U.S. patent application number 12/254513 was filed with the patent office on 2009-03-19 for materials and methods for inhibiting wip1.
This patent application is currently assigned to Government of the United States of America, Represented by the Secretary, Department of Health. Invention is credited to Ettore Appella, Dmitry Bulavin, Albert J. Fornace, JR., Anne Kallioniemi.
Application Number | 20090074724 12/254513 |
Document ID | / |
Family ID | 28675296 |
Filed Date | 2009-03-19 |
United States Patent
Application |
20090074724 |
Kind Code |
A1 |
Bulavin; Dmitry ; et
al. |
March 19, 2009 |
MATERIALS AND METHODS FOR INHIBITING WIP1
Abstract
Isolated or purified oligonucleotides and isolated or purified
morpholino oligomers; a method of detecting cancer or a
predisposition to cancer in a mammal, comprising comparing the
level of expression of Wip1 in the mammal to a control; a method of
treating cancer in a mammal that expresses the same or a higher
level of Wip1 as compared to a mammal of the same species that does
not have cancer, comprising administering to the mammal a
cancer-treating effective amount of a Wip1 inhibitor; a method of
screening an oligonucleotide or morpholino oligomer for the ability
to inhibit the expression of Wip1; a method of determining the
efficacy with which a test oligonucleotide or morpholino oligomer
inhibits Wip1 expression; a method of screening a compound for
Wip1-inhibiting activity; and a method of determining the efficacy
with which a test compound inhibits Wip1.
Inventors: |
Bulavin; Dmitry; (Singapore,
SG) ; Appella; Ettore; (Chevy Chase, MD) ;
Fornace, JR.; Albert J.; (Bethesda, MD) ;
Kallioniemi; Anne; (Tampere, FI) |
Correspondence
Address: |
LEYDIG, VOIT & MAYER, LTD.
TWO PRUDENTIAL PLAZA, SUITE 4900, 180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6731
US
|
Assignee: |
Government of the United States of
America, Represented by the Secretary, Department of Health
Rockville
MD
|
Family ID: |
28675296 |
Appl. No.: |
12/254513 |
Filed: |
October 20, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11868794 |
Oct 8, 2007 |
7456268 |
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12254513 |
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10507701 |
Oct 14, 2004 |
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PCT/US03/08997 |
Mar 21, 2003 |
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11868794 |
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60366883 |
Mar 22, 2002 |
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Current U.S.
Class: |
424/93.2 ;
435/18; 435/6.14; 514/231.2; 514/44R |
Current CPC
Class: |
A61P 35/04 20180101;
A61P 35/00 20180101; C07K 14/82 20130101 |
Class at
Publication: |
424/93.2 ;
435/18; 435/6; 514/44; 514/231.2 |
International
Class: |
C12Q 1/42 20060101
C12Q001/42; C12Q 1/68 20060101 C12Q001/68; A61K 31/7088 20060101
A61K031/7088; A61K 48/00 20060101 A61K048/00; A61K 31/5375 20060101
A61K031/5375; A61P 35/04 20060101 A61P035/04 |
Claims
1.-2. (canceled)
3. A method of detecting cancer or a predisposition to cancer in a
mammal, which method comprises comparing the level of expression of
Wip1 in a test sample comprising Wip1 obtained from the mammal to
the level of expression of Wip1 in a control sample, wherein a
higher level of expression of Wip1 in the test sample as compared
to the control sample is indicative of cancer or a predisposition
to cancer in the mammal.
4. The method of claim 3, wherein Wip1 is in the form of nucleic
acid in the test sample and the control sample.
5. The method of claim 3, wherein Wip1 is in the form of protein in
the test sample and the control sample.
6. The method of claim 3, wherein the cancer is breast cancer.
7. A method of treating cancer in a mammal that expresses the same
level or a higher level of Wip1 as compared to a mammal of the same
species that does not have cancer, which method comprises
administering to the mammal a cancer-treating effective amount of a
Wip1 inhibitor, whereupon the mammal is treated for cancer.
8. The method of claim 7, wherein the inhibitor is an
oligonucleotide or a morpholino oligomer, either one of which
hybridizes to any part of a nucleic acid encoding Wip1.
9. The method of claim 8, wherein the oligonucleotide or morpholino
oligomer comprises at least 18 genetic nitrogenous bases.
10. The method of claim 8, wherein the oligonucleotide or
morpholino oligomer hybridizes to exon 1 of a nucleic acid encoding
Wip1.
11. The method of claim 10, wherein the oligonucleotide or
morpholino oligomer comprises SEQ ID NO: 1.
12. The method of claim 8, wherein the oligonucleotide or
morpholino oligomer hybridizes to any part of the 5' untranslated
region (UTR) of a nucleic acid encoding Wip1.
13. The method of claim 12, wherein the oligonucleotide or
morpholino oligomer comprises SEQ ID NO: 2.
14. The method of claim 8, wherein the oligonucleotide or
morpholino oligomer hybridizes to any part of the 3' UTR of a
nucleic acid encoding Wip1.
15. The method of claim 8, wherein the oligonucleotide comprises at
least one modified oligodeoxynucleotide.
16. The method of claim 8, wherein the oligonucleotide or
morpholino oligomer is administered in the form of a liposome.
17. The method of claim 8, wherein the oligonucleotide or
morpholino oligomer is administered in the form of a vector.
18. The method of claim 17, wherein the vector is a plasmid
expression vector, a retroviral vector, or an adenoviral
vector.
19. The method of claim 8, wherein the cancer is in the form of a
tumor and the oligonucleotide or morpholino oligomer is
administered peritumorally or intratumorally.
20. The method of claim 8, wherein the oligonucleotide or
morpholino oligomer is administered with the carrier ethoxylated
polyethylenimine (EPEI).
