U.S. patent application number 13/671118 was filed with the patent office on 2013-05-23 for cancer-cell-specific cytostatic agent.
This patent application is currently assigned to GENECARE RESEARCH INSTITUTE CO., LTD.. The applicant listed for this patent is GENECARE RESEARCH INSTITUTE CO., LTD.. Invention is credited to Yasuhiro Furuichi, Ayumi Sato, Akira Shimamoto, Motoki Takagi.
Application Number | 20130131331 13/671118 |
Document ID | / |
Family ID | 36407163 |
Filed Date | 2013-05-23 |
United States Patent
Application |
20130131331 |
Kind Code |
A1 |
Takagi; Motoki ; et
al. |
May 23, 2013 |
CANCER-CELL-SPECIFIC CYTOSTATIC AGENT
Abstract
The present inventors discovered that although suppressing
expression of the RecQ1 gene, a RecQ helicase family gene, shows
suppressive effects on cell proliferation in cancer cells, such
effects are not observed in human TIG3 cells (a normal diploid
fibroblast cell line), which are normal cells. Hence, the present
inventors discovered that siRNAs against RecQ1 gene have cancer
cell-specific cell proliferation-suppressing effects that are
mediated by suppression of the expression of said gene.
Inventors: |
Takagi; Motoki;
(Kamakura-shi, JP) ; Shimamoto; Akira;
(Kamakura-shi, JP) ; Furuichi; Yasuhiro;
(Kamakura-shi, JP) ; Sato; Ayumi; (Kamakura-shi,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENECARE RESEARCH INSTITUTE CO., LTD.; |
Kamakura-shi |
|
JP |
|
|
Assignee: |
GENECARE RESEARCH INSTITUTE CO.,
LTD.
Kamakura-shi
JP
|
Family ID: |
36407163 |
Appl. No.: |
13/671118 |
Filed: |
November 7, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11791129 |
Mar 16, 2009 |
8314073 |
|
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PCT/JP2005/021099 |
Nov 17, 2005 |
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13671118 |
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Current U.S.
Class: |
536/24.5 ;
435/320.1 |
Current CPC
Class: |
A61P 43/00 20180101;
C12N 2310/14 20130101; C12N 15/1137 20130101; A61P 35/02 20180101;
A61P 35/00 20180101 |
Class at
Publication: |
536/24.5 ;
435/320.1 |
International
Class: |
C12N 15/113 20100101
C12N015/113 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 19, 2004 |
JP |
2004-336742 |
Claims
1. A double-stranded RNA that can suppress the expression of an
RecQ1 gene by an RNAi effect, wherein the RNA comprises a structure
in which an RNA comprising the nucleotide sequence of SEQ ID NO: 40
and an RNA comprising a sequence complementary to said RNA are
hybridized.
2. The double-stranded RNA of claim 1, which comprises a structure
in which one or more DNAs or RNAs overhang at an end.
3. A DNA vector that can express an RNA comprising the nucleotide
sequence of SEQ ID NO: 40.
4. A cancer cell-specific cell proliferation inhibitor which
comprises the RNA of claim 1 or 2, or the DNA of claim 3.
5. An anticancer agent comprising the cancer cell-specific cell
proliferation inhibitor of claim 4 as an active ingredient.
Description
TECHNICAL FIELD
[0001] The present invention relates to compounds that suppress
expression of RecQ1 genes, and particularly relates to cell
proliferation inhibitors comprising siRNAs that exhibit the effect
of suppressing expression of these genes.
BACKGROUND ART
[0002] Genes belonging to the RecQ DNA helicase family are widely
present in organisms ranging from prokaryotes such as Escherichia
coli (E. coli) to higher eukaryotes including humans. Conserved in
the evolution process, these genes diversified along with the
multicellularization of organisms. The E. coli RecQ gene was the
first of the RecQ family genes to be discovered. This gene was
identified as a gene participating in zygotic recombination and in
the RecF pathway for UV damage repair (see Non-Patent Document 1).
The E. coli RecQ gene has been revealed to have the function of
suppressing incorrect recombinations (see Non-Patent Document 2).
The budding yeast SGS1 gene and the fission yeast Rqh1 gene are the
only known RecQ homologues in these yeasts. Both of these genes
mainly suppress recombination and play important roles in genome
stabilization (see Non-Patent Documents 3 and 4). Higher eukaryotes
carry a number of RecQ homologues. In humans, there are five types
of genes known to belong to the RecQ family: the RecQL1 (see
Non-Patent Document 6), BLM, WRN, RTS, and RecQL5 genes. Of these
five, the RTS gene (see Non-Patent Document 5 and Patent Documents
1 and 2) and the RecQL5 gene (see Non-Patent Document 5 and Patent
Document 3) were identified by the present inventors. The BLM, WRN,
and RTS genes respectively cause Bloom's syndrome (see Non-Patent
Document 7), Werner's syndrome (see Non-Patent Document 8), and
Rothmund-Thomson syndrome (see Non-Patent Document 9). These genes
all play important roles in genome stabilization in cells.
[0003] In fibroblast cells and lymphocytic cell lines derived from
patients with Werner's syndrome, chromosomal translocation and
deletion, which are indexes for genome instability, have been
reported to occur with high frequency (see Non-Patent Document 10).
Chromosomal breakage and sister chromatid exchange (SCE) are
frequently detected in cells derived from patients with Bloom's
syndrome (see Non-Patent Document 11). Trisomies of human
chromosome 2 and 8 are frequently found in lymphocytes derived from
patients with Rothmund-Thomson syndrome (see Non-Patent Document
12). These findings suggest that the WRN helicase, BLM helicase,
and RTS helicase encoded by the various causative genes of these
three genetic diseases play important roles in genome stabilization
in cells.
[0004] Telomere length abnormalities are seen in lymphocytic cell
lines derived from patients with Werner's syndrome as compared to
cell lines derived from normal healthy subjects (see Non-Patent
Document 13). In addition, cell immortalization was not observed in
lymphocytic cell lines derived from patients with Werner's
syndrome, although about 15% of cell lines derived from normal
healthy subjects were immortalized after passaging (see Non-Patent
Document 14). This finding indicates that WRN helicase contributes
to telomere structure maintenance, and is thus essential for the
immortalization (canceration) of lymphocytic cell lines.
[0005] It has been suggested that WRN helicase is associated with
homologous recombination-mediated repair, because the helicase
forms foci in the nucleus in response to DNA-damaging agents, and
these foci are co-localized with the single-stranded DNA-binding
protein RPA (which is a WRN-binding protein) and with the
recombination repair factor RAD51 (see Non-Patent Document 15). In
addition, WRN helicase has been known to bind to the DNA-dependent
protein kinase complex (DNA-PK) and to flap endonuclease 1 (FEN-1).
By binding to DNA-PK, WRN helicase plays an important role in the
processing of terminals generated by DNA double strand breaks,
which are repaired in the pathway of non-homologous end joining
(see Non-Patent Document 16). WRN helicase is believed to activate
FEN-1 by binding to it, and to provide a site for precise
reconstruction of the replication fork through homologous
recombination by processing Okazaki fragments (see Non-Patent
Document 17). The above findings suggest that WRN helicase plays an
important role in DNA repair during DNA replication. BLM helicase
is localized in the PML body, a specific structure found in the
nucleus, and it binds to topoisomerase III (see Non-Patent Document
18). The helicase has the unwinding activity of the G-quadruplex
structure, and thus is considered to contribute to telomere
maintenance (see Non-Patent Document 19). Furthermore, the helicase
has been reported to unwind the Holliday junction and to interact
with the Rad51 protein (see Non-Patent Document 20). These findings
suggest that BLM helicase cooperates with other DNA-metabolizing
enzymes and plays an important role in recombinational DNA repair
and telomere maintenance.
[0006] Of the five human proteins belonging to the RecQ DNA
helicase family (RecQ1, BLM, WRN, RTS, and RecQ5), RecQ1, BLM, WRN,
and RTS are expressed at negligible levels in resting cells, but
are expressed at high levels in cells whose proliferation has been
enhanced by transformation with viruses (see Non-Patent Document
21). Furthermore, when the carcinogenic promoter TPA is added to
resting cells, the expression of RecQ1, BLM, WRN, and RTS is
induced along with the induction of cell division (see Non-Patent
Document 21). These findings suggest the importance of the RecQ DNA
helicase family in cell proliferation.
[0007] Taken collectively, these findings suggest that the RecQ DNA
helicase family members may be potential target molecules for
anti-cancer therapy because the family members participate in
genomic repair in cells (BLM, WRN and RTS) and also in the
maintenance of telomere structure (BLM and WRN), that they play
important roles in the immortalization of certain cells (WRN), and
that their expression is induced following cell division (RecQ1,
BLM, WRN and RTS).
[0008] However, even if a compound can suppress the proliferation
of cancer cells, if it has similar proliferation-suppressing
effects on normal cells, that compound cannot be expected to be a
useful anticancer agent. So far, nothing is known concerning how
compounds that suppress expression of RecQ1 genes act on normal
cells, or whether such compounds have cancer cell-specific cell
proliferation-suppressing effects.
[0009] [Patent Document 1] Japanese Patent Application No.
H09-200387.
[0010] [Patent Document 2] Japanese Patent Application No.
H11-11218.
[0011] [Patent Document 3] Japanese Patent Application No.
H10-81492 (Japanese Patent Application Kokai Publication No. (JP-A)
H11-276173 (unexamined, published Japanese patent
application)).
[0012] [Non-Patent Document 1] Nakayama H, Nakayama K, Nakayama R,
Irino N, Nakayama Y, Hanawalt P C, "Isolation and genetic
characterization of a thymineless death-resistant mutant of
Escherichia coli K12: identification of a new mutation (recQ1) that
blocks the RecF recombination pathway", Mol Gen Genet., 1984, Vol.
