -- -- " " " 5 nM -- -- " " " 25 nM
35% -- " " " 100 nM 76% 1% 21873 52 stop 1 nM -- 53% " " " 5 nM --
31% " " " 25 nM 54% 28% " " " 100 nM 92% 25% 21875 54 3'-UTR 1 nM
-- 11% " " " 5 nM -- 16% " " " 25 nM 33% 2% " " " 100 nM 72% 4%
Example 5
Mouse p38.beta. Oligonucleotide Sequences
[0119] Antisense oligonucleotides were designed to target mouse
p38.beta.. Target sequence data are from a mouse EST sequence;
Genbank accession number AI119044, provided herein as SEQ ID NO:
61. Oligonucleotides was synthesized as 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 in the wings are phosphodiester
(P.dbd.O). Internucleoside linkages in the central gap are
phosphorothioate (P.dbd.S). All 2'-MOE cytosines and 2'-OH
cytosines were 5-methyl-cytosines. These oligonucleotide sequences
are shown in Table 10.
10TABLE 10 Nucleotide Sequences of Mouse p38.beta. Chimeric (deoxy
gapped) Phosphorothioate Oligonucleotides TARGET GENE NUCLEOTIDE
NUCLEOTIDE SEQUENCE.sup.1 CO- ISIS NO. (5' -> 3') SEQ ID NO:
ORDINATES.sup.2 100800 CoAoCoAoGsAsAsGsCsAsGsCsTsGsGsAoGoC- oGoA 63
0051-0070 100801 ToGoCoGoGsCsAsCsCsTsCsCsCsAsTsAo- CoToGoT 64
0119-0138 100802 CoCoCoToGsCsAsGsCsCsGsCsTsGsCs- GoGoCoAoC 65
0131-0150 100803 GoCoAoGoAsCsTsGsAsGsCsCsGsTs- AsGoGoCoGoC 66
0171-0190 100804 ToToAoCoAsGsCsCsAsCsCsTsTs- CsTsGoGoCoGoC 67
0211-0230 100805 GoToAoToGsTsCsCsTsCsCsTs- CsGsCsGoToGoGoA 68
0261-0280 100806 AoToGoGoAsTsGsTsGsGsCsCsGsGsCsGoToGoAoA 69
0341-0360 100807 GoAoAoToTsGsAsAsCsAsTsGsCsTsCsAoToCoGoC 70
0441-0460 100808 AoCoAoToTsGsCsTsGsGsGsCsTsTsCsAoGoGoToC 71
0521-0540 100809 AoToCoCoTsCsAsGsCsTsCsCsCsAsGsToCoCoToC 72
0551-0570 100810 ToAoCoCoAsCsCsGsTsGsTsGsGsCsCsAoCoAoToA 73
0617-0636 100811 CoAoGoToTsTsAsGsCsAsTsGsAsTsCsToCoToGoG 74
0644-0663 100812 CoAoGoGoCsCsAsCsAsGsAsCsCsAsGsAoToGoToC 75
0686-0705 100813 CoCoToToCsCsAsGsCsAsGsTsTsCsAsAoGoCoCoA 76
0711-0730 101123 CoAoGoCoAsCsCsAsTsGsGsAsCsGsCsGoGoAoAoC 77 21871
mismatch .sup.1 Emboldened residues, 2'-methoxyethoxy-residues
(others are 2'-deoxy-), including 2'-MOE and 2'-deoxy residues,
5-methyl-cytosines; "s" linkages are phosphorothioate linkages, "o"
linkages are phosphodiester. .sup.2 Co-ordinates from Genbank
Accession No. AI119044, locus name "AI119044", SEQ ID NO. 61.
[0120] Mouse p38.beta. antisense sequences were screened in bEND.3
cells as described in Example 4. Results are shown in Table 11.
[0121] Oligonucleotides 100800 (SEQ ID NO. 63), 100801 (SEQ ID NO.
