U.S. patent application number 12/616484 was filed with the patent office on 2010-03-11 for protein kinase c zeta as a drug target for arthritis and other inflammatory diseases.
This patent application is currently assigned to WYETH. Invention is credited to Maya Arai, Lisa A. Collins-Racie, Edward R. LaVallie.
Application Number | 20100062448 12/616484 |
Document ID | / |
Family ID | 33479253 |
Filed Date | 2010-03-11 |
United States Patent
Application |
20100062448 |
Kind Code |
A1 |
LaVallie; Edward R. ; et
al. |
March 11, 2010 |
PROTEIN KINASE C ZETA AS A DRUG TARGET FOR ARTHRITIS AND OTHER
INFLAMMATORY DISEASES
Abstract
The present invention is based on the discovery that .zeta.PKC
expression is increased in the tissues of arthritis patients as
compared to normal individuals. Accordingly, the present invention
provides methods of diagnosing, prognosing, and monitoring the
course of arthritis in a patient based on increased .zeta.PKC gene
expression in arthritic tissue. The present invention further
provides compounds that inhibit the expression of .zeta.PKC for use
as remedies in the treatment of arthritis, including, but not
limited to, inhibitory polynucleotides and polypeptides, small
molecules, and peptide inhibitors. In addition, the present
invention provides pharmaceutical formulations and routes of
administration for such remedies, as well as methods for assessing
their efficacy.
Inventors: |
LaVallie; Edward R.;
(Harvard, MA) ; Collins-Racie; Lisa A.; (Acton,
MA) ; Arai; Maya; (Brookline, MA) |
Correspondence
Address: |
WYETH LLC;PATENT LAW GROUP
5 GIRALDA FARMS
MADISON
NJ
07940
US
|
Assignee: |
WYETH
Madison
NJ
|
Family ID: |
33479253 |
Appl. No.: |
12/616484 |
Filed: |
November 11, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10842142 |
May 10, 2004 |
7638482 |
|
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12616484 |
|
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60468987 |
May 8, 2003 |
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60491274 |
Jul 31, 2003 |
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Current U.S.
Class: |
435/6.16 ;
435/15; 435/29; 514/44A; 536/24.5 |
Current CPC
Class: |
G01N 2333/91215
20130101; G01N 2800/102 20130101; G01N 2500/00 20130101; A61P 19/02
20180101; G01N 33/564 20130101; G01N 33/6872 20130101 |
Class at
Publication: |
435/6 ; 435/29;
435/15; 514/44.A; 536/24.5 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12Q 1/02 20060101 C12Q001/02; C12Q 1/48 20060101
C12Q001/48; A61K 31/7088 20060101 A61K031/7088; C07H 21/02 20060101
C07H021/02 |
Claims
1. A method for use in the diagnosis of arthritis in a subject
comprising the steps of: detecting a test amount of a .zeta.PKC
gene product in a sample from the subject; and comparing the test
amount with a normal amount of the .zeta.PKC gene product in a
control sample, whereby a finding that the test amount is greater
than the normal amount provides a positive indication in the
diagnosis of arthritis.
2. The method of claim 1, wherein the sample comprises
chondrocytes.
3. The method of claim 1, wherein the .zeta.PKC gene product
comprises RNA or cDNA.
4. The method of claim 1, wherein the .zeta.PKC gene product is
.zeta.PKC polypeptide.
5. A method for use in the prognosis of arthritis in a subject
comprising the steps of: detecting a test amount of a .zeta.PKC
gene product in a sample from the subject; and comparing the test
amount with prognostic amounts of the .zeta.PKC gene product in
control samples, whereby a comparison of the test amount with the
prognostic amounts provides an indication of the prognosis of
arthritis.
6. The method of claim 5, wherein the sample comprises
chondrocytes.
7. The method of claim 5, wherein the .zeta.PKC gene product
comprises RNA or cDNA.
8. The method of claim 5, wherein the .zeta.PKC gene product is
.zeta.PKC polypeptide.
9. A method for use in monitoring the course of arthritis in a
subject comprising the steps of: detecting a first test amount of a
.zeta.PKC gene product in a sample from the subject at a first
time; detecting a second test amount of the .zeta.PKC gene product
in a sample from the subject at a second, later time; and comparing
the first test amount and the second test amount, whereby an
increase in the amount of the .zeta.PKC gene product in the second
test amount as compared with the first test amount indicates
progression of arthritis, and whereby a decrease in the amount of
the PKC gene product in the second test amount as compared with the
first test amount indicates remission of arthritis.
10. The method of claim 9, wherein the sample comprises
chondrocytes.
11. The method of claim 9, wherein the PKC gene product comprises
RNA or cDNA.
12. The method of claim 9, wherein the .zeta.PKC gene product is
.zeta.PKC polypeptide.
13. A method for assessing the efficacy of a treatment for
arthritis in a subject comprising the steps of: detecting a first
test amount of a .zeta.PKC gene product in a sample from the
subject prior to treatment; detecting a second test amount of the
.zeta.PKC gene product in a sample from the subject after
treatment; and comparing the first test amount and the second test
amount, whereby a decrease in the amount of the .zeta.PKC gene
product in the second test amount as compared with the first test
amount indicates that the treatment for arthritis is
efficacious.
14. The method of claim 13, wherein the sample comprises
chondrocytes.
15. The method of claim 13, wherein the .zeta.PKC gene product
comprises RNA or cDNA.
16. The method of claim 13, wherein the .zeta.PKC gene product is
.zeta.PKC polypeptide.
17. A method of screening for a compound capable of inhibiting
arthritis in a subject comprising the steps of: providing a first
sample and a second sample containing equivalent amounts of
.zeta.PKC; contacting the first sample with the compound; and
determining whether the activity of .zeta.PKC in the first sample
is decreased relative to the activity of .zeta.PKC in the second
sample not contacted with the compound, whereby a decrease in the
activity of .zeta.PKC in the first sample as compared with the
second sample indicates that the compound inhibits arthritis in the
subject.
18. The method of claim 17, wherein the compound inhibits the
activity of PKC in chondrocytes.
19. The method of claim 17, wherein the compound is a small
molecule.
20. The method of claim 17, wherein the activity of .zeta.PKC is
determined by use of an enzymatic protein kinase assay.
21. The method of claim 17, wherein the activity of .zeta.PKC is
determined by use of a chondrocyte pellet assay.
22. The method of claim 17, wherein the activity of .zeta.PKC is
determined by use of an assay measuring proteoglycan
degradation.
23. The method of claim 17, wherein the activity of .zeta.PKC is
determined by use of an assay measuring NF-.kappa.B activity.
24. A method of screening for a compound capable of inhibiting
arthritis in a subject comprising the steps of: providing a first
sample and a second sample containing equivalent amounts of cells
that express .zeta.PKC; contacting the first sample with the
compound; and determining whether the expression of .zeta.PKC gene
product in the first sample is decreased relative to the expression
of .zeta.PKC gene product in the second sample not contacted with
the compound, whereby a decrease in the expression of .zeta.PKC
gene product in the first sample as compared with the second sample
indicates that the compound inhibits arthritis in the subject.
25. The method of claim 24, wherein the compound inhibits the
expression of PKC gene product in chondrocytes.
26. The method of claim 24, wherein the compound is a small
molecule.
27. The method of claim 24, wherein the expression of .zeta.PKC
gene product is determined by use of an enzymatic protein kinase
assay.
28. The method of claim 24, wherein the expression of .zeta.PKC
gene product is determined by use of a chondrocyte pellet
assay.
29. The method of claim 24, wherein the expression of .zeta.PKC
gene product is determined by use of an assay measuring
proteoglycan degradation.
30. The method of claim 24, wherein the expression of .zeta.PKC
gene product is determined by use of an assay measuring NF-.kappa.B
activity.
31. A method for the treatment of arthritis in a subject comprising
administering to the subject a compound that inhibits the activity
of .zeta.PKC in the subject.
32. The method of claim 31, wherein the compound inhibits the
activity of PKC in chondrocytes.
33. The method of claim 31, wherein the compound is an antisense
polynucleotide.
34. The method of claim 31, wherein the compound is a small
molecule.
35. The method of claim 31, wherein the compound is a siRNA
molecule.
36. The method of claim 35, wherein the siRNA molecule is selected
from the group consisting of siRNA molecules shown in FIG. 1.
37. A method for the treatment of arthritis in a subject comprising
administering to the subject a compound that inhibits the
expression of .zeta.PKC in the subject.
38. The method of claim 37, wherein the compound inhibits the
expression of .zeta.PKC in chondrocytes.
39. The method of claim 37, wherein the compound is an antisense
polynucleotide.
40. The method of claim 37, wherein the compound is a small
molecule.
41. The method of claim 37, wherein the compound is a siRNA
molecule.
42. The method of claim 41, wherein the siRNA molecule is selected
from the group consisting of siRNA molecules shown in FIG. 1.
43. A siRNA molecule that inhibits the expression or activity of
.zeta.PKC.
44. The siRNA molecule of claim 43, wherein the siRNA molecule is
selected from the group consisting of siRNA molecules shown in FIG.
1.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/842,142, filed May 10, 2004, which claims
the benefit of U.S. Provisional Application Ser. No. 60/468,987,
filed May 8, 2003, and U.S. Provisional Application Ser. No.
60/491,274, filed Jul. 31, 2003, all of which are incorporated
herein by reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention is directed to methods of diagnosing,
prognosing, and monitoring the course of arthritis in a subject
based on increased protein kinase C zeta (.zeta.PKC) gene
expression in arthritic tissue. The present invention further
provides compounds that inhibit the expression of .zeta.PKC for use
as remedies in the treatment of arthritis.
[0004] 2. Related Background Art
[0005] Protein kinase C zeta (.zeta.PKC) is emerging as an
important signal transduction component. There is growing
literature suggesting that .zeta.PKC is involved in the NF-.kappa.B
and AP-1 pathways. For example, a .zeta.PKC knockout mouse is fully
viable but displays a phenotype reminiscent of the tumor necrosis
factor (TNF) receptor and lymphotoxin receptor knockouts, with
severe impairment of NF-.kappa.B-dependent transcriptional activity
(Leitges et al. (2001) Mol. Cell. 8:771-80). Other investigators
(Lallena et al. (1999) Mol. Cell. Biol. 19:2180-88) have shown a
role for .zeta.PKC in activating I.kappa.B and, thereby, activating
NF-.kappa.B.
[0006] NF-.kappa.B activation has been implicated in numerous
inflammatory disorders, including asthma, inflammatory bowel
disease, and arthritis (reviewed in Roshak et al. (2002) Curr.
Opin. Pharmacol. 2:316-21). NF-.kappa.B has been shown to play an
essential role in the secretion of various matrix
metalloproteinases (MMPs) from various cell types (Bond et al.
(1998) FEBS Lett. 435:29-34; Bond et al. (1999) Biochem. Biophys.
Res. Commun. 264:561-67; Bond et al. (2001) Cardiovasc. Res.
50:556-65). In arthritis, cytokines such as TNF and interleukin-1
(IL-1) increase the production and synthesis of MMPs and other
degradative enzymes above levels that can be naturally controlled,
resulting in disease (reviewed in Smith (1999) Front. Biosci.
4:D704; Mort and Billington (2001) Arthritis Res. 3:337-41;
Catterall and Cawston (2003) Arthritis Res. Ther. 5:12-24).
[0007] To date, there has been no direct evidence linking .zeta.PKC
to arthritis. If PKC were expressed in affected tissues, however,
it would help to explain the degradative actions of TNF and IL-1 by
transducing the extracellular receptor binding of these factors to
the intracellular induction of synthesis of degradative enzymes by
NF-.kappa.B. In this regard, inhibitors of .zeta.PKC may block TNF
and IL-1 action and serve as treatments for arthritis and other
inflammatory diseases. Such .zeta.PKC inhibitors should be more
efficacious than traditional cytokine and MMP inhibitors because
they should ultimately affect more than just one target (Roshak,
supra; Smith, supra). Such .zeta.PKC inhibitors should also be
safer than NF-.kappa.B inhibitors because .zeta.PKC is only one of
many effectors in the NF-.kappa.B pathway.
SUMMARY OF THE INVENTION
[0008] The present invention is based on the discovery that
.kappa.PKC expression is increased in the tissues of arthritis
patients as compared to normal individuals. The present invention
provides compounds that inhibit the expression of .zeta.PKC in
arthritic tissue including, but not limited to, inhibitory
polynucleotides and polypeptides, small molecules, and peptide
inhibitors. The present invention further provides methods of
diagnosing, prognosing, and monitoring the course of arthritis
based on aberrant .zeta.PKC gene expression in arthritic tissue, as
well as therapies for use as remedies for such aberrant expression.