195, p. 474-480.
[0013] [Non-Patent Document 2] Hanada K, Ukita T, Kohno Y, Saito K,
Kato J, Ikeda H, "RecQ DNA helicase is a suppressor of illegitimate
recombination in Escherichia coli", Proc Natl Acad Sci USA., 1997,
Vol. 94, p. 3860-3865.
[0014] [Non-Patent Document 3] Myung K, Datta A, Chen C, Kolodner R
D, "SGS1, the Saccharomyces cerevisiae homologue of BLM and WRN,
suppresses genome instability and homologous recombination", Nat
Genet., 2001, Vol. 27, p. 113-116.
[0015] [Non-Patent Document 4] Doe C L, Dixon J, Osman F, Whitby M
C, "Partial suppression of the fission yeast rqh1(-) phenotype by
expression of a bacterial Holliday junction resolvase", EMBO J.,
2000, Vol. 19, p. 2751-2762.
[0016] [Non-Patent Document 5] Kitao S, Ohsugi I, Ichikawa K, Goto
M, Furuichi Y, Shimamoto A, "Cloning of two new human helicase
genes of the RecQ family: biological significance of multiple
species in higher eukaryotes", Genomics., 1998, Vol. 54, p.
443-452.
[0017] [Non-Patent Document 6] Seki, M., Miyazawa, H., Tada, S.,
Yanagisawa, J., Yamaoka, T., Hoshino, S., Ozawa, K., Eki, T.,
Nogami, M., Okumura K., et al, "Molecular cloning of cDNA encoding
human DNA helicase Q1 which has homology to Escherichia coli Rec Q
helicase and localization of the gene at chromosome 12p12", Nucleic
Acids Res., 1994, Vol. 22, No. 22, p. 4566-4573.
[0018] [Non-Patent Document 7] Ellis N A, Groden J, Ye T Z,
Straughen J, Lennon D J, Ciocci S, Proytcheva M, German J, "The
Bloom's syndrome gene product is homologous to RecQ helicases",
Cell, 1995, Vol. 83, p. 655-666.
[0019] [Non-Patent Document 8] Yu C E, Oshima J, Fu Y H, Wijsman E
M, Hisama F, Alisch R, Matthews S, Nakura J, Miki T, Ouais S,
Martin G M, Mulligan J, Schellenberg G D, "Positional cloning of
the Werner's syndrome gene", Science, 1996, Vol. 272, p.
258-262.
[0020] [Non-Patent Document 9] Kitao S, Shimamoto A, Goto M, Miller
R W, Smithson W A, Lindor N M, Furuichi Y, "Mutations in RECQL4
cause a subset of cases of Rothmund-Thomson syndrome", Nat Genet.,
1999, Vol. 22, p. 82-84.
[0021] [Non-Patent Document 10] Goto M, "Hierarchical deterioration
of body systems in Werner's syndrome: implications for normal
ageing", Mech. Ageing Dev., 1997, Vol. 98, p. 239-254.
[0022] [Non-Patent Document 11] Ellis N A, German J, "Molecular
genetics of Bloom's syndrome", Hum Mol Genet., 1996, Vol. 5, p.
1457-1463.
[0023] [Non-Patent Document 12] Lindor N M, Devries E M, Michels V
V, Schad C R, Jalal S M, Donovan K M, Smithson W A, Kvols L K,
Thibodeau S N, Dewald G W, "Rothmund-Thomson syndrome in siblings:
evidence for acquired in vivo mosaicism", Clin Genet., 1996, Vol.
49, p. 124-129.
[0024] [Non-Patent Document 13] Tahara H, Tokutake Y, Maeda S,
Kataoka H, Watanabe T, Satoh M, Matsumoto T, Sugawara M, Ide T,
Goto M, Furuichi Y, Sugimoto M, "Abnormal telomere dynamics of
B-lymphoblastoid cell strains from Werner's syndrome patients
transformed by Epstein-Barr virus", Oncogene, 1997, Vol. 15, p.
1911-1920.
[0025] [Non-Patent Document 14] Sugimoto M, Furuichi Y, Ide T, Goto
M, "Incorrect us of "immortalization" for B-lymphoblastoid cell
lines transformed by Epstein-Barr virus", Virol., 1999, Vol. 73, p.
9690-9691.
[0026] [Non-Patent Document 15] Sakamoto S, Nishikawa K, Heo S J,
Goto M, Furuichi Y, Shimamoto A, "Werner helicase relocates into
nuclear foci in response to DNA damaging agents and co-localizes
with RPA and Rad51", Genes Cells., 2001, Vol. 6, p. 421-430.
[0027] [Non-Patent Document 16] Yannone S M, Roy S, Chan D W,
Murphy M B, Huang S, Campisi J, Chen D J, "Werner syndrome protein
is regulated and phosphorylated by DNA-dependent protein kinase", J
Biol Chem., 2001, Vol. 276, p. 38242-38248.
[0028] [Non-Patent Document 17] Brosh R M Jr, von Kobbe C, Sommers
J A, Karmakar P, Opresko P L, Piotrowski J, Dianova I, Dianov G L,
Bohr V A, "Werner syndrome protein interacts with human flap
endonuclease 1 and stimulates its cleavage activity", EMBO J.,
2001, Vol. 20, p. 5791-5801.
[0029] [Non-Patent Document 18] Johnson F B, Lombard D B, Neff N F,
Mastrangelo M A, Dewolf W, Ellis N A, Marciniak R A, Yin Y,
Jaenisch R, Guarente L, "Association of the Bloom syndrome protein
with topoisomerase III alpha in somatic and meiotic cells", Cancer
Res., 2000, Vol. 60, p. 1162-1167.
[0030] [Non-Patent Document 19] Mohaghegh P, Karow J K, Brosh Jr R
M Jr, Bohr V A, Hickson I D, "The Bloom's and Werner's syndrome
proteins are DNA structure-specific helicases", Nucleic Acids Res.,
2001, Vol. 29, p. 2843-2849.
[0031] [Non-Patent Document 20] Wu L, Davies S L, Levitt N C,
Hickson I D, "Potential role for the BLM helicase in
recombinational repair via a conserved interaction with RAD51", J
Biol Chem., 2001, Vol. 276, p. 19375-19381.
[0032] [Non-Patent Document 21] Kawabe, T., Tsuyama, N., Kitao, S.,
Nishikawa, K., Shimamoto, A., Shiratori, M., Matsumoto, T., Anno,
K., Sato, T., Mitsui, Y., Seki, M., Enomoto, T., Goto, M., Ellis,
N. A., Ide, T., Furuichi, Y., and Sugimoto, M., "Differential
regulation of human RecQ family helicases in cell transformation
and cell cycle", Oncogene., 2000, Vol. 19, No. 41, p.
4764-4772.
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0033] The present invention was achieved in view of the above
circumstances. An objective of the present invention is to provide
cancer cell-specific cell proliferation inhibitors aimed at
suppressing expression of RecQ1 helicase genes.
Means to Solve the Problems
[0034] The expression level of the RecQ DNA helicase family was
found to be significantly high in tumor cells and methods of
screening for compounds that suppress tumor growth using the
suppression of expression of RecQ DNA helicase family genes as an
index are known. It has also been suggested that compounds
suppressing RecQ helicase gene expression may suppress cancer cell
growth (see Japanese Patent Application Kokai Publication No.
(JP-A) 2000-166600 (unexamined, published Japanese patent
application)).
[0035] However, the relationship between suppression of RecQ1 gene
expression and cancer cell-specific cell proliferation suppression
has until now been unknown.
[0036] Even if a certain compound is found to have cancer cell
proliferation-suppressing effects, if it is unclear whether the
compound has a proliferation-suppressing effect on normal cells,
that compound would not be an effective pharmaceutical. This is
because when such a compound also shows a proliferation-suppressing
effect on normal cells, it carries the risk of side effects. In
fact, to date, findings indicating that various anticancer agents
have side effects have been reported (for example, Komarov P. G. et
al., Science Vol. 285, 1733-1737, 1999; Kamarova E. A. and Gudkov
A. V. Biochemistry (Moscow) Vol. 65, 41-48, 2000; Botchkarev V. A.
Cancer Research Vol. 60, 5002-5006, 2000). If it is possible to
develop pharmaceutical agents that have cancer cell-specific cell
proliferation-suppressing effects and do not act on normal cells,
these agents will be expected to be very useful anticancer agents
with few side effects.
[0037] The present inventors carried out dedicated research to
achieve the above-mentioned objectives. The expression of genes
from the RecQ DNA helicase family is known to be increased in tumor
cell systems (for example, cancer cells). The present inventors
used siRNAs that exhibit the effect of suppressing expression of
the RecQ1 gene, which belongs to the human RecQ helicase family
genes, to examine the effect of suppressing RecQ1 gene expression
on cancer cell proliferation. As a result, the present inventors
discovered that, although suppressing the expression of the RecQ1
gene leads to observation of cell proliferation-suppressing effects
in cancer cells, such effects are not seen in human TIG3 cells
(normal diploid fibroblast cell line), which are normal cells.
Hence, the present inventors discovered for the first time that a
cancer cell-specific cell proliferation-suppressing effect is
observed as a result of suppressing RecQ1 gene expression.
Therefore, the RecQ1 gene may be a target molecule for excellent
carcinostatic agents with few side effects. Furthermore, the
present inventors succeeded in finding siRNA molecules with cancer
cell-specific cell proliferation-suppressing effects.
Pharmaceutical agents comprising such molecules are expected to be
effective pharmaceuticals for treating cancers with few side
effects.
[0038] As described above, many of the existing anticancer agents
have side effects; therefore, it would be very difficult to predict
in advance that a molecule having the effect of suppressing cancer
cell proliferation will not act on normal cells, similarly to the
siRNA molecules of the present application against the RecQ1 gene.