21. The method of claim 7, wherein the inhibitor is a compound that
inhibits Wip1 phosphatase activity.
22. The method of claim 21, wherein the inhibitor is a small
molecular weight compound.
23. The method of claim 7, wherein the cancer is breast cancer.
24. A method of screening an oligonucleotide or morpholino oligomer
for the ability to inhibit the expression of Wip1, which method
comprises comparing the level of expression of Wip1 in a test
sample obtained from Wip1-expressing cells that have been contacted
with the oligonucleotide or morpholino oligomer to the level of
expression of Wip1 in a control sample obtained from
Wip1-expressing cells that have not been contacted with the
oligonucleotide or morpholino oligomer, respectively, wherein a
lower level of expression of Wip1 in the test sample as compared to
the control sample is indicative of the ability of the
oligonucleotide or morpholino oligomer to inhibit the expression of
Wip1.
25. The method of claim 24, wherein Wip1 is in the form of a
nucleic acid in the test sample and the control sample.
26. The method of claim 24, wherein Wip1 is in the form of protein
in the test sample and the control sample.
27. A method of determining the efficacy with which a test
oligonucleotide or morpholino oligomer inhibits Wip1 expression,
which method comprises comparing the level of expression of Wip1 in
a test sample obtained from Wip1-expressing cells that have been
contacted with the test oligonucleotide or morpholino oligomer to
the level of expression of Wip1 in a control sample obtained from
Wip1-expressing cells that have been contacted with an
oligonucleotide or morpholino oligomer that is known to inhibit the
expression of Wip1, wherein a lower level of expression of Wip1 in
the test sample as compared to the control sample is indicative of
the test oligonucleotide or morpholino oligomer having a greater
efficacy for inhibiting the expression of Wip1 than the known
oligonucleotide or morpholino oligomer and wherein a higher level
of expression of Wip1 in the test sample as compared to the control
sample is indicative of the test oligonucleotide or morpholino
oligomer having a lower efficacy for inhibiting the expression of
Wip1 than the known oligonucleotide or morpholino oligomer.
28. The method of any of claim 27, wherein Wip1 is in the form of
nucleic acid in the test sample and the control sample.
29. The method of claim 27, wherein Wip1 is in the form of protein
in the test sample and the control sample.
30. A method of screening a compound for Wip1-inhibiting activity,
which method comprises comparing the level of Wip1 phosphatase
activity in a test sample obtained from Wip1-expressing cells that
have been contacted with the compound to the level of Wip1
phosphatase activity in a control sample obtained from
Wip1-expressing cells that have not been contacted with the
compound, wherein a lower level of Wip1 phosphatase activity in the
test sample as compared to the control sample is indicative of the
ability of the compound to inhibit Wip1.
31. A method of determining the efficacy with which a test compound
inhibits Wip1, which method comprises comparing the level of Wip1
phosphatase activity in a test sample obtained from Wip1-expressing
cells that have been contacted with the test compound to the level
of Wip1 phosphatase activity in a control sample obtained from
Wip1-expressing cells that have been contacted with a compound that
is known to inhibit Wip1, wherein a lower level of Wip1 phosphatase
activity in the test sample as compared to the control sample is
indicative of the test compound having a greater efficacy for
inhibiting Wip1 than the known compound and wherein a higher level
of Wip1 phosphatase activity in the test sample as compared to the
control sample is indicative of the test compound having a lower
efficacy for inhibiting Wip1 than the known compound.
Description
FIELD OF THE INVENTION
[0001] This invention pertains to isolated or purified
oligonucleotides, isolated or purified morpholino oligomers, a
method of detecting cancer or a predisposition to cancer, as well
as a method of treating cancer. The present invention further
pertains to a method of screening an oligonucleotide or morpholino
oligomer for the ability to inhibit Wip1 expression and a method of
screening a compound for inhibiting Wip1 activity, in addition to a
method of determining the efficacy with which an oligonucleotide or
morpholino oligomer inhibits Wip1 expression and a method of
determining the efficacy with which a test compound inhibits Wip1
phosphatase activity.
BACKGROUND OF THE INVENTION
[0002] Wild-type p53-induced phosphatase 1 (Wip1) is a
Mg.sup.2+-dependent serine/threonine protein phosphatase that is
expressed in response to ionizing or ultra-violet (UV) radiation in
a manner that is dependent on the tumor suppressor gene product
p53. Its role in cancer was first suggested by Fiscella et al.,
Proceedings of the National Academy of Sciences, U.S.A. 94:
6048-6053 (1997), which reported Wip1 as an important inhibitor of
growth, since ectopic expression of WIP1 (also known as PPMD1) in a
human glioblastoma cell line (T98G) resulted in fewer colonies of
cells. In contrast to these results, Wip1 was shown by Takekawa et
al., EMBO Journal 19(23): 6517-6526 (2000), to dephosphorylate the
kinase p38, which functions to activate p53 for the induction of
apoptosis and transcription in response to environmental stress,
thereby rendering Wip1 anti-apoptotic as opposed to
anti-proliferative.
[0003] Consistent with Takekawa et al., Wip1 has been shown by the
present inventors to be a positive regulator of tumorigenesis.
WIP1, located at chromosome 17q22/q23 by FISH analysis, was found
amplified in human breast tumor cell lines as well as in
approximately 11% of primary breast tumors as determined by
Northern blot analysis, Southern blot analysis, and tissue
microarray analysis. Furthermore, exogeneous expression of WIP1 in
cells expressing H-Ras-V12 resulted in a decrease in p53-mediated
apoptosis and a partial rescue of these cells from cell cycle
arrest. Moreover, Wip1 was demonstrated as a negative regulator of
p38, since UV-induced activation of p38 kinase was significantly
attenuated in breast cell lines in which the Wip1 gene was
amplified and overexpressed (BT-474 and MCF7) compared to a breast
line (MA-MB436) without Wip1 amplification. Taken together, these
results indicate that WIP1 is a candidate proto-oncogene involved
in tumorigenesis and, thus, represents an attractive new target for
cancer therapy.
