U.S. patent application number 16/237875 was filed with the patent office on 2019-10-31 for sugar chain-related gene and use thereof.
This patent application is currently assigned to STELIC INSTITUTE & CO.. The applicant listed for this patent is STELIC INSTITUTE & CO.. Invention is credited to Masato Fujii, Jun Koyama, Hiroyuki YONEYAMA.
Application Number | 20190330637 16/237875 |
Document ID | / |
Family ID | 40823968 |
Filed Date | 2019-10-31 |
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United States Patent
Application |
20190330637 |
Kind Code |
A1 |
YONEYAMA; Hiroyuki ; et
al. |
October 31, 2019 |
SUGAR CHAIN-RELATED GENE AND USE THEREOF
Abstract
As a result of dedicated studies, the present inventors
succeeded in discovering, for the first time, that fibrogenesis
could be suppressed at the physiological tissue level by inhibiting
sulfation at position 4 or 6 of GalNAc, which is a sugar that
constitutes sugar chains. Furthermore, the present inventors
conducted studies using various disease model animals, and as a
result, successfully demonstrated that inhibitors of sulfation at
position 4 or 6 of GalNAc had therapeutic effects on diseases
caused by tissue fibrogenesis (tissue fibrogenic disorders).
Inventors: |
YONEYAMA; Hiroyuki; (Tokyo,
JP) ; Koyama; Jun; (Tokyo, JP) ; Fujii;
Masato; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
STELIC INSTITUTE & CO. |
Tokyo |
|
JP |
|
|
Assignee: |
STELIC INSTITUTE & CO.
Tokyo
JP
|
Family ID: |
40823968 |
Appl. No.: |
16/237875 |
Filed: |
January 2, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15216231 |
Jul 21, 2016 |
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16237875 |
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14704333 |
May 5, 2015 |
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15216231 |
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14075919 |
Nov 8, 2013 |
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14704333 |
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12809969 |
Sep 1, 2010 |
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PCT/JP2008/004025 |
Dec 26, 2008 |
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14075919 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/08 20180101;
A61P 29/00 20180101; A61P 25/16 20180101; A61P 1/18 20180101; A61P
11/00 20180101; A61P 13/08 20180101; G01N 33/5023 20130101; A61P
3/10 20180101; A61P 19/02 20180101; A61P 19/04 20180101; A61P 21/00
20180101; A61P 9/06 20180101; A61P 37/02 20180101; A61P 7/00
20180101; C12N 9/13 20130101; C12N 2310/14 20130101; G01N
2333/91194 20130101; A61P 7/02 20180101; A61P 9/10 20180101; A61P
27/02 20180101; A61P 31/14 20180101; A61P 13/12 20180101; C12Q 1/48
20130101; A61P 3/00 20180101; A61K 31/7105 20130101; A61P 25/00
20180101; A61P 25/18 20180101; Y10T 436/143333 20150115; C12N
15/1137 20130101; A61P 1/16 20180101; A61P 25/28 20180101; A61P
25/02 20180101; A61P 17/00 20180101; A61P 9/00 20180101; A61P 1/00
20180101; A61P 9/04 20180101; A61P 13/10 20180101; A61P 43/00
20180101; A61P 15/10 20180101; A61P 31/20 20180101; C12Q 1/485
20130101; A61P 21/04 20180101; A61P 1/04 20180101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; G01N 33/50 20060101 G01N033/50; C12Q 1/48 20060101
C12Q001/48; A61K 31/7105 20060101 A61K031/7105; C12N 9/10 20060101
C12N009/10 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 27, 2007 |
JP |
2007-336518 |
Claims
1: A tissue fibrogenesis suppressing-agent, comprising: an
inhibitor of sulfation at position 4 or 6 of N-acetylgalactosamine,
wherein the inhibitor is an siRNA that suppresses expression of the
GalNAc4S-6ST gene.
2-11. (canceled)
12: A method of producing a pharmaceutical composition comprising:
(a) selecting, as a tissue fibrogenesis suppressing-agent, a
compound that inhibits sulfation at position 4 or 6 of
N-acetylgalactosamine that forms a sugar chain; and (b) combining
the compound with a pharmaceutically acceptable carrier.
13: A method of treating a fibrogenic disorder, comprising:
identifying a subject having a fibrogenic disorder of a tissue
selected from the group consisting of cardiac tissue,
gastrointestinal tissue, lung tissue, pancreatic tissue, kidney
tissue, ocular tissue, cranial nerve tissue, and skin tissue; and
administering to the subject a therapeutically effective amount of
an siRNA that suppresses expression of a gene encoding a
sulfotransferase that transfers a sulfate to position 4 or 6 of
N-acetylgalactosamine; wherein the siRNA suppresses expression of a
nucleotide sequence selected from the group consisting of a
GalNAc4S-6ST gene, a GalNAc4ST-1 gene, a GalNAc4ST-2 gene, a C4ST-1
gene, a C4ST-2 gene, a C4ST-3 gene, a C6ST-1 gene, a C6ST-2 gene,
and a D4ST gene.
14: The method of claim 13, wherein the subject has a fibrogenic
disorder of gastrointestinal tissues.
15: The method of claim 14, wherein the siRNA suppresses expression
of the GalNAc4S-6ST gene.
16: The method of claim 13, wherein the gene encoding a
sulfotransferase is a human gene.
17: The tissue fibrogenesis suppressing-agent of claim 1, wherein
the inhibitor is siRNA comprising the sequences of SEQ ID NO: 25
and SEQ ID NO: 26.
18: The tissue fibrogenesis suppressing-agent of claim 1,
comprising: the inhibitor; and a pharmaceutically acceptable
carrier.
19: The tissue fibrogenesis suppressing-agent of claim 17,
comprising: the inhibitor; and a pharmaceutically acceptable
carrier.
20: A siRNA comprising the sequences of SEQ ID NO: 25 and SEQ ID
NO: 26.
21: A composition, comprising: the siRNA of claim 20; and a
pharmaceutically acceptable carrier.
22: A composition, comprising: the siRNA of claim 20; and a
pharmaceutically acceptable carrier, wherein the composition has a
therapeutic effect on a tissue fibrogenic disorder.
23: A composition, comprising: the siRNA of claim 20; and a
pharmaceutically acceptable carrier, wherein the composition has a
therapeutic effect on an inflammatory bowel disease.
24: A composition, comprising: the siRNA of claim 20; and a
pharmaceutically acceptable carrier, wherein the composition has a
therapeutic effect on Crohn's disease.
25: A composition, comprising: the siRNA of claim 20; and a
pharmaceutically acceptable carrier, wherein the composition has a
therapeutic effect on ulcerative colitis.
Description
TECHNICAL FIELD
[0001] The present invention relates to inhibitors of fibrogenesis
at the physiological tissue level by inhibiting sugar chain-related
genes.
BACKGROUND ART
[0002] Intensive studies have been conducted on nucleic acids and
proteins, revealing many findings. However, these studies also
showed that there are only about 22,000 human genes and also that
post-translational modification of proteins plays an important role
in vivo. They also suggested limitations of conventional study
approaches. In recent years, the importance of sugar chains has
been rediscovered with the post-genome and post-proteomics trends
(the journal "Nature" extensively featured sugar chains in Vol. 446
published in Apr. 26, 2007 (Non-patent Documents 1 to 7). Sugar
chains have not been analyzed intensively because of the difficulty
to perform structural analysis, etc. Although they are assumed to
be involved in cancer, inflammation, immunity, viral infection,
etc., at present, little is known about their roles and such, and
therefore, elucidation is being awaited.
[0003] There are various known sugars (monosaccharides) that
constitute sugar chains. Such known sugars include, for example,
glucose (Glc), galactose (Gal), mannose (Man), glucuronic acid
(GlcUA), iduronic acid (IdoA), fucose (Fuc), glucosamine (GlcN),
N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc),
xylose (Xyl), and sialic acid (SA).
[0004] Furthermore, it has been reported that sugars constituting
sugar chains are subject to a variety of chemical modifications.
Such chemical modifications include, for example, methylation,
acetylation, formylation, myristoylation, amidation,
ubiquitination, acylation, phosphorylation, epimerization, and
sulfation. Examples of chemical modifications also include
sialylation, asialylation, fucosylation, glycosylation,
galactosylation, lactosylation, and mannosylation.
[0005] Sugars have a number of sites for such chemical
modifications. For example, it is known that GlcNAc can be
chemically-modified in any of carbons at positions 1 to 6. It is
reported that other sugars are also chemically-modified at various
sites. [0006] [Non-patent Document 1] Danica P. Galonic and David
Y. Gin, Nature 446: 1000-1007 (2007) [0007] [Non-patent Document 2]
Christopher J. Thibodeaux et al., Nature 446: 1008-1016 (2007)
[0008] [Non-patent Document 3] Gerald W. Hart et al., Nature 446:
1017-1022 (2007) [0009] [Non-patent Document 4] Ajit Varki, Nature
446: 1023-1029 (2007) [0010] [Non-patent Document 5] Joseph R.
Bishop et al., Nature 446: 1030-1037 (2007) [0011] [Non-patent
Document 6] Christopher N. et al., Nature 446: 1038-1045 (2007)
[0012] [Non-patent Document 7] Peter H. Seeberger and Daniel B.
Werz, Nature 446: 1046-1051 (2007)
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0013] The present invention is based on a new finding obtained
through studies on sugar chain-related genes. An objective of the
present invention is to provide novel uses of sugar chain-related
genes. Specifically, the objective is to provide agents that
suppress fibrogenesis at the physiological tissue level by
inhibiting the function of sugar chain-related genes, and methods
of screening for the agents.
Means for Solving the Problems
[0014] Sugar chains have been suggested to play a very important
role in vivo. However, little is known about the in vivo functions
of sugar chains.
[0015] As described above, several types of sugars constituting
sugar chains are known. Sugars are variously chemically-modified at
multiple sites, and such modifications are considered to assume
important physiological effects in vivo.
[0016] As described above, there are various types of sugars that
constitute sugar chains, many types of chemical modifications that
target sugars, and many chemical modification sites in the sugars.
This suggests that possible sugar chain variations are innumerable.
Thus, it is very difficult to determine a sugar chain structure
that plays a certain role, and it is also extremely difficult to
reveal the relationship between a pathological condition caused by
a disease and a specific action of a sugar chain.
[0017] The present inventors conducted dedicated studies, and as a
result succeeded for the first time in discovering that tissue
fibrogenesis can be suppressed at the physiological level by
inhibiting sulfation at position 4 or 6 of GalNAc, a sugar that
constitutes sugar chains. Furthermore, by studies using various
animal disease models, the present inventors demonstrated that
inhibitors of sulfation at position 4 or 6 of GalNAc produce
therapeutic effects against diseases caused by tissue fibrogenesis
(tissue fibrogenic disorders).
[0018] The present invention relates to agents that suppress
fibrogenesis at the physiological tissue level by inhibiting the
functions of sugar chain-related genes, and methods of screening
for the agents. Specifically, the present invention provides:
[0019] [1] a tissue fibrogenesis suppressing-agent, which comprises
as an ingredient an inhibitor of sulfation at position 4 or 6 of
N-acetylgalactosamine;
[0020] [2] the agent of [1], which has an effect of suppressing
fibrogenesis of a physiological tissue;
[0021] [3] the agent of [1] or [2], wherein the inhibitor has the
activity of inhibiting the function of a sulfotransferase that
transfers a sulfate to position 4 or 6 of
N-acetylgalactosamine;
[0022] [4] the agent of [3], wherein the inhibitor is an siRNA that
suppresses the expression of the sulfotransferase that transfers a
sulfate to position 4 or 6 of N-acetylgalactosamine;
[0023] [5] the agent of [1] or [2], wherein the inhibitor is a
desulfating enzyme that desulfates at position 4 or 6 of
N-acetylgalactosamine;
[0024] [6] the agent of any one of [1] to [5] for treating or
preventing a fibrogenic disorder;
[0025] [7] a method of screening for a tissue fibrogenesis
suppressing-agent, which comprises the step of selecting a compound
that inhibits sulfation at position 4 or 6 of N-acetylgalactosamine
that constitutes a sugar chain;
[0026] [8] a method of screening for a tissue fibrogenesis
suppressing-agent, which comprises the steps of:
[0027] contacting a test compound with N-acetylgalactosamine or a
sugar chain comprising N-acetylgalactosamine;
[0028] (b) determining the degree of sulfation at position 4 or 6
of N-acetylgalactosamine; and
[0029] (c) selecting a compound that reduces the degree of
sulfation as compared to when the test compound is not
contacted;
[0030] [9] a method of screening for a tissue fibrogenesis
suppressing-agent, which comprises the steps of:
[0031] (a) contacting a test compound with a sulfotransferase that
transfers a sulfate to position 4 or 6 of
N-acetylgalactosamine;
[0032] (b) determining the sulfotransferase activity of the enzyme;
and
[0033] (c) selecting a compound that reduces the activity as
compared to when the test compound is not contacted;
[0034] [10] a method of screening for a tissue fibrogenesis
suppressing-agent, which comprises the steps of:
[0035] (a) contacting a test compound with a cell expressing a gene
encoding a sulfotransferase that transfers a sulfate to position 4
or 6 of N-acetylgalactosamine;
[0036] (b) determining the expression level of the gene in the
cell; and
[0037] (c) selecting a compound that reduces the expression level
of the gene as compared to when the test compound is not
contacted;
[0038] [11] a method of screening for a tissue fibrogenesis
suppressing-agent, which comprises the steps of:
[0039] (a) contacting a test compound with a cell or cell extract
containing a DNA wherein a reporter gene is operably linked to the
transcriptional regulatory region of the gene encoding a
sulfotransferase that transfers a sulfate to position 4 or 6 of
N-acetylgalactosamine;
[0040] (b) determining the expression level of the reporter gene;
and
[0041] (c) selecting a compound that reduces the expression level
of the reporter gene as compared to when the test compound is not
contacted; and
[0042] [12] a method of producing a pharmaceutical composition for
treating or preventing a fibrogenic disorder, which comprises the
steps of:
[0043] (a) selecting a tissue fibrogenesis suppressing-agent from
test compounds by the method of any one of claims 7 to 11; and
[0044] (b) combining the agent with a pharmaceutically acceptable
carrier.
[0045] The present invention also provides:
[0046] [13] a method of suppressing tissue fibrogenesis, which
comprises the step of administering an inhibitor of sulfation at
position 4 or 6 of N-acetylgalactosamine to an individual;
[0047] [14] use of an inhibitor of sulfation at position 4 or 6 of
N-acetylgalactosamine in the manufacture of a tissue fibrogenesis
suppressing-agent; and
[0048] [15] an inhibitor of sulfation at position 4 or 6 of
N-acetylgalactosamine for use in suppressing tissue
fibrogenesis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] FIG. 1 shows the result of quantitative reverse
transcription PCR method for gene expressions of cardiomyopathy
model mice in which cardiomyopathy was induced by intratracheal
administration of DOX. The examined items were GalNAc4S-6ST as an
siRNA target gene, and .alpha.-SMA and type I collagen, both of
which are fibrogenesis markers. The graph indicates relative ratios
between a target gene and a house keeping gene (ribosome 18S).
*P<0.001 (t-test)
[0050] FIG. 2 shows the result analyzing the heart weight/body
weight ratio of cardiomyopathy model mice in which cardiomyopathy
was induced by intraperitoneal administration of Doxorubicin
hydrochloride (DOX; Kyowa Hakko). *P<0.05 (t-test)
[0051] FIG. 3 depicts photographs showing histological observations
indicating the suppressive effect on type I collagen deposition in
an siRNA-treated group of cardiomyopathy model mice in which
cardiomyopathy was induced by intratracheal administration of DOX.
The magnification is 100 fold.
[0052] FIG. 4 depicts photographs showing histological observations
indicating the suppressive effect on type III collagen deposition
in an siRNA-treated group of cardiomyopathy model mice in which
cardiomyopathy was induced by intratracheal administration of DOX.
The magnification is 100 fold.
[0053] FIG. 5 depicts photographs showing the suppressive effect on
fibroblast accumulation in an siRNA-treated group of cardiomyopathy
model mice in which cardiomyopathy was induced by intratracheal
administration of DOX. The magnification is 50 fold.
[0054] FIG. 6 depicts graphs showing clinical features of a mouse
intestinal fibrogenesis model, in which intestinal fibrogenesis was
induced by DSS. The administration of GalNAc4S-6ST siRNA resulted
in significant reduction of clinical score (left) and colon
shortening (right).
[0055] FIG. 7 depicts graphs showing the expression of
fibrogenesis-related genes in a mouse intestinal fibrogenesis
model. GalNAc4S-6ST, and .alpha.-SMA and type I collagen, both of
which are fibrogenesis markers, were assessed for the expression in
colonic tissue. The graphs indicate relative ratios between a
target gene and a house keeping gene (ribosome 18S). The enhanced
expression of type I collagen (left, bottom) and .alpha.-SMA
(right, bottom) is significantly suppressed due to the silencing
effect of GalNAc4S-6ST siRNA (upper).
[0056] FIG. 8 depicts photographs showing collagen deposition in a
mouse intestinal fibrogenesis model. Images of Masson-stained
colonic tissues (blue). The magnification is 100 fold. GalNAc4S-6ST
siRNA reduces the collagen deposition in tissues.
[0057] FIG. 9 depicts photographs showing fibroblast infiltration
in a mouse model for intestinal fibrogenesis. Images of stained
fibroblasts (brown) in colonic tissues. The magnification is 100
fold. GalNAc 4S-6ST siRNA suppresses the full-thickness
infiltration of fibroblasts.
[0058] FIG. 10 depicts photographs showing images of macrophage
infiltration in a mouse intestinal fibrogenesis model. Images of
stained macrophages (brown) in colonic tissues. Magnification:
100.times.. GalNAc4S-6ST siRNA suppresses the full-thickness
infiltration of macrophages.
[0059] FIG. 11 depicts a graph showing colon lengths in a mouse
intestinal fibrogenesis model. GalNAc4ST siRNA significantly
suppresses colon shortening.
[0060] FIG. 12 depicts photographs showing clinical images of a
mouse emphysema model, in which emphysema was induced by PPE.
C6ST-1 siRNA administration significantly suppresses the
histological disruption of the pulmonary alveolar wall.
Magnification: 100.times..
[0061] FIG. 13 depicts graphs showing the expression of
fibrogenesis-related genes in a mouse emphysema model.
GalNAc4S-6ST, and .alpha.-SMA and type I collagen, both of which
are fibrogenesis markers, were assessed for the expression in
pulmonary tissues. The graphs indicate relative ratios between a
target gene and a house keeping gene (ribosome 18S). The enhanced
expression of type I collagen (middle) and .alpha.-SMA (right) is
significantly suppressed due to the silencing effect of C6ST-1
siRNA (left).
[0062] FIG. 14 depicts photographs showing fibroblast infiltration
in a mouse emphysema model. Images of stained fibroblasts (brown)
in pulmonary tissues. Magnification: 200.times.. C6ST-1 siRNA
suppresses fibroblast infiltration into the pulmonary alveolar
interstitium.
[0063] FIG. 15 depicts photographs showing images of macrophage
infiltration in a mouse emphysema model. Images of stained
macrophages (brown) in pulmonary tissues. Magnification:
200.times.. C6ST-1 siRNA suppresses macrophage infiltration into
the pulmonary alveolar interstitium.
[0064] FIG. 16 depicts a graph showing static lung compliance (Cst)
in a mouse emphysema model. C6ST-1 siRNA significantly reduces
Cst.
[0065] FIG. 17 depicts a graph showing the right lung volume
(.mu.l) in a mouse emphysema model. C6ST-1 siRNA significantly
reduces lung volume.
[0066] FIG. 18 depicts graphs showing the expression of
fibrogenesis-related genes in a mouse emphysema model. .alpha.-SMA,
type I collagen, and TGF-.beta., which are fibrogenesis markers,
were assessed for the expression in pulmonary tissues. The graphs
indicate relative ratios between a target gene and a house keeping
gene (ribosome 18S). The enhanced expression of each fibrogenesis
marker is significantly suppressed due to the silencing effect of
GalNAcST siRNA.
[0067] FIG. 19 depicts a graph showing static lung compliance (Cst)
in a mouse emphysema model. GalNAcST siRNA significantly reduces
Cst.
[0068] FIG. 20 depicts a graph showing the right lung volume
(.mu.l) in a mouse emphysema model. GalNAcST siRNA significantly
reduces lung volume.
[0069] FIG. 21 depicts a graph showing obesity changes in a mouse
type 2 diabetes model. C4ST-1, C4ST-2, and C4ST-3 siRNAs tends to
suppress obesity. C4ST-2 and C4ST-3 siRNAs produce a significant
anti-obesity effect.
[0070] FIG. 22 depicts a graph showing insulin resistance in a
mouse type 2 diabetes model. C4ST-1, C4ST-2, and C4ST-3 siRNAs
significantly improve insulin resistance.
[0071] FIG. 23 depicts photographs showing gene expressions in
pancreatic tissues in a mouse type 2 diabetes model. C4ST-1,
C4ST-2, and C4ST-3 siRNAs suppress the expression of C4ST-1,
C4ST-2, and C4ST-3 genes in pancreatic tissues.
[0072] FIG. 24 depicts photographs showing APP deposition in
pancreatic islets in a mouse type 2 diabetes model. C4ST-2 siRNA
suppresses the deposition of APP (green) in islets. Cell nucleus
(red); magnification: 400.times..
[0073] FIG. 25 depicts photographs showing fibroblast infiltration
in pancreatic islets in a mouse type 2 diabetes model. C4ST-1,
C4ST-2, C4ST-3 siRNAs suppress fibroblast infiltration (brown) into
islets. Magnification: 200.times..
[0074] FIG. 26 depicts photographs showing macrophage infiltration
into pancreatic islets in a mouse type 2 diabetes model. C4ST-1,
C4ST-2, and C4ST-3 siRNAs suppress macrophage infiltration (brown)
into islets. Magnification: 200.times..
[0075] FIG. 27 depicts a graph showing insulin resistance in a
mouse type 2 diabetes model. GalNAcST siRNA administration
decreases the blood glucose level well after insulin loading.
Specifically, this shows the improvement of insulin resistance.
[0076] FIG. 28 depicts a graph and photographs showing the
accumulation of fibroblasts in the interstitium of kidney tissue in
a mouse diabetic nephropathy model. C4ST-1 siRNA significantly
suppresses the accumulation of fibroblasts (brown). Magnification:
200.times..
[0077] FIG. 29 depicts a graph and photographs showing the
accumulation of macrophages in the interstitium of kidney tissue in
a mouse model for diabetic nephropathy. C4ST-1 siRNA significantly
suppresses the accumulation of macrophages (brown). Magnification:
200.times..
[0078] FIG. 30 depicts photographs showing the accumulation of
.alpha.SMA-positive cells in the interstitium of kidney tissue in a
mouse diabetic nephropathy model. C4ST-1 siRNA significantly
suppresses the accumulation of .alpha.SMA-positive cells.
Magnification: 200.times..
[0079] FIG. 31 depicts graphs showing an anti-fibrogenic effect in
a mouse diabetic nephropathy model. GalNAc4S-6ST (G#1) siRNA
administration significantly suppresses the enhanced expression of
GalNAc4S-6ST (G#1), .alpha.SMA, and TGF.beta. in kidney tissues.
ARB; angiotensin receptor antagonist (Valsartan).
[0080] FIG. 32 depicts a graph and photographs showing fibroblast
accumulation in the interstitium of kidney tissue in a mouse
diabetic nephropathy model. GalNAc4S-6ST (G#1) siRNA significantly
suppresses the accumulation of fibroblasts (brown). Magnification:
200.times..
[0081] FIG. 33 depicts a graph and photographs showing macrophage
accumulation in the interstitium of kidney tissue in a mouse
diabetic nephropathy model. GalNAc4S-6ST (G#1) siRNA significantly
suppresses the accumulation of macrophages (brown). Magnification:
200.times..
[0082] FIG. 34 depicts a graph and photographs showing the
accumulation of type IV collagen in a mouse diabetic nephropathy
model. GalNAc4S-6ST (G#1) siRNA significantly suppresses the
thickening of glomerular basement membrane, which can be confirmed
by the positivity for type IV collagen (brown). Magnification:
400.times..
[0083] FIG. 35 depicts graphs showing the renal protective effect
in a mouse diabetic nephropathy model. GalNAc4S-6ST (G#1) siRNA
significantly suppresses the enhanced expression of angiotensinogen
and ACE in kidney tissues.
[0084] FIG. 36 depicts a graph showing the renal function
protective effect in a mouse diabetic nephropathy model.
GalNAc4S-6ST (G#1) siRNA significantly suppresses the increase of
serum creatinine, i.e., suppresses decline of renal function.
[0085] FIG. 37 depicts graphs showing gene expression in a mouse
model for diabetic nephropathy. GalNAcST siRNA significantly
suppresses the enhanced expression of GalNAc4ST-1, GalNAc4ST-2, and
GalNAc4S-6ST in kidney tissues.
[0086] FIG. 38 depicts graphs showing an anti-fibrogenic effect in
a mouse diabetic nephropathy model. GalNAcST siRNA significantly
suppresses the enhanced expression of CTGF, .alpha.SMA, type I
collagen, and ACE in kidney tissues.
[0087] FIG. 39 depicts a graph showing gene expression in mice with
drug-induced interstitial nephritis. GalNAc4S-6ST (G#1) siRNA
significantly suppresses the enhanced expression of GalNAc4S-6ST
(G#1) in kidney tissues.
[0088] FIG. 40 depicts photographs showing collagen deposition in
mice with drug-induced interstitial nephritis. GalNAc4S-6ST (G#1)
siRNA significantly decreases the deposition of type I collagen
(brown) in renal interstitium. Magnification: 200.times..
[0089] FIG. 41 depicts graphs showing an anti-fibrogenic effect in
a mouse UUO fibrogenesis model. C6ST siRNA significantly suppresses
the enhanced expression of C6ST-2 (G#10), TGF.beta., .alpha.SMA,
type I collagen, and CTGF in kidney tissues.
[0090] FIG. 42 depicts a graph and photographs showing fibroblast
accumulation in the interstitium in a mouse UUO fibrogenesis model.
C6ST siRNA significantly suppresses fibroblast accumulation (brown)
in juxtaglomerular and interstitial area. Magnification:
200.times..
[0091] FIG. 43 depicts a graph and photographs showing macrophage
accumulation in the interstitium in a mouse UUO fibrogenesis model.
C6ST siRNA significantly suppresses the accumulation of macrophages
(brown) in juxtaglomerular and interstitial area. Magnification:
200.times..
[0092] FIG. 44 depicts a graph and photographs showing collagen
deposition in a mouse UUO fibrogenesis model. C6ST siRNA
significantly suppresses the thickening of glomerular basement
membrane, which can be confirmed by the positivity of type IV
collagen (brown). Magnification: 400.times..
[0093] FIG. 45 depicts photographs showing the activation of
fibroblasts accumulated in tissues in a mouse UUO fibrogenesis
model. C6ST siRNA significantly suppresses the accumulation of
.alpha.SMA-positive cells (brown) in juxtaglomerular and
interstitial area. Magnification: 400.times..
[0094] FIG. 46 depicts photographs showing the accumulation of
ACE-producing cells in the interstitium in a mouse UUO fibrogenesis
model. C6ST siRNA significantly suppresses the accumulation of
ACE-producing cells (brown) in juxtaglomerular and interstitial
area. Magnification: 400.times..
[0095] FIG. 47 depicts photographs showing the tissue accumulation
of type IV collagen in a mouse diabetic retinopathy model.
GalNac4S-6ST (G#1) siRNA significantly suppresses the accumulation
of type IV collagen. Magnification: 200.times.. GCL, ganglion cell
layer; INL, inner nuclear layer; ONL, external granular layer.
[0096] FIG. 48 depicts photographs showing the tissue accumulation
of CSPG in a mouse diabetic retinopathy model. GalNac4S-6ST (G#1)
siRNA significantly suppresses CSPG accumulation (brown). In
particular, the suppressing effect is prominent in GCL, ONL, and
pigment epithelial cell layer. Magnification: 200.times..
[0097] FIG. 49 depicts photographs showing GFAP-positive cells in a
mouse diabetic retinopathy model. GalNac4S-6ST (G#1) siRNA
significantly increases GFAP-positive cells (brown) in the region
from INL to GCL. Magnification: 200.times..
[0098] FIG. 50 depicts a graph showing the number of gangliocytes
in a mouse diabetic retinopathy model. Counts show the number of
gangliocytes in GGL. GalNac4S-6ST (G#1) siRNA significantly
suppresses the reduction in gangliocyte number.
[0099] FIG. 51 depicts graphs showing the optic nerve regeneration
effect in a mouse diabetic retinopathy model. GalNac4S-6ST (G#1)
siRNA significantly increases the expression of GS in ocular
tissues.
[0100] FIG. 52 depicts a graph showing gene expression in a mouse
fatty liver injury model. The enhanced expression of GalNAc4S-6ST
in liver tissues, and significant suppression of the expression by
GalNAcST siRNA are shown.
[0101] FIG. 53 depicts graphs showing the expression of
fibrogenesis-related genes in a mouse fatty liver injury model. The
enhanced expression of type I collagen and OSAM in liver tissues,
and significant suppression of the expression by GalNAcST siRNA are
shown.
[0102] FIG. 54 depicts photographs showing the infiltration of
fibrogenic cells in a mouse fatty liver injury model. GalNAcST
siRNA suppresses the bridge-like accumulation of fibroblasts
(brown) in liver tissues. Magnification: 100.times..
[0103] FIG. 55 depicts a graph showing clinical scores for
fibrogenesis in a mouse fatty liver injury model. GalNAcST siRNA
significantly suppresses the increase in the fibrogenesis score in
liver tissues.
[0104] FIG. 56 depicts photographs showing macrophage infiltration
in a mouse fatty liver injury model. GalNAcST siRNA significantly
suppresses the accumulation of macrophages (brown) in liver
tissues. Magnification: 100.times..
[0105] FIG. 57 depicts graphs showing the expression of lipid
metabolism-related genes in a mouse fatty liver injury model.
GalNAcST siRNA significantly suppresses the increased expression of
ChREBP and ACC2 in liver tissues.
[0106] FIG. 58 depicts photographs showing the infiltration of
fibrogenic cells in a mouse fatty liver injury model. C4ST-1,
C4ST-2, and C4ST-3 siRNAs suppress the bridge-like accumulation of
fibroblasts (brown) in liver tissues. Magnification:
100.times..
[0107] FIG. 59 depicts a graph showing clinical scores for
fibrogenesis in a mouse fatty liver injury model. C4ST-1, C4ST-2,
and C4ST-3 siRNAs significantly suppress the increase in the
fibrogenesis score in liver tissues.
[0108] FIG. 60 depicts a graph showing clinical hepatic disorder in
a mouse fatty liver injury model. C4ST-1, C4ST-2, and C4ST-3 siRNAs
suppress the increase in ALT, which is an indicator of hepatocyte
disorder.
[0109] FIG. 61 depicts photographs showing the infiltration of
fibroblasts in a mouse hepatic fibrosis model. C6ST siRNA
significantly suppresses the accumulation of fibroblasts (brown) in
liver tissues. Magnification: 50.times..
[0110] FIG. 62 depicts a graph showing clinical scores for
fibrogenesis in a mouse hepatic fibrosis model. C6ST siRNA
significantly suppresses the increase in the clinical fibrogenesis
score in liver tissues.
[0111] FIG. 63 depicts graphs showing the anti-fibrogenic effect in
a mouse hepatic fibrosis model. C6ST siRNA significantly suppresses
the expression of .alpha.SMA, type I collagen, CTGF, and
TGF.beta..
[0112] FIG. 64 depicts graphs showing the anti-fibrogenic effect in
a mouse Parkinson's disease model. GalNAc4S-6ST siRNA significantly
suppresses the expression of GalNAc4S-6ST, TGF.beta., type I
collagen, and .alpha.SMA in brain tissues.
[0113] FIG. 65 depicts photographs showing the accumulation of
fibroblasts in a mouse Parkinson's disease model. GalNAc4S-6ST
siRNA drastically decreases the accumulation of fibroblasts (brown)
in brain tissues. Magnification: 200.times..
[0114] FIG. 66 depicts graphs showing the nerve protective effect
in a mouse Parkinson's disease model. GalNAc4S-6ST siRNA
significantly enhances the expression of GDNF and Nurr1.
[0115] FIG. 67 depicts photographs showing the dopamine neuron
regeneration effect in a mouse Parkinson's disease model.
GalNAc4S-6ST siRNA suppresses the degeneration of TH-positive
dopamine neurons (green). Magnification: 200.times..
[0116] FIG. 68 depicts photographs showing the dopamine neuron
regeneration effect in a mouse Parkinson's disease model. GalNAc4ST
siRNA suppresses the degeneration of TH-positive dopamine neurons
(green). Magnification: 200.times..
[0117] FIG. 69 depicts photographs showing the CSPG-reducing effect
of C4-sulfatase in a mouse type 2 diabetic retinopathy model. The
photographs show images of stained CSPG (CS56) (brown, arrow) in
the retina of type 2 diabetic retinopathy model mice.
[0118] FIG. 70 depicts photographs showing the
angiogenesis-suppressing effect of C4-sulfatase in a mouse type 2
diabetic retinopathy model. The photographs show images of stained
vascular endothelial cells (CD31) (brown, arrow) in the retina of
type 2 diabetic retinopathy model mice.
[0119] FIG. 71 depicts photographs showing the collagen
augmentation-suppressing effect of C4-sulfatase in a mouse type 2
diabetic retinopathy model. The photographs show images of stained
type IV collagen (brown, arrow) in the retina of type 2 diabetic
retinopathy model mice.
[0120] FIG. 72 depicts photographs showing the suppressive effect
of C4-sulfatase on fibroblast accumulation in the liver of a mouse
type 2 diabetes model. The magnification is 50 or 100 fold.
[0121] FIG. 73 depicts photographs showing the macrophage
infiltration-suppressing effect in the liver of a mouse type 2
diabetes model. The magnification is 50 or 100 fold.
[0122] FIG. 74 depicts graphs showing the result of serum
biochemical tests (AST, ALT, and TG) in a mouse type 2 diabetes
model. In the graphs: unt, untreated group; nor, control group;
C4sul, C4-sulfatase.
[0123] FIG. 75 depicts photographs showing the localization of CSPG
in brain tissues. The photographs show the result of analyzing the
dynamics of CSPG expression in brain tissues using an
enzyme-antibody immunostaining method. Assay was carried out using
CS-56 (Seikagaku Co.) for primary antibody and Mouse Stain Kit for
staining.
[0124] FIG. 76 depicts photographs showing the localization of
dopaminergic neurons in brain tissues. The photographs show an
analysis result obtained by a fluorescence immunostaining
method.
[0125] FIG. 77 depicts a graph showing the result of TNF-.alpha.
gene expression analysis, which was obtained by Real-time PCR
method. The graph shows the result of real-time PCR for the
expression of TGF-3 as a fibrosis marker, and TNF-.alpha. as an
indicator for inflammation associated with macrophage
infiltration.
[0126] FIG. 78 depicts a graph showing the result of Nurr1 gene
expression analysis by real-time PCR method. The graph shows the
result for Nurr1 gene expression in brain tissues, which was
obtained using Cyber premix kit (Takara Bio) and Real-time PCR
thermal cycler DICE (Takara Bio). The graph indicates relative
ratios between Nurr1 and a house keeping gene (.beta.-actin).
MODE FOR CARRYING OUT THE INVENTION
[0127] The present invention will be specifically described
below.
[0128] The present invention relates to tissue fibrogenesis
inhibitors based on the mechanism of inhibiting sulfation at
position 4 or 6 of N-acetylgalactosamine, a sugar constituting
sugar chains.
[0129] Specifically, the present invention provides tissue
fibrogenesis inhibitors comprising as an ingredient an inhibitor of
sulfation at position 4 or 6 of N-acetylgalactosamine (herein
sometimes also referred to as "inhibitors of the present invention"
or simply as "inhibitors").
[0130] Herein, "N-acetylgalactosamine (GalNAc)" refers to the
N-acetylated form of galactosamine, which is a hexosamine.
[0131] Furthermore, it is known that N-acetylgalactosamine can be
chemically-modified at positions 1 to 6.
[0132] The present invention is characterized by inhibiting
sulfation at position 4 or 6 of N-acetylgalactosamine.
[0133] Specifically, the sites where the sulfation is inhibited by
the inhibitors of the present invention are indicated by arrow in
the following formula of GalNAc.
##STR00001##
[0134] The sites where the sulfation is inhibited by the inhibitors
of the present invention are positions 4 or 6 of GalNAc. In the
present invention, the sulfation of GalNAc may be inhibited at both
positions 4 and 6. In the present invention, preferred GalNAc is a
sugar in chondroitin sulfate proteoglycan (CSPG).
[0135] Herein, the inhibition of sulfation refers to inhibition of
transfer of a sulfate group to position 4 or 6 in GalNAc,
elimination of a sulfate group from a site where GalNAc has been
already sulfated, or substitution of a sulfate group with other
chemically-modified group.
[0136] The agents for suppressing tissue fibrogenesis of the
present invention (herein sometimes referred to as "agents of the
present invention") preferably have an in vivo
fibrogenesis-suppressing effect.
[0137] Tissues where fibrogenesis is suppressed by the agents of
the present invention are not particularly limited. Such tissues
include, for example, cardiac tissues, gastrointestinal tissues,
lung tissues, pancreatic tissues, kidney tissues, ocular tissues,
liver tissues, cranial nerve tissues, and skin tissues.
[0138] Herein, "fibrogenesis" may be referred to as "fibrosis".
Alternatively, "fibrogenesis" may be synonymous with other phrases
such as "fibrogenic lesion in tissues", "fibrogenic tissue
alteration", and "neofibrogenesis".
[0139] The inhibitors of the present invention are not particularly
limited as long as they are substances having the activity of
inhibiting the sulfation at position 4 or 6 of
N-acetylgalactosamine.
[0140] A preferred embodiment of inhibitors of the present
invention includes, for example, substances having the activity of
inhibiting the function of sulfotransferase that transfers a
sulfate to position 4 or 6 of N-acetylgalactosamine. Preferred
embodiments of the above-described substances include, for example,
compounds (nucleic acids) selected from the group consisting
of:
[0141] (a) antisense nucleic acids against transcripts of the genes
encoding the sulfotransferases that transfer a sulfate to position
4 or 6 of N-acetylgalactosamine, or portions thereof;
[0142] (b) nucleic acids with the ribozyme activity of specifically
cleaving transcripts of genes encoding the sulfotransferases that
transfer a sulfate to position 4 or 6 of N-acetylgalactosamine;
and
[0143] (c) nucleic acids with the activity of using RNAi effect to
inhibit the expression of genes encoding the sulfotransferases that
transfer a sulfate to position 4 or 6 of N-acetylgalactosamine
(siRNAs that suppress the expression of sulfotransferase
genes).
[0144] The "substances with the activity of inhibiting sulfation"
also include, for example, compounds selected from the group
consisting of:
[0145] (a) antibodies that bind to sulfotransferases that transfer
a sulfate to position 4 or 6 of N-acetylgalactosamine;
[0146] (b) sulfotransferase variants for sulfotransferases that
transfer a sulfate to position 4 or 6 of N-acetylgalactosamine;
and
[0147] (c) low-molecular-weight compounds that bind to
sulfotransferases that transfer a sulfate to position 4 or 6 of
N-acetylgalactosamine.
[0148] Another embodiment of inhibitors of the present invention
includes, for example, substances having the activity of
desulfating a sulfate group at position 4 or 6 of
N-acetylgalactosamine. Such substances include, for example,
enzymes that desulfate the sulfate group (desulfating enzymes) at
position 4 or 6 of N-acetylgalactosamine.
[0149] The "desulfating" sulfate group at position 4 or 6 of
N-acetylgalactosamine means that a sulfate group at position 4 or 6
is eliminated from N-acetylgalactosamine.
[0150] Such desulfating enzymes include, for example,
chondroitin-4-sulfatase (C4-sulfatase) and
chondroitin-6-sulfatase.
[0151] Sulfotransferases of the present invention are not
particularly limited as long as enzymes have an activity of
transferring a sulfate to position 4 or 6 of GalNAc, but include,
for example:
[0152] 1) GalNAc4ST-1: N-acetylgalactosamine
4-sulfotransferase-1
[0153] Alias CHST8:Carbohydrate (N-acetylgalactosamine 4-O)
sulfotransferase 8
[0154] 2) GalNAc4ST-2
[0155] Alias CHST9: Carbohydrate (N-acetylgalactosamine 4-O)
sulfotransferase 9
[0156] 3) C4ST-1: chondroitin-4-O-sulfotransferase-1
[0157] Alias CHST11: Carbohydrate (chondroitin 4) sulfotransferase
11
[0158] 4) C4ST-2
[0159] Alias CHST12
[0160] 5) C4ST-3
[0161] Alias CHST13
[0162] 6) C6ST-1: chondroitin-6-O-sulfotransferase-1
[0163] Alias CHST3: Carbohydrate (chondroitin 6) sulfotransferase
3
[0164] 7) GalNAc4S-6ST: N-acetylgalactosamine 4-sulfate 6-0
sulfotransferase
[0165] 8) D4ST-1:dermatan 4 sulfotransferase 1
[0166] 9) C6ST-2: chondroitin-6-O-sulfotransferase-2
[0167] Alias CHST7: Carbohydrate (chondroitin 6) sulfotransferase
7
[0168] Further, on a genomic DNA level, such groups of enzymes
sharing features do not necessarily correspond to single genes. For
example, both chondroitin-4-sulfatase and chondroitin-6-sulfatase
can be retrieved from the public gene database GenBank as sequences
referred to by multiple accession numbers (for example, GenBank
accession Nos: NT_039500 (a portion thereof is shown under
accession No: CAAA01098429 (SEQ ID NO: 1)), NT_078575, NT_039353,
NW_001030904, NW_001030811, NW_001030796, and NW_000349).
[0169] Specifically, below are examples of sulfotransferases of the
present invention with accession numbers in the public gene
database GenBank, nucleotide sequences, and amino acid
sequences:
[0170] GalNAc4ST-1 (Accession number NM_175140; nucleotide
sequence: SEQ ID NO: 2; amino acid sequence, SEQ ID NO: 3)
[0171] GalNAc4ST-2 (Accession number NM_199055; nucleotide
sequence: SEQ ID NO: 4; amino acid sequence, SEQ ID NO: 5)
[0172] C4ST-1 (Accession number NM_021439; nucleotide sequence: SEQ
ID NO: 6, amino acid sequence, SEQ ID NO: 7)
[0173] C4ST-2 (Accession number NM_021528; nucleotide sequence: SEQ
ID NO: 8; amino acid sequence, SEQ ID NO: 9)
[0174] C4ST-3 (Accession number XM_355798; nucleotide sequence: SEQ
ID NO: 10; amino acid sequence, SEQ ID NO: 11)
[0175] D4ST (Accession number NM_028117; nucleotide sequence: SEQ
ID NO: 12; amino acid sequence, SEQ ID NO: 13)
[0176] C6ST-1 (Accession number NM_016803; nucleotide sequence: SEQ
ID NO: 14; amino acid sequence, SEQ ID NO: 15)
[0177] C6ST-2 (Accession number AB046929; nucleotide sequence: SEQ
ID NO: 16; amino acid sequence, SEQ ID NO: 17)
[0178] GalNAc4S-6ST (Accession number NM_015892; nucleotide
sequence: SEQ ID NO: 18; amino acid sequence, SEQ ID NO: 19)
[0179] In addition to the proteins listed above, the proteins of
the present invention include those exhibiting high homology
(typically 70% or higher, preferably 80% or higher, more preferably
90% or higher, and most preferably 95% or higher) to sequences
shown in the Sequence Listing and having a function of the proteins
listed above (for example, the function of binding to intracellular
components). The proteins listed above are, for example, proteins
comprising an amino acid sequence with an addition, deletion,
substitution, or insertion of one or more amino acids in any of the
amino acid sequences of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, and 17,
in which the number of altered amino acids is typically 30 amino
acids or less, preferably ten amino acids or less, more preferably
five amino acids or less, and most preferably three amino acids or
less.
[0180] The above-described genes of the present invention include,
for example, endogenous genes of other organisms which correspond
to DNAs comprising any of the nucleotide sequences of SEQ ID NOs:
2, 4, 6, 8, 10, 12, 14, and 16 (homologues to the human genes
described above, or the like).
[0181] Each of the endogenous DNAs of other organisms which
correspond to DNAs comprising any of the nucleotide sequences of
SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16 are generally highly
homologous to a DNA of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,
and 16. High homology means 50% or higher homology, preferably 70%
or higher homology, more preferably 80% or higher homology, and
still more preferably 90% or higher homology (for example, 95% or
higher, or 96%, 97%, 98%, or 99% or higher). Homology can be
determined using the mBLAST algorithm (Altschul, et al. Proc. Natl.
Acad. Sci. USA, 1990, 87, 2264-8; Karlin and Altschul, Proc. Natl.
Acad. Sci. USA, 1993, 90, 5873-7). When the DNAs have been isolated
from the body, each of them may hybridize under stringent
conditions to a DNA of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, and 16.
Herein, stringent conditions include, for example, "2.times.SSC,
0.1% SDS, 50.degree. C.", "2.times.SSC, 0.1% SDS, 42.degree. C.",
and "1.times.SSC, 0.1% SDS, 37.degree. C."; more stringent
conditions include "2.times.SSC, 0.1% SDS, 65.degree. C.",
"0.5.times.SSC, 0.1% SDS, 42.degree. C.", and "0.2.times.SSC, 0.1%
SDS, 65.degree. C.".
[0182] Those skilled in the art can appropriately obtain proteins
functionally equivalent to the above-described proteins from the
above-described highly homologous proteins by using methods for
assaying the activity of desulfating or inhibiting the sulfation at
position 4 or 6 of N-acetylgalactosamine.
[0183] Further, based on the nucleotide sequences of the
above-described genes, those skilled in the art can appropriately
obtain endogenous genes of other organisms that correspond to the
above-described genes. In the present invention, the
above-described proteins and genes in non-human organisms, which
correspond to the above-described proteins and genes, or the
above-described proteins and genes that are functionally equivalent
to the above-described proteins and genes, may simply be referred
to using the above-described names.
[0184] The proteins of the present invention can be prepared not
only as natural proteins but also as recombinant proteins using
genetic recombination techniques. The natural proteins can be
prepared by, for example, methods of subjecting cell extracts
(tissue extracts) that may express the above-described proteins to
affinity chromatography using antibodies against the
above-described proteins. On the other hand, the recombinant
proteins can be prepared, for example, by culturing cells
transformed with DNAs encoding the proteins described above. The
above-described proteins of the present invention can be suitably
used, for example, in the screening methods described herein
below.
[0185] In the present invention, "nucleic acids" refer to both RNAs
and DNAs. Chemically synthesized nucleic acid analogs, such as
so-called "PNAs" (peptide nucleic acids), are also included in the
nucleic acids of the present invention. PNAs are nucleic acids in
which the fundamental backbone structure of nucleic acids, the
pentose-phosphate backbone, is replaced by a polyamide backbone
with glycine units. PNAs have a three-dimensional structure quite
similar to that of nucleic acids.
[0186] Methods for inhibiting the expression of specific endogenous
genes using antisense technology are well known to those skilled in
the art. There are a number of causes for the action of antisense
nucleic acids in inhibiting target gene expression, including:
[0187] inhibition of transcription initiation by triplex
formation;
[0188] transcription inhibition by hybrid formation at a site with
a local open loop structure generated by an RNA polymerase;
[0189] transcription inhibition by hybrid formation with the RNA
being synthesized;
[0190] splicing inhibition by hybrid formation at an intron-exon
junction;
[0191] splicing inhibition by hybrid formation at the site of
spliceosome formation;
[0192] inhibition of transport from the nucleus to the cytoplasm by
hybrid formation with mRNA;
[0193] splicing inhibition by hybrid formation at the capping site
or poly(A) addition site;
[0194] inhibition of translation initiation by hybrid formation at
the translation initiation factor binding site;
[0195] inhibition of translation by hybrid formation at the
ribosome binding site adjacent to the start codon;
[0196] inhibition of peptide chain elongation by hybrid formation
in the translational region of mRNA or at the polysome binding site
of mRNA; and
[0197] inhibition of gene expression by hybrid formation at the
protein-nucleic acid interaction sites. Thus, antisense nucleic
acids inhibit the expression of target genes by inhibiting various
processes, such as transcription, splicing, and translation
(Hirashima and Inoue, Shin Seikagaku Jikken Koza 2 (New Courses in
Experimental Biochemistry 2), Kakusan (Nucleic Acids) IV: "Idenshi
no Fukusei to Hatsugen (Gene replication and expression)", Ed. The
Japanese Biochemical Society, Tokyo Kagakudojin, 1993, pp.
