U.S. patent application number 16/634675 was filed with the patent office on 2021-04-01 for pharmaceutical composition for preventing or treating liver cancer.
The applicant listed for this patent is LEMONEX INC.. Invention is credited to Cheolhee WON.
Application Number | 20210093654 16/634675 |
Document ID | / |
Family ID | 1000005300762 |
Filed Date | 2021-04-01 |
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United States Patent
Application |
20210093654 |
Kind Code |
A1 |
WON; Cheolhee |
April 1, 2021 |
PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING LIVER
CANCER
Abstract
The present invention provides siRNA or dsRNA, which can
effectively inhibit the expression of three highly expressed
markers in liver cancer, and a pharmaceutical composition including
the same can obtain excellent effects of preventing or treating
liver cancer through RNAi. A pharmaceutical composition for
preventing or treating liver cancer according to an embodiment of
the present invention includes at least one of siRNA which includes
a sense RNA having at least one sequence selected from the group
consisting of sequences of SEQ ID NOs: 5 to 157, and an antisense
RNA having a complementary sequence thereto and dsRNA having at
least one sequence selected from the group consisting of sequences
of SEQ ID NOs: 158 to 310.
Inventors: |
WON; Cheolhee; (Seoul,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LEMONEX INC. |
Seoul |
|
KR |
|
|
Family ID: |
1000005300762 |
Appl. No.: |
16/634675 |
Filed: |
July 30, 2018 |
PCT Filed: |
July 30, 2018 |
PCT NO: |
PCT/KR2018/008611 |
371 Date: |
January 28, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62538034 |
Jul 28, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/713 20130101;
A61P 35/00 20180101; C12N 15/113 20130101 |
International
Class: |
A61K 31/713 20060101
A61K031/713; C12N 15/113 20060101 C12N015/113; A61P 35/00 20060101
A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 30, 2018 |
KR |
10-2018-0088375 |
Claims
1. A pharmaceutical composition for preventing or treating liver
cancer, comprising at least one of the followings: siRNA which
includes a sense RNA having at least one sequence selected from the
group consisting of sequences of SEQ ID NOs