In addition, the present invention provides pharmaceutical
formulations and routes of administration for such remedies, as
well as methods for assessing the efficacy of such remedies.
[0009] In one embodiment, the invention provides a method for use
in the diagnosis of arthritis in a subject comprising the steps of
detecting a test amount of a .zeta.PKC gene product in a sample
from the subject; and comparing the test amount with a normal
amount of the .zeta.PKC gene product in a control sample, whereby a
finding that the test amount is greater than the normal amount
provides a positive indication in the diagnosis of arthritis. In a
preferred embodiment, the sample comprises chondrocytes. In some
other preferred embodiments, the .zeta.PKC gene product comprises
RNA or cDNA, or is .zeta.PKC polypeptide.
[0010] In another embodiment, the invention provides a method for
use in the prognosis of arthritis in a subject comprising the steps
of detecting a test amount of a .zeta.PKC gene product in a sample
from the subject; and comparing the test amount with prognostic
amounts of the .zeta.PKC gene product in control samples, whereby a
comparison of the test amount with the prognostic amounts provides
an indication of the prognosis of arthritis. In a preferred
embodiment, the sample comprises chondrocytes. In some other
preferred embodiments, the .zeta.PKC gene product comprises RNA or
cDNA, or is .zeta.PKC polypeptide.
[0011] In another embodiment, the invention provides a method for
use in monitoring the course of arthritis in a subject comprising
the steps of detecting a first test amount of a .zeta.PKC gene
product in a sample from the subject at a first time; detecting a
second test amount of the .zeta.PKC gene product in a sample from
the subject at a second, later time; and comparing the first test
amount and the second test amount, whereby an increase in the
amount of the .zeta.PKC gene product in the second test amount as
compared with the first test amount indicates progression of
arthritis, and whereby a decrease in the amount of the .zeta.PKC
gene product in the second test amount as compared with the first
test amount indicates remission of arthritis. In a preferred
embodiment, the sample comprises chondrocytes. In some other
preferred embodiments, the .zeta.PKC gene product comprises RNA or
cDNA, or is .zeta.PKC polypeptide.
[0012] In another embodiment, the invention provides a method for
assessing the efficacy of a treatment for arthritis in a subject
comprising the steps of detecting a first test amount of a
.zeta.PKC gene product in a sample from the subject prior to
treatment; detecting a second test amount of the .zeta.PKC gene
product in a sample from the subject after treatment; and comparing
the first test amount and the second test amount, whereby a
decrease in the amount of the .zeta.PKC gene product in the second
test amount as compared with the first test amount indicates that
the treatment for arthritis is efficacious. In a preferred
embodiment, the sample comprises chondrocytes. In some other
preferred embodiments, the .zeta.PKC gene product comprises RNA or
cDNA, or is .zeta.PKC polypeptide.
[0013] In another embodiment, the invention provides a method of
screening for a compound capable of inhibiting arthritis in a
subject comprising the steps of providing a first sample and a
second sample containing equivalent amounts of .zeta.PKC;
contacting the first sample with the compound; and determining
whether the activity of .zeta.PKC in the first sample is decreased
relative to the activity of .zeta.PKC in the second sample not
contacted with the compound, whereby a decrease in the activity of
.zeta.PKC in the first sample as compared with the second sample
indicates that the compound inhibits arthritis in the subject. In a
preferred embodiment, the compound inhibits the activity of
.zeta.PKC in chondrocytes. In another preferred embodiment, the
compound is a small molecule. In other preferred embodiments, the
activity of .zeta.PKC is determined by use of an enzymatic protein
kinase assay, a chondrocyte pellet assay, an assay measuring
proteoglycan degradation, or an assay measuring NF-.kappa.B
activity.
[0014] In another embodiment, the invention provides a method of
screening for a compound capable of inhibiting arthritis in a
subject comprising the steps of providing a first sample and a
second sample containing equivalent amounts of cells that express
.zeta.PKC; contacting the first sample with the compound; and
determining whether the expression of .zeta.PKC gene product in the
first sample is decreased relative to the expression of .zeta.PKC
gene product in the second sample not contacted with the compound,
whereby a decrease in the expression of .zeta.PKC gene product in
the first sample as compared with the second sample indicates that
the compound inhibits arthritis in the subject. In a preferred
embodiment, the compound inhibits the expression of .zeta.PKC gene
product in chondrocytes. In another preferred embodiment, the
compound is a small molecule. In other preferred embodiments, the
expression of .zeta.PKC gene product is determined by use of an
enzymatic protein kinase assay, a chondrocyte pellet assay, an
assay measuring proteoglycan degradation, or an assay measuring
NF-.kappa.B activity.
[0015] In another embodiment, the invention provides a method for
the treatment of arthritis in a subject comprising administering to
the subject a compound that inhibits the activity of .zeta.PKC in
the subject. In a preferred embodiment, the compound inhibits the
activity of .zeta.PKC in chondrocytes. In another preferred
embodiment, the compound is an antisense polynucleotide. In another
preferred embodiment, the compound is a small molecule. In another
preferred embodiment, the compound is a siRNA molecule. In a
further preferred embodiment, the siRNA molecule is selected from
the group consisting of siRNA molecules shown in FIG. 1.
[0016] In another embodiment, the invention provides a method for
the treatment of arthritis in a subject comprising administering to
the subject a compound that inhibits the expression of .zeta.PKC in
the subject. In a preferred embodiment, the compound inhibits the
expression of .zeta.PKC in chondrocytes. In another preferred
embodiment, the compound is an antisense polynucleotide. In another
preferred embodiment, the compound is a small molecule. In another
preferred embodiment, the compound is a siRNA molecule. In a
further preferred embodiment, the siRNA molecule is selected from
the group consisting of siRNA molecules shown in FIG. 1.
[0017] In another embodiment, the invention provides a siRNA
molecule that inhibits the expression or activity of .zeta.PKC. In
a preferred embodiment, the siRNA molecule is selected from the
group consisting of siRNA molecules shown in FIG. 1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows preferred siRNA molecules targeted to human
.zeta.PKC mRNA for use in RNAi. Target segments [SEQ ID NOs:9-20;
45-59; 90-109; and 150-154] of the .zeta.PKC transcripts are
grouped according to their first two nucleotides (AA, CA, GA, or
TA) and are shown in the 5'->3' orientation. "GC Ratio" refers
to the percentage of total G+C nucleotides in each target segment;
"Position" refers to the nucleotide position in the human .zeta.PKC
cDNA (SEQ ID NO:1) immediately preceding the beginning of each
target segment. Preferred siRNA molecules (siRNA duplexes) are
shown on the right side of the figure. Both the sense strand for
each siRNA duplex [SEQ ID NOs:21-32; 60-74; 110-129; and 155-159]
and the corresponding antisense strand [SEQ ID NOs:33-44; 75-89;
130-149; and 160-164] are shown in the 5'->3' orientation. For
example, the siRNA molecule directed to the first target segment
presented in the figure (i.e., SEQ ID NO:9) is the siRNA duplex of
the sense and antisense strands identified (i.e., SEQ ID NO:21 and
SEQ ID NO:33, respectively).
[0019] FIG. 2 is a graph depicting the effects of the NF-.kappa.B
blocker SN50 (300 .mu.g/ml), or its inactive analog SN50M (300
.mu.g/ml), on TNF- or IL-1-mediated proteoglycan degradation in
primary bovine chondrocytes in culture. The top panel shows
proteoglycan content released in the media (.mu.g/0.5 ml); the
bottom panel shows proteoglycan content retained in the cell pellet
(.mu.g/ml).
[0020] FIG. 3 is a graph depicting the effects of a myristoylated
.zeta.PKC pseudosubstrate peptide (2089) or .zeta.PKC small
molecule inhibitor Ro-31-8220 (RO31) on TNF-mediated proteoglycan
degradation in primary bovine chondrocytes in culture. The top
panel shows proteoglycan content released in the media (.mu.g/0.5
ml); the bottom panel shows proteoglycan content retained in the
cell pellet (.mu.g/ml).
[0021] FIG. 4 is a graph depicting the dose-dependent effects of a
myristoylated .zeta.PKC pseudosubstrate peptide (2089) on TNF- or
IL-1-mediated proteoglycan degradation in primary bovine
chondrocytes in culture. The top panel shows proteoglycan content
released in the media (.mu.g/0.5 ml); the bottom panel shows
proteoglycan content retained in the cell pellet (.mu.g/ml).
[0022] FIG. 5 shows that .zeta.PKC is upregulated in human
osteoarthritic articular cartilage. Panel A shows .zeta.PKC mRNA
levels using the HG-U95Av2 Affymetrix GeneChip.RTM. Array; panel B
shows .zeta.PKC mRNA levels using TaqMan PCR analysis.
[0023] FIG. 6 shows that adenoviral-mediated expression of
.zeta.PKC increases proteoglycan degradation. Panel A shows
proteoglycan released into the media in the chondrocyte pellet
assay in response to overexpression of .zeta.PKC and GFP; panel B
shows the effects of stimulation with suboptimal levels of the
cytokine TNF.alpha..
[0024] FIG. 7 demonstrates that .zeta.PKC is responsible for
TNF.alpha.-mediated proteoglycan release in articular chondrocytes.
TNF.alpha. was added (100 ng/ml; denoted by *) to some cultures in
the chondrocyte pellet assay. Two inhibitors were added at various
doses: bisindolylmaleimide (BIS), a pan-PKC inhibitor; and
chelerythrine chloride (CC), a competitive inhibitor of the phorbol
ester-binding site that does not inhibit .zeta.PKC. Proteoglycan
release into the media is shown on the y-axis as .mu.g/ml.
[0025] FIG. 8 shows the effects of the inhibitors BIS and CC on
TNF.alpha.-induced activation of NF-.kappa.B. Activation of
NF-.kappa.B was measured in an immortalized human chondrocyte cell
line into which a luciferase reporter gene under the control of an
NF-.kappa.B response element was introduced; activity (i.e., units
on the y-axis) is expressed as "relative luciferase activity."
DETAILED DESCRIPTION OF THE INVENTION
[0026] We have discovered that .zeta.PKC expression is upregulated
in the tissues of arthritis patients as compared to normal
individuals. The discovery that this enzyme is upregulated in
arthritic tissue enables methods for diagnosing arthritis by
detecting an increase in .zeta.PKC expression and methods for
treating arthritis by downregulating .zeta.PKC expression. In
addition, this discovery enables the identification of new
.zeta.PKC inhibitors useful in the treatment of arthritis.
Methods for Diagnosing, Prognosing, and Monitoring the Progress of
Arthritis
Introduction
[0027] The present invention provides methods for diagnosing
arthritis by detecting the upregulation of .zeta.PKC. "Diagnostic"
or "diagnosing" means identifying the presence or absence of a
pathologic condition. Diagnostic methods involve detecting
upregulation of .zeta.PKC by determining a test amount of .zeta.PKC
gene product (e.g., mRNA, cDNA, or polypeptide, including fragments
thereof) in a biological sample from a subject (human or nonhuman
mammal), and comparing the test amount with a normal amount or
range (i.e., an amount or range from an individual(s) known not to
suffer from arthritis) for the .zeta.PKC gene product. While a
particular diagnostic method may not provide a definitive diagnosis
of arthritis, it suffices if the method provides a positive
indication that aids in diagnosis.
[0028] The present invention also provides methods for prognosing
arthritis by detecting the upregulation of .zeta.PKC. "Prognostic"
or "prognosing" means predicting the probable development and/or
severity of a pathologic condition. Prognostic methods involve
determining the test amount of a .zeta.PKC gene product in a
biological sample from a subject, and comparing the test amount to
a prognostic amount or range (i.e., an amount or range from
individuals with varying severities of arthritis) for the .zeta.PKC
gene product. Various amounts of the .zeta.PKC gene product in a
test sample are consistent with certain prognoses for arthritis.
The detection of an amount of .zeta.PKC gene product at a
particular prognostic level provides a prognosis for the
subject.
[0029] The present invention also provides methods for monitoring
the course of arthritis by detecting the upregulation of .zeta.PKC.
Monitoring methods involve determining the test amounts of a
.zeta.PKC gene product in biological samples taken from a subject
at a first and second time, and comparing the amounts. A change in
amount of .zeta.PKC gene product between the first and second time
indicates a change in the course of arthritis, with a decrease in
amount indicating remission of arthritis, and an increase in amount
indicating progression of arthritis. Such monitoring assays are
also useful for evaluating the efficacy of a particular therapeutic
intervention (e.g., disease attenuation vs. reversal) in patients
being treated for arthritis.