319-347).
[0198] The antisense nucleic acids used in the present invention
may inhibit the expression and/or function of genes encoding any of
the sulfotransferases described above, based on any of the actions
described above.
[0199] In one embodiment, antisense sequences designed to be
complementary to an untranslated region adjacent to the 5' end of
an mRNA for a gene encoding an above-described sulfotransferase may
be effective for inhibiting translation of the gene. Sequences
complementary to a coding region or 3'-untranslated region can also
be used. Thus, the antisense nucleic acids to be used in the
present invention include not only nucleic acids comprising
sequences antisense to the coding regions, but also nucleic acids
comprising sequences antisense to untranslated regions of genes
encoding the above-described sulfotransferases. Such antisense
nucleic acids to be used are linked downstream of adequate
promoters and are preferably linked with transcription termination
signals on the 3' side. Nucleic acids thus prepared can be
introduced into desired animals (cells) using known methods. The
sequences of the antisense nucleic acids are preferably
complementary to a gene or portion thereof encoding a
sulfotransferase that is endogenous to the animals (cells) to be
transformed with them. However, the sequences need not be perfectly
complementary, as long as the antisense nucleic acids can
effectively suppress expression of a gene. The transcribed RNAs
preferably have 90% or higher, and most preferably 95% or higher
complementarity to target gene transcripts. To effectively inhibit
target gene expression using antisense nucleic acids, the antisense
nucleic acids are preferably at least 15 nucleotides long, and less
than 25 nucleotides long. However, the lengths of the antisense
nucleic acids of the present invention are not limited to the
lengths mentioned above, and they may be 100 nucleotides or more,
or 500 nucleotides or more.
[0200] The antisense nucleic acids of the preset invention are not
particularly limited, and can be prepared, for example, based on
the nucleotide sequence of C4ST-1 (GenBank Accession No: NM_021439;
SEQ ID NO: 6), C4ST-2 (GenBank Accession No: NM_021528; SEQ ID NO:
8), C4ST-3 (GenBank Accession No: XM_355798; SEQ ID NO: 10), or
such.
[0201] Expression of the above-mentioned genes encoding
sulfotransferases can also be inhibited using ribozymes or
ribozyme-encoding DNAs. Ribozymes refer to RNA molecules with
catalytic activity. There are various ribozymes with different
activities. Among others, studies that focused on ribozymes
functioning as RNA-cleaving enzymes have enabled the design of
ribozymes that cleave RNAs in a site-specific manner. Some
ribozymes have 400 or more nucleotides, such as group I intron type
ribozymes and M1 RNA, which is comprised by RNase P, but others,
called hammerhead and hairpin ribozymes, have a catalytic domain of
about 40 nucleotides (Koizumi, M. and Otsuka E., Tanpakushitsu
Kakusan Koso (Protein, Nucleic Acid, and Enzyme), 1990, 35,
2191).
[0202] For example, the autocatalytic domain of a hammerhead
ribozyme cleaves the sequence G13U14C15 at the 3' side of C15. Base
pairing between U14 and A9 has been shown to be essential for this
activity, and the sequence can be cleaved when C15 is substituted
with A15 or U15 (Koizumi, M. et al., FEBS Lett., 1988, 228, 228).
Restriction enzyme-like RNA-cleaving ribozymes that recognize the
sequence UC, UU, or UA in target RNAs can be created by designing
their substrate-binding sites to be complementary to an RNA
sequence adjacent to a target site (Koizumi, M. et al., FEBS Lett.,
1988, 239, 285; Koizumi, M, and Otsuka, E., Tanpakushitsu Kakusan
Koso (Protein, Nucleic Acid, and Enzyme), 1990, 35, 2191; and
Koizumi, M. et al., Nucl Acids Res., 1989, 17, 7059).
[0203] In addition, hairpin ribozymes are also useful for the
purposes of the present invention. Such ribozymes are found in, for
example, the minus strand of satellite RNAs of tobacco ring spot
viruses (Buzayan, J. M., Nature, 1986, 323, 349). It has been shown
that target-specific RNA-cleaving ribozymes can also be created
from hairpin ribozymes (Kikuchi, Y. and Sasaki, N., Nucl Acids
Res., 1991, 19, 6751; and Kikuchi, Y. Kagaku to Seibutsu (Chemistry
and Biology), 1992, 30, 112). Thus, the expression of the
above-described genes encoding sulfotransferases can be inhibited
by using ribozymes to specifically cleave the gene transcripts.
[0204] The expression of endogenous genes can also be suppressed by
RNA interference (hereinafter abbreviated as "RNAi"), using
double-stranded RNAs comprising a sequence the same as or similar
to a target gene sequence.
[0205] A great many disease-related genes have been rapidly
identified since the entire human nucleotide sequence was revealed
upon the recent completion of the genome project, and currently
specific gene-targeted therapies and drugs are being actively
developed. Of these, the application to gene therapy of small
interfering RNAs (siRNAs), which produce the effect of specific
post-transcriptional suppression, has been drawing attention.
[0206] RNAi is a phenomenon discovered by Fire et al. in 1998
(Fire, A., Nature (1998) 391: 806-811), where double strand RNA
strongly suppresses expression of homologous target genes. RNAi has
been drawing attention recently as a method applicable in gene
therapy, because it is simpler than conventional gene transfer
methods using vectors or such, and its target specificity is high.
Furthermore, in mammalian cells, RNAi can be induced using short
dsRNAs (siRNAs) and has many advantages: compared to knockout mice,
RNAi has a stable effect, is easy to experiment with, has a low
cost, and so on.
[0207] Nucleic acids with inhibitory activity based on the RNAi
effect are generally referred to as siRNAs or shRNAs. RNAi is a
phenomenon in which, when cells or such are introduced with short
double-stranded RNAs (hereinafter abbreviated as "dsRNAs")
comprising sense RNAs that have sequences homologous to the mRNAs
of a target gene, and antisense RNAs that comprise sequences
homologous a sequence complementary thereto, the dsRNAs bind
specifically and selectively to the target gene mRNAs, induce their
disruption, and cleave the target gene, thereby effectively
inhibiting (suppressing) target gene expression. For example, when
dsRNAs are introduced into cells, the expression of genes with
sequences homologous to the RNAs is suppressed (the genes are
knocked down). As described above, RNAi can suppress the expression
of target genes, and is thus drawing attention as a method
applicable to gene therapy, or as a simple gene knockout method
replacing conventional methods of gene disruption, which are based
on complicated and inefficient homologous recombination.
[0208] In the present invention, the RNAs to be used in RNAi are
not necessarily perfectly identical to the genes or portions
thereof that encode an above-described sulfotransferase; however,
the RNAs are preferably perfectly homologous to the genes or
portions thereof. Furthermore, the terminal portion may include an
overhang of about two bases.
[0209] The targets of the siRNAs to be designed are not
particularly limited, as long as they are genes encoding an
above-described sulfotransferase. Any region of the gene can be a
candidate for a target.
[0210] For example, siRNAs may be prepared based on a nucleotide
sequence of C4ST-1 gene (SEQ ID NO: 6), C4ST-2 gene (SEQ ID NO: 8),
C4ST-3 gene (SEQ ID NO: 10), and such. More specifically, partial
regions of such sequences may be used as candidates for the
targets. For example, siRNAs may be prepared based on portions of
the nucleotide sequences of C4ST-1 gene (SEQ ID NO: 20), C4ST-2
gene (SEQ ID NO: 21), C4ST-3 gene (SEQ ID NO: 22), C6ST-1 gene (SEQ
ID NO: 23), C6ST-2 gene (SEQ ID NO: 24), or such. More
specifically, examples of the siRNAs also include those targeted to
the DNA sequences (SEQ ID NOs: 25, 26, 35 to 50, 55 to 65, and 82
to 88) specifically shown herein.
[0211] The siRNAs can be introduced into cells by adopting methods
of introducing cells with plasmid DNAs linked with siRNAs
synthesized in vitro or methods that comprise annealing two RNA
strands.
[0212] The two RNA molecules described above may be closed at one
end or, for example, may be siRNAs with hairpin structures
(shRNAs). shRNAs refer to short hairpin RNAs, which are RNA
molecules with a stem-loop structure, since a portion of the single
strand constitutes a strand complementary to another portion. Thus,
molecules capable of forming an intramolecular RNA duplex structure
are also included in the siRNAs of the present invention.
[0213] In a preferred embodiment of the present invention, the
siRNAs of the present invention also include, for example,
double-stranded RNAs with additions or deletions of one or a few
RNAs in an siRNA which targets a specific DNA sequence (SEQ ID NOs:
25, 26, 35 to 50, 55 to 65, and 82 to 88) shown herein and which
can suppress the expression of C4ST-1, C4ST-2, C4ST-3, or such via
RNAi effect, as long as the double-stranded RNAs have the function
of suppressing the expression of a gene encoding an above-described
sulfotransferase.
[0214] The RNAs used in RNAi (siRNAs) do not need to be perfectly
identical (homologous) to the genes encoding the above proteins or
portions thereof; however, the RNAs are preferably perfectly
identical (homologous).
[0215] Some details of the RNAi mechanism still remain unclear, but
it is understood that an enzyme called "DICER" (a member of the
RNase III nuclease family) is contacted with a double-stranded RNA
and degrades it in to small fragments, called "small interfering
RNAs" or "siRNAs". The double-stranded RNAs of the present
invention that have RNAi effect include such double-stranded RNAs
prior to being degraded by DICER. Specifically, since even long
RNAs that have no RNAi effect when intact can be degraded into
siRNAs which have RNAi effect in cells, the length of the
double-stranded RNAs of the present invention is not particularly
limited.
[0216] For example, long double-stranded RNAs covering the
full-length or near full-length mRNA of a gene encoding an
above-described sulfotransferase can be pre-digested, for example,
by DICER, and then the degradation products can be used as agents
of the present invention. These degradation products are expected
to contain double-stranded RNA (siRNA) molecules with an RNAi
effect. With this method, it is not necessary to specifically
select the mRNA regions expected to have RNAi effect. In other
words, it is not necessary to accurately determine regions with
RNAi effect in the mRNAs of the genes described above.
[0217] The above-described "double-stranded RNAs capable of
suppression via RNAi effect" can be suitably prepared by those
skilled in the art based on nucleotide sequences of the
above-described sulfotransferases, which are targeted by the
double-stranded RNAs. For example, the double-stranded RNAs of the
present invention can be prepared based on the nucleotide sequence
of SEQ ID NO: 25. In other words, it is within the range of
ordinary experimentation for those skilled in the art to select an
arbitrary consecutive RNA region in an mRNA that is a transcript of
the nucleotide sequence of SEQ ID NO: 25, and prepare
double-stranded RNA corresponding to the region. Those skilled in
the art can also use known methods to properly select siRNA
sequences with stronger RNAi effect from the mRNA sequence, which
is the transcript of the nucleotide sequence of SEQ ID NO: 25. When
one of the strands is already identified, those skilled in the art
can readily determine the nucleotide sequence of the other strand
(complementary strand). Those skilled in the art can appropriately
prepare siRNAs using a commercially available nucleic acid
synthesizer. Alternatively, general custom synthesis services may
be used to synthesize desired RNAs.
[0218] The siRNAs of the present invention are not necessarily
single pairs of double-stranded RNAs directed to target sequences,
but may be mixtures of multiple double-stranded RNAs directed to
regions that cover the target sequence. Herein, those skilled in
the art can appropriately prepare the siRNAs as nucleic acid
mixtures matched to a target sequence by using a commercially
available nucleic acid synthesizer or DICER enzyme. Meanwhile,
general custom synthesis services may be used to synthesize desired
RNAs. The siRNAs of the present invention include so-called "siRNA
cocktails".
[0219] All nucleotides in the siRNAs of the present invention do
not necessarily need to be ribonucleotides (RNAs). Specifically,
one or more of the ribonucleotides constituting the siRNAs of the
present invention may be replaced with corresponding
deoxyribonucleotides. The term "corresponding" means that although
the sugar moieties are structurally differently, the nucleotide
residues (adenine, guanine, cytosine, or thymine (uracil)) are the
same. For example, deoxyribonucleotides corresponding to
ribonucleotides with adenine refer to deoxyribonucleotides with
adenine. The term "or more" described above is not particularly
limited, but preferably refers to a small number of about two to
five ribonucleotides.
[0220] Furthermore, DNAs (vectors) capable of expressing the RNAs
of the present invention are also included in the preferred
embodiments of compounds capable of suppressing the expression of
the genes encoding the above-described proteins of the present
invention. The DNAs (vectors) capable of expressing the
double-stranded RNAs of the present invention are, for example,
DNAs structured such that a DNA encoding one strand of a
double-stranded RNA and a DNA encoding the other strand of the
double-stranded RNA are linked with promoters so that each DNA can
be expressed. The above DNAs of the present invention can be
appropriately prepared by those skilled in the art using standard
genetic engineering techniques. More specifically, the expression
vectors of the present invention can be prepared by adequately
inserting DNAs encoding the RNAs of the present invention into
various known expression vectors.
[0221] Furthermore, the expression-inhibiting substances of the
present invention also include compounds that inhibit the
expression of the above-described sulfotransferases by binding to
an expression regulatory region of a gene encoding the
above-described sulfotransferases (for example, a promoter region).
Such compounds can be obtained, for example, using a fragment of a
promoter DNA of the gene encoding an above-described
sulfotransferase to perform screening methods using as an indicator
the activity of binding to the DNA fragment. Those skilled in the
art can appropriately determine whether compounds of interest
inhibit the expression of the above-described genes encoding
sulfotransferases by using known methods, for example, reporter
assays and such.
[0222] Furthermore, DNAs (vectors) capable of expressing the
above-described RNAs of the present invention are also included in
preferred embodiments of the compounds capable of inhibiting the
expression of a gene encoding an above-described sulfotransferase
of the present invention. For example, DNAs (vectors) capable of
expressing the above-described double-stranded RNAs of the present
invention are structured such that a DNA encoding one strand of a
double-stranded RNA and a DNA encoding the other strand of the
double-stranded RNA are linked to promoters so that both can be
expressed. Those skilled in the art can appropriately prepare the
above-described DNAs of the present invention using standard
genetic engineering techniques. More specifically, the expression
vectors of the present invention can be prepared by appropriately
inserting DNAs encoding the RNAs of the present invention into
various known expression vectors.
[0223] Preferred embodiments of the above-described vector of the
present invention include vectors expressing RNAs (siRNAs) that can
suppress the expression of C4ST-1, C4ST-2, C4ST-3, or the like by
the RNAi effect.
[0224] Antibodies that bind to the above-described
sulfotransferases can be prepared by methods known to those skilled
in the art. Polyclonal antibodies can be obtained, for example, by
the following procedure: small animals such as rabbits are
immunized with an above-described natural protein or a recombinant
protein expressed in microorganisms as a fusion protein with GST,
or a partial peptide thereof. Sera are obtained from these animals
and purified by, for example, ammonium sulfate precipitation,
Protein A or G column, DEAE ion exchange chromatography, affinity
column coupled with the sulfotransferase described above, synthetic
peptide, or such, to prepare antibodies. Monoclonal antibodies can
be obtained by the following procedure: small animals such as mice
are immunized with an above-described sulfotransferase, or a
partial peptide thereof. Spleens are removed from the mice and
crushed to isolate cells. The cells are fused with mouse myeloma
cells using a reagent such as polyethylene glycol. Clones producing
antibodies that bind to an above-described sulfotransferase are
selected from among the resulting fused cells (hybridomas). The
obtained hybridomas are then transplanted into the peritoneal
cavities of mice, and ascites collected. The obtained monoclonal
antibodies can be purified by, for example, ammonium sulfate
precipitation, Protein A or G columns, DEAE ion exchange
chromatography, affinity columns coupled with an above-described
sulfotransferase, synthetic peptides, or such.
[0225] The antibodies of the present invention are not particularly
limited as long as they bind to an above-described sulfotransferase
of the present invention. The antibodies of the present invention
may be human antibodies, humanized antibodies created by gene
recombination, fragments or modified products of such antibodies,
in addition to the polyclonal and monoclonal antibodies described
above.
[0226] The proteins of the present invention used as sensitizing
antigens to prepare antibodies are not limited in terms of the
animal species from which the proteins are derived. However, the
proteins are preferably derived from mammals, for example, mice and
humans. Human-derived proteins are particularly preferred. The
human-derived proteins can be appropriately obtained by those
skilled in the art using the gene or amino acid sequences disclosed
herein.
[0227] In the present invention, the proteins to be used as
sensitizing antigens may be whole proteins or partial peptides
thereof. Such partial peptides of the proteins include, for
example, amino-terminal (N) fragments and carboxyl-terminal (C)
fragments of the proteins. Herein, "antibodies" refer to antibodies
that react with a full-length protein or fragment thereof.
[0228] In addition to immunizing nonhuman animals with antigens to
obtain the above hybridomas, human lymphocytes, for example, EB
virus-infected human lymphocytes, can be sensitized in vitro with
the proteins or with cells expressing the proteins, or with lysates
thereof, and the sensitized lymphocytes can be fused with
human-derived myeloma cells with the ability to divide permanently,
for example, U266, to obtain hybridomas that produce desired human
antibodies with binding activity to the proteins.
[0229] It is expected that antibodies against the above-described
sulfotransferases of the present invention exhibit the effect of
inhibiting protein expression or function by binding to the
proteins. When using the prepared antibodies for human
administration (antibody therapy), the antibodies are preferably
human or humanized antibodies in order to reduce
immunogenicity.
[0230] Furthermore, in the present invention, low-molecular-weight
substances (low-molecular-weight compounds) that bind to the
above-described sulfotransferases are also included in the
substances capable of inhibiting the function of the
above-described sulfotransferases. Such low-molecular-weight
substances may be natural or artificial compounds. In general, the
compounds can be produced or obtained by methods known to those
skilled in the art. The compounds of the present invention can also
be obtained by the screening methods described below.
[0231] In addition, the substances of the present invention capable
of inhibiting the expression or function of the above-described
sulfotransferases include dominant-negative mutants
(dominant-negative proteins) for the above-described
sulfotransferases. The "dominant-negative protein mutants for the
above sulfotransferases that transfer a sulfate to position 4 or 6
of N-acetylgalactosamine" refer to proteins with the function of
reducing or abolishing the activity of endogenous wild-type
proteins by expressing the genes encoding the sulfotransferases
that transfer a sulfate to position 4 or 6 of
N-acetylgalactosamine.
[0232] The inhibitors of the present invention that inhibit
sulfation at position 4 or 6 of N-acetylgalactosamine have
therapeutic or preventive effect for fibrogenic disorders.
Therefore, in a preferred embodiment, the agents of the present
invention are therapeutic or preventive agents for fibrogenic
disorders.
[0233] Herein, "therapeutic or preventive" does not necessarily
refer to a perfect therapeutic or preventive effect on organs or
tissues with tissue fibrogenesis, and may refer to a partial
effect.
[0234] The tissue fibrogenesis-suppressing agents of the present
invention have the activity of suppressing fibrogenesis through
inhibiting the sulfation at position 4 or 6 of
N-acetylgalactosamine, which is a cause of fibrogenesis. Thus,
preferred embodiments of the present invention provide, for
example, therapeutic or preventive agents for tissue fibrogenic
disorders which comprise as an active ingredient a tissue
fibrogenesis-suppressing agent of the present invention.
[0235] The "therapeutic agents for tissue fibrogenic disorders" of
the present invention can also be referred to as "improving agents
for tissue fibrogenic disorders", "anti-tissue fibrogenesis
agents", or the like. Meanwhile, the agents of the present
invention can also be referred to as "pharmaceutical agents",
"pharmaceutical compositions", "therapeutic medicines", or the
like.
[0236] The "treatments" of the present invention also comprise
preventive effects that can suppress the onset of fibrogenesis in
advance. The treatments are not limited to those producing a
complete therapeutic effect on fibrogenic organs (tissues), and the
effects may be partial.
[0237] The agents of the present invention can be combined with
physiologically acceptable carriers, excipients, diluents and such,
and orally or parenterally administered as pharmaceutical
compositions. Oral agents may be in the form of granules, powders,
tablets, capsules, solutions, emulsions, suspensions, or the like.
The dosage forms of parenteral agents can be selected from
injections, infusions, external preparations, inhalants
(nebulizers), suppositories, and the like. Injections include
preparations for subcutaneous, intramuscular, intraperitoneal,
intracranial, and intranasal injections, and the like. The external
preparations include nasal preparations, ointments, and such.
Techniques for formulating the above-described dosage forms that
contain the agents of the present invention as primary ingredients
are known.
[0238] For example, tablets for oral administration can be produced
by compressing and shaping the agents of the present invention in
combination with excipients, disintegrants, binders, lubricants,
and the like. Excipients commonly used include lactose, starch,
mannitol, and the like. Commonly used disintegrants include calcium
carbonate, carboxymethylcellulose calcium, and the like. Binders
include gum arabic, carboxymethylcellulose, and
polyvinylpyrrolidone. Known lubricants include talc, magnesium
stearate, and such.
[0239] Known coatings can be applied to tablets comprising the
agents of the present invention to prepare enteric coated
formulations or for masking. Ethylcellulose, polyoxyethylene
glycol, or such can be used as a coating agent.
[0240] Meanwhile, injections can be prepared by dissolving the
agents of the present invention, which are chief ingredients,
together with an appropriate dispersing agent, or dissolving or
dispersing the agents in a dispersion medium. Both water-based and
oil-based injections can be prepared, depending on the selection of
dispersion medium. When preparing water-based injections, the
dispersing agent is distilled water, physiological saline, Ringer's
solution or such. For oil-based injections, any of the various
vegetable oils, propylene glycols, or such is used as a dispersing
agent. If required, a preservative such as paraben may be added at
this time. Known isotonizing agents such as sodium chloride and
glucose can also be added to the injections. In addition, soothing
agents such as benzalkonium chloride and procaine hydrochloride can
be added.
[0241] Alternatively, the agents of the present invention can be
formed into solid, liquid, or semi-solid compositions to prepare
external preparations. Such solid or liquid compositions can be
prepared as the same compositions as described above and then used
as external preparations. The semi-solid compositions can be
prepared using an appropriate solvent, to which a thickener is
added if required. Water, ethyl alcohol, polyethylene glycol, and
the like can be used as the solvent. Commonly used thickeners are
bentonite, polyvinyl alcohol, acrylic acid, methacrylic acid,
polyvinylpyrrolidone, and the like. Preservatives such as
benzalkonium chloride can be added to these compositions.
Alternatively, suppositories can be prepared by combining the
compositions with carriers, like oil bases such as cacao butter, or
aqueous gel bases such as cellulose derivatives.
[0242] When the agents of the present invention are used as gene
therapy agents, the agents may be directly administered by
injection, or vectors carrying the nucleic acid may be
administered. Such vectors include adenovirus vectors,
adeno-associated virus vectors, herpes virus vectors, vaccinia
virus vectors, retroviral vectors, and lentivirus vectors. These
vectors allow efficient administration.
[0243] Alternatively, the agents of the present invention can be
encapsulated into phospholipid vesicles such as liposomes, and then
the vesicles can be administered. Vesicles carrying siRNAs or
shRNAs are introduced into given cells by lipofection. The
resulting cells are then systemically administered, for example,
intravenously or intra-arterially. The cells can also be locally
administered into tissues or such with fibrogenesis. siRNAs exhibit
a quite superior and specific post-transcriptional suppression
effect in vitro; however, in vivo they are rapidly degraded due to
serum nuclease activity, and thus, their time was limited. There is
therefore demand for the development of optimized and effective
delivery systems. As one example, Ochiya et al. have reported that
atelocollagen, a bio-affinity material, is a highly suitable siRNA
carrier because it has the activity of protecting nucleic acids
from nucleases in the body when mixed with the nucleic acids to
form a complex (Ochiya, T. et al., Nat. Med., 1999, 5, 707-710;
Ochiya, T. et al., Curr. Gene Ther., 2001, 1, 31-52); however, the
methods for introducing drugs of the present invention are not
limited thereto.
[0244] The agents of the present invention are administered to
mammals including humans at required (effective) doses, within a
dose range considered to be safe. Ultimately, the doses of the
agents of the present invention can be appropriately determined by
medical practitioners or veterinarians after considering the dosage
form and administration method, and the patient's age and weight,
symptoms, and the like. For example, adenoviruses are administered
once a day at a dose of about 106 to 1013 viruses every one to
eight weeks, although the doses vary depending on the age, sex,
symptoms, administration route, administration frequency, and
dosage form.
[0245] Commercially available gene transfer kits (for example:
AdenoExpress.TM., Clontech) may be used to introduce siRNAs or
shRNAs into target tissues or organs.
[0246] Diseases to be treated or prevented by the agents of the
present invention is not particularly limited as long as they are
caused by tissue fibrogenesis, but preferably include, cardiac
disorders, intestinal diseases, liver diseases, hepatic disorders,
kidney disorders, cranial nerve diseases, eye disorders, pancreas
disorders.
[0247] The "diseases caused by fibrogenesis" in the present
invention is not particularly limited, and specifically include,
for example, elastosis, scleroderma, chronic peritonitis, and
retroperitoneal fibrosis in integumentary and epithelial tissues
such as skin;
[0248] polymyositis, dermatomyositis, polyarteritis nodosa, soft
tissue fibrosis, chronic rheumatoid arthritis, palmar fibromatosis,
tendinitis, tenovaginitis, Achilles tendinitis, mycetoma pedis, and
such in supportive tissues such as connective tissues and
muscles;
[0249] myelofibrosis, hypersplenism, vasculitis, bradyarrhythmia,
arteriosclerosis, obstructive thrombotic angiitis, nodular
fibrosis, angina pectoris, dilated congestive cardiomyopathy, heart
failure, restrictive cardiomyopathy, diffuse nonobstructive
cardiomyopathy, obstructive cardiomyopathy, cor pulmonale, mitral
stenosis, aortic valve stenosis, chronic pericarditis, endocardial
fibrosis, endomyocardial fibrosis, and such in blood tissues and
vascular system such as bone marrow and heart;
[0250] chronic pancreatitis, Crohn's disease, ulcerative colitis,
alcoholic hepatitis, chronic hepatitis B, chronic hepatitis C,
Wilson's disease, cirrhosis, viral hepatitis, Gaucher's disease,
glycogen storage disease, alpha 1-antitrypsin deficiency,
hemochromatosis, tyrosinemia, levulosemia, galactosemia, Zellweger
syndrome, congenital hepatic fibrosis, portal hypertension, hepatic
granulomatosis, Budd-Chiari syndrome, primary sclerosing
cholangitis, fatty liver, nonalcoholic hepatitis, hepatic fibrosis,
congenital hepatic fibrosis, alcoholic cirrhosis, viral cirrhosis,
parasitic cirrhosis, toxic cirrhosis, trophopathic cirrhosis,
congestive cirrhosis, hepatic sclerosis, Charcot's cirrhosis,
Todd's cirrhosis, secondary biliary cirrhosis, unilobar cirrhosis,
cirrhosis resulting from chronic nonsuppurative destructive
cholangitis, obstructive cirrhosis, cholangiolitic cirrhosis,
biliary cirrhosis, atrophic cirrhosis, postnecrotic cirrhosis,
posthepatitic cirrhosis, nodular cirrhosis of the liver, mixed
cirrhosis, micronodular cirrhosis, compensatory cirrhosis,
decompensated cirrhosis, macronodular cirrhosis, septal cirrhosis,
cryptogenic cirrhosis, periportal cirrhosis, portal cirrhosis,
primary biliary cirrhosis, and such in the gastrointestinal system
such as liver;
[0251] coccidioidomycosis, blastomycosis, allergic bronchopulmonary
aspergillosis, Goodpasture's syndrome, pulmonary fibrosis
associated with adult respiratory distress syndrome, chronic
obstructive pulmonary disease, pulmonary atelectasis, pneumonia,
chalicosis, asbestosis, hypersensitivity pneumonitis, lymphocytic
interstitial pneumonia, Langerhans-cell granulomatosis, cystic
fibrosis, pustular fibrosis, pulmonary fibrosis, idiopathic
pulmonary fibrosis, fibrosing pulmonary alveolitis, interstitial
fibrosis, diffuse pulmonary fibrosis, chronic interstitial
pneumonia, bronchiectasis, bronchiolar fibrosis, peribronchial
fibrosis, pleural fibrosis, and such in the respiratory system such
as lung;
[0252] male hypogonadism, myotonic dystrophy, fibrosis such as
associated with Peyronie's disease, chronic tubulointerstitial
nephritis, autosomal recessive cystic kidney, myeloma kidney,
hydronephrosis, rapidly progressive glomerulonephritis, nephrotoxic
diseases, xanthogranulomatous pyelonephritis, sickle cell
nephropathy, nephrogenic diabetes insipidus, autosomal dominant
polycystic kidney disease, chronic glomerular nephritis, IgA
nephropathy, renal sclerosis, focal glomerulosclerosis, membranous
nephritis, membranoproliferative glomerulonephritis, chronic
pyelonephritis, renal amyloidosis, polycystic kidney disease,
retroperitoneal fibrosis, pathology in the kidney associated with a
connective tissue disease such as lupus nephritis, diabetic
nephropathy, chronic prostatitis, and urocystitis associated with
schistosomiasisin the urogenital system such as kidney;
[0253] fibrotic breast disease, mammary fibroadenoma, and such;
[0254] congenital torticollis, ankylosing spondylitis, spinal cord
disorders such as neurofibroma and neurological dysfunction after
spinal cord injury, and cranial nerve diseases such as Parkinson's
disease and Alzheimer's disease in the nervous system such as
spinal cord;
[0255] retrolental fibrosis and proliferative retinopathy in the
eyeball; and
[0256] sarcoidosis that develops systemic involvement, fibrosis and
systemic scleroderma associated with systemic lupus erythematosus,
polymyositis, dermatomyositis, and such. However, in the present
invention, the "disease caused by fibrogenesis" is not limited
thereto, and includes diseases caused by fibrosis in each body
tissue such as skin and organs.
[0257] The present invention also relates to methods of screening
for agents for suppressing tissue fibrogenesis (herein sometimes
referred to as "methods of the present invention"), which use as an
indicator the degree of sulfation at position 4 or 6 of
N-acetylgalactosamine.
[0258] A preferred embodiment of methods of the present invention
is the methods comprising the step of selecting compounds that
inhibit the sulfation at position 4 or 6 of N-acetylgalactosamine
that constitute sugar chains.
[0259] Using the screening methods of the present invention, tissue
fibrogenesis-suppressing agents or candidate compounds for agents
for treating or preventing fibrogenic disorders can be efficiently
acquired.
[0260] Preferred embodiments of the screening methods of the
present invention are methods of screening for tissue
fibrogenesis-suppressing agents that comprise the steps of (a) to
(c):
[0261] (a) contacting test compounds with N-acetylgalactosamines or
sugar chain having N-acetylgalactosamines;
[0262] (b) measuring a degree of sulfation at position 4 or 6 of
N-acetylgalactosamines; and
[0263] (c) selecting compounds that reduce the degree of sulfation
as compared with those without contacting with the test
compounds.
[0264] Embodiments of the screening methods of the present
invention are exemplified below. In the embodiments described
below, N-acetylgalactosamines, sulfotransferases, desulfatases, or
such to be used include those derived from humans, mice, rats, and
others, but are not particularly limited thereto.
[0265] The test compounds to be used in the embodiments described
below are not particularly limited, but include, for example,
single compounds, such as natural compounds, organic compounds,
inorganic compounds, proteins, and peptides, as well as compound
libraries, expression products of gene libraries, cell extracts,
cell culture supernatants, products of fermenting microorganisms,
extracts of marine organisms, and plant extracts.
[0266] In the methods of the present invention, the "contact" with
test compounds is typically achieved by mixing the test compounds
with N-acetylgalactosamines, sulfotransferases, or desulfatases,
but the "contact" is not limited to this methods. For example, the
"contact" can also be achieved by contacting test compounds with
cells expressing these proteins or portions thereof.
[0267] In the embodiments described below, the "cells" include
those derived from humans, mice, rats, and such, but are not
limited thereto. Cells of microorganisms, such as Escherichia coli
and yeasts, which are transformed to express the proteins used in
each embodiment, can also be used. For example, the "cells that
express genes encoding sulfotransferases that transfer a sulfate to
position 4 or 6 of N-acetylgalactosamine" include cells that
express endogenous genes encoding sulfotransferases that transfer a
sulfate to position 4 or 6 of N-acetylgalactosamine, or cells that
express introduced foreign genes encoding sulfotransferases that
transfer a sulfate to position 4 or 6 of N-acetylgalactosamine.
Such cells that express foreign genes encoding sulfotransferases
that transfer a sulfate to position 4 or 6 of N-acetylgalactosamine
can typically be prepared by introducing host cells with expression
vectors carrying a gene encoding a sulfotransferase that transfers
a sulfate to position 4 or 6 of N-acetylgalactosamine as an insert.
The expression vectors can be prepared using standard genetic
engineering techniques.
[0268] In addition, the degree of sulfation in the methods of the
present invention can be determined by methods known to those
skilled in the art. For example, the degree of sulfation can be
determined by measuring the amount of label using a labeled
compound, antibody, or such that binds to a sulfated structure at
position 4 or 6 of N-acetylgalactosamine, or a portion thereof.
Alternatively, the degree of sulfation can be detected by
chromatography, mass spectrometry, or the like.
[0269] Those skilled in the art can appropriately evaluate the
degree of sulfation at position 4 or 6 of N-acetylgalactosamine,
for example, by the following known methods:
[0270] (1) method based on quantitative dye binding using a
labeling dye (1-9-dimethylene blue) (Nature. 1998 Feb. 26; 391
(6670): 908-11)
[0271] (2) method based on photo-affinity labeling using
[.sup.32P]3',5'-ABP (Mandon, E. C., Milla, M. E., Kempner, E., and
Hirschberg, C. B. (1994) Proc. Natl. Acad. Sci. USA, 91,
10707-10711)
[0272] (3) method based on photo-affinity labeling using
3'-[.sup.32P]-.beta. methyleneb PAPS (Ozeran, J. D., Wesley, J.,
and Schwarz, N. B. (1996) Biochemistry, 35, 3695-3703)
[0273] (4) method using anion exchange resins (method for isolating
sulfated glycoproteins) (Vol. 16, No. 2 (19860430) pp. 69-72,
Kitasato University, ISSN: 03855449)
[0274] (5) colorimetric staining of sGAG with Alcian blue (Anal
Biochem. 1998 Feb. 15; 256(2): 229-37)
[0275] Furthermore, those skilled in the art can readily evaluate
by the above-listed methods or the like whether a substance is an
inhibitor of sulfation at position 4 or 6 of N-acetylgalactosamine
of the present invention.
[0276] Another embodiment of screening methods of the present
invention includes the methods comprising the step of selecting
substances (compounds) which reduce the activity of
sulfotransferases that transfer a sulfate to position 4 or 6 of
N-acetylgalactosamine.
[0277] The above-described methods of the present invention
comprise, for example, the steps of:
[0278] (a) contacting a test compound with sulfotransferases that
transfer a sulfate to position 4 or 6 of N-acetylgalactosamine;
[0279] (b) measuring the sulfotransferase activity of the enzymes;
and
[0280] (c) selecting a compound that reduces the activity as
compared to when the test compound is not contacted.
[0281] In the above-described methods, first, a test compound is
contacted with a sulfotransferase that transfers a sulfate to
position 4 or 6 of N-acetylgalactosamine.
[0282] Then, the sulfotransferase activity of the enzyme is
measured. Next, a compound that reduces the activity as compared to
without contact with the test compound is selected. Such a compound
that reduces the activity can be used as a fibrogenesis inhibitor
or a therapeutic agent for fibrogenic disorders.
[0283] Methods that enable evaluation (determination) of whether a
test compound has the above-described sulfotransferase activity
include, for example, the methods described below.
[0284] Various test compounds are mixed during a set period of
culture of cells or cell lines that promote the sulfation at
position 4 or 6 of N-acetylgalactosamine, and the degree of
sulfation before and after the culture can be easily determined by,
for example, using an antibody that recognizes sulfation at
position 4 (clone: LY111, 2H6) or an antibody that recognizes
sulfation at position 6 (clone: MC21C, M0225, and CS-56) (all from
Seikagaku Co.). Fluorescence values may be compared between before
and after the culture by using fluorescently labeled antibodies.
Alternatively, the same detection method can be conducted using
2-B-6 or 3-B-3 antibodies before and after culture. Compounds that
suppress an increase in the sulfation after cell culture (an
increase in the fluorescence value for LY111 or MC21C), or
compounds that promote the progression of desulfation after cell
culture (an increase in the fluorescence value for 2-B-6 or 3-B-3)
can be selected as a desired candidate compound in the methods of
the present invention.
[0285] As a further option, cell lines that constitutively express
sulfotransferase genes such as C4ST-1 and C6ST-1 can be prepared by
introducing the genes into CHO cells, L cells, or such by
well-known methods. The use of such cell lines that constitutively
add sulfate groups allows a more clear determination of candidates
for therapeutic compounds.
[0286] Another preferred embodiment of the screening methods for a
tissue fibrogenesis inhibitor of present invention includes methods
comprising the step of selecting compounds that reduce the
expression of N-acetylgalactosamine sulfotransferase genes of the
present invention. The above methods of the present invention
comprise, for example, the steps of:
[0287] (a) contacting a test compound with cells expressing genes
encoding sulfotransferases that transfer a sulfate to position 4 or
6 of N-acetylgalactosamine;
[0288] (b) measuring the gene expression level of the cells;
and
[0289] (c) selecting a compound that reduces the gene expression
level as compared to when the test compound is not contacted.
[0290] In the above methods, test compounds are first contacted
with cells expressing a gene encoding a sulfotransferase that
transfers a sulfate to position 4 or 6 of
N-acetylgalactosamine.
[0291] Next, the expression level of the gene encoding the
sulfotransferases that transfer a sulfate to position 4 or 6 of
N-acetylgalactosamine is measured. Herein, "expression of the gene"
includes both transcription and translation. Gene expression level
can be measured by methods known to those skilled in the art.
[0292] For example, mRNAs are extracted from cells expressing any
one of the above-described proteins by conventional methods, and
these mRNAs can be used as templates in Northern hybridization,
RT-PCR, DNA arrays, or such to measure the transcription level of
the gene. Alternatively, protein fractions are collected from cells
expressing a gene encoding any of the above-described proteins, and
expression of the protein can be detected by electrophoresis such
as SDS-PAGE to measure the level of gene translation.
Alternatively, the level of gene translation can be measured by
detecting the expression of any of the above-described proteins by
Western blotting using an antibody against the proteins. Such
antibodies for use in detecting the proteins are not particularly
limited, as long as they are detectable. For example, both
monoclonal and polyclonal antibodies can be used.
[0293] Next, the expression level is compared with that in the
absence of the test compounds (the control).
[0294] Then, compounds that reduce (suppress) the expression level
of the gene as compared to when the test compounds are absent are
selected. The compounds resulting in a reduction (suppression) can
be agents for suppressing tissue fibrogenesis or candidate
compounds for treating fibrogenic disorders.
[0295] Furthermore, an embodiment of the screening methods of the
present invention includes methods of selecting the present
invention's compounds that reduce the expression level of a gene
encoding a sulfotransferase that transfers a sulfate to position 4
or 6 of N-acetylgalactosamine, using as an indicator, the amount
(level) of reporter gene expression. The above-described methods
comprise, for example, the steps of:
[0296] (a) contacting a test compound with cells or cell extracts
containing a DNA structured such that a reporter gene is operably
linked to a transcriptional regulatory region of a gene encoding a
sulfotransferase that transfer a sulfate to position 4 or 6 of
N-acetylgalactosamine;
[0297] (b) measuring the expression amount (level) of the reporter
gene; and
[0298] (c) selecting a compound that reduces the expression amount
(level) of the reporter gene as compared to when the test compound
is not contacted.
[0299] In the above methods, test compounds are first contacted
with cells or cell extracts containing DNAs structured such that a
reporter gene is operably linked with a transcriptional regulatory
region of a gene encoding a sulfotransferase that transfers a
sulfate to position 4 or 6 of N-acetylgalactosamine.
[0300] Herein, "operably linked" means that a reporter gene is
linked with a transcriptional regulatory region of a gene encoding
a sulfotransferase that transfers a sulfate to position 4 or 6 of
N-acetylgalactosamine, such that expression of the reporter gene is
induced upon binding of transcriptional factors to the
transcriptional regulatory region. Therefore, the meaning of
"operably linked" also includes cases where a reporter gene is
linked with a different gene and produces a fusion protein with a
different gene product, as long as expression of the fusion protein
is induced upon the binding of transcriptional factors to the
transcriptional regulatory region of the gene encoding the
sulfotransferase that transfers a sulfate to position 4 or 6 of
N-acetylgalactosamine. Those skilled in the art can obtain the
transcriptional regulatory regions of genes encoding
sulfotransferases that transfer a sulfate to position 4 or 6 of
N-acetylgalactosamine that are present in the genome, based on the
cDNA nucleotide sequences of the genes encoding the
sulfotransferases that transfer a sulfate to position 4 or 6 of
N-acetylgalactosamine.
[0301] The reporter genes for use in these methods are not
particularly limited, as long as their expression is detectable.
The reporter genes include, for example, the CAT gene, the lacZ
gene, the luciferase gene, and the GFP gene. The "cells containing
a DNA structured such that a reporter gene is operably linked with
a transcriptional regulatory region of a gene encoding a
sulfotransferase that transfers a sulfate to position 4 or 6 of
N-acetylgalactosamine" include, for example, cells introduced with
vectors carrying such structures as inserts. Such vectors can be
prepared by methods well known to those skilled in the art. The
vectors can be introduced into cells by standard methods, for
example, calcium phosphate precipitation, electroporation,
lipofection, and microinjection. The "cells containing a DNA
structured such that a reporter gene is operably linked with a
transcriptional regulatory region of a gene encoding a
sulfotransferase that transfers a sulfate to position 4 or 6 of
N-acetylgalactosamine" include cells in which the structure has
been integrated into the chromosomes. A DNA structure can be
integrated into chromosomes by methods generally used by those
skilled in the art, for example, gene transfer methods using
homologous recombination.
[0302] The "cell extracts containing a DNA structured such that a
reporter gene is operably linked with a transcriptional regulatory
region of a gene encoding a sulfotransferase that transfers a
sulfate to position 4 or 6 of N-acetylgalactosamine" include, for
example, mixtures of cell extracts included in commercially
available in vitro transcription-translation kits and DNAs
structured such that a reporter gene is operably linked with the
transcriptional regulatory region of the gene encoding a
sulfotransferase that transfers a sulfate to position 4 or 6 of
N-acetylgalactosamine.
[0303] "Contact" can be achieved by adding test compounds to a
culture medium of "cells containing a DNA structured such that a
reporter gene is operably linked with a transcriptional regulatory
region of a gene encoding a sulfotransferase that transfers a
sulfate to position 4 or 6 of N-acetylgalactosamine", or by adding
test compounds to the above-described commercially available cell
extracts containing the DNAs. When the test compound is a protein,
contact may also be achieved, for example, by introducing a DNA
vector expressing the protein into the cells.
[0304] In the above methods, the expression level of the reporter
gene is then measured. The expression level of the reporter gene
can be measured by methods known to those skilled in the art,
depending on the type of the reporter gene. When the reporter gene
is the CAT gene, its expression can be determined, for example, by
detecting the acetylation of chloramphenicol by the gene product.
When the reporter gene is the lacZ gene, its expression level can
be determined by detecting the color development of chromogenic
compounds due to the catalytic action of the gene expression
product. Alternatively, when the reporter gene is the luciferase
gene, its expression level can be determined by detecting the
fluorescence of fluorogenic compounds due to the catalytic action
of the gene expression product. Furthermore, when the reporter gene
is the GFP gene, its expression level can be determined by
detecting the fluorescence of the GFP protein.
[0305] In the above methods, the expression amount (level) of the
reporter gene is then compared with that in the absence of the test
compounds (the control). Compounds that reduce (suppress) the
expression level of the reporter gene as compared with a control
are then selected, where the reporter gene is operably linked with
a gene encoding a sulfotransferase that transfers a sulfate to
position 4 or 6 of N-acetylgalactosamine. Compounds resulting in a
reduction (suppression) can be agents for suppressing tissue
fibrogenesis or candidate compounds for treating fibrogenic
disorders.
[0306] The tissue fibrogenesis inhibitors that are found by the
screening methods of the present invention are preferably
therapeutic or preventive agents for fibrogenic disorders.
[0307] The present invention also provides methods of producing
pharmaceutical compositions for treating or preventing fibrogenic
disorders. The above-described production methods of the present
invention comprise, for example, the steps of:
[0308] (a) selecting a tissue fibrogenesis inhibitor from test
samples by the above-described methods of screening for tissue
fibrogenesis inhibitors; and
[0309] (b) combining the agent with a pharmaceutically acceptable
carrier.
[0310] In these methods, first, a tissue fibrogenesis inhibitor is
selected from test samples by the above-described methods of
screening for tissue fibrogenesis inhibitors.
[0311] Then, the selected agent is combined with a pharmaceutically
acceptable carrier. The pharmaceutically acceptable carrier
includes, for example, those described above.
[0312] The present invention also provides kits comprising various
agents, reagents, and the like, which are used to conduct the
screening methods of the present invention.
[0313] The kits of the present invention can be prepared, for
example, by selecting adequate reagents from the above-described
various reagents, depending on the screening method to be
conducted. The kits of the present invention may contain, for
example, the sulfotransferases that transfer a sulfate to position
4 or 6 of N-acetylgalactosamine of the present invention. The kits
of the present invention may further contain various reagents,
vessels, and the like to be used in the methods of the present
invention. The kits may appropriately contain, for example,
antibodies, probes, various reaction reagents, cells, culture
media, control samples, buffers, and instruction manuals containing
a description of how to use the kits.
[0314] The present invention also provides therapeutic or
preventive methods for fibrogenic disorders, which comprise the
step of administering the agents of the present invention to
individuals (for example, to patients and such).
[0315] The individuals subjected to the therapeutic or preventive
methods of the present invention are not particularly limited, as
long as they are organisms that can develop a fibrogenic disorder;
however, humans are preferred.
[0316] In general, administration to individuals can be achieved,
for example, by methods known to those skilled in the art, such as
intraarterial injections, intravenous injections, and subcutaneous
injections. The administered dose varies depending on the patient's
weight and age, and the administration method or such; however,
those skilled in the art (medical practitioners, veterinarians,
pharmacists, and the like) can appropriately select a suitable
dose.
[0317] The present invention also relates to the uses of agents of
the present invention in producing tissue fibrogenesis
inhibitors.
[0318] All prior art documents cited in this specification are
incorporated herein by reference.
EXAMPLES
[0319] Herein below, the present invention will be specifically
described with reference to Examples, but the technical scope of
the present invention is not to be construed as being limited
thereto.
[0320] Herein, occasionally, an siRNA structure (sequence) is
presented by showing a DNA region of a target gene. Those skilled
in the art can readily understand the structure of an siRNA
comprising double-stranded RNA corresponding to the DNA sequence
based on the information of DNA sequence described as a target
sequence.
[Cardiac Tissue]
[Example 1] Assessment of the Target Sugar Chain-Related Gene
Knockdown Effect of siRNAs and Anti-Fibrogenic Effect at the Gene
Level in a Mouse Cardiomyopathy Model
[0321] A model prepared by intraperitoneal administration of
Doxorubicin hydrochloride (DOX; Kyowa Hakko), as a standard mouse
cardiomyopathy model, was used in this Example and those below.
This mouse model is classical, but highly reproducible and simple.
Thus, the model has been widely used as a cardiomyopathy model for
elucidating pathological conditions, experimenting new
therapeutics, or such (Longhu Li, Circulation (2006) 113: 535-543;
Xiaoming Yi, Am J Physiol Heart Circ Physiol (2006) 290:
H1098-H1102; Kang Y J, J Biol Chem. 2000 May 5; 275(18): 13690-8;
Nozaki N, Circulation (2004) 110: 2869-2874; Fisher P W,
Circulation (2005) 111: 1601-1610).