Therefore, the siRNA molecules provided by the present invention
have advantageous effects (cell proliferation-suppressing effects
that are specific to cancer cells and do not affect normal cells)
that cannot be predicted even by those skilled in the art.
[0039] Thus, the present invention relates to cancer cell-specific
cell proliferation inhibitors that target RecQ1 helicase gene
expression, and particularly relates to cancer cell-specific cell
proliferation inhibitors comprising siRNAs with the effect of
suppressing RecQ1 gene expression. More specifically, the present
invention provides the following:
[0040] [1] a double-stranded RNA that can suppress the expression
of an RecQ1 gene by an RNAi effect, wherein the RNA comprises a
structure in which an RNA comprising the nucleotide sequence of any
one of SEQ ID NOs: 1 to 32 or SEQ ID NOs: 40 to 43 and an RNA
comprising a sequence complementary to said RNA are hybridized;
[0041] [2] the double-stranded RNA of [1], which comprises a
structure in which one or more DNAs or RNAs overhang at an end;
[0042] [3] a DNA vector that can express an RNA comprising the
nucleotide sequence of any one of SEQ ID NOs: 1 to 32 or SEQ ID
NOs: 40 to 43;
[0043] [4] a cancer cell-specific cell proliferation inhibitor
which comprises the RNA of [1] or [2], or the DNA of [3]; and
[0044] [5] an anticancer agent comprising the cancer cell-specific
cell proliferation inhibitor of [4] as an active ingredient. The
above-mentioned cancer cells preferably refer to human cancer cells
(cancer cells of human origin).
[0045] Furthermore, the present invention relates to:
[0046] [6] a method for suppressing cell proliferation cancer
cell-specifically (a method for treating a cancer), which comprises
the step of administering the RNA of [1] or [2] or the DNA of [3]
to an individual (a subject, test subject, patient, etc.); and
[0047] [7] a use of the RNA of [1] or [2] or the DNA of [3] in the
production of a cancer cell-specific anticancer agent (a cancer
cell-specific cell proliferation inhibitor).
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1 shows the nucleotide sequences of the siRNAs against
RecQ1 gene that were used in the Examples. All of the sequences are
RNAs, and the overhang sequence of all of the siRNAs is the
deoxynucleotides `TT`.
[0049] FIG. 2 shows the expression levels of the RecQ1 gene in HeLa
cells into which siRNAs against the RecQ1 gene have been
introduced.
[0050] FIG. 3 shows the survival rates of HeLa cells 96 hours after
siRNAs against the RecQ1 gene have been introduced.
[0051] FIG. 4 is a graph showing results of introducing siRNAs
against the RecQ1 gene into TIG3 cells, and then quantifying the
expression of mRNAs 48 hours later by semi-quantitative RT-PCR. NS
is a control siRNA. 15 and 24 are the SEQ ID NOs of siRNAs shown in
FIG. 1. The gene expression obtained when a non-silencing siRNA was
introduced was taken as 100%.
[0052] FIG. 5 is a graph indicating the survival rate of TIG3 cells
96 hours after introduction of siRNAs against the RecQ1 gene. NS is
a control siRNA. 15 and 24 are the SEQ ID NOs of siRNAs shown in
FIG. 1. The graph shows the number of cells when the number of
cells after introduction of a non-silencing siRNA was taken as
100%.
[0053] FIG. 6 shows the evaluation results of the medicinal effect
of the siRNAs against the RecQ1 gene. NT refers to untreated
cancer-bearing mice.
BEST MODE FOR CARRYING OUT THE INVENTION
[0054] The present inventors discovered that, by suppressing the
expression of the RecQ1 gene, which belongs to the RecQ DNA
helicase family genes, cell proliferation is suppressed cancer cell
(tumor cell)-specifically. Further, the present inventors
discovered RNA molecules that exhibit effective cancer
cell-specific cell proliferation-suppressing effects through the
suppression of RecQ1 gene expression by RNAi effects.
[0055] Therefore, firstly, the present invention provides RNAs
(siRNAs and shRNAs) that can suppress RecQ1 gene expression by RNAi
effects. Such RNAs have cancer cell-specific cell
proliferation-suppressing effects. In the present invention, the
term "cancer cell-specific" refers to action against cancer cells
but substantial inaction (not showing effective action) against
normal cells. Cases in which the effect against normal cells is
significantly less than the effect against cancer cells are also
comprised in the term "cancer cell-specific" of the present
invention.
[0056] Those skilled in the art can readily obtain information on
the nucleotide sequences of the RecQ1 genes of the present
invention from public gene databases (for example, GenBank).
Exemplary GenBank accession numbers of the genes described above
are listed below:
[0057] RecQ1 gene: NM.sub.--002907 (SEQ ID NO: 33), NM.sub.--032941
(SEQ ID NO: 34), BC001052 (SEQ ID NO: 35), D37984 (SEQ ID NO: 36),
and L36140 (SEQ ID NO: 37).
[0058] An example of an amino acid sequence of a protein encoded by
a RecQ1 gene of the present invention is indicated in SEQ ID NO:
38.
[0059] The RecQ1 genes of the present invention typically include,
but are not limited to, those derived from animals, more preferably
those derived from mammals, and most preferably those derived from
humans.
[0060] The RNAs of the present invention that can suppress the
expression of RecQ1 genes by RNAi (RNA interference) effects (may
be simply referred to as "the siRNAs of the present invention" in
this application) are more specifically, for example, RNAs
comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to
32. Furthermore, examples of preferred embodiments of the siRNAs of
the present invention include double-stranded RNAs (siRNAs) that
include RNAs comprising the nucleotide sequence of any one of SEQ
ID NOs: 1 to 32 as one of the strands.
[0061] The present invention provides double-stranded RNAs which
are RNAs (siRNAs) that can suppress RecQ1 gene expression by RNAi
effects, and which comprise structures in which an RNA comprising
the nucleotide sequence of any one of SEQ ID NOs: 1 to 32 and an
RNA comprising a sequence complementary to this RNA are
hybridized.
[0062] For example, the siRNAs of the present invention that
comprise the nucleotide sequence of SEQ ID NO: 1
(5'-cuacggcuuuggagauaua-3') may be RNA molecules structured as
below:
##STR00001##
(herein, "I" indicates a hydrogen bond).
[0063] The above-mentioned RNA molecules that are structured such
that one end is closed, for example, siRNAs comprising a hairpin
structure (shRNAs), are also included in the present invention.
Hence, molecules that can form an intramolecular double-stranded
RNA structure are also comprised in the present invention.
[0064] For example, molecules such as 5'-cuacggcuuuggagauaua-3'
(SEQ ID NO: 1) (xxxx)n uauaucuccaaagccguag (SEQ ID NO: 39)-3' are
also comprised in the present invention. (The aforementioned
"(xxxx)n" indicates a polynucleotide comprising any nucleotide and
any number of sequences.)
[0065] Preferred embodiments of the siRNAs of the present invention
are preferably double-stranded RNAs which are RNAs (siRNAs) that
can suppress RecQ1 gene expression by RNAi effects, and which
comprise a structure in which an RNA comprising the nucleotide
sequence of any one of SEQ ID NOs: 1 to 32 and an RNA comprising a
sequence complementary to this RNA are hybridized. Double-stranded
RNAs structured such that, for example, there are one or more RNA
additions or deletions at the end of such a double-stranded RNA are
also comprised in the present invention. In such cases, the RNAs
forming the double strand are preferably homologous to a partial
sequence of a RecQ1 gene. The length of the region of the RNA
forming a double strand in an siRNA of the present invention is
ordinarily 15 to 30 bp, preferably 15 to 27 bp or so, more
preferably 19 to 21 bp, and most preferably 19 bp (for example, an
siRNA in which one of the strands is an RNA of any one of SEQ ID
NOs: 1 to 32), but the length is not necessarily limited
thereto.
[0066] All of the nucleotides in the siRNAs of the present
invention are not necessarily required to be ribonucleotides
(RNAs). Namely, in the present invention, one or more of the
ribonucleotides composing the siRNAs may be corresponding
deoxyribonucleotides. "Corresponding" means that the nucleotides
have identical base species (adenine, guanine, cytosine, and
thymine (uracil)), but that the structure of the sugar portion is
different. For example, the deoxyribonucleotide corresponding to a
ribonucleotide with adenine means a deoxyribonucleotide with
adenine. In addition, the above "more" is not limited to a
particular number but preferably means a small number around two to
five.
[0067] In general, the term "RNAi" refers to a phenomenon where
target gene expression is inhibited by inducing disruption of the
target gene mRNAs. This disruption is caused by introducing into
cells a double-stranded RNA that comprises, a) a sense RNA
comprising a sequence homologous to a target gene mRNA sequence,
and b) an antisense RNA comprising a sequence complementary to the
sense RNA. While the precise RNAi mechanism remains unclear, it is
thought that an enzyme called DICER (a member of the RNase III
nuclease family) contacts the double-stranded RNA, degrading it
into small fragments called "small interfering RNAs" or "siRNAs".
The double-stranded RNAs of the present invention comprising the
RNAi effects preferably refer to these siRNAs.
[0068] In a preferred embodiment of the present invention, the
double-stranded RNAs are RNAs that can suppress RecQ1 gene
expression by RNAi effects and that comprise a structure in which
an RNA comprising the nucleotide sequence of any one of SEQ ID NOs:
1 to 32 and an RNA comprising a sequence complementary to this RNA
are hybridized.