Biological Sample Collection
[0030] Increased expression of .zeta.PKC can be detected in a
variety of biological samples, including cells (e.g., whole cells,
cell fractions, and cell extracts) and tissues. Biological samples
also include sections of tissue such as biopsies and frozen
sections taken for histological purposes. Preferred biological
samples include articular cartilage (i.e., chondrocytes), synovium,
and synovial fluid.
Normal, Diagnostic, and Prognostic Values
[0031] In the diagnostic and prognostic assays of the present
invention, the .zeta.PKC gene product is detected and quantified to
yield a test amount. The test amount is then compared to a normal
amount or range. An amount above the normal amount or range (e.g.,
a 30% or greater increase (with p<0.01), or a 100% or greater
increase (with p<0.05)) is a positive sign in the diagnosis of
arthritis. Particular methods of detection and quantitation of
.zeta.PKC gene products are described below.
[0032] Normal amounts or baseline levels of .zeta.PKC gene products
can be determined for any particular sample type and population.
Generally, baseline (normal) levels of .zeta.PKC protein or mRNA
are determined by measuring the amount of .zeta.PKC protein or mRNA
in a biological sample type from normal (i.e., healthy) subjects.
Alternatively, normal values of .zeta.PKC gene product can be
determined by measuring the amount in healthy cells or tissues
taken from the same subject from which the diseased (or possibly
diseased) test cells or tissues were taken. The amount of .zeta.PKC
gene product (either the normal amount or the test amount) can be
determined or expressed on a per cell, per total protein, or per
volume basis. To determine the cell amount of a sample, one can
measure the level of a constitutively expressed gene product or
other gene product expressed at known levels in cells of the type
from which the biological sample was taken.
[0033] It will be appreciated that the assay methods of the present
invention do not necessarily require measurement of absolute values
of .zeta.PKC gene product because relative values are sufficient
for many applications of these methods. It will also be appreciated
that in addition to the quantity or abundance of .zeta.PKC gene
products, variant or abnormal .zeta.PKC gene products or their
expression patterns (e.g., mutated transcripts, truncated
polypeptides) may be identified by comparison to normal gene
products and expression patterns.
Assays for .zeta.PKC Gene Products
[0034] The diagnostic, prognostic, and monitoring assays of the
present invention involve detecting and quantifying .zeta.PKC gene
products in biological samples. .zeta.PKC gene products include,
for example, .zeta.PKC mRNA and .zeta.PKC polypeptide, and both can
be measured using methods well known to those skilled in the
art.
[0035] For example, .zeta.PKC mRNA can be directly detected and
quantified using hybridization-based assays, such as Northern
hybridization, in situ hybridization, dot and slot blots, and
oligonucleotide arrays. Hybridization-based assays refer to assays
in which a probe nucleic acid is hybridized to a target nucleic
acid. In some formats, the target, the probe, or both are
immobilized. The immobilized nucleic acid may be DNA, RNA, or
another oligonucleotide or polynucleotide, and may comprise
naturally or nonnaturally occurring nucleotides, nucleotide
analogs, or backbones. Methods of selecting nucleic acid probe
sequences for use in the present invention are based on the nucleic
acid sequence of .zeta.PKC and are well known in the art.
[0036] Alternatively, .zeta.PKC mRNA can be amplified before
detection and quantitation. Such amplification-based assays are
well known in the art and include polymerase chain reaction (PCR),
reverse-transcription-PCR(RT-PCR), PCR-enzyme-linked immunosorbent
assay (PCR-ELISA), and ligase chain reaction (LCR). Primers and
probes for producing and detecting amplified .zeta.PKC gene
products (e.g., mRNA or cDNA) may be readily designed and produced
without undue experimentation by those of skill in the art based on
the nucleic acid sequence of .zeta.PKC. Amplified .zeta.PKC gene
products may be directly analyzed, e.g., by gel electrophoresis; by
hybridization to a probe nucleic acid; by sequencing; by detection
of a fluorescent, phosphorescent, or radioactive signal; or by any
of a variety of well-known methods. In addition, methods are known
to those of skill in the art for increasing the signal produced by
amplification of target nucleic acid sequences. One of skill in the
art will recognize that whichever amplification method is used, a
variety of quantitative methods known in the art (e.g.,
quantitative PCR) may be used if quantitation of .zeta.PKC gene
products is desired.
[0037] .zeta.PKC polypeptide (or fragments thereof) can be detected
and quantified using various well-known enzymatic and immunological
assays. Enzymatic assays refer to assays that utilize .zeta.PKC
substrates to detect protein kinase activity. Various natural and
artificial substrates useful for detecting and quantifying
.zeta.PKC activity are known, and include myristoyl alanine-rich C
kinase substrate (MARCKS) peptide (Herget et al. (1995) Eur. J.
Biochem. 233:448-57), p47phox (Dang et al. (2001) J. Immunol.
166:1206-13), myelin basic protein (Kim et al. (2002) J. Biol.
Chem. 277:30375-81), protamine sulfate (McGlynn et al. (1992) J.
Cell. Biochem. 49:239-50), nucleolin (Zhou et al. (1997) J. Biol.
Chem. 272:31130-37); heterogeneous ribonucleoprotein AI (hnRNPA1)
(Municio et al. (1995) J. Biol. Chem. 270:15884-91),
.zeta.PKC-derived peptide (Kochs et al. (1993) Eur. J. Biochem.
216:597-606), and .zeta.PKC-derived peptide (Standaert et al.
(1999) J. Biol. Chem. 274:14074-78). Numerous enzymatic assay
protocols (radioactive and nonradioactive) suitable for detecting
and quantifying .zeta.PKC activity are described in the literature
and/or are commercially available in kit form from, e.g., PanVera
(Madison, Wis.), Promega (Madison, Wis.), Transbio (Baltimore,
Md.), Upstate (Waltham, Mass.), and Research & Diagnostic
Antibodies (Benicia, Calif.).
[0038] Immunological assays refer to assays that utilize an
antibody (e.g., polyclonal, monoclonal, chimeric, humanized, scFv,
and fragments thereof) that specifically binds to .zeta.PKC
polypeptide (or a fragment thereof). A number of well-established
immunological assays suitable for the practice of the present
invention are known, and include ELISA, radioimmunoassay (RIA),
immunoprecipitation, immunofluorescence, and Western blotting.
[0039] The anti-.zeta.PKC antibodies (preferably anti-mammalian
.zeta.PKC; more preferably anti-human .zeta.PKC) to be used in the
immunological assays of the present invention are commercially
available from, e.g., Sigma-Aldrich (St. Louis, Mo.), Upstate
(Waltham, Mass.), and Research Diagnostics (Flanders, N.J.).
Alternatively, anti-.zeta.PKC antibodies may be produced by methods
well known to those skilled in the art. For example, monoclonal
antibodies to .zeta.PKC (preferably mammalian; more preferably
human (e.g., GenBank Acc. No. Q05513; SEQ ID NO:2)) can be produced
by generation of hybridomas in accordance with known methods.
Hybridomas formed in this manner are then screened using standard
methods, such as ELISA, to identify one or more hybridomas that
produce an antibody that specifically binds to .zeta.PKC.
Full-length .zeta.PKC may be used as the immunogen, or,
alternatively, antigenic peptide fragments of .zeta.PKC may be
used.
[0040] As an alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal antibody to .zeta.PKC may be identified
and isolated by screening a recombinant combinatorial
immunoglobulin library (e.g., an antibody phage display library) to
thereby isolate immunoglobulin library members that bind to
.zeta.PKC. Kits for generating and screening phage display
libraries are commercially available from, e.g., Dyax Corp.
(Cambridge, Mass.) and Maxim Biotech (South San Francisco, Calif.).
Additionally, examples of methods and reagents particularly
amenable for use in generating and screening antibody display
libraries can be found in the literature.
[0041] Polyclonal sera and antibodies may be produced by immunizing
a suitable subject, such as a rabbit, with .zeta.PKC (preferably
mammalian; more preferably human) or an antigenic fragment thereof.
The antibody titer in the immunized subject may be monitored over
time by standard techniques, such as with ELISA, using immobilized
marker protein. If desired, the antibody molecules directed against
.zeta.PKC may be isolated from the subject or culture media and
further purified by well-known techniques, such as protein A
chromatography, to obtain an IgG fraction.
[0042] Fragments of antibodies to .zeta.PKC may be produced by
cleavage of the antibodies in accordance with methods well known in
the art. For example, immunologically active F(ab') and
F(ab').sub.2 fragments may be generated by treating the antibodies
with an enzyme such as pepsin. Additionally, chimeric, humanized,
and single-chain antibodies to .zeta.PKC, comprising both human and
nonhuman portions, may be produced using standard recombinant DNA
techniques. Humanized antibodies to .zeta.PKC may also be produced
using transgenic mice that are incapable of expressing endogenous
immunoglobulin heavy and light chain genes, but which can express
human heavy and light chain genes.
[0043] In the immunological assays of the present invention, the
.zeta.PKC polypeptide is typically detected directly (i.e., the
anti-.zeta.PKC antibody is labeled) or indirectly (i.e., a
secondary antibody that recognizes the anti-.zeta.PKC antibody is
labeled) using a detectable label. The particular label or
detectable group used in the assay is usually not critical, as long
as it does not significantly interfere with the specific binding of
the antibodies used in the assay.
[0044] The immunological assays of the present invention may be
competitive or noncompetitive. In competitive assays, the amount of
.zeta.PKC in a sample is measured indirectly by measuring the
amount of added (exogenous) .zeta.PKC displaced from a capture
agent (i.e., an anti-.zeta.PKC antibody) by the .zeta.PKC in the
sample. In noncompetitive assays, the amount of .zeta.PKC in a
sample is directly measured. In a preferred noncompetitive
"sandwich" assay, the capture agent (e.g., a first anti-.zeta.PKC
antibody) is bound directly to a solid support (e.g., membrane,
microtiter plate, test tube, dipstick, glass or plastic bead) where
it is immobilized. The immobilized agent then captures any
.zeta.PKC polypeptide present in the sample. The immobilized
.zeta.PKC can then be detected using a second labeled
anti-.zeta.PKC antibody. Alternatively, the second anti-.zeta.PKC
antibody can be detected using a labeled secondary antibody that
recognizes the second anti-.zeta.PKC antibody.
Screening Methods for Identifying Compounds that Inhibit .zeta.PKC
Expression and/or Activity
Introduction
[0045] The present invention provides methods (also referred to
herein as "screening assays") for identifying novel compounds
(e.g., small molecules) that inhibit expression of .zeta.PKC in
arthritic tissue. In one embodiment, cells that express PKC (either
naturally or recombinantly) are contacted with a test compound to
determine whether the compound inhibits expression of a .zeta.PKC
gene product (e.g., mRNA or polypeptide), with a decrease in
expression (as compared to an untreated sample of cells) indicating
that the compound inhibits .zeta.PKC in arthritic tissue. Changes
in .zeta.PKC gene expression can be determined by any method known
in the art or described above. In a preferred embodiment, cells
transfected with a reporter construct comprising a marker gene
(e.g., luciferase or green fluorescent protein (GFP)) downstream of
a NF-.kappa.B binding site are contacted with a test compound to
determine whether the compound can inhibit expression of the marker
protein when the cells are treated with cytokines. Compounds
identified that inhibit .zeta.PKC or marker protein expression are
candidates as drugs for the prophylactic and therapeutic treatment
of arthritis.
[0046] Alternatively, compounds can be identified that inhibit the
kinase activity of .zeta.PKC in vitro using assays described
previously. Purified (or partially purified) .zeta.PKC is contacted
with a test compound to determine whether the compound inhibits the
kinase activity of .zeta.PKC (as compared to an untreated sample of
enzyme). Compounds identified that inhibit .zeta.PKC activity could
then be tested in in vitro and in vivo models of arthritis. Several
in vitro models are described in the Examples below. In vivo models
of arthritis include, but are not limited to, the anterior cruciate
ligament resection models in the dog and rabbit, and the partial
meniscectomy models in the rabbit and mouse. Exemplary methods and
assays for directly and indirectly measuring the activity of
.zeta.PKC and/or for determining inhibition of the activity of
.zeta.PKC include, but are not limited to, enzymatic protein kinase
activity assays (as detailed above), chondrocyte pellet assays,
assays measuring proteoglycan degradation, and assays measuring
NF-.kappa.B activity.