[0004] In view of the foregoing, the present invention provides
materials and methods for treating cancer in a mammal that
expresses elevated levels of Wip1. These and other advantages of
the invention, as well as additional inventive features, will be
apparent from the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
[0005] The present invention provides an isolated or purified
oligonucleotide consisting essentially of the nucleotide sequence
of SEQ ID NO: 1 or SEQ ID NO: 2, as well as an isolated or purified
morpholino oligomer consisting essentially of the sequence of SEQ
ID NO: 1 or SEQ ID NO: 2.
[0006] The present invention further provides a method of detecting
cancer or a predisposition to cancer in a mammal. The method
comprises comparing the level of expression of Wip1 in a test
sample comprising Wip1 obtained from the mammal to the level of
expression of Wip1 in a control sample. A higher level of
expression of Wip1 in the test sample as compared to the control
sample is indicative of cancer or a predisposition to cancer in the
mammal.
[0007] Further provided by the present invention is a method of
treating cancer in a mammal that expresses the same level or a
higher level of Wip1 as compared to a mammal of the same species
that does not have cancer. The method comprises administering to
the mammal a cancer-treating effective amount of a Wip1
inhibitor.
[0008] The present invention also provides a method of screening an
oligonucleotide or morpholino oligomer for the ability to inhibit
the expression of Wip1. The method comprises comparing the level of
expression of Wip1 in a test sample obtained from Wip1-expressing
cells that have been contacted with the oligonucleotide or
morpholino oligomer to the level of expression of Wip1 in a control
sample obtained from Wip1-expressing cells that have not been
contacted with the oligonucleotide or morpholino oligomer. A lower
level of expression of Wip1 in the test sample as compared to the
control sample is indicative of the ability of the oligonucleotide
or morpholino oligomer to inhibit the expression of Wip1.
[0009] A method of determining the efficacy with which a test
oligonucleotide or morpholino oligomer inhibits Wip1 expression is
further provided by the present invention. The method comprises
comparing the level of expression of Wip1 in a test sample obtained
from Wip1-expressing cells that have been contacted with the test
oligonucleotide or morpholino oligomer to the level of expression
of Wip1 in a control sample obtained from Wip1-expressing cells
that have been contacted with an oligonucleotide or morpholino
oligomer that is known to inhibit the expression of Wip1. A lower
level of expression of Wip1 in the test sample as compared to the
control sample is indicative of the test oligonucleotide or
morpholino oligomer having a greater efficacy for inhibiting the
expression of Wip1 than the known oligonucleotide or morpholino
oligomer, whereas an higher level of expression of Wip1 in the test
sample as compared to the control sample is indicative of the test
oligonucleotide or morpholino oligomer having a lower efficacy for
inhibiting the expression of Wip1 than the known oligonucleotide or
morpholino oligomer.
[0010] Further provided is a method of screening a compound for
Wip1-inhibiting activity. The method comprises comparing the level
of Wip1 phosphatase activity in a test sample obtained from
Wip1-expressing cells that have been contacted with the compound to
the level of Wip1 phosphatase activity in a control sample obtained
from Wip1-expressing cells that have not been contacted with the
compound. A lower level of Wip1 phosphatase activity in the test
sample as compared to the control sample is indicative of the
ability of the compound to inhibit Wip1.
[0011] The present invention also provides a method of determining
the efficacy with which a test compound inhibits Wip1. The method
comprises comparing the level of Wip1 phosphatase activity in a
test sample obtained from Wip1-expressing cells that have been
contacted with the test compound to the level of Wip1 phosphatase
activity in a control sample obtained from Wip1-expressing cells
that have been contacted with a compound that is known to inhibit
Wip1. A lower level of Wip1 phosphatase activity in the test sample
as compared to the control sample is indicative of the test
compound having a greater efficacy for inhibiting Wip1 than the
known compound, whereas a higher level of Wip1 phosphatase activity
in the test sample as compared to the control sample is indicative
of the test compound having a lower efficacy for inhibiting Wip1
than the known compound.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 represents a table of sequences (5'.fwdarw.3' when
read from left to right) of the oligonucleotides or morpholino
oligomers of the present invention and the regions to which they
hybridize.
DETAILED DESCRIPTION OF THE INVENTION
[0013] The present invention provides isolated or purified
oligonucleotides consisting essentially of the nucleotide sequence
of SEQ ID NO: 1 or SEQ ID NO: 2, which can be used in the methods
described herein. The term "isolated" as used herein means having
been removed from its natural environment. The term "purified" as
used herein means having been increased in purity, wherein "purity"
is a relative term, and not to be construed as absolute purity. The
term "oligonucleotide" as used herein means a polymer of DNA or
RNA, (i.e., a polynucleotide), which can be single-stranded or
double-stranded, synthesized or obtained from natural sources, and
which can contain natural, non-natural or altered nucleotides. With
respect to the isolated or purified oligonucleotide of the present
invention, it is preferred that no insertions, deletions,
inversions, and/or substitutions are present in the
oligonucleotide. However, it may be suitable in some instances for
the isolated or purified oligonucleotide of the present invention
to comprise one or more insertions, deletions, inversions, and/or
substitutions. It is, furthermore, preferred that the isolated or
purified oligonucleotides of the present invention are synthesized,
single-stranded polymers of DNA.