[0069] Furthermore, DNAs that allow the expression of the siRNAs
(double-stranded RNAs) of the present invention are also included
in the present invention. Specifically, the present invention
provides DNAs (vectors) that allow the expression of
double-stranded RNAs of the present invention. These DNAs (vectors)
that allow the expression of double-stranded RNAs of the present
invention are typically DNAs comprising a structure where a DNA
encoding one strand of the double-stranded RNA and a DNA encoding
the other strand of the double-stranded RNA are operably linked to
a promoter. Those skilled in the art can readily prepare an
above-described DNA of the present invention with common genetic
engineering techniques. More specifically, expression vectors of
the present invention can be prepared by appropriately inserting
DNAs encoding RNAs of the present invention into various known
expression vectors.
[0070] Generally, the double-stranded RNAs having an RNAi effect
are double-stranded RNAs comprising a sense RNA, which comprises a
sequence homologous to a continuous RNA region in the mRNA of a
target gene whose expression is to be suppressed, and an antisense
RNA, which comprises a sequence complementary to the sense RNA.
[0071] In general, since double-stranded RNAs with an overhang of
several nucleotides on one end have strong RNAi effects, the
double-stranded RNAs of the present invention preferably comprise
an overhang of several nucleotides on an end. The length of the
nucleotides forming the overhang as well as the sequence are not
particularly limited. This overhang may be DNA or RNA. For example,
the overhang preferably has two nucleotides. A double-stranded RNA
comprising an overhang of, for example, TT (a thymine doublet), UU
(a uracil doublet), or some other nucleotide (most preferably, a
molecule comprising a double-stranded RNA of 19 nucleotides and an
overhang of two nucleotides (TT)) can be suitably used in the
present invention. The double-stranded RNAs of the present
invention also include molecules in which the overhanging
nucleotides are DNAs.
[0072] Examples of the siRNA molecules of the present invention
where the nucleotides of the overhang portion are TT include
molecules having TT added to their 3' side, such as the molecule
indicated below:
##STR00002##
[0073] The above-mentioned "double-stranded RNAs having an RNAi
effect on RecQ1 genes" of the present invention can be suitably
produced by those skilled in the art based on the nucleotide
sequences disclosed in the present description. Specifically, the
double-stranded RNAs of the present invention can be produced based
on the nucleotide sequence of any one of SEQ ID NOs: 1 to 32. If
one of the strands has been determined (for example, a nucleotide
sequence described in any one of SEQ ID NOs: 1 to 32), the
nucleotide sequence of the other strand (the complementary strand)
can be easily determined by those skilled in the art. siRNAs of the
present invention can be suitably produced by those skilled in the
art using commercially available nucleic acid synthesizers. Common
custom synthesis services can also be used to synthesize desired
RNAs.
[0074] Since the siRNAs of the present invention (for example, a
double-stranded RNA molecule in which one of the strands has the
nucleotide sequence of any one of SEQ ID NOs: 1 to 32) have cancer
cell-specific cell proliferation-suppressing effects, the present
invention provides cancer cell-specific cell proliferation
inhibitors that comprise an siRNA of the present invention as an
active ingredient.
[0075] If the cancer cell proliferation-suppressing effect in the
present invention arises from the induction of apoptosis, the
siRNAs of the present invention will be expected to be cancer
cell-specific apoptosis-inducing agents.
[0076] The term "apoptosis" generally refers to cell death actively
induced by the cell itself under physiological condition. The
morphological features of apoptosis include, for example,
chromosome condensation in the cell nucleus, nuclear fragmentation,
loss of microvilli on the cell surface, and cytoplasmic shrinkage.
Thus, as used herein, the term "apoptosis-inducing effect" refers
to, for example, the effect of inducing in cells the
above-described morphological features of apoptosis, but is not
limited to those described above. One skilled in the art can
appropriately assess whether or not apoptosis is being induced in
cells.
[0077] For example, the present invention's apoptosis inducers
specific for cancer cells are expected to be anticancer agents
(carcinostatic agents) having apoptosis-inducing activity as their
mechanism of action.
[0078] The present invention provides anticancer agents
(pharmaceutical compositions for cancer therapy) that comprise a
cancer cell-specific cell proliferation inhibitor of the present
invention as an active ingredient.
[0079] Pharmaceutical agents of the present invention can be
provided as a mixture with a pharmaceutically acceptable carrier.
Such pharmaceutically acceptable carriers can include, but are not
limited to, for example, detergents, excipients, coloring agents,
flavoring agents, preservatives, stabilizers, buffers, suspensions,
isotonizing agents, binders, disintegrating agents, lubricants,
fluidizing agents, and correctives. Other conventional carriers can
be also used appropriately.
[0080] The pharmaceutical agents of the present invention can be
formulated by adding the above-indicated carriers as required and
according to conventional methods. Specifically, such carriers
include: light anhydrous silicic acid, lactose, crystalline
cellulose, mannitol, starch, carmellose calcium, carmellose sodium,
hydroxypropyl cellulose, hydroxypropylmethyl cellulose,
polyvinylacetaldiethylamino acetate, polyvinylpyrrolidone, gelatin,
medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated
castor oil 60, saccharose, carboxymethyl cellulose, cornstarch, and
inorganic salts.
[0081] The dosage forms for the agents described above include, for
example, oral forms, such as tablets, powders, pills, dispersing
agents, granules, fine granules, soft and hard capsules,
film-coated tablets, pellets, sublingual tablets, and pastes; and
parenteral forms, such as injections, suppositories, endermic
liniments, ointments, plasters, and liquids for external use. Those
skilled in the art can select the optimal dosage form depending on
the administration route, subject, and such.
[0082] Viral vectors such as retroviruses, adenoviruses, and Sendai
viruses and non-viral vectors such as liposomes can be used to
administer DNAs expressing the siRNAs of the present invention that
suppress the RecQ1 genes into living bodies. Alternatively,
non-viral vectors such as liposomes, polymer micelles, or cationic
carriers, may be used to administer synthetic siRNAs of the present
invention that suppress the RecQ1 genes into living bodies. The
administration methods include, for example, in-vivo and ex-vivo
methods.
[0083] The present invention also comprises the above-described
pharmaceutical compositions having cancer cell-specific cell
proliferation-suppressing effect. Ultimately, the doses of the
pharmaceutical agents or pharmaceutical compositions of the present
invention can be appropriately determined by a physician
considering the type of dosage form, administration method,
patient's age, weight, symptoms, and so on.
[0084] The types of cancers for which a cell
proliferation-suppressing effect is expected in the present
invention are not particularly limited, but examples include breast
cancers, lung cancers, osteosarcomas, cervical cancers,
fibrosarcomas, ovarian teratocarcinomas, embryonal cancers, bladder
cancers, chronic myeloid leukemias, acute lymphoblastic leukemias,
glioblastomas, liver cancers, glioblastomas, melanomas, kidney
cancers, pancreatic cancers, stomach cancers, prostate cancers, and
such.
[0085] Furthermore, the present invention relates to methods for
suppressing cancer cancer cell-specifically (cancer cell-specific
methods for treating cancer) and methods for suppressing cell
proliferation cancer cell-specifically, which comprise the step of
administering an RNA or DNA of the present invention or a
pharmaceutical agent of the present invention to individuals (for
example, patients) or to cellular tissues (cancer cell tissues and
such). The individuals in the methods of the present invention are
preferably humans, but are not particularly limited thereto, and
they may be non-human animals.
[0086] In general, administration to individuals can be carried out
by methods known to those skilled in the art, examples of which
include intra-arterial injection, intravenous injection, and
subcutaneous injection. Although the dosage varies depending on the
weight and age of the subject (patient and such), the
administration method, and so on, suitable dosages can be
appropriately selected by those skilled in the art.
[0087] Moreover, the present invention relates to the uses of the
RNAs or DNAs of the present invention, or to uses of the
pharmaceutical agents of the present invention, in the production
of cancer cell-specific cell proliferation inhibitors or anticancer
agents.
[0088] All prior art references cited herein are incorporated by
reference into this description.
EXAMPLES
[0089] The present invention will be described in detail below with
reference to Examples, but is not to be construed as being limited
thereto.
Example 1
Cell Cultures
[0090] HeLa cells (human cervical cancer cells) were used as human
cancer cells, and TIG3 cells (normal diploid fibroblast cells) were
used as normal human cells. HeLa cells and TIG3 cells were cultured
at 37.degree. C. under 5% CO.sub.2 using Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum and 50 .mu.g/mL
gentamicin.
Example 2
siRNA Design
[0091] Thirty-two siRNAs against RecQ1 gene were designed according
to the method of Elbasher et al. (Elbasher, M. S. et al. Duplexes
of 21-nucleotide RNAs mediate RNA interference in cultured
mammalian cells. Nature 411, 494-498 (2001)) and the method of
Reynolds et al. (Reynolds A. et al., Rational siRNA design for RNA
interference. Nat. Biotechnol. 3, 326-30 (2004)). FIG. 1 shows each
of the siRNA sequences. The siRNAs were synthesized at Qiagen.
Example 3
Cancer Cell-Specific Cell Proliferation-Suppressing Effects Due to
the Suppression of RecQ1 Gene Expression
[0092] (1) Suppression of RecQ1 Gene Expression by siRNAs
[0093] Cells were plated onto 24-well plates at a density of
0.8-1.5.times.10.sup.4 cells/well 24 hours before transfection, and
siRNAs were transfected under the condition of 20-50% confluency.
10 pmol of siRNA was transfected per well using Oligofectamine
(Invitrogen) or Lipofectamine 2000 (Invitrogen) following the
manufacturer's protocol. Expression of the RecQ1 gene mRNA 24 hours
after introduction of siRNA was quantified using Taqman PCR.