64), 100803 (SEQ ID NO. 66), 100804 (SEQ ID NO. 67), 100805 (SEQ ID
NO. 68), 100807 (SEQ ID NO. 70), 100808 (SEQ ID NO. 71), 100809
(SEQ ID NO. 72), 100810 (SEQ ID NO. 73), 100811 (SEQ ID NO.74), and
100813 (SEQ ID NO. 76) resulted in at least 50% inhibition of
p38.beta. mRNA expression. Oligonucleotides 100801 (SEQ ID NO.64),
100803 (SEQ ID NO. 66), 100804 (SEQ ID NO. 67), 100805 (SEQ ID NO.
68), 100809 (SEQ ID NO. 72), and 100810 (SEQ ID NO. 73) resulted in
at least 70% inhibition and are preferred. Oligonucleotides 100801
(SEQ ID NO. 64), 100805 (SEQ ID NO. 68), and 100811 (SEQ ID NO. 74)
resulted in significant inhibition of p38.alpha. mRNA expression in
addition to their effects on p38.beta..
11TABLE 11 Inhibition of Mouse p38 mRNA expression in bEND.3 Cells
by Chimeric (deoxy gapped) Mixed Backbone p38.beta. Antisense
Oligonucleotides SEQ ID % p38.beta. mRNA % p38.alpha. mRNA ISIS No:
NO: INHIBITION INHIBITION control -- 0% 0% 100800 63 51% -- 100801
64 74% 31% 100802 65 35% -- 100803 66 74% 18% 100804 67 85% 18%
100805 68 78% 58% 100806 69 22% 3% 100807 70 64% -- 100808 71 53%
13% 100809 72 84% 14% 100810 73 72% 1% 100811 74 60% 43% 100812 75
36% 17% 100813 76 54% --
Example 6
Effect of p38 MAPK Antisense Oligonucleotides on IL-6 Secretion
[0122] p38 MAPK antisense oligonucleotides were tested for their
ability to reduce IL-6 secretion. bEND.3 cells were cultured and
treated as described in Example 4 except that 48 hours after
oligonucleotide treatment, cells were stimulated for 6 hours with 1
ng/mL recombinant mouse IL-1 (R&D Systems, Minneapolis, Minn.).
IL-6 was measured in the medium using an IL-6 ELISA kit (Endogen
Inc., Woburn, Mass.).
[0123] Results are shown in Table 12. oligonucleotides targeting a
specific p38 MAPK isoform were effective in reducing IL-6 secretion
greater than approximately 50%.
12TABLE 12 Effect of p38 Antisense Oligonucleotides on IL-6
secretion SEQ ID GENE DOSE % IL-6 ISIS No: NO: TARGET (.mu.M)
INHIBITION control -- -- 0% 21873 52 p38.alpha. 100 49% 100804 67
p38.beta. 100 57% 21871 50 p38.alpha. 200 23% and p38.beta.
Example 7
Activity of p38.beta. Antisense Oligonucleotides in Rat
Cardiomyocytes
[0124] Rat p38.alpha. antisense oligonucleotides were screened in
Rat A-10 cells. A-10 cells (American Type Culture Collection,
Manassas, Va.) were grown in high-glucose DMEM (Life Technologies,
Gaithersburg, Md.) medium containing 10% fetal calf serum (FCS).
Cells were treated with oligonucleotide as described in Example 2.
Oligonucleotide concentration was 200 nM. mRNA was isolated 24
hours after time zero and quantitated by Northern blot as described
in Example 2.
[0125] Results are shown in Table 13. Oligonucleotides 21845 (SEQ
ID NO. 48), 21846 (SEQ ID NO. 49), 21871 (SEQ ID NO. 50), 21872
(SEQ ID NO. 51), 21873 (SEQ ID NO. 52), 21874 (SEQ ID NO. 53),
21875 (SEQ ID NO. 54), 21877 (SEQ ID NO. 56), 21878 (SEQ ID NO.
57), 21879 (SEQ ID NO. 58), and 21881 (SEQ ID NO. 60) inhibited
p38.beta. mRNA expression by 65% or greater in this assay.
Oligonucleotides 21846 (SEQ ID NO. 49), 21871 (SEQ ID NO. 50),
21872 (SEQ ID NO. 51), 21877 (SEQ ID NO. 56), and 21879 (SEQ ID NO.
58) inhibited p38.alpha. mRNA expression by greater than 85% and
are preferred.