[0322] The model mouse histologically develops fibrogenesis of the
myocardial interstitium. This pathological findings is commonly
observed in dilated cardiomyopathy, restrictive cardiomyopathy,
hypertrophic cardiomyopathy, and arrhythmogenic right ventricular
cardiomyopathy (ARVC), as well as left ventricular remodeling after
acute myocardial infarction, stable angina pectoris, unstable
angina pectoris, myocarditis, valvular heart disease, arrhythmia,
or hypertension. The fibrogenesis is a pathological feature
responsible for the myocardial dysfunction in chronic heart failure
caused by the above-listed diseases (Jugdutt B I, Circulation. 108:
1395-1403, 2003).
[0323] First, the method of preparing the mouse model is described
below. DOX (15 mg/kg; Kyowa Hakko) is administered to the
peritoneal cavities of C57BL6/J mice (male, eight weeks old, CLEA
Japan Inc.). The mice were reared for one week after
administration, and then heart tissues were collected from them. As
a control group, similar mice were also purchased and reared around
the same time without DOX administration.
[0324] The GalNac4S-6ST siRNA agent was administered by the
following procedure: 1 .mu.g of GalNac4S-6ST siRNA (Hokkaido System
Science, Co., Ltd.) was combined with 200 .mu.l of 1% atelocollagen
(Koken Co.) as a vehicle, and the mixture was intraperitoneally
administered to each mouse 24 hours before DOX administration. The
nucleotide sequence of the GalNac4S-6ST siRNA agent used in this
Example is shown below, but the sequence is not limited to this
Example.
[0325] [human GalNac4S-6ST siRNA] (Gene Bank accession number
NM_015892)
[0326] (Hokkaido System Science, Co., Ltd.)
[0327] 5'-ggagcagagcaagaugaauacaauc-ag-3' (SEQ ID NO: 25)
[0328] 3'-ua-ccucgucucguucuacuuauguuag-5' (SEQ ID NO: 26)
[0329] 1 ml of RNA iso (TAKARA BIO INC.) was added to 50 mg each of
organs (heart) excised from cardiomyopathy model mouse. The organs
were crushed using an electrical homogenizer (DIGITAL HOMOGENIZER;
AS ONE), then, 200 .mu.l of chloroform (Sigma-Aldrich Japan) was
added to the resulting suspension. The mixture was gently mixed and
then cooled on ice for about five minutes, and centrifuged in a
centrifuge (Centrifuge 5417R; Eppendorf) at 12,000 rpm and
4.degree. C. for 15 minutes. After centrifugation, 500 .mu.l of the
supernatant was transferred to a fresh Eppendorf tube, and an equal
volume of isopropanol (500 .mu.l; Sigma-Aldrich Japan) was added
thereto. The solution was mixed, and then 1 .mu.l of glycogen
(Invitrogen) was added thereto. The mixture was cooled on ice for
15 minutes, and then centrifuged at 12,000 rpm and 4.degree. C. for
15 minutes. Next, RNA precipitate obtained after washing three
times with 1,000 .mu.l of 75% ethanol (Sigma-Aldrich Japan) was
air-dried for 30 minutes to one hour, and then dissolved in Otsuka
distilled water (Otsuka Pharmaceutical Co., Ltd). The solution was
100 times diluted with Otsuka distilled water. The RNA
concentrations of extracted samples in UV plates (Corning Costar)
were determined using a plate reader (POWER Wave XS; BIO-TEK).
[0330] Next, reverse transcription reaction (cDNA synthesis) is
conducted by the following procedure. The concentrations of the
obtained RNA samples were adjusted to 500 ng/20 .mu.l. The samples
were heated at 68.degree. C. for three minutes in a BLOCK INCUBATOR
(ASTEC), and cooled on ice for ten minutes. After cooling on ice,
80 .mu.l of RT PreMix solution (composition: 18.64 .mu.l of 25 mM
MgCl.sub.2 (Invitrogen), 20 .mu.l of 5.times. Buffer (Invitrogen),
6.6 .mu.l of 0.1 M DTT (Invitrogen), 10 .mu.l of 10 mM dNTP mix
(Invitrogen), 2 .mu.l of RNase Inhibitor (Invitrogen), 1.2 .mu.l of
MMLV Reverse Transcriptase (Invitrogen), 2 .mu.l of Random primer
(Invitrogen), and 19.56 .mu.l of sterile distilled water (Otsuka
distilled water; Otsuka Pharmaceutical Co., Ltd.)), which had been
prepared in advance, was added to the samples. The mixtures were
heated in a BLOCK INCUBATOR (ASTEC) at 42.degree. C. for one hour
and at 99.degree. C. for five minutes, and then cooled on ice. 100
.mu.l of desired cDNAs were prepared and quantitative PCR reaction
was carried out using the prepared cDNAs in the following
composition. For quantitative PCR, SYBR Premix Kit (TAKARA BIO
INC.) and Real-time PCR thermal cycler DICE (TAKARA BIO INC.) were
used. Conditions of PCR reaction was: 95.degree. C. for 10 seconds,
40 cycles of 95.degree. C. for 5 seconds and 60.degree. C. for 30
seconds, finally, melting curve analysis was conducted. Nucleotide
sequences of primers used in the quantitative PCR were described
below.
TABLE-US-00001 [Quantitative PCR Primer sequences] *mouse
GalNAc4S-6ST (TAKARA BIO INC.) Forward: (SEQ ID NO: 27)
5'-GTGAGTTCTGCTGCGGTCCA-3' Reverse: (SEQ ID NO: 28)
5'-AGTCCATGCTGATGCCCAGAG-3' *mouse procollagen Type 1 alpha 2
(TAKARA BIO INC.) Forward: (SEQ ID NO: 29)
5'-ACCCGATGGCAACAATGGA-3' Reverse: (SEQ ID NO: 30)
5'-ACCAGCAGGGCCTTGTTCAC-3' *mouse .alpha.-SMA (TAKARA BIO INC.)
Forward: (SEQ ID NO: 31) 5'-CATCCGTAAAGACCTCTATGCCAAC-3' Reverse:
(SEQ ID NO: 32) 5'-ATGGAGCCACCGATCCACA-3' *mouse rRibosome 18S
(TAKARA BIO INC.) Forward: (SEQ ID NO: 33)
5'-TTCTGGCCAACGGTCTAGACAAC-3' Reverse: (SEQ ID NO: 34)
5'-CCAGTGGTCTTGGTGTGCTGA-3'
[0331] As shown in FIG. 2, the expressions of GalNAc4S-6ST, type I
collagen, and .alpha.-SMA genes were determined, and the result
showed that the expression of GalNac4S-6ST was significantly
suppressed in the GalNAc4S-6ST siRNA-treated group as compared to
the untreated group (P<0.001; when compared to the untreated
group). Furthermore, the expressions of .alpha.-SMA and type I
collagen genes were measured as indicators for fibrogenesis, which
is an important pathological condition of cardiomyopathy. As a
result, the significant reduction of expression were confirmed in
the GalNAc4S-6ST siRNA-treated group as compared to the untreated
group (P<0.001; when compared to the untreated group). This
result demonstrates that the target knockdown effect of the
GalNAc4S-6ST siRNA results in suppression of the progression of
myocardial fibrogenesis at the gene expression level.
[0332] The agents of the present invention are thus useful, for
example, as myocardial fibrogenesis inhibitors.
[Example 2] Cardiac Hypertrophy-Suppressing Effect of GalNAc4S-6ST
siRNA in a Mouse Cardiomyopathy Model
[0333] In this Example, the heart weights (mg) and body weights (g)
of cardiomyopathy model mice were measured to calculate the
heart/body weight ratio which is an indicator for cardiac
hypertrophy. The cardiac hypertrophy-suppressing effect of the
GalNAc4S-6ST (GalNac) siRNA was evaluated. Cardiac hypertrophy also
serves as an indicator for tissue fibrotic change.
[0334] FIG. 1 shows the result of calculating the heart weight
(mg)/body weight (g) ratios in the siRNA-treated group (n=4) and
untreated group (n=4). The result showed that the ratio was
6.376.+-.0.484 and 5.442.+-.0.203 in the untreated and
siRNA-treated groups, respectively. Thus, the significant reduction
of the ratio was found in the siRNA-treated group as compared to
the untreated group (p<0.05; t-test). This suggests that
GalNac4S-6ST siRNA has the effect of suppressing pathological
cardiac hypertrophy.
[0335] The agents of the present invention are thus useful, for
example, as cardiac hypertrophy-suppressing agents (therapeutic
agents for cardiac hypertrophy).
[Example 3] Assessment of Type I Collagen Deposition-Suppressing
Effect of GalNAc4S-6ST siRNA in a Mouse Cardiomyopathy Model
[0336] In this Example, the type I collagen deposition (an
indicator of fibrogenesis)-suppressing effect of GalNac4S-6ST siRNA
was assessed using heart samples of cardiomyopathy model mice.
Cardiac tissue samples were collected from the same mice as
described in Example 1, and embedded in OCT compound (Miles), an
embedding medium for cryosectioning. The samples were sliced into
thin sections using Cryostat (Carl Zeiss). The resulting sections
were fixed with acetone (Sigma Aldrich Japan) for ten minutes, and
then washed with phosphate buffer. A rabbit antiserum anti-type I
collagen (rabbit polyclonal antibody, 1:2,000 dilution; LSL) was
added as the primary antibody, and the sections were incubated at
room temperature for one hour. Then, a peroxidase-labeled goat
anti-rabbit IgG antibody (1:200 dilution; Cappel) was added as the
secondary antibody, and the sections were incubated at room
temperature for 30 minutes. After incubation, DAB substrate
(Nichirei) was added to the samples. The samples were observed
under a light microscope (Leica Microsystems).
[0337] The histological findings were shown in FIG. 3. Very intense
positive signals for type I collagen were observed between
myocardial fibers in the untreated group. Meanwhile, in the
siRNA-treated group, the type I collagen-positive signals were
considerably weaker than those of the untreated group. The
above-described type I collagen immunostaining result demonstrates
that the GalNac4S-6ST siRNA has the effect of suppressing the
excessive deposition of type I collagen in myocardial tissues. This
result correlates with the result of quantitative PCR described in
Example 1.
[0338] The agents of the present invention are thus useful, for
example, as agents for suppressing type I collagen deposition in
myocardial tissues.
[Example 4] Assessment of Type III Collagen Deposition-Suppressing
Effect of GalNAc4S-6ST siRNA in a Mouse Cardiomyopathy Model
[0339] In this Example, the type III collagen deposition (an
indicator of fibrogenesis activity)-suppressing effect of
GalNac4S-6ST siRNA was assessed using heart samples of
cardiomyopathy model mice. Tissue sections obtained by the same
method as described in Example 3 were fixed with acetone (Sigma
Aldrich Japan) for ten minutes, and then washed with phosphate
buffer. A rabbit antiserum anti-type III collagen (rabbit
polyclonal antibody, 1:2000 dilution; LSL) was added as the primary
antibody, and the sections were incubated at room temperature for
one hour. Then, a peroxidase-labeled goat anti-rabbit IgG antibody
(1:200 dilution; Cappel) was added as the secondary antibody, and
the sections were incubated at room temperature for 30 minutes.
After incubation, DAB substrate (Nichirei) was added to the
samples. The samples were observed under a light microscope (Leica
Microsystems).
[0340] The histological findings are shown in FIG. 4. Moderately
strong positive signals for type III collagen were observed between
myocardial fibers in the untreated group. Meanwhile, in the
siRNA-treated group, the type III collagen-positive signals were
comparable to those of the control group. The above-described
result of type III collagen immunostaining demonstrates that the
GalNac4S-6ST siRNA has the effect of suppressing the type III
collagen deposition in heart tissues, implying that the siRNA is
also effective in suppressing active collagen deposition.
[0341] The agents of the present invention are thus useful, for
example, as agents for suppressing type III collagen deposition in
myocardial tissues.
[Example 5] Assessment of Fibroblast Infiltration-Suppressing
Effect of GalNAc4S-6ST siRNA in a Mouse Cardiomyopathy Model
[0342] This Example assesses the pharmacological effect of
GalNAc4S-6ST siRNA on the kinetics of fibroblasts that infiltrate
into cardiac tissues of cardiomyopathy model mice due to DOX
administration. Tissue sections obtained by the same method as
described in Example 3 were fixed with acetone (Sigma Aldrich
Japan) for ten minutes, and then washed with phosphate buffer. An
anti-mouse fibroblast antibody (ER-TR7, rat monoclonal antibody,
1:400 dilution; BMA Biomedicals Ltd.) was added as the primary
antibody, and the sections were incubated at room temperature for
one hour. Then, a peroxidase-labeled goat anti-rat immunoglobulin
antibody (1:200 dilution; Biosource International, Inc.) was added
as the secondary antibody, and the sections were incubated at room
temperature for 30 minutes. After incubation, DAB substrate
(Nichirei Biosciences) was added to the samples. The samples were
observed under a light microscope (Leica Microsystems).
[0343] The histological findings were shown in FIG. 5. The
photograph focuses on the ventricular septum. Infiltration of
numerous fibroblasts was observed in the untreated group as
compared to the control group. In contrast, the degree of
fibroblast infiltration in the siRNA-treated group was less as
compared to the untreated group. The above-described result shows
that GalNac4S-6ST siRNA has the pharmacological effect of
suppressing the fibroblast infiltration into myocardial tissues and
this activity contributes to the anti-fibrogenic effect.
[0344] The agents of the present invention are thus useful, for
example, as agents for suppressing fibroblast infiltration into
myocardial tissues.
[Gastrointestinal Tissue]
[Example 6] Clinical Fibrogenesis-Suppressing Effect of
GalNAc4S-6ST in a Mouse Intestinal Fibrosis Model
[0345] The colitis model mice were prepared by allowing C57BL/6J
mice (female, six weeks old; CLEA Japan Inc.) to freely drink
high-concentration chlorine water containing 3% dextran sulfate
sodium (DSS; Wako Pure Chemical Industries Ltd.) for eight days.
The DSS-induced colitis model has excellent reproducibility, and is
thus widely used as a typical experimental mouse model for
inflammatory bowel diseases such as ulcerative colitis or Crohn's
disease, as well as a model with full-thickness inflammation and
fibrotic changes, and muscle layer thickening, which are
histological characteristics of the narrowing of colon lumen
(Sasaki N, J Inflamm. 2005 2: 13, Review: Pucilowska J B et al. Am
J Physiol Gastroenterol Liver Physiol. 279: G653-G659, 2000).
Therefore, the histological findings are commonly and widely
observed in inflammatory bowel diseases as well as pathological
conditions with the histological narrowing of the intestinal lumen,
specifically diseases such as intestinal Behcet's disease (simple
ulcer), irritable bowel syndrome, ischemic enteritis, drug-induced
enteritis, radiation enteritis, esophagus achalasia,
esophagostenosis associated with scleroderma, narrowing of the
colon lumen associated with systemic lupus erythematosus (SLE),
Hirschsprung's disease, stenosis after removal of intestine
(postoperative stricture), narrowing of the intestinal lumen after
endoscopic mucosal resection for gastrointestinal cancer (tongue
cancer, epipharynx carcinoma, pharyngeal cancer, esophageal cancer,
stomach cancer, small intestinal cancer, colon cancer, and rectal
cancer), and ileus.
[0346] Simultaneously to feeding mice with 3% DSS water, the same
GalNAc4S-6ST siRNA (1 .mu.g/head) as described in Example 1 was
combined with atelocollagen (Koken Co.) prediluted 10-fold with PBS
and 200 .mu.l of the mixture was injected to the peritoneal
cavities of the mice. The group of mice treated as described above
was named "GalNAc4S-6ST siRNA group", while a group treated with
atelocollagen alone without combining GalNAc4S-6ST siRNA was named
the "control group". The body weight and the disease activity index
(DAI) score were recorded during seven days of 3% DSS water feeding
(Kihara M., Gut. 2003, 52, 713-9). The evaluation criteria for DAI
are shown below.
TABLE-US-00002 Index Weight loss Stool consistency Fecal blood 0
None Normal Normal 1 1-5% Hem occult (+) 2 5-10% Loose Stool Hem
occult (++) 3 10-20% Hem occult (+++) 4 >20% Diarrhea Gross
Bleeding
[0347] The result of scoring the DAI of each mouse, setting the
score on the first day of DSS water feeding (day 0) as 1, is shown
in FIG. 6. On the third day, the GalNAc4S-6ST siRNA-administered
group exhibited a significantly lower score as compared to the
control group (p<0.001; t-test). This result suggests that
suppression of GalNAc4S-6ST gene expression produces the effect of
suppressing inflammatory activity at relatively earlier stages.
[0348] Furthermore, the mice were sacrificed and their colon
lengths were measured on the fifth day. The colon shortening was
significantly suppressed in the GalNAc4S-6ST siRNA-administered
group (p<0.005; t-test) (FIG. 6). The colon length is a
definitive indicator that reflects intestinal fibrogenesis or
stenosis. Thus, it was also clinically demonstrated that the
fibrotic change of intestine was suppressed in the GalNAc4S-6ST
siRNA-administered group.
[0349] The agents of the present invention are thus useful, for
example, as agents for suppressing fibrotic changes of the
intestine.
[Example 7] Intestinal Fibrogenesis-Suppressing Effect of
GalNAc4S-6ST siRNA in a Mouse Intestinal Fibrosis Model
[0350] In this Example, the expression of fibrogenesis-related
genes in the colon after GalNAc4S-6ST siRNA administration was
assessed by the quantitative real-time PCR method.
[0351] The intestinal fibrogenesis model was prepared by the same
method as described in Example 6. The mice were sacrificed on day
7. A part of the collected colon was placed in 1.5-ml tubes, and
frozen with liquid nitrogen. cDNA was synthesized by the same
method as that described in Example 1, and quantitative PCR was
carried out. The primer sequences and the number of PCR cycle
conditions were the same as those described in Example 1.
[0352] The result is shown in FIG. 7. The expression of
GalNAc4S-6ST gene was enhanced in this model. The significant
knockdown of the gene was confirmed by GalNAc4S-6ST siRNA treatment
(p<0.001; t-test). Furthermore, the enhanced expression of type
I collagen and .alpha.-SMA as indicators of fibrogenesis were
significantly suppressed by GalNAc4S-6ST siRNA (for both genes,
p<0.001; t-test). The result suggests that the enhanced fibrotic
changes of the colon can be effectively suppressed by suppressing
the expression of GalNAc4S-6ST.
[0353] The agents of the present invention are thus useful, for
example, as agents for suppressing the fibrotic changes of the
colon.
[Example 8] Tissue Fibrogenesis-Suppressing Effect of GalNAc4S-6ST
siRNA in a Mouse Intestinal Fibrosis Model
[0354] The intestinal fibrogenesis model was prepared by the same
method as described in Example 6. The mice were sacrificed on day
7. Cryoblocks and tissue sections were prepared from the collected
colon by the same method as described in Example 3. Masson-stained
images of colonic tissue sections are shown in FIG. 8. Masson
staining serves as an indicator to assess fibrotic change of
tissues by visualizing collagen fibers. In the GalNAc4S-6ST
siRNA-administered group, the full-thickness (lamina propria
mucosae, submucosa, and muscle layer) collagen fiber deposition was
significantly suppressed as compared to the control group.
[0355] The agents of the present invention are thus useful, for
example, as agents for suppressing the full-thickness (lamina
propria mucosae, submucosa, and muscle layer) collagen fiber
deposition.
[Example 9] Histological Fibroblast Infiltration-Suppressing Effect
of GalNAc4S-6ST siRNA in a Mouse Intestinal Fibrosis Model
[0356] The intestinal fibrogenesis model was prepared by the same
method as described in Example 6. The mice were sacrificed on day
7. Cryoblocks and tissue sections were prepared from the collected
colon by the same method as described in Example 3. The resulting
sections were fixed with acetone (Wako Pure Chemical Industries)
for ten minutes, and then washed with phosphate buffer. An
anti-ER-TR7 antibody (rat monoclonal antibody, 1 .mu.g/ml; BMA) was
added as the primary antibody, and the sections were incubated at
room temperature for one hour. Then, the secondary antibody
reaction was carried out by adding a peroxidase-labeled anti-rat
IgG antibody (1:200 dilution), which was followed by color
development by adding DAB substrate (Nichirei Biosciences). Then,
the nucleus was stained by Lillie-Mayer hematoxylin (Muto Pure
Chemicals Co.). The samples were observed under a light microscope
(Leica Microsystems). The antibody binding was detected by
visualizing it as brown signals.
[0357] As a result, the full-thickness infiltration of fibroblasts
was significantly suppressed in the GalNAc4S-6ST siRNA-treated
group as compared to the control group (FIG. 9). This result
demonstrates that the inhibition of GalNAc4S-6ST gene expression
results in suppression of the infiltration and retention of
fibroblasts in focal tissue lesion and thereby reduces the enhanced
fibrotic change.
[0358] The agents of the present invention are thus useful, for
example, as agents for suppressing the infiltration or retention of
fibroblasts.
[Example 10] Histological Macrophage Infiltration-Suppressing
Effect of GalNAc4S-6ST siRNA in a Mouse Intestinal Fibrosis
Model
[0359] The intestinal fibrogenesis model was prepared by the same
method as described in Example 6. The mice were sacrificed on day
7. Cryoblocks and tissue sections were prepared from the collected
colon by the same method as described in Example 3. The resulting
sections were fixed with acetone (Wako Pure Chemical Industries)
for ten minutes, and then washed with phosphate buffer. An
anti-F4/80 antibody (clone A3-1, rat monoclonal antibody, 2
.mu.g/ml: CALTAG LABORATORIES) was added as the primary antibody,
and the sections were incubated at room temperature for one hour.
Then, the secondary antibody reaction was carried out by adding a
peroxidase-labeled anti-rat IgG antibody (1:200 dilution), which
was followed by color development by adding DAB substrate (Nichirei
Biosciences). Then, the nucleus was stained by Lillie-Mayer
hematoxylin (Muto Pure Chemicals Co.). The samples were observed
under a light microscope (Leica Microsystems). The antibody binding
was detected by visualizing it as brown signals.
[0360] The result showed that the full-thickness macrophage
infiltration was significantly suppressed in the GalNAc4S-6ST
siRNA-treated group as compared to the control group (FIG. 10).
This result demonstrates that the inhibition of GalNAc4S-6ST gene
expression resulted in suppression of the infiltration of
macrophages and fibroblasts, which are cell groups responsible for
the persistent or enhanced fibrotic changes, and thereby
comprehensively suppressed the tissue fibrotic changes.
[0361] The agents of the present invention are thus useful, for
example, as agents for suppressing the infiltration of macrophages
or fibroblasts.
[Example 11] Histological Macrophage Infiltration-Suppressing
Effect of GalNAcST siRNA in a Mouse Intestinal Fibrosis Model
[0362] GalNAc4S-6ST is an enzyme that transfers a sulfate group to
position 6 in N-acetylgalactosamine sulfated at position 4.
GalNAc-4ST1 and GalNAc-4ST2, which belong to the
4-O-sulfotransferase family, were assessed in this Example. The
intestinal fibrogenesis model was prepared by the same method as
described in Example 6. The mice were sacrificed on day 7. In this
Example, GalNAc4S-6ST siRNA, GalNAc4ST-1, and GalNAc4ST-2
(GeneWorld) were combined together; 1 .mu.g of the mixture was
combined with 200 .mu.l of 1% atelocollagen (Koken Co.), which is a
vehicle, and administered intraperitoneally to each mouse. A group
administered with the siRNA is referred to as "GalNAc ST
siRNA-administered group". The control group is the same as
described in Example 6. The siRNA nucleotide sequences of
GalNAc4S-6ST, GalNAc4ST-1, and GalNAc4ST-2 used in this Example are
shown below, but the sequence are not limited the Example.
TABLE-US-00003 [GalNAc4ST-1 siRNA cocktail sequences] (GenBank
accession number NM_175140) (GeneWorld) (SEQ ID NO: 35)
5'-ACCCCCAACTCGGAACGATGCGGCT-3' (SEQ ID NO: 36)
5'-TGCATGTTCTCGTCCATCCTGCTG-3' (SEQ ID NO: 37)
5'-CGCCACCGTGTACTGTACTGTGAAGT-3' (SEQ ID NO: 38) 5'-AGGCT
GCTCCAACTG GAAGAGGGTG-3' [GalNAc4ST-2 siRNA cocktail sequences]
(GenBank accession number NM_199055) (GeneWorld) (SEQ ID NO: 39)
5'-ATATAGTATCTAGGATATATGTAG-3' (SEQ ID NO: 40)
5'-GAAGTACCAAAAGCTGGCTGCTCTA-3' (SEQ ID NO: 41)
5'-TTCTATCACTTGGACTATTTGATGTT-3' (SEQ ID NO: 42)
5'-TACACAACTCCACATTTGTAATTTG-3' [GALNac4S-6ST siRNA cocktail
sequences] (GenBank accession number NM_029935) (GeneWorld) (SEQ ID
NO: 43) 5'-CCAGAAGCCAAGCTCATTGTTATG-3' (SEQ ID NO: 44)
5'-CTGTGGAGAGGTTGTACTCAGACTA-3' (SEQ ID NO: 45)
5'-ATTTGCCTGGAAGACAACGTGAGAGC-3' (SEQ ID NO: 46)
5'-GTCCCTTCTGCAGAAGCTGGGCCCACT-3'
[0363] As investigated in detail in Examples 6 to 10, the tissue
fibrotic changes in the mouse intestinal fibrogenesis model can be
assessed representatively by the colon length. Accordingly, the
colon length on day 7 is shown as an essential evaluation item in
this Example. In the GalNAc ST siRNA-administered group, the
shortening of colon was significantly suppressed as compared to the
control group (p<0.01; t-test) (FIG. 11). It was thus
demonstrated that the intestinal fibrogenesis was inhibited by
suppressing the expression of the GalNAc4ST-1 and GalNAc4ST-2
genes.
[0364] The agents of the present invention are thus useful, for
example, as intestinal fibrogenesis inhibitors.
[Lung Tissue]
[Example 12] Effect of C6ST-1 siRNA on Pulmonary Alveolar
Interstitium in a Mouse Pulmonary Emphysema Model
[0365] A basic mouse pulmonary emphysema model, which is prepared
by intratracheal administration of porcine pancreatic elastase
(PPE), is used in this Example. This mouse model is classical, but
highly reproducible and simple. Thus, this mouse model has been
used commonly as a pulmonary emphysema model. Inflammatory cell
infiltration to the pulmonary alveolar interstitium is observed as
a histological feature. This histological finding is commonly
detected in chronic obstructive pulmonary disease (COPD) such as
emphysema and chronic bronchitis as well as diseases causing
chronic respiratory failure, such as idiopathic interstitial
pneumonias (IIPs), coniosis, and pulmonary tuberculosis sequelae
(Karlinsky J B et al., Am Rev Respir Dis 1978; 117: 1109-1133;
Otto-Verberne C J et al., Protective effect of pulmonary surfactant
on elastase-induced emphysema in mice. Eur Respir J 1992; 5:
1223-1230; Janoff A et al., Prevention of elastase-induced
experimental emphysema by oral administration of a synthetic
elastase inhibitor. Am Rev Respir Dis 1980; 121: 1025-1029;
Christensen T G, et al., Irreversible bronchial goblet cell
metaplasia in hamsters with elastase-induced panacinar emphysema. J
Clin Invest 1977; 59: 397-404; Lucey E C, et al., Remodeling of
alveolar walls after elastase treatment of hamsters: results of
elastin and collagen mRNA in situ hybridization. Am J Respir Crit
Care Med 1998; 158: 555-564; Snider G L, Lucey E C, Stone P J.
Animal models of emphysema. Am Rev Respir Dis 1986; 133:
149-169).
[0366] In this Example, the effect of C6ST-1 siRNA in suppressing
emphysematous lesions was examined by hematoxylin-eosin staining
(HE staining) of lung tissue samples from pulmonary emphysema model
mice.
[0367] First, the model mice were prepared. PPE (4 units;
Calbiochem-Novabiochem) was administered intratracheally to
C57BL6/J mice (female, 5- to 6-weeks old; CLEA Japan). The mice
were grown for three weeks after administration, and then lung
tissues were collected from them. Mice that did not have PPE
administration were used as the control group.
[0368] The C6ST-1 siRNA was administered by the same procedure as
shown in Example 1: 1 .mu.g of C6ST-1 siRNA (Ambion) was combined
with 1% atelocollagen (Koken Co.), which is an siRNA vehicle, and
administered to the peritoneal cavities once a week after PPE
administration. The dose was 200 al/head.
TABLE-US-00004 *[C6ST-1 siRNA cocktail sequences] (GenBank
accession number NM_016803) (SEQ ID NO: 47)
5'-gcgccccctctccccatggagaaag-3' (SEQ ID NO: 48)
5'-gctttgcctcaggatttccgggacc-3' (SEQ ID NO: 49)
5'-ggttcagccttggtctaccgtgatgtc-3' (SEQ ID NO: 50)
5'-gcagttgttgctatgcgacctgtat-3'
[0369] The collected right lung tissues were embedded in the OCT
compound (Miles), an embedding medium for cryosectioning, and
cryoblocks were prepared using liquid nitrogen. The cryoblocks were
sliced into 6-.mu.m sections using cryostat (Microm).
[0370] The resulting sections were fixed with 1% glutaraldehyde
(Nacalai Tesque) for 10 minutes, and further fixed with
formol-calcium solution for 10 minutes. The sections were washed
with phosphate buffer, and then stained with Lillie-Mayer's
hematoxylin solution (Sigma Aldrich Japan) at room temperature for
5 minutes. The sections were washed gently with a decolorizing
solution (70% ethanol containing 0.5% HCl; prepared using reagents
from Nacalai Tesque), and then washed with water for 10 minutes.
The sections were stained with eosin-alcohol at room temperature
for 5 minutes, and then washed with water for 10 minutes. The
sections were washed gently with 100% ethanol, and then allowed to
stand for 3 minutes. The sections were further washed gently with
xylene (Nacalai Tesque), and allowed to stand for 10 minutes. This
sample was histologically observed using a light microscope (Leica
Microsystems).
[0371] The obtained histological images are shown in FIG. 12. The
histological features of a normal lung parenchyma with
characteristic faveolate alveolar septal walls are found in the
control group (PPE non-administered mice). Meanwhile, emphysematous
lesions due to destruction and abolishment of alveolar septal walls
and enlargement of air space (characteristics of pulmonary
emphysema) can be observed in the untreated group (administered
with PPE but not with C6ST-1 siRNA) shown in the middle photograph.
On the other hand, in the enzyme-treated group (administered with
PPE and C6ST-1 siRNA), slight abolishment of alveolar septal walls
and emphysematous lesions can be observed; however, their levels
are significantly improved.
[Example 13] Pulmonary Interstitial Fibrosis-Suppressing Effect of
C6ST-1 siRNA in a Mouse Pulmonary Emphysema Model
[0372] In this Example, the expression of fibrosis-related genes in
the pulmonary alveolar interstitium after C6ST-1 siRNA
administration is assessed by quantitative real-time PCR
method.
[0373] The COPD model was prepared by the same method as described
in Example 12. A part of the collected lung tissue were placed in
1.5-ml tubes, and frozen with liquid nitrogen. cDNA synthesis was
carried out by the same method as described in Example 1, and
quantitative PCR was conducted. The primer sequences for type I
collagen and .alpha.-SMA and PCR conditions were also the same as
described in Example 1. The sequences of C6ST-1 primers are shown
below.
TABLE-US-00005 [Quantitative PCR primer sequences] *mouse C6ST-1
(Takara Bio) Forward: (SEQ ID NO: 51) 5'-TGTTCCTGGCATTTGTGGTCATA-3'
Reverse: (SEQ ID NO: 52) 5'-CCAACTC GCTCAGGGACAAGA-3'
[0374] The result is shown in FIG. 13. The expression of C6ST-1
gene is enhanced in this model. Significant knockdown of the C6ST-1
gene was confirmed by C6ST-1 siRNA treatment (p<0.01; t-test).
Furthermore, the enhanced expression of type I collagen and
.alpha.-SMA as indicators for fibrogenesis were significantly
suppressed by C6ST-1 siRNA (for both genes, p<0.01; t-test).
This result suggests that the enhanced fibrotic changes of
pulmonary interstitium can be effectively suppressed by inhibiting
the expression of C6ST-1.
[0375] The agents of the present invention are thus useful, for
example, as agents for suppressing fibrotic changes of the
pulmonary interstitium.
[Example 14] Histological Fibroblast Cell Infiltration-Suppressing
Effect of C6ST-1 siRNA in a Mouse Pulmonary Emphysema Model
[0376] The intestinal fibrogenesis model was prepared by the same
method as described in Example 12. Cryoblocks and tissue sections
were prepared from the collected lung tissues by the same method as
described in Example 3. The resulting sections were fixed with
acetone (Wako Pure Chemical Industries) for ten minutes, and then
washed with phosphate buffer. An anti-ER-TR7 antibody (rat
monoclonal antibody, 1 .mu.g/ml; BMA) was added as the primary
antibody, and the sections were incubated at room temperature for
one hour. Then, the secondary antibody reaction was carried out by
adding a peroxidase-labeled anti-rat IgG antibody (1:200 dilution),
which was followed by color development by adding DAB substrate
(Nichirei Biosciences). Then, the nucleus was stained by
Lillie-Mayer hematoxylin (Muto Pure Chemicals Co.). The samples
were observed under a light microscope (Leica Microsystems). The
antibody binding was detected by visualizing it as brown
signals.
[0377] As a result, a strong accumulation of fibroblasts was
observed in the interstitium with damaged alveolar septa in the
control group (FIG. 14; left panel for the control group).
Furthermore, accumulation of many fibroblasts was also observed in
the interstitium where the alveolar septa were being damaged (FIG.
14; right panel for the control group). Thus, the present inventors
obtained the unexpected result that excessive accumulation of
fibroblasts lead to the alveolar wall damaging process in
pathological conditions such as COPD. Meanwhile, fibroblast
infiltration to the interstitium of alveolar tissue was obviously
suppressed in the C6ST-1 siRNA-treated group (FIG. 14). It was thus
demonstrated that the suppression of C6ST-1 gene expression
resulted in inhibition of fibroblast infiltration and retention in
the interstitium of alveolar tissue and thereby reduced the
enhanced fibrotic changes.
[0378] The agents of the present invention are thus useful, for
example, as agents for suppressing fibroblast infiltration and
retention in the interstitium of alveolar tissue.
[Example 15] Histological Macrophage Infiltration-Suppressing
Effect of C6ST-1 siRNA in a Mouse Pulmonary Emphysema Model
[0379] The COPD model was prepared by the same method as described
in Example 12. The collected lung tissues were processed by the
same method as described in Example 3 to prepare tissue cryoblocks
and sections. The resulting sections were fixed with acetone (Wako
Pure Chemical Industries) for ten minutes, and then washed with
phosphate buffer. An anti-F4/80 antibody (clone A3-1, rat
monoclonal antibody, 2 .mu.g/ml; CALTAG LABORATORIES) was added as
the primary antibody, and the sections were incubated at room
temperature for one hour. Then, the secondary antibody reaction was
carried out by adding a peroxidase-labeled anti-rat IgG antibody
(1:200 dilution), which was followed by color development by adding
DAB substrate (Nichirei Biosciences). Then, the nucleus was stained
by Lillie-Mayer hematoxylin (Muto Pure Chemicals Co.). The samples
were observed under a light microscope (Leica Microsystems). The
antibody binding was detected by visualizing it as brown
signals.
[0380] The result showed that the macrophage infiltration to the
pulmonary alveolar interstitium was clearly suppressed in the
C6ST-1 siRNA-treated group as compared to the control group (FIG.
15). It was thus demonstrated that the suppression of C6ST-1 gene
expression resulted in suppression of the infiltration of
macrophages and fibroblasts, which are cell groups responsible for
the persistent or enhanced fibrotic changes, and thereby
comprehensively suppressed the tissue fibrotic changes.
[0381] The agents of the present invention are thus useful, for
example, as agents for suppressing the infiltration of macrophages
to the pulmonary alveolar interstitium.
[Example 16] Respiratory Function-Preserving Effect of C6ST-1 siRNA
in a Mouse Pulmonary Emphysema Model
[0382] In this Example, C6ST-1 siRNA was assessed for its influence
on respiratory function using static lung compliance (static
compliance (Cst)) as an indicator to evaluate the clinical effect
of C6ST-1 siRNA in pulmonary emphysema model mice. Cst represents a
measure of lung tissue flexibility. Cst is increased in pulmonary
emphysema, which is a disease with alveolar tissue damage.
[0383] Pulmonary emphysema model mice were prepared by the same
procedure described in Example 12, and then treated with C6ST-1
siRNA. An anesthetic agent was given to the mice to stop
spontaneous respiration, and then their Cst was monitored using
FlexiVent (SCIREQ) respiratory function analyzer in the PV loop
mode. Mice were connected to the FlexiVent by the following
procedure: a median incision was performed after stopping
spontaneous respiration, and then a special cannula was inserted
into the trachea, which was followed by peribronchial ligation.
[0384] The result of this Example is shown in FIG. 16. Cst was
42.62.+-.2.25 .mu.l/cm H2O in the control group, while it was
51.22.+-.5.2 .mu.l/cm H2O in the untreated group (when compared to
the control group, P=0.03; t-test). Thus, a statistically
significant increase was observed in the untreated group. In
contrast, Cst was 42.92.+-.1.82 .mu.l/cm H2O (when compared to the
untreated group, P=0.03; t-test) and thus significantly decreased
in the C6ST-1 siRNA-administered group when compared to the
untreated group.
[0385] It was thus demonstrated that the C6ST-1 siRNA-administered
group significantly suppressed the increase in Cst caused by
pulmonary emphysema induced by intratracheal administration of PPE.
Furthermore, the result described in this Example suggests that the
suppression of C6ST-1 gene expression not only suppresses the
alveolar damage caused by fibrotic changes of the interstitium of
lung tissue but also improves the actual clinical symptoms
(respiratory conditions).
[0386] The agents of the present invention are thus useful, for
example, as agents for suppressing alveolar damage.
[Example 17] Tissue-Preserving Effect of C6ST-1 siRNA in a Mouse
Pulmonary Emphysema Model
[0387] In pulmonary emphysema, whose characteristic pathological
feature is enlarged alveolar air space, the lung capacity is
increased with progression of emphysematous lesions. The purpose of
this Example is to prove that the therapeutic effect of C6ST-1
siRNA is not only suppression of damage at the cell level but also
the effect of morphological maintenance and preservation of the
organ.
[0388] The pulmonary tissues used in this Example were the right
lungs of the same lung tissues as used in Example 12. The lung
tissues isolated from mice were gently washed with phosphate buffer
and then immersed into phosphate buffer saturated in a glass
container. The glass container filled with phosphate buffer was
weighed in advance. After the lung tissues were added to the
container, lung capacity was calculated by converting the increased
weight into a liquid volume.
[0389] The result of this Example is shown in FIG. 17. The lung
capacity was 277.5.+-.61.85 .mu.l in the control group, while it
was 413.33.+-.77.67 .mu.l in the untreated group (when compared to
the control group, P=0.024; t-test). Thus, a statistically
significant increase of lung capacity was observed in the untreated
group. In contrast, the lung capacity was 292.5.+-.51.23 .mu.l and
thus significantly decreased in the C6ST-1 siRNA-treated group
(when compared to the untreated group P=0.027; t-test) when
compared to the untreated group.
[0390] These results revealed that the inhibition of C6ST-1 gene
expression effectively suppresses the increase in the lung capacity
associated with pulmonary emphysema induced by intratracheal
administration of PPE. This suggests that the effect is not only
the suppression of the damage at the cell level but also the effect
of morphological maintenance and preservation of the organ or
effect of repairing damaged tissues.
[0391] The agents of the present invention are thus useful, for
example, as agents for suppressing the increase of lung capacity
caused by pulmonary emphysema.
[Example 18] Pulmonary Interstitial Fibrosis-Suppressing Effect of
GalNAcST siRNA in a Mouse Pulmonary Emphysema Model
[0392] The importance of sulfation at position 4 and 6 is
demonstrated with an additional Example. In this Example, the
expression of fibrosis-related genes in the pulmonary alveolar
interstitium after GalNAcST siRNA administration was assessed by
quantitative real-time PCR method using the same method as
described in Example 11. The siRNA sequences are the same as shown
in Example 11.
[0393] The pulmonary emphysema model was prepared by the same
method as described in Example 12. A part of the collected lung
tissues were placed into 1.5-ml tubes, and frozen with liquid
nitrogen. cDNA synthesis was carried out by the same method as
described in Example 1, and assessed by quantitative PCR method.
The primer sequences and of PCR conditions were the same as
described in Examples 1 and 13. The primer sequences for TGF-.beta.
are shown below.
TABLE-US-00006 [Primer sequences used in quantitative PCR] *mouse
TGF-.beta. (Takara Bio Inc.) Forward: (SEQ ID NO: 53)
5'-GTGTGGAGCAACATGTGGAACTCTA-3' Reverse: (SEQ ID NO: 54)
5'-TTGGTTCAGCCACTGCCGTA-3'
[0394] The result is shown in FIG. 18. The enhanced expression of
type I collagen, .alpha.-SMA, and TGF-3 as indicators for
fibrogenesis were also significantly suppressed by GalNAcST siRNA
(for all genes, p<0.01; t-test) in this Example. This result
demonstrates that the suppression of GAlNAc4ST-1, GAlNAc4ST-2, and
GAlNAc4S-6ST expression can effectively inhibit the enhanced
fibrotic changes of pulmonary interstitium.
[0395] The agents of the present invention are thus useful, for
example, as agents for suppressing fibrotic changes in lung
interstitium.
[Example 19] Respiratory Function-Preserving Effect of GalNAcST
siRNA in a Mouse Pulmonary Emphysema Model
[0396] In this Example, GAlNAcST siRNA was assessed for its
influence on respiratory function using static lung compliance
(static compliance (Cst)) as an indicator to evaluate the clinical
effect of GAlNAcST siRNA in pulmonary emphysema model mice.
[0397] Pulmonary emphysema model mice were prepared by the same
procedure described in Example 12, and then treated with GAlNAcST
siRNA. The spontaneous respiration of mice was ceased by an
anesthetic agent, and then their Cst was monitored using FlexiVent
(SCIREQ) respiratory function analyzer in the PV loop mode. Mice
were connected to the FlexiVent by the following procedure: a
median incision was performed after stopping spontaneous
respiration, and then a special cannula was inserted into the
trachea, which was followed by peribronchial ligation.
[0398] The result of this Example is shown in FIG. 18. Cst was
42.62.+-.2.25 .mu.l/cm H2O in the control group, while it was
51.22.+-.5.2 .mu.l/cm H2O in the untreated group (when compared to
the control group, P=0.03; t test). Thus, a statistically
significant increase was observed in the untreated group. In
contrast, Cst was 44.15.+-.2.29 .mu.l/cm H2O (when compared to the
untreated group, P=0.0018; t-test) and thus significantly decreased
in the C6ST-1 siRNA-administered group when compared to the
untreated group.
[0399] It was thus demonstrated that the increase in Cst caused by
pulmonary emphysema induced by intratracheal administration of PPE
was significantly suppressed in the GalNAcST siRNA-administered
group. Furthermore, the result described in this Example suggests
that the inhibition of GAlNAc4ST-1, GAlNAc4ST-2, and GAlNAc4S-6ST
gene expression not only suppresses the alveolar damage caused by
the fibrotic change of interstitium of lung tissue but also
improves the actual clinical symptom (respiratory condition).
[0400] The agents of the present invention are thus useful, for
example, as agents for suppressing alveolar damage or as an agent
for improving respiratory conditions.
[Example 20] Tissue-Preserving Effect of GalNAcST siRNA in a Mouse
Pulmonary Emphysema Model
[0401] The objective of this Example is to prove that the
therapeutic effect of GalNAcST siRNA is not only the suppression at
the cell level but also the effect of morphological maintenance and
preservation of the organ.
[0402] The pulmonary tissues used in this Example were the right
lungs of the same lung tissues as used in Example 12. The lung
tissues isolated from mice were gently washed with phosphate buffer
and then immersed in phosphate buffer saturated in a glass
container. The glass container filled with phosphate buffer was
weighed in advance. After the lung tissues were added to the
container, lung capacity was calculated by converting the increased
weight into a liquid volume.
[0403] The result of this Example is shown in FIG. 20. The lung
capacity was 277.5.+-.61.85 .mu.l in the control group, while it
was 413.33.+-.77.67 .mu.l in the untreated group (when compared to
the control group, P=0.024; t-test). Thus, a statistically
significant increase of lung capacity was observed in the untreated
group. In contrast, the lung capacity was 315.+-.51.96 .mu.l and
thus significantly decreased in the GalNAcST siRNA-treated group
(when compared to the untreated group P=0.049; t-test).
[0404] When taken together, these results reveal that the
inhibition of GAlNAc4ST-1, GAlNAc4ST-2, and GAlNAc4S-6ST gene
expressions effectively suppresses the increase in the lung
capacity associated with pulmonary emphysema induced by
intratracheal administration of PPE. This suggests that the effect
is not only the suppression of the damage at the cell level but
also the effect of maintaining and preserving the morphology of the
organ or the effect of repairing damaged tissues.
[0405] The agents of the present invention are thus useful, for
example, as agents for maintaining or preserving lung
morphology.
[Pancreatic Tissue]
[0406] The Examples below describe preparation of a type 2 diabetes
model by administration of Streptozotocin to C57BL/6JcL mice
(female; CLEA Japan Inc.) on day 2 after birth and assessment of
the mice for weight and blood glucose changes, gene expression, and
fibrotic change of pancreatic tissues caused by treatment with
chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 1 (C4ST-1)
siRNA, chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 2
(C4ST-2) siRNA, and chondroitin
D-N-acetylgalactosamine-4-O-sulfotransferase 3 (C4ST-3) siRNA. Each
siRNA was administered by the same method as described in Example
1. Sequences are shown below.
TABLE-US-00007 *[C4ST-1 siRNA cocktail sequences] C4ST1
(Chondroitin D-N-acetylgalactosamine-4-O- sulfotransferase 1)
(GenBank accession number NM_021439) (SEQ ID NO: 55)
5'-ACAAAGCCATGAAGCCGGCGCTGCTGGAAGTGATGAGGATGAACAGA ATT-3' (SEQ ID
NO: 56) 5'-CAACCTGAAGACCCTTAACCAGTACA-3' (SEQ ID NO: 57)
5'-GCATCCCAGAGATCAACCACCGCTTG-3' *[C4ST-2 siRNA cocktail sequences]
C4ST2 (Chondroitin D-N-acetylgalactosamine-4-O- sulfotransferase 2)
(GenBank accession number NM_021528) (SEQ ID NO: 58)
5'-GCCAGGAGTGGGCCCAGCCCAGGGC-3' (SEQ ID NO: 59)
5'-ATGACCAAGCCGCGGCTCTTCCGGCTG-3' (SEQ ID NO: 60)
5'-AGAGCCTGCTGGACCGGGGCAGCCCCTA-3' (SEQ ID NO: 61)
5'-GAGACCCCCTGGACATCCCCCGGGAACA-3' *[C4ST-3 siRNA cocktail
sequences] C4ST3 (Chondroitin D-N-acetylgalactosamine-4-O-
sulfotransferase 3) (GenBank accession number XM_355798) (SEQ ID
NO: 62) 5'-ATGACTGTCGCCTGCCACGCGTGCCA-3' (SEQ ID NO: 63)
5'-CAGCATGGGAAGACGCTCCTGTTGCA-3' (SEQ ID NO: 64)
5'-TCCAAGCGCAATCCCTGCGCACGAGGCG-3' (SEQ ID NO: 65)
5'-GCCTGGCCTGCTGCCCTCGCTGGCC-3'
[0407] First, sample preparation was conducted as described
below.