Specifically, total RNA was extracted from cells at 24 hours after
siRNA transfection using an RNeasy Mini Kit (Qiagen). ABI PRISM
7000 Sequence Detection System (Applied Biosystems) was used for
quantitative PCR. RT-PCR primers for the RecQ1 gene and
.beta.-actin gene, and TaqMan probes were purchased from Applied
Biosystems. RT-PCR reactions were performed using a QuantiTect
Probe RT-PCR Kit (Qiagen) according to the manual. Expression of
RecQ1 mRNA was quantitatively compared using 13-actin as a
standard. The expression level of the RecQ1 gene mRNA in cells into
which control siRNAs that do not affect RecQ1 gene expression had
been transfected was defined as 100%, and the RecQ1 mRNA
expressions in cells into which each siRNA had been introduced were
compared.
(2) Cell Proliferation Assays
[0094] siRNA transfection was performed under the same conditions
as described above, and 96 hours later, viable cells were measured
using a viable cell count reagent SF (Nakalai Tesque). The
experiment was carried out at N=3, and average values were
calculated. The viable cell count of cells into which control siRNA
that does not affect RecQ1 gene expression was introduced was
defined as 100%, and the viable cell counts for cells into which
each siRNA was introduced were calculated.
(3) Results
[0095] Using HeLa cells, which are human cervical cancer cells, the
effects on cell proliferation of suppression of RecQ1 gene
expression by siRNAs were investigated. As a result of individually
transfecting the 32 types of siRNAs against the RecQ1 gene into
HeLa cells, a gene expression-suppressing effect of 70% or more was
observed for all of the siRNAs (FIG. 2). Under such conditions,
when the number of viable HeLa cells after 96 hours was compared to
that of the NS-siRNA-treated group, a proliferation suppression of
30% or more was observed in all of the siRNA-treated groups (FIG.
3).
[0096] Next, the effects on the proliferation of normal cells were
investigated using TIG3 cells. When the siRNAs of SEQ ID NOs: 15
and 24, which showed strong proliferation-suppressing effects in
HeLa cells, were individually introduced into TIG3 cells, each of
them suppressed RecQ1 gene expression by approximately 70% (FIG.
4). Under such conditions, when the number of viable TIG3 cells
after 96 hours was compared to that of the NS-siRNA-treated group,
no effect on the proliferation of TIG3 cells was recognized (FIG.
5).
[0097] These results proved that RecQ1-siRNA strongly inhibits the
proliferation of cancer cells, but hardly affects the proliferation
of normal cells.
Example 4
Proliferation Inhibition of Tumor Cells by siRNAs in Cancer-Bearing
Animal Models
[0098] The sequences of the siRNAs and 27 mer dsRNA used in the
animal studies are shown below:
TABLE-US-00001 TABLE 1 SEQUENCES OF SIRNAS AGAINST RECQ1 USED IN
ANIMAL STUDIES siRNA sequence 24 GGGCAAUCAGGAAUCAUAU (SEQ ID NO:
24) 33 GCUUGAAACUAUUAACGUA (SEQ ID NO: 40) 34 UAAGACCACAGUUCAUAGA
(SEQ ID NO: 41) 35 GUUAUCCAUCAUUCAAUGA (SEQ ID NO: 42)
All siRNA sequences are RNAs.
[0099] The overhang sequence of all of the siRNAs is the
deoxynucleotides `TT`.
[0100] The 27 mer dsRNA sequence against RecQ1 used in animal
studies
TABLE-US-00002 (SEQ ID NO: 43) 36 GGAAAAGUUCAGACCACUUCAGCUUGA
The dsRNA sequence is all RNA and does not have an overhang.
[0101] The RecQ1 gene expression levels in HeLa cells treated with
the above RecQ1-siRNAs are the following:
TABLE-US-00003 TABLE 2 Gene expression level 33 3% 34 19% 35 18% 36
6% NS 100%
[0102] The present inventors also examined whether proliferation
inhibition of tumor cells by siRNAs against RecQ1 helicase will
also occur in cancer-bearing animal models. The siRNAs and 27 mer
dsRNA against the RecQ1 gene shown above were used.
[0103] Male BALB/cA nude mice were purchased from CLEA Japan, Inc.
A549 cells (5.times.10.sup.6 cells/0.1 mL) were subcutaneously
transplanted into the back of nude mice (seven weeks old). siRNA
administration began on the eighth day after tumor cell
transplantation. With regard to RecQ1-siRNA, 22 .mu.g of siRNA with
phosphorylated 5' end was mixed with 5 .mu.g of polyethylenimine
(molecular weight of 10,000, Wako) in 50 .mu.L of physiological
saline. This mixture was subcutaneously injected six times, once
every three days (on days 8, 11, 14, 17, 20, and 23), into the
uppermost part of the solid tumor. The tumor volume was measured
using calipers. The equation for calculating tumor volume was
L.times.W.sup.2/2. Herein, L is the major axis and W is the minor
axis of the tumor. Statistical significance of the tumor volume was
analyzed using t-tests.
[0104] As a result, all RecQ1-siRNAs suppressed tumor growth, but
NS-siRNA (an siRNA which does not affect the expression of human
and mouse genes), which was similarly mixed with polyethylenimine,
had no effect and tumor volume increased (FIG. 6). Mice
administered with a mixture of RecQ1-siRNA and polyethylenimine did
not show a reduction in weight compared to non-cancer-bearing mice,
which indicated that this treatment does not have serious side
effects.
[0105] The studies by the present inventors revealed that silencing
of RecQ1 helicase expression causes suppression of tumors in
cancer-bearing animal models.
INDUSTRIAL APPLICABILITY
[0106] Even if a certain compound is found to have the effect of
suppressing cancer cell proliferation, use of that compound as a
pharmaceutical is difficult when it is unclear whether it also has
the effect of suppressing the proliferation of normal cells. This
is because when such a compound also shows cell
proliferation-suppressing effect on normal cells, it carries with
it the risk of side effects. Hence, if the cell
proliferation-suppressing effect is not cancer cell-specific, it
would ordinarily be difficult to actually use the compound as a
pharmaceutical. The pharmaceutical agents of the present invention
(nucleic acids having RNAi effects) can be said to be very
practical and highly effective pharmaceutical agents, since their
cell proliferation-suppressing effect is cancer cell-specific.
[0107] The various embodiments described above can be combined to
provide further embodiments. All of the U.S. patents, U.S. patent
application publications, U.S. patent applications, foreign
patents, foreign patent applications and non-patent publications
referred to in this specification and/or listed in the Application
Data Sheet are incorporated herein by reference, in their entirety.
Aspects of the embodiments can be modified, if necessary to employ
concepts of the various patents, applications and publications to
provide yet further embodiments.
[0108] These and other changes can be made to the embodiments in
light of the above-detailed description. In general, in the
following claims, the terms used should not be construed to limit
the claims to the specific embodiments disclosed in the
specification and the claims, but should be construed to include
all possible embodiments along with the full scope of equivalents
to which such claims are entitled. Accordingly, the claims are not