13TABLE 13 Inhibition of Rat p38.alpha. mRNA expression in A-10
Cells by Chimeric (deoxy gapped) Mixed Backbone p38.alpha.
Antisense Oligonucleotides SEQ % p38.alpha. % p38.alpha. ID GENE
mRNA mRNA ISIS No: NO: TARGET REGION EXPRESSION INHIBITION control
-- -- 100% 0% 21844 47 AUG 75% 25% 21845 48 coding 25% 75% 21846 49
coding 8% 92% 21871 50 coding 12% 88% 21872 51 coding 13% 87% 21873
52 stop 19% 81% 21874 53 3'-UTR 22% 78% 21875 54 3'-UTR 26% 74%
21876 55 3'-UTR 61% 39% 21877 56 3'-UTR 12% 88% 21878 57 3'-UTR 35%
65% 21879 58 3'-UTR 11% 89% 21881 60 3'-UTR 31% 69%
[0126] The most active oligonucleotide in this screen (SEQ ID NO.
49) was used in rat cardiac myocytes prepared from neonatal rats
(Zechner, D., et. al., J. Cell Biol., 1997, 139, 115-127). Cells
were grown as described in Zechner et al. and transfected with
oligonucleotide as described in Example 2. Oligonucleotide
concentration was 1 .mu.M. mRNA was isolated 24 hrs after time zero
and quantitated using Northern blotting as described in Example 2.
An antisense oligonucleotide targeted to JNK-2 was used as a
non-specific target control.
[0127] Results are shown in Table 14. Oligonucleotide 21846 (SEQ ID
NO. 49) was able to reduce p38.alpha. expression in rat cardiac
myocytes by nearly 60%. The JNK-2 antisense oligonucleotide had
little effect on p38.alpha. expression.
14TABLE 14 Inhibition of Rat p38.alpha. mRNA expression in Rat
Cardiac Myocytes by A Chimeric (deoxy gapped) Mixed Backbone
p38.alpha. Antisense Oligonucleotide SEQ GENE % p38.alpha. ID
TARGET mRNA % p38.alpha. mRNA ISIS No: NO: REGION EXPRESSION
INHIBITION control -- -- 100% 0% 21846 49 coding 41% 59%
Example 8
Additional Human p38.alpha. Oligonucleotide Sequences
[0128] Additional antisense oligonucleotides were designed to
target human p38.beta. based on active rat sequences. Target
sequence data are from the p38 MAPK cDNA sequence; Genbank
accession number L35253, provided herein as SEQ ID NO: 1.
Oligonucleotides was synthesized as 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 2'-MOE cytosines and 2'-OH
cytosines were 5-methyl-cytosines. These oligonucleotide sequences
are shown in Table 15.
15TABLE 15 Additional Nucleotide Sequences of Human p38.alpha. Chi-
meric (deoxy gapped) Phosphorothioate Oligonucleo- tides TARGET
GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE.sup.1 ID CO-
TARGET NO. (5' -> 3') NO: ORDINATES.sup.2 REGION 100860
CTGAGACATTTTCCAGCGGC 78 0284-0303 Start 100861 ACGCTCGGGCACCTCCCAGA
79 0344-0363 coding 100862 AGCTTCTTCACTGCCACACG 80 0439-0458 coding
100863 AATGATGGACTGAAATGGTC 81 0464-0483 coding 100864
TCCAACAGACCAATCACATT 82 0538-0557 coding 100865
TGTAAGCTTCTGACATTTCA 83 0644-0663 coding 100866
TGAATGTATATACTTTAGAC 84 0704-0723 coding 100867
CTCACAGTCTTCATTCACAG 85 0764-0783 coding 100868
CACGTAGCCTGTCATTTCAT 86 0824-0843 coding 100869
CATCCCACTGACCAAATATC 87 0907-0926 coding 100870
TATGGTCTGTACCAGGAAAC 88 0960-0979 coding 100871
AGTCAAAGACTGAATATAGT 89 1064-1083 coding 100872
TTCTCTTATCTGAGTCCAAT 90 1164-1183 coding 100873
CATCATCAGGATCGTGGTAC 91 1224-1243 coding 100874
TCAAAGGACTGATCATAAGG 92 1258-1277 coding 100875
GGCACAAAGCTGATGACTTC 93 1324-1343 coding 100876
AGGTGCTCAGGACTCCATCT 94 1364-1383 stop 100877 GCAACAAGAGGCACTTGAAT
95 1452-1471 3'-UTR .sup.1 Emboldened residues,
2'-methoxyethoxy-residues (others are 2'-deoxy-) including "C" and
"C" residues, 5-methyl-cytosines; all linkages are phosphorothioate
linkages. .sup.2 Co-ordinates from Genbank Accession No. L35253,
locus name "HUMMAPKNS", SEQ ID NO. 1.