[0014] Isolated or purified morpholino oligomers consisting
essentially of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 are
further provided by the present invention. The term "morpholino
oligomer" as used herein refers to a polymer of the genetic
nitrogeneous bases, adenine, guanine, cytosine, and thymine, in
which the nitrogenous bases are linked to a 6-membered morpholine
ring, as opposed to ribose or deoxyribose as in RNA or DNA. Also,
each unit of the morpholino oligomer that comprises the
nitrogeneous base and the morpholine ring is bridged to neighboring
units via phosphorodiamidate linkages, in contrast to the
phosphodiester linkages of oligonucleotides. For more description
of morpholino oligomers, see Summerton et al., Antisense &
Nucleic Acid Drug Development 7:187-195 (1997).
[0015] The sequence of the nitrogeneous bases of the morpholino
oligomers is essential for their use in the methods described
herein. Like the oligonucleotides of the present invention, it is
preferred that the morpholino oligomers do not comprise any
insertions, deletions, inversions, and/or substitutions. However,
it may be suitable in some instances for one or more insertions,
deletions, inversions, and/or substitutions to be present in the
morpholino oligomers of the present invention. It is, furthermore,
preferred that the isolated or purified morpholino oligomers of the
present invention are single-stranded.
[0016] A variety of techniques used to synthesize the
oligonucleotides of the present invention are known in the art.
See, for example, Lemaitre et al., Proceedings of the National
Academy of the Sciences 84: 648-652 (1987). Likewise, a variety of
techniques employed for the synthesis of morpholino oligomers are
known in the art. See, for example, U.S. Pat. No. 5,185,444.
Alternatively, the oligonucleotides or morpholino oligomers of the
present invention can be purchased from companies, such as
Eurogentec, Belgium (for oligonucleotides) and Gene Tools,
Philomath, Oreg. (for morpholino oligomers).
[0017] The present invention further provides a method of detecting
cancer or a predisposition to cancer in a mammal. The method
comprises comparing the level of expression of Wip1 in a test
sample comprising Wip1 obtained from the mammal to the level of
expression of Wip1 in a control sample, wherein the control sample
is a sample comprising Wip1 taken from a mammal, desirably of the
same species, which is known to not have cancer or a predisposition
to cancer. In this method, a higher level of expression of Wip1 in
the test sample as compared to the control sample is indicative of
cancer or a predisposition to cancer in the mammal.
[0018] With respect to the above method, in addition to the other
methods of the present invention, wherein the method comprises
comparing the level of expression of Wip1, a variety of techniques
known in the art can be used to compare the level of expression of
Wip1. For example, Western blotting can be used to compare the
levels of Wip1 protein expressed in two different cell populations.
Alternatively, Northern blotting can be used to compare the levels
of Wip1 mRNA expressed in two different cell populations. Finally,
Southern blotting can be used to compare the number of copies of
the Wip1 gene found in two different cell populations. These
processes are described in Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2.sup.nd Ed., Cold Spring Harbor Press, Cold
Spring Harbor, N.Y. (1989).
[0019] For purposes of the present invention, mammals include, but
are not limited to, the order Rodentia, such as mice, and the order
Logomorpha, such as rabbits. It is preferred that the mammals are
from the order Camivora, including Felines (cats) and Canines
(dogs). It is more preferred that the mammals are from the order
Artiodactyla, including Bovines (cows) and Suines (pigs) or of the
order Perssodactyla, including Equines (horses). It is most
preferred that the mammals are of the order Primates, Ceboids, or
Simoids (monkeys) or of the order Anthropoids (humans and apes). An
especially preferred mammal is the human.
[0020] In a preferred embodiment of the present inventive method of
detecting cancer or a predisposition to cancer, Wip1 is in the form
of nucleic acid in the test sample and the control sample. The term
"nucleic acid" as used herein refers to a chain of nucleotides, as
in DNA and RNA. In another preferred embodiment of the present
inventive method, Wip1 is in the form of protein in the test sample
and the control sample.
[0021] The present invention also provides a method of treating
cancer in a mammal that expresses the same level or a higher level
of Wip1 as compared to a mammal of the same species that does not
have cancer. The method comprises administering to the mammal a
cancer-treating effective amount of a Wip1 inhibitor. A
"cancer-treating effective amount" of a Wip1 inhibitor is an amount
sufficient to inhibit the progression of cancer to any degree. It
is understood by one of ordinary skill in the art that the
inhibition mediated by an inhibitor does not require complete
inhibition, as a beneficial or therapeutic effect can be realized
with any degree of inhibition. Rather, there are varying degrees of
inhibition. In this regard, any suitable inhibitor of Wip1 can be
used.
[0022] With respect to the present inventive methods, wherein
cancer or a predisposition to cancer is either detected or treated,
the cancer can be cancer of any tissue from a mammal. Preferably,
the cancer is cancer of the breast.
[0023] In a preferred embodiment of the above inventive method, the
Wip1 inhibitor is an oligonucleotide or a morpholino oligomer that
inhibits Wip1 by preventing the expression of either Wip1 mRNA or
Wip1 protein, such as by hybridizing to any part of a nucleic acid
encoding (i.e., DNA or RNA) Wip1. The oligonucleotide or morpholino
oligomer for use in the present inventive method desirably
comprises any sequence that is substantially complementary to the
sequence of the untranslated region (UTR) of a nucleic acid
encoding Wip1 or is substantially complementary to the sequence of
a nucleic acid encoding Wip1, such that the oligonucleotide or
morpholino oligomer selectively hybridizes to the Wip1 gene or Wip1
mRNA, thereby inhibiting transcription of the gene or translation
of the mRNA. It is desirable for the selective hybridization to be
highly stringent. In other words, the oligonucleotides or
morpholino oligomers specifically hybridize to target sequences of
Wip1 mRNA or the Wip1 gene in an amount that is detectably stronger
than non-specific hybridization.