limited by the disclosure.
Sequence CWU 1
1
43119RNAArtificialan artificially synthesized sequence 1cuacggcuuu
ggagauaua 19219RNAArtificialan artificially synthesized sequence
2gaacuggauu cuauaacca 19319RNAArtificialan artificially synthesized
sequence 3uuaccaguua ccagcauua 19419RNAArtificialan artificially
synthesized sequence 4ugagguuugu uauccauca 19519RNAArtificialan
artificially synthesized sequence 5aaauggucag ccaaugaaa
19619RNAArtificialan artificially synthesized sequence 6gaggaacugg
auucuauaa 19719RNAArtificialan artificially synthesized sequence
7gcaaccaugu uaaaugcuu 19819RNAArtificialan artificially synthesized
sequence 8ggagcauguu aaauggguu 19919RNAArtificialan artificially
synthesized sequence 9gcccucaaac acugaagau 191019RNAArtificialan
artificially synthesized sequence 10gguaguagug gcaacuguu
191119RNAArtificialan artificially synthesized sequence
11gcagucuggu ucuaagaau 191219RNAArtificialan artificially
synthesized sequence 12gcccauugau cucucuuau 191319RNAArtificialan
artificially synthesized sequence 13ggaauucaug caggugcuu
191419RNAArtificialan artificially synthesized sequence
14gggaauugau aagccagau 191519RNAArtificialan artificially
synthesized sequence 15ggaacucaga agcauguaa 191619RNAArtificialan
artificially synthesized sequence 16gacaccggac agucaaaca
191719RNAArtificialan artificially synthesized sequence
17gucaaacacc ggagaguua 191819RNAArtificialan artificially
synthesized sequence 18ggccaccaaa gccuguuua 191919RNAArtificialan
artificially synthesized sequence 19ggaagaccaa uuaaugguu
192019RNAArtificialan artificially synthesized sequence
20cgaguuaaag cugauuuau 192119RNAArtificialan artificially
synthesized sequence 21cggcagaagc ccucaaaca 192219RNAArtificialan
artificially synthesized sequence 22gauauuguaa agcucauua
192319RNAArtificialan artificially synthesized sequence
23cauuaauggg agauacaaa 192419RNAArtificialan artificially
synthesized sequence 24gggcaaucag gaaucauau 192519RNAArtificialan
artificially synthesized sequence 25gaacaaguua cgguuaguu
192619RNAArtificialan artificially synthesized sequence
26caggucgaga ugacaugaa 192719RNAArtificialan artificially
synthesized sequence 27cucagaagca uguaacaaa 192819RNAArtificialan
artificially synthesized sequence 28gcagagaucu aaucaagau
192919RNAArtificialan artificially synthesized sequence
29cauacaaucg ucuuaaguu 193019RNAArtificialan artificially
synthesized sequence 30cauggucugg uaaaguuaa 193119RNAArtificialan
artificially synthesized sequence 31ggcucaacau uuugaugaa
193219RNAArtificialan artificially synthesized sequence
32gguucaugcu gaaauggua 19332866DNAHomo sapiensGenBank/NM_002907
33gagtagcgga aagatctgct cgaggcctgg gtgctttggt gtcggagatc cgagagtcgg
60agatcggaga gtcggacaca ggacagtcgg acaccggaca gtcaaacacc ggagagttag
120actgggcttc tcggtgggga gaggctctgg gataactact gttacagctt
tgaagggtca 180agggtgtgcg ctttttgttt catccttccc tttcctgctg
cagggcgagg ccggtctgta 240gcggatcact tcctttcgcc cacacattgg
cggaggagaa accggaaagt taatcactgc 300cctgctctga gaactcgggc
ctttaggggc acgttcgcct gctgaccggt cttctgatct 360ccccattctt
ttccatgcag gaggattggc caccaaagcc tgtttattag cagctgccat
420ttgttgaaag aaatttggat tattttagaa acaaatttgg aaagaaaaag
aatggcgtcc 480gtttcagctc taactgagga actggattct ataaccagtg
agctacatgc agtagaaatt 540caaattcaag aacttacgga aaggcaacaa
gagcttattc agaaaaaaaa agtcctgaca 600aagaaaataa agcagtgttt
agaggattct gatgccgggg caagcaatga atatgattct 660tcacctgccg
cttggaataa agaagatttt ccatggtctg gtaaagttaa agatattctg
720caaaatgtct ttaaactgga aaagttcaga ccacttcagc ttgaaactat
taacgtaaca 780atggctggaa aggaggtatt tcttgttatg cctacaggag
gtggaaagag cttatgttac 840cagttaccag cattatgttc agatggtttt
acactcgtca tttgcccatt gatctctctt 900atggaagacc aattaatggt
tttaaaacaa ttaggaattt cagcaaccat gttaaatgct 960tctagttcta
aggagcatgt taaatgggtt catgctgaaa tggtaaataa aaactccgag
1020ttaaagctga tttatgtgac tccagagaaa attgcaaaaa gcaaaatgtt
tatgtcaaga 1080ctagagaaag cctatgaagc aaggagattt actcgaattg
ctgtggatga agttcactgc 1140tgtagtcagt ggggacatga tttcagacct
gattataagg cacttggtat cttaaagcgg 1200cagttcccta acgcatcact
aattgggctg actgcaactg caacaaatca cgttttgacg 1260gatgctcaga
aaattttgtg cattgaaaag tgttttactt ttacagcttc ttttaatagg
1320ccaaatctat attatgaggt tcggcagaag ccctcaaaca ctgaagattt
tattgaggat 1380attgtaaagc tcattaatgg gagatacaaa gggcaatcag
gaatcatata ttgtttttct 1440cagaaagact ctgaacaagt tacggttagt
ttgcagaatc tgggaattca tgcaggtgct 1500taccatgcca atttggagcc
agaagataag accacagttc atagaaaatg gtcagccaat 1560gaaattcagg
tagtagtggc aactgttgca tttggtatgg gaattgataa gccagatgtg
1620aggtttgtta tccatcattc aatgagtaaa tccatggaaa attattacca
agagagtgga 1680cgtgcaggtc gagatgacat gaaagcagac tgtattttgt
actacggctt tggagatata 1740ttcagaataa gttcaatggt ggtgatggaa
aatgtgggac agcagaagct ttatgagatg 1800gtatcatact gtcaaaacat
aagcaaatgt cgtcgtgtgt tgatggctca acattttgat 1860gaagtatgga
actcagaagc atgtaacaaa atgtgcgata actgctgtaa agacagtgca
1920tttgaaagaa agaacataac agagtactgc agagatctaa tcaagatcct
gaagcaggca 1980gaggaactga atgaaaaact cactccattg aaactgattg
attcttggat gggaaagggt 2040gcagcaaaac tgagagtagc aggtgttgtg
gctcccacac ttcctcgtga agatctggag 2100aagattattg cacactttct
aatacagcag tatcttaaag aagactacag ttttacagct 2160tatgctacca
tttcgtattt gaaaatagga cctaaagcta accttctgaa caatgaggca
2220catgctatta ctatgcaagt gacaaagtcc acgcagaact ctttcagggc
tgaatcgtct 2280caaacttgtc attctgaaca aggtgataaa aagatggagg
aaaaaaattc aggcaacttc 2340cagaagaagg ctgcaaacat gcttcagcag
tctggttcta agaatacagg agctaagaaa 2400agaaaaatcg atgatgcctg
atatgaatgt tactaaattt tctaattaaa gatggtttat 2460gcatgtatat
gccattattt ttgtagttag acaatagttt ttaaaagaat ttcatagata
2520ttttatatgt atggatctat attttcagag cttatctctg aagatctaaa
cttttgagaa 2580tgtttgaaaa ttagagatca tgaattatat aattttccag
tataaaacaa gggaaaaatt 2640tttatgtaaa accctttaaa tgtaaaatat
ttgagaataa gttcatacaa tcgtcttaag 2700ttttttatgc ctttatatac
ttagctatat tttttctttt gacataacta tctttttgaa 2760agcaatatta
tactgacaga ggctcactga gtgatacttt aagttaaata tgtagatcaa
2820gggatgtcca atcttttggc ttccctgagc cagcgaattg tgcaca
2866342286DNAHomo sapiensGenBank/NM_032941 34gagtagcgga aagatctgct
cgaggcctgg gtgctttggt gtcggagatc cgagagtcgg 60agatcggaga gtcggacaca
ggacagtcgg acaccggaca gtcaaacacc ggagagttag 120actgggcttc
tcggtgggga gaggctctgg gataactact gttacagctt tgaagggtca
180agggaggatt ggccaccaaa gcctgtttat tagcagctgc catttgttga
aagaaatttg 240gattatttta gaaacaaatt tggaaagaaa aagaatggcg
tccgtttcag ctctaactga 300ggaactggat tctataacca gtgagctaca
tgcagtagaa attcaaattc aagaacttac 360ggaaaggcaa caagagctta
ttcagaaaaa aaaagtcctg acaaagaaaa taaagcagtg 420tttagaggat
tctgatgccg gggcaagcaa tgaatatgat tcttcacctg ccgcttggaa
480taaagaagat tttccatggt ctggtaaagt taaagatatt ctgcaaaatg
tctttaaact 540ggaaaagttc agaccacttc agcttgaaac tattaacgta
acaatggctg gaaaggaggt 600atttcttgtt atgcctacag gaggtggaaa
gagcttatgt taccagttac cagcattatg 660ttcagatggt tttacactcg
tcatttgccc attgatctct cttatggaag accaattaat 720ggttttaaaa
caattaggaa tttcagcaac catgttaaat gcttctagtt ctaaggagca
780tgttaaatgg gttcatgctg aaatggtaaa taaaaactcc gagttaaagc
tgatttatgt 840gactccagag aaaattgcaa aaagcaaaat gtttatgtca
agactagaga aagcctatga 900agcaaggaga tttactcgaa ttgctgtgga
tgaagttcac tgctgtagtc agtggggaca 960tgatttcaga cctgattata