[0129] For an initial screen of human p38.alpha. antisense
oligonucleotides, T-24 cells, a human transitional cell bladder
carcinoma cell line, were obtained from the American Type Culture
Collection (ATCC) (Manassas, Va.). T-24 cells were routinely
cultured in complete McCoy's 5A basal media (Gibco/Life
Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf
serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100
units per mL, and streptomycin 100 micrograms per mL (Gibco/Life
Technologies, Gaithersburg, Md.). Cells were routinely passaged by
trypsinization and dilution when they reached 90% confluence. Cells
were seeded into 96-well plates (Falcon-Primaria #3872) at a
density of 7000 cells/well for use in RT-PCR analysis. A control
oligonucleotide ISIS 118965 (TTATCCTAGCTTAGACCTAT, herein
incorporated as SEQ ID NO: 96) was synthesized as chimeric
oligonucleotide ("gapmer") 20 nucleotides in length, composed of a
central "gap" region consisting of ten 2'-deoxynucleotides, which
is flanked on both sides (5' and 3' directions) by five-nucleotide
"wings." The wings are composed of 2'-methoxyethyl (2'-MOE)
nucleotides. The internucleoside (backbone) linkages are
phosphorothioate (P.dbd.S) throughout the oligonucleotide. All
2'-MOE cytosines and 2'-OH cytosines were 5-methyl-cytosines.
[0130] 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.
mRNA was measured by Northern blot. Results are shown in Table 16.
Oligonucleotides 100861 (SEQ ID NO. 79), 100862 (SEQ ID NO. 80),
100863 (SEQ ID NO. 81), 100866 (SEQ ID NO. 84), 100867 (SEQ ID NO.
85), 100868 (SEQ ID NO. 86), 100870 (SEQ ID NO. 88), 100871 (SEQ ID
NO. 89), 100872 (SEQ ID NO. 90), 100873 (SEQ ID NO. 91), and 100874
(SEQ ID (NO. 92), 100875 (SEQ ID NO. 93) and 100877 (SEQ ID NO. 95)
gave than approximately 40% inhibition and are preferred.
16TABLE 16 Inhibition of Human p38.alpha. mRNA expression in T-24
Cells by Chimeric (deoxy gapped) Phosphorothioate Oligonucleotides
SEQ % P38.alpha. % P38.beta. ID GENE TARGET mRNA mRNA ISIS No: NO:
REGION EXPRESSION EXPRESSION 100860 78 0284-0303 73% 71% 100861 79
0344-0363 60% 47% 100862 80 0439-0458 56% 45% 100863 81 0464-0483
49% 67% 100864 82 0538-0557 66% 70% 100865 83 0644-0663 64% 63%
100866 84 0704-0723 55% 65% 100867 85 0764-0783 58% 33% 100868 86
0824-0843 47% 60% 100869 87 0907-0926 61% 100% 100870 88 0960-0979
51% No data 100871 89 1064-1083 57% 96% 100872 90 1164-1183 37% 77%
100873 91 1224-1243 34% 70% 100874 92 1258-1277 42% 76% 100875 93
1324-1343 39% 90% 100876 94 1364-1383 77% 93% 100877 95 1452-1471
47% 95%
[0131] Oligonucleotides 100872 (SEQ ID NO. 90), 100873 (SEQ ID NO.
91), 100874 (SEQ ID NO. 92), and 100875 (SEQ ID NO. 93) were chosen
for dose response studies.
[0132] Results are shown in Table 17. The effect of these
oligonucleotides on human p38.beta. was also determined.