[0024] Preferably, the oligonucleotide or morpholino oligomer
comprises at least 18 genetic nitrogenous bases. More preferably,
the oligonucleotide or morpholino oligomer comprises 25 genetic
nitrogenous bases. Preferably, the oligonucleotide or morpholino
oligomer hybridizes to exon 1 of a nucleic acid encoding Wip1. In
an even more preferred embodiment of the present invention, the
oligonucleotide or morpholino oligomer comprises the sequence
5'-CTCCCAGCGAGTACAGCCCCGCCAT-3. (SEQ ID NO: 1). Alternatively and
also preferably, the oligonucleotide or morpholino oligomer
hybridizes to anypart of an UTR of a nucleic acid encoding Wip1.
The oligonucleotide or morpholino oligomer of the present invention
can hybridize to any part of the 3' UTR, which is the region
located downstream of the stop codon of the nucleic acid encoding
Wip1. Alternatively, the oligonucleotide or morpholino oligomer
hybridizes to the 5' UTR, which is the region located upstream of
the initiation codon of the nucleic acid encoding Wip1. In this
instance, it is most preferred that the oligonucleotide or
morpholino oligomer comprises the sequence
5'-CGGTCCCACGCAGCCCGCCGAATCC-3' (SEQ ID NO: 2).
[0025] The oligonucleotide can comprise at least one modified
oligodeoxynucleotide. For example, the oligonucleotide can contain
at least one variant linkage that bridges one nucleotide to
another, such as a phosphoroamidate linkage or a phosphorothioate
linkage instead of the phosphodiester linkages found in unmodified
oligonucleotides.
[0026] The inhibitor alternatively can be a compound that inhibits
Wip1 phosphatase activity. It is to be understood that the
inhibition of Wip1 phosphatase activity does not require complete
inhibition, as a beneficial or therapeutic effect can be realized
with any degree of inhibition. Rather, there are varying degrees of
inhibition. The compound that inhibits Wip1 phosphatase activity
can be a small molecular weight compound. As used herein, the term
"small molecular weight compound" refers to a compound having a
molecular weight of less than about 10 kDa as measured by, for
example, gel filtration chromatography. One skilled in the art will
appreciate that a small molecular weight compound is, generally, a
non-peptidic compound that is cell permeable and resistant to
degradation. The term "non-peptidic" as used herein refers to not
being derived from a protein.
[0027] The Wip1 inhibitor can be administered in a variety of
forms. For example, when the Wip1 inhibitor is an oligonucleotide
or morpholino oligomer, the oligonucleotide or morpholino oligomer
can be administered in the form of a liposome. Alternatively, the
oligonucleotide or morpholino oligomer can be administered in the
form of a vector. One of ordinary skill in the art will appreciate
that any of a number of vectors known in the art are suitable for
use in the invention. Examples of suitable vectors include, for
instance, plasmids, plasmid-liposome complexes, and viral vectors,
e.g., parvoviral-based vectors (i.e., adeno-associated virus
(AAV)-based vectors), retroviral vectors, herpes simplex virus
(HSV)-based vectors, and adenovirus-based vectors. Any of these
expression constructs can be prepared using standard recombinant
DNA techniques described in, e.g., Sambrook et al. (1989), supra
and Ausubel et al., Current Protocols in Molecular Biology, Greene
Publishing Associates and John Wiley & Sons, New York, N.Y.
(1994).
[0028] In addition, the Wip1 inhibitor can be administered with a
carrier. The carrier can be any suitable carrier. Preferably, the
carrier is pharmaceutically acceptable. With respect to the
oligonucleotides or morpholino oligomers of the present invention,
the carrier can be any of those conventionally used and is limited
only by chemico-physical considerations, such as solubility and
lack of reactivity with the oligonucleotides or morpholino
oligomers of the present invention, and by the route of
administration. The pharmaceutically acceptable carriers described
herein, for example, vehicles, adjuvants, excipients, and diluents,
are well-known to those skilled in the art and are readily
available to the public. Typically, the pharmaceutical composition
comprising the carrier and the oligonucleotide or morpholino
oligomer can comprise a physiological saline solution; dextrose or
other saccharide solution; or ethylene, propylene, polyethylene, or
other glycol. It is preferred that the pharmaceutically acceptable
carrier be one which is chemically inert to the oligonucleotides or
morpholino oligomers and one which has no detrimental side effects
or toxicity under the conditions of use. The choice of carrier will
be determined, in part, by the particular Wip1 inhibitor and by the
particular method used to administer the resulting composition.
Preferably, the oligonucleotides or morpholino oligomers are
administered with the carrier ethoxylated polyethylenimine (EPEI),
which is a weakly basic delivery reagent that electrostatically
binds to the anionic charges of oligonucleotides and
morpholino/oligonucleotide duplexes. See Morcos, Genesis 30: 94-102
(2001), for more description on this method.
[0029] One skilled in the art will appreciate that suitable methods
of administering a Wip1 inhibitor, such as an oligonucleotide or a
morpholino oligomer of the present invention, to a mammal, such as
a human, are known, and, although more than one route can be used
to administer a particular composition, a particular route can
provide a more immediate and more effective reaction than another
route. If the cancer is in the form of a tumor, preferably the Wip1
inhibitor is administered peritumorally or intratumorally.
[0030] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the
oligonucleotide or morpholino oligomer dissolved in diluents, such
as water or saline, (b) capsules, sachets or tablets, each
containing a predetermined amount of the active ingredient, as
solids or granules, (c) suspensions in an appropriate liquid, and
(d) suitable emulsions.