[Example 21] Assessment of Anti-Obesity Effect of C4ST-1, C4ST-2,
and C4ST-3 siRNA Treatment in Streptozocin-Induced C57BL/6JcL Type
2 Diabetes Model Mice
[0408] Gestational Day 14 C57BL/6JcL mice (CLEA Japan Inc.) were
reared and allowed to deliver. 10 mg/ml Streptozocin (SIGMA) was
subcutaneously administered at l/head to Day 2 postnatal female
C57BL/6JcL mice. The mice were reared with sterile water and CE-2
Diet (CLEA Japan Inc.) until they were four weeks old, and then on
sterile water and a High Fat Diet (CLEA Japan Inc.) for the next
two weeks. On the second week, 1 .mu.g of a mixture of chondroitin
D-N-acaetylgalactosamine-4-O-sulfotransferase 1 (C4ST-1),
chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 2
(C4ST-2), and chondroitin
D-N-acetylgalactosamine-4-O-sulfotransferase 3 (C4ST-3) siRNAs
(GeneWorld) were combined with 1% atelocollagen (Koken Co.), which
is a vehicle for siRNA, and 200 .mu.l of the mixture was
administered into peritoneal cavities once a week, twice in total
(for two weeks). On Day 14 of this experiment, 100 .mu.l of 5 mg/ml
BrdU (ZyMED Laboratory Inc.) was administered into the tail vein,
and one hour after administration the mice were dissected and the
liver, pancreas, kidney, testis, ovary, and muscle were isolated to
obtain samples for immunostaining and gene expression analysis. The
body weight change was monitored over time for 14 days of this
Example.
[0409] The result is shown in FIG. 21. The vertical axis indicates
the body weight (g), while the horizontal axis indicates the
monitoring days. FIG. 21 shows that the weight increase tended to
be suppressed 10 days after treatment in the C4ST-1 siRNA-treated
group, C4ST-2 siRNA-treated group, and C4ST-3 siRNA-treated group
as compared to the control group. The weight increase was
significantly suppressed on day 18 in the C4ST-2 siRNA-treated
group and C4ST-3 siRNA-treated group (both groups; p<0.05). This
result suggests that obesity associated with type 2 diabetes can be
suppressed by inhibiting the expression of C4ST-1, C4ST-2, and
C4ST-3.
[0410] The agents of the present invention are thus useful, for
example, as agents for suppressing body weight increase, or as
agents for suppressing obesity associated with type 2 diabetes.
[Example 22] Assessment of Insulin Resistance after Treatment with
C4ST-1, C4ST-2, and C4ST-3 siRNAs in Streptozocin-Induced
C57BL/6JcL Type 2 Diabetes Model Mice
[0411] As an insulin-tolerance test, human crystalline insulin
(0.75 U/kg) was administered into peritoneal cavities the day
before dissection, and 0, 15, and 60 minutes after administration,
the blood glucose levels were measured and evaluated using a blood
glucose monitor, Glu-Test Ace (BOMBYX Medicine co.). The blood
glucose level changes monitored at 0, 15, and 60 minutes after
siRNA treatment are shown in FIG. 22. The vertical axis indicates
the blood glucose level (mg/dl), while the horizontal axis
indicates 0, 15, and 60 minutes after insulin-tolerance test.
[0412] FIG. 22 shows that the blood glucose level was not
significantly decreased 0 and 15 minutes after treatment in any of
the C4ST-1 siRNA-treated group, C4ST-2 siRNA-treated group, and
C4ST-3 siRNA-treated group as compared to the control group, while
it significantly decreased 60 minutes after treatment in each of
the C4ST-1 siRNA-treated group, C4ST-2 siRNA-treated group, and
C4ST-3 siRNA-treated group. This result suggests that insulin
resistance, which is an essential functional disorder in type 2
diabetes, can be effectively improved by suppressing the expression
of C4ST-1, C4ST-2, and C4ST-3.
[0413] The agents of the present invention are thus useful, for
example, as agents for improving insulin resistance in type 2
diabetes.
[Example 23] Assessment of Gene Expression in Pancreatic Tissues
after Treatment with C4ST-1, C4ST-2, and C4ST-3 siRNAs in
Streptozocin-Induced C57BL/6JcL Type 2 Diabetes Model Mice
[0414] cDNA was prepared by the same method as described in Example
1 from 50 mg of each organ (pancreas) isolated from
Streptozocin-induced female C57BL/6JcL mice. PCR was conducted in
the following composition.
[0415] 2 .mu.l of PCR Buffer [composition: 166 mM
(NH.sub.4).sub.2SO.sub.4 (Sigma Aldrich Japan), 670 mM Tris pH8.8
(Invitrogen), 67 mM MgCl.sub.2.6H.sub.2O (Sigma Aldrich Japan), 100
mM 2-mercaptoethanol (WAKO)], 0.8 .mu.l of 25 mM dNTP mix
(Invitrogen), 0.6 .mu.l of DMSO (Sigma Aldrich Japan), 0.2 .mu.l of
Primer Forward (GeneWorld), 0.2 .mu.l of Primer Reverse
(GeneWorld), 15.7 .mu.l of Otsuka distilled water (Otsuka
Pharmaceuticals, Inc.), 0.1 .mu.l of Taq polymerase (Perkin Elmer),
and 1 .mu.l of cDNA obtained as described above were combined, and
reacted using Authorized Thermal Cycler (eppendorf) at 35 cycles of
94.degree. C. for 45 seconds, 56.degree. C. for 45 seconds, and
72.degree. C. for 60 seconds. After the reaction, the obtained PCR
products were combined with 2 .mu.l of Loading Dye (Invitrogen).
1.5% agarose gel was prepared using UltraPure Agarose (Invitrogen),
and the samples were electrophoresed in a Mupid-2 plus (ADVANCE) at
100 V for 20 minutes. After electrophoresis, the gel was shaken for
20-30 minutes in a stain solution prepared by 10,000 times diluting
Ethidium Bromide (Invitrogen) with 1.times. LoTE (composition: 3 mM
Tris-HCl (pH 7.5) (Invitrogen), 0.2 mM EDTA (pH 7.5) (Sigma Aldrich
Japan)). The gel was photographed with EXILIM (CASIO) positioned on
I-Scope WD (ADVANCE) and the gene expression was confirmed.
[0416] Primers (Forward and Reverse) (GeneWorld) used are described
in the following.
TABLE-US-00008 [Primer sequences] *GAPDH Forward: (SEQ ID NO: 66)
5'-CTGCCAAGTATGACATCA-3' Reverse: (SEQ ID NO: 67)
5'-TACTCCTTGGAGGCCATGTAG-3' *C4ST1 (Chondroitin
D-N-acetylgalactosamine-4-O- sulfotransferase 1) Forward: (SEQ ID
NO: 68) 5'-gtggatgaggaccacgaact-3' Reverse: (SEQ ID NO: 69)
5'-cttttcaagcggtggttgat-3' *C4ST2 (Chondroitin
D-N-acetylgalactosamine-4-O- sulfotransferase 2) Forward: (SEQ ID
NO: 70) 5'-acctcctagacccacacacg-3' Reverse: (SEQ ID NO: 71)
5'-ggatgttggcaaaccagtct-3' *C4ST3 (Chondroitin
D-N-acetylgalactosamine-4-O- sulfotransferase 3) Forward: (SEQ ID
NO: 72) 5'-atgagcccttcaacgaacac-3' Reverse: (SEQ ID NO: 73)
5'-tggtagaaggggctgatgtc-3'
[0417] Result is shown in FIG. 23. GAPDH expression, which is used
as a positive control, was confirmed by RT-PCR in the control
group, C4ST1 (Chondroitin
D-N-acetylgalactosamine-4-O-sulfotransferase 1), C4ST2 (Chondroitin
D-N-acetylgalactosamine-4-O-sulfotransferase 2), and C4ST3
(Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 3).
Compared to the control group, expression was reduced in C4ST1
(Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 1)
siRNA-, C4ST2 (Chondroitin
D-N-acetylgalactosamine-4-O-sulfotransferase 2) siRNA-, C4ST3
(Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 3)
siRNA-treated groups, and C4ST1 (Chondroitin
D-N-acetylgalactosamine-4-O-sulfotransferase 1), C4ST2 (Chondroitin
D-N-acetylgalactosamine-4-O-sulfotransferase 2), C4ST3 (Chondroitin
D-N-acetylgalactosamine-4-O-sulfotransferase 3) gene knockdown was
confirmed by administration of Atellocollagen-mediated C4ST1
(Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 1) siRNA,
C4ST2 (Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 2)
siRNA, C4ST3 (Chondroitin
D-N-acetylgalactosamine-4-O-sulfotransferase 3) siRNA.
[Example 24] Assessment of Accumulation of Amyloid Precursor
Protein in Pancreas after Treatment with C4ST-1, C4ST-2, and C4ST-3
siRNAs in Streptozocin-Induced C57BL/6JcL Type 2 Diabetes Model
Mice
[0418] In this Example, the amyloid precursor protein (APP)
deposition-suppressing effect of C4ST-1, C4ST-2, and C4ST-3 siRNAs
was assessed using pancreatic tissue samples of type 2 diabetes
model mice. The deposition of APP and amyloid fibers in islets
including 3 cells has long been known to be an important
histopathological feature of type 2 diabetes. In recent years, APP
has been demonstrated to be involved in Alzheimer's disease
(Johnson K H et al. N Eng J Med 321: 513, 1989; Rhodes C J. Science
307: 380, 2005; Haan M N. Nat Clin Pract Neurol 3: 159, 2006;
Prentki M et al. J Clin Invest 116: 1802, 2006).
[0419] The prepared sections of tissue samples were stained with a
goat anti-amyloid precursor protein antibody (Calbiochem) by the
same method as described in Example 3 to assess its expression at
the tissue level. FIG. 24 shows histological images of the normal
mouse group, control group, and C4ST-2 siRNA-treated group. The APP
deposition is enhanced in the islets of type 2 diabetes model mice.
The deposition was demonstrated to be clearly suppressed in the
C4ST-2 siRNA-treated group as compared to the control group.
[0420] The agents of the present invention are thus useful, for
example, as agents for suppressing amyloid fiber deposition.
[Example 25] Histological Fibroblast Infiltration-Suppressing
Effect of C4ST-1, C4ST-2, and C4ST-3 siRNA Treatment in
Streptozocin-Induced C57BL/6JcL Type 2 Diabetes Model Mice
[0421] Cryoblocks and tissue sections were prepared from the
collected pancreatic tissues by the same method as described in
Example 3. The resulting sections were fixed with acetone (Wako
Pure Chemical Industries) for ten minutes, and then washed with
phosphate buffer. An anti-ER-TR7 antibody (rat monoclonal antibody,
1 .mu.g/ml; BMA) was added as the primary antibody, and the
sections were incubated at room temperature for one hour. Then, the
secondary antibody reaction was carried out by adding a
peroxidase-labeled anti-rat IgG antibody (1:200 dilution), and
color development was performed by adding DAB substrate (Nichirei
Biosciences). Then, the nucleus was stained by Lillie-Mayer
hematoxylin (Muto Pure Chemicals Co.). The samples were observed
under a light microscope (Leica Microsystems). The antibody binding
was detected by visualizing it as brown signals.
[0422] The result showed that the fibroblast infiltration to
pancreatic islets was significantly suppressed in each of the
C4ST-1 siRNA-, C4ST-2 siRNA-, and C4ST-3 siRNA-treated groups as
compared to the control group (FIG. 25). This result demonstrates
that the inhibition of C4ST-1, C4ST-2, and C4ST-3 gene expression
reduces the enhanced tissue fibrotic change by suppressing the
fibroblast infiltration and retention in islets containing 3
cells.
[0423] The agents of the present invention are thus useful, for
example, as agents for suppressing fibroblast infiltration to
islets.
[Example 26] Histological Macrophage Infiltration-Suppressing
Effect of Treatment with C4ST-1, C4ST-2, and C4ST-3 siRNAs in
Streptozocin-Induced C57BL/6JcL Type 2 Diabetes Model Mice
[0424] Cryoblocks and tissue sections were prepared from the
collected pancreatic tissues by the same method as described in
Example 3. The resulting sections were fixed with acetone (Wako
Pure Chemical Industries) for ten minutes, and then washed with
phosphate buffer. An anti-F4/80 antibody (clone A3-1, rat
monoclonal antibody, 2 .mu.g/ml; CALTAG LABORATORIES) was added as
the primary antibody, and the sections were incubated at room
temperature for one hour. Then, the secondary antibody reaction was
carried out by adding a peroxidase-labeled anti-rat IgG antibody
(1:200 dilution), and color development was performed by adding DAB
substrate (Nichirei). Then, the nucleus was stained by Lillie-Mayer
hematoxylin (Muto Pure Chemicals Co.). The samples were observed
under a light microscope (Leica Microsystems). The antibody binding
was detected by visualizing it as brown signals.
[0425] The result showed that the macrophage infiltration to the
pancreatic islets was significantly suppressed in each of the
C4ST-1 siRNA-, C4ST-2 siRNA-, and C4ST-3 siRNA-treated groups as
compared to the control group (FIG. 26). This finding demonstrates
that the suppression of C4ST-1, C4ST-2, and C4st-3 gene expression
resulted in general inhibition of the tissue fibrotic changes via
suppression of the infiltration of macrophages and fibroblasts,
which are cell groups responsible for the persistent or enhanced
fibrotic changes.
[0426] The agents of the present invention are thus useful, for
example, as agents for suppressing the infiltration of macrophages
to the pancreatic islets.
[Example 27] Assessment of Insulin Resistance after GalNAcST siRNA
Treatment in Streptozocin-Induced C57BL/6JcL Type 2 Diabetes Model
Mice
[0427] The importance of sulfation at position 4 and 6 is
demonstrated with an additional Example. In this Example, the
improvement of insulin resistance by GalNAcST siRNA administration
is assessed using the same methods as described in Examples 11 and
18. The type 2 diabetes model was prepared by the same method as
described in Example 21, and the insulin resistance was tested by
the same method as described in Example 22. The result is shown in
FIG. 27.
[0428] GalNAcST siRNA administration exhibited a good
antihyperglycemic effect after insulin loading. This result
suggests that the insulin resistance arising from fibrotic changes
in islets of pancreatic tissues can be effectively improved by
suppressing the expression of GalNAc4ST-1, GalNAc4ST-2, and
GalNAc4S-6ST.
[0429] The agents of the present invention are thus useful, for
example, as hypoglycemic agents or as agents for improving the
insulin resistance of pancreatic tissue.
[Kidney Tissue]
[0430] Fibrotic changes of kidney tissue are thought to be the
endpoints of various kidney diseases. (1) From a classical point of
view, tubulointerstitial disease is understood as a representative
disease caused by renal fibrogenesis. This disease includes
Sjogren's syndrome, transplant rejection (chronic allograft
nephropathy, etc.), and graft-versus-host reaction
(graft-versus-host disease, etc.), in addition to drug-induced,
infective, radiation-induced, and heavy metal-induced interstitial
renal disorders. (2) Renal fibrogenesis also includes renovascular
disease. Renovascular disease includes nephrosclerosis associated
with hypertension. In recent years, this disease also includes
fibrogenesis associated with arteriosclerosis and metabolic
syndrome. Furthermore, interstitial fibrogenesis also occurs as a
result of proteinuria or the like in primary glomerular disease,
which leads to renal failure. Thus, renal fibrotic changes can also
be developed in: (3) primary glomerular disease and (4) secondary
glomerular diseases. The diseases of (3) include IgA nephropathy,
minimal lesion nephrotic syndrome, membranous nephropathy,
membranoproliferative glomerulonephritis, and focal segmental
glomerulosclerosis (FGFS). The diseases of (4) include diabetic
nephropathy, lupus nephritis associated with systemic lupus
erythematosus (SLE), nephropathy associated with chronic rheumatoid
arthritis, amyloid nephropathy, and nephropathy associated with
type B or C hepatitis. (5) Finally, renal fibrogenesis also
includes interstitial kidney diseases caused by urinary
obstruction, including urinary lithiasis, tumors, and neurogenic
bladder dysfunctions.
[0431] Recently, the group of diseases listed above has been
collectively named "chronic kidney disease (CKD)" as a clinical
concept which is classified and diagnosed based on the decree of
kidney function. CKD gradually progresses through interstitial
fibrotic changes, leading to chronic renal failure, and then
end-stage renal disease (ESRD). Conventionally, only dialysis has
been a definitive treatment for ESRD. Although the possibility of
slowing down progression to ESRD has been suggested by using an
antihypertensive agent that act on the angiotensin system,
therapeutic methods that target renal fibrogenesis itself have not
been established. There is a desperate need for new CKD treatment.
In terms of the progression process of pathological conditions,
treatment targeting fibrotic changes is of the greatest
significance as a common therapeutic strategy regardless of the
type of primary disease. It has been reported that there are 500
million CKD patients worldwide. It is predicted that the number of
patients will continue to increase in the future due to altered
lifestyle habits. In addition, a large-scale trial revealed the
very high risk of death from a cardiovascular disorder before
receiving dialysis. Thus, considering CKD as a disease of the 21st
century, active treatment of CKD is a significant challenge in the
medical community (Sergio A et al., Hypertension 38: 635, 2001;
Weiner D E et al., JASN 15: 1307, 2004; Anavekar N S et al., N Eng
J Med 351: 1285, 2004; Remuzzi G et al., J Clin Invest 116: 288,
2006; Tonelli M et al., BMJ 332: 1426, 2006; Khwaja A et al.,
Kidney International doi: 10.1038/sj.ki.5002489).
[0432] In the next Examples, the inhibition of sugar chain-related
genes that modify the sulfate group at position 4 or 6 was assessed
for the effect on histological fibrotic changes in the renal
interstitium.
[Example 28] Assessment of Anti-Fibrogenic Effect of C4ST-1 siRNA
in a Mouse Diabetic Nephropathy Model
[0433] A type 2 diabetes model was prepared by the same method as
described in Example 21. Gestational Day 14 of C57BL/6JcL mice
(CLEA Japan Inc.) were reared and allowed to deliver.
Streptozotocin (STZ; Sigma) was administered to Day 2 postnatal
C57BL/6JcL mice to prepare a STZ-induced diabetes model. The mice
were subcutaneously injected with 20 .mu.l of STZ (10 mg/ml) three
times for two days. Thus, a total of 60 .mu.l was administered to
the mice. Together with their mothers, the mice were fed with a
normal diet until they are four weeks old. After weanling at the
age of four completed weeks, the mice were fed with a High Fat Diet
(CLEA Japan Inc.) for two weeks. In the second week, 1 .mu.g of
chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 1 (C4ST-1)
siRNA (GeneWorld) was combined with 0.1% atelocollagen (Koken Co.),
which is a vehicle for siRNA, and 200 .mu.l of the resulting
mixture was administered into peritoneal cavities once a week (one
shot/week) twice in total (for two weeks). On day 14 of this
experiment, the mice were dissected and their kidneys were isolated
to obtain samples for immunostaining. Gene expression, body weight,
and effect on insulin resistance are described in Examples 21, 22,
and 23.
[0434] The prepared sections of kidney tissue samples were fixed
with acetone (Wako Pure Chemical Industries) for ten minutes, and
then washed with phosphate buffer. An anti-F4/80 antibody (clone
A3-1, rat monoclonal antibody, 2 .mu.g/ml; CALTAG LABORATORIES) was
added as the primary antibody, and the sections were incubated at
room temperature for one hour. Then, the secondary antibody
reaction was carried out by adding a peroxidase-labeled anti-rat
IgG antibody (1:200 dilution), and color development was performed
by adding DAB substrate (Nichirei Biosciences). Then, the nucleus
was stained by Lillie-Mayer hematoxylin (Muto Pure Chemicals Co.).
The samples were observed under a light microscope (Leica
Microsystems). The antibody binding was detected by visualizing it
as brown signals.
[0435] The result showed that the infiltration of fibroblasts
(ER-TR7-positive cells) in the renal cortex and medulla was less in
the C4ST-1 siRNA-treated group as compared to that in the control
group (FIG. 28; the original images are in color). The
ER-TR7-positive cells were counted to quantify the fibrogenesis in
inflammatory cells. Each sample was observed with ten microscopic
optical fields under a microscope at a magnification of 400 fold,
and the number of positive cell were counted and compared between
the control group and C4ST-1 siRNA-treated group. The result showed
that the ER-TR7 positivity ratio was significantly reduced in the
C4ST-1 siRNA-treated group as compared to the control group
(p<0.001).
[Example 29] Assessment of C4ST-1 siRNA for its Effect on
Macrophage Infiltration in a Mouse Diabetic Nephropathy Model
[0436] The prepared sections of kidney tissue samples were fixed
with acetone (Wako Pure Chemical Industries) for ten minutes, and
then washed with phosphate buffer. An anti-ER-TR7 antibody (rat
monoclonal antibody, 1 .mu.g/ml; BMA) was added as the primary
antibody, and the sections were incubated at room temperature for
one hour. Then, the secondary antibody reaction was carried out by
adding a peroxidase-labeled anti-rat IgG antibody (1:200 dilution),
and color development was performed by adding DAB substrate
(Nichirei Biosciences). Then, the nucleus was stained by
Lillie-Mayer hematoxylin (Muto Pure Chemicals Co.). The samples
were observed under a light microscope (Leica Microsystems). The
antibody binding was detected by visualizing it as brown
signals.
[0437] The result showed that the infiltration of macrophages
(F4/80-positive cells) in the renal cortex and medulla was less in
the C4ST-1 siRNA-treated group as compared to that in the control
group (FIG. 29; the original images are in color). Furthermore, the
tissue lesions were quantified by the same method as described in
Example 28. The result showed that the macrophage positivity ratio
was significantly reduced in the C4ST-1 siRNA-treated group as
compared to the control group (p<0.001).
[Example 30] Assessment of C4ST-1 siRNA for Fibroblast Activation
in Tissues in a Mouse Diabetic Nephropathy Model
[0438] The sections of kidney tissue samples were fixed with
acetone (Wako Pure Chemical Industries) for ten minutes, and washed
with phosphate buffer. An anti-human smooth muscle actin antibody
(.alpha.SMA: mouse monoclonal antibody, 1:100; DACO) was added as
the primary antibody, and the sections were incubated at room
temperature for one hour. Then, the secondary antibody reaction was
carried out using Histofine Mouse Stain kit (Nichirei), and color
development was performed by adding DAB substrate (Nichirei
Biosciences). Then, the nucleus was stained by Lillie-Mayer
hematoxylin (Muto Pure Chemicals Co.). The samples were observed
under a light microscope (Leica Microsystems). The antibody binding
was detected by visualizing it as brown signals.
[0439] The result showed that .alpha.SMA-positive cells retained in
juxtaglomerular and interstitial areas were significantly reduced
in the C4ST-1 siRNA-treated group as compared to the control group
(FIG. 30; the original images are in color). .alpha.SMA serves as a
functional marker for fibroblast activation. This result
demonstrates that inhibition of C4ST-1 gene expression suppress
activation of fibroblasts infiltrating in the tissue.
[0440] The agents of the present invention are thus useful, for
example, as fibroblast activation inhibitors.
[Example 31] Assessment of GalNAc4S-6ST siRNA for Tissue Fibrogenic
Alteration in a Mouse Diabetic Nephropathy Model
[0441] The diabetic nephropathy model was prepared by the same
method as described in Example 28 to assess the effect of
GalNAc4S-6ST siRNA. In this Example, the follow-up examination was
carried out over a longer period of time. The sequence of
GalNAc4S-6ST siRNAs was the same as shown in Example 1. The siRNAs
were administered into peritoneal cavities once at the age of eight
weeks and once again at age of nine weeks. Furthermore, as a
control for comparison, Valsartan, an angiotensin II receptor
blocker (ARB), was orally administered at a dose of 30 mg/kg on the
same schedule. Kidney tissues were collected at the age of ten
weeks to conduct immunohistochemical and gene expression tests. To
assess the gene expression in the kidney tissues, quantitative PCR
was carried out by the same method as described in Example 1. In
this Example, 36B4 was used as an internal control. The sequence of
36B4 is shown below.
TABLE-US-00009 [Primer sequences used in quantitative PCR] *mouse
36B4 (Takara Bio) Forward: (SEQ ID NO: 74)
5'-TTCCAGGCTTTGGGCATCA-3' Reverse: (SEQ ID NO: 75)
5'-ATGTTCAGCATGTTCAGCAGTGTG-3'
[0442] The result is shown in FIG. 31 (GalNAc4S-6ST is abbreviated
as G#1 in this figure). In the diabetic nephropathy model, the
expression of GalNAc4S-6ST in kidney tissues is enhanced. The
administration of GalNAc4S-6ST siRNA significantly suppressed not
only the expression of GalNAc4S-6ST gene in kidney tissues but also
the enhanced expression of .alpha.SMA and TGF.beta., which are
fibrosis markers. The therapeutic effect was evaluated by comparing
with that of ARB, and the result showed that the
TGF.beta.-suppressing effect was observed in both groups and there
was no significant difference between the two while the
.alpha.SMA-suppressing effect was significant in the GalNAc4S-6ST
siRNA-administered group as compared to the ARB-administered group.
This result suggests that the markers for fibrotic changes in
kidney tissues can be suppressed by inhibiting the expression of
GalNAc4S-6ST gene. The result also demonstrates that GalNAc4S-6ST
siRNA showed a markedly superior effect in terms of fibroblast
activation (enhancement of .alpha.SMA).
[Example 32] Assessment of GalNAc4S-6ST siRNA for Fibroblast
Infiltration in a Mouse Diabetic Nephropathy Model
[0443] The degree of fibroblast infiltration into kidney tissues
was quantitatively evaluated by conducting an immunohistochemical
study using the same method as described in Example 28. The result
is shown in FIG. 32. Fibroblast infiltration to kidney tissues was
significantly suppressed by GalNAc4S-6ST siRNA administration. A
quantitative evaluation over the entire interstitium did not show
any significant difference between GalNAc4S-6ST siRNA
administration and ARB treatment. Meanwhile, when the evaluation
was restricted to fibroblasts infiltrating in juxtaglomerular
areas, a significant suppressing effect was observed in the
GalNAc4S-6ST siRNA-administered group, as shown in the figure.
[0444] The agents of the present invention are thus useful, for
example, as agents for suppressing fibroblast infiltration into
renal interstitium.
[Example 33] Assessment of GalNAc4S-6ST siRNA in Macrophage
Infiltration in a Mouse Diabetic Nephropathy Model
[0445] The degree of fibroblast infiltration into kidney tissues
was quantitatively evaluated by conducting an immunohistochemical
study using the same method as described in Example 29. The result
is shown in FIG. 33. Macrophage infiltration into kidney tissues
was significantly suppressed by GalNAc4S-6ST administration. A
quantitative evaluation over the entire interstitium did not show
any significant difference between GalNAc4S-6ST siRNA
administration and ARB treatment. Meanwhile, when the evaluation
was restricted to macrophages infiltrating in juxtaglomerular
areas, a significant suppressing effect was observed in the
GalNAc4S-6ST siRNA-administered group, as shown in the figure.
[0446] The agents of the present invention are thus useful, for
example, as agents for suppressing macrophage infiltration to renal
interstitium.
[Example 34] Assessment of GalNAc4S-6ST siRNA in Glomerular
Basement Membrane Thickening in a Mouse Diabetic Nephropathy
Model
[0447] Type IV collagen was immunostained by the same method as
described in Examples 32 and 33. A rabbit anti-mouse type IV
collagen antiserum (LSL) was added as the primary antibody, and the
sections were incubated at room temperature for one hour. Then, the
secondary antibody reaction was carried out by adding a
peroxidase-labeled anti-rabbit IgG antibody (1:200 dilution), and
color development was performed by adding DAB substrate (Nichirei
Biosciences). Then, the nucleus was stained by Lillie-Mayer
hematoxylin (Muto Pure Chemicals Co.). The samples were observed
under a light microscope (Leica Microsystems). The antibody binding
was detected by visualizing it as brown signals.
[0448] The thickness of collagen visualized as brown signals
surrounding glomeruli was measured to quantify the accumulation of
type IV collagen. 15 to 20 glomeruli were assessed for each sample.
The thickness of the thickest portion around a glomerulus was
measured on a display monitor using vernier calipers. The result
showed that glomerular basement membrane (GBM) thickening was
significantly suppressed in the GalNAc4S-6ST siRNA-administered
group as compared to the control group (FIG. 34). The thickening
tended to be suppressed more markedly in the GalNAc4S-6ST
siRNA-administered group as compared to ARB.
[0449] The agents of the present invention are thus useful, for
example, as agents for suppressing glomerular basement membrane
thickening.
[Example 35] Assessment of GalNAc4S-6ST siRNA in the Angiotensin
Pathway in a Mouse Diabetic Nephropathy Model
[0450] Angiotensin II has been reported to be involved in
fibrogenesis in diabetic nephropathy. In this Example, the
angiotensin pathway in kidney tissues was assessed by quantitative
PCR. The expression of angiotensinogen and angiotensin converting
enzyme (ACE) is enhanced in this model (FIG. 35). The enhanced
expression was speculated to be a factor responsible for the renal
enhancement of angiotensin II. The effect on suppressing
angiotensinogen and ACE was observed in the GalNAc4S-6ST
siRNA-administered group (FIG. 35). The result demonstrates that
GalNAc4S-6ST siRNA also produces an improving effect on the
angiotensin pathway through suppression of fibroblast infiltration
and activation by suppressing the GalNAc4S-6ST gene.
[0451] The agents of the present invention are thus useful, for
example, as agents for suppressing the expression of
angiotensinogen or as an angiotensin converting enzyme
inhibitor.
[Example 36] Assessment of GalNAc4S-6ST siRNA in Serum Creatinine
Concentration in a Mouse Diabetic Nephropathy Model
[0452] Serum creatinine is a most commonly used clinical marker for
renal function. The serum creatinine concentration is elevated due
to impaired renal function during the process from diabetic
nephropathy to ESRD. However, it has been revealed that functional
nephrons are already functionally impaired by 50% when the serum
creatinine level is elevated. Protecting renal function before the
creatinine level increases is a clinically important challenge.
[0453] In this model as well, elevation of serum creatinine level
was observed eventually at the age of 18 weeks when histological
fibrogenesis had already progressed (FIG. 36). The elevation of
serum creatinine was suppressed in the GalNAc4S-6ST
siRNA-administered group as compared to the control group (FIG.
36). This result suggests that inhibition of the GalNAc4S-6ST gene
results in suppression of fibrotic changes of renal tissue and
thereby suppresses the elevation of serum creatinine, i.e., the
deterioration of renal function. Thus, suppression of the
GalNAc4S-6ST gene produces a very beneficial renal protective
effect.
[0454] The agents of the present invention are thus useful, for
example, as agents for suppressing the deterioration of renal
function, or as a renal protective agent.
[Example 37] Assessment of Anti-Fibrogenic Effect of GalNAcST siRNA
in a Mouse Diabetic Nephropathy Model
[0455] The importance of sulfation at position 4 and 6 is shown
with an additional Example. The effect of GalNAcST siRNA
administration on fibrotic changes in the renal interstitium was
assessed by the same method as described in Example 11. The
schedule of GalNAcST siRNA administration is the same as described
in Example 28. Quantitative PCR was carried out using kidney
tissues by the same method as described Example 31. In this
Example, .beta.-actin was used as an internal control. The sequence
of .beta.-actin is shown below.
TABLE-US-00010 [Quantitative PCT primer sequences] *mouse .beta.
actin (Takara Bio) Forward: (SEQ ID NO: 76)
5'-CATCCGTAAAGACCTCTATGCCAAC-3' Reverse: (SEQ ID NO: 77)
5'-ATGGAGCCACCGATCCACA-3'
[0456] As shown in FIG. 37, the expression of GalNAc4ST-1,
GalNAc4ST-2, and GalNAc4S-6ST was enhanced in kidney tissues in the
diabetic nephropathy model. The expression of all the genes was
significantly suppressed by administering GalNAcST siRNA.
[Example 38] Assessment of Anti-Fibrogenic and Kidney-Protecting
Effects of GalNAcST siRNA in a Mouse Diabetic Nephropathy Model
[0457] The enhanced expression of .alpha.SMA, TGF.beta., and CTGF,
which are fibrogenesis markers for kidney tissue fibrogenesis, was
significantly suppressed in the GalNAcST siRNA-administered group
as compared to the control group (FIG. 38). The enhanced expression
of ACE was also significantly suppressed in the GalNAcST
siRNA-administered group. This result demonstrates that inhibition
of GalNAc4ST-1, GalNAc4ST-2, and GalNAc4S-6ST expression results in
suppression of kidney tissue fibrogenesis and renal protection.
Together with the result of Example 35, the fact that ACE was
markedly reduced by suppressing any of the genes demonstrates that
they have a hypotensive effect.
[0458] The agents of the present invention are thus useful, for
example, as antihypertensive agents.
[Example 39] Assessment of GalNAc4S-6ST siRNA for Gene Expression
in a Mouse Drug-Induced Interstitial Nephritis Model
[0459] This Example assesses the effect of GalNAc4S-6ST siRNA on a
typical drug-induced interstitial nephritis. First, the mouse model
is prepared as described below. Adriamycin (15 mg/kg; Kyowa Hakko)
was administered to the peritoneal cavities of C57BL6/J mice (male,
eight weeks old, CLEA Japan Inc.). The mice were reared for one
week after administration, and then kidney tissues were collected
from them. As a control group, mice of the same lineage and age
were also purchased and reared in the same period, but Adriamycin
was not given to them.
[0460] GalNac 4S-6ST siRNA was administered by the same method as
described in Example 1: 1 .mu.g of GalNac 4S-6ST siRNA (Hokkaido
System Science Co.) was combined with 200 .mu.l of 1% atelocollagen
(Koken Co.), which is a vehicle, and the resulting mixture was
administered intraperitoneally to each mouse 24 hours before
Adriamycin administration. The expression of GalNac4S-6ST is also
enhanced in kidney tissues in the typical drug-induced interstitial
nephritis model described in this Example (FIG. 39). The expression
was significantly suppressed by administering GalNac4S-6ST
siRNA.
[Example 40] Assessment of GalNAc4S-6ST siRNA in Gene Expression in
a Mouse Drug-Induced Interstitial Nephritis Model
[0461] In this Example, immunostaining of type I collagen was
carried out by the same method as described in Example 34. A rabbit
anti-rat type I collagen antiserum (LSL) was added as the primary
antibody, and the sections were incubated at room temperature for
one hour. Then, the secondary antibody reaction was carried out by
adding a peroxidase-labeled anti-rabbit IgG antibody (1:200
dilution), and color development was performed by adding DAB
substrate (Nichirei Biosciences). Then, the nucleus was stained by
Lillie-Mayer hematoxylin (Muto Pure Chemicals Co.). The samples
were observed under a light microscope (Leica Microsystems). The
antibody binding was detected by visualizing it as brown
signals.
[0462] The result is shown in FIG. 40. Diffuse deposition of type I
collagen was observed over juxtaglomerular and interstitial areas
in the control group, while the deposition was markedly suppressed
in the GalNac4S-6ST-administered group. This result suggests that
fibrotic changes in kidney tissues can also be suppressed by
suppressing the expression of GalNac4S-6ST in the drug-induced
interstitial nephritis.
[0463] The agents of the present invention are thus useful, for
example, as agents for suppressing fibrotic changes in kidney
tissues.
[Example 41] Assessment of Anti-Fibrosis Effect of C6ST siRNA in
Renal Fibrosis Model Mice Induced by Unilateral Ureteral
Obstruction (UUO)
[0464] First, a mouse model for renal fibrosis is prepared as
described below. Renal fibrosis model mouse was prepared by
conducting unilateral ureteral obstruction (UUO) to C57BL/6JcL mice
(female, eight weeks old; CLEA Japan Inc.). This model has
excellent reproducibility, and is thus widely used as an
experimental mouse renal fibrosis model (American Journal of
Pathology 2003 163 (4): 1261-1273). Mice were subjected to
laparotomy under Ketalar/xylazine anesthesia. The ureters were
exposed and the right ureter was ligated at two sites with 4-0
surgical suture. The peritoneum and skin were closed with 1-0
surgical suture.
[0465] The effect of inhibiting C6ST expression by C6ST siRNA
administration was checked by PCR method using renal fibrosis model
mice prepared by unilateral ureteral obstruction (UUO), as a
typical example of renal fibrosis model mice. The renal fibrosis
model was prepared by conducting UUO to C57BL/6JcL mice (female,
eight weeks old; CLEA Japan Inc.). A mixture of C6ST-1 and C6ST-2
siRNAs (1 .mu.g/head; GeneWorld) or PBS was combined with 0.1%
atelocollagen (Koken Co.), which is a vehicle for siRNA, and 200
.mu.l of the resulting mixture was injected into the peritoneal
cavity of each mouse. Groups of mice treated as described above
were named C6ST siRNA group and control group. On day 8 of the
experiment, the mice were dissected to excise the UUO-treated
kidney. Thus, samples for immunostaining and gene expression
analysis were obtained from the mice. Quantitative PCR was carried
out by the same method as described in Example 1.
[0466] C6ST-1 siRNA, C6ST-2 primers (Forward, Reverse) (GeneWorld)
used herein are shown below.
TABLE-US-00011 [Primer sequences] *C6ST1 (Chondroitin
6-sulfotransferase-1) Forward: (SEQ ID NO: 78)
5'-tgtgtggacacacctcccta-3' Reverse: (SEQ ID NO: 79)
5'-cttcaaaggtccccttcctc-3' *C6ST2 (Chondroitin
6-sulfotransferase-2) Forward: (SEQ ID NO: 80)
5'-cagcttgagccatttcaaca-3' Reverse: (SEQ ID NO: 81)
5'-gggtgaggcctttaggaaac-3' [C6ST-1 cocktail sequences] (Gene Bank
accession number NM_016803) (GeneWorld) (SEQ ID NO: 82)
5'-gcgccccctctccccatggagaaag-3' (SEQ ID NO: 83)
5'-gctttgcctcaggatttccgggacc-3' (SEQ ID NO: 84)
5'-ggttcagccttggtctaccgtgatgtc-3' (SEQ ID NO: 85)
5'-gcagttgttgctatgcgacctgtat-3' [C6ST-2 cocktail sequences] (Gene
Bank accession number NM_021715) (GeneWorld) (SEQ ID NO: 86)
5'-tggggagagtgaggattcggtgaa-3' (SEQ ID NO: 87)
5'-cggacgtgggactcgtcgaggacaaag-3' (SEQ ID NO: 88)
5'-cgaaagtacctgcccgcccgtttcgc-3'
[0467] The result showed that the expression of C6ST-2 (G#10) was
enhanced in the kidney (FIG. 41; C6ST-2 is abbreviated as G#10).
The expression level was decreased in the C6ST siRNA-treated group.
The C6ST-2 gene knockdown was confirmed in the
atelocollagen-mediated C6ST siRNA administration (FIG. 41). The
C6ST siRNA administration also significantly suppressed the
enhanced expression of fibrogenesis markers: TGF.beta., .alpha.SMA,
type I collagen, and CTGF.
[Example 42] Assessment of C6ST siRNA for Fibroblast Infiltration
in Renal Fibrosis Model Mice Induced by Unilateral Ureteral
Obstruction (UUO)
[0468] The isolated tissues were embedded in OCT compound (Sakura
Finetechnical Co.), an embedding medium for cryosectioning.
Cryoblocks were prepared using liquid nitrogen, and sliced into
6-.mu.m thick sections using Cryostat (Micro-edge Instruments Co.).
The resulting sections were immunostained by the same method as
described in Example 28 using an anti-ER-TR7 antibody. The samples
were observed under a light microscope (Leica Microsystems). The
antibody binding was detected by visualizing it as brown signals
and quantified.
[0469] The result showed that the accumulation of fibroblasts in
the renal interstitium was significantly suppressed in the
UUO-treated kidney in the C6ST siRNA-treated group as compared to
the control group (FIG. 42).
[0470] The agents of the present invention are thus useful, for
example, as agents for suppressing fibroblast accumulation in
kidney interstitium.
[Example 43] Assessment of C6ST siRNA in Macrophage Infiltration in
Renal Fibrosis Model Mice Induced by Unilateral Ureteral
Obstruction (UUO)
[0471] Immunostaining and quantitation was carried out by the same
method as described in Example 42 using an anti-F4/80 antibody. The
result showed that the accumulation of macrophages in the renal
interstitium was significantly suppressed in the UUO-treated kidney
in the C6ST siRNA-treated group as compared to the control group
(FIG. 43).
[0472] The agents of the present invention are thus useful, for
example, as agents for suppressing macrophage accumulation in
kidney interstitium.
[Example 44] Assessment of C6ST siRNA in Collagen Accumulation in a
Renal Fibrosis Mouse Model Included by Unilateral Ureteral
Obstruction (UUO)
[0473] Immunostaining was carried out by the same method as
described in Example 42 using an anti-type IV collagen antibody,
and quantitation was achieved by the same method as described in
Example 34. The result showed that the fibrous thickening of
glomerular basement membrane in the UUO-treated kidney was
significantly suppressed in the C6ST siRNA-treated group as
compared to the control group (FIG. 44). This suggests that the
infiltration and fibrogenesis of inflammatory cells can be
suppressed by inhibiting the expression of C6ST-2 gene.
[0474] The agents of the present invention are thus useful, for
example, as agents for suppressing the infiltration of inflammatory
cells.
[Example 45] Assessment of C6ST siRNA for Fibroblast Activation in
a Renal Fibrosis Mouse Model Induced by Unilateral Ureteral
Obstruction (UUO)
[0475] Immunostaining was carried out by the same method as
described in Example 30 using an .alpha.SMA antibody. The result
showed that .alpha.SMA-positive cells were clearly decreased in the
interstitium, juxtaglomerular areas in particular, of the
UUO-treated kidneys in the C6ST siRNA-treated group as compared to
the control group (FIG. 45). This result suggests that the
activation of fibroblasts accumulating in the renal interstitium is
suppressed by inhibiting the expression of C6ST-1 and C6ST-2
genes.
[0476] The agents of the present invention are thus useful, for
example, as agents for suppressing the activation of fibroblasts in
the renal interstitium.
[Example 46] Assessment of C6ST siRNA in ACE Expression in a Renal
Fibrosis Mouse Model Induced by Unilateral Ureteral Obstruction
(UUO)
[0477] Immunostaining was carried out by the same method using a
rabbit anti-human ACE antibody (Santa Cruz). The result showed that
ACE-positive cells were clearly decreased in the interstitium,
juxtaglomerular areas in particular, of the UUO-treated kidneys in
the C6ST siRNA-treated group as compared to the control group (FIG.
46). This result demonstrates that inhibition of C6ST-2 gene
expression results in the suppression of ACE expression with the
suppression of the activation of fibroblasts accumulated in the
renal interstitium. Suppression of ACE produces a hypotensive
effect. Thus, the result described above strongly suggests that
C6ST siRNA has antihypertensive activity or
arteriosclerosis-suppressing effect.
[0478] The agents of the present invention are thus useful, for
example, as arteriosclerosis-suppressing agents.
[Ocular Tissue]
[0479] Like other organs, fibrotic changes in ocular tissues occur
as a result of invasion due to various causes. This leads to
impairment and/or loss of vision. Such major diseases include
diabetic retinopathy, retinal vein occlusion, retinopathy of
prematurity, age-related macular degeneration, and retinitis
pigmentosa. The diseases also include fibrogenesis associated with
corneal inflammation, glaucoma, or cataract (reviews: Fiedlander M.
J Clin Invest 117: 576-586, 2007; Harada T et al. Genes and Dev.
21: 367-378, 2007). From the histopathological viewpoint,
damage/decrease of photoreceptor cells caused by fibrogenesis is
the major cause of visual loss. Thus, suppressing fibrogenesis in
ocular tissues has been expected as a novel therapeutic strategy to
prevent visual loss in all ocular diseases.
[0480] Next Examples focus on retinal fibrogenesis and the
resulting loss of photoreceptor cells in a diabetic retinopathy
model.
[Example 47] Assessment of GalNAc4S-6ST (G#1) siRNA in Collagen
Accumulation in a Mouse Diabetic Retinopathy Model
[0481] Gestational Day 14 C57BL6J/JcL mice (CLEA Japan Inc.) were
reared and allowed to deliver. 10 mg/ml Streptozocin (SIGMA) was
subcutaneously administered at l/head to Day 2 postnatal female
C57BL6J/JcL mice. The mice were reared with sterile water and CE-2
Diet (CLEA Japan Inc.) until they were four weeks old, and then
with sterile water and a High Fat Diet (CLEA Japan Inc.) for
subsequent two weeks. 1 .mu.g of GalNac4S-6ST (G#1) siRNA (Hokkaido
System Science Co.) was combined with 200 .mu.l of 1% atelocollagen
(Koken Co.), which is a vehicle, and the resulting mixture was
administered into the peritoneal cavity of each mouse in the eighth
and ninth week by the same method as described in Example 11. In
the 18th week, eye balls were excised from mice of the two groups
and immunohistochemically examined to assess the effect of
GalNac4S-6ST (G#1) siRNA.
[0482] Cryoblocks and sections were prepared from the excised eye
balls. The sections were fixed with acetone (Sigma Aldrich Japan)
for ten minutes, and then washed with phosphate buffer. A rabbit
anti-type IV collagen antiserum (1:2000 dilution; LSL) was added as
the primary antibody, and the sections were incubated at room
temperature for one hour. Then, a peroxidase-labeled anti-rabbit
IgG antibody (1:25 dilution; Cappel) was added as the secondary
antibody, and the samples were incubated at room temperature for 30
minutes. After incubation, an enzyme-mediated chromogenic reaction
was conducted by adding DAB substrate (Nichirei Biosciences). The
samples were observed under a light microscope (Leica
Microsystems).
[0483] Obtained histology of the retina is shown in FIG. 47. In the
control group, the deposition of type IV collagen was increased
over the region from ganglion cell layer (GCL) to inner nuclear
layer (INL), which is essential for vision. By contrast, the
increase of collagen in GCL in particular, was significantly
suppressed in the GalNac4S-6ST (G#1) siRNA-administered group.
[0484] The agents of the present invention are thus useful, for
example, as agents for suppressing collagen increase in the
ganglion cell layer.
[Example 48] Assessment of GalNac4S-6ST (G#1) siRNA in the
Accumulation of Sodium Chondroitin Sulfate Proteoglycan in a Mouse
Diabetic Retinopathy Model
[0485] The retina was immunohistochemically assessed using the same
method as described in Example 47. An anti-chondroitin sulfate
proteoglycan (CSPG) antibody (clone CS56, mouse monoclonal
antibody, 1:100; Seikagaku Co.) was added as the primary antibody,
and the sections were incubated at room temperature for one hour.
Then, the secondary antibody reaction was carried out using
Histofine Mouse Stain kit (Nichirei; used for mouse monoclonal
antibody).
[0486] As shown in FIG. 48, a significant enhancement of
CS56-positive signals were observed in GCL, and the segment from
outer nuclear layer (ONL) to pigmented cell layer of retina in the
control group. In contrast, the CS56 signal intensity was markedly
reduced in the GalNac4S-6ST (G#1) siRNA-administered group. Thus,
gangliocytes and retinal pigment epithelial cells were
morphologically well preserved in GCL.
[0487] The result described above demonstrates that in vivo
administration of GalNac4S-6ST (G#1) siRNA significantly suppresses
the induced CSPG deposition in retinal tissues in this model mouse.
Together with collagen, CSPG is considered to be essential for the
formation of fibrogenic lesions. Furthermore, CSPG has been
reported to inhibit the process of axon extension of gangliocyte
(Brittis P A et al., Science 255: 733, 1992). Previously published
reports only describe results of in vitro experiments and
developmental process. Thus, the role in in vivo pathological
lesions still remains unknown. However, the result described herein
for the first time suggests the role of CSPG in lesional
tissues.
[Example 49] Assessment of GalNac4S-6ST (G#1) siRNA in the
Accumulation of Glial Cells in a Mouse Diabetic Retinopathy
Model
[0488] Optic nerve regeneration has been reported to serve as a
biological defense mechanism after retinal damage. Meanwhile, it is
reported that the optic nerve progenitor cells responsible for such
regeneration after injury are glial cells (Fischer A J et al.,
Nature neuroscience 4: 247, 2001; Ooto S et al., PNAS 101: 13645,
2004). In this Example, immunostaining was carried out using a goat
anti-GFAP antibody (Santa Cruz) as a glial cell marker by the same
method as described in Example 47.
[0489] The result is shown in FIG. 49. The number of GFAP-positive
glial cells was not altered in both normal and control groups. In
contrast, the cell count was markedly increased in the area from
INL to GCL in the GalNac4S-6ST (G#1) siRNA-administered group.
Optic nerve regeneration has been reported to occur from INL toward
GCL. Thus, the result described above suggests the process of
active optic nerve regeneration induced by GalNac4S-6ST (G#1)
siRNA.
[0490] The agents of the present invention are thus useful, for
example, as agents for regenerating the optic nerve.