Sources of .zeta.PKC
[0047] The .zeta.PKC (preferably mammalian; more preferably human
(e.g., GenBank Acc. No. Q05513; SEQ ID NO:2)) to be used in the
screening assays of the current invention are commercially
available from, e.g., Sigma-Aldrich, (St. Louis, Mo.), Research
Diagnostics (Flanders, N.J.), ProQinase (Freiburg, Germany), and
PanVera (Madison, Wis.). Alternatively, .zeta.PKC can be purified
or partially purified from various tissues (preferably mammalian;
more preferably human), including brain, placenta, testes and lung,
using known purification processes such as gel filtration and ion
exchange chromatography. Purification may also include affinity
chromatography with agents known to bind .zeta.PKC (e.g.,
anti-.zeta.PKC antibodies). These purification processes may also
be used to purify .zeta.PKC from recombinant sources.
[0048] Polynucleotides encoding .zeta.PKC (or enzymatic portions
thereof) may be operably linked to an appropriate expression
control sequence for recombinant production of .zeta.PKC. The
.zeta.PKC polynucleotides are preferably of mammalian origin (e.g.,
mouse .zeta.PKC cDNA (GenBank Acc. No. M94632); rat .zeta.PKC cDNA
(GenBank Acc. No. J04532); rabbit .zeta.PKC cDNA (GenBank Acc. No.
U78768)), and more preferably of human origin (e.g., human
.zeta.PKC cDNA (GenBank Acc. No. NM.sub.--002744; SEQ ID NO:1)).
General methods for expressing these recombinant .zeta.PKC
polynucleotides are well known in the art.
[0049] A number of cell lines may act as suitable host cells for
recombinant expression of .zeta.PKC. Mammalian host cell lines
include, for example, COS cells, CHO cells, 293 cells, A431 cells,
3T3 cells, CV-1 cells, HeLa cells, L cells, BHK21 cells, HL-60
cells, U937 cells, HaK cells, and Jurkat cells, as well as normal
diploid cells, cell strains derived from in vitro culture of
primary tissue, and primary explants.
[0050] Alternatively, .zeta.PKC (or enzymatic portions thereof) may
be recombinantly produced in lower eukaryotes such as yeast or in
prokaryotes. Potentially suitable yeast strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces
strains, and Candida strains. Potentially suitable bacterial
strains include Escherichia coli, Bacillus subtilis, and Salmonella
typhimurium. If the polypeptides of the present invention are made
in yeast or bacteria, it may be necessary to modify them by, for
example, phosphorylation or glycosylation of appropriate sites, in
order to obtain functionality. Such covalent attachments may be
accomplished using well-known chemical or enzymatic methods.
[0051] .zeta.PKC (or enzymatic portions thereof) may also be
recombinantly produced using insect expression vectors, such as
baculovirus vectors, and employing an insect cell expression
system. Materials and methods for baculovirus/Sf9 expression
systems are commercially available in kit form (e.g., the
MaxBac.RTM. kit, Invitrogen, Carlsbad, Calif.).
[0052] In order to facilitate purification, .zeta.PKC (or enzymatic
portions thereof) may be recombinantly expressed as fusions with
proteins such as maltose-binding protein (MBP),
glutathione-S-transferase (GST), or thioredoxin (TRX). Kits for
expression and purification of such fusion proteins are
commercially available from New England BioLabs (Beverly, Mass.),
Pharmacia (Piscataway, N.J.), and Invitrogen (Carlsbad, Calif.),
respectively. .zeta.PKC can also be tagged with a small epitope and
subsequently identified or purified using a specific antibody to
the epitope. One such epitope is the FLAG epitope, which is
commercially available from Eastman Kodak (New Haven, Conn.).
[0053] .zeta.PKC (or enzymatic portions thereof) may also be
produced by known conventional chemical synthesis. Methods for
chemically synthesizing polypeptides are well known to those
skilled in the art. Such chemically synthetic .zeta.PKC should
possess biological properties in common with the naturally produced
form, and thus can be employed as a biologically active or
immunological substitute for natural .zeta.PKC.
Sources and Screening of Test Compounds
[0054] The test compounds of the present invention may be obtained
from a number of sources. For example, combinatorial libraries of
molecules are available for screening. Using such libraries,
thousands of molecules can be screened for inhibitory activity.
Preparation and screening of compounds can be screened as described
above or by other methods well known to those of skill in the art.
The compounds thus identified can serve as conventional "lead
compounds" or can be used as the actual therapeutics.
Methods of Treatment
Introduction
[0055] The present invention provides both prophylactic and
therapeutic methods for the treatment of arthritis by inhibiting
expression and/or activity of .zeta.PKC. The methods involve
contacting cells (either in vitro, in vivo, or ex vivo) with an
agent in an amount effective to inhibit expression and/or activity
of .zeta.PKC. The agent can be any molecule that inhibits
expression and/or activity of .zeta.PKC, including, but not limited
to, inhibitory polynucleotides, small molecules, inhibitory protein
biologics, and peptide inhibitors.
Inhibitory Polynucleotides
[0056] Decreased expression of .zeta.PKC in an organism afflicted
with (or at risk for) arthritis, or in an involved cell from such
an organism, may be achieved through the use of various inhibitory
polynucleotides, such as antisense polynucleotides and ribozymes,
that bind and/or cleave the mRNA transcribed from the .zeta.PKC
gene (e.g., Galderisi et al. (1999) J. Cell Physiol. 181:251-57;
Sioud (2001) Curr. Mol. Med. 1:575-88).
[0057] The antisense polynucleotides or ribozymes of the invention
can be complementary to an entire coding strand of .zeta.PKC, or to
a portion thereof. Alternatively, antisense polynucleotides or
ribozymes can be complementary to a noncoding region of the coding
strand of .zeta.PKC. The antisense polynucleotides or ribozymes can
be constructed using chemical synthesis and enzymatic ligation
reactions using procedures well known in the art. The nucleoside
linkages of chemically synthesized polynucleotides can be modified
to enhance their ability to resist nuclease-mediated degradation,
as well as to increase their sequence specificity. Such linkage
modifications include, but are not limited to, phosphorothioate,
methylphosphonate, phosphoroamidate, boranophosphate, morpholino,
and peptide nucleic acid (PNA) linkages (Galderisi et al., supra;
Heasman (2002) Dev. Biol. 243:209-14; Micklefield (2001) Curr. Med.
Chem. 8:1157-79). Alternatively, these molecules can be produced
biologically using an expression vector into which a polynucleotide
of the present invention has been subcloned in an antisense (i.e.,
reverse) orientation.
[0058] The inhibitory polynucleotides of the present invention also
include triplex-forming oligonucleotides (TFOs) which bind in the
major groove of duplex DNA with high specificity and affinity
(Knauert and Glazer (2001) Hum. Mol. Genet. 10:2243-51). Expression
of .zeta.PKC can be inhibited by targeting TFOs complementary to
the regulatory regions of the .zeta.PKC gene (i.e., the promoter
and/or enhancer sequences) to form triple helical structures that
prevent transcription of the .zeta.PKC gene.
[0059] In a preferred embodiment, the inhibitory polynucleotides of
the present invention are short interfering RNA (siRNA) molecules.
siRNA molecules are short (preferably 19-25 nucleotides; most
preferably 19 or 21 nucleotides), double-stranded RNA molecules
that cause sequence-specific degradation of target mRNA. This
degradation is known as RNA interference (RNAi) (e.g., Bass (2001)
Nature 411:428-29). Originally identified in lower organisms, RNAi
has been effectively applied to mammalian cells and has recently
been shown to prevent fulminant hepatitis in mice treated with
siRNAs targeted to Fas mRNA (Song et al. (2003) Nature Med.
9:347-51). In addition, intrathecally delivered siRNA has recently
been reported to block pain responses in two models
(agonist-induced pain model and neuropathic pain model) in the rat
(Dorn et al. (2004) Nucleic Acids Res. 32(5):e49).
[0060] The siRNA molecules of the present invention can be
generated by annealing two complementary single-stranded RNA
molecules together (one of which matches a portion of the target
mRNA) (Fire et al., U.S. Pat. No. 6,506,559) or through the use of
a single hairpin RNA molecule that folds back on itself to produce
the requisite double-stranded portion (Yu et al. (2002) Proc. Natl.
Acad. Sci. USA 99:6047-52). The siRNA molecules can be chemically
synthesized (Elbashir et al. (2001) Nature 411:494-98) or produced
by in vitro transcription using single-stranded DNA templates (Yu
et al., supra). Alternatively, the siRNA molecules can be produced
biologically, either transiently (Yu et al., supra; Sui et al.
(2002) Proc. Natl. Acad. Sci. USA 99:5515-20) or stably (Paddison
et al. (2002) Proc. Natl. Acad. Sci. USA 99:1443-48), using an
expression vector(s) containing the sense and antisense siRNA
sequences. Recently, reduction of levels of target mRNA in primary
human cells, in an efficient and sequence-specific manner, was
demonstrated using adenoviral vectors that express hairpin RNAs,
which are further processed into siRNAs (Arts et al. (2003) Genome
Res. 13:2325-32).
[0061] The siRNA molecules targeted to the polynucleotides of the
present invention can be designed based on criteria well known in
the art (e.g., Elbashir et al. (2001) EMBO J. 20:6877-88). For
example, the target segment of the target mRNA preferably should
begin with AA (most preferred), TA, GA, or CA; the GC ratio of the
siRNA molecule preferably should be 45-55%; the siRNA molecule
preferably should not contain three of the same nucleotides in a
row; the siRNA molecule preferably should not contain seven mixed
G/Cs in a row; and the target segment preferably should be in the
ORF region of the target mRNA and preferably should be at least 75
by after the initiation ATG and at least 75 by before the stop
codon. Based on these criteria, preferred siRNA molecules of the
present invention, targeted to human .zeta.PKC mRNA, have been
designed and are shown in FIG. 1. Other siRNA molecules targeted to
.zeta.PKC mRNAs can be designed by one of ordinary skill in the art
using the aforementioned criteria or other known criteria (e.g.,
Reynolds et al. (2004) Nature Biotechnol. 22:326-30).
Small Molecules
[0062] Decreased expression of .zeta.PKC in an organism afflicted
with (or at risk for) arthritis, or in an involved cell from such
an organism, may also be achieved through the use of small
molecules (usually organic small molecules) that bind to and
inhibit the activity of .zeta.PKC. Small molecules known to inhibit
the activity of PKC (preferably isoform specific) can be used in
the treatment methods of the present invention. Numerous small
molecules that inhibit PKC are known in the art (including ones
approved for treatment of disease, as well as others in clinical
trials), and include both natural (e.g., staurosporine) and
artificial (e.g., LY333531) compounds (reviewed in Goekjian and
Jirousek (2001) Expert. Opin. Investing. Drugs 10:2117-40; Way et
al. (2000) Trends Pharmacol. Sci. 21:181-87, both of which are
incorporated by reference herein). These molecules can be used
directly or can serve as starting compounds for the development of
improved .zeta.PKC inhibitors (preferably isoform specific).
Alternatively, novel small molecules (preferably isoform specific)
identified by the screening methods described above may be
used.
Inhibitory Protein Biologics
[0063] Decreased activity of .zeta.PKC in an organism afflicted
with (or at risk for) arthritis, or in an involved cell from such
an organism, may also be achieved using protein biologics
Inhibitory protein biologics refer to protein molecules having
inhibitory biological activity in a cell or organism. Preferred
inhibitory protein biologics for use in the treatment methods of
the present invention include Par4 and kinase-defective
dominant-negative (DN) mutant forms of .zeta.PKC. Par4 is a
naturally occurring protein that binds to .zeta.PKC, which serves
to inhibit its enzymatic function (Diaz-Meco et al. (1996) Cell
86:777-86). DN mutant forms of .zeta.PKC, such as rat .zeta.PKC
with a lysine 281 to tryptophan point mutation (Bandyopadhyay et
al. (1997) J. Biol. Chem. 272:2551-58), reduce the activity of
endogenous .zeta.PKC by competing for substrate and can be made
using well-known site-directed mutagenesis techniques. Any variant
of .zeta.PKC that lacks kinase activity but still inhibits
.zeta.PKC-mediated signal transduction may be used as a DN mutant.
These inhibitory protein biologics may be generated in cells
(preferably chondrocytes) in situ using the above-described
expression techniques.