[0031] Tablet forms can include one or more of lactose, mannitol,
cornstarch, potato starch, microcrystalline cellulose, acacia,
gelatin, colloidal silicon dioxide, croscarmellose sodium, talc,
magnesium stearate, stearic acid, and other excipients, colorants,
diluents, buffering agents, moistening agents, preservatives,
flavoring agents, and pharmacologically compatible carriers.
Lozenge forms can comprise the active ingredient in a flavor,
usually sucrose and acacia or tragacanth, as well as pastilles
comprising the active ingredient in an inert base, such as gelatin
and glycerin or sucrose and acacia emulsions, gels, and the like
containing, in addition to the active ingredient, such carriers as
are known in the art.
[0032] Formulations suitable for parenteral administration include
aqueous and non-aqueous solutions, isotonic sterile injection
solutions, which can contain anti-oxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. The formulations
can be presented in unit-dose or multi-dose sealed containers, such
as ampules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid carrier, for example, water, for injections, immediately
prior to use. Extemporaneous injection solutions and suspensions
can be prepared from sterile powders, granules, and tablets of the
kind previously described.
[0033] The Wip1 inhibitor, such as the oligonucleotides or
morpholino oligomers of the present invention, can be administered
alone or in combination with other suitable components. Such
components include other active agents, such as anti-cancer agents
and agents that help the Wip1 inhibitor inhibit expression of Wip1
protein or Wip1 mRNA or Wip1 phosphatase activity more
effectively.
[0034] The amount or dose of the Wip1 inhibitor administered to a
mammal, particularly a human, in the context of the present
invention should be sufficient to effect a therapeutic response in
the animal over a reasonable time frame. The dose will be
determined by the efficacy of the particular Wip1 inhibitor and the
condition of the animal (e.g., human), as well as the body weight
of the animal (e.g., human) to be treated. The size of the dose
also will be determined by the existence, nature and extent of any
adverse side effects that might accompany the administration of a
particular Wip1 inhibitor. Ultimately, the attending physician will
decide the dosage of the Wip1 inhibitor of the present invention
with which to treat each individual patient, taking into
consideration a variety of factors, such as age, body weight,
general health, diet, sex, inhibitor to be administered, route of
administration, and the severity of the cancer being treated.
[0035] A method of screening an oligonucleotide or morpholino
oligomer for the ability to inhibit the expression of Wip1 is
further provided by the present invention. The method comprises
comparing the level of expression of Wip1 in a test sample obtained
from Wip1-expressing cells that have been contacted with the
oligonucleotide or morpholino oligomer to the level of expression
of Wip1 in a control sample obtained from Wip1-expressing cells
that have not been contacted with the oligonucleotide or morpholino
oligomer. In this method, a lower level of expression of Wip1 in
the test sample as compared to the control sample is indicative of
the ability of the oligonucleotide or morpholino oligomer to
inhibit the expression of Wip1.
[0036] Further provided by the present invention is a method of
determining the efficacy with which a test oligonucleotide or
morpholino oligomer inhibits Wip1 expression. The method comprises
comparing the level of expression of Wip1 in a test sample obtained
from Wip1-expressing cells that have been contacted with the test
oligonucleotide or morpholino oligomer to the level of expression
of Wip1 in a control sample obtained from Wip1-expressing cells
that have been contacted with an oligonucleotide or morpholino
oligomer that is known to inhibit the expression of Wip1. A lower
level of expression of Wip1 in the test sample as compared to the
control sample is indicative of the test oligonucleotide or
morpholino oligomer having a greater efficacy for inhibiting the
expression of Wip1 than the known oligonucleotide or morpholino
oligomer, whereas a higher level of expression of Wip1 in the test
sample as compared to the control sample is indicative of the test
oligonucleotide or morpholino oligomer having a lower efficacy for
inhibiting the expression of Wip1 than the known oligonucleotide or
morpholino oligomer.
[0037] Wip1 can be in the form of nucleic acid in the test sample
and the control sample. Alternatively, Wip1 can be in the form of
protein in the test sample and the control sample.
[0038] With respect to the method of screening an oligonucleotide
or morpholino oligomer and the method of determining the efficacy
with which a test oligonucleotide or morpholino oligomer inhibits
Wip1 expression, a variety of techniques known in the art can be
used to compare the level of expression of Wip1. For example,
Western blotting can be used to compare the levels of Wip1 protein
expressed in two different cell populations. Alternatively,
Northern blotting can be used to compare the levels of Wip1 mRNA
expressed in two different cell populations. Finally, Southern
blotting can be used to compare the number of copies of the Wip1
gene found in two different cell populations. These processes are
described in Sambrook et al. (1989), supra.
[0039] The present invention also provides a method of screening a
compound for Wip1-inhibiting activity. The method comprises
comparing the level of Wip1 phosphatase activity in a test sample
obtained from Wip1-expressing cells that have been contacted with
the compound to the level of Wip1 phosphatase activity in a control
sample obtained from Wip1-expressing cells that have not been
contacted with the compound. A lower level of Wip1 phosphatase
activity in the test sample as compared to the control sample is
indicative of the ability of the compound to inhibit Wip1.
[0040] A method of determining the efficacy with which a test
compound inhibits Wip1 is also provided by the present invention.
The method comprises comparing the level of Wip1 phosphatase
activity in a test sample obtained from Wip1-expressing cells that
have been contacted with the test compound to the level of Wip1
phosphatase activity in a control sample obtained from
Wip1-expressing cells that have been contacted with a compound that
is known to inhibit Wip1. In this method, a lower level of Wip1
phosphatase activity in the test sample as compared to the control
sample is indicative of the test compound having a greater efficacy
for inhibiting Wip1 than the known compound, whereas a higher level
of Wip1 phosphatase activity in the test sample as compared to the
control sample is indicative of the test compound having a lower
efficacy for inhibiting Wip1 than the known compound.