[Example 50] Assessment of GalNac4S-6ST (G#1) siRNA in Gangliocytes
in a Mouse Diabetic Retinopathy Model
[0491] Gangliocytes were quantified using samples prepared in
Example 47. The result revealed that GCL gangliocytes were reduced
in the diabetic retinopathy model but the loss was significantly
recovered by GalNac4S-6ST (G#1) siRNA administration (FIG. 50).
[Example 51] Assessment of GalNac4S-6ST (G#1) siRNA in Gene
Expression in a Mouse Diabetic Retinopathy Model
[0492] RNA was extracted from ocular tissues and quantitative PCR
was carried out by the same method as described in Example 1. The
ability to regenerate the optic nerve was assessed by analyzing
changes in the expression of glutamate synthetase (GS), which is a
Muller cell marker. The sequences of PCR primers are shown
below.
TABLE-US-00012 [Quantitative PCR primer sequences] *mouse GS
(Takara Bio Inc.) Forward: (SEQ ID NO: 89)
5'-CTGTGAGCCCAAGTGTGTGGA-3' Reverse: (SEQ ID NO: 90)
5'-GTCTCGAAACATGGCAACAGGA-3'
[0493] The result is shown in FIG. 51. Administration of
GalNac4S-6ST (G#1) siRNA could significantly inhibit the enhanced
expression of GalNAc4S-6ST in ocular tissues. The expression
inhibition resulted in a significant increase in the expression of
GS in ocular tissues in the GalNac4S-6ST (G#1) siRNA-administered
group. The result described above shows that GalNac4S-6ST (G#1)
siRNA administration results in Muller cell regeneration, i.e.,
optic nerve restoration.
[0494] This Example revealed that fibrotic changes of retinal
tissues could be suppressed by inhibiting the expression of
GalNAc4S-6ST gene, which led to the prevention of photoreceptor
cell loss through the regeneration of gangliocytes. This
histological feature is commonly observed in a wide variety of
ocular diseases with fibrosis including glaucoma and diabetic
retinopathy.
[0495] The agents of the present invention are thus useful, for
example, as agents for regenerating Muller cells or
gangliocytes.
[Liver Tissue]
[0496] Fibrotic changes in liver tissues are the progressive or
terminal stage of various liver diseases. Liver fibrogenesis
results from various liver diseases, including viral hepatitis
(type A, B, C, D, E, and G viral hepatitis), alcohol liver disease,
nonalcoholic fatty liver diseases (NAFLD and NASH), metabolic liver
disease, drug-induced liver disease, idiopathic portal
hypertension, Budd-Chiari's syndrome, autoimmune hepatitis, primary
biliary cirrhosis, primary sclerosing cholangitis, biliary
disorders (including biliary atresia and biliary dilation), biliary
atresia caused by pancreatic diseases such as tumor,
graft-versus-host reaction, and chronic rejection (Bataller R et
al., J Clin Invest 115: 209, 2005; Iredale J P. J Clin Invest 117:
539, 2007). Fibrotic changes of liver at the tissue level were
assessed in the next Examples.
[Example 52] Assessment of GalNAcST siRNA in Gene Expression in a
Mouse Fatty Liver Disease Model
[0497] Gestational day 14 C57BL/6JcL mice (CLEA Japan Inc.) were
reared and allowed to deliver. Streptozocin (STZ; SIGMA) was
administered to Day 2 postnatal C57BL/6JcL mice. STZ (10 mg/ml) was
subcutaneously administered at 20 .mu.l/head three times for two
days. Thus, a total of 60 .mu.l was administered to the mice.
Together with their mothers, the mice were fed with a normal diet
until they were four weeks old. After weanling at the age of four
weeks, the mice were fed with a High Fat Diet (CLEA Japan Inc.) for
two weeks. In the second week, 200 .mu.l of GalNAcST siRNA
described in Example 11 was administered into peritoneal cavities
once a week (one shot/week) twice in total (for two weeks). On day
14 of the experiment, the mice were dissected and their livers were
isolated to prepare samples for gene expression analysis and
immunostaining.
[0498] Quantitative PCR was carried out by the same method as
described in Example 1 using liver tissues. The result is shown in
FIG. 52. The expression of GalNAc4S-6ST is enhanced in liver
tissues in this model. GalNAcST siRNA administration resulted in
significant suppression of the expression.
[Example 53] Assessment of Anti-Fibrogenic Effect of GalNAcST siRNA
in a Mouse Fatty Liver Disease Model
[0499] The expression of fibrogenesis markers in liver tissues was
assessed by the same method as described in Example 52. The
expression of type I collagen and .alpha.SMA was significantly
enhanced in this model (FIG. 53), suggesting enhanced fibrotic
changes in liver tissues. Meanwhile, the enhanced expression of the
fibrogenesis markers was significantly suppressed by administering
GalNAcST siRNA.
[Example 54] Assessment of GalNAcST siRNA in Fibroblast
Infiltration in a Mouse Fatty Liver Disease Model
[0500] Next, liver tissues were immunostained by the same method as
described in Example 3. A rat anti-mouse fibroblast antibody (clone
ER-TR7, 1:500 dilution; BMA) was added as the primary antibody, and
the sections were incubated at room temperature for one hour. Then,
a peroxidase-labeled anti-rat IgG antibody (1:200 dilution;
Biosource International, Inc.) was added as the secondary antibody,
and the sections were incubated at room temperature for 30 minutes.
After incubation, DAB substrate (Nichirei Biosciences) was added,
and the samples were observed under a light microscope (Leica
Microsystems). The antibody binding was detected by visualizing it
as brown signals.
[0501] Examples of obtained images of immunostained liver tissues
are shown in FIG. 53. The accumulation of fibroblasts was clearly
observed and a bridge formation was confirmed in the histological
picture in the control group. In contrast, there was almost no
accumulation of fibroblasts in the GalNAcST siRNA-administered
group.
[Example 55] Assessment of GalNAcST siRNA in Fibrogenic Score in a
Mouse Fatty Liver Disease Model
[0502] Each of the samples immunohistochemically stained in Example
54 was assessed for the degree of live fibrogenesis using
fibrogenesis scores based on previous reports (Dai K, et al., World
J Gactroenterol. 31: 4822-4826, 2005; Hillebrandt S, et al., Nature
Genetics 37: 835-843, 2005). The fibrogenesis scores were defined
according to the following criteria: 0, normal; 1, few collagen
fibrils extend from the central vein; 2, extension of collagen
fibrils is apparent but collagen fibrils have not yet encompassed
the whole liver; 3, extension of collagen fibrils is apparent and
collagen fibrils have encompassed the whole liver; 4, diffuse
extension of collagen fibrils is observed in the whole liver and
pseudolobules are formed.
[0503] The result is shown as a graph in FIG. 55. Each bar
indicates mean.+-.standard deviation of the fibrogenesis score in
each group. The fibrogenesis was statistically significantly
reduced in the GalNAcST siRNA-administered group as compared to the
control group (p<0.01; t-test). This finding suggests that
GalNAcST siRNA administration also clinically produces a superior
liver fibrogenesis-suppressing effect.
[Example 56] Assessment of GalNAcST siRNA in Macrophage
Infiltration in a Mouse Fatty Liver Disease Model
[0504] Next, liver tissues were immunostained by the same method as
described in Example 54. A rat anti-mouse F4/80 antibody (clone
A3-1, 2 .mu.g/ml; CALTAG LABORATORIES) was added as the primary
antibody, and the sections were incubated at room temperature for
one hour. Then, a peroxidase-labeled anti-rat IgG antibody (1:200
dilution; Biosource International, Inc.) was added as the secondary
antibody, and the sections were incubated at room temperature for
30 minutes. After incubation, DAB substrate (Nichirei Biosciences)
was added, and the samples were observed under a light microscope
(Leica Microsystems). The antibody binding was detected by
visualizing it as brown signals.
[0505] Examples of obtained images of immunostained liver tissues
are shown in FIG. 56. The accumulation of macrophages was clearly
observed in the control group. Formation of inflammatory
accumulation lesions were seen in the histological pictures. In
contrast, there was no excessive accumulation of macrophages in the
GalNAcST siRNA-administered group.
[Example 57] Assessment of GalNAcST siRNA in the Hepatic Lipid
Metabolism in a Mouse Fatty Liver Disease Model
[0506] The expression of lipid metabolism-related genes in the
liver was assessed by the same method as described in Example 52.
The enhanced expression of carbohydrate response element-binding
protein (ChREBP) and acetyl-CoA carxylase-2 (ACC2) was observed.
Meanwhile, the enhanced expression was significantly suppressed in
the GalNAcST siRNA-administered group (FIG. 57). This result shows
that the glycolipid metabolism can be improved via suppression of
fibrotic changes in liver tissues by inhibiting the expression of
GAlNAc4ST-1, GalNAc4ST-2, and GalNAc4S-6ST genes.
[0507] The agents of the present invention are thus useful, for
example, as agents for improving glycolipid metabolism.
[Example 58] Assessment of C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3
siRNA for Fibroblast Accumulation in a Mouse Fatty Liver Disease
Model
[0508] A fatty liver disease model was prepared by the same method
as described in Example 52. C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3
siRNA described in Example 21 were administered according to the
same administration protocol. Liver tissues were collected from the
mice.
[0509] Next, liver tissues were immunostained by the same method as
described in Example 3. A rat anti-mouse fibroblast antibody (clone
ER-TR7, 1:500 dilution; BMA) was added as the primary antibody, and
the sections were incubated at room temperature for one hour. Then,
a peroxidase-labeled anti-rat IgG antibody (1:200 dilution;
Biosource International, Inc.) was added as the secondary antibody,
and the sections were incubated at room temperature for 30 minutes.
After incubation, DAB substrate (Nichirei Biosciences) was added,
and the samples were observed under a light microscope (Leica
Microsystems). The antibody binding was detected by visualizing it
as brown signals.
[0510] Examples of obtained images of immunostained liver tissues
are shown in FIG. 58. The accumulation of fibroblasts was clearly
observed in the control group. A bridge formation was seen in the
histological pictures. In contrast, fibroblast accumulation was not
observed in any of the C4ST-1 siRNA-, C4ST-2 siRNA-, and C4ST-3
siRNA-administered group.
[Example 59] Assessment of C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3
siRNA in the Fibrogenic Score in a Mouse Fatty Liver Disease
Model
[0511] Each sample was assessed for the degree of liver
fibrogenesis using the fibrogenesis scores based on the
immunohistochemical staining carried out in Example 58. The result
is shown in FIG. 59. Each bar indicates mean.+-.standard deviation
of the fibrogenesis score in each group. The fibrogenesis was
statistically significantly reduced in all of the C4ST-1 siRNA-,
C4ST-2 siRNA-, C4ST-3 siRNA-administered groups as compared to the
control group (p<0.001; t-test). This finding suggests that the
administration of siRNAs against C4ST-1, C4ST-2, or C4ST-3 also
clinically produces a superior liver fibrogenesis-suppressing
effect.
[Example 60] Assessment of C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3
siRNA in Hepatocyte Disorders in a Mouse Fatty Liver Disease
Model
[0512] When mice were sacrificed, blood was collected from them
according to the protocol as described in Example 58. The blood
samples were custom-assayed for the serum alanine transferase (ALT)
levels through SRL Inc. The result is shown in FIG. 60. The serum
ALT level is the most widely used clinical indicator for hepatocyte
destruction. The serum ALT level was elevated in the control group.
Meanwhile, the mean value was decreased to 50% or less in each of
the C4ST-1 siRNA-, C4ST-2 siRNA-, and C4ST-3 siRNA-administered
groups as compared to the control group (FIG. 60). This result
demonstrates that hepatocyte damages can be reduced via suppression
of fibrotic changes by inhibiting the expression of C4ST-1, C4ST-2,
or C4ST-3 gene in liver tissues.
[0513] The agents of the present invention are thus useful, for
example, as agents for reducing hepatocyte damage.
[Example 61] Assessment of the Anti-Fibrogenic Effect of C6ST siRNA
in a Mouse Hepatic Fibrosis Model
[0514] In this Example, experiments were carried out using a mouse
cirrhosis model induced by carbon tetrachloride, which is the most
widely used cirrhosis model. First, the mouse model was prepared as
described below. Carbon tetrachloride (25 al/100 g body weight;
Sigma-Aldrich) was injected into peritoneal cavities of C57BL6/J
mice (female, five or six weeks old; CLEA Japan Inc.) twice a week
for four weeks (eight times) to induce hepatic fibrosis. Then,
carbon tetrachloride was additionally administered twice a week for
two weeks (a total of 12 times) to induce cirrhosis. Mice with
induced cirrhosis were sacrificed, and their livers were collected
(cirrhotic livers). Meanwhile, in the control experiment, livers
were collected from C57BL6/J mice (female, CLEA Japan Inc.) of the
same age without carbon tetrachloride administration (normal
livers).
[0515] The same C6ST siRNA as described in Example 41 and a control
were administered into peritoneal cavities four times at the same
time of additional carbon tetrachloride administration (a total
four times from the ninth to twelfth administration). After the
additional administration, mice in the both groups were sacrificed.
Liver tissue sections were prepared from the mice and
immunohistochemically assessed. A rat anti-mouse fibroblast
antibody (clone ER-TR7, 1:500 dilution; BMA) was added as the
primary antibody, and the sections were incubated at room
temperature for one hour. Then, a peroxidase-labeled anti-rat IgG
antibody (1:200 dilution; Biosource International, Inc.) was added
as the secondary antibody, and the sections were incubated at room
temperature for 30 minutes. After incubation, DAB substrate
(Nichirei Biosciences) was added, and the samples were observed
under a light microscope (Leica Microsystems). The antibody binding
was detected by visualizing it as brown signals.
[0516] Examples of obtained images of immunostained liver tissues
are shown in FIG. 61. The accumulation of fibroblasts was clearly
observed in the control group. A bridge formation was shown in the
histological pictures. In contrast, there was no accumulation of
fibroblasts in any of the C6ST siRNA-administered groups.
[Example 62] Assessment of C6ST siRNA in the Fibrogenesis Score in
a Mouse Hepatic Fibrosis Model
[0517] The degree of liver fibrogenesis in each sample was assessed
using the fibrogenesis scores based on the immunohistochemical
staining carried out in Example 61. The result is shown in FIG. 62.
Each bar indicates mean.+-.standard deviation of the fibrogenesis
score in each group. The fibrogenesis was statistically
significantly reduced in the C6ST siRNA-administered group as
compared to the control group (p<0.05; t-test). This finding
suggests that the administration of C4ST-1 siRNA, C4ST-2 siRNA, or
C4ST-3 siRNA also produces a clinically beneficial effect of
suppressing liver fibrogenesis.
[Example 63] Assessment of the Anti-Fibrogenic Effect of C6ST siRNA
in a Mouse Hepatic Fibrosis Model
[0518] RNA was extracted from liver tissues and quantitative PCR
was carried out by the same method as described in Example 1. The
result is shown in FIG. 63. The expression of .alpha.SMA, type I
collagen, CTGF, and TGF.beta. as fibrogenesis markers was enhanced
in the control group. Meanwhile, the expression was significantly
suppressed in the C6ST siRNA-administered group. This result
suggests that fibrotic changes in liver tissues can be suppressed
by inhibiting the expression of C6ST-1 and C6ST-2 genes.
[Cranial Nerve Tissue]
[0519] In these Examples, an MPTP-induced Parkinson's disease model
was used as a fundamental mouse model for Parkinson's disease. This
model is a classical but highly reproducible and simple model, and
thus has been widely used as a Parkinson's disease model. The
histological features are: infiltration of inflammatory cells into
brain parenchymal tissue and reduction in the number of
dopamine-producing neurons. Classically, neurofibrillary tangles
were thought to be a cause of the reduction in the number of
neurons in such pathological conditions.
[0520] Thus, such diseases include pathological conditions with
neuronal disorders, specifically, not only representative
neurodegenerative diseases such as Parkinson's disease, progressive
supranuclear palsy, corticobasal degeneration, Alzheimer's disease,
polyglutamine disease, amyotrophic lateral sclerosis (ALS), spinal
progressive muscular atrophy, spinobulbar muscular atrophy,
Huntington's disease, and multiple sclerosis, but also other
diseases such as multiple system atrophy (striatonigral
degeneration, olivopontocerebellar atrophy, and Shy-Drager
syndrome), adrenoleukodystrophy, Guillain-Barre syndrome,
myasthenia gravis, Fisher syndrome, chronic inflammatory
demyelinating polyneuropathy, Lewis-Sumner syndrome, Crow-Fukase
syndrome, normal pressure hydrocephalus, syringomyelia, prion
disease (Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker
disease, and fatal familial insomnia), subacute sclerosing
panencephalitis (SSPE), progressive multifocal leukoencephalopathy
(PML), and spinocerebellar degeneration. Such diseases also include
posttraumatic cerebral sequelae, sequelae of cerebrovascular
disease (cerebral infarction and hemorrhage), sequelae of viral
encephalitis, sequelae of bacterial meningitis, sequelae of spinal
cord injury, and neurofibrillary tangle in the spinal nerve,
peripheral nerve, auditory nerve, and optical nerve, etc. In
particular, the above-listed sequelae have long been speculated as
a basis for psychiatric disorders such as depression, and thus
neurofibrillary tangle is important as a cause of psychiatric
symptoms.
[Example 64] Assessment of the Anti-Fibrogenic Effect of
GalNAc4S-6ST siRNA Treatment in a C57BL/6JcL Mouse Parkinson's
Disease Model Induced by MPTP
[0521] In this Example, a mouse Parkinson's disease model was
prepared by selectively degenerating dopamine neurons using
1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) (Amende et al.,
(2005) Journal of NeuroEngineering and Rehabilitation 2(20): 1-13).
The mice were administered with GalNAc4S-6ST siRNA, and the gene
expression and histological features after treatment was
assessed.
[0522] Gestational day 14 C57BL/6JcL mice (CLEA Japan Inc.) were
reared and allowed to deliver. 1 .mu.g of the same GalNAc4S-6ST
siRNA (GeneWorld) as described in Example 1 was combined with 1%
atelocollagen (Koken Co.), which is a vehicle for siRNA, and the
mixture was administered at 200 .mu.l/head to the peritoneal
cavities of C57BL/6JcL mice (female, eight weeks old; CLEA Japan
Inc.). Two, three, and four days after administration, MPTP (Sigma
Aldrich Japan), which selectively destroys dopamine neurons, was
administered to the mice at 30 mg/kg (three times in total). The
mice were reared, and on day 8 of the experiment 100 .mu.l of 5
mg/ml BrdU (ZyMED Laboratory Inc.) was administered into the tail
vein. After one hour, the mice were dissected and their brains were
isolated to prepare samples for immunostaining and gene expression
analysis.
[0523] The gene expression was assessed quantitatively by the same
method as described in Example 1. The result is shown in FIG. 64.
The expressions of GalNAc4S-6ST, and TGF.beta., type I collagen,
and .alpha.SMA, which are fibrogenesis markers, are enhanced in
brain tissues in the Parkinson's disease model. Meanwhile, the
expression was significantly suppressed by GalNAc4S-6ST siRNA
administration. This demonstrates that fibrotic changes in brain
tissues can be suppressed by inhibiting the expression of
GalNAc4S-6ST.
[0524] The agents of the present invention are thus useful, for
example, as agents for suppressing fibrotic changes in brain
tissues.
[Example 65] Assessment of GalNAc4S-6ST siRNA Treatment for
Fibroblast Accumulation in a C57BL/6JcL Mouse Parkinson's Disease
Model Induced by MPTP
[0525] Brain tissue samples were treated by the same method as
described in Example 3 to compare histological features after in
vivo administration of GalNAc4S-6ST against fibrogenesis of neurons
in the brain. The resulting sections were fixed with 4%
PFA-phosphate buffer (Nacalai Tesque) for ten minutes, and then
washed with deionized water. An anti-fibroblast antibody (ER-TR7,
1:100 dilution; BMA) was added as the primary antibody, and the
sections were incubated at 4.degree. C. overnight. Then, an
Alexa488-labeled goat anti-rat IgG antibody (1:200 dilution;
Invitrogen) was added as the secondary antibody, and the sections
were incubated at room temperature for 30 minutes.
[0526] Images of tissues obtained by the method described above are
shown in FIG. 65. The strong positive signals in the untreated
group suggest intracranial infiltration of fibroblasts around the
granular cortex in the splenium of posterior corpus callosum as
compared to the control group. The positive signals of fibroblasts
were drastically decreased in the GalNAc4S-6ST siRNA-treated group.
The result described above demonstrates that the feature
represented by ER-TR7-positive signals in brain tissues induced in
the mouse Parkinson's disease model was significantly suppressed by
in vivo administration of GalNAc4S-6ST siRNA.
[Example 66] Assessment of the Neuroprotective Effect of
GalNAc4S-6ST siRNA Treatment in a C57BL/6JcL Mouse Parkinson's
Disease Model Induced by MPTP
[0527] Next, to assess whether the above-described fibrogenesis was
associated with the decrease of neurons, the expression of nerve
regeneration-related genes in brain tissues was quantified by the
same method as described in Example 64. The administration of
GalNAc4S-6ST siRNA resulted in enhanced expression of GDNF, which
is a factor that regulates the survival and differentiation of
dopamine neurons as well as enhances the regeneration of the
neurons, and Nurr1, which is a factor for forming dopamine neurons
(FIG. 66). This result suggests that the regeneration of dopamine
neurons in brain tissues can be stimulated by inhibiting the
expression of GalNAc4S-6ST.
[0528] The agents of the present invention are thus useful, for
example, as agents for stimulating the regeneration of dopamine
neurons in brain tissues.
[Example 67] Assessment of the Neuroprotective Effect of
GalNAc4S-6ST siRNA Treatment in a C57BL/6JcL Mouse Parkinson's
Disease Model Induced by MPTP
[0529] To finally verify the results described above in the
Examples, histological features of the prepared tissue sample
sections were weighed by staining dopamine neurons of the sections
with an antibody against tyrosine hydroxylase, which is a marker
for dopamine neuron. Tyrosine hydroxylase (TH) is an enzyme that
converts the dopamine precursor into dopamine. The sections
prepared by the same method as described in Example 64 were fixed
with 4% PFA-phosphate buffer (Nacalai Tesque) for ten minutes, and
then washed with deionized water. A rabbit anti-tyrosine
hydroxylase polyclonal antibody (1:50 dilution; Calbiochem) was
added as the primary antibody, and the sections were incubated at
room temperature for one hour. An Alexa488-labeled donkey
anti-rabbit antibody (1:200 dilution; Invitrogen) was added as the
secondary antibody, and the sections were incubated at room
temperature for 30 minutes.
[0530] Histological images for the control, untreated, and
GalNAc4S-6ST siRNA-treated groups are shown in FIG. 67 (the
original images are in color). Normal expression of tyrosine
hydroxylase was confirmed in the superior colliculus of midbrain in
the control group. Meanwhile, the signal for the expression was
negative in the untreated group. This finding suggests that MPTP
selectively destroyed dopamine neurons. On the other hand, a
stronger signal was confirmed in the GalNAc4S-6ST siRNA-treated
group as compared to the untreated group. In sum, it was concluded
that functional recovery of dopamine neurons can be achieved via
suppression of fibrotic changes by in vivo administration of
GalNAc4S-6ST siRNA.
[0531] The agents of the present invention are thus useful, for
example, as agents for recovering the function of dopamine
neuron.
[Example 68] Assessment of Neuroprotective Effect of GalNAcST siRNA
Treatment in a C57BL/6JcL Mouse Parkinson's Disease Model Induced
by MPTP
[0532] By the same procedure as described in Example 64,
gestational day 14 of C57BL/6JcL mice (CLEA Japan Inc.) were reared
and allowed to deliver. 1 .mu.g of GalNAcST (a mixture of
GalNAc4ST-1, GalNAc4ST-2, and GALNAC4S-6ST cocktail sequences)
siRNAs (GeneWorld) was combined with 1% atelocollagen (Koken Co.),
which is a vehicle for siRNA, and the resulting mixture was
administered at 200 .mu.l/head to the peritoneal cavities of
C57BL/6JcL mice (female, eight weeks old; CLEA Japan Inc.). Two,
three, and four days after administration, MPTP (Sigma Aldrich
Japan), which selectively destroys dopamine neurons, was
administered to the mice at 30 mg/kg (three times in total). The
mice were reared, and on day 8 of the experiment 100 .mu.l of 5
mg/ml BrdU (ZyMED Laboratory Inc.) was administered into the tail
vein. After one hour, the mice were dissected and their brains were
isolated to prepare samples for immunostaining and gene expression
analysis.
[0533] The resulting sections were immunostained with an anti-TH
antibody by the same method as described in Example 67. The result
is shown in FIG. 68. The result demonstrated that the reduction of
TH-positive dopamine neurons was suppressed in the GalNAcST
siRNA-treated group. Specifically, like Example 67, it was
concluded that function recovery of dopamine neurons can be
achieved via suppression of neurofibrillary tangle by inhibiting
the expression of GalNAc4ST-1, GalNAc4ST-2, and GalNAc4S-6ST.
[0534] The agents of the present invention are thus useful, for
example, as agents for suppressing neurofibrillary tangle.
[Example 69] Assessment of the Effect of C4-Sulfatase in a Mouse
Type 2 Diabetic Retinopathy Model Induced by Streptozotocin:
Reduction of Sulfated CSPG
[0535] Gestational day 14 C57BL/6JcL mice (CLEA Japan Inc.) were
reared and allowed to deliver. 10 mg/ml Streptozocin (SIGMA) was
subcutaneously administered at 20 .quadrature.l/head to postnatal
Day 2 female C57BL/6JcL mice. The mice were reared with sterile
water and CE-2 Diet (CLEA Japan Inc.) until they were four weeks
old, and then with sterile water and a High Fat Diet (CLEA Japan
Inc.) for subsequent two weeks. In the second week,
chondro-4-desulfating enzyme (C4-sulfatase) (20 units/ml; Seikagaku
Co.) was administered at 4 units/head or medium (phosphate buffer)
was administered into the peritoneal cavities twice a week four
times (two weeks). On day 14, eye balls were isolated from mice of
both groups and immunohistochemically assayed to assess the effect
of C4-sulfatase.
[0536] Cryoblocks and sections were prepared from the isolated eye
balls. The sections were fixed with acetone (Sigma Aldrich Japan)
for ten minutes, and then washed with phosphate buffer. Then, an
anti-chondroitin sulfate proteoglycan (CSPG) antibody (clone CS56,
mouse monoclonal antibody, 10 .mu.g/ml; Seikagaku Co.) was added as
the primary antibody, and the sections were incubated at room
temperature for one hour. Next, the secondary antibody reaction was
carried out using a Histofine Mouse Stain kit (Nichirei
Biosciences; used for mouse monoclonal antibody), and color
development was performed by adding DAB substrate (Nichirei
Biosciences). The samples were observed under a light microscope
(Leica Microsystems).
[0537] The obtained histological pictures are shown in FIG. 69 (the
original images are in color). In the untreated group,
CS56-positive signals are newly found in the vitreous side of the
retina. In contrast, the intensity of CS56 is reduced in the
enzyme-treated group. CS56 is an antibody that recognizes a sulfate
group, and the reduction is suggested to reflect a decrease of
sulfate groups at position 4. The result described above
demonstrates that the induced deposition of CSPG in retinal tissues
is suppressed via modification by in vivo administration of
C4-sulfatase.
[0538] C4-sulfatase is an enzyme that desulfates GalNAc at position
4. Thus, it was demonstrated that tissue fibrogenesis at the
biological level could be suppressed by inhibiting the sulfation of
GalNAc at position 4. Specifically, desulfating enzymes for the
sulfate group of GalNAc at position 4 are useful as tissue
fibrogenesis inhibitors.
[Example 70] Assessment of the Effect of C4-Sulfatase in a Mouse
Type 2 Diabetic Retinopathy Model Induced by Streptozotocin:
Suppression of Vascular Endothelial Cell Proliferation
[0539] The sections prepared by the same method as described in
Example 69 were fixed with acetone (Sigma Aldrich Japan) for ten
minutes, and then washed with phosphate buffer. A rat anti-vascular
endothelial cell antibody (CD31; 1:200 dilution; Phamingen) was
added as the primary antibody, and the sections were incubated at
room temperature for one hour. Then, a donkey peroxidase-labeled
anti-rat IgG antibody (1:200 dilution; Biosource International,
Inc.) was added as the secondary antibody, and the sections were
incubated at room temperature for 30 minutes. After incubation, DAB
substrate (Nichirei Biosciences) was added to the samples. The
samples were observed under a light microscope (Leica
Microsystems). The obtained histological pictures are shown in FIG.
70 (the original images are in color). The images show that the
number of CD31-positive cells was increased in the vitreous side of
the retina and some of them were protruded into the vitreum in the
untreated group. This indicates that the model reflects the stage
of preproliferative retinopathy in diabetic retinopathy and shows
the effectiveness of this model. Meanwhile, the number of
CD31-positive cells was significantly reduced in the corresponding
region in the enzyme-treated group. In sum, it is suggested that
the number of vascular endothelial cells is increased in the
vitreous side of the retina in type 2 diabetes model, and that such
proliferation of blood vessels can be suppressed by administering
C4-sulfatase.
[0540] Specifically, desulfating enzymes for the sulfate group at
position 4 of GalNAc are useful as an agent for suppressing
proliferation of blood vessels.
[Example 71] Assessment of the Effect of C4-Sulfatase in a Mouse
Type 2 Diabetic Retinopathy Model Induced by Streptozotocin:
Suppression of Collagen-Proliferative Alteration
[0541] The sections prepared by the same method as described in
Example 69 were fixed with acetone (Sigma Aldrich Japan) for ten
minutes, and then washed with phosphate buffer. A rabbit anti-type
IV collagen antibody (1:250 dilution; Sigma) was added as the
primary antibody, and the sections were incubated at room
temperature for one hour. A peroxidase-labeled anti-rabbit IgG
antibody (1:200 dilution; Jackson ImmunoResearch) was added as the
secondary antibody, and the sections were incubated at room
temperature for 30 minutes. After incubation, DAB substrate
(Nichirei) was added to the samples. The samples were observed
under a light microscope (Leica Microsystems).
[0542] The obtained histological pictures are shown in FIG. 71 (the
original images are in color). In the untreated group, type IV
collagen-positive signals were increased in the vitreous side of
the retina and arranged parallel to the internal limiting membrane
of the retina. This suggests morphological aberration of vein and
collagen proliferation, i.e., fibrotic changes. In contrast, type
IV collagen proliferation was markedly suppressed in the
corresponding region in the enzyme-treated group. This demonstrated
that retinal collagen proliferation is observed in the type 2
diabetes model but the collagen proliferation can be suppressed by
administering C4-sulfatase.
[0543] Specifically, desulfating enzymes for the sulfate group at
position 4 of GalNAc are useful as agents for treating type 2
diabetic retinopathy.
[Example 72] Localization of Fibroblasts in the Liver of Type 2
Diabetes Model Mouse
[0544] The anti-fibrogenic effect (using tissue infiltration of
fibroblasts as an indicator) of C4-sulfatase on the liver was
assessed in Example 72 using the livers collected from the
above-described type 2 diabetes model mice (Examples 69-71).
Cryoblock preparation, immunostaining, and such were all achieved
by the methods described above. As shown in FIG. 72, infiltration
of many fibroblasts was observed in the untreated group. Meanwhile,
the degree of fibroblast infiltration was reduced in the
C4-sulfatase-treated group as compared to the untreated group. This
suggests that C4-sulfatase has the pharmacological effect of
suppressing fibroblast infiltration and this effect contributes to
the anti-fibrogenic effect.
[0545] Specifically, desulfating enzymes for the sulfate group at
position 4 of GalNAc are useful as agents for suppressing
fibroblast infiltration.
[Example 73] Localization of Macrophages in the Liver of Type 2
Diabetes Model Mouse
[0546] The anti-inflammatory effect (using tissue infiltration of
macrophages as an indicator) of C4-sulfatase on the liver was
assessed using the livers collected from the above-described type 2
diabetes model mice (Examples 69-71). Cryoblock preparation,
immunostaining, and such were all achieved by the methods described
above. As shown in FIG. 73, infiltration of many macrophages
accompanying spot formation was observed in the untreated group.
Meanwhile, the degree of macrophage infiltration was reduced in the
C4-sulfatase-treated group as compared to the untreated group. This
result suggests that C4-sulfatase has the pharmacological effect of
suppressing macrophage infiltration and this effect contributes to
the anti-inflammatory effect.
[0547] Specifically, desulfating enzymes for the sulfate group at
position 4 of GalNAc are useful as agents for suppressing
macrophage infiltration or as anti-inflammatory agents.
[Example 74] Serum Biochemical Test Findings on Type 2 Diabetes
Model Mice
[0548] An additional analysis was carried out in Example 74 to
supplement the results of Examples 72 and 73. This Example assayed
aspartate aminotransferase (AST) and alanine aminotransferase (ALT)
as indicators for liver function, and triacylglycerol (TG) as an
indicator for lipid metabolism, using sera collected from the
above-described type 2 diabetes model mice (Example 69-71). The
result is shown in FIG. 74. Biochemical tests were outsourced. AST,
ALT, and TG values all tended to be increased in the untreated
group (unt) as compared to the control group (nor). Meanwhile, the
increases in the AST, ALT, and TG values tended to be suppressed in
the C4-sulfatase-treated group (C4sul) as compared to the untreated
group (unt). The result described above supports the results of
Examples 72 and 73, suggesting that the liver function is preserved
due to the anti-fibrogenic and anti-inflammatory effects of
C4-sulfatase.
[0549] Specifically, desulfating enzymes for the sulfate group at
position 4 of GalNAc are useful as agents for treating liver
function disorders.
[Example 75] Comparison of CSPG Expression in Brain Tissues in a
C57BL/6JcL Mouse Model for Parkinson's Disease Induced by MPDP
[0550] The experiments in this Example was performed by preparing a
model using MPDP, which is a metabolite of
1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP).
[0551] On Day 0 and 2, 100 .mu.l of 4 U/ml C4-sulfatase (Seikagaku
Co.) was administered into the peritoneal cavity of each of
C57BL/6JcL mice (female, eight weeks old; CLEA Japan Inc.).
Alternatively, 200 .mu.l of a mixture of GalNAc4S-6ST siRNA (1
.mu.g; Hokkaido System Science Co.) and 1% atelocollagen was
pre-administered on Day 0. On Day 2 to 4, MPDP (Sigma Aldrich
Japan) was administered at 30 mg/kg every day.
[0552] The nucleotide sequence of mouse GalNac4S-6ST siRNA agent
used in this Example is shown below, but the sequences are not
limited to these examples.
TABLE-US-00013 [mouse GalNac4-6ST siRNAs] (Gene Bank accession
number NM_029935) (Hokkaido System Science, Co., Ltd.) (SEQ ID NO:
91) 5'-gcagcccagcaagaugaauaagauc-ag-3' (SEQ ID NO: 92)
3'-ua-cgucgggucguucuacuuauucuag-5'
[0553] On day 8, 100 .mu.l of BrdU 5 .mu.g/mL (ZyMED
Laboratory.Inc) was administered into mouse tail vein. One hour
after administration, mice were dissected and their brains were
isolated to prepare samples for immunostaining and gene expression
analysis. 1 ml of RNA-Bee (TEL-TEST) was added to 50 mg each of
excised organs (brains). The organs were crushed using an
electrical homogenizer (DIGITAL HOMOGENIZER; AS ONE), then, 200
.mu.l of chloroform (Sigma-Aldrich Japan) was added to the
resulting suspension. The mixture was gently mixed and then cooled
on ice for about five minutes, and centrifuged in a centrifuge
(Centrifuge 5417R; Eppendorf) at 12,000 rpm and 4.degree. C. for 15
minutes. After centrifugation, 500 .mu.l of the supernatant was
transferred to a fresh eppendorf tube, and an equal volume of
isopropanol (500 .mu.l; Sigma-Aldrich Japan) was added thereto. The
solution was mixed, and then 1 .mu.l of glycogen (Invitrogen) was
added thereto. The mixture was cooled on ice for 15 minutes, and
then centrifuged at 12,000 rpm and 4.degree. C. for 15 minutes.
Next, RNA precipitate obtained after washing three times with 1,000
.mu.l of 75% ethanol (Sigma-Aldrich Japan) was air-dried for 30
minutes to one hour, and then dissolved in Otsuka distilled water
(Otsuka Pharmaceutical Co., Ltd). The solution was 100 times
diluted with Otsuka distilled water. The RNA concentrations of
extracted samples in UV plates (Corning Costar) were determined
using a plate reader (POWER Wave XS; BIO-TEK).
[0554] Next, an RT reaction (cDNA synthesis) was conducted by the
following procedure. The concentrations of the obtained RNA samples
were adjusted to 500 ng/20 .mu.l. The samples were heated at
68.degree. C. for three minutes in a BLOCK INCUBATOR (ASTEC), and
cooled on ice for ten minutes. After cooling on ice, 80 .mu.l of RT
PreMix solution (composition: 18.64 .mu.l of 25 mM MgCl.sub.2
(Invitrogen), 20 .mu.l of 5.times. Buffer (Invitrogen), 6.6 .mu.l
of 0.1 M DTT (Invitrogen), 10 .mu.l of 10 mM dNTP mix (Invitrogen),
2 .mu.l of RNase Inhibitor (Invitrogen), 1.2 .mu.l of MMLV Reverse
Transcriptase (Invitrogen), 2 .mu.l of Random primer (Invitrogen),
and 19.56 .mu.l of sterile distilled water (Otsuka distilled water;
Otsuka Pharmaceutical Co., Ltd.)), which had been prepared in
advance, was added to the samples. The mixtures were heated in a
BLOCK INCUBATOR (ASTEC) at 42.degree. C. for one hour and at
99.degree. C. for five minutes, and then cooled on ice. 100 .mu.l
of desired cDNAs were prepared and PCR reaction was carried out
using the prepared cDNAs in the following composition.
[0555] 2 .mu.l of PCR Buffer [composition: 166 mM
(NH.sub.4).sub.2SO.sub.4 (Sigma Aldrich Japan), 670 mM Tris pH8.8
(Invitrogen), 67 mM MgCl.sub.2.6H.sub.2O (Sigma Aldrich Japan), 100
mM 2-mercaptoethanol (WAKO)], 0.8 .mu.l of 25 mM dNTP mix
(Invitrogen), 0.6 .mu.l of DMSO (Sigma Aldrich Japan), 0.2 .mu.l of
Primer Forward (GeneWorld), 0.2 .mu.l of Primer Reverse
(GeneWorld), 15.7 .mu.l of Otsuka distilled water (Otsuka
Pharmaceuticals, Inc.), 0.1 .mu.l of Taq polymerase (Perkin Elmer),
and 1 .mu.l of cDNA obtained as described above were combined, and
reacted using Authorized Thermal Cycler (eppendorf) at 30 cycles of
94.degree. C. for 45 seconds, 55.degree. C. for 45 seconds,
72.degree. C. for 60 seconds. After the reaction, the obtained PCR
products were combined with 2 .mu.l of Loading Dye (Invitrogen).
1.5% agarose gel was prepared using UltraPure Agarose (Invitrogen),
and the samples were electrophoresed in a Mupid-2 plus (ADVANCE) at
100 V for 20 minutes. After electrophoresis, the gel was shaken for
20 minutes in a stain solution prepared by 10,000 times diluting
Ethidium Bromide (Invitrogen) with 1.times. LoTE (composition: 3 mM
Tris-HCl (pH 7.5) (Invitrogen), 0.2 mM EDTA (pH 7.5) (Sigma Aldrich
Japan)). The gel was photographed with EXILIM (CASIO) positioned on
I-Scope WD (ADVANCE) and confirmed the gene expression. Cryoblock
preparation, immunostaining, and such were all achieved by the
methods described above.
[0556] The expression of CSPG in the MPDP-induced Parkinson's
disease model was assessed by immunostaining using antibody against
CS-56 (an anti-CSPG antibody, 1:100 dilution; Seikagaku Co.). The
result is shown in FIG. 75. Strong CSPG-positive signals were
observed in the untreated group, as shown in FIG. 75. Meanwhile,
the positive signals were reduced in the C4-sulfatase-treated group
and gene therapy group.
[Example 76] Localization of Dopaminergic Neurons in Brain Tissues
in a C57BL/6JcL Mouse Model for Parkinson's Disease Induced by
MPDP
[0557] To assess the pharmacological effects of C4-sulfatase and
GalNAc4S-6ST siRNA on dopaminergic neurons, localization of
dopaminergic neurons in the MPDP-induced Parkinson's disease model
was analyzed by fluorescent immunostaining using an anti-tyrosine
hydroxylase (TH) antibody (1:20 dilution) and an Alexa-488-labeled
anti-rabbit IgG antibody (1:200 dilution; Invitrogen) for the
secondary antibody. As shown in FIG. 76, the positive signals for
dopaminergic neurons localized in brain tissues were weaker in the
untreated group as compared to the other groups. Meanwhile, there
was no great difference of the positive signal intensity in the
C4-sulfatase-treated group and gene therapy group compared to the
control group. The result suggests that the protective effect or
regeneration/repair-promoting effect on dopaminergic neuron is
produced by the administration of C4-sulfatase or the siRNA.
[0558] Specifically, inhibitors that act on GalNAc at position 4 or
6 (desulfating enzymes for the sulfate group at position 4 of
GalNAc, and GalNAc4S-6ST siRNA) are useful as agents for protecting
dopaminergic neurons or as agents for promoting the
regeneration/repair of dopaminergic neurons.
[Example 77] Analysis of Inflammation-Related Gene Expression in
Brain Tissues in a C57BL/6JcL Mouse Parkinson's Disease Model
Induced by MPDP
[0559] To compare the anti-inflammatory effects of C4-sulfatase and
GalNAc siRNA, total RNAs were extracted by the method described
above from the same samples used to prepare tissue sections
described in Examples 75 and 76, and the expression of
TNF-.quadrature. was analyzed by quantitative PCR. For quantitative
PCR, SYBR Premix Kit (TAKARA BIO INC.) and Real-time PCR thermal
cycler DICE (TAKARA BIO INC.) were used. Conditions of PCR reaction
was: 95.degree. C. for 10 seconds, 40 cycles of 95.degree. C. for 5
seconds and 60.degree. C. for 30 seconds, finally, melting curve
analysis was conducted. Nucleotide sequences of primers used in the
quantitative PCR were described below.
TABLE-US-00014 mouse .beta. actin (TAKARA BIO INC.) Forward: (SEQ
ID NO: 93) 5'-CATCCGTAAAGACCTCTATGCCAAC-3' Reverse: (SEQ ID NO: 94)
5'-ATGGAGCCACCGATCCACA-3' Tumor Necrosis Factor (TNF-.alpha.)
(TAKARA BIO INC.) Forward: (SEQ ID NO: 95)
5'-CAGGAGGGAGAACAGAAACTCCA-3 Reverse: (SEQ ID NO: 96)
5'-CCTGGTTGGCTGCTTGCTT-3'
[0560] As shown in FIG. 77, the expression of TNF-.alpha. was
suppressed in the C4-sulfatase-treated group as compared to the
untreated group. Meanwhile, the gene therapy group only showed a
tendency of suppressing the expression. The result described above
demonstrates that C4-sulfatase has the activity of suppressing
inflammation.
[0561] Specifically, desulfating enzymes for the sulfate group at
position 4 of GalNAc are useful as anti-inflammatory agents.
[Example 78] Analysis of Inflammation-Related Genes for their
Expression in Brain Tissues in a C57BL/6JcL Mouse Parkinson's
Disease Model Induced by MPDP
[0562] To complement the results described in Example 77,
Examination was conducted using Nurr1, which is a gene involved in
the generation of dopaminergic neurons, as a marker in this
Example. The effects of C4-sulfatase and GalNAc4S-6ST were
evaluated by the above-described quantitative PCR. The result is
described below. The nucleotide sequences of primers used are as
follows:
TABLE-US-00015 Nuclear receptor subfamily 4 Group A member 2
(Nurr1): (TAKARA BIO INC.) Forward: (SEQ ID NO: 97)
5'-CTGCCCTGGCTATGGTCACA-3' Reverse: (SEQ ID NO: 98)
5'-AGACAGGTAGTTGGGTCGGTTCA-3'
[0563] As shown in FIG. 78, the expression level of Nurr1 was
significantly elevated in the C4-sulfatase-treated group and gene
therapy group as compared to the untreated group (P<0.001). This
result supports the result described in Example 76 and shows the
dopaminergic neuron-protecting effect or dopaminergic neuron
regeneration/repair-promoting effect by administration of
C4-sulfatase or the siRNA.
[0564] Specifically, inhibitors that act on GalNAc at position 4 or
6 (desulfating enzymes for the sulfate group at position 4 of
GalNAc, and GalNAc4S-6ST siRNA) are useful as agents for protecting
dopaminergic neurons or as agents for promoting the
regeneration/repair of dopaminergic neurons.
INDUSTRIAL APPLICABILITY
[0565] The present invention provides agents for suppressing
fibrogenesis at the physiological tissue level through inhibiting
the functions of sugar chain-related genes. The agents are useful
for treating or preventing tissue fibrogenic disorders.
[0566] Chronic tissue fibrogenesis (fibrogenic tissue alterations)
can develop in any organ in the body, and is a general term for a
group of diseases that cause organ dysfunctions, leading to death
(review: Wynn T A, J. Clin. Invest. 117: 524-529, 2007).
[0567] Fibrogenic disorders are thought to be a terminal stage of
chronic inflammation and can develop in any organ of the body.
"Fibrogenic disorders" is a general term for a group of diseases
that cause organ dysfunctions, leading to death. Fibrogenesis and
the resulting dysfunctions are suspected to be the root of diseases
with a high mortality rate, such as cardiovascular and
cerebrovascular diseases. There is a view that 45% of deaths are
caused by fibrogenesis in Western countries. Under this view,
causes of individual death from disease are summarized into three
groups: cancer, infections, and fibrogenesis. In addition to the
conventional definitions of fibrogenic disorders such as cirrhosis,
pulmonary fibrosis, and nephrosclerosis, a wide variety of diseases
(mainly chronic diseases and excluding cancer and infections), can
be defined as "fibrogenic disorders". Particularly, recent changes
in lifestyle habits (commonly called "Westernization") have led to
a rapid increase of new life-threatening diseases (disease
concepts) such as nonalcoholic steatohepatitis (NASH) and chronic
kidney disease (CKD). The finding that the diseases are caused by
tissue fibrogenesis suggests the urgency of establishing
therapeutic agents for "fibrogenic disorders". To date, however,
only transplantation and artificial organs are available and there
is no fundamental therapeutic method. This is a very urgent problem
to be solved.
[0568] The present invention provides techniques for suppressing
fibrogenic disorders (fibrogenic tissue lesions and resulting
dysfunctions) based on a completely new method that targets the
recently-identified sugar chain-related genes, functions of which
were unknown.
[0569] Fibrogenic disorders that lead to clinically intractable
dysfunctions are not only a deciding factor of death, but are also
disorders that severely impair the daily quality of life (QOL).
Thus, it is very important to establish tissue
fibrogenesis-suppressing agents. However, no such agents are
commercially available to date. Dedicated experimental studies are
being conducted to assess TGF-3 inhibitors, angiotensin inhibitors,
inflammatory cytokine inhibitors, TLR inhibitors, MMP inhibitors,
and the like. Still, however, their efficacy has not yet been
established (review: Wynn T A, J. Clin. Invest. 117: 524-529,
2007).
[0570] The present inventors administered inhibitors that target
sugar chain-related genes to subjects, which were model animals for
fibrogenic disorders of various organs, in order to relieve
fibrogenic lesions and to restore organ function.
[0571] The fibrogenic disorders were definitively confirmed based
on histopathological features (including immunostaining for
fibroblasts or collagen, and Masson staining). The therapeutic
effect (fibrogenesis inhibitory effect) was determined based on
clinical symptoms and the expression level of collagen in each
organ, in addition to the histopathological features.
[0572] The therapeutic effect was proven by using as a technical
method direct gene knockdown with nucleic acid pharmaceuticals
(siRNA) against sugar chain-related genes.