Peptide Inhibitors
[0064] Decreased activity of .zeta.PKC in an organism afflicted
with (or at risk for) arthritis, or in an involved cell from such
an organism, may also be achieved using peptide inhibitors that
bind to and inhibit the activity of .zeta.PKC. Peptide inhibitors
include peptide pseudosubstrates that prevent .zeta.PKC from
interacting with its substrates, as well as peptides that bind to
either .zeta.PKC or its substrates and block PKC-mediated
phosphorylation. Peptide inhibitors that inhibit .zeta.PKC are
known in the literature and include SIYRRGARRWRKL (SEQ ID NO:3),
SIYRRGARRWRKLYRAN (SEQ ID NO:4), and RRGARRWRK (SEQ ID NO:5) (e.g.,
Dang et al., supra; Zhou et al., supra). Preferably these peptide
inhibitors are myristoylated (SEQ ID NOs:6, 7, and 8, respectively)
to improve cell permeability (e.g., Standaert et al., supra; for
SEQ ID NO:6). Myristoylated and nonmyristoylated .zeta.PKC peptide
inhibitors can be chemically synthesized and are commercially
available from, e.g., Quality Controlled Biochemical (Hopkinton,
Mass.) and BioSource International, Inc., USA (Camarillo, Calif.).
One can provide a cell (preferably a chondrocyte) with a peptide
inhibitor in vitro, in vivo, or ex vivo using the techniques
described above.
Administration
[0065] Any of the compounds described herein (preferably a small
molecule) can be administered in vivo in the form of a
pharmaceutical composition for the treatment of arthritis. The
pharmaceutical compositions may be administered by any number of
routes, including, but not limited to, oral, nasal, rectal,
topical, sublingual, intravenous, intramuscular, intraarterial,
intramedullary, intrathecal, intraventricular, intraperitoneal,
intraarticular, or transdermal routes. In addition to the active
ingredients, the pharmaceutical compositions may contain
pharmaceutically acceptable carriers comprising excipients,
coatings, and auxiliaries known in the art.
[0066] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture or in animal models. The
therapeutically effective dose refers to the amount of active
ingredient that ameliorates the condition or its symptoms.
Therapeutic efficacy and toxicity in cell cultures or animal models
may be determined by standard pharmaceutical procedures (e.g.,
ED50: the dose therapeutically effective in 50% of the population;
LD50: the dose lethal to 50% of the population). The dose ratio
between therapeutic and toxic effects is the therapeutic index, and
can be expressed as the ratio ED50/LD50. Pharmaceutical
compositions that exhibit large therapeutic indexes are
preferred.
[0067] The data obtained from cell culture and animal models can
then be used to formulate a range of dosage for the compound for
use in mammals, preferably humans. The dosage of such a compound
preferably lies within a range of concentrations that include the
ED50 with little to no toxicity. The dosage may vary within this
range depending upon the composition form employed and the
administration route utilized.
EXAMPLES
[0068] The Examples which follow are set forth to aid in the
understanding of the invention but are not intended to, and should
not be construed to, limit its scope in any way. The Examples do
not include detailed descriptions of conventional methods, such as
those employed in the construction of vectors and plasmids, the
insertion of genes encoding polypeptides into such vectors and
plasmids, the introduction of such vectors and plasmids into host
cells, or the expression of polypeptides from such vectors and
plasmids in host cells. Such methods, and other conventional
methods, are well known to those of ordinary skill in the art.
Example 1
.zeta.PKC Expression is Upregulated in Arthritis
Example 1.1
Experimental Design
[0069] To identify transcripts differentially expressed between
arthritic and normal articular cartilage, tissue samples were
obtained from arthritis patients with end-stage knee replacement
and nonarthritic amputee individuals. The presence or absence of
arthritis was confirmed by histology.
Example 1.2
Oligonucleotide Array Hybridization
[0070] The Human Genome U95Av2 (HG-U95Av2) GeneChip.RTM. Array
(Affymetrix, Santa Clara, Calif.) was used for expression
profiling. The HG-U95Av2 chip contains 25-mer oligonucleotide
probes representing .about.12,000 primarily full-length sequences
(.about.16 probe pairs/sequence) derived from the human genome. For
each probe designed to be perfectly complimentary to a target
sequence, a partner probe is generated that is identical except for
a single base mismatch in its center. These probe pairs allow for
signal quantitation and subtraction of nonspecific noise.
[0071] RNA was extracted from individual articular cartilage
tissue, converted to biotinylated cRNA, and fragmented according to
the Affymetrix protocol. The fragmented cRNAs were diluted in
1.times.MES buffer containing 100 .mu.g/ml herring sperm DNA and
500 .mu.g/ml acetylated BSA and denatured for 5 min at 99.degree.
C. followed immediately by 5 min at 45.degree. C. Insoluble
material was removed from the hybridization mixtures by a brief
centrifugation, and the hybridization mix was added to each array
and incubated at 45.degree. C. for 16 hr with continuous rotation
at 60 rpm. After incubation, the hybridization mix was removed and
the chips were extensively washed with 6.times.SSPET and stained
with SAPE solution as described in the Affymetrix protocol.
Example 1.3
Oligonucleotide Array Data Analysis
[0072] The raw florescent intensity value of each transcript was
measured at a resolution of 6 mm with a Hewlett-Packard Gene Array
Scanner. GeneChip.RTM. software 3.2 (Affymetrix), which uses an
algorithm to determine whether a gene is "present" or "absent," as
well as the specific hybridization intensity values or "average
differences" of each gene on the array, was used to evaluate the
fluorescent data. The average difference for each gene was
normalized to frequency values by referral to the average
differences of 11 control transcripts of known abundance that were
spiked into each hybridization mix according to the procedure of
Hill et al. ((2000) Science 290:809-12). The frequency of each gene
was calculated and represents a value equal to the total number of
individual gene transcripts per 10.sup.6 total transcripts.
[0073] The frequency of each transcript was evaluated, and the
transcript was included in the study if it met the following three
criteria. First, transcripts which were called "present" by the
GeneChip.RTM. software in at least one of the arrays for both
arthritis and normal cartilage were included in the analysis.
Second, for comparison between arthritis and normal cartilage, a
t-test was applied to identify the subset of transcripts that had a
significant (p<0.05) increase or decrease in frequency values.
Third, average-fold changes in frequency values across the
statistically significant subset of transcripts were required to be
2.4-fold or greater. These criteria were established based upon
replicate experiments that estimated the intraarray
reproducibility.
[0074] Based on these criteria, 602 transcripts were identified
that were differentially expressed in arthritic and normal
articular cartilage. One such transcript identified was
.zeta.PKC.
Example 2
Inhibition of .zeta.PKC Activity Inhibits Extracellular Matrix
(ECM) Degradation
Example 2.1
Primary Bovine Chondrocyte Isolation and Culture
[0075] Full-thickness bovine articular cartilage slices were
dissected under aseptic conditions, rinsed four times in PBS, and
subjected to pronase and collagenase digestion (1 mg/ml pronase
(Calbiochem, San Diego, Calif.) for 30 minutes and 1 mg/ml
Collagenase P (Roche Diagnostics Corporation, Indianapolis, Ind.)
overnight at 37.degree. C. in DME without serum) to isolate
chondrocytes embedded in the cartilage extracellular matrix. The
digest was filtered through a 70 micron Falcon.TM. cell strainer
(BD Biosciences, San Jose, Calif.) and washed twice in DME
containing 10% FBS. Typically 2-4.times.10.sup.8 cells were
obtained from a calf metacarpophalangeal joint surface. Cells were
plated in monolayer in six-well plates at density of
2.times.10.sup.6 cells/well. For pellet culture, cells were
resuspended in growth media [HL-1 media (Cambrex Corporation, East
Rutherford, N.J.), penicillin+streptomycin, glutamine, 50 ng/ml
ascorbate, and 10% FBS] at 1.times.10.sup.6 cells/ml, and 1 ml
aliquots of cells were transferred to 15 ml sterile Falcon
centrifuge tubes. The cells were centrifuged at 200.times.g for 5
min at 4.degree. C. and the resulting cell pellets were cultured as
described previously (Xu et al. (1996) Endocrinology 137:3557-65).
Cell media were collected and stored for collagen and proteoglycan
assays, and cells were refed with fresh media (3 ml/well, 1
ml/tube) every 3-4 days. Pellet cultures were maintained for 3
weeks, at which time the pellets were harvested and either digested
with 0.5 ml of 300 .mu.g/ml papain at 65.degree. C. for 3-6 hrs for
dimethylmethylene blue (DMMB) dye assays or prepared for
histology.
Example 2.2
Peptide Blocker of NF-.kappa.B Can Inhibit TNF- or IL-1-Mediated
Proteoglycan Degradation
[0076] To demonstrate that blocking NF-.kappa.B activity can
inhibit proteoglycan degradation in our culture system, primary
bovine chondrocytes were cultured with the NF-.kappa.B blocker SN50
for 4 days at a concentration of 300 .mu.g/ml in the presence or
absence of 10 ng/ml TNF or 1 ng/ml IL-1. Cells were incubated with
the inhibitor for 3 hrs prior to the addition of either TNF or
IL-1. SN50 is a peptide that contains the nuclear localization
signal of NF-.kappa.B coupled to a stretch of hydrophobic amino
acids to facilitate transport across lipid bilayers, and has been
shown to block NF-.kappa.B-mediated transcription (e.g., Lin et al.
(1995) J. Biol. Chem. 270:14255-58). SN50M, which served as a
negative control, is the same peptide with amino acid changes to
abolish NF-.kappa.B-blocking activity. SN50 and SN50M are available
from, e.g., Biomol Research Laboratories, Inc. (Plymouth Meeting,
Pa.).
[0077] As shown in FIG. 2, SN50M was ineffective at preventing TNF-
and IL-1-mediated proteoglycan degradation in bovine chondrocytes,
as measured by proteoglycan release into the media and decreased
recovery in the cell pellet. In contrast, SN50 completely inhibited
the cytokine-mediated degradation of proteoglycan. In addition,
SN50 even prevented proteoglycan degradation in the absence of
cytokine treatment, as compared to SN50M. These results demonstrate
that blocking NF-.kappa.B, which controls the cytokine-mediated
synthesis of collagenases and aggrecanases, inhibits proteoglycan
degradation in primary bovine chondrocytes.
Example 2.3
.zeta.PKC Inhibitors Block TNF-Mediated Proteoglycan
Degradation
[0078] To determine whether .zeta.PKC inhibitors can inhibit
TNF-mediated proteoglycan degradation, primary bovine chondrocytes
were cultured with various concentrations of TNF for 5 days with or
without (1) the myristoylated .zeta.PKC peptide pseudosubstrate
[herein termed "2089"; synthesized in-house; equivalent to SEQ ID
NO:6; available from, e.g., BioSource International, Inc., USA
(Camarillo, Calif.)], or (2) the small molecule inhibitor
Ro-31-8220 (Sigma-RBI, Natick, Mass.). The inhibitors were added 3
hrs prior to addition of TNF.
[0079] As shown in FIG. 3, TNF at 10 and 100 ng/ml caused
significant proteoglycan degradation, as measured by proteoglycan
release into the media and decreased recovery in the cell pellet.
This proteoglycan degradation was significantly inhibited by both
50 .mu.M 2089 and 10 .mu.M Ro-31-8220 (RO31). In addition, these
compounds inhibited proteoglycan degradation even in the absence of
TNF, suggesting a blockade of constitutive levels of proteases.
Ro-31-8220 at a concentration of 5 .mu.M was relatively ineffective
at preventing proteoglycan degradation. These results demonstrate
that both 2089 and Ro-31-8220 penetrate the cell membrane of
primary chondrocytes and effectively block TNF-mediated
proteoglycan degradation. Trypan blue staining of the chondrocytes
and lactate assays on the culture media were performed to rule out
cytotoxicity as a possible explanation for the results. These
experiments confirmed that the compounds do not cause appreciable
cytotoxicity at the doses used in these experiments. In addition,
control peptides were synthesized and tested in the same assay
system to address the possibility that nonspecific effects may be
causing the observed results. A nonmyristoylated version of the
pseudosubstrate peptide, as well as a "scrambled control" peptide
containing the same amino acid content as the pseudosubstrate
peptide, but with a scrambled sequence, were tested, and both were
found to be ineffective at blocking proteoglycan degradation.
Example 2.4
Myristoylated .zeta.PKC Peptide Pseudosubstrate 2089 Blocks Both
TNF- and IL-1-Mediated Proteoglycan Degradation in a Dose-Dependent
Manner
[0080] To determine whether myristoylated .zeta.PKC peptide
pseudosubstrate 2089 can inhibit cytokine-mediated proteoglycan
degradation in a dose-dependent manner, primary bovine chondrocytes
were cultured with either 10 ng/ml TNF or 1 ng/ml IL-1 for 4 days
after addition of various concentrations of 2089.