[0041] A variety of techniques known in the art can be used to
compare levels of phosphatase activity. An example of a Wip1
phosphatase activity assay can be found in Fiscella et al. (1997),
supra.
[0042] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
EXAMPLES
Abbreviations
[0043] For convenience, the following abbreviations are used
herein: Wip1, wild-type p53-induced phosphatase; UV, ultra-violet;
PPMD1, protein phosphatase, magnesium-dependent 1; FISH,
fluorescent in situ hybridization; mRNA, messenger RNA; UTR,
untranslated region; HSV, herpes simplex virus; AAV,
adeno-associated virus; EPEI, ethoxylated polyethylenimine; GAPDH,
glyceraldehyde 3-phosphate dehydrogenase; GADD34, growth arrest and
DNA damage-inducible 34; and PCR, polymerase chain reaction; cDNA,
complementary DNA; BAC, bacteria artificial chromosome; dUTP,
deoxyuridine triphosphate; MEF, mouse embryo fibroblasts.
[0044] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by
reference:
[0045] Birren et al., Genome Analysis: A Laboratory Manual Series,
Volume 1, Analyzing DNA, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y. (1997),
[0046] Birren et al., Genome Analysis: A Laboratory Manual Series,
Volume 2, Detecting Genes, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1998),
[0047] Birren et al., Genome Analysis: A Laboratory Manual Series,
Volume 3, Cloning Systems, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1999),
[0048] Birren et al., Genome Analysis: A Laboratory Manual Series,
Volume 4, Mapping Genomes, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1999),
[0049] Harlow et al., Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988),
[0050] Harlow et al., Using Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1999),
[0051] Hoffman, Cancer and the Search for Selective Biochemical
Inhibitors, CRC Press (1999),
[0052] Pratt, The Anticancer Drugs, 2nd edition, Oxford University
Press, NY (1994),
[0053] QIAexpress Detection and Assay Handbook, 2nd edition, QIAGEN
Inc., 28159 Avenue Stanford, Valencia, Calif. 91355 (April 1999),
and
[0054] Sambrook et al. (1989), supra.
Example 1
[0055] This example demonstrates the expression levels of Wip1 mRNA
in human tumor cell lines and tumor samples.
[0056] Total RNA was extracted from 67 human tumor cell lines of
which 58 are listed in O'Connor et al., Cancer Research 57:
4285-4300 (1997) and 11 are IMR-90, NDA-N, Sum 52, NCI-ADR, BT-474,
MCF7, MCI-H % 22, ACHN, CAKI-1, MOLT4, and OVCAR4, in addition to
11 tumor samples National Cancer Institute, National Institutes of
Health), using the RNeasy kit (Qiagen, Inc., Valencia, Calif.).
Wip1 mRNA levels in primary breast tumors were determined after
reverse transcription coupled to the Real-Time PCR procedure using
an ABI PRISM.RTM. 7700 Sequence Detection System (Applied
Biosystems, Foster City, Calif.) and the SYBR Green PCR Master Mix
(Applied Biosystems, Foster City, Calif.). Expression levels were
determined relative to the expression of GAPDH and GADD34. The
following primers were used: WIP1:
5'-tgcccgggagcacttgt-3'/5'-ggcagcgcaaaccttagc-3' and
5'-gacccgaaggatgactttgtc-3'/5'-gcttctgagggtcaagagtg-3'; GADD34,
5'-cctctacttctgccttgtctcca-3'/5'-ccgtggcttgattctcttcct-3'; and
GAPDH, 5-gaaggtgaaggtcggagtc-3'/5'-gaagatggtgatgggatttc-3' mRNA
levels in the panel of human tumor cell lines were analyzed using a
dot-blotting procedure described in Koch-Paiz et al., Biotechniques
29: 706-714 (2000). polyU served as a control for relative mRNA
content.
[0057] In the human tumor cell lines tested, Wip1 mRNA levels were
4.7 to 9.4 times higher in the four breast tumor cell lines
MDA-MB361, BT474, MCF-7 and KPL-1 as compared to IMR-90 cells. This
analysis demonstrated that Wip1 mRNA was overexpressed in tumor
samples.
Example 2
[0058] This example demonstrates that WP1 is amplified in some
breast cancer cell lines.
[0059] Genomic DNA from IMR-90, NDA-N, Sum52, NCI-ADR, BT-474,
MCF7, MCI-H % 22, ACHN, CAKI-1, MOLT4, and OVCAR4 cell lines were
digested with PvuII enzyme (New England Biolabs, Beverly, Mass.)
The digested DNA was Southern blotted according to Sambrook et al.
(1989), supra, using probes comprising the full-length cDNA
sequence of Wip1, which has the GenBank accession number, U78305.
From this analysis, it was shown that MCF7 and BT-474 breast cancer
cell lines, and not the other cell lines tested, overexpressed
WIP1. This analysis demonstrated that Wip1 mRNA was overexpressed
in breast cancer.
Example 3
[0060] This example demonstrates the amplification of WIP1 in
primary tumors.