Sequence CWU 1
1
9819120DNAMus musculus 1tgtatatctg agtatctgtg tatgtgcata tatgtgtgtc
tatgtgtatg tgtgggtttg 60tgcatgtgtg tctgtgtgta catgtctgtg tgtctctgtg
tgtgtctgtg tatgtgtgtt 120tacatgtgtg tgtgtgtttc tgtgtggacg
catgtgtatg tgtctccgtg tgcctgcatg 180tgtgtttgtg tatgtgtgta
ggcatgagta catcatggtg tacatgtaga aattgagaga 240gactcttggg
tgtggttctc atctcccacc atatctgaag taaatgtctt attcacacca
300ctgtgcacat cagaaagcta gccttgacct ccaaggaact ctgtctctgt
ctcctgtctt 360attgtaggta cctggggttt ataagcctat gctgctgcat
atggcttcac ttgggttctg 420gggatttgaa cttaggtcct cacagtttta
gggcaagcat tttattcact gaacaatctc 480ccatgatccc tagagtttat
tgtaggcaac ttaaccttta gtcgtcattg agatttagca 540ttgatttcag
ttccagttgt attctgatgt tgagcgctac agcagaggta cccagggact
600gctgattttc tgtggccatg aatcctctag tgattaataa atttcatcat
aatgttctgt 660ttatttctaa atgtccagac aactctaatt tcaaattaat
ctcatcctaa acagcacagg 720ataaaagtta cagattgtcc tatcttcatt
catgtgatgc ctcaaatcag atgtttaatc 780ctttagtcag ttatttgtga
aacggaacct aattacacac actcgcatgc gcgtgcgcgc 840acacacacac
acacacacac acacacacac acacacacac acttgtagat ttgcccacac
900agctgcttaa gcaatttgga gcaaatgtag aaatattatg aaatttcaca
taaaaatggg 960ttttcctctg tcttcttgag aaatgtgtga gtttcaatat
gacagtaatc ttctggtgtt 1020ggactataac cacttgaacc cctgaggtac
taagtgtgta cctactacgt gcctggattc 1080catcatttcc ctacttatat
aagcttctcc atcctgtaga tcagcactgt gtaatggagt 1140aacacaagtc
attgaatcat gcaatcattt aatcaattta attggcactt ctgataagag
1200aagtgggcac atatttagtt gtgtttgcag cctgtgctat gtcatttttc
tctatatata 1260ctttaagatt tttttctttt ttccatttac gggggggggg
ttattccatt taatttacat 1320gcttaatgac atataatttg catatcaggt
gttcctttct ttagagttaa cttcttttag 1380gaccattgtt tataatggat
gtatatcatc cctaaattaa atcagccatt gtctacttaa 1440cacaccagtt
tctctgatgt ccctctggct cttcctcctc tgaggttcta gttataggaa
1500agtctggcat tttgatgttt ttccatggct tcgtgggctc tgatcatcca
cttacagtgt 1560actttcatct cttgtctaca aactgcatat catctattga
tctgcgttca aggctactac 1620attttcatgc aattccactt tgctgttaaa
ccttttctat gatccgtttt tatacttata 1680aacaatccaa gtgttgtact
ctttgggtat aaatgttctg tatttcacag gtcttaactt 1740cttttcagac
tatacaaaga aagattttct atttttttta tttgttgtat gcattttatg
1800catttagcac tgggatttcc ataacaggca gttaagatca ttgaatacat
actttgtgtg 1860gtaaatataa tacggcttat ttgattctcc cccttaaaat
gctcccatct tctcagtcat 1920gtcaggttgc tatggagtcc accctgagca
ccatgagtgt gatgctgtac agacactcct 1980gatatgcctt ctgttatttg
aactccacct gcaagttttc attcaagcat agctcctaat 2040cccattcagt
gtgcactttt cttaattgat ttgctggtag agagctatat gtaagcatgg
2100cttagcatca aattgaactt tctcctctcc ctagcttctt tccagtatat
tcttcctcac 2160cttcatcaga ttttgaggtc ttgagatcta aaccttcatg
ttacaatttg cagagatacc 2220aggatctcta tcagacttga tgtgtctcca
tgtaattcta gtatcagcct ttcataagat 2280aattgcaaaa tatagaaccc
cccctctctc cttttgacta ttacagcttt gtttctctcc 2340caagttaata
cagatgtatg caaatcaact aagaaaagca aacactgaat gggataagga
2400gctatactca aaagaaagct tgcagttgga gttcaaataa cacaagtgga
accaagagta 2460tctgcacagc atcagactca ctgtgctcag gatggaatcc
atagcaacct cccataactg 2520agaggatgtg agcccctttt aaagggaaaa
tcaaataagc agtattatat taaccacatg 2580atgtgtatat tcaatgtaga
ttgggctgca ccgaagttat gattttagta atgagtattt 2640ctccttgggt
tttcttctgg accaccctgg ttttcctttt aaaaatagta aaatagcagc
2700aatgactgct tctcttaaaa tctgaacaac acagagcact tcagcatcct
ttcaacctat 2760ctaaatccat tgtagacctg cactgacttg ctgtgagcta
ttgtgtcaca actattattg 2820tagctaaaca ccttgaattt tcaaagctgc
tctggactgt gtcttctttt agaattaaac 2880ttgctaaaga tattggactc
tgagatgctt attcaccact ttctataaaa gagtaaggat 2940gacaatgctt
tgagtagaag aagattgcat acgagggtac cagcatgtca ttatgtcccc
3000agcagtcagt aaagtaagga tccagtgatg gtagagttat tatgtccatt
atggaactaa 3060tgtaaatgtt tatcaatgac tagaatggct gtaatcacca
atccttagac aagtgtgagt 3120aacatcatgc tcttttctta taataataga
tagtcacaca caccatggtc ttaaacatta 3180cattttgatt tttctctttg
tgtctgtctt ttcgtacctc tactccactt acctgagcct 3240tcagctaggg
ctcctccaag acggcagtca cagtactagt gaggataatt ctcatatgga
3300gacctgaagc agaataacct tcttccaagg aaaccaaaat cctagtcaac
atcttctcca 3360ctctttggtt atttattttt atttctattt ctattttgcc
atttaagaat ttttaaactc 3420ttttctgttt tcttatatat attctttttt
ttacaacttt tgatattctt tatcaaatga 3480tttatcccat tgcctttcct
cctgcagcta tctgtgagtt ttagcatgga tatttagaat 3540acaattggaa
attatgaagg tcgaataaaa gttctctaag agccaggacc tcactagcct
3600agttagttgc ttagtttcta atataaggca tggtttatct cctactgtat
ggcccttaaa 3660tccactagac atctgttggc ttccaacagc atacaattgc
tgctattgta tgtttaggta 3720tatattttta tgaaacacta tattctgatt
ggtgttcatt cttcctatga aggggaagat 3780tttattgcct gctcaaattc
aagtcagtcc tttttgagta gacaaaggtt gtccaatctt 3840gctaaaccct
tttctttttt gcttggggaa tttctttagg gaaaattctc aaccatgaac
3900caagcctaga gattgataat agctgaaggc agagtagcca gtcttagtaa
gggatgagag 3960aaagactgac caaagactga ataggaaatg gttgatagca
ttcattggga ctagacaata 4020aagttagcta ccccaacatg ggcaaatgga
gaaaatgggg agagtcatat ggatatgtag 4080ggaggagaag aaaggaaggg
gtgagtttga agtatggaag agcctttaaa gcatttgtaa 4140gatttgtaag
aaaaagcttt tgagagacag agtattacct gccttcaaat cccttcaatt
4200actgtcccta caagacatac ctgtgctcag ctgctcttgg accacaaggg
ggcctaagag 4260catgtttgtg tccctgtctt tatacaggct tctcatctgt
atttctcaaa cgtaaataca 4320aatgggggag ataacaaatg atatttctaa
gtggtacaca ggtatgagaa ctgcacacaa 4380tctatttata tgtataatgg
ccagagctct cagcagggaa cagaggaagc ctaggtaact 4440cctgctccag
agcatgtctg tatggagacc tcagttcagc aactatgaaa tctgtgttca
4500aagggtcatc actgggaaca gtttctggaa tagcagacat attcattctc
tctctctctc 4560tctctctctc tctctctctc tctctctctc tctctctctc
tctctgtctg tctctgtctc 4620tctctgtctg tctgtctgtc tctcccttct
gtcaggggta tgcatacatg ggctgggaat 4680tctatcttcc ctgcctgctg
tctttggtgt ctccctaatg tggtgccatg tttacttaac 4740ctgtctgaaa
tggaggataa cctctggtct gataactgtc aagttactga aatgtcaggg
4800aattgtgaat gaacccctct gaatgctggg cagatgtttt gagcacgtgg
gtatccatag 4860attttttttt cagcaggatt tccttcttta atatattgtg
ggaacgaaac attgaagttt 4920ccaacactaa gaaaagcaat gctctgggtt
tgcaaagaag tctccaaagc tgtgggagag 4980gcaatggctt tgcctcacaa
aggtgatcct gatttggatt gggtgttcct cagtctccct 5040catcctttgt
ttgtggacca aacctctgat caaataatca gtatacagac aagtactgac
5100tgactcctgt cactgtttcc catgtaaatt ctcaaccatg tgtgtcagaa
ttgtttgagt 5160catcaaagtt ccgtgatgct caggatggtg aataccagaa
actgaagtct gaaatccaat 5220ttggcctttc tcgggcactg taaactgagt
gcaaatgtgc ccagttccct cgtggcccat 5280ttatagcaca ataacacagt
gcttgcttac aaaatctagt cccgattaaa gtatttattt 5340aaagttgctt
tagacaaata tatttctaaa tggaaggatg aaacagaaag aatattccaa
5400ctgtacagtc tatattttgt ttctaaaaat ttttattaga attctgactt
ttagaaaaca 5460aatagatatt caaaaactac ttctttaatt aaataataac
atcattatag acattctcca 5520gccatttaga agtagcatga ttacaaagca
ttcatgttcc ctttgttttt tgaaacagaa 5580gtatttatat gacaaataaa
aagcaaaaat ctcatatatg tgatagctat agatgcattg 5640ataggaatta
aatttaatca aaaagaactt ttaatggcaa taatgtggct actaacttca
5700ttgtttgtaa taatgctgca aatgtttaat gaaaactgtt actaacaata
caggagaaga 5760ttacagatat gattaggtcg tgttgataga aatggaaaga
gaaaggtcac tcactacctc 5820agccacagaa cttgaggaat gtgtgagggt
ttttgaggaa aaaagagaac aatgtaacat 5880cacagcaaca tcttaatctg
aaagtagacg acaaaggttt cagagtaggg gatcagaaat 5940ttaagtatgc
agagaagcaa ggcatatacg gcttagacaa tgtatgaaga acagcagcag
6000acagcaagtg aaagaaatca cagtccacat ggaaatcaca cagaccacag
tgtgcacaca 6060gagacagaaa tcagcaaggg acacacggct tccagttatg
aaagttagac agcaaaattt 6120aagttgtccc acctaaaaca tgatgtatcg
tctctcaaat ggggaagggg aaggagcagc 6180aaacatctgg atatgtctaa
aacctcacag agaacatctg ggatctttgc atttctccct 6240ctgactttct
caatatgtta gctacttcta gatagtttca ataatggtct ctaattgact
6300atggagagac aggtctcatg tatgtgaaca tccttaacca ggagatggaa
tgtgtttcct 6360tgtcactcat tttctaagga agtgaattcc ttctgcattg
cccaggattg gatgttacgt 6420gaaactaaaa atcattgatt aaaaagaaat
gtcattacca cagttgggtt gaatcaatca 6480agacttatcc tagttacagg
catcacctcc ccttaagcac aaatactgag aacagctaag 6540ctcactttga
aatctgggat gaggaggctt taggtcaagc atctgacaat gtgtgtgtgt
6600atgtgtgtgt gtgtgtgtgt gtgtagtatg attatatgta tatatgtaga
tgtgtgtata 6660tatttatatg tatatatgta tatgtgagta tgtatatatg
tatatgtgtg tatgtgtatg 6720agtgtgtgta tgtatgacta tgagtgtgta
tgtattgtgc aagtgtgtgt atatatgtgt 6780gtatatgttt atgtatatgt
gtattgtatg ggtgtgaatg tattatatat gtgtgtaggt 6840acatatgtgt
atatgtttat gcgtgtatat gtgtatgcat tatatgtatg tgtgtatgtg
6900tgtatgtttt catgtgtatg tagcatatgt atatgcatgt gtgtgtctgt
gtgtgtaagc 6960atgtatttac taagaaaaaa tcagtttctc tagctaatga
gtcattgaag tgaagaaacc 7020ttacgctatc acattactat gtttccagca
taaattgcaa tatcagtaag acattcccca 7080tcataactta atggacactt
agattaggtc atgcagcagt aaattatgaa agcgtctatg 7140ctgagctgag
gtgggaaatc gtctttcctg cctccttcct cctgttcttt ctcctaaagg
7200ccctcatcct ctttctcccc ttcctttctt ttaaaatatt ctatcattga
tccaagctaa 7260ctaacccttc ccttgctcac atgtgacctg cccagaggaa
tctatttata gtcaacaata 7320acaggccaag gacatgcgaa caaatgactt
tacttagccc cacttggtga atctattggg 7380tgaataggta aggggttatt
tataggagct tgactgactg gcacagctgt atctctgaaa 7440agtcccccaa
agcatcatgg gtgggaacta aggaaagccg gaaatgctgg agctggaatc
7500ccaacctgca gtggctacat gatgaatgtg tctcctttcc tactaaatat
ttaagactct 7560gggcaggctg gttagtcttg taaaagtctt gtgggtttct
ggagtcctgg gaggctcact 7620gtccctgcag aagcttgtta ttattgtata
tgaaagaaag tgctcaacac tgagtcacaa 7680aagaagcccc cgagaatcgg
tggcagaggt ggaaatagaa gaatccgatg cagttcccga 7740ggcaagagtt
gatcctctcc atggtgacaa gaaaacaaga gccacagaag cctgaaggct
7800aagactatga cctgtgaggt tctcctctcc taggacttga atgcacttaa
ctctgagggc 7860tagcagggca gagattcagg agcgttctca gaggttgtcc
cttctccttg tatgattcta 7920ggagaagctc agtgcatagg cagaagttgc
aaactctaac acattaaccc acatcctgaa 7980ttgttttata tcaattgttt
tataactaca tgttttaact tttgttttgg tttttgaatt 8040ttgtttgttt
gtttgttttc tatttttcat atgtgtgcat gtgagccaga agacaggcta
8100agggactctg ttctctcttt ccagcatgtg ggtttcaggg atcaaagtta
ggtcccaagg 8160cttcactggg caacgggacc cttagcctct gagtcatctt
cttaatcttt ttctttgaaa 8220ctttttcatt acattgtgtc agtgcacaca
cacacacaca cacacatgca tatacatcca 8280catgcatata catccacatg
ccatatgtca tggcacatgt gtggaaagaa atgggtgcct 8340tgaggaaatc
agttttcttc ttccatcacg tggatcttat gacttcaggt aacccatctc
8400tgctccccaa gcatggctac aagcatttac ctgccaagtc atctccctgg
ctcccacatt 8460aatttataat aatacaccct ttcagcctag agagatgact
cagtggttaa gaactctggc 8520tgtgcttcag aaggttctga gttcaattcc
cagcagctat aaggtggctc acaactatct 8580ctaatgttgt tttctgtcct
cttctggcat gcacttgtat gtgcagatag agtacttata 8640tgcataaata
aatataaata cattaataat cttttaaaaa gaaggaaaga ttaaaagaac
8700aatacaccct ttcttttaag taagaaaaca tgcttagtat atagaataaa
tgctggtgtt 8760gaaaggtgaa attcagaaga aattataggt tggaaatatt
tgaactcttt tgtgtcaaac 8820caaatggtaa agatgatgtg ataataaggt
aaggttttct tcctacacat atacttgttt 8880aataaccagt ataagactat
gagtgaacat aactaaaaat acatgaaaaa aaactatttt 8940aaaaaaacat
cactttcaga tgactctggt gaaactcact gataaccagc aatctctttg
9000atagaccaga gtgtccagtc tgaagttaat tcttttgctt tattctcaaa
caatgttgca 9060gaagtatgaa ccatagtatc aagatacact gtccacatat
gcacctggcc tgacttgtgt 912022105DNAMus musculusCDS(418)..(1671)
2gagccttccc ggcgcgtgag ccggatccgg tggcaccgcg gggaagagac aggaccgggc
60ggtggcggca gagacagggg gacgcacccg gtgcagaaga tccaatagga gcacgccgcc
120gcaacctctc ccgcgcgctc cggtcgccga ctctacgccg atcgcccact
ccccgcacct 180tggactacac cgggaaaaag acgcagcctg gcccgaactg
gggcggagat ccttcttgct 240tcccaagagc tcgggaggga agattctccc
gccgccgaga accccgccgg actggaggaa 300cccgtggcct ggagagcgct
ggctgtgcca gaccccagcc tgatggatgt ctggtgtgga 360taatgaggga
agaacgtgcc ttttacaccc aagaggtgac cccggagcgt gccccgg 417atg acc cca
caa ctc gga acg atg cgg cta gcc tgc atg ttc tcg tcc 465Met Thr Pro
Gln Leu Gly Thr Met Arg Leu Ala Cys Met Phe Ser Ser1 5 10 15atc ctg
ctg ttt gga gct gcg ggc ctg ctc ctc ttc atc agc ctc cag 513Ile Leu
Leu Phe Gly Ala Ala Gly Leu Leu Leu Phe Ile Ser Leu Gln 20 25 30gac
cct ata gag ctc agc ccc cag caa gtt cca ggt ata aag ttc agc 561Asp
Pro Ile Glu Leu Ser Pro Gln Gln Val Pro Gly Ile Lys Phe Ser 35 40
45atc agg ccc cag caa ccc cag cat gat agc cac ttg agg ata tcc aca
609Ile Arg Pro Gln Gln Pro Gln His Asp Ser His Leu Arg Ile Ser Thr
50 55 60gaa aag ggc aca cga gat tca ccc agc ggg tcg cca aga ggc ctc
cag 657Glu Lys Gly Thr Arg Asp Ser Pro Ser Gly Ser Pro Arg Gly Leu
Gln65 70 75 80ctg caa gcg cct gac caa cct cga cct cac ccg aag gca
gcg gga tct 705Leu Gln Ala Pro Asp Gln Pro Arg Pro His Pro Lys Ala
Ala Gly Ser 85 90 95cct ttg cgc ctc cgg cag cgc agg cgg aga ctg ctc
atc aaa aag atg 753Pro Leu Arg Leu Arg Gln Arg Arg Arg Arg Leu Leu
Ile Lys Lys Met 100 105 110cca gcc gca ggg act aac caa ggc aac aac
tcg tcc gaa acc ttt atc 801Pro Ala Ala Gly Thr Asn Gln Gly Asn Asn
Ser Ser Glu Thr Phe Ile 115 120 125cag ccg aga ccc cgc acc atg gac
agt cgt tgg gtc agc ctg cac cag 849Gln Pro Arg Pro Arg Thr Met Asp
Ser Arg Trp Val Ser Leu His Gln 130 135 140acc caa cag gag cgc aag
cgt gtg atg cga gaa gcc tgc gct aaa tac 897Thr Gln Gln Glu Arg Lys
Arg Val Met Arg Glu Ala Cys Ala Lys Tyr145 150 155 160agg gcc agc
agc agc cgc aga gct gtc act ccc cgc cac gtc tcc cgc 945Arg Ala Ser
Ser Ser Arg Arg Ala Val Thr Pro Arg His Val Ser Arg 165 170 175atc
ttc gtg gag gac cgc cac cgt gta ctg tac tgt gaa gta ccc aag 993Ile
Phe Val Glu Asp Arg His Arg Val Leu Tyr Cys Glu Val Pro Lys 180 185
190gca ggc tgc tcc aac tgg aag agg gtg ctc atg gtg ctg gca ggg tta
1041Ala Gly Cys Ser Asn Trp Lys Arg Val Leu Met Val Leu Ala Gly Leu
195 200 205gcc tca tcc acg gca gat atc caa cac aac acc gtc cac tat
ggc agc 1089Ala Ser Ser Thr Ala Asp Ile Gln His Asn Thr Val His Tyr
Gly Ser 210 215 220gcc ctt aag cgc ctg gat act ttt gac cgg cag ggc
ata gtg cac cgc 1137Ala Leu Lys Arg Leu Asp Thr Phe Asp Arg Gln Gly
Ile Val His Arg225 230 235 240ctc agt acc tac acc aag atg ctc ttt
gtc cgg gaa ccc ttt gag cgg 1185Leu Ser Thr Tyr Thr Lys Met Leu Phe
Val Arg Glu Pro Phe Glu Arg 245 250 255ctg gtc tct gct ttc cga gac
aag ttt gag cat cct aac agc tac tat 1233Leu Val Ser Ala Phe Arg Asp
Lys Phe Glu His Pro Asn Ser Tyr Tyr 260 265 270cat cct gtc ttt ggc
aag gct atc ctg gcc cgg tac cgc gcc aac gcc 1281His Pro Val Phe Gly
Lys Ala Ile Leu Ala Arg Tyr Arg Ala Asn Ala 275 280 285tcg cgg gag
gca ctg cgg act ggc tcc ggt gtg cag ttc ccc gag ttc 1329Ser Arg Glu
Ala Leu Arg Thr Gly Ser Gly Val Gln Phe Pro Glu Phe 290 295 300gtc
cag tac ctg ttg gat gtc cac cgg ccc gtg ggc atg gac atc cac 1377Val
Gln Tyr Leu Leu Asp Val His Arg Pro Val Gly Met Asp Ile His305 310
315 320tgg gac cat gtt agc cgg ctg tgc agc ccc tgc ctc atc gac tat
gac 1425Trp Asp His Val Ser Arg Leu Cys Ser Pro Cys Leu Ile Asp Tyr
Asp 325 330 335ttt gtg ggc aag ttc gag agc atg gaa gac gat gcc aac
ttc ttc ctg 1473Phe Val Gly Lys Phe Glu Ser Met Glu Asp Asp Ala Asn
Phe Phe Leu 340 345 350cgt ctc atc cat gcg ccc ggg aac ctg act ttc
ccg agg ttc aag gac 1521Arg Leu Ile His Ala Pro Gly Asn Leu Thr Phe
Pro Arg Phe Lys Asp 355 360 365agg cac tcc gag gag gcg cgg acc aca
tcg aga atc acc cat cag tac 1569Arg His Ser Glu Glu Ala Arg Thr Thr
Ser Arg Ile Thr His Gln Tyr 370 375 380ttc gct cag ctc tcc tcg ctg
cag cga cag cga acc tac gac ttc tac 1617Phe Ala Gln Leu Ser Ser Leu
Gln Arg Gln Arg Thr Tyr Asp Phe Tyr385 390 395 400tac atg gat tac
ctg atg ttc aac tac tcc aaa cct ttc tcg gac ctg 1665Tyr Met Asp Tyr
Leu Met Phe Asn Tyr Ser Lys Pro Phe Ser Asp Leu 405 410 415tac tga
gggcggggcc tgctggtcag gggcggggtc tgccggtcat gcccactcac
1721Tyrctgcgcatag gcggcctccg gggactgagc tctgaggatg tgaggccttg
tggctgtggc 1781cctagggtgg gccacagagg cccagacaat ggaccttgac
ccttgtccca cacccatttc 1841ctcattgggt tggctgagtt tgagacggag
cacgactcgg atggatgctt taagaactca 1901gctgagctat gctgagctat
gctgtccagg gaagcctgag acccagaaga gggccccagc 1961gtcgagggat
gtcctacatc cccttatcct ttgccttgta ccaaaccacg tggtttgctg
2021cttttctatg acccagggtc atctgaataa agcacatggt tttcagagca
aaaaaaaaaa 2081aaaaaaaaaa aaaaaaaaaa aaaa 21053417PRTMus musculus
3Met Thr Pro Gln Leu Gly Thr Met Arg Leu Ala Cys Met Phe Ser Ser1 5
10 15Ile Leu Leu Phe Gly Ala Ala Gly Leu Leu Leu Phe Ile Ser Leu
Gln 20 25 30Asp Pro Ile Glu Leu Ser Pro Gln Gln Val Pro Gly Ile Lys
Phe Ser 35 40 45Ile Arg Pro Gln Gln Pro Gln His Asp Ser His Leu Arg
Ile Ser Thr 50 55 60Glu Lys Gly Thr Arg Asp Ser Pro Ser Gly Ser Pro
Arg Gly Leu Gln65 70 75 80Leu Gln Ala Pro Asp Gln Pro Arg Pro His
Pro Lys Ala Ala Gly Ser 85 90 95Pro Leu Arg Leu Arg Gln Arg Arg Arg
Arg Leu Leu Ile Lys Lys Met 100 105 110Pro Ala Ala Gly Thr Asn Gln
Gly Asn Asn Ser Ser Glu Thr Phe Ile 115 120 125Gln Pro Arg Pro Arg
Thr Met Asp Ser Arg Trp Val Ser Leu His Gln 130 135 140Thr Gln Gln
Glu Arg Lys Arg
Val Met Arg Glu Ala Cys Ala Lys Tyr145 150 155 160Arg Ala Ser Ser
Ser Arg Arg Ala Val Thr Pro Arg His Val Ser Arg 165 170 175Ile Phe
Val Glu Asp Arg His Arg Val Leu Tyr Cys Glu Val Pro Lys 180 185
190Ala Gly Cys Ser Asn Trp Lys Arg Val Leu Met Val Leu Ala Gly Leu
195 200 205Ala Ser Ser Thr Ala Asp Ile Gln His Asn Thr Val His Tyr
Gly Ser 210 215 220Ala Leu Lys Arg Leu Asp Thr Phe Asp Arg Gln Gly
Ile Val His Arg225 230 235 240Leu Ser Thr Tyr Thr Lys Met Leu Phe
Val Arg Glu Pro Phe Glu Arg 245 250 255Leu Val Ser Ala Phe Arg Asp
Lys Phe Glu His Pro Asn Ser Tyr Tyr 260 265 270His Pro Val Phe Gly
Lys Ala Ile Leu Ala Arg Tyr Arg Ala Asn Ala 275 280 285Ser Arg Glu
Ala Leu Arg Thr Gly Ser Gly Val Gln Phe Pro Glu Phe 290 295 300Val
Gln Tyr Leu Leu Asp Val His Arg Pro Val Gly Met Asp Ile His305 310
315 320Trp Asp His Val Ser Arg Leu Cys Ser Pro Cys Leu Ile Asp Tyr
Asp 325 330 335Phe Val Gly Lys Phe Glu Ser Met Glu Asp Asp Ala Asn
Phe Phe Leu 340 345 350Arg Leu Ile His Ala Pro Gly Asn Leu Thr Phe
Pro Arg Phe Lys Asp 355 360 365Arg His Ser Glu Glu Ala Arg Thr Thr
Ser Arg Ile Thr His Gln Tyr 370 375 380Phe Ala Gln Leu Ser Ser Leu
Gln Arg Gln Arg Thr Tyr Asp Phe Tyr385 390 395 400Tyr Met Asp Tyr
Leu Met Phe Asn Tyr Ser Lys Pro Phe Ser Asp Leu 405 410
415Tyr41369DNAMus musculusCDS(40)..(1281) 4attcagacat gaagagagac
acagtggtct gaagtggtc atg aaa gcc aaa caa 54 Met Lys Ala Lys Gln 1
5gtc ttc ttt tct gtc ctg ctg ttt ggg aca gca ggg ctt ctg ctc ttc
102Val Phe Phe Ser Val Leu Leu Phe Gly Thr Ala Gly Leu Leu Leu Phe
10 15 20atg tac ttg caa gca tgg att gaa gaa cat cat aca ggg aaa ata
gag 150Met Tyr Leu Gln Ala Trp Ile Glu Glu His His Thr Gly Lys Ile
Glu 25 30 35aag aaa agg gat cag aaa gga gta tcg gtg act acg gga aaa
atc cag 198Lys Lys Arg Asp Gln Lys Gly Val Ser Val Thr Thr Gly Lys
Ile Gln 40 45 50aaa cag atc acg aat cag aac tct gag gtt cac atg cct
gaa gat ctg 246Lys Gln Ile Thr Asn Gln Asn Ser Glu Val His Met Pro
Glu Asp Leu 55 60 65aag aag aaa ggg gga gat ctg ctc aac cta ggg agt
cca aca agg gtt 294Lys Lys Lys Gly Gly Asp Leu Leu Asn Leu Gly Ser
Pro Thr Arg Val70 75 80 85tta agg aag atc agc cat tca caa agg gag
aac gga gct tac aga tca 342Leu Arg Lys Ile Ser His Ser Gln Arg Glu
Asn Gly Ala Tyr Arg Ser 90 95 100act gaa gca cat caa gga gct aaa
att gaa gtt ttt cag aaa ccc atc 390Thr Glu Ala His Gln Gly Ala Lys
Ile Glu Val Phe Gln Lys Pro Ile 105 110 115cag atg gac tgg cca ctg
gtc act cag ccc tta aac aaa agt ttg gtc 438Gln Met Asp Trp Pro Leu
Val Thr Gln Pro Leu Asn Lys Ser Leu Val 120 125 130caa ggc aac aaa
tgg aag aaa gca gat gca acc caa gag aag cgt cgg 486Gln Gly Asn Lys
Trp Lys Lys Ala Asp Ala Thr Gln Glu Lys Arg Arg 135 140 145tca ttc
ctt cat gag ttt tgc aag aaa tat ggt aga gta aat gat ccc 534Ser Phe
Leu His Glu Phe Cys Lys Lys Tyr Gly Arg Val Asn Asp Pro150 155 160
165aag ttc aac ctt ttt cat ata gta tct agg ata tat gta gaa gac aaa
582Lys Phe Asn Leu Phe His Ile Val Ser Arg Ile Tyr Val Glu Asp Lys
170 175 180cac aaa atc ctg tac tgt gaa gta cca aaa gct ggc tgc tct
aat tgg 630His Lys Ile Leu Tyr Cys Glu Val Pro Lys Ala Gly Cys Ser
Asn Trp 185 190 195aaa aga att ctg atg gtc cta aat gga ttg gct tcc
tct gca tac aat 678Lys Arg Ile Leu Met Val Leu Asn Gly Leu Ala Ser
Ser Ala Tyr Asn 200 205 210atc tcc cat gat act gtg cac tat ggg aag
cat ctg aaa aca ctg gat 726Ile Ser His Asp Thr Val His Tyr Gly Lys
His Leu Lys Thr Leu Asp 215 220 225agt ttt gac tta aaa gga gta cac
atg cgt ttg aat aca tat acc aaa 774Ser Phe Asp Leu Lys Gly Val His
Met Arg Leu Asn Thr Tyr Thr Lys230 235 240 245gct gtg ttt gtt aga
gat ccc atg gaa aga tta gtc tcc gca ttt agg 822Ala Val Phe Val Arg
Asp Pro Met Glu Arg Leu Val Ser Ala Phe Arg 250 255 260gat aaa ttt
gag cat ccc aat agt tac tac cat ccg gtg ttt gga aag 870Asp Lys Phe
Glu His Pro Asn Ser Tyr Tyr His Pro Val Phe Gly Lys 265 270 275gca
att atc aag aaa tat cga cca aat gcc tct gca gaa gca tta aat 918Ala
Ile Ile Lys Lys Tyr Arg Pro Asn Ala Ser Ala Glu Ala Leu Asn 280 285
290aat gga tct gga gtc aaa ttc aaa gaa ttc gcc tac tat ttg ctg gat
966Asn Gly Ser Gly Val Lys Phe Lys Glu Phe Ala Tyr Tyr Leu Leu Asp
295 300 305gct cac cgt cca gta gga atg gat att cac tgg gaa aga gtc
agc aaa 1014Ala His Arg Pro Val Gly Met Asp Ile His Trp Glu Arg Val
Ser Lys310 315 320 325ctg tgt tat ccg tgt ttg atc aac tat gac ttt
gta ggg aag ttt gag 1062Leu Cys Tyr Pro Cys Leu Ile Asn Tyr Asp Phe
Val Gly Lys Phe Glu 330 335 340acc tta gga gag gat gcc aat tac ttt
cta cag ttg att ggt gct cca 1110Thr Leu Gly Glu Asp Ala Asn Tyr Phe
Leu Gln Leu Ile Gly Ala Pro 345 350 355aaa gag ttg aca ttt cca aac
ttt aag gat agg cac tcc tct gat gaa 1158Lys Glu Leu Thr Phe Pro Asn
Phe Lys Asp Arg His Ser Ser Asp Glu 360 365 370aga acc aat gcc cac
gtg gta agg cag tat tta aag gac ctg agc aca 1206Arg Thr Asn Ala His
Val Val Arg Gln Tyr Leu Lys Asp Leu Ser Thr 375 380 385gcc gaa aga
cag ctc atc tat gac ttc tat cac ttg gac tat ttg atg 1254Ala Glu Arg
Gln Leu Ile Tyr Asp Phe Tyr His Leu Asp Tyr Leu Met390 395 400
405ttt aat tac aca act cca cat ttg taa tttgcattca tttttctaaa
1301Phe Asn Tyr Thr Thr Pro His Leu 410gccctacata gatttaatga
tgatggcctc aaataagcta ctgtaattgt cctacaattc 1361tctgtatg
13695413PRTMus musculus 5Met Lys Ala Lys Gln Val Phe Phe Ser Val
Leu Leu Phe Gly Thr Ala1 5 10 15Gly Leu Leu Leu Phe Met Tyr Leu Gln
Ala Trp Ile Glu Glu His His 20 25 30Thr Gly Lys Ile Glu Lys Lys Arg
Asp Gln Lys Gly Val Ser Val Thr 35 40 45Thr Gly Lys Ile Gln Lys Gln
Ile Thr Asn Gln Asn Ser Glu Val His 50 55 60Met Pro Glu Asp Leu Lys
Lys Lys Gly Gly Asp Leu Leu Asn Leu Gly65 70 75 80Ser Pro Thr Arg
Val Leu Arg Lys Ile Ser His Ser Gln Arg Glu Asn 85 90 95Gly Ala Tyr
Arg Ser Thr Glu Ala His Gln Gly Ala Lys Ile Glu Val 100 105 110Phe
Gln Lys Pro Ile Gln Met Asp Trp Pro Leu Val Thr Gln Pro Leu 115 120
125Asn Lys Ser Leu Val Gln Gly Asn Lys Trp Lys Lys Ala Asp Ala Thr
130 135 140Gln Glu Lys Arg Arg Ser Phe Leu His Glu Phe Cys Lys Lys
Tyr Gly145 150 155 160Arg Val Asn Asp Pro Lys Phe Asn Leu Phe His
Ile Val Ser Arg Ile 165 170 175Tyr Val Glu Asp Lys His Lys Ile Leu
Tyr Cys Glu Val Pro Lys Ala 180 185 190Gly Cys Ser Asn Trp Lys Arg
Ile Leu Met Val Leu Asn Gly Leu Ala 195 200 205Ser Ser Ala Tyr Asn
Ile Ser His Asp Thr Val His Tyr Gly Lys His 210 215 220Leu Lys Thr
Leu Asp Ser Phe Asp Leu Lys Gly Val His Met Arg Leu225 230 235
240Asn Thr Tyr Thr Lys Ala Val Phe Val Arg Asp Pro Met Glu Arg Leu
245 250 255Val Ser Ala Phe Arg Asp Lys Phe Glu His Pro Asn Ser Tyr
Tyr His 260 265 270Pro Val Phe Gly Lys Ala Ile Ile Lys Lys Tyr Arg
Pro Asn Ala Ser 275 280 285Ala Glu Ala Leu Asn Asn Gly Ser Gly Val
Lys Phe Lys Glu Phe Ala 290 295 300Tyr Tyr Leu Leu Asp Ala His Arg
Pro Val Gly Met Asp Ile His Trp305 310 315 320Glu Arg Val Ser Lys
Leu Cys Tyr Pro Cys Leu Ile Asn Tyr Asp Phe 325 330 335Val Gly Lys
Phe Glu Thr Leu Gly Glu Asp Ala Asn Tyr Phe Leu Gln 340 345 350Leu
Ile Gly Ala Pro Lys Glu Leu Thr Phe Pro Asn Phe Lys Asp Arg 355 360
365His Ser Ser Asp Glu Arg Thr Asn Ala His Val Val Arg Gln Tyr Leu
370 375 380Lys Asp Leu Ser Thr Ala Glu Arg Gln Leu Ile Tyr Asp Phe
Tyr His385 390 395 400Leu Asp Tyr Leu Met Phe Asn Tyr Thr Thr Pro
His Leu 405 41065532DNAMus musculusCDS(369)..(1427) 6ccagtgcacg
gcggcggcgg caccttcctt ctcgcatccc ggtcgcccgg cccgtgagga 60ggcagcggcg
gccgcaggcg ccagcagagg aggcggcgga gcatcgagca gaggcgaggc
120ggaccggcag gagcgcgcgg ccggagccac gcatcctcac acttgcacac
caactcctgc 180gcctttcacc ctctagacca gagtcggggc ctcggagagc
ctcggtgaag ctagcgggag 240ccagctccgg agcccagcca gctctgttct
gagcctcccc ggtgctggta cccccgcggc 300ggggtccccg cttctgcgct
cgggacgcgc ctcccggaca gccgggtccc cgcggccagg 360acaaagcc atg aag ccg
gcg ctg ctg gaa gtg atg agg atg aac aga att 410 Met Lys Pro Ala Leu
Leu Glu Val Met Arg Met Asn Arg Ile 1 5 10tgc cgg atg gtg ctg gcc
act tgc ttc gga tcc ttt atc ttg gtc atc 458Cys Arg Met Val Leu Ala
Thr Cys Phe Gly Ser Phe Ile Leu Val Ile15 20 25 30ttc tat ttc caa
agt atg ttg cac cca gtc atg cgg agg aac ccc ttc 506Phe Tyr Phe Gln
Ser Met Leu His Pro Val Met Arg Arg Asn Pro Phe 35 40 45ggt gtg gac
atc tgc tgc cgg aag gga tcg aga agt ccc ctg cag gag 554Gly Val Asp
Ile Cys Cys Arg Lys Gly Ser Arg Ser Pro Leu Gln Glu 50 55 60ctc tac
aat ccc atc cag ctg gag cta tcc aac act gcc atc ctg cac 602Leu Tyr
Asn Pro Ile Gln Leu Glu Leu Ser Asn Thr Ala Ile Leu His 65 70 75cag
atg aga cgg gac cag gtg aca gac acc tgc cgg gcc aac agt gcc 650Gln
Met Arg Arg Asp Gln Val Thr Asp Thr Cys Arg Ala Asn Ser Ala 80 85
90atg agc cgc aag cgc agg gtg ctg acc ccc aac gac ctg aag cac ctg
698Met Ser Arg Lys Arg Arg Val Leu Thr Pro Asn Asp Leu Lys His
Leu95 100 105 110gtg gtg gat gag gac cac gaa ctc atc tac tgc tat
gtg ccc aag gta 746Val Val Asp Glu Asp His Glu Leu Ile Tyr Cys Tyr
Val Pro Lys Val 115 120 125gcg tgc acc aac tgg aag agg ctc atg atg
gtc ctg agt ggc cgg ggc 794Ala Cys Thr Asn Trp Lys Arg Leu Met Met
Val Leu Ser Gly Arg Gly 130 135 140aag tac agc gat ccc atg gag atc
cca gcc aac gaa gcc cac gtg tcg 842Lys Tyr Ser Asp Pro Met Glu Ile
Pro Ala Asn Glu Ala His Val Ser 145 150 155gcc aac ctg aag acc ctt
aac cag tac agc atc cca gag atc aac cac 890Ala Asn Leu Lys Thr Leu
Asn Gln Tyr Ser Ile Pro Glu Ile Asn His 160 165 170cgc ttg aaa agc
tac atg aag ttc ctg ttc gtg cgg gaa ccc ttc gag 938Arg Leu Lys Ser
Tyr Met Lys Phe Leu Phe Val Arg Glu Pro Phe Glu175 180 185 190agg
ctg gtg tct gcc tac cgc aac aag ttc acg cag aag tac aac acc 986Arg
Leu Val Ser Ala Tyr Arg Asn Lys Phe Thr Gln Lys Tyr Asn Thr 195 200
205tcc ttc cac aag cgc tac ggc acc aag atc atc cga cgc cag cgg aag
1034Ser Phe His Lys Arg Tyr Gly Thr Lys Ile Ile Arg Arg Gln Arg Lys
210 215 220aac gcc acg cag gag gcc ctg cgc aag ggg gac gat gtc aag
ttc gag 1082Asn Ala Thr Gln Glu Ala Leu Arg Lys Gly Asp Asp Val Lys
Phe Glu 225 230 235gag ttc gtg gcc tac ctc atc gac ccc cac acc cag
cgg gag gag ccc 1130Glu Phe Val Ala Tyr Leu Ile Asp Pro His Thr Gln
Arg Glu Glu Pro 240 245 250ttc aac gag cac tgg cag acg gtc tac tct
ctc tgc cac ccg tgc cac 1178Phe Asn Glu His Trp Gln Thr Val Tyr Ser
Leu Cys His Pro Cys His255 260 265 270atc cac tac gac ctc gtg ggc
aag tat gag aca ctg gag gag gac tcc 1226Ile His Tyr Asp Leu Val Gly
Lys Tyr Glu Thr Leu Glu Glu Asp Ser 275 280 285aat tac gta ctg cag
ctg gcc gga gtg agc ggc tac ctg aag ttc ccc 1274Asn Tyr Val Leu Gln
Leu Ala Gly Val Ser Gly Tyr Leu Lys Phe Pro 290 295 300acc tat gca
aag tcc acc cga act acc gac gag atg acc acg gag ttc 1322Thr Tyr Ala
Lys Ser Thr Arg Thr Thr Asp Glu Met Thr Thr Glu Phe 305 310 315ttc
cag aac atc agc gcc gag cac cag aca cag ctg tac gaa gtc tac 1370Phe
Gln Asn Ile Ser Ala Glu His Gln Thr Gln Leu Tyr Glu Val Tyr 320 325
330aaa ctg gac ttt tta atg ttc aac tac tca gtg cca aac tac ctg aag
1418Lys Leu Asp Phe Leu Met Phe Asn Tyr Ser Val Pro Asn Tyr Leu
Lys335 340 345 350ttg gat tag agggctgcgg aggggagggg gaggggtggt
tgggggagag 1467Leu Aspggagagaatc ctgcttttta atttaagatt tttatttgtc
aaaagaattc tatggagact 1527gggttatttt gtaagttaat atgtcttcgg
gggagatgct gcgagcggca tggttaagaa 1587ttatttaaaa attctccacg
gggaaggaca gctgtctttg caggggagcg gggtggaata 1647tccctgtttt
tagaagtgga tactgcaaca ctgtctccaa ggtgtccttg tgttctggtg
1707aagtccacaa actgcattcc ataaagtcta atgaatctta tttatagtta
tttaaacgtg 1767gtctgtggca gcagcctgct cctgtctgtg cagaggagag
tacagtctgt gcttcgctgg 1827ctatggctca ttgggggagg ggagggggtt
atctccccca ctgaccgtgt gtagaatccc 1887atggtgacag ctgcgggatg
tgtgcctgtg gccattcaag aaaccctgct agaaagaagt 1947caattgtcct
tgcactaaga aacagggatg agactggttc aaaccgattg ctagcaaccc
2007caaagtccct tgtagtaaag aaatgcaaat ttttttaacc caagaagaga
gcagatgata 2067gcgatttctc tttaccgaaa ttagcaggct ataagaatag
gctttctgtg gaaatcttta 2127agtggcactt tccagtgcta gcgaaggcag
aataatggtt gttttctagt caataagtat 2187tgagcatcta ctattctcca
ggcttgactg cagatagcac attgagtgaa accaggagcc 2247ttggccttca
gcaagcttta tgtgaacaca gggcatccag ctcaacagtg aaccaacacc
2307cccatccctt tggtcctcac acagacttta gactattctc aagtggtcca
gagggtccag 2367gaccctagca aatttgcgta tttctacagt tcaaatttga
aagccacaca tttggatcag 2427gccacttaac tagctaagta gttcattgcc
caacacaaga aatgacagaa acccttttga 2487tcatgtatat ctagtgcagt
gtcttcatag tagaagccta taagccacaa tattctccaa 2547gcgcaaggat
ggaggactgc tgtgattgat tagtgatgtc tgccttgact aaggaaaggg
2607aggaacattt ctcaagggcc tcctactgtg atctccagtc cacatcttgt
cctgcctgcc 2667acttaaaaca gtgcactgca tacatttgga gactggcgga
gttcaagtgt gaaggtcaaa 2727ggtggagtgt tggtacccac aggcacatgc
ccaccctcca tgtcctgcat tggcagagca 2787tccaggacct ccctctcctc
cagaaggttg gaaatttgct ctttcctgtt ccttgctgat 2847caatgcccaa
acctattcct catttctagt tgtttctgat actttgcggt tgacatttga
2907gccacattcc catgacagcc agtgtccggg tacgtagcag atgctgtaca
tagttctgat 2967gataagggag ctggtaggct gtaaattttg ctttttgtgt
ttttgtgggg ttttgtttgt 3027tttgatgcag tatagagtga agggggtaag
aatattctga aaaacataaa attgagtaat 3087ttattcacag aaactattaa
aatggtattg ttaggctcac aaggaggctg ggggcagcgg 3147taactgctac
actgtctcgc agaggaaatt attttattta atgtgagaga aatgtgagcc
3207agagtggctg cctgaggcta tttataaaac gcaggtacct tccccaattt
ctctcccctc 3267gcctttaata aatagattta tgtcatgtgt gtgaccaaga
acagtgacag atcttggctg 3327tccctggaat agagaggcag tcatggtgct
caggccctgg gcgtggtgta gctgatgcat 3387gtcccaccga gcccaggctc
tccaaggcat cagtttgtgg tcacatatcc ttagaaaaca 3447aaattaagtc
aggttcctgt ctgcaggagt tttcagagcc ttgaatacat agtctgcctt
3507gtgaaactgg gagaatgtag catttctggg ctgcatgacc taggtatcct
gtatggcaga 3567ccatgtctta gctggttttt cacacagtag ccttcagtga
gggtgatcta gcctgtctga 3627ggtctgcaaa gattgacagc tgcttcctag
atccttccac aaatcagcag aaacagtgtc 3687catctgggaa ctttatgaga
gtgttgcttt acctagttgg ggtgtcttca tctggatact 3747gccccagaga
gtgatcccgt ccctccaagc acagcggagg gaagcctagg gtgttatgga
3807gtggggcagg tggcttaagg acaccttaaa ggccacttgg tggggataat
ggcctcgtgg 3867ggatactcct cacttccacc cacagtttgg aactgttcac
tgtgtgcttc aggagtttta 3927aatggagatt cgtgttgatt tccatggtcc
gcatctgttt gtcttccgtc tgtggtccag 3987tgctgtgtgc agatggtact
tactcccttt ctctctctta cagggagaaa gactgtcctt 4047ctggggagag
tgtatcaata cgcttccaga atgagggagt ctttctcctg tcttgtgttc
4107acacttccta gtgacaattt
tcaaaatatg tatcggcttg actgtttatt gtagagtgta 4167gaaaggtttc
tgtgttcctg gctaaagaca tccattggtg aaatgtgctt ggaaatccaa
4227gtgttttcta ctgaggaaaa aaaaatgttg gaaaagcttt ctcctgcatg
cctcttgtgc 4287ttagctagaa aggggagctg ccagcgtccc agctctgagc
cactgttcaa aggtgcagct 4347gtgttttagg actaggtaca ggcagagtga
taaggacatg cctgcttagt gtactcattt 4407atcgagaaag tttagactat
gagccacagg ggtccctgcc tgcagtccct atagcctccc 4467cctgccccag
tgcctttctc agctggtctg ggaatcaggg cagcccattg aggggggctc
4527agacaaccct ttcttcctgg ttctgggagt aaggctttct ctgcagccac
aaggagaaca 4587tgggaaccag aaacttctct ttggtcagcc cttctgagca