17TABLE 17 Dose Response of p38.alpha. in T-24 cells to human
p38.alpha. Chimeric (deoxy gapped) Phosphorothioate
Oligonucleotides % p38.alpha. % p38.beta. SEQ ID ASO Gene mRNA mRNA
ISIS # NO: Target Dose Expression Inhibition Control 96 -- -- 94%
80% 118965 100872 90 coding 50 nM 45% 108% " " " 100 nM 18% 91% " "
" 200 nM 17% 92% 100873 91 coding 50 nM 19% 90% " " " 100 nM 12%
78% " " " 200 nM 8% 44% 100874 92 coding 50 nM 47% 107% " " " 100
nM 27% 101% " " " 200 nM 13% 51% 100875 93 coding 50 nM 30% 105% "
" " 100 nM 13% 92% " " " 200 nM 8% 69%
Example 9
Additional Human p38.beta. Oligonucleotide Sequences
[0133] Additional antisense oligonucleotides were designed to
target human p38.beta. based on active rat sequences. Target
sequence data are from the p38 MAPK cDNA sequence; Genbank
accession number U53442, provided herein as SEQ ID NO: 23.
[0134] Oligonucleotides was synthesized as 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 in the wings
are phosphodiester (P.dbd.O). Internucleoside linkages in the
central gap are phosphorothioate (P.dbd.S). All 2'-MOE cytosines
and 2'-OH cytosines were 5-methyl-cytosines. These oligonucleotide
sequences are shown in Table 18. A control oligonucleotide ISIS
118966 (GTTCGATCGGCTCGTGTCGA), herein incorporated as SEQ ID NO:
107) was synthesized as chimeric oligonucleotide ("gapmer") 20
nucleotides in length, composed of a central "gap" region
consisting of ten 2'-deoxynucleotides, which is flanked on both
sides (5' and 3' directions) by five-nucleotide "wings." The wings
are composed of 2'-methoxyethyl (2'-MOE) nucleotides. The
internucleoside (backbone) linkages are phosphorothioate (P.dbd.S)
in the gap and phosphodiester in the wings. All 2'-MOE cytosines
and 2'-OH cytosines were 5-methyl-cytosines.
18TABLE 18 Additional Nucleotide Sequences of Human p38.beta. Chi-
meric (deoxy gapped) Mixed-Backbone Phosphorothio- ate
Oligonucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE
SEQUENCE.sup.1 ID CO- TARGET NO. (5' -> 3') NO. ORDINATES.sup.2
REGION 107869 ACAGACGGAGCCGTAGGCGC 97 117-136 coding 107870
CACCGCCACCTTCTGGCGCA 98 156-175 coding 107871 GTACGTTCTGCGCGCGTGGA
99 207-226 coding 107872 ATGGACGTGGCCGGCGTGAA 100 287-306 coding
107873 CAGGAATTGAACGTGCTCGT 101 414-433 coding 107874
ACGTTGCTGGGCTTCAGGTC 102 491-510 coding 107875 TACCAGCGCGTGGCCACATA
103 587-606 coding 107876 CAGTTGAGCATGATCTCAGG 104 614-633 coding
107877 CGGACCAGATATCCACTGTT 105 649-668 coding 107878
TGCCCTGGAGCAGCTCAGCC 106 682-701 coding .sup.1 Emboldened residues,
2'-methoxyethoxy-residues (others are 2'-deoxy-) including "C" and
"C" residues, 5-methyl-cytosines. .sup.2 Co-ordinates from Genbank
Accession No. U53442, SEQ ID NO.23.
[0135] For an initial screen of human p38.beta. antisense
oligonucleotides, T-24 cells, a human transitional cell bladder
carcinoma cell line, were obtained from the American Type Culture
Collection (ATCC) (Manassas, Va.). T-24 cells were routinely
cultured in complete McCoy's 5A basal media (Gibco/Life
Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf
serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100
units per mL, and streptomycin 100 micrograms per mL (Gibco/Life
Technologies, Gaithersburg, Md.). Cells were routinely passaged by
trypsinization and dilution when they reached 90% confluence. Cells
were seeded into 96-well plates (Falcon-Primaria #3872) at a
density of 7000 cells/well for use in RT-PCR analysis. A control
oligonucleotide ISIS 118966 (TTATCCTAGCTTAGACCTAT, herein
incorporated as SEQ ID NO: 106) was synthesized as chimeric
oligonucleotide ("gapmer") 20 nucleotides in length, composed of a
central "gap" region consisting of ten 2'-deoxynucleotides, which
is flanked on both sides (5' and 3' directions) by five-nucleotide
"wings." The wings are composed of 2'-methoxyethyl (2'-MOE)
nucleotides. The internucleoside (backbone) linkages are
phosphorothioate (P.dbd.S) in the gap and phosphodiester in the
wings. All 2'-MOE cytosines and 2'-OH cytosines were
5-methyl-cytosines.