aggcacttgg tatcttaaag cggcagttcc ctaacgcatc 1020actaattggg
ctgactgcaa ctgcaacaaa tcacgttttg acggatgctc agaaaatttt
1080gtgcattgaa aagtgtttta cttttacagc ttcttttaat aggccaaatc
tatattatga 1140ggttcggcag aagccctcaa acactgaaga ttttattgag
gatattgtaa agctcattaa 1200tgggagatac aaagggcaat caggaatcat
atattgtttt tctcagaaag actctgaaca 1260agttacggtt agtttgcaga
atctgggaat tcatgcaggt gcttaccatg ccaatttgga 1320gccagaagat
aagaccacag ttcatagaaa atggtcagcc aatgaaattc aggtagtagt
1380ggcaactgtt gcatttggta tgggaattga taagccagat gtgaggtttg
ttatccatca 1440ttcaatgagt aaatccatgg aaaattatta ccaagagagt
ggacgtgcag gtcgagatga 1500catgaaagca gactgtattt tgtactacgg
ctttggagat atattcagaa taagttcaat 1560ggtggtgatg gaaaatgtgg
gacagcagaa gctttatgag atggtatcat actgtcaaaa 1620cataagcaaa
tgtcgtcgtg tgttgatggc tcaacatttt gatgaagtat ggaactcaga
1680agcatgtaac aaaatgtgcg ataactgctg taaagacagt gcatttgaaa
gaaagaacat 1740aacagagtac tgcagagatc taatcaagat cctgaagcag
gcagaggaac tgaatgaaaa 1800actcactcca ttgaaactga ttgattcttg
gatgggaaag ggtgcagcaa aactgagagt 1860agcaggtgtt gtggctccca
cacttcctcg tgaagatctg gagaagatta ttgcacactt 1920tctaatacag
cagtatctta aagaagacta cagttttaca gcttatgcta ccatttcgta
1980tttgaaaata ggacctaaag ctaaccttct gaacaatgag gcacatgcta
ttactatgca 2040agtgacaaag tccacgcaga actctttcag ggctgaatcg
tctcaaactt gtcattctga 2100acaaggtgat aaaaagatgg aggaaaaaaa
ttcaggcaac ttccagaaga aggctgcaaa 2160catgcttcag cagtctggtt
ctaagaatac aggagctaag aaaagaaaaa tcgatgatgc 2220ctgatatgaa
tgttactaaa ttttctaatt aaagatggtt tatgcaaaaa aaaaaaaaaa 2280aaaaaa
2286352251DNAHomo sapiensGenBank/BC001052 35ggcacgaggg agatccgaga
gtcggagatc ggagagtcgg acacaggaca gtcggacacc 60ggacagtcaa acaccggaga
gttagactgg gcttctcggt ggggagaggc tctgggataa 120ctactgttac
agctttgaag ggtcaaggga ggattggcca ccaaagcctg tttattagca
180gctgccattt gttgaaagaa atttggatta ttttagaaac aaatttggaa
agaaaaagaa 240tggcgtccgt ttcagctcta actgaggaac tggattctat
aaccagtgag ctacatgcag 300tagaaattca aattcaagaa cttacggaaa
ggcaacaaga gcttattcag aaaaaaaaag 360tcctgacaaa gaaaataaag
cagtgtttag aggattctga tgccggggca agcaatgaat 420atgattcttc
acctgccgct tggaataaag aagattttcc atggtctggt aaagttaaag
480atattctgca aaatgtcttt aaactggaaa agttcagacc acttcagctt
gaaactatta 540acgtaacaat ggctggaaag gaggtatttc ttgttatgcc
tacaggaggt ggaaagagct 600tatgttacca gttaccagca ttatgttcag
atggttttac actcgtcatt tgcccattga 660tctctcttat ggaagaccaa
ttaatggttt taaaacaatt aggaatttca gcaaccatgt 720taaatgcttc
tagttctaag gagcatgtta aatgggttca tgctgaaatg gtaaataaaa
780actccgagtt aaagctgatt tatgtgactc cagagaaaat tgcaaaaagc
aaaatgttta 840tgtcaagact agagaaagcc tatgaagcaa ggagatttac
tcgaattgct gtggatgaag 900ttcactgctg tagtcagtgg ggacatgatt
tcagacctga ttataaggca cttggtatct 960taaagcggca gttccctaac
gcatcactaa ttgggctgac tgcaactgca acaaatcacg 1020ttttgacgga
tgctcagaaa attttgtgca ttgaaaagtg ttttactttt acagcttctt
1080ttaataggcc aaatctatat tatgaggttc ggcagaagcc ctcaaacact
gaagatttta 1140ttgaggatat tgtaaagctc attaatggga gatacaaagg
gcaatcagga atcatatatt 1200gtttttctca gaaagactct gaacaagtta
cggttagttt gcagaatctg ggaattcatg 1260caggtgctta ccatgccaat
ttggagccag aagataagac cacagttcat agaaaatggt 1320cagccaatga
aattcaggta gtagtggcaa ctgttgcatt tggtatggga attgataagc
1380cagatgtgag gtttgttatc catcattcaa tgagtaaatc catggaaaat
tattaccaag 1440agagtggacg tgcaggtcga gatgacatga aagcagactg
tattttgtac tacggctttg 1500gagatatatt cagaataagt tcaatggtgg
tgatggaaaa tgtgggacag cagaagcttt 1560atgagatggt atcatactgt
caaaacataa gcaaatgtcg tcgtgtgttg atggctcaac 1620attttgatga
agtatggaac tcagaagcat gtaacaaaat gtgcgataac tgctgtaaag
1680acagtgcatt tgaaagaaag aacataacag agtactgcag agatctaatc
aagatcctga 1740agcaggcaga ggaactgaat gaaaaactca ctccattgaa
actgattgat tcttggatgg 1800gaaagggtgc agcaaaactg agagtagcag
gtgttgtggc tcccacactt cctcgtgaag 1860atctggagaa gattattgca
cactttctaa tacagcagta tcttaaagaa gactacagtt 1920ttacagctta
tgctaccatt tcgtatttga aaataggacc taaagctaac cttctgaaca
1980atgaggcaca tgctattact atgcaagtga caaagtccac gcagaactct
ttcagggctg 2040aatcgtctca aacttgtcat tctgaacaag gtgataaaaa
gatggagaaa aaaaattcag 2100gcaacttcca gaagaaggct gcaaacatgc
ttcagcagtc tggttctaag aatacaggag 2160ctaagaaaag aaaaatcgat
gatgcctgat atgaatgtta ctaaattttc taattaaaga 2220tggtttatgc
aaaaaaaaaa aaaaaaaaaa a 2251362449DNAHomo sapiensGenBank/D37984
36tcggcgtccg tttcagctct aactgaggaa ctggattcta taaccagtga gctacatgca
60gtagaaattc aaattcaaga acttacggaa aggcaacaag agcttattca gaaaaaaaaa
120gtcctgacaa agaaaataaa gcagtgttta gaggattctg atgccggggc
aagcaatgaa 180tatgattctt cacctgccgc ttggaataaa gaagattttc
catggtctgg taaagttaaa 240gatattctgc aaaatgtctt taaactggaa
aagttcagac cacttcagct tgaaactatt 300aacgtaacaa tggctggaaa
ggaggtattt cttgttatgc ctacaggagg tggaaagagc 360ttatgttacc
agttaccagc attatgttca gatggtttta cactcgtcat ttgcccattg
420atctctctta tggaagacca attaatggtt ttaaaacaat taggaatttc
agcaaccatg 480ttaaatgctt ctagttctaa ggagcatgtt aaatgggttc
atgctgaaat ggtaaataaa 540aactccgagt taaagctgat ttatgtgact
ccagagaaaa ttgcaaaaag caaaatgttt 600atgtcaagac tagagaaagc
ctatgaagca aggagattta ctcgaattgc tgtggatgaa 660gttcactgct
gtagtcagtg gggacatgat ttcagacctg attataaggc acttggtatc
720ttaaagcggc agttccctaa cgcatcacta attgggctga ctgcaactgc
aacaaatcac 780gttttgacgg atgctcagaa aattttgtgc attgaaaagt
gttttacttt tacagcttct 840tttaataggc caaatctata ttatgaggtt
cggcagaagc cctcaaacac tgaagatttt 900attgaggata ttgtaaagct
cattaatggg agatacaaag ggcaatcagg aatcatatat 960tgtttttctc
agaaagactc tgaacaagtt acggttagtt tgcagaatct gggaattcat
1020gcaggtgctt accatgccaa tttggagcca gaagataaga ccacagttca
tagaaaatgg 1080tcagccaatg aaattcaggt agtagtggca actgttgcat
ttggtatggg aattgataag 1140ccagatgtga ggtttgttat ccatcattca
atgagtaaat ccatggaaaa ttattaccaa 1200gagagtggac gtgcaggtcg
agatgacatg aaagcagact gtattttgta ctacggcttt 1260ggagatatat
tcagaataag ttcaatggtg gtgatggaaa atgtgggaca gcagaagctt
1320tatgagatgg tatcatactg tcaaaacata agcaaatgtc gtcgtgtgtt
gatggctcaa 1380cattttgatg aagtatggaa ctcagaagca tgtaacaaaa
tgtgcgataa ctgctgtaaa 1440gacagtgcat ttgaaagaaa gaacataaca
gagtactgca gagatctaat caagatcctg 1500aagcaggcag aggaactgaa
tgaaaaactc actccattga aactgattga ttcttggatg 1560ggaaagggtg
cagcaaaact gagagtagca ggtgttgtgg ctcccacact tcctcgtgaa
1620gatctggaga agattattgc acactttcta atacagcagt atcttaaaga
agactacagt 1680tttacagctt atgctaccat ttcgtatttg aaaataggac
ctaaagctaa tcttctgaac 1740aatgaggcac atgctattac tatgcaagtg
acaaagtcca cgcagaactc tttcagggct 1800gaatcgtctc aaacttgtca
ttctgaacaa ggtgataaaa agatggagga aaaaaattca 1860ggcaacttcc
agaagaaggc tgcaaacatg cttcagcaat ctggttctaa gaatacagga
1920gctaagaaaa gaaaaatcga tgatgcctga tatgactgtt actaaatttt
ctaattaaag 1980atggtttatg catgtatatg ccattatttt tgtagttaga
caatagtttt taaaagaatt 2040tcatagatat tttatatgta tggatctata
ttttcagagc ttatctctga agatctaaac 2100ttttggagaa tgtttggaaa
attagagatc atgaattata taattttcca gtataaaaca 2160agggaaaaat
ttttatgtaa aaccctttaa atgtaaaata tttgagaata agttcataca
2220atcgtcttaa gttttttatg cctttatata cttagctata ttttttcttt
tgacataacc 2280atctttttga aagcaatatt atactgacag aggttcactg
agtgatactt taagttaaat 2340atgtagatca gggatgtcca atcttttggc
ttccctgagc cacattggaa gaagaattgt 2400cttgggccgc acataaaata
tgctaacact gacgatagct gatgagctt 2449372925DNAHomo