cacaggttta gcccatgctc 4647caagaatgct cctgagcatt gtataagccc
ctaggactct tagctcatgt gataatgatt 4707gcattactat cctgctttaa
ctgacaaaac cacagagaat tttgcttgct ttcctactct 4767cttcctctgc
ccaggcctcc tccatacgta tacatacaca catgggcatg tatacacaca
4827catgcacaat agacatgcac acacacatgc acacatgggt atacatatac
acacatgcac 4887aatagacatg catatataca tatacatgca cacattgata
ggcatacaca catgcacagt 4947agacatgcac acatgggtat acatagacac
acatgcacag acatgcatac acacacatgc 5007acacataaat gtgcacacac
acatacacaa attgcccaag cctgtgcact cctggtaccc 5067atgtcccatc
ctggcccacc tctttgtcag caggactgac tccatcactc cctcaggttt
5127tgcccaccag caccccccag ctttccccca gcagagctaa acagcccctc
tgaatagaca 5187acaggacctt ctagaaacag ctgaaccctt ggacagcagc
aggcagattt tgatctgctc 5247tcatcaggta caaaaggtgg tatccgtatt
ctcttctgag catgctcagt aagcatatgg 5307acatagaatt taacatctct
gtggagtgtg tgttttttac atatttgtat gcagtcgagg 5367agggcctgtt
gtagaattct ctccctgtat cttactatac tgttaaagaa gctgaattct
5427atgttgccaa cagatgcgtg aaatgttcct ccaggaaaag ccattcaagc
ctgattattt 5487ttctaagtaa cttcaattaa attgaagaag aaaaaaaaaa aaaaa
55327352PRTMus musculus 7Met Lys Pro Ala Leu Leu Glu Val Met Arg
Met Asn Arg Ile Cys Arg1 5 10 15Met Val Leu Ala Thr Cys Phe Gly Ser
Phe Ile Leu Val Ile Phe Tyr 20 25 30Phe Gln Ser Met Leu His Pro Val
Met Arg Arg Asn Pro Phe Gly Val 35 40 45Asp Ile Cys Cys Arg Lys Gly
Ser Arg Ser Pro Leu Gln Glu Leu Tyr 50 55 60Asn Pro Ile Gln Leu Glu
Leu Ser Asn Thr Ala Ile Leu His Gln Met65 70 75 80Arg Arg Asp Gln
Val Thr Asp Thr Cys Arg Ala Asn Ser Ala Met Ser 85 90 95Arg Lys Arg
Arg Val Leu Thr Pro Asn Asp Leu Lys His Leu Val Val 100 105 110Asp
Glu Asp His Glu Leu Ile Tyr Cys Tyr Val Pro Lys Val Ala Cys 115 120
125Thr Asn Trp Lys Arg Leu Met Met Val Leu Ser Gly Arg Gly Lys Tyr
130 135 140Ser Asp Pro Met Glu Ile Pro Ala Asn Glu Ala His Val Ser
Ala Asn145 150 155 160Leu Lys Thr Leu Asn Gln Tyr Ser Ile Pro Glu
Ile Asn His Arg Leu 165 170 175Lys Ser Tyr Met Lys Phe Leu Phe Val
Arg Glu Pro Phe Glu Arg Leu 180 185 190Val Ser Ala Tyr Arg Asn Lys
Phe Thr Gln Lys Tyr Asn Thr Ser Phe 195 200 205His Lys Arg Tyr Gly
Thr Lys Ile Ile Arg Arg Gln Arg Lys Asn Ala 210 215 220Thr Gln Glu
Ala Leu Arg Lys Gly Asp Asp Val Lys Phe Glu Glu Phe225 230 235
240Val Ala Tyr Leu Ile Asp Pro His Thr Gln Arg Glu Glu Pro Phe Asn
245 250 255Glu His Trp Gln Thr Val Tyr Ser Leu Cys His Pro Cys His
Ile His 260 265 270Tyr Asp Leu Val Gly Lys Tyr Glu Thr Leu Glu Glu
Asp Ser Asn Tyr 275 280 285Val Leu Gln Leu Ala Gly Val Ser Gly Tyr
Leu Lys Phe Pro Thr Tyr 290 295 300Ala Lys Ser Thr Arg Thr Thr Asp
Glu Met Thr Thr Glu Phe Phe Gln305 310 315 320Asn Ile Ser Ala Glu
His Gln Thr Gln Leu Tyr Glu Val Tyr Lys Leu 325 330 335Asp Phe Leu
Met Phe Asn Tyr Ser Val Pro Asn Tyr Leu Lys Leu Asp 340 345
35081785DNAMus musculusCDS(149)..(1408) 8ggcgatttcg gctgcagaat
cagcatcacc agcaacagca gcagcggcgg tgactgtggc 60gggcgctagg tccgtctcct
agggaccatg tcccagctgt gcacaaggct gaagtgaagg 120gccaggagtg
ggcccagccc agggcagc atg acc aag ccg cgg ctc ttc cgg 172 Met Thr Lys
Pro Arg Leu Phe Arg 1 5ctg tgg ctg gta cta ggg tcg gct ctc atg atc
ctt ttg atc att gta 220Leu Trp Leu Val Leu Gly Ser Ala Leu Met Ile
Leu Leu Ile Ile Val 10 15 20tat tgg gac aac gtg gga acc gcc cac ttc
tat ctg cac acg tct ctc 268Tyr Trp Asp Asn Val Gly Thr Ala His Phe
Tyr Leu His Thr Ser Leu25 30 35 40tcc agg cca cac atc cta gaa ccc
ctt ccc acc cag gga ttg gtg gag 316Ser Arg Pro His Ile Leu Glu Pro
Leu Pro Thr Gln Gly Leu Val Glu 45 50 55gag aac gtg ttc aca tct gac
gtg gat gag ttt ttg gat act ctc ctt 364Glu Asn Val Phe Thr Ser Asp
Val Asp Glu Phe Leu Asp Thr Leu Leu 60 65 70agt tct gac gcg aag cac
aac gac ctt tcc agg aga aaa act gag cag 412Ser Ser Asp Ala Lys His
Asn Asp Leu Ser Arg Arg Lys Thr Glu Gln 75 80 85ccc ccg gcg ccc gcc
ccc agc aag cca gtc ttg agc cac atg gag gag 460Pro Pro Ala Pro Ala
Pro Ser Lys Pro Val Leu Ser His Met Glu Glu 90 95 100aac gtg aga
ggc tac gac tgg tcc act cat gat gcc cat cag aac cct 508Asn Val Arg
Gly Tyr Asp Trp Ser Thr His Asp Ala His Gln Asn Pro105 110 115
120gac cgg gac agg cag cag gcc gag agg agg agc ctg ctg aga gac ttc
556Asp Arg Asp Arg Gln Gln Ala Glu Arg Arg Ser Leu Leu Arg Asp Phe
125 130 135tgt gcc aac gcc agc ctg gca ttc ccc acc aag gac cgc tct
ttt gac 604Cys Ala Asn Ala Ser Leu Ala Phe Pro Thr Lys Asp Arg Ser
Phe Asp 140 145 150gac atc ccc aac tac gaa ctg aac cac ctg atc gtg
gac gac cgc cac 652Asp Ile Pro Asn Tyr Glu Leu Asn His Leu Ile Val
Asp Asp Arg His 155 160 165ggg gtc atc tac tgc tac gtg ccc aag gtg
gcc tgc acc aac tgg aag 700Gly Val Ile Tyr Cys Tyr Val Pro Lys Val
Ala Cys Thr Asn Trp Lys 170 175 180cga gtg atg atc gtg ctg agc gag
agc ctg ctg gac cgg ggc agc ccc 748Arg Val Met Ile Val Leu Ser Glu
Ser Leu Leu Asp Arg Gly Ser Pro185 190 195 200tac cga gac ccc ctg
gac atc ccc cgg gaa cac gtg cac aac acc agc 796Tyr Arg Asp Pro Leu
Asp Ile Pro Arg Glu His Val His Asn Thr Ser 205 210 215acg cac ctc
acc ttc aac aag ttc tgg cgc cgc tac gga aag ttc tcc 844Thr His Leu
Thr Phe Asn Lys Phe Trp Arg Arg Tyr Gly Lys Phe Ser 220 225 230cgt
cac ctc atg aag gtg aag ctg aag aag tac acc aag ttc ctg ttc 892Arg
His Leu Met Lys Val Lys Leu Lys Lys Tyr Thr Lys Phe Leu Phe 235 240
245gtg cgc gac ccc ttt gtg cgc ctc atc tca gcc ttc cgc agc aag ttc
940Val Arg Asp Pro Phe Val Arg Leu Ile Ser Ala Phe Arg Ser Lys Phe
250 255 260gag ctg gag aac gaa gag ttt tac cgc aag ttc gcg gtg ccc
atg ctc 988Glu Leu Glu Asn Glu Glu Phe Tyr Arg Lys Phe Ala Val Pro
Met Leu265 270 275 280cga ctg tac gcc aac cac acc agc ctg ccc gcc
tcg gtg agt gag gct 1036Arg Leu Tyr Ala Asn His Thr Ser Leu Pro Ala
Ser Val Ser Glu Ala 285 290 295ttc agc gcc ggg ctc aag gtc tcc ttc
gcc aac ttc atc cag tac ctc 1084Phe Ser Ala Gly Leu Lys Val Ser Phe
Ala Asn Phe Ile Gln Tyr Leu 300 305 310cta gac cca cac acg gag aag
ctg gcg cct ttc aac gag cac tgg cga 1132Leu Asp Pro His Thr Glu Lys
Leu Ala Pro Phe Asn Glu His Trp Arg 315 320 325cag gtg tac cgc ctc
tgc cac ccg tgc cag ata gac tat gac ttc gtg 1180Gln Val Tyr Arg Leu
Cys His Pro Cys Gln Ile Asp Tyr Asp Phe Val 330 335 340ggg aag ctg
gag acg ctc gat gag gac gct gcc cag ctc ctg agg ttc 1228Gly Lys Leu
Glu Thr Leu Asp Glu Asp Ala Ala Gln Leu Leu Arg Phe345 350 355
360ctc aag gta gac tcc cag ctc cac ttc ccc ccc agt tat cgg aac agg
1276Leu Lys Val Asp Ser Gln Leu His Phe Pro Pro Ser Tyr Arg Asn Arg
365 370 375acg gcc agc agc tgg gag gaa gac tgg ttt gcc aac atc ccc
ctg gca 1324Thr Ala Ser Ser Trp Glu Glu Asp Trp Phe Ala Asn Ile Pro
Leu Ala 380 385 390tgg agg caa cag ctc tat aaa ctc tac gag gcc gac
ttt gtt ctc ttt 1372Trp Arg Gln Gln Leu Tyr Lys Leu Tyr Glu Ala Asp
Phe Val Leu Phe 395 400 405ggc tac ccc aag cca gaa aac ctg ctc agg
gac tga gcccccagaa 1418Gly Tyr Pro Lys Pro Glu Asn Leu Leu Arg Asp
410 415gccctcacgc tgcccccaac aaattgaatg gctgtcccat gaggccgtcc
tttgaggatg 1478ggaccctgtg gcctcctggg ttctctcctg gcttcctttg
cttcctggtg tgacaggcag 1538aggattccac gcccccccct cgcatctgga
gaccgtggta cagccaagac cgaagcacct 1598cactctccag agttttgcgc
tccccacccc cgcccttttg caatctggat ttgtttactc 1658cacagcctgt
attcatggaa cactgtgtta aatactgttt tctaagatta atatatttca
1718gatatattta atacgaaagt gggaggaagc tggagtaaag tgtggcgccc
gcaaaaaaaa 1778aaaaaaa 17859419PRTMus musculus 9Met Thr Lys Pro Arg
Leu Phe Arg Leu Trp Leu Val Leu Gly Ser Ala1 5 10 15Leu Met Ile Leu
Leu Ile Ile Val Tyr Trp Asp Asn Val Gly Thr Ala 20 25 30His Phe Tyr
Leu His Thr Ser Leu Ser Arg Pro His Ile Leu Glu Pro 35 40 45Leu Pro
Thr Gln Gly Leu Val Glu Glu Asn Val Phe Thr Ser Asp Val 50 55 60Asp
Glu Phe Leu Asp Thr Leu Leu Ser Ser Asp Ala Lys His Asn Asp65 70 75
80Leu Ser Arg Arg Lys Thr Glu Gln Pro Pro Ala Pro Ala Pro Ser Lys
85 90 95Pro Val Leu Ser His Met Glu Glu Asn Val Arg Gly Tyr Asp Trp
Ser 100 105 110Thr His Asp Ala His Gln Asn Pro Asp Arg Asp Arg Gln
Gln Ala Glu 115 120 125Arg Arg Ser Leu Leu Arg Asp Phe Cys Ala Asn
Ala Ser Leu Ala Phe 130 135 140Pro Thr Lys Asp Arg Ser Phe Asp Asp
Ile Pro Asn Tyr Glu Leu Asn145 150 155 160His Leu Ile Val Asp Asp
Arg His Gly Val Ile Tyr Cys Tyr Val Pro 165 170 175Lys Val Ala Cys
Thr Asn Trp Lys Arg Val Met Ile Val Leu Ser Glu 180 185 190Ser Leu
Leu Asp Arg Gly Ser Pro Tyr Arg Asp Pro Leu Asp Ile Pro 195 200
205Arg Glu His Val His Asn Thr Ser Thr His Leu Thr Phe Asn Lys Phe
210 215 220Trp Arg Arg Tyr Gly Lys Phe Ser Arg His Leu Met Lys Val
Lys Leu225 230 235 240Lys Lys Tyr Thr Lys Phe Leu Phe Val Arg Asp
Pro Phe Val Arg Leu 245 250 255Ile Ser Ala Phe Arg Ser Lys Phe Glu
Leu Glu Asn Glu Glu Phe Tyr 260 265 270Arg Lys Phe Ala Val Pro Met
Leu Arg Leu Tyr Ala Asn His Thr Ser 275 280 285Leu Pro Ala Ser Val
Ser Glu Ala Phe Ser Ala Gly Leu Lys Val Ser 290 295 300Phe Ala Asn
Phe Ile Gln Tyr Leu Leu Asp Pro His Thr Glu Lys Leu305 310 315
320Ala Pro Phe Asn Glu His Trp Arg Gln Val Tyr Arg Leu Cys His Pro
325 330 335Cys Gln Ile Asp Tyr Asp Phe Val Gly Lys Leu Glu Thr Leu
Asp Glu 340 345 350Asp Ala Ala Gln Leu Leu Arg Phe Leu Lys Val Asp
Ser Gln Leu His 355 360 365Phe Pro Pro Ser Tyr Arg Asn Arg Thr Ala
Ser Ser Trp Glu Glu Asp 370 375 380Trp Phe Ala Asn Ile Pro Leu Ala
Trp Arg Gln Gln Leu Tyr Lys Leu385 390 395 400Tyr Glu Ala Asp Phe
Val Leu Phe Gly Tyr Pro Lys Pro Glu Asn Leu 405 410 415Leu Arg
Asp101728DNAMus musculusCDS(1)..(1116) 10atg act gtc gcc tgc cac
gcg tgc cag gca cag cat ggg aag acg ctc 48Met Thr Val Ala Cys His
Ala Cys Gln Ala Gln His Gly Lys Thr Leu1 5 10 15ctg ttg cag gcg gcc
ctt gcc ggt ggt ggc aag tct ggg tgc tgc act 96Leu Leu Gln Ala Ala
Leu Ala Gly Gly Gly Lys Ser Gly Cys Cys Thr 20 25 30cct gct cct gtg
cgc cct gcg tcc cgg gta acc aca gga aag gat gcc 144Pro Ala Pro Val
Arg Pro Ala Ser Arg Val Thr Thr Gly Lys Asp Ala 35 40 45cag gac act
gaa tgg cag ggc tcc cca aaa gcc ctt ttg ggg gtt ccg 192Gln Asp Thr
Glu Trp Gln Gly Ser Pro Lys Ala Leu Leu Gly Val Pro 50 55 60aca ttt
gaa aat aaa gct ctg ggc tcc agc tgg ttc ggt gga gtg agg 240Thr Phe
Glu Asn Lys Ala Leu Gly Ser Ser Trp Phe Gly Gly Val Arg65 70 75
80aag agt ccc cta cag ctg ttg cgt gac ctg gac cag ggt cca cgc tcc
288Lys Ser Pro Leu Gln Leu Leu Arg Asp Leu Asp Gln Gly Pro Arg Ser
85 90 95gcg atg gcc gag gtg cac cag cag cgg cgt gag ctg ctg cgc cgg
gcc 336Ala Met Ala Glu Val His Gln Gln Arg Arg Glu Leu Leu Arg Arg
Ala 100 105 110tgc agc cgc cac acg cga cgc caa cgc ctg ctg cag ccg
gag gac ctg 384Cys Ser Arg His Thr Arg Arg Gln Arg Leu Leu Gln Pro
Glu Asp Leu 115 120 125cgt cac gtg ctg gtg gac gac gcg cac cgg ctg
ctg tac tgc tac gtg 432Arg His Val Leu Val Asp Asp Ala His Arg Leu
Leu Tyr Cys Tyr Val 130 135 140cct aag gtg gcc tgc acc aac tgg aag
cgt gtg atg ctg gcg ttg cgc 480Pro Lys Val Ala Cys Thr Asn Trp Lys
Arg Val Met Leu Ala Leu Arg145 150 155 160ggc cgt ggg gat cca agc
gca atc cct gcg cac gag gcg cat gcg cct 528Gly Arg Gly Asp Pro Ser
Ala Ile Pro Ala His Glu Ala His Ala Pro 165 170 175ggc ctg ctg ccc
tcg ctg gcc gac ttt gcg ccg gct gag gtc aac tgg 576Gly Leu Leu Pro
Ser Leu Ala Asp Phe Ala Pro Ala Glu Val Asn Trp 180 185 190cgg ctg
cgc gac tac ctg acc ttt ctc ttc gtg cgg gag ccc ttc gag 624Arg Leu
Arg Asp Tyr Leu Thr Phe Leu Phe Val Arg Glu Pro Phe Glu 195 200
205cgc ctg gcg tca gcc tac cgc aac aag ctg gcg cgg cca cac agc gcg
672Arg Leu Ala Ser Ala Tyr Arg Asn Lys Leu Ala Arg Pro His Ser Ala
210 215 220gcc ttc cag cgg cgc tat ggc aca cgc atc gtg cgt cgc cta
cga cca 720Ala Phe Gln Arg Arg Tyr Gly Thr Arg Ile Val Arg Arg Leu
Arg Pro225 230 235 240cac gcg cag ccc gat gcg ctg gcc cgc ggc cac
gac gtg cgc ttc gcc 768His Ala Gln Pro Asp Ala Leu Ala Arg Gly His
Asp Val Arg Phe Ala 245 250 255gag ttc ctg gcc tac ctg ctc gac ccg
cgc acg cgc cgt cat gag ccc 816Glu Phe Leu Ala Tyr Leu Leu Asp Pro
Arg Thr Arg Arg His Glu Pro 260 265 270ttc aac gaa cac tgg gag cgc
gca cac gcg ctg tgc cat ccg tgc cta 864Phe Asn Glu His Trp Glu Arg
Ala His Ala Leu Cys His Pro Cys Leu 275 280 285gtg cgc tat gat gta
gtg ggc aag ttt gag acg ata gca gat gat gct 912Val Arg Tyr Asp Val
Val Gly Lys Phe Glu Thr Ile Ala Asp Asp Ala 290 295 300gcc ttc gtg
ctg gac ctg gtg ggt gag cct ggg cta cgt ttc cct gct 960Ala Phe Val
Leu Asp Leu Val Gly Glu Pro Gly Leu Arg Phe Pro Ala305 310 315
320cca ccg ctg agg cca gag aag gac ctt acg cgt gag cag gcc cgg cgc
1008Pro Pro Leu Arg Pro Glu Lys Asp Leu Thr Arg Glu Gln Ala Arg Arg
325 330 335ctt ttc cag gac atc agc ccc ttc tac cag cgt cgc ctc ttt
aac ctc 1056Leu Phe Gln Asp Ile Ser Pro Phe Tyr Gln Arg Arg Leu Phe
Asn Leu 340 345 350tat aag atg gac ttt ctg ctc ttc aat tac tct gcc
cct tcc tac ctg 1104Tyr Lys Met Asp Phe Leu Leu Phe Asn Tyr Ser Ala
Pro Ser Tyr Leu 355 360 365cga ctg caa taa gggtgttggg tgcaatagag
ccagtggctg ctgtgaccag 1156Arg Leu Gln 370gaggccacca ggaggtctgg
aacgaacctg gttgtgtgga ttggagacct tatccagtgg 1216gcctgaccaa
gagtctggcc actggtcaca ctcattccga ctgggtaggg tacaggttgc
1276tttaggtgac cataaccttg tcaggccgtt tctgctgtta gtttgatgtg
tgtctcttcc 1336tcccactctg cagatgtcag gcttcttcct aggactccag
gtttgtagtt ctttggtttg 1396gtttgagagg ccatttctca gtcttgtctg
tgtagaacct gtccgtggca tggtgctaca 1456agacagaatg tcatggcttg
gttcagtgtg gcctaaggtc ttgtcagcat ttactgctta 1516ggagttaaca
tgagctgcct gccaccccag cagtcacagg atggtgagca ctaccactcc
1576acatctacct
ggctgatcta cctttgatct cagccttgca agaggctgga tcttcccctg
1636tgtcagcaaa ggccaagatg caatactgtg gcagcttttc cagctcactt
ttattttttt 1696tgttgttttt ttaaataaat atgttttgtt ac 172811371PRTMus
musculus 11Met Thr Val Ala Cys His Ala Cys Gln Ala Gln His Gly Lys
Thr Leu1 5 10 15Leu Leu Gln Ala Ala Leu Ala Gly Gly Gly Lys Ser Gly
Cys Cys Thr 20 25 30Pro Ala Pro Val Arg Pro Ala Ser Arg Val Thr Thr
Gly Lys Asp Ala 35 40 45Gln Asp Thr Glu Trp Gln Gly Ser Pro Lys Ala
Leu Leu Gly Val Pro 50 55 60Thr Phe Glu Asn Lys Ala Leu Gly Ser Ser
Trp Phe Gly Gly Val Arg65 70 75 80Lys Ser Pro Leu Gln Leu Leu Arg
Asp Leu Asp Gln Gly Pro Arg Ser 85 90 95Ala Met Ala Glu Val His Gln
Gln Arg Arg Glu Leu Leu Arg Arg Ala 100 105 110Cys Ser Arg His Thr
Arg Arg Gln Arg Leu Leu Gln Pro Glu Asp Leu 115 120 125Arg His Val
Leu Val Asp Asp Ala His Arg Leu Leu Tyr Cys Tyr Val 130 135 140Pro
Lys Val Ala Cys Thr Asn Trp Lys Arg Val Met Leu Ala Leu Arg145 150
155 160Gly Arg Gly Asp Pro Ser Ala Ile Pro Ala His Glu Ala His Ala
Pro 165 170 175Gly Leu Leu Pro Ser Leu Ala Asp Phe Ala Pro Ala Glu
Val Asn Trp 180 185 190Arg Leu Arg Asp Tyr Leu Thr Phe Leu Phe Val
Arg Glu Pro Phe Glu 195 200 205Arg Leu Ala Ser Ala Tyr Arg Asn Lys
Leu Ala Arg Pro His Ser Ala 210 215 220Ala Phe Gln Arg Arg Tyr Gly
Thr Arg Ile Val Arg Arg Leu Arg Pro225 230 235 240His Ala Gln Pro
Asp Ala Leu Ala Arg Gly His Asp Val Arg Phe Ala 245 250 255Glu Phe
Leu Ala Tyr Leu Leu Asp Pro Arg Thr Arg Arg His Glu Pro 260 265
270Phe Asn Glu His Trp Glu Arg Ala His Ala Leu Cys His Pro Cys Leu
275 280 285Val Arg Tyr Asp Val Val Gly Lys Phe Glu Thr Ile Ala Asp
Asp Ala 290 295 300Ala Phe Val Leu Asp Leu Val Gly Glu Pro Gly Leu
Arg Phe Pro Ala305 310 315 320Pro Pro Leu Arg Pro Glu Lys Asp Leu
Thr Arg Glu Gln Ala Arg Arg 325 330 335Leu Phe Gln Asp Ile Ser Pro
Phe Tyr Gln Arg Arg Leu Phe Asn Leu 340 345 350Tyr Lys Met Asp Phe
Leu Leu Phe Asn Tyr Ser Ala Pro Ser Tyr Leu 355 360 365Arg Leu Gln
370122073DNAMus musculusCDS(129)..(1259) 12ccggccctgg tccctgcctg
caccccggga gctggccacc cactatccct cccctcccga 60gacctccagc ccctgctgca
gtcacctccc ctgcagcctc gaggtcggcg aggtctggcc 120gcagcacc atg ttt ccc
cgc cct ctg acc cca ctg gct gcc ccg aaa agc 170 Met Phe Pro Arg Pro
Leu Thr Pro Leu Ala Ala Pro Lys Ser 1 5 10gcg gag acc ctg ggc cgc
acg cca agg cgg gcc cca ttg ggc cgg gcc 218Ala Glu Thr Leu Gly Arg
Thr Pro Arg Arg Ala Pro Leu Gly Arg Ala15 20 25 30cgg gct ggg ctc
ggg ggg ccg ccc ctg ctg ctg ccg tcc atg ctg atg 266Arg Ala Gly Leu
Gly Gly Pro Pro Leu Leu Leu Pro Ser Met Leu Met 35 40 45ttc gct gta
atc gtg gcc tcc agc gga ctg ctg ctc atg atc gag cga 314Phe Ala Val
Ile Val Ala Ser Ser Gly Leu Leu Leu Met Ile Glu Arg 50 55 60ggc atc
cta tcg gag atg aaa ccc ctt ccc ctg cac cct ccc agc cac 362Gly Ile
Leu Ser Glu Met Lys Pro Leu Pro Leu His Pro Pro Ser His 65 70 75aaa
ggc gcg gcc tgg agc ggg aca gat cct aag cct aga ggc cta tcc 410Lys
Gly Ala Ala Trp Ser Gly Thr Asp Pro Lys Pro Arg Gly Leu Ser 80 85
90ttg gat gct ggg gac tcg gac ttg caa gtg agg gag gac atc cga aac
458Leu Asp Ala Gly Asp Ser Asp Leu Gln Val Arg Glu Asp Ile Arg
Asn95 100 105 110cgg acc ttg agg gcc gtg tgc gga caa cca ggc atg
ccc cgg gac ccc 506Arg Thr Leu Arg Ala Val Cys Gly Gln Pro Gly Met
Pro Arg Asp Pro 115 120 125tgg gac ttg ccg gtg gga cag cgg cgc acc
ctg ctg cgc cac att ctc 554Trp Asp Leu Pro Val Gly Gln Arg Arg Thr
Leu Leu Arg His Ile Leu 130 135 140gta agt gac cgc tac cgc ttc ctc
tac tgc tat gtc ccc aaa gtg gcc 602Val Ser Asp Arg Tyr Arg Phe Leu
Tyr Cys Tyr Val Pro Lys Val Ala 145 150 155tgc tct aac tgg aaa cgt
gtg ctg aag gtg ctg gct ggc gtc ctg aac 650Cys Ser Asn Trp Lys Arg
Val Leu Lys Val Leu Ala Gly Val Leu Asn 160 165 170aac gtg gat gtc
cgc ctc aag atg gac cac ccc agt gac ttg gtg ttt 698Asn Val Asp Val
Arg Leu Lys Met Asp His Pro Ser Asp Leu Val Phe175 180 185 190ctg
gca gac ctg cgg cct gag gag att cgc tac cgt ctg cag cac tac 746Leu
Ala Asp Leu Arg Pro Glu Glu Ile Arg Tyr Arg Leu Gln His Tyr 195 200
205ttc aag ttc ctg ttt gtg cga gac ccc ttg gaa cgc ctc ctg tct gct
794Phe Lys Phe Leu Phe Val Arg Asp Pro Leu Glu Arg Leu Leu Ser Ala
210 215 220tac cgt aac aag ttt gga gag atc cga gag tac cag cag cga
tat ggg 842Tyr Arg Asn Lys Phe Gly Glu Ile Arg Glu Tyr Gln Gln Arg
Tyr Gly 225 230 235gcc gaa att gtc agg cgc tac agg gct gga gct ggt
ccc agc cct gca 890Ala Glu Ile Val Arg Arg Tyr Arg Ala Gly Ala Gly
Pro Ser Pro Ala 240 245 250ggg gac gat gtc acc ttc cca gag ttc ctg
aga tac ctg gtg gat gag 938Gly Asp Asp Val Thr Phe Pro Glu Phe Leu
Arg Tyr Leu Val Asp Glu255 260 265 270gat cct gaa cat atg aat gag
cat tgg atg cct gtg tac cac ctg tgc 986Asp Pro Glu His Met Asn Glu
His Trp Met Pro Val Tyr His Leu Cys 275 280 285caa cca tgt gct gtg
cac tac gac ttc gtg ggt tcc tat gag agg ctg 1034Gln Pro Cys Ala Val
His Tyr Asp Phe Val Gly Ser Tyr Glu Arg Leu 290 295 300gag gct gat
gcc aac cag gtg ctg gag tgg gtg cgg gcc cca ccc cat 1082Glu Ala Asp
Ala Asn Gln Val Leu Glu Trp Val Arg Ala Pro Pro His 305 310 315gtc
cgg ttc cca gct cgc cag gcc tgg tac cgg cca gcc agc cca gaa 1130Val
Arg Phe Pro Ala Arg Gln Ala Trp Tyr Arg Pro Ala Ser Pro Glu 320 325
330agt ctg cat tac cac ttg tgc aat gtt cca cgg gcc ctg ctt caa gat
1178Ser Leu His Tyr His Leu Cys Asn Val Pro Arg Ala Leu Leu Gln
Asp335 340 345 350gtg cta cct aag tat atc ctg gac ttc tcc ctc ttt
gct tac cca ctg 1226Val Leu Pro Lys Tyr Ile Leu Asp Phe Ser Leu Phe
Ala Tyr Pro Leu 355 360 365ccc aat gtc acc aag gaa gcc tgt cac caa
tga cagtaggcca gcaccttttg 1279Pro Asn Val Thr Lys Glu Ala Cys His
Gln 370 375gagtttgggt ttaatgatat cagctttggg atgtctttca gagaaactcc
tggctctggg 1339tggcttcctg gtttctctag gtgtctccat atctcagtgg
taaggactgt ccttggaggt 1399ccttgtccac agtggctcag aggacagagc
tagaaaggag gcctgctgct ttcactggtg 1459aactgcctct cttaggggcc
tgtggtatcc gtgtctgcag ggcaccagtg gttattaaag 1519ccatatgttt
gatcgaaaga ctgacttcag ccccctggct gctgggtcta tgcagtccac
1579ctggtctgtt gtaatttaac ctgtggccaa atcccaaata tgacactagc
caagcacatg 1639atcatgccta ggaccaatgg ctgtgacccc ctattcaccc
atcccatgga cctcaggact 1699ggagtgagct gtggtgcctt agaaatgaaa
tgtgtgcaat tctactccag acttttacat 1759ttcctcctct tgctaggtct
gaatcatttt tctaaggaaa gagaaacgga agtggggccc 1819ttacctcgaa
gctctaaagc ccagcccctc aagcatccaa agacgcctgt gcctgacctc
1879ttcctagggc tcctggagca tcttcaataa gcctcccttc cctacaaacc
tttggagact 1939atgtgagact gtatggccca tatatctggc tgtcacttgt
ctaatgcatt tatttaaaat 1999gtgtatattt taataggatc cttgtaaggg
ctgactttta ataaagcttt ttcatataca 2059aaaaaaaaaa aaaa
207313376PRTMus musculus 13Met Phe Pro Arg Pro Leu Thr Pro Leu Ala
Ala Pro Lys Ser Ala Glu1 5 10 15Thr Leu Gly Arg Thr Pro Arg Arg Ala
Pro Leu Gly Arg Ala Arg Ala 20 25 30Gly Leu Gly Gly Pro Pro Leu Leu
Leu Pro Ser Met Leu Met Phe Ala 35 40 45Val Ile Val Ala Ser Ser Gly
Leu Leu Leu Met Ile Glu Arg Gly Ile 50 55 60Leu Ser Glu Met Lys Pro
Leu Pro Leu His Pro Pro Ser His Lys Gly65 70 75 80Ala Ala Trp Ser
Gly Thr Asp Pro Lys Pro Arg Gly Leu Ser Leu Asp 85 90 95Ala Gly Asp
Ser Asp Leu Gln Val Arg Glu Asp Ile Arg Asn Arg Thr 100 105 110Leu
Arg Ala Val Cys Gly Gln Pro Gly Met Pro Arg Asp Pro Trp Asp 115 120
125Leu Pro Val Gly Gln Arg Arg Thr Leu Leu Arg His Ile Leu Val Ser
130 135 140Asp Arg Tyr Arg Phe Leu Tyr Cys Tyr Val Pro Lys Val Ala
Cys Ser145 150 155 160Asn Trp Lys Arg Val Leu Lys Val Leu Ala Gly
Val Leu Asn Asn Val 165 170 175Asp Val Arg Leu Lys Met Asp His Pro
Ser Asp Leu Val Phe Leu Ala 180 185 190Asp Leu Arg Pro Glu Glu Ile
Arg Tyr Arg Leu Gln His Tyr Phe Lys 195 200 205Phe Leu Phe Val Arg
Asp Pro Leu Glu Arg Leu Leu Ser Ala Tyr Arg 210 215 220Asn Lys Phe
Gly Glu Ile Arg Glu Tyr Gln Gln Arg Tyr Gly Ala Glu225 230 235
240Ile Val Arg Arg Tyr Arg Ala Gly Ala Gly Pro Ser Pro Ala Gly Asp
245 250 255Asp Val Thr Phe Pro Glu Phe Leu Arg Tyr Leu Val Asp Glu
Asp Pro 260 265 270Glu His Met Asn Glu His Trp Met Pro Val Tyr His
Leu Cys Gln Pro 275 280 285Cys Ala Val His Tyr Asp Phe Val Gly Ser
Tyr Glu Arg Leu Glu Ala 290 295 300Asp Ala Asn Gln Val Leu Glu Trp
Val Arg Ala Pro Pro His Val Arg305 310 315 320Phe Pro Ala Arg Gln
Ala Trp Tyr Arg Pro Ala Ser Pro Glu Ser Leu 325 330 335His Tyr His
Leu Cys Asn Val Pro Arg Ala Leu Leu Gln Asp Val Leu 340 345 350Pro
Lys Tyr Ile Leu Asp Phe Ser Leu Phe Ala Tyr Pro Leu Pro Asn 355 360
365Val Thr Lys Glu Ala Cys His Gln 370 375146000DNAMus
musculusCDS(496)..(1914) 14cttagccact gaaccatctc tccagcctct
gttttggggt gtttgtttga gacagaatca 60aaaggctggt ctaaagtaca tattgtagct
aaagatggct tccaactcct gccttgcctc 120cagagtgtta gggtgtgacc
tttaaatgac ctattaaaga aaccccaaca ggtgtagagt 180acacctgtga
tcccagctct taggaggttg gacagatgga taaagaggtc caggtgtccc
240ggactacata gagagaccct atctcaaaag agaggcgaag acttaggttt
tgagagtggg 300cctgaggtct ctcttcaaaa gttttataga aagatgtctc
tgttccctgt ctatgaacac 360ggatggcctg agcacctgtc tcttcacagg
atcagagtgt cccccacctg aagagggctg 420attgggtccc aagctatgct
cctgagctga gtgcctgcag ccagtctgag gaactccatg 480gcgccccctc tcccc atg
gag aaa gga ctc gct ttg cct cag gat ttc cgg 531 Met Glu Lys Gly Leu
Ala Leu Pro Gln Asp Phe Arg 1 5 10gac ctt gta cac agc cta aag att
cga ggc aga tac gtc ttg ttc ctg 579Asp Leu Val His Ser Leu Lys Ile
Arg Gly Arg Tyr Val Leu Phe Leu 15 20 25gca ttt gtg gtc ata gtt ttt
atc ttc att gaa aag gaa aat aaa atc 627Ala Phe Val Val Ile Val Phe
Ile Phe Ile Glu Lys Glu Asn Lys Ile 30 35 40ata tcc agg gtc tcc gac
aag ctg aag cag atc cct cat ttt gtg gca 675Ile Ser Arg Val Ser Asp
Lys Leu Lys Gln Ile Pro His Phe Val Ala45 50 55 60gat gcc aac agc
act gac cca gcc ctg ctc tta tcg gag aat gca tct 723Asp Ala Asn Ser
Thr Asp Pro Ala Leu Leu Leu Ser Glu Asn Ala Ser 65 70 75ctc ttg tcc
ctg agc gag ttg gat tcc acc ttt tcc cat ctg cgg agc 771Leu Leu Ser
Leu Ser Glu Leu Asp Ser Thr Phe Ser His Leu Arg Ser 80 85 90cgc ctg
cac aac ctg agc ctg cag ctg ggc gtg gag cca gca atg gag 819Arg Leu
His Asn Leu Ser Leu Gln Leu Gly Val Glu Pro Ala Met Glu 95 100
105agc cag gag gct ggg gca gag aag cca tcc cag cag gct gga gca ggg
867Ser Gln Glu Ala Gly Ala Glu Lys Pro Ser Gln Gln Ala Gly Ala Gly
110 115 120acc cgg cgc cac gtg ctt ctc atg gcc acc acc cgc acg ggt
tcc tcg 915Thr Arg Arg His Val Leu Leu Met Ala Thr Thr Arg Thr Gly
Ser Ser125 130 135 140ttc gtg ggc gag ttc ttc aac cag cag ggc aat
atc ttc tac ctc ttc 963Phe Val Gly Glu Phe Phe Asn Gln Gln Gly Asn
Ile Phe Tyr Leu Phe 145 150 155gag cca ctg tgg cac atc gag cgc acc
gtg ttc ttc cag cag cga ggc 1011Glu Pro Leu Trp His Ile Glu Arg Thr
Val Phe Phe Gln Gln Arg Gly 160 165 170gcc agc gcg gct ggt tca gcc
ttg gtc tac cgt gat gtc ctc aag cag 1059Ala Ser Ala Ala Gly Ser Ala
Leu Val Tyr Arg Asp Val Leu Lys Gln 175 180 185ttg ttg cta tgc gac
ctg tat gtg ctg gag ccc ttc atc agc cct ccg 1107Leu Leu Leu Cys Asp
Leu Tyr Val Leu Glu Pro Phe Ile Ser Pro Pro 190 195 200ccc gag gac
cac ttg act cag ttc ctg ttc cgc cgg gga tcc agc cgt 1155Pro Glu Asp
His Leu Thr Gln Phe Leu Phe Arg Arg Gly Ser Ser Arg205 210 215
220tca ctc tgc gag gat ccg gtg tgc aca ccc ttc gtc aag aag gtc ttt
1203Ser Leu Cys Glu Asp Pro Val Cys Thr Pro Phe Val Lys Lys Val Phe
225 230 235gag aag tac cac tgc agg aac cgt cgc tgc ggg cca ctc aac
gtg acc 1251Glu Lys Tyr His Cys Arg Asn Arg Arg Cys Gly Pro Leu Asn
Val Thr 240 245 250ttg gcg ggc gag gcc tgc cgc cgc aag gac cac gtg
gcc ctc aag gct 1299Leu Ala Gly Glu Ala Cys Arg Arg Lys Asp His Val
Ala Leu Lys Ala 255 260 265gtg cgc atc cgt cag ctg gag ttc ctg cag
ccg cta gtt gag gac ccg 1347Val Arg Ile Arg Gln Leu Glu Phe Leu Gln
Pro Leu Val Glu Asp Pro 270 275 280agg ttg gat cta cga gtc att cag
ctg gtg cgc gac ccc cgg gcc gtg 1395Arg Leu Asp Leu Arg Val Ile Gln
Leu Val Arg Asp Pro Arg Ala Val285 290 295 300ctg gct tca cgc ata
gtg gcc ttt gcg ggc aag tat gag aac tgg aag 1443Leu Ala Ser Arg Ile
Val Ala Phe Ala Gly Lys Tyr Glu Asn Trp Lys 305 310 315aag tgg ctg
tcc gag ggg cag gac cag ctg agc gag gat gag gtg cag 1491Lys Trp Leu
Ser Glu Gly Gln Asp Gln Leu Ser Glu Asp Glu Val Gln 320 325 330cga
ttg cgg ggc aac tgt gag agc atc cgc ctg tct gca gag ctg ggc 1539Arg
Leu Arg Gly Asn Cys Glu Ser Ile Arg Leu Ser Ala Glu Leu Gly 335 340
345ttg cgg cag cca gcc tgg ctg cgc ggt cgt tac atg ctg gtg cgc tat
1587Leu Arg Gln Pro Ala Trp Leu Arg Gly Arg Tyr Met Leu Val Arg Tyr
350 355 360gag gat gtg gca cgc agg cca ctg cag aag gcc cga gag atg
tac agc 1635Glu Asp Val Ala Arg Arg Pro Leu Gln Lys Ala Arg Glu Met
Tyr Ser365 370 375 380ttt gcg ggc atc ccc ttg acc ccg cag gtg gag
gac tgg atc cag aag 1683Phe Ala Gly Ile Pro Leu Thr Pro Gln Val Glu
Asp Trp Ile Gln Lys 385 390 395aac acg cag gcg aca cgc gac agc agc
gat gtc tac tcc act cag aaa 1731Asn Thr Gln Ala Thr Arg Asp Ser Ser
Asp Val Tyr Ser Thr Gln Lys 400 405 410aac tct tct gag cag ttt gag
aag tgg cgc ttc agc atg cct ttc aag 1779Asn Ser Ser Glu Gln Phe Glu
Lys Trp Arg Phe Ser Met Pro Phe Lys 415 420 425ctg gca cag gtg gta
cag gct gcc tgt ggc ccg acc atg cac ctc ttt 1827Leu Ala Gln Val Val
Gln Ala Ala Cys Gly Pro Thr Met His Leu Phe 430 435 440ggc tac aag
ttg gcc agg gat gcc gcc tca ctc acc aac cgc tcc atc 1875Gly Tyr Lys
Leu Ala Arg Asp Ala Ala Ser Leu Thr Asn Arg Ser Ile445 450 455
460agc ctg ctg gag gag cgg ggc acc ttc tgg gtc acg tag tgggggatgt
1924Ser Leu Leu Glu Glu Arg Gly Thr Phe Trp Val Thr 465
470ctgggaccct tggaactcct tcttgtgaaa ggctggccct gcttccctca
cacccagcct 1984ggcagtgaga catagccctg gcagaaagtc aaaatgggga
gcgatgatgg gaacatagcc 2044cctggctgta gcggtagggc cccctgccag
ctagactccc cagagcagct acagctaggg 2104ccttgggctc ctctgaggac
accctgtctc cttagtggta ctggtcataa ggtgtcctca 2164ccctatgacc
tgcagtgtcc cgagcaggtc aaggtaggtt cctgtgtgtg gacacacctc
2224cctagctctt tctcacacag tctacacgaa gcctgtaaag gcctgtaggt
tgtgtgggct 2284ggagtccaag tatttaacaa ccagaagggg tgtagaccct
ctgcagcctc tcagacctcc
2344tactgtatta agtgttaacc tcttccgctc tgagtcagag aagctgggat
ctggctgagt 2404cctgggaagg aggaggacag cctagggtga ggaaggggac
ctttgaagct cctcagggaa 2464cagtggctgt gtaccagctc atagaaaatg
gtttgatcag ctgttctagt cactcatgag 2524tatcaatcag cctgtgtaga
gcaagacaca caaagtgcat tgaaaacaga caaggccagg 2584ggtgtggctc
agcggtaaag cccttgcctc tctcatgccc agggccctag gtgtgattct
2644tagcactata aatataaggg aaaataagcc atacatacgt gtgcagtaca
gagagagaca 2704gaaaagcgta ataaacattc ccccctctca taagcacacg
ctctgcctct gtctctgtct 2764ctgtctctct ctgtctctgt ctctctctgt
ctctctctct cactctctct ctctctctct 2824ctctctcaca cacacacaca
cacacacaca cacaaacaca cacacacaca ctggtacaga 2884aaagcttccc
ttctccactc tttatgctca ccatgttttt aggatttttg tttgtttgtt
2944ttgttttggg tttctatata tctaaacaag gagccttaaa ttacatcttt
ttgggattac 3004gtggcgaggg ggagcagcag ggagcatttg cttctggtaa
aaggatagac cagaaaggac 3064ttccccttgg atgatcggag tcatgcagag
cccccagggt cccacatgtt ctgtgtgaac 3124ttcatgtgga atgactcaca
gaagtgacta ccttaggcgc tcgctctgta gctcaatgtg 3184cgtagagttt
gtctattgta taggaagccc tgggttctgt gccctgcccc acaccatata
3244ggtatgatgg cgtgtaccca tatcccagca ttcaggaaat caaaggttgg
cggtcaaaag 3304ttcaaggtgc agctatgtat cttttgagat gtccgagtct
ataagctccc tctactgaga 3364gctgctagta ttccttctca agaacaggtt
atttgaaaat ctcttcttgt aagccccaac 3424atttggggga ggggaggatg
ggggaactct tgactgagca gctaggtttc cccaagcaga 3484agttcttctc
tcccagtgca agctgaggtt tagtgccccg gctagcctcg gaactggctc
3544agtgggggtt ggcacgctga cttcctggac aattgctgca tctgacactg
tgagcgccca 3604ggacaccgga tgtgtagcat ggatgtgggc ctagcacagg
tgtggaagag gacagtcagg 3664acactcactg ttggtgtttg ttatccacct
gttcagccgc cgggtcccac acataggtca 3724tctttcgtca ctcaagccgg
cctctgtttt tgattttaac aatccaagag caagtgtgta 3784ggggacaaag
agacaccagc tcttcatact tgatggagac cgggacagga tgctgcaggc
3844aggctcggga gtgcattatc gtttcttctg agctcttcat ccaggccata
ttcccttttc 3904tgcatcgcat ctggggtggg aggggctgag ctgggagttt
ccgtccttct tccctgtggg 3964ggtgggagat ggggctcaag gtctccatct
ctcggctccc tgaggacaga acccccacag 4024tggacctttg ggccttctca
ggacattgac aatgttgtgt gcactgcaag ttgactttat 4084ttattttgta
ggaaaaagag atggtattct ttagcaggat ctgaaactgt accctagtca
4144agaagtacaa ttaataacat tattattaat aacaattggg atgattcaaa
ggtcacacac 4204acaccagacc acccaagaac catgtaggag atgaggcgga
gccaggtcat ggaagcccag 4264aatgctacag agattctgtg ggtctattat
tgatcagaaa atacaacatg ggggggcctt 4324ttctatggca taactcaggt
gtctgcaaag acagagcagc tcaggtgggt ggataggcag 4384gcagatggag
ccttgaatca catcccctga ggctcagcca gccagtgcga atcctcaagg
4444ttcggaaagg gacagtgtag cggcaaacta ctgggcatct tctctgatgc
ttagaggctc 4504ttaccaagga gcttgatgga gaaagtggcc atgttggtgg
catgggacaa ctgctcaccc 4564aacttcaaag atcaaagggc acccaggtgg
cctatgacag tgacactctt tcctaccatt 4624aggggtgtct gcccccactg
aaagccatga aatttctggc cctaaggggg taggaaggac 4684ttaggagtag
cagatggttt gatcccatcc cccccacaca cacacacaca tccctcagct
4744gtcttccaca ttagagccac ttcagttgtc catggacttg tccctttgag
acatcctgga 4804ttttgaagga tagaaacatc cccaagatgg tctcgtgtta
atcccacaac agaggcagaa 4864gggtcatact gagagagaga gagagagaga
gagagagaga gagaggagtg ggggccagac 4924cctcaagaag ccaagtgggt
ctggtcaacc tgtgcacatg agaaggaggg aacatcacta 4984aaatcagggc
ctgggctggt gtgttgttgg tgagatcccg tggagtggct ggctagatat
5044ggatgagttt tctgagcatg ctcacacacc cccaacttca cattcttaga
aatagcacaa 5104ccataatgcc ttacctcaaa aggatgaggc aaagcttgca
attaattccc agtgtctgaa 5164gacctccggg atcctttgag ctgtgtgtcc
ttgtcagtac atggggacag gctccttagg 5224tattcctgac atagaggtaa
ggtgctgccc ttgctgcctt gcataagctg tgacaagctt 5284cttactgggc
acatagaacg gctctgtcat ctgcttccac taaatttggg cttggacttt
5344gcctcctgcc aaatctccat ctctgctgga tagtctagtc cctagggtct
aaccaccacc 5404ctccacttcc tggtgggtcc tacaagcgct gtctttgtgc
cagtacccgg atggtgtcct 5464gcctcatggc taaatggtac aggacatctt
cccagactga gtggtcatgg catgtgcatg 5524tatgttcatg agtacatgca
tggtgtgtgg gggtggatgg gtgcattctg ttctgcttct 5584ggtgcagcta
cacagccaca acctctttct gtcattgacc ttcttggtct tcttgtagcc
5644acagataatc ttccaatgcc cgattctgct gttctcatct gagagctgac
aacccagcgc 5704tcagagtaga gttcatgacc taggaaaccc ttctcctggt
agcttactga acttatttaa 5764ttaaaaacga acatataggg gttggagaga
tggttcattg gttaagagca ccggctgctt 5824ttgcaaagga cctgagttca