[0136] 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.
mRNA was measured by Northern blot. Results are shown in Table 19.
For comparison, ISIS 17893 and ISIS 17899, both targeting human
p38.beta. (SEQ ID NO: 27) and ISIS 100802 targeting mouse p38.beta.
(SEQ ID NO: 65) described in Examples 3 and 5 above, respectively,
were included in the screen.
[0137] Oligonucleotides 107869 (SEQ ID NO. 97), 107871 (SEQ ID NO.
99), 107872 (SEQ ID NO. 100), 107873 (SEQ ID NO. 101), 107878 (SEQ
ID NO.106), 17893 (SEQ ID NO. 27), 17899 (SEQ ID NO. 33) and 100802
(SEQ ID NO.65, targeted to mouse p38.beta.) gave greater than
approximately 40% inhibition and are preferred.
19TABLE 19 Inhibition of Human p38.beta. mRNA expression in T-24
Cells by Chimeric (deoxy gapped) Mixed-Backbone Phosphorothioate
Oligonucleotides SEQ ID GENE TARGET % p38.beta. mRNA % p38.alpha.
mRNA ISIS No: NO: REGION EXPRESSION EXPRESSION 107869 97 Coding 60%
93% 107870 98 Coding 74% 97% 107871 99 Coding 60% 111% 107872 100
Coding 57% 123% 107873 101 Coding 58% 120% 107874 102 Coding 61%
100% 107875 103 Coding 92% 112% 107876 104 Coding 127% 137% 107877
105 Coding No data No data 107878 106 Coding 54% 112% 17893 27
Coding 31% 61% 17899 33 Coding 56% 117% 100802 65 Coding 47%
78%
[0138] Oligonucleotides 107871, 107872, 107873, 107874, 107875,
107877, 107878, 17893 and 17899 were chosen for dose response
studies.
[0139] Results are shown in Table 20. The effect of these
oligonucleotides on human p38.alpha. was also determined.
20TABLE 20 Dose Response of p38.beta. in T-24 cells to human
p38.beta. Chimeric (deoxy gapped) Mixed-backbone Phosphorothioate
Oligonucleotides % p38.beta. % p38.alpha. SEQ ID ASO Gene mRNA mRNA
ISIS # NO: Target Dose Expression Inhibition Control 107 -- -- 100%
100% 118966 107871 99 coding 50 nM 41% 105% " " " 100 nM 42% 132% "
" " 200 nM 10% 123% 107872 100 coding 50 nM 71% 124% " " " 100 nM
13% 84% " " " 200 nM 22% 102% 107873 101 coding 50 nM 69% 132% " "
" 100 nM 41% 119% " " " 200 nM 23% 131% 107874 102 coding 50 nM 75%
109% " " " 100 nM 34% 99% " " " 200 nM 23% 87% 107875 103 coding 50
nM 82% 93% " " " 100 nM 38% 101% " " " 200 nM 40% 91% 107877 105
coding 50 nM 50% 127% " " " 100 nM 34% 125% " " " 200 nM 22% 106%
107878 106 coding 50 nM 70% 110% " " " 100 nM 43% 109% " " " 200 nM
27% 116% 17893 27 coding 50 nM 28% 88% " " " 100 nM 27% 115% " " "
200 nM 16% 108% 17899 33 coding 50 nM 89% 87% " " " 100 nM 36% 104%
" " " 200 nM 15% 80%
[0140] These data show that the oligonucleotides designed to target
human p38.beta., do so in a target-specific and dose-dependent
manner.
Sequence CWU 0
0
* * * * *