sapiensGenBank/L36140 37cttttttttt tttttttttt tttttataag attattagta
taaaatttta gataggtagg 60agtagcgaaa agatctgctc gaggcctggg tgctttggtg
tcggagatcc gagagtcgga 120gatcggagag tcggacacag gacagtcgga
caccggacag tcaaacaccg gagagttaga 180ctgggcttct cggtggggac
aggctctggg ataactactg ttacagcttt gaagggtcaa 240gggtgtgcgc
tttttctttc atccttccct ttcctgctgc aggcgaggcc ggtctgatgc
300ggatcacttc ctttcgccca cacattggcg gaggagaaac cggaaagtta
atcactgccc 360tgctctgaga actcgggcct ttaggggcac gttcgcctgc
tgaccggtct tctgatctcc 420ccattctttt ccatgcagga ggattggcca
ccaaagcctg tttattagca gctgccattt 480gttaaagaaa tttggattat
tttagaaaca atttggaaag aaaaagaatg gcgtccgttt 540cagctctaac
tgaggaactg gattctataa ccagtgagct acatgcagta gaaattcaaa
600ttcaagaact tacggaaagg caacaagagc ttattcagaa aaaaaaagtc
ctgacaaaga 660aaataaagca gtgtttagag gattctgatg ccggggcaag
caatgaatat gattcttcac 720ctgccgcttg gaataaagaa gattttccat
ggtctggtaa agttaaagat attctgcaaa 780atgtctttaa actggaaaag
ttcagaccac ttcagcttga aactattaac gtaacaatgg 840ctggaaagga
ggtatttctt gttatgccta caggaggtgg aaagagctta tgttaccagt
900taccagcatt atgttcagat ggttttacac tcgtcatttg cccattgatc
tctcttatgg 960aagaccaatt aatggtttta aaacaattag gaatttcagc
aaccatgtta aatgcttcta 1020gttctaagga gcatgttaaa tgggttcatg
atgaaatggt
aaataaaaac tccgagttaa 1080agctgattta tgtgactcca gagaaaattg
caaaaagcaa aatgtttatg tcaagactag 1140agaaagccta tgaagcaagg
agatttactc gaattgctgt ggatgaagtt cactgctgta 1200gtcagtgggg
acatgatttc agacctgatt ataaggcact tggtatctta aagcggcagt
1260tccctaacgc atcactaatt gggctgactg caactgcaac aaatcacgtt
ttgacggatg 1320ctcagaaaat tttgtgcatt gaaaagtgtt ttacttttac
agcttctttt aataggccaa 1380atctatatta tgaggttcgg cagaagccct
caaacactga agattttatt gaggatattg 1440taaagctcat taatgggaga
tacaaagggc aatcaggaat catatattgt ttttctcaga 1500aagactctga
acaagttacg gttagtttgc agaatctggg aattcatgca ggtgcttacc
1560atgccaattt ggagccagaa gataagacca cagttcatag aaaatggtca
gccaatgaaa 1620ttcaggtagt agtggcaact gttgcatttg gtatgggaat
tgataagcca gatgtgaggt 1680ttgttatcca tcattcaatg agtaaatcca
tggaaaatta ttaccaagag agtggacgtg 1740caggtcgaga tgacatgaaa
gcagactgta ttttgtacta cggctttgga gatatattca 1800gaataagttc
aatggtggtg atggaaaatg tgggacagca gaagctttat gagatggtat
1860catactgtca aaacataagc aaatctcgtc gtgtgttgat ggctcaacat
tttgatgaag 1920tatggaactc agaagcatgt aacaaaatgt gcgataactg
ctgtaaagac agtgcatttg 1980aaagaacgaa cataacagag tactgcagag
atctaatcaa gatcctgaag caggcagagg 2040aactgaatga aaaactcact
ccattgaaac tgattgattc ttggatggga aagggtgcag 2100caaaactgag
agtagcaggt gttgtggctc ccacacttcc tcgtgaagat ctggagaaga
2160ttattgcaca ctttctaata cagcagtatc ttaaagaaga ctacagtttt
acagcttatg 2220ctgccatttc gtatttgaaa ataggaccta aagctaatct
tctgaacaat gaggcacatg 2280ctattactat gcaagtgaca aagtccacgc
agaactcttt cagggctgaa tcgtctcaaa 2340cttgtcattc tgaacaaggt
gataaaaaga atggaggaaa aaaaattcag gcaacttcca 2400gaagaaggct
gcaaacatgc ttcagcaatc tggttctaag aatacaggag ctaagaaaag
2460aaaaatcgat gatgcctgat atgaatgtta ctaaattttc taattaaaga
tggtttatgc 2520atgtatatgc cattattttt gtagttagac aatagttttt
aaaagaattt catagatatt 2580ttatatgtat ggatctatat tttcagagct
tatctctgaa gatctaaact tttgagaatg 2640tttgaaaatt agagatcatg
aattatataa ttttccagtg taaaacaagg gaaaaatttt 2700tatgtaaaac
cctttaaatg taaaatattt gagaataagt tcatacaatc gtcttaagtt
2760ttttatgcct ttatatactt agctatattt tttcttttga cataactatc
tttttgaaag 2820caatattata ctgacagagg cttcactgag tgatacttta
agttaaatat gtagatcaag 2880ggatgtccaa tcttttggct tccctgagcc
agcgaattgt gcaca 292538649PRTHomo sapiensGenBank/NM_002907 38Met
Ala Ser Val Ser Ala Leu Thr Glu Glu Leu Asp Ser Ile Thr Ser 1 5 10
15 Glu Leu His Ala Val Glu Ile Gln Ile Gln Glu Leu Thr Glu Arg Gln
20 25 30 Gln Glu Leu Ile Gln Lys Lys Lys Val Leu Thr Lys Lys Ile
Lys Gln 35 40 45 Cys Leu Glu Asp Ser Asp Ala Gly Ala Ser Asn Glu
Tyr Asp Ser Ser 50 55 60 Pro Ala Ala Trp Asn Lys Glu Asp Phe Pro
Trp Ser Gly Lys Val Lys 65 70 75 80 Asp Ile Leu Gln Asn Val Phe Lys
Leu Glu Lys Phe Arg Pro Leu Gln 85 90 95 Leu Glu Thr Ile Asn Val
Thr Met Ala Gly Lys Glu Val Phe Leu Val 100 105 110 Met Pro Thr Gly
Gly Gly Lys Ser Leu Cys Tyr Gln Leu Pro Ala Leu 115 120 125 Cys Ser
Asp Gly Phe Thr Leu Val Ile Cys Pro Leu Ile Ser Leu Met 130 135 140
Glu Asp Gln Leu Met Val Leu Lys Gln Leu Gly Ile Ser Ala Thr Met 145
150 155 160 Leu Asn Ala Ser Ser Ser Lys Glu His Val Lys Trp Val His
Ala Glu 165 170 175 Met Val Asn Lys Asn Ser Glu Leu Lys Leu Ile Tyr
Val Thr Pro Glu 180 185 190 Lys Ile Ala Lys Ser Lys Met Phe Met Ser
Arg Leu Glu Lys Ala Tyr 195 200 205 Glu Ala Arg Arg Phe Thr Arg Ile
Ala Val Asp Glu Val His Cys Cys 210 215 220 Ser Gln Trp Gly His Asp
Phe Arg Pro Asp Tyr Lys Ala Leu Gly Ile 225 230 235 240 Leu Lys Arg
Gln Phe Pro Asn Ala Ser Leu Ile Gly Leu Thr Ala Thr 245 250 255 Ala
Thr Asn His Val Leu Thr Asp Ala Gln Lys Ile Leu Cys Ile Glu 260 265
270 Lys Cys Phe Thr Phe Thr Ala Ser Phe Asn Arg Pro Asn Leu Tyr Tyr
275 280 285 Glu Val Arg Gln Lys Pro Ser Asn Thr Glu Asp Phe Ile Glu
Asp Ile 290 295 300 Val Lys Leu Ile Asn Gly Arg Tyr Lys Gly Gln Ser
Gly Ile Ile Tyr 305 310 315 320 Cys Phe Ser Gln Lys Asp Ser Glu Gln
Val Thr Val Ser Leu Gln Asn 325 330 335 Leu Gly Ile His Ala Gly Ala
Tyr His Ala Asn Leu Glu Pro Glu Asp 340 345 350 Lys Thr Thr Val His
Arg Lys Trp Ser Ala Asn Glu Ile Gln Val Val 355 360 365 Val Ala Thr
Val Ala Phe Gly Met Gly Ile Asp Lys Pro Asp Val Arg 370 375 380 Phe
Val Ile His His Ser Met Ser Lys Ser Met Glu Asn Tyr Tyr Gln 385 390
395 400 Glu Ser Gly Arg Ala Gly Arg Asp Asp Met Lys Ala Asp Cys Ile
Leu 405 410 415 Tyr Tyr Gly Phe Gly Asp Ile Phe Arg Ile Ser Ser Met
Val Val Met 420 425 430 Glu Asn Val Gly Gln Gln Lys Leu Tyr Glu Met
Val Ser Tyr Cys Gln 435 440 445 Asn Ile Ser Lys Cys Arg Arg Val Leu
Met Ala Gln His Phe Asp Glu 450 455 460 Val Trp Asn Ser Glu Ala Cys
Asn Lys Met Cys Asp Asn Cys Cys Lys 465 470 475 480 Asp Ser Ala Phe
Glu Arg Lys Asn Ile Thr Glu Tyr Cys Arg Asp Leu 485 490 495 Ile Lys
Ile Leu Lys Gln Ala Glu Glu Leu Asn Glu Lys Leu Thr Pro 500 505 510
Leu Lys Leu Ile Asp Ser Trp Met Gly Lys Gly Ala Ala Lys Leu Arg 515
520 525 Val Ala Gly Val Val Ala Pro Thr Leu Pro Arg Glu Asp Leu Glu
Lys 530 535 540 Ile Ile Ala His Phe Leu Ile Gln Gln Tyr Leu Lys Glu
Asp Tyr Ser 545 550 555 560 Phe Thr Ala Tyr Ala Thr Ile Ser Tyr Leu
Lys Ile Gly Pro Lys Ala 565 570 575 Asn Leu Leu Asn Asn Glu Ala His
Ala Ile Thr Met Gln Val Thr Lys 580 585 590 Ser Thr Gln Asn Ser Phe
Arg Ala Glu Ser Ser Gln Thr Cys His Ser 595 600 605 Glu Gln Gly Asp
Lys Lys Met Glu Glu Lys Asn Ser Gly Asn Phe Gln 610 615 620 Lys Lys
Ala Ala Asn Met Leu Gln Gln Ser Gly Ser Lys Asn Thr Gly 625 630 635
640 Ala Lys Lys Arg Lys Ile Asp Asp Ala 645 3919RNAArtificial
SequenceAn artificially synthesized siRNA sequence 39uauaucucca
aagccguag 194019RNAArtificialAn artificially synthesized siRNA
sequence 40gcuugaaacu auuaacgua 194119RNAArtificialAn artificially
synthesized siRNA sequence 41uaagaccaca guucauaga
194219RNAArtificialAn artificially synthesized siRNA sequence
42guuauccauc auucaauga 194327RNAArtificialan artificially
synthesized nucleotide sequence 43ggaaaaguuc agaccacuuc agcuuga
27
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