[0081] As shown in FIG. 4, 2089 inhibited both TNF- and
IL-1-mediated proteoglycan degradation in a dose-dependent manner,
with the highest dose (100 .mu.M) completely inhibiting
proteoglycan release into the media. Again, increased cell pellet
retention of proteoglycan and decreased release of proteoglycan
into the media was achieved with 2089 even in the absence of
cytokine. Cumulatively, these results indicate that inhibition of
.zeta.PKC in chondrocytes inhibits cytokine-mediated proteolytic
degradation of proteoglycan. This, along with the fact that the
.zeta.PKC knockout mouse has a very benign phenotype (Leitges et
al., supra), indicates that inhibition of .zeta.PKC may be a safe,
effective treatment for arthritis, as well as other inflammatory
diseases.
Example 3
.zeta.PKC mRNA is Upregulated in Human Osteoarthritic (OA)
Cartilage
[0082] Transcriptional profiling data on human articular cartilage
from osteoarthritic (OA) patients showed a statistically
significant increase in .zeta.PKC mRNA as compared with human
non-OA cartilage. In panel A of FIG. 5, RNA was extracted from
frozen pulverized articular cartilage tissue from clinical samples,
and subjected to expression profiling analysis using the HG-U95Av2
chip. Three groups were analyzed: normal (non-OA) cartilage [13
samples]; severe OA cartilage (nonlesional areas) [29 samples]; and
severe OA cartilage (lesional areas) [26 samples]. Levels of
.zeta.PKC mRNA were elevated in severe OA samples as compared with
normal samples. In panel B of FIG. 5, TaqMan.RTM. Q-PCR (Applied
Biosystems, Foster City, Calif.) analysis of the same set of
samples showed significantly higher levels of .zeta.PKC mRNA in
severe OA samples as compared with normal samples; TaqMan.RTM.
Q-PCR protocols were conducted according to the manufacturer's
instructions.
Example 4
.zeta.PKC Protein is Expressed in Chondrocytes
[0083] An anti-.zeta.PKC antibody (nPKC.zeta.(C-20); Santa Cruz
Biotechnology, Inc., Santa Cruz, Calif.) was used to compare
production of .zeta.PKC protein in chondrocytes and Jurkat cells.
The lysates of bovine chondrocytes and Jurkat cells (human cell
line) were prepared by similar methods: cells were washed with cold
phosphate-buffered saline and immediately placed in cell lysis
buffer (Cell Signaling Technology, Inc., Beverly, Mass.) containing
phosphatase inhibitors. Cells were incubated for 5 min on ice, and
then centrifuged at 12,000 rpm for 10 min at 4.degree. C. Samples
were resolved by 12% SDS-polyacrylamide gel electrophoresis under
reducing conditions. A Western blot showed that chondrocytes
expressed a substantial amount of .zeta.PKC protein; the same blot
did not show appreciable expression of .zeta.PKC protein in Jurkat
cells.
Example 5
Adenoviral-Mediated Expression of .zeta.PKC Increases Proteoglycan
Degradation
[0084] Primary bovine chondrocytes were isolated and cultured as
described above (in pellet format) in Example 2.1. Cells were
cultured in 0.5 ml growth media (HL-1) containing 2% FBS prior to
the addition of adenovirus (in 15 ml Falcon tubes). Adenovirus
vectors containing .zeta.PKC or GFP (green fluorescent protein)
were prepared (Alden et al. (1999) Hum. Gene Ther. 10:2245-53), and
cultures of chondrocytes were infected immediately following
isolation and prior to pelleting. The adenovirus expressing GFP or
.zeta.PKC was added directly into the culture at a multiplicity of
infection (MOI) of 5000. As seen in panel A of FIG. 6,
overexpression of full-length .zeta.PKC in primary bovine
chondrocytes in culture (without addition of cytokines) resulted in
a modest but statistically significant increase in proteoglycan
degradation (as measured by proteoglycan released into the media in
the chondrocyte pellet assay) as compared with overexpression of
GFP. Medium containing 10% FBS was added to the culture after a
2-hour incubation at 37.degree. C. (in a humidified atmosphere of
5% CO.sub.2). The serum composition was gradually decreased every 3
days (sequentially to 5%, 2.5% and finally to 0% (serum-free) with
every feeding of the chondrocyte pellets) to wean the cells from
serum. Proteoglycan released into the media represents total
proteoglycan released over 25 days.
[0085] In panel B of FIG. 6, proteoglycan was measured in the media
over 4 days, with or without addition of cytokines, after cells had
been weaned from serum. Addition of suboptimal levels of TNF.alpha.
significantly enhanced the amount of proteoglycan released into the
media in response to overexpression of .zeta.PKC, as compared with
overexpression of GFP or absence of adenovirus infection (FIG. 6,
panel B).
Example 6
.zeta.PKC is Responsible for TNF.alpha.-Mediated Proteoglycan
Release in Chondrocytes
[0086] Articular bovine chondrocytes were prepared as previously
detailed for the pellet assay. As shown in FIG. 7, TNF.alpha. was
added (100 ng/ml; bars labeled with *) to some cultures in the
chondrocyte pellet assay. Two inhibitors were added at various
doses. One inhibitor, bisindolylmaleimide (BIS), is a pan-PKC
inhibitor, reported to block the activity of all isoforms of
.zeta.PKC, including .zeta.PKC (e.g., Toullec et al. (1991) J.
Biol. Chem. 266:15771-81). The other inhibitor, chelerythrine
chloride (CC), is a competitive inhibitor of the phorbol
ester-binding site. CC competes for the phorbol ester-binding
domain of the conventional and novel .zeta.PKC family members and
inhibits them; however, as the atypical .zeta.PKCs (e.g.,
.zeta.PKC; Ca.sup.++-independent and diacylglycerol-independent
.zeta.PKCs) lack this binding domain, they are not inhibited by CC
(e.g., Herbert et al. (1990) Biochem. Biophys. Res. Commun.
172:993-99). Cytokine (TNF.alpha.)-mediated release of proteoglycan
into the media in the chondrocyte pellet assay was blocked by BIS
(at 20-40 .mu.M), but was not blocked by CC (FIG. 7), indicating
that selective inhibition of .zeta.PKC blocks cytokine-mediated
proteoglycan degradation.
Example 7
.zeta.PKC is Responsible for TNF.alpha.-Induced Activation of
NF-.kappa.B in Chondrocytes
[0087] Activation of NF-.kappa.B was measured in an immortalized
human chondrocyte cell line (C28/I2; see, e.g., Finger et al.
(2003) Arthritis Rheum. 48:3395-403; Goldring (1994) J. Clin.
Invest. 94:2307-16) into which a luciferase reporter gene under the
control of an NF-.kappa.B response element was introduced. The
cells were cultured in DMEM/Ham's F12 supplemented with 10% FBS;
the cells were split into 96 wells (1.times.10.sup.5 cells/well)
and infected with adenovirus expressing NF-.kappa.B luciferase
construct (100 MOI) 24 hrs prior to assay. The chondrocytes were
incubated with inhibitors in serum-free media 2 hrs prior to the
addition of TNF. As shown in FIG. 8, TNF.alpha. was added (1 ng/ml
or 10 ng/ml) to some cultures in the chondrocyte pellet assay. BIS
(20 .mu.M), a pan-PKC inhibitor, was added to some cultures, and CC
(8 .mu.M), a competitive inhibitor of the phorbol ester binding
site in some forms of .zeta.PKC (but not the atypical .zeta.PKCs,
e.g., .zeta.PKC), was added to other cultures. Cytokine
(TNF.alpha.)-mediated activation of NF-.kappa.B was blocked by BIS,
but was not blocked by CC, indicating that selective inhibition of
.zeta.PKC blocks cytokine-mediated activation of NF-.kappa.B.
Sequence CWU 1
1
16412164DNAHomo sapiensCDS(1)..(1779) 1atg ccc agc agg acc gac ccc
aag atg gaa ggg agc ggc ggc cgc gtc 48Met Pro Ser Arg Thr Asp Pro
Lys Met Glu Gly Ser Gly Gly Arg Val1 5 10 15cgc ctc aag gcg cat tac
ggg ggg gac atc ttc atc acc agc gtg gac 96Arg Leu Lys Ala His Tyr
Gly Gly Asp Ile Phe Ile Thr Ser Val Asp 20 25 30gcc gcc acg acc ttc
gag gag ctc tgt gag gaa gtg aga gac atg tgt 144Ala Ala Thr Thr Phe
Glu Glu Leu Cys Glu Glu Val Arg Asp Met Cys 35 40 45cgt ctg cac cag
cag cac ccg ctc acc ctc aag tgg gtg gac agc gaa 192Arg Leu His Gln
Gln His Pro Leu Thr Leu Lys Trp Val Asp Ser Glu 50 55 60ggt gac cct
tgc acg gtg tcc tcc cag atg gag ctg gaa gag gct ttc 240Gly Asp Pro
Cys Thr Val Ser Ser Gln Met Glu Leu Glu Glu Ala Phe65 70 75 80cgc
ctg gcc cgt cag tgc agg gat gaa ggc ctc atc att cat gtt ttc 288Arg
Leu Ala Arg Gln Cys Arg Asp Glu Gly Leu Ile Ile His Val Phe 85 90
95ccg agc acc cct gag cag cct ggc ctg cca tgt ccg gga gaa gac aaa
336Pro Ser Thr Pro Glu Gln Pro Gly Leu Pro Cys Pro Gly Glu Asp Lys
100 105 110tct atc tac cgc cgg gga gcc aga aga tgg agg aag ctg tac
cgt gcc 384Ser Ile Tyr Arg Arg Gly Ala Arg Arg Trp Arg Lys Leu Tyr
Arg Ala 115 120 125aac ggc cac ctc ttc caa gcc aag cgc ttt aac agg
aga gcg tac tgc 432Asn Gly His Leu Phe Gln Ala Lys Arg Phe Asn Arg
Arg Ala Tyr Cys 130 135 140ggt cag tgc agc gag agg ata tgg ggc ctc
gcg agg caa ggc tac agg 480Gly Gln Cys Ser Glu Arg Ile Trp Gly Leu
Ala Arg Gln Gly Tyr Arg145 150 155 160tgc atc aac tgc aaa ctg ctg
gtc cat aag cgc tgc cac ggc ctc gtc 528Cys Ile Asn Cys Lys Leu Leu
Val His Lys Arg Cys His Gly Leu Val 165 170 175ccg ctg acc tgc agg
aag cat atg gat tct gtc atg cct tcc caa gag 576Pro Leu Thr Cys Arg
Lys His Met Asp Ser Val Met Pro Ser Gln Glu 180 185 190cct cca gta
gac gac aag aac gag gac gcc gac ctt cct tcc gag gag 624Pro Pro Val