[0061] The BLASTN program (http://www.ncbi.nlm.nih.gov/BLAST/) was
used to localize the WIP1 gene to three overlapping bacteria
artificial chromosome (BAC) clones (RP11-15E18, RP11-634F5, and
RP11-1081E4) in the draft human genome sequence that map to 17q23
(http://www.ncbi.nlm.nih.gov/genome/guide/human/). BAC clone
RP11-634F5, representing WIP1, labeled with
SpectrumOrange-deoxyuridine triphosphate (dUTP) (Applied
Biosystems, Foster City, Calif.) and centromere specific,
SpectrumGreen-dUTP-labeled chromosome 17 probe (Applied Biosystems,
Foster City, Calif.) were hybridized to a tissue microarray
containing primary breast tumors (Kononen et al., Nature Medicine
4: 844-847 (1998)). Nuclei were stained with 4',
6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, Mo.). Tumor
samples with at least a 3-fold increase in the number of WIP1
signals, as compared with chromosome 17 centromere signals, were
considered to be amplified. Accordingly, 37 of the 326 (11.3%)
tumors tested had WIP1 region amplified. From this example, it is
evident that the WIP1 is amplified in some primary tumors.
Example 4
[0062] This example demonstrates the effect of WIP1 overexpression
on p38-mediated phosphorylation of p53.
[0063] IMR-90 cells were infected with plasmids encoding Wip1,
which were made by cloning the Wip1 cDNA into the PINCO vector
(Grignani, et al., Cancer Research 58: 14-19 (1998)) using
BamHI/NotI sites, and with H-RasV12, which was obtained from S.
Lowe (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). p53
was immunoprecipitated from 1 mg of the total protein extract from
these cells and phosphorylation on Ser33 or Ser46 of p53 was
assessed by Western blotting with antibodies specific for
phosphorylated p53 as described in Sakaguchi et al., Genes
Development 12: 2831-2841 (1998). In cells that overexpressed Wip1,
p53 had little, if any, phosphorylation on Ser 33 or Ser46 as
compared to cells infected with H-RasV12 alone. From this example,
it is evident that the overexpression of WIP1 results in less
phosphorylation of p53 on sites phosphorylated by p38 kinase.
Example 5
[0064] This example demonstrates that WIP1 phosphatase complements
different oncogene proteins for growth in soft agar.
[0065] Retroviruses containing WIP1 and different oncogenes,
including H-RasV12, MYC or NEU1, were co-infected into wild-type
mouse embryo fibroblasts (MEFs) and were analyzed for
anchorage-independent growth and the ability to form foci in soft
agar. The IRES c-myc (human) retroviral vector, pBabeMN Sgfpmyc,
(Oster et al., Molecular and Cellular Biology 20: 6768-6778 (2000))
was kindly provided by L. Z. Penn (Toronto University, Toronto,
Ontario); pbabe-c-neu (Bargmann et al., Cell 45: 649-657 (1986))
(rat) was obtained from P. Sicinski (Dana-Farber Cancer Institute,
Boston, Mass.). Suppression of colony formation was determined 2-3
weeks after infection of MEFs in 100 mm dishes with the designated
retroviruses. Colonies were selected with 500 .mu.g/ml G418
(Sigma-Aldrich, St. Louis, Mo.), and fixed and stained with 0.1%
crystal violet (Sigma-Aldrich, St. Louis, Mo.). For soft agar
cloning, 20,000 puromycin-(Sigma-Aldrich, St. Louis, Mo.) selected,
infected cells were seeded in 0.5% agar into each well of 6-well
plates. Samples were analyzed in triplicate. The results of this
assay are shown below in Table 1.
TABLE-US-00001 TABLE 1 MEF type Retrovirus Growth in soft agar
Wild-type Puro - H-rasV12 - neu - myc - Wip1 - H-rasV12 + Wip1 +
neu + Wip1 + myc + Wip1 + ras + myc ++ ras + neu + p53.sup.-/- Puro
- H-rasV12 ++ Wip1 - H-rasV12 + Wip1 ++ Neu +++ myc +++ Score: + =
10-30 colonies; ++ = 30-100 colonies; +++ = more than 100 colonies
per well of a 6-well plate.
[0066] In both assays, WIP1 complemented H-RasV12 for
transformation of wild-type MEFs. From this analysis, it became
evident that WIP1 is a proto-oncogene.
Example 6
[0067] This example demonstrates a method of delivering morpholino
oligomers to cells in culture.
[0068] Morpholino oligomers comprising the sequence of SEQ ID NO: 1
or SEQ ID NO: 2 were synthesized by and purchased from Gene Tools
(Philomath, Oreg.). Sterile water (600 microliters (.mu.l)) was
added to one vial containing 300 nM of Special Delivery
morpholino/DNA (Gene Tools, Philomath, Oreg.) to make a 0.5
millimolar (mM) stock solution. Sterile water (200 .mu.l) was added
to 100 nM Special Delivery Standard Control morpholino oligomers
(Gene Tools, Philomath, Oreg.) to make a 0.5 stock solution. In a
15 milliliter (ml) centrifuge tube, sterile water (188.8 .mu.l) was
mixed with 5.6 .mu.l of the 0.5 mM Special Delivery morpholino
oligomer/DNA stock solution and 5.6 .mu.l 200 micromolar (.mu.M)
EPEI Special Delivery solution (Gene Tools, Philomath, Oreg.) and
vortexed immediately upon mixing. The mixture was then incubated at
room temperature for exactly 20 minutes. Serum-free medium (1.8 ml)
was added to the mixture and vortexed immediately upon addition.
This mixture (500 .mu.l) was then added to media-free cells and
incubated in an incubator for 3 hours. Afterwards, the mixture was
removed from the cells via centrifugation and fresh media
containing serum was added to the cells. The cells were then
incubated for at least 16 hours before testing for delivery of the
morpholino oligomers. This example demonstrates a method of
delivering morpholino oligomers to cultured cells.
[0069] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0070] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0071] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
2125DNAArtificial SequenceSynthetic 1ctcccagcga gtacagcccc gccat
25225DNAArtificial SequenceSynthetic 2cggtcccacg cagcccgccg aatcc
25
* * * * *
References