aatcccagca accatatggt ggcttacaac catctgtaat 5884gagttctggt
gccctcttct ggcatgcagg tgtacatgca gatagagcac tcctatgcat
5944aaaataaata aatcttttaa ataaataaat aaatgaacat gaaaaaaaaa aaaaaa
600015472PRTMus musculus 15Met Glu Lys Gly Leu Ala Leu Pro Gln Asp
Phe Arg Asp Leu Val His1 5 10 15Ser Leu Lys Ile Arg Gly Arg Tyr Val
Leu Phe Leu Ala Phe Val Val 20 25 30Ile Val Phe Ile Phe Ile Glu Lys
Glu Asn Lys Ile Ile Ser Arg Val 35 40 45Ser Asp Lys Leu Lys Gln Ile
Pro His Phe Val Ala Asp Ala Asn Ser 50 55 60Thr Asp Pro Ala Leu Leu
Leu Ser Glu Asn Ala Ser Leu Leu Ser Leu65 70 75 80Ser Glu Leu Asp
Ser Thr Phe Ser His Leu Arg Ser Arg Leu His Asn 85 90 95Leu Ser Leu
Gln Leu Gly Val Glu Pro Ala Met Glu Ser Gln Glu Ala 100 105 110Gly
Ala Glu Lys Pro Ser Gln Gln Ala Gly Ala Gly Thr Arg Arg His 115 120
125Val Leu Leu Met Ala Thr Thr Arg Thr Gly Ser Ser Phe Val Gly Glu
130 135 140Phe Phe Asn Gln Gln Gly Asn Ile Phe Tyr Leu Phe Glu Pro
Leu Trp145 150 155 160His Ile Glu Arg Thr Val Phe Phe Gln Gln Arg
Gly Ala Ser Ala Ala 165 170 175Gly Ser Ala Leu Val Tyr Arg Asp Val
Leu Lys Gln Leu Leu Leu Cys 180 185 190Asp Leu Tyr Val Leu Glu Pro
Phe Ile Ser Pro Pro Pro Glu Asp His 195 200 205Leu Thr Gln Phe Leu
Phe Arg Arg Gly Ser Ser Arg Ser Leu Cys Glu 210 215 220Asp Pro Val
Cys Thr Pro Phe Val Lys Lys Val Phe Glu Lys Tyr His225 230 235
240Cys Arg Asn Arg Arg Cys Gly Pro Leu Asn Val Thr Leu Ala Gly Glu
245 250 255Ala Cys Arg Arg Lys Asp His Val Ala Leu Lys Ala Val Arg
Ile Arg 260 265 270Gln Leu Glu Phe Leu Gln Pro Leu Val Glu Asp Pro
Arg Leu Asp Leu 275 280 285Arg Val Ile Gln Leu Val Arg Asp Pro Arg
Ala Val Leu Ala Ser Arg 290 295 300Ile Val Ala Phe Ala Gly Lys Tyr
Glu Asn Trp Lys Lys Trp Leu Ser305 310 315 320Glu Gly Gln Asp Gln
Leu Ser Glu Asp Glu Val Gln Arg Leu Arg Gly 325 330 335Asn Cys Glu
Ser Ile Arg Leu Ser Ala Glu Leu Gly Leu Arg Gln Pro 340 345 350Ala
Trp Leu Arg Gly Arg Tyr Met Leu Val Arg Tyr Glu Asp Val Ala 355 360
365Arg Arg Pro Leu Gln Lys Ala Arg Glu Met Tyr Ser Phe Ala Gly Ile
370 375 380Pro Leu Thr Pro Gln Val Glu Asp Trp Ile Gln Lys Asn Thr
Gln Ala385 390 395 400Thr Arg Asp Ser Ser Asp Val Tyr Ser Thr Gln
Lys Asn Ser Ser Glu 405 410 415Gln Phe Glu Lys Trp Arg Phe Ser Met
Pro Phe Lys Leu Ala Gln Val 420 425 430Val Gln Ala Ala Cys Gly Pro
Thr Met His Leu Phe Gly Tyr Lys Leu 435 440 445Ala Arg Asp Ala Ala
Ser Leu Thr Asn Arg Ser Ile Ser Leu Leu Glu 450 455 460Glu Arg Gly
Thr Phe Trp Val Thr465 470162080DNAMus musculusCDS(147)..(1601)
16caccgcccgg ctgctccact ccgctccacc caacattgag ggagacccga agaggccgga
60gccgaggact ttgggctggg ttttctggac agaaccagca ggcgcctact ctgctctggg
120tggggagagt gaggattcgg tgaact atg aag ggc cgg cgg cgg cgg cgc cga
173 Met Lys Gly Arg Arg Arg Arg Arg Arg 1 5aag tat tgc aag ttc acg
ctg ctc ttg gcg ctg tac acg ctt ttg cta 221Lys Tyr Cys Lys Phe Thr
Leu Leu Leu Ala Leu Tyr Thr Leu Leu Leu10 15 20 25ctt ctt gtc ccc
tct gta ctg gac agc cac agc gag cag gac aag ggc 269Leu Leu Val Pro
Ser Val Leu Asp Ser His Ser Glu Gln Asp Lys Gly 30 35 40agg aac tgc
ccc ggc ctg cag cgc agc ttg ggt gtg tgg agc ctg gag 317Arg Asn Cys
Pro Gly Leu Gln Arg Ser Leu Gly Val Trp Ser Leu Glu 45 50 55gcg gcg
gcg gcc ggg gaa cgt gag cag ggc gct gag gtg cgg tcc ctg 365Ala Ala
Ala Ala Gly Glu Arg Glu Gln Gly Ala Glu Val Arg Ser Leu 60 65 70gcc
gaa gga aac ccg gat cga tcc ccc ggg tcc ccc ggc aac ctc agc 413Ala
Glu Gly Asn Pro Asp Arg Ser Pro Gly Ser Pro Gly Asn Leu Ser 75 80
85gcc gtc ggt gag gcg gtg acc cag gaa aag caa cac atc tat gtg cat
461Ala Val Gly Glu Ala Val Thr Gln Glu Lys Gln His Ile Tyr Val
His90 95 100 105gcc acc tgg cgc acc ggc tcg tcc ttc ttg ggc gaa ctc
ttc aac cag 509Ala Thr Trp Arg Thr Gly Ser Ser Phe Leu Gly Glu Leu
Phe Asn Gln 110 115 120cac ccg gac gtt ttc tac ttg tac gac ccc atg
tgg cat ctg tgg cag 557His Pro Asp Val Phe Tyr Leu Tyr Asp Pro Met
Trp His Leu Trp Gln 125 130 135gca ctg tat ccg ggc gac gcg gag agc
ctg cag ggc gca cta aga gac 605Ala Leu Tyr Pro Gly Asp Ala Glu Ser
Leu Gln Gly Ala Leu Arg Asp 140 145 150atg ctg cgc tcc ctc ttc cgc
tgt gat ttc tct gtg ctg cgc ctg tac 653Met Leu Arg Ser Leu Phe Arg
Cys Asp Phe Ser Val Leu Arg Leu Tyr 155 160 165gcg cag cct ggg gac
cct ggg gag cga gca ccg gac tcg gcc aac ctc 701Ala Gln Pro Gly Asp
Pro Gly Glu Arg Ala Pro Asp Ser Ala Asn Leu170 175 180 185acc acg
gcc atg ctt ttc cgc tgg cgg acc aac aag gtc atc tgc tcg 749Thr Thr
Ala Met Leu Phe Arg Trp Arg Thr Asn Lys Val Ile Cys Ser 190 195
200ccg cct ctg tgc ccc gcc gcg ccc cgg gca cgc gcg gac gtg gga ctc
797Pro Pro Leu Cys Pro Ala Ala Pro Arg Ala Arg Ala Asp Val Gly Leu
205 210 215gtc gag gac aaa gcc tgc gaa agt acc tgc ccg ccc gtt tcg
ctc cgc 845Val Glu Asp Lys Ala Cys Glu Ser Thr Cys Pro Pro Val Ser
Leu Arg 220 225 230gcc ctg gag gcc gag tgc cgc aag tac ccg gtg gtg
gtc atc aaa gac 893Ala Leu Glu Ala Glu Cys Arg Lys Tyr Pro Val Val
Val Ile Lys Asp 235 240 245gtg cgg cta ctg gac ctg gga gtg ctg gtc
cct ctg ctg cgt gac cca 941Val Arg Leu Leu Asp Leu Gly Val Leu Val
Pro Leu Leu Arg Asp Pro250 255 260 265ggc ctc aac cta aag gtg gtg
caa ctc ttc cga gac cct cgg gcc gtg 989Gly Leu Asn Leu Lys Val Val
Gln Leu Phe Arg Asp Pro Arg Ala Val 270 275 280cac aac tcg cgc ctc
aag tcg agg cag gga ctg ctg cgc gaa agc atc 1037His Asn Ser Arg Leu
Lys Ser Arg Gln Gly Leu Leu Arg Glu Ser Ile 285 290 295cag gtg ctg
cgc acg cgc cag agg ggc gac cac ttc cac cgg gtg ctg 1085Gln Val Leu
Arg Thr Arg Gln Arg Gly Asp His Phe His Arg Val Leu 300 305 310ctg
gcg cat gga gtg gat gcc cgt ccg gga ggc cag gcc cgg gct ctg 1133Leu
Ala His Gly Val Asp Ala Arg Pro Gly Gly Gln Ala Arg Ala Leu 315 320
325ccc tcg gcg cca cgc gct gat ttc ttc tta acc agc gcg ctt gag gtg
1181Pro Ser Ala Pro Arg Ala Asp Phe Phe Leu Thr Ser Ala Leu Glu
Val330 335 340 345atc tgt gaa gcg tgg ctt cgc gac ctg cta ttc acc
cgc ggc gcg ccc 1229Ile Cys Glu Ala Trp Leu Arg Asp Leu Leu Phe Thr
Arg Gly Ala Pro 350 355 360gcc tgg ctg agg cgt cgc tac ctg cgg ctg
cgt tat gag gac ctg gtg 1277Ala Trp Leu Arg Arg Arg Tyr Leu Arg Leu
Arg Tyr Glu Asp Leu Val 365 370 375tgg cag ccc caa gcc cag ctg cgc
cgc ctg ctg cgc ttc tct ggg ttg 1325Trp Gln Pro Gln Ala Gln Leu Arg
Arg Leu Leu Arg Phe Ser Gly Leu 380 385 390cgg aca ctc gcc gcg ctt
gat gcc ttc gca ttc aat atg acg cgg ggc 1373Arg Thr Leu Ala Ala Leu
Asp Ala Phe Ala Phe Asn Met Thr Arg Gly 395 400 405tcg gcc tac ggc
gcc gat cgt ccc ttc cac ttg tct gcg cgg gac gcc 1421Ser Ala Tyr Gly
Ala Asp Arg Pro Phe His Leu Ser Ala Arg Asp Ala410 415 420 425cga
gag gct gtg cac gcc tgg cgc gaa cgt ctg agc caa gag cag gtg 1469Arg
Glu Ala Val His Ala Trp Arg Glu Arg Leu Ser Gln Glu Gln Val 430 435
440cgc caa gtg gaa acc gcc tgc gcc cct gcc atg cgt ctg ctt gcc tac
1517Arg Gln Val Glu Thr Ala Cys Ala Pro Ala Met Arg Leu Leu Ala Tyr
445 450 455cct cga agt ggg gac gaa cgc gac agg aag acc gtc agg gaa
ggg gag 1565Pro Arg Ser Gly Asp Glu Arg Asp Arg Lys Thr Val Arg Glu
Gly Glu 460 465 470aca cca ctg gag acc aag gcc aat tgg gct gtg taa
taccctgatc 1611Thr Pro Leu Glu Thr Lys Ala Asn Trp Ala Val 475
480cctgaaccct gccccggggc gtattcaggt cagtggccat aaaaaggtga
actcagcatg 1671ctgcccccgc actggagagg ctgcacggtg gaggcgatct
atcacactgt gagacactgg 1731gactgatttg gtatcaactg ctgtgccatt
ctcctggtca ggagcatcac aagctgttaa 1791gtaatgacag acaccttggc
tgagatgaag tttccagaaa ggaagtaaca gtgcaatgtg 1851gatatttgtg
accacaacat aggaaaagct gtacttccca ggctgaactt ggctcagctt
1911gagccatttc aacaaggcat cctcacaata atgaagagat gtgatctggt
ttcctttcac 1971atcagccaag atgtctggac aaaaccatca atgtgaataa
gggccaagtg cagttgtgtc 2031tctcttgatt aaattacttc atattaaata
aaaaaaaaaa aaaaaaaaa 208017484PRTMus musculus 17Met Lys Gly Arg Arg
Arg Arg Arg Arg Lys Tyr Cys Lys Phe Thr Leu1 5 10 15Leu Leu Ala Leu
Tyr Thr Leu Leu Leu Leu Leu Val Pro Ser Val Leu 20 25 30Asp Ser His
Ser Glu Gln Asp Lys Gly Arg Asn Cys Pro Gly Leu Gln 35 40 45Arg Ser
Leu Gly Val Trp Ser Leu Glu Ala Ala Ala Ala Gly Glu Arg 50 55 60Glu
Gln Gly Ala Glu Val Arg Ser Leu Ala Glu Gly Asn Pro Asp Arg65 70 75
80Ser Pro Gly Ser Pro Gly Asn Leu Ser Ala Val Gly Glu Ala Val Thr
85 90 95Gln Glu Lys Gln His Ile Tyr Val His Ala Thr Trp Arg Thr Gly
Ser 100 105 110Ser Phe Leu Gly Glu Leu Phe Asn Gln His Pro Asp Val
Phe Tyr Leu 115 120 125Tyr Asp Pro Met Trp His Leu Trp Gln Ala Leu
Tyr Pro Gly Asp Ala 130 135 140Glu Ser Leu Gln Gly Ala Leu Arg Asp
Met Leu Arg Ser Leu Phe Arg145 150 155 160Cys Asp Phe Ser Val Leu
Arg Leu Tyr Ala Gln Pro Gly Asp Pro Gly 165 170 175Glu Arg Ala Pro
Asp Ser Ala Asn Leu Thr Thr Ala Met Leu Phe Arg 180 185 190Trp Arg
Thr Asn Lys Val Ile Cys Ser Pro Pro Leu Cys Pro Ala Ala 195 200
205Pro Arg Ala Arg Ala Asp Val Gly Leu Val Glu Asp Lys Ala Cys Glu
210 215 220Ser Thr Cys Pro Pro Val Ser Leu Arg Ala Leu Glu Ala Glu
Cys Arg225 230 235 240Lys Tyr Pro Val Val Val Ile Lys Asp Val Arg
Leu Leu Asp Leu Gly 245 250 255Val Leu Val Pro Leu Leu Arg Asp Pro
Gly Leu Asn Leu Lys Val Val 260 265 270Gln Leu Phe Arg Asp Pro Arg
Ala Val His Asn Ser Arg Leu Lys Ser 275 280 285Arg Gln Gly Leu Leu
Arg Glu Ser Ile Gln Val Leu Arg Thr Arg Gln 290 295 300Arg Gly Asp
His Phe His Arg Val Leu Leu Ala His Gly Val Asp Ala305 310 315
320Arg Pro Gly Gly Gln Ala Arg Ala Leu Pro Ser Ala Pro Arg Ala Asp
325 330 335Phe Phe Leu Thr Ser Ala Leu Glu Val Ile Cys Glu Ala Trp
Leu Arg 340 345 350Asp Leu Leu Phe Thr Arg Gly Ala Pro Ala Trp Leu
Arg Arg Arg Tyr 355 360 365Leu Arg Leu Arg Tyr Glu Asp Leu Val Trp
Gln Pro Gln Ala Gln Leu 370 375 380Arg Arg Leu Leu Arg Phe Ser Gly
Leu Arg Thr Leu Ala Ala Leu Asp385 390 395 400Ala Phe Ala Phe Asn
Met Thr Arg Gly Ser Ala Tyr Gly Ala Asp Arg 405 410 415Pro Phe His
Leu Ser Ala Arg Asp Ala Arg Glu Ala Val His Ala Trp 420 425 430Arg
Glu Arg Leu Ser Gln Glu Gln Val Arg Gln Val Glu Thr Ala Cys 435 440
445Ala Pro Ala Met Arg Leu Leu Ala Tyr Pro Arg Ser Gly Asp Glu Arg
450 455 460Asp Arg Lys Thr Val Arg Glu Gly Glu Thr Pro Leu Glu Thr
Lys Ala465 470 475 480Asn Trp Ala Val184713DNAHomo
sapiensCDS(514)..(2199) 18ggaaatctgg cattttttaa agtttgcgcc
ccacaaagag gaaatattcc aaaggtactc 60aggatgtaaa aggggagatc
ttcacagatg cctccgtgga tggcatggca atccatccat 120caatgagaag
accatgattt cttttaattt tctgtgtgtt tccacattcc ccagtgagaa
180ttcttccacc tttttttgtg ccatgggaaa aacctgaagg gcaggcagag
ctgctcccga 240acttgtgacc ttctctgagg ttgcagcggc tcttgtagaa
catgactctg ggacatcact 300tccttttgtt ttctttcgga gctgaaccaa
agaatgtgca ccctctttct ctagtgctgt 360ggtgtctgct tatttttgta
tttgtgcttt ccatccatct tctgtgatca caaggcattc 420ttaaggtttt
ctagcacgac ttgcggacat ccagactcgt ggggggccca cccatggctc
480ggtaagccag cagcccaggg cactggcact acc atg agg cac tgc att aat tgc
534 Met Arg His Cys Ile Asn Cys 1 5tgc ata cag ctg tta ccc gac ggc
gca cac aag cag cag gtc aac tgc 582Cys Ile Gln Leu Leu Pro Asp Gly
Ala His Lys Gln Gln Val Asn Cys 10 15 20caa ggg ggc ccc cat cac ggt
cac cag gcg tgc ccc acg tgc aaa gga 630Gln Gly Gly Pro His His Gly
His Gln Ala Cys Pro Thr Cys Lys Gly 25 30 35gaa aac aaa att ctg ttt
cgt gtg gac agt aag cag atg aac ttg ctt 678Glu Asn Lys Ile Leu Phe
Arg Val Asp Ser Lys Gln Met Asn Leu Leu40 45 50 55gct gtt ctc gaa
gtg agg act gaa ggg aac gaa aac tgg ggt ggg ttt 726Ala Val Leu Glu
Val Arg Thr Glu Gly Asn Glu Asn Trp Gly Gly Phe 60 65 70ttg cgc ttc
aaa aag ggg aag cga tgt agc ctc gtt ttt gga ctg ata 774Leu Arg Phe
Lys Lys Gly Lys Arg Cys Ser Leu Val Phe Gly Leu Ile 75 80 85ata atg
acc ttg gta atg gct tct tac atc ctt tct ggg gcc cac caa 822Ile Met
Thr Leu Val Met Ala Ser Tyr Ile Leu Ser Gly Ala His Gln 90 95
100gag ctt ctg atc tca tca cct ttc cat tac gga ggc ttc ccc agc aac
870Glu Leu Leu Ile Ser Ser Pro Phe His Tyr Gly Gly Phe Pro Ser Asn
105 110 115ccc agc ttg atg gac agc gaa aac cca agt gac aca aag gag
cat cac 918Pro Ser Leu Met Asp Ser Glu Asn Pro Ser Asp Thr Lys Glu
His His120 125 130 135cac caa tcc tct gta aat aat att tca tac atg
aag gac tat cca agc 966His Gln Ser Ser Val Asn Asn Ile Ser Tyr Met
Lys Asp Tyr Pro Ser 140 145 150att aaa tta att atc aac agc atc aca
act agg att gag ttc acg acc 1014Ile Lys Leu Ile Ile Asn Ser Ile Thr
Thr Arg Ile Glu Phe Thr Thr 155 160 165aga cag ctc cca gac tta gaa
gac ctt aag aag cag gag ttg cat atg 1062Arg Gln Leu Pro Asp Leu Glu
Asp Leu Lys Lys Gln Glu Leu His Met 170 175 180ttt tca gtc atc ccc
aac aaa ttc ctt cca aac agt aag agc ccc tgt 1110Phe Ser Val Ile Pro
Asn Lys Phe Leu Pro Asn Ser Lys Ser Pro Cys 185 190 195tgg tac gag
gag ttc tcg ggg cag aac acc acc gac ccc tac ctc acc 1158Trp Tyr Glu
Glu Phe Ser Gly Gln Asn Thr Thr Asp Pro Tyr Leu Thr200 205 210
215aac tcc tac gtg ctc tac tcc aag cgc ttc cgc tcc acc ttc gac gcc
1206Asn Ser Tyr Val Leu Tyr Ser Lys Arg Phe Arg Ser Thr Phe Asp Ala
220 225 230ctg cgc aag gcc ttc tgg ggc cac ctg gcg cac gcg cac ggg
aag cac 1254Leu Arg Lys Ala Phe Trp Gly His Leu Ala His Ala His Gly
Lys His 235 240 245ttc cgc ctg cgc tgc ctg ccg cac ttc tac atc ata
ggg cag ccc aag 1302Phe Arg Leu Arg Cys Leu Pro His Phe Tyr Ile Ile
Gly Gln Pro Lys 250 255 260tgc ggg acc aca gac ctc tat gac cgc ctg
cgg ctg cac cct gag gtc 1350Cys Gly Thr Thr Asp Leu Tyr Asp Arg Leu
Arg Leu His Pro Glu Val 265 270 275aag ttc tcc gcc atc aag gag cca
cac tgg tgg acc cgg aag cgc ttt 1398Lys Phe Ser Ala Ile Lys Glu Pro
His Trp Trp Thr Arg Lys Arg Phe280 285 290 295gga atc gtc cgc cta
aga gat ggg ctg cga gac cgc tat ccc gtg gaa 1446Gly Ile Val Arg Leu
Arg Asp Gly Leu Arg Asp Arg Tyr Pro Val Glu 300 305 310gat tat ctg
gac ctc ttt gac ctg gcc gca cac cag atc cat caa gga 1494Asp Tyr Leu
Asp Leu Phe Asp Leu Ala Ala His Gln Ile His Gln Gly 315 320 325ctg
cag gcc agc tct gca aag gag cag agc aag atg aat aca atc att 1542Leu
Gln Ala Ser Ser Ala Lys Glu Gln Ser Lys Met Asn Thr Ile Ile 330 335
340atc ggg gag gcc agt gcc tcc acg atg tgg gat aat aat gcc tgg acg
1590Ile Gly Glu Ala Ser Ala Ser Thr Met Trp Asp Asn Asn Ala Trp Thr
345 350 355ttc ttc tac gac aac agc acg gat ggc gag cca ccg ttt ctg
acg cag 1638Phe Phe Tyr Asp Asn Ser Thr Asp Gly Glu Pro Pro Phe Leu
Thr Gln360 365 370 375gac ttc atc cac gcc ttt cag cca aat gcc aga
ctg att gtc atg ctc 1686Asp Phe Ile His Ala Phe Gln Pro Asn Ala Arg
Leu Ile Val Met Leu 380 385 390agg gac cct gtg gag agg ttg tac tca
gac tat ctc tac ttt gca agt 1734Arg Asp Pro Val Glu Arg Leu Tyr Ser
Asp Tyr Leu Tyr Phe Ala Ser 395 400 405tcg aat aaa tcc gcg gac gac
ttc cat gag aaa gtg aca gaa gca ctg 1782Ser Asn Lys Ser Ala Asp Asp
Phe His Glu Lys Val Thr Glu Ala Leu 410 415 420cag ctg ttt gaa aat
tgc atg ctt gat tat tca ctg cgc gcc tgc gtc 1830Gln Leu Phe Glu Asn
Cys Met Leu Asp Tyr Ser Leu Arg Ala Cys Val 425 430 435tac aac aac
acc ctc aac aac gcc atg cct gtg agg ctc cag gtt ggg 1878Tyr Asn Asn
Thr Leu Asn Asn Ala Met Pro Val Arg Leu Gln Val Gly440 445 450
455ctc tat gct gtg tac ctt ctg gac tgg ctc agc gtt ttt gac aag caa
1926Leu Tyr Ala Val Tyr Leu Leu Asp Trp Leu Ser Val Phe Asp Lys Gln
460 465 470cag ttt ctc att ctt cgc ctg gaa gat cat gca tcc aac gtc
aag tac 1974Gln Phe Leu Ile Leu Arg Leu Glu Asp His Ala Ser Asn Val
Lys Tyr 475 480 485acc atg cac aag gtc ttc cag ttt ctg aac cta ggg
ccc tta agt gag 2022Thr Met His Lys Val Phe Gln Phe Leu Asn Leu Gly
Pro Leu Ser Glu 490 495 500aag cag gag gct ttg atg acc aag agc ccc
gca tcc aat gca cgg cgt 2070Lys Gln Glu Ala Leu Met Thr Lys Ser Pro
Ala Ser Asn Ala Arg Arg 505 510 515ccc gag gac cgg aac ctg ggg ccc
atg tgg ccc atc aca cag aag att 2118Pro Glu Asp Arg Asn Leu Gly Pro
Met Trp Pro Ile Thr Gln Lys Ile520 525 530 535ctg cgg gat ttc tac
agg ccc ttc aac gct agg ctg gcg cag gtc ctc 2166Leu Arg Asp Phe Tyr
Arg Pro Phe Asn Ala Arg Leu Ala Gln Val Leu 540 545 550gcg gat gag
gcg ttt gcg tgg aag acg acg tga gagctgaatt gttgctgcac 2219Ala Asp
Glu Ala Phe Ala Trp Lys Thr Thr 555 560gtgctgggcc cgccaatgcc
gtcatcatca ggattttaca aatctctttg cggggaactg 2279tttcactcat
ggtatggaaa accccaggac tctgccactc taggcacaca tgaattataa
2339ccattttgga atttccttcg tgatgttcga gagctcagca atggacccct
cacagagctc 2399ctctatccga ggccattgga gaccccagtt tctcaagaat
tcagctctgc tctgagcgtc 2459ctggagcttg gggatgcagc cagctggcct
gcactgggtg tggagagaac acctagggaa 2519ggcagcctgg ccctgcccgc
ctccgccttc tggagagcct ctgggttctg agtcagcaag 2579ccagaggtca
tgccacaggc ctggctggaa cttacacttc acgttccctt tttttccccc
2639tagagatggg gtctcgccgt gttgcacaga ctgtctgtat tcaatggcta
tcttcacagg 2699tgtgatcata ccacattcac ttctgaaaca ctcttgttgc
gatcgctaac ctcactggga 2759cagagaaccg cagtctttcg agaatggagg
ctcttcattt tttttttctc ctttactcca 2819aactcagccc tccagtttct
tcagatgtaa accctgttaa cgtcactgtt tccaaaagga 2879aaaaaataag
tcagtttttg gcagcacctt catctttctg acctcctcct attctgtcct
2939tgtggactta tgtttaacat agaaaatgaa tgcgtttaaa acaaaaccac
tttctgcatt 2999taaccagtcc tggctctctc tctgctgcct cttcatacgt
tttctcaaga acttcagttt 3059ataattggaa gagaaatttt tgctgttaat
gccagaatga gcaacctcaa ggaattgaac 3119acttcttgga aaatctaggt
aattcaagcc ctcatcaggt ttacaagatc atcagagaaa 3179cagaggattt
taatttttag ttctggccgg ctacaggctc catttctctg ccttcccatt
3239ggaaatagtt tatttccaca ttctccactg cgtgtggtca aagttcctca
cccagcaagg 3299gactatagat actcgtgtcc caattccaaa acacaatgca
caagctgaac ttgggctgaa 3359cgtggcgtgt tgagatttgg aatgaggttt
ctaagagccg tgttcttcat ggaattttcc 3419aggccacttg gcagcttggt
ttaccgatgg atgggctaga gatcttgtcg tttcttggaa 3479gtcacaggga
agattgaaga gaacgcttga gcatccttgg caacagccca ggtgggacct
3539ggatgaagct ttgcactcaa gtattgtcaa gggaagcttc ctgtgaacca
aagttctcag 3599gccaaggtct cgcccaccaa agccagaaag tgcaagcacc
cgtctaccca gctctaactt 3659gtatgtgtga gacagaccag gcttcggggg
taggaggatc tgcagttgtt cagccgtctt 3719tctgctggtg ttgtctttct
gccatcagag aagggacaca cagcccgttc gaaggtgtgc 3779agagggctct
gagcgccagg atggccaggg ctgtttttgc tactgaagga gcgtgtgtcc
3839tgaactccca cttgcaggga cagtccccac cttctctata gccggcactg
ggagcagccg 3899ccagcaggga aatctggcct gagcacaagg atgctttagg
gagagatcac ttcagtgtgt 3959gtgtatattt atttgcagta cagtgcgcgc
gtgtgtgtgt gtgtacgcgc acgtgtgggt 4019gagtgcgtct tctgagtggg
ttctgttcag ttgctaatga ggctcctccg ctctggacac 4079aaccctttta
tagattaatt tctctgccaa ttaacttgtc attttcagta catattttac
4139tattccacac caaccataat tacaacaagg gatttttctt atgcactcct
atgcatgtga 4199ataacatgtg gtgtaattct gcttcttaca gaagtattac
tgaaggtatt atttccaata 4259ttatttggtt tattatgcgg atctttttta
tatatgcagt cccatccctt ctgtgccact 4319caatgccatc cagacatggt
ttttccctcc aggggccttt ctctccagag ggcacttcgg 4379ctgcctctgc
ttcctctcat tcgaggcccg gctcttgctg acagaatagg ttccgttctg
4439ggcggtggtt ctcgagcctg ccattcaaaa ccaaagcaaa ttggagcatt
tctcacaaca 4499tggtattgaa gttccttttt gttctcaaaa gttgtgaccg
tgttaaattg tactccctta 4559gtcctgtaag gtatgttaag tgaatcgcag
ttacgctgta cttttattaa tatttaacat 4619aattaaagat ggacccataa
gagtgacgcc tgtggagcgc gtgctcttcc tctgcagcca 4679aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaa 471319561PRTHomo sapiens 19Met Arg His
Cys Ile Asn Cys Cys Ile Gln Leu Leu Pro Asp Gly Ala1 5 10 15His Lys
Gln Gln Val Asn Cys Gln Gly Gly Pro His His Gly His Gln 20 25 30Ala
Cys Pro Thr Cys Lys Gly Glu Asn Lys Ile Leu Phe Arg Val Asp 35 40
45Ser Lys Gln Met Asn Leu Leu Ala Val Leu Glu Val Arg Thr Glu Gly
50 55 60Asn Glu Asn Trp Gly Gly Phe Leu Arg Phe Lys Lys Gly Lys Arg
Cys65 70 75 80Ser Leu Val Phe Gly Leu Ile Ile Met Thr Leu Val Met
Ala Ser Tyr 85 90 95Ile Leu Ser Gly Ala His Gln Glu Leu Leu Ile Ser
Ser Pro Phe His 100 105 110Tyr Gly Gly Phe Pro Ser Asn Pro Ser Leu
Met Asp Ser Glu Asn Pro 115 120 125Ser Asp Thr Lys Glu His His His
Gln Ser Ser Val Asn Asn Ile Ser 130 135 140Tyr Met Lys Asp Tyr Pro
Ser Ile Lys Leu Ile Ile Asn Ser Ile Thr145 150 155 160Thr Arg Ile
Glu Phe Thr Thr Arg Gln Leu Pro Asp Leu Glu Asp Leu 165 170 175Lys
Lys Gln Glu Leu His Met Phe Ser Val Ile Pro Asn Lys Phe Leu 180 185
190Pro Asn Ser Lys Ser Pro Cys Trp Tyr Glu Glu Phe Ser Gly Gln Asn
195 200 205Thr Thr Asp Pro Tyr Leu Thr Asn Ser Tyr Val Leu Tyr Ser
Lys Arg 210 215 220Phe Arg Ser Thr Phe Asp Ala Leu Arg Lys Ala Phe
Trp Gly His Leu225 230 235 240Ala His Ala His Gly Lys His Phe Arg
Leu Arg Cys Leu Pro His Phe 245 250 255Tyr Ile Ile Gly Gln Pro Lys
Cys Gly Thr Thr Asp Leu Tyr Asp Arg 260 265 270Leu Arg Leu His Pro
Glu Val Lys Phe Ser Ala Ile Lys Glu Pro His 275 280 285Trp Trp Thr
Arg Lys Arg Phe Gly Ile Val Arg Leu Arg Asp Gly Leu 290 295 300Arg
Asp Arg Tyr Pro Val Glu Asp Tyr Leu Asp Leu Phe Asp Leu Ala305 310
315 320Ala His Gln Ile His Gln Gly Leu Gln Ala Ser Ser Ala Lys Glu
Gln 325 330 335Ser Lys Met Asn Thr Ile Ile Ile Gly Glu Ala Ser Ala
Ser Thr Met 340 345 350Trp Asp Asn Asn Ala Trp Thr Phe Phe Tyr Asp
Asn Ser Thr Asp Gly 355 360 365Glu Pro Pro Phe Leu Thr Gln Asp Phe
Ile His Ala Phe Gln Pro Asn 370 375 380Ala Arg Leu Ile Val Met Leu
Arg Asp Pro Val Glu Arg Leu Tyr Ser385 390 395 400Asp Tyr Leu Tyr
Phe Ala Ser Ser Asn Lys Ser Ala Asp Asp Phe His 405 410 415Glu Lys
Val Thr Glu Ala Leu Gln Leu Phe Glu Asn Cys Met Leu Asp 420 425
430Tyr Ser Leu Arg Ala Cys Val Tyr Asn Asn Thr Leu Asn Asn Ala Met
435 440 445Pro Val Arg Leu Gln Val Gly Leu Tyr Ala Val Tyr Leu Leu
Asp Trp 450 455 460Leu Ser Val Phe Asp Lys Gln Gln Phe Leu Ile Leu
Arg Leu Glu Asp465 470 475 480His Ala Ser Asn Val Lys Tyr Thr Met
His Lys Val Phe Gln Phe Leu 485 490 495Asn Leu Gly Pro Leu Ser Glu
Lys Gln Glu Ala Leu Met Thr Lys Ser 500 505 510Pro Ala Ser Asn Ala
Arg Arg Pro Glu Asp Arg Asn Leu Gly Pro Met 515 520 525Trp Pro Ile
Thr Gln Lys Ile Leu Arg Asp Phe Tyr Arg Pro Phe Asn 530 535 540Ala
Arg Leu Ala Gln Val Leu Ala Asp Glu Ala Phe Ala Trp Lys Thr545 550
555 560Thr20536DNAMus musculus 20acaaagccat gaagccggcg ctgctggaag
tgatgaggat gaacagaatt tgccggatgg 60tgctggccac ttgcttcgga tcctttatct
tggtcatctt ctatttccaa agtatgttgc 120acccagtcat gcggaggaac
cccttcggtg tggacatctg ctgccggaag ggatcgagaa 180gtcccctgca
ggagctctac aatcccatcc agctggagct atccaacact gccatcctgc
240accagatgag acgggaccag gtgacagaca cctgccgggc caacagtgcc
atgagccgca 300agcgcagggt gctgaccccc aacgacctga agcacctggt
ggtggatgag gaccacgaac 360tcatctactg ctatgtgccc aaggtagcgt
gcaccaactg gaagaggctc atgatggtcc 420tgagtggccg gggcaagtac
agcgatccca tggagatccc agccaacgaa gcccacgtgt 480cggccaacct
gaagaccctt aaccagtaca gcatcccaga gatcaaccac cgcttg 53621660DNAMus
musculus 21gccaggagtg ggcccagccc agggcagcat gaccaagccg cggctcttcc
ggctgtggct 60ggtactaggg tcggctctca tgatcctttt gatcattgta tattgggaca
acgtgggaac 120cgcccacttc tatctgcaca cgtctctctc caggccacac
atcctagaac cccttcccac 180ccagggattg gtggaggaga acgtgttcac
atctgacgtg gatgagtttt tggatactct 240ccttagttct gacgcgaagc
acaacgacct ttccaggaga aaaactgagc agcccccggc 300gcccgccccc
agcaagccag tcttgagcca catggaggag aacgtgagag gctacgactg
360gtccactcat gatgcccatc agaaccctga ccgggacagg cagcaggccg
agaggaggag 420cctgctgaga gacttctgtg ccaacgccag cctggcattc
cccaccaagg accgctcttt 480tgacgacatc cccaactacg aactgaacca
cctgatcgtg gacgaccgcc acggggtcat 540ctactgctac gtgcccaagg
tggcctgcac caactggaag cgagtgatga tcgtgctgag 600cgagagcctg
ctggaccggg gcagccccta ccgagacccc ctggacatcc cccgggaaca
66022600DNAMus musculus 22atgactgtcg cctgccacgc gtgccaggca
cagcatggga agacgctcct gttgcaggcg 60gcccttgccg gtggtggcaa gtctgggtgc
tgcactcctg ctcctgtgcg ccctgcgtcc 120cgggtaacca caggaaagga
tgcccaggac actgaatggc agggctcccc aaaagccctt 180ttgggggttc
cgacatttga aaataaagct ctgggctcca gctggttcgg tggagtgagg
240aagagtcccc tacagctgtt gcgtgacctg gaccagatgt ttggcagctg
tgagctatgg 300gtagtcagtg gggagcggca gaaagtgggt ccacgctccg
cgatggccga ggtgcaccag 360cagcggcgtg agctgctgcg ccgggcctgc
agccgccaca cgcgacgcca acgcctgctg 420cagccggagg acctgcgtca
cgtgctggtg gacgacgcgc accggctgct gtactgctac 480gtgcctaagg
tggcctgcac caactggaag cgtgtgatgc tggcgttgcg cggccgtggg
540gatccaagcg caatccctgc gcacgaggcg catgcgcctg gcctgctgcc
ctcgctggcc 60023600DNAMus musculus 23gcgccccctc tccccatgga
gaaaggactc gctttgcctc aggatttccg ggaccttgta 60cacagcctaa agattcgagg
cagatacgtc ttgttcctgg catttgtggt catagttttt 120atcttcattg
aaaaggaaaa taaaatcata tccagggtct ccgacaagct gaagcagatc
180cctcattttg tggcagatgc caacagcact gacccagccc tgctcttatc
ggagaatgca 240tctctcttgt ccctgagcga gttggattcc accttttccc
atctgcggag ccgcctgcac 300aacctgagcc tgcagctggg cgtggagcca
gcaatggaga gccaggaggc tggggcagag 360aagccatccc agcaggctgg
agcagggacc cggcgccacg tgcttctcat ggccaccacc 420cgcacgggtt
cctcgttcgt gggcgagttc ttcaaccagc agggcaatat cttctacctc
480ttcgagccac tgtggcacat cgagcgcacc gtgttcttcc agcagcgagg
cgccagcgcg 540gctggttcag ccttggtcta ccgtgatgtc ctcaagcagt
tgttgctatg cgacctgtat 60024720DNAMus musculus 24tggggagagt
gaggattcgg tgaactatga agggccggcg gcggcggcgc cgagagtatt 60gcaagttcac
gctgctcttg gcgctgtaca cgcttttgct acttcttgtc ccctctgtac
120tggacagcca cagcgagcag gacaagggca ggaactgccc cggcctgcag
cgcagcttgg 180gtgtgtggag cctggaggcg gcggcggccg gggaacgtga
gcagggcgct gaggtgcggt 240ccctggccga aggaaacccg gatcgatccc
ccgggtcccc cggcaacctc agcgccgtcg 300gtgaggcggt gacccaggaa
aagcaacaca tctatgtgca tgccacctgg cgcaccggct 360cgtccttctt
gggcgaactc ttcaaccagc acccggacgt tttctacttg tacgagccca
420tgtggcatct gtggcaggca ctgtatccgg gcgacgcgga gagcctgcag
ggcgcactaa 480gagacatgct gcgctccctc ttccgctgtg atttctctgt
gctgcgcctg tacgcgcagc 540ctggggaccc tggggagcga gcaccggact
cggccaacct caccacggcc atgcttttcc 600gctggcggac caacaaggtc
atctgctcgc cgcctctgtg ccccgccgcg ccccgggcac 660gcgcggacgt
gggactcgtc gaggacaaag cctgcgaaag tacctgcccg cccgtttcgc
7202527RNAArtificial SequenceSynthetic polynucleotide 25ggagcagagc
aagaugaaua caaucag 272627RNAArtificial SequenceSynthetic
polynucleotide 26gauuguauuc aucuugcucu gcuccau 272720DNAArtificial
SequenceSynthetic primer 27gtgagttctg ctgcggtcca
202821DNAArtificial SequenceSynthetic primer 28agtccatgct
gatgcccaga g 212919DNAArtificial SequenceSynthetic primer
29acccgatggc aacaatgga 193020DNAArtificial SequenceSynthetic primer
30accagcaggg ccttgttcac 203125DNAArtificial SequenceSynthetic
primer 31catccgtaaa gacctctatg ccaac 253219DNAArtificial
SequenceSynthetic primer 32atggagccac cgatccaca 193323DNAArtificial
SequenceSynthetic primer 33ttctggccaa cggtctagac aac
233421DNAArtificial SequenceSynthetic primer 34ccagtggtct
tggtgtgctg a 213525DNAArtificial SequenceSynthetic polynucleotide
35acccccaact cggaacgatg cggct 253624DNAArtificial SequenceSynthetic
polynucleotide 36tgcatgttct cgtccatcct gctg 243726DNAArtificial
SequenceSynthetic polynucleotide 37cgccaccgtg tactgtactg tgaagt
263825DNAArtificial SequenceSynthetic polynucleotide 38aggctgctcc
aactggaaga gggtg 253924DNAArtificial SequenceSynthetic
polynucleotide 39atatagtatc taggatatat gtag 244025DNAArtificial
SequenceSynthetic polynucleotide 40gaagtaccaa aagctggctg ctcta
254126DNAArtificial SequenceSynthetic polynucleotide 41ttctatcact
tggactattt gatgtt 264225DNAArtificial SequenceSynthetic
polynucleotide 42tacacaactc cacatttgta atttg 254324DNAArtificial
SequenceSynthetic polynucleotide 43ccagaagcca agctcattgt tatg
244425DNAArtificial SequenceSynthetic polynucleotide 44ctgtggagag
gttgtactca gacta 254526DNAArtificial SequenceSynthetic
polynucleotide 45atttgcctgg aagacaacgt gagagc
264627DNAArtificialSynthetic polynucleotide 46gtcccttctg cagaagctgg
gcccact 274725DNAArtificial SequenceSynthetic polynucleotide
47gcgccccctc tccccatgga gaaag 254825DNAArtificial SequenceSynthetic
polynucleotide 48gctttgcctc aggatttccg ggacc 254927DNAArtificial
SequenceSynthetic polynucleotide 49ggttcagcct tggtctaccg tgatgtc
275025DNAArtificial SequenceSynthetic polynucleotide 50gcagttgttg
ctatgcgacc tgtat 255123DNAArtificial SequenceSynthetic primer
51tgttcctggc atttgtggtc ata 235221DNAArtificial SequenceSynthetic
primer 52ccaactcgct cagggacaag a 215325DNAArtificial
SequenceSynthetic primer 53gtgtggagca acatgtggaa ctcta
255420DNAArtificial SequenceSynthetic primer 54ttggttcagc
cactgccgta 205550DNAArtificial SequenceSynthetic polynucleotide
55acaaagccat gaagccggcg ctgctggaag tgatgaggat gaacagaatt
505626DNAArtificial SequenceSynthetic polynucleotide 56caacctgaag
acccttaacc agtaca 265726DNAArtificial SequenceSynthetic
polynucleotide 57gcatcccaga gatcaaccac cgcttg 265825DNAArtificial
SequenceSynthetic polynucleotide 58gccaggagtg ggcccagccc agggc
255927DNAArtificial SequenceSynthetic polynucleotide 59atgaccaagc
cgcggctctt ccggctg 276028DNAArtificial SequenceSynthetic
polynucleotide 60agagcctgct ggaccggggc agccccta 286128DNAArtificial
SequenceSynthetic polynucleotide 61gagaccccct ggacatcccc cgggaaca
286226DNAArtificial SequenceSynthetic polynucleotide 62atgactgtcg
cctgccacgc gtgcca 266326DNAArtificial SequenceSynthetic
polynucleotide 63cagcatggga agacgctcct gttgca 266428DNAArtificial
SequenceSynthetic polynucleotide 64tccaagcgca atccctgcgc acgaggcg
286525DNAArtificial SequenceSynthetic polynucleotide 65gcctggcctg
ctgccctcgc tggcc 256618DNAArtificial SequenceSynthetic primer
66ctgccaagta tgacatca 186721DNAArtificial SequenceSynthetic primer
67tactccttgg aggccatgta g 216820DNAArtificial SequenceSynthetic
primer 68gtggatgagg accacgaact 206920DNAArtificial
SequenceSynthetic primer 69cttttcaagc ggtggttgat
207020DNAArtificial SequenceSynthetic primer 70acctcctaga
cccacacacg 207120DNAArtificial SequenceSynthetic primer
71ggatgttggc aaaccagtct 207220DNAArtificial SequenceSynthetic
primer 72atgagccctt caacgaacac 207320DNAArtificial
SequenceSynthetic primer 73tggtagaagg ggctgatgtc
207419DNAArtificial SequenceSynthetic primer 74ttccaggctt tgggcatca
197524DNAArtificial SequenceSynthetic primer 75atgttcagca
tgttcagcag tgtg 247625DNAArtificial SequenceSynthetic primer
76catccgtaaa gacctctatg ccaac 257719DNAArtificial SequenceSynthetic
primer 77atggagccac cgatccaca 197820DNAArtificial SequenceSynthetic
primer 78tgtgtggaca cacctcccta 207920DNAArtificial
SequenceSynthetic primer 79cttcaaaggt ccccttcctc
208020DNAArtificial SequenceSynthetic primer 80cagcttgagc
catttcaaca 208120DNAArtificial SequenceSynthetic primer
81gggtgaggcc tttaggaaac 208225DNAArtificial SequenceSynthetic
polynucleotide 82gcgccccctc tccccatgga gaaag 258325DNAArtificial
SequenceSynthetic polynucleotide 83gctttgcctc aggatttccg ggacc
258427DNAArtificial SequenceSynthetic polynucleotide 84ggttcagcct
tggtctaccg tgatgtc 278525DNAArtificial SequenceSynthetic
polynucleotide 85gcagttgttg ctatgcgacc tgtat 258624DNAArtificial
SequenceSynthetic polynucleotide 86tggggagagt gaggattcgg tgaa
248727DNAArtificial SequenceSynthetic polynucleotide 87cggacgtggg
actcgtcgag gacaaag 278826DNAArtificial SequenceSynthetic
polynucleotide 88cgaaagtacc tgcccgcccg tttcgc 268921DNAArtificial
SequenceSynthetic primer 89ctgtgagccc aagtgtgtgg a
219022DNAArtificial SequenceSynthetic primer 90gtctcgaaac
atggcaacag ga 229127RNAArtificial SequenceSynthetic siRNA
91gcagcccagc aagaugaaua agaucag 279227RNAArtificial
SequenceSynthetic siRNA 92gaucuuauuc aucuugcugg gcugcau
279325DNAArtificial SequenceSynthetic primer 93catccgtaaa
gacctctatg ccaac 259419DNAArtificial SequenceSynthetic primer
94atggagccac cgatccaca 199523DNAArtificial SequenceSynthetic primer
95caggagggag aacagaaact cca 239619DNAArtificial SequenceSynthetic
primer 96cctggttggc tgcttgctt 199720DNAArtificial SequenceSynthetic
primer 97ctgccctggc tatggtcaca 209823DNAArtificial
SequenceSynthetic primer 98agacaggtag ttgggtcggt tca 23
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