Asp Asp Lys Asn Glu Asp Ala Asp Leu Pro Ser Glu Glu 195 200 205aca
gat gga att gct tac att tcc tca tcc cgg aag cat gac agc att 672Thr
Asp Gly Ile Ala Tyr Ile Ser Ser Ser Arg Lys His Asp Ser Ile 210 215
220aaa gac gac tcg gag gac ctt aag cca gtt atc gat ggg atg gat gga
720Lys Asp Asp Ser Glu Asp Leu Lys Pro Val Ile Asp Gly Met Asp
Gly225 230 235 240atc aaa atc tct cag ggg ctt ggg ctg cag gac ttt
gac cta atc aga 768Ile Lys Ile Ser Gln Gly Leu Gly Leu Gln Asp Phe
Asp Leu Ile Arg 245 250 255gtc atc ggg cgc ggg agc tac gcc aag gtt
ctc ctg gtg cgg ttg aag 816Val Ile Gly Arg Gly Ser Tyr Ala Lys Val
Leu Leu Val Arg Leu Lys 260 265 270aag aat gac caa att tac gcc atg
aaa gtg gtg aag aaa gag ctg gtg 864Lys Asn Asp Gln Ile Tyr Ala Met
Lys Val Val Lys Lys Glu Leu Val 275 280 285cat gat gac gag gat att
gac tgg gta cag aca gag aag cac gtg ttt 912His Asp Asp Glu Asp Ile
Asp Trp Val Gln Thr Glu Lys His Val Phe 290 295 300gag cag gca tcc
agc aac ccc ttc ctg gtc gga tta cac tcc tgc ttc 960Glu Gln Ala Ser
Ser Asn Pro Phe Leu Val Gly Leu His Ser Cys Phe305 310 315 320cag
acg aca agt cgg ttg ttc ctg gtc att gag tac gtc aac ggc ggg 1008Gln
Thr Thr Ser Arg Leu Phe Leu Val Ile Glu Tyr Val Asn Gly Gly 325 330
335gac ctg atg ttc cac atg cag agg cag agg aag ctc cct gag gag cac
1056Asp Leu Met Phe His Met Gln Arg Gln Arg Lys Leu Pro Glu Glu His
340 345 350gcc agg ttc tac gcg gcc gag atc tgc atc gcc ctc aac ttc
ctg cac 1104Ala Arg Phe Tyr Ala Ala Glu Ile Cys Ile Ala Leu Asn Phe
Leu His 355 360 365gag agg ggg atc atc tac agg gac ctg aag ctg gac
aac gtc ctc ctg 1152Glu Arg Gly Ile Ile Tyr Arg Asp Leu Lys Leu Asp
Asn Val Leu Leu 370 375 380gat gcg gac ggg cac atc aag ctc aca gac
tac ggc atg tgc aag gaa 1200Asp Ala Asp Gly His Ile Lys Leu Thr Asp
Tyr Gly Met Cys Lys Glu385 390 395 400ggc ctg ggc cct ggt gac aca
acg agc act ttc tgc gga acc ccg aat 1248Gly Leu Gly Pro Gly Asp Thr
Thr Ser Thr Phe Cys Gly Thr Pro Asn 405 410 415tac atc gcc ccc gaa
atc ctg cgg gga gag gag tac ggg ttc agc gtg 1296Tyr Ile Ala Pro Glu
Ile Leu Arg Gly Glu Glu Tyr Gly Phe Ser Val 420 425 430gac tgg tgg
gcg ctg gga gtc ctc atg ttt gag atg atg gcc ggg cgc 1344Asp Trp Trp
Ala Leu Gly Val Leu Met Phe Glu Met Met Ala Gly Arg 435 440 445tcc
ccg ttc gac atc atc acc gac aac ccg gac atg aac aca gag gac 1392Ser
Pro Phe Asp Ile Ile Thr Asp Asn Pro Asp Met Asn Thr Glu Asp 450 455
460tac ctt ttc caa gtg atc ctg gag aag ccc atc cgg atc ccc cgg ttc
1440Tyr Leu Phe Gln Val Ile Leu Glu Lys Pro Ile Arg Ile Pro Arg
Phe465 470 475 480ctg tcc gtc aaa gcc tcc cat gtt tta aaa gga ttt
tta aat aag gac 1488Leu Ser Val Lys Ala Ser His Val Leu Lys Gly Phe
Leu Asn Lys Asp 485 490 495ccc aaa gag agg ctc ggc tgc cgg cca cag
act gga ttt tct gac atc 1536Pro Lys Glu Arg Leu Gly Cys Arg Pro Gln
Thr Gly Phe Ser Asp Ile 500 505 510aag tcc cac gcg ttc ttc cgc agc
ata gac tgg gac ttg ctg gag aag 1584Lys Ser His Ala Phe Phe Arg Ser
Ile Asp Trp Asp Leu Leu Glu Lys 515 520 525aag cag gcg ctc cct cca
ttc cag cca cag atc aca gac gac tac ggt 1632Lys Gln Ala Leu Pro Pro
Phe Gln Pro Gln Ile Thr Asp Asp Tyr Gly 530 535 540ctg gac aac ttt
gac aca cag ttc acc agc gag ccc gtg cag ctg acc 1680Leu Asp Asn Phe
Asp Thr Gln Phe Thr Ser Glu Pro Val Gln Leu Thr545 550 555 560cca
gac gat gag gat gcc ata aag agg atc gac cag tca gag ttc gaa 1728Pro
Asp Asp Glu Asp Ala Ile Lys Arg Ile Asp Gln Ser Glu Phe Glu 565 570
575ggc ttt gag tat atc aac cca tta ttg ctg tcc acc gag gag tcg gtg
1776Gly Phe Glu Tyr Ile Asn Pro Leu Leu Leu Ser Thr Glu Glu Ser Val
580 585 590tga ggccgcgtgc gtctctgtcg tggacacgcg tgattgaccc
tttaactgta 1829tccttaacca ccgcatatgc atgccaggct gggcacggct
ccgagggcgg ccagggacag 1889acgcttgcgc cgagaccgca gagggaagcg
tcagcgggcg ctgctgggag cagaacagtc 1949cctcacacct ggcccggcag
gcagcttcgt gctggaggaa cttgctgctg tgcctgcgtc 2009gcggcggatc
cgcggggacc ctgccgaggg ggctgtcatg cggtttccaa ggtgcacatt
2069ttccacggaa acagaactcg atgcactgac ctgctccgcc aggaaagtga
gcgtgtagcg 2129tcctgaggaa taaaatgttc cgatgaaaaa aaaaa
21642592PRTHomo sapiens 2Met Pro Ser Arg Thr Asp Pro Lys Met Glu
Gly Ser Gly Gly Arg Val1 5 10 15Arg Leu Lys Ala His Tyr Gly Gly Asp
Ile Phe Ile Thr Ser Val Asp 20 25 30Ala Ala Thr Thr Phe Glu Glu Leu
Cys Glu Glu Val Arg Asp Met Cys 35 40 45Arg Leu His Gln Gln His Pro
Leu Thr Leu Lys Trp Val Asp Ser Glu 50 55 60Gly Asp Pro Cys Thr Val
Ser Ser Gln Met Glu Leu Glu Glu Ala Phe65 70 75 80Arg Leu Ala Arg
Gln Cys Arg Asp Glu Gly Leu Ile Ile His Val Phe 85 90 95Pro Ser Thr
Pro Glu Gln Pro Gly Leu Pro Cys Pro Gly Glu Asp Lys 100 105 110Ser
Ile Tyr Arg Arg Gly Ala Arg Arg Trp Arg Lys Leu Tyr Arg Ala 115 120
125Asn Gly His Leu Phe Gln Ala Lys Arg Phe Asn Arg Arg Ala Tyr Cys
130 135 140Gly Gln Cys Ser Glu Arg Ile Trp Gly Leu Ala Arg Gln Gly
Tyr Arg145 150 155 160Cys Ile Asn Cys Lys Leu Leu Val His Lys Arg
Cys His Gly Leu Val 165 170 175Pro Leu Thr Cys Arg Lys His Met Asp
Ser Val Met Pro Ser Gln Glu 180 185 190Pro Pro Val Asp Asp Lys Asn
Glu Asp Ala Asp Leu Pro Ser Glu Glu 195 200 205Thr Asp Gly Ile Ala
Tyr Ile Ser Ser Ser Arg Lys His Asp Ser Ile 210 215 220Lys Asp Asp
Ser Glu Asp Leu Lys Pro Val Ile Asp Gly Met Asp Gly225 230 235
240Ile Lys Ile Ser Gln Gly Leu Gly Leu Gln Asp Phe Asp Leu Ile Arg
245 250 255Val Ile Gly Arg Gly Ser Tyr Ala Lys Val Leu Leu Val Arg
Leu Lys 260 265 270Lys Asn Asp Gln Ile Tyr Ala Met Lys Val Val Lys
Lys Glu Leu Val 275 280 285His Asp Asp Glu Asp Ile Asp Trp Val Gln
Thr Glu Lys His Val Phe 290 295 300Glu Gln Ala Ser Ser Asn Pro Phe
Leu Val Gly Leu His Ser Cys Phe305 310 315 320Gln Thr Thr Ser Arg
Leu Phe Leu Val Ile Glu Tyr Val Asn Gly Gly 325 330 335Asp Leu Met
Phe His Met Gln Arg Gln Arg Lys Leu Pro Glu Glu His 340 345 350Ala
Arg Phe Tyr Ala Ala Glu Ile Cys Ile Ala Leu Asn Phe Leu His 355 360
365Glu Arg Gly Ile Ile Tyr Arg Asp Leu Lys Leu Asp Asn Val Leu Leu
370 375 380Asp Ala Asp Gly His Ile Lys Leu Thr Asp Tyr Gly Met Cys
Lys Glu385 390 395 400Gly Leu Gly Pro Gly Asp Thr Thr Ser Thr Phe
Cys Gly Thr Pro Asn 405 410 415Tyr Ile Ala Pro Glu Ile Leu Arg Gly
Glu Glu Tyr Gly Phe Ser Val 420 425 430Asp Trp Trp Ala Leu Gly Val
Leu Met Phe Glu Met Met Ala Gly Arg 435 440 445Ser Pro Phe Asp Ile
Ile Thr Asp Asn Pro Asp Met Asn Thr Glu Asp 450 455 460Tyr Leu Phe
Gln Val Ile Leu Glu Lys Pro Ile Arg Ile Pro Arg Phe465 470 475
480Leu Ser Val Lys Ala Ser His Val Leu Lys Gly Phe Leu Asn Lys Asp
485 490 495Pro Lys Glu Arg Leu Gly Cys Arg Pro Gln Thr Gly Phe Ser
Asp Ile 500 505 510Lys Ser His Ala Phe Phe Arg Ser Ile Asp Trp Asp
Leu Leu Glu Lys 515 520 525Lys Gln Ala Leu Pro Pro Phe Gln Pro Gln
Ile Thr Asp Asp Tyr Gly 530 535 540Leu Asp Asn Phe Asp Thr Gln Phe
Thr Ser Glu Pro Val Gln Leu Thr545 550 555 560Pro Asp Asp Glu Asp
Ala Ile Lys Arg Ile Asp Gln Ser Glu Phe Glu 565 570 575Gly Phe Glu
Tyr Ile Asn Pro Leu Leu Leu Ser Thr Glu Glu Ser Val 580 585
590313PRTArtificialpeptide inhibitor 3Ser Ile Tyr Arg Arg Gly Ala
Arg Arg Trp Arg Lys Leu1 5 10417PRTArtificialpeptide inhibitor 4Ser
Ile Tyr Arg Arg Gly Ala Arg Arg Trp Arg Lys Leu Tyr Arg Ala1 5 10
15Asn59PRTArtificialpeptide inhibitor 5Arg Arg Gly Ala Arg Arg Trp
Arg Lys1 5613PRTArtificialpeptide inhibitor 6Ser Ile Tyr Arg Arg
Gly Ala Arg Arg Trp Arg Lys Leu1 5 10717PRTArtificialpeptide
inhibitor 7Ser Ile Tyr Arg Arg Gly Ala Arg Arg Trp Arg Lys Leu Tyr
Arg Ala1 5 10 15Asn89PRTArtificialpeptide inhibitor 8Arg Arg Gly
Ala Arg Arg Trp Arg Lys1 5921DNAHomo sapiens 9aagtgagaga catgtgtcgt
c 211021DNAHomo sapiens 10aagatggagg aagctgtacc g 211121DNAHomo
sapiens 11aaggctacag gtgcatcaac t 211221DNAHomo sapiens
12aactgctggt ccataagcgc t 211321DNAHomo sapiens 13aagagcctcc
agtagacgac a 211421DNAHomo sapiens 14aagacgactc ggaggacctt a
211521DNAHomo sapiens 15aagagctggt gcatgatgac g 211621DNAHomo
sapiens 16aagtcggttg ttcctggtca t 211721DNAHomo sapiens
17aagctcacag actacggcat g 211821DNAHomo sapiens 18aagaggatcg
accagtcaga g 211921DNAHomo sapiens 19aactgtatcc ttaaccaccg c
212021DNAHomo sapiens 20aaccaccgca tatgcatgcc a
212121RNAArtificialsiRNA polynucleotide, synthesized 21gugagagaca
ugugucgucu u 212221RNAArtificialsiRNA polynucleotide, synthesized
22gauggaggaa gcuguaccgu u 212321RNAArtificialsiRNA polynucleotide,
synthesized 23ggcuacaggu gcaucaacuu u 212421RNAArtificialsiRNA
polynucleotide, synthesized 24cugcuggucc auaagcgcuu u
212521RNAArtificialsiRNA polynucleotide, synthesized 25gagccuccag
uagacgacau u 212621RNAArtificialsiRNA polynucleotide, synthesized
26gacgacucgg aggaccuuau u 212721RNAArtificialsiRNA polynucleotide,
synthesized 27gagcuggugc augaugacgu u 212821RNAArtificialsiRNA
polynucleotide, synthesized 28gucgguuguu ccuggucauu u
212921RNAArtificialsiRNA polynucleotide, synthesized 29gcucacagac
uacggcaugu u 213021RNAArtificialsiRNA polynucleotide, synthesized
30gaggaucgac cagucagagu u 213121RNAArtificialsiRNA polynucleotide,
synthesized 31cuguauccuu aaccaccgcu u 213221RNAArtificialsiRNA
polynucleotide, synthesized 32ccaccgcaua ugcaugccau u
213321RNAArtificialsiRNA polynucleotide, synthesized 33gacgacacau
gucucucacu u 213421RNAArtificialsiRNA polynucleotide, synthesized
34cgguacagcu uccuccaucu u 213521RNAArtificialsiRNA polynucleotide,
synthesized 35aguugaugca ccuguagccu u 213621RNAArtificialsiRNA
polynucleotide, synthesized 36agcgcuuaug gaccagcagu u
213721RNAArtificialsiRNA polynucleotide, synthesized 37ugucgucuac
uggaggcucu u 213821RNAArtificialsiRNA polynucleotide, synthesized
38uaagguccuc cgagucgucu u 213921RNAArtificialsiRNA polynucleotide,
synthesized 39cgucaucaug caccagcucu u 214021RNAArtificialsiRNA
polynucleotide, synthesized 40augaccagga acaaccgacu u
214121RNAArtificialsiRNA polynucleotide, synthesized 41caugccguag
ucugugagcu u 214221RNAArtificialsiRNA polynucleotide, synthesized
42cucugacugg ucgauccucu u 214321RNAArtificialsiRNA polynucleotide,
synthesized 43gcggugguua aggauacagu u 214421RNAArtificialsiRNA
polynucleotide, synthesized 44uggcaugcau augcgguggu u 214521DNAHomo
sapiens 45cagaagatgg aggaagctgt a 214621DNAHomo sapiens
46caaggctaca ggtgcatcaa c 214721DNAHomo sapiens 47cagtagacga
caagaacgag g 214821DNAHomo sapiens 48cagacgacaa gtcggttgtt c
214921DNAHomo sapiens 49caagtcggtt gttcctggtc a 215021DNAHomo
sapiens 50cacatcaagc tcacagacta c 215121DNAHomo sapiens
51catcaagctc acagactacg g 215221DNAHomo sapiens 52caagctcaca
gactacggca t 215321DNAHomo sapiens 53cacagactac ggcatgtgca a
215421DNAHomo sapiens 54catgaacaca gaggactacc t
215521DNAHomo sapiens 55cattccagcc acagatcaca g 215621DNAHomo
sapiens 56cacagatcac agacgactac g 215721DNAHomo sapiens
57cagatcacag acgactacgg t 215821DNAHomo sapiens 58cagacgatga
ggatgccata a 215921DNAHomo sapiens 59cattattgct gtccaccgag g
216021RNAArtificialsiRNA polynucleotide, synthesized 60gaagauggag
gaagcuguau u 216121RNAArtificialsiRNA polynucleotide, synthesized
61aggcuacagg ugcaucaacu u 216221RNAArtificialsiRNA polynucleotide,
synthesized 62guagacgaca agaacgaggu u 216321RNAArtificialsiRNA
polynucleotide, synthesized 63gacgacaagu cgguuguucu u
216421RNAArtificialsiRNA polynucleotide, synthesized 64agucgguugu
uccuggucau u 216521RNAArtificialsiRNA polynucleotide, synthesized
65caucaagcuc acagacuacu u 216621RNAArtificialsiRNA polynucleotide,
synthesized 66ucaagcucac agacuacggu u 216721RNAArtificialsiRNA
polynucleotide, synthesized 67agcucacaga cuacggcauu u
216821RNAArtificialsiRNA polynucleotide, synthesized 68cagacuacgg
caugugcaau u 216921RNAArtificialsiRNA polynucleotide, synthesized
69ugaacacaga ggacuaccuu u 217021RNAArtificialsiRNA polynucleotide,
synthesized 70uuccagccac agaucacagu u 217121RNAArtificialsiRNA
polynucleotide, synthesized 71cagaucacag acgacuacgu u
217221RNAArtificialsiRNA polynucleotide, synthesized 72gaucacagac
gacuacgguu u 217321RNAArtificialsiRNA polynucleotide, synthesized
73gacgaugagg augccauaau u 217421RNAArtificialsiRNA polynucleotide,
synthesized 74uuauugcugu ccaccgaggu u 217521RNAArtificialsiRNA
polynucleotide, synthesized 75uacagcuucc uccaucuucu u
217621RNAArtificialsiRNA polynucleotide, synthesized 76guugaugcac
cuguagccuu u 217721RNAArtificialsiRNA polynucleotide, synthesized
77ccucguucuu gucgucuacu u 217821RNAArtificialsiRNA polynucleotide,
synthesized 78gaacaaccga cuugucgucu u 217921RNAArtificialsiRNA
polynucleotide, synthesized 79ugaccaggaa caaccgacuu u
218021RNAArtificialsiRNA polynucleotide, synthesized 80guagucugug
agcuugaugu u 218121RNAArtificialsiRNA polynucleotide, synthesized
81ccguagucug ugagcuugau u 218221RNAArtificialsiRNA polynucleotide,
synthesized 82augccguagu cugugagcuu u 218321RNAArtificialsiRNA
polynucleotide, synthesized 83uugcacaugc cguagucugu u
218421RNAArtificialsiRNA polynucleotide, synthesized 84agguaguccu
cuguguucau u 218521RNAArtificialsiRNA polynucleotide, synthesized
85cugugaucug uggcuggaau u 218621RNAArtificialsiRNA polynucleotide,
synthesized 86cguagucguc ugugaucugu u 218721RNAArtificialsiRNA
polynucleotide, synthesized 87accguagucg ucugugaucu u
218821RNAArtificialsiRNA polynucleotide, synthesized 88uuauggcauc
cucaucgucu u 218921RNAArtificialsiRNA polynucleotide, synthesized
89ccucggugga cagcaauaau u 219021DNAHomo sapiens 90gagctctgtg
aggaagtgag a 219121DNAHomo sapiens 91gaggaagtga gagacatgtg t
219221DNAHomo sapiens 92gaagtgagag acatgtgtcg t 219321DNAHomo
sapiens 93gagagacatg tgtcgtctgc a 219421DNAHomo sapiens
94gaagatggag gaagctgtac c 219521DNAHomo sapiens 95gacctgcagg
aagcatatgg a 219621DNAHomo sapiens 96gaggagacag atggaattgc t
219721DNAHomo sapiens 97gaggacctta agccagttat c 219821DNAHomo
sapiens 98gatgacgagg atattgactg g 219921DNAHomo sapiens
99gattacactc ctgcttccag a 2110021DNAHomo sapiens 100gacgacaagt
cggttgttcc t 2110121DNAHomo sapiens 101gacaagtcgg ttgttcctgg t
2110221DNAHomo sapiens 102gacctgatgt tccacatgca g 2110321DNAHomo
sapiens 103gatgttccac atgcagaggc a 2110421DNAHomo sapiens
104gactacggca tgtgcaagga a 2110521DNAHomo sapiens 105gacatgaaca
cagaggacta c 2110621DNAHomo sapiens 106gacttgctgg agaagaagca g
2110721DNAHomo sapiens 107gatcacagac gactacggtc t 2110821DNAHomo
sapiens 108gaggatcgac cagtcagagt t 2110921DNAHomo sapiens
109gatcgaccag tcagagttcg a 2111021RNAArtificialsiRNA
polynucleotide, synthesized 110gcucugugag gaagugagau u
2111121RNAArtificialsiRNA polynucleotide, synthesized 111ggaagugaga
gacauguguu u 2111221RNAArtificialsiRNA polynucleotide, synthesized
112agugagagac augugucguu u 2111321RNAArtificialsiRNA
polynucleotide, synthesized 113gagacaugug ucgucugcau u
2111421RNAArtificialsiRNA polynucleotide, synthesized 114agauggagga
agcuguaccu u 2111521RNAArtificialsiRNA polynucleotide, synthesized
115ccugcaggaa gcauauggau u 2111621RNAArtificialsiRNA
polynucleotide, synthesized 116ggagacagau ggaauugcuu u
2111721RNAArtificialsiRNA polynucleotide, synthesized 117ggaccuuaag
ccaguuaucu u 2111821RNAArtificialsiRNA polynucleotide, synthesized
118ugacgaggau auugacuggu u 2111921RNAArtificialsiRNA
polynucleotide, synthesized 119uuacacuccu gcuuccagau u
2112021RNAArtificialsiRNA polynucleotide, synthesized 120cgacaagucg
guuguuccuu u 2112121RNAArtificialsiRNA polynucleotide, synthesized
121caagucgguu guuccugguu u 2112221RNAArtificialsiRNA
polynucleotide, synthesized 122ccugauguuc cacaugcagu u
2112321RNAArtificialsiRNA polynucleotide, synthesized 123uguuccacau
gcagaggcau u 2112421RNAArtificialsiRNA polynucleotide, synthesized
124cuacggcaug ugcaaggaau u 2112521RNAArtificialsiRNA
polynucleotide, synthesized 125caugaacaca gaggacuacu u
2112621RNAArtificialsiRNA polynucleotide, synthesized 126cuugcuggag
aagaagcagu u 2112721RNAArtificialsiRNA polynucleotide, synthesized
127ucacagacga cuacggucuu u 2112821RNAArtificialsiRNA
polynucleotide, synthesized 128ggaucgacca gucagaguuu u
2112921RNAArtificialsiRNA polynucleotide, synthesized 129ucgaccaguc
agaguucgau u 2113021RNAArtificialsiRNA polynucleotide, synthesized
130ucucacuucc ucacagagcu u 2113121RNAArtificialsiRNA
polynucleotide, synthesized 131acacaugucu cucacuuccu u
2113221RNAArtificialsiRNA polynucleotide, synthesized 132acgacacaug
ucucucacuu u 2113321RNAArtificialsiRNA polynucleotide, synthesized
133ugcagacgac acaugucucu u 2113421RNAArtificialsiRNA
polynucleotide, synthesized 134gguacagcuu ccuccaucuu u
2113521RNAArtificialsiRNA polynucleotide, synthesized 135uccauaugcu
uccugcaggu u 2113621RNAArtificialsiRNA polynucleotide, synthesized
136agcaauucca ucugucuccu u 2113721RNAArtificialsiRNA
polynucleotide, synthesized 137gauaacuggc uuaagguccu u
2113821RNAArtificialsiRNA polynucleotide, synthesized 138ccagucaaua
uccucgucau u 2113921RNAArtificialsiRNA polynucleotide, synthesized
139ucuggaagca ggaguguaau u 2114021RNAArtificialsiRNA
polynucleotide, synthesized 140aggaacaacc gacuugucgu u
2114121RNAArtificialsiRNA polynucleotide, synthesized 141accaggaaca
accgacuugu u 2114221RNAArtificialsiRNA polynucleotide, synthesized
142cugcaugugg aacaucaggu u 2114321RNAArtificialsiRNA
polynucleotide, synthesized 143ugccucugca uguggaacau u
2114421RNAArtificialsiRNA polynucleotide, synthesized 144uuccuugcac
augccguagu u 2114521RNAArtificialsiRNA polynucleotide, synthesized
145guaguccucu guguucaugu u 2114621RNAArtificialsiRNA
polynucleotide, synthesized 146cugcuucuuc uccagcaagu u
2114721RNAArtificialsiRNA polynucleotide, synthesized 147agaccguagu
cgucugugau u 2114821RNAArtificialsiRNA polynucleotide, synthesized
148aacucugacu ggucgauccu u 2114921RNAArtificialsiRNA
polynucleotide, synthesized 149ucgaacucug acuggucgau u
2115021DNAHomo sapiens 150tagacgacaa gaacgaggac g 2115121DNAHomo
sapiens 151tacagacaga gaagcacgtg t 2115221DNAHomo sapiens
152tacactcctg cttccagacg a 2115321DNAHomo sapiens 153tattgctgtc
caccgaggag t 2115421DNAHomo sapiens 154taaccaccgc atatgcatgc c
2115521RNAArtificialsiRNA polynucleotide, synthesized 155gacgacaaga
acgaggacgu u 2115621RNAArtificialsiRNA polynucleotide, synthesized
156cagacagaga agcacguguu u 2115721RNAArtificialsiRNA
polynucleotide, synthesized 157cacuccugcu uccagacgau u
2115821RNAArtificialsiRNA polynucleotide, synthesized 158uugcugucca
ccgaggaguu u 2115921RNAArtificialsiRNA polynucleotide, synthesized
159accaccgcau augcaugccu u 2116021RNAArtificialsiRNA
polynucleotide, synthesized 160cguccucguu cuugucgucu u
2116121RNAArtificialsiRNA polynucleotide, synthesized 161acacgugcuu
cucugucugu u 2116221RNAArtificialsiRNA polynucleotide, synthesized
162ucgucuggaa gcaggagugu u 2116321RNAArtificialsiRNA
polynucleotide, synthesized 163acuccucggu ggacagcaau u
2116421RNAArtificialsiRNA polynucleotide, synthesized 164ggcaugcaua
ugcggugguu u 21
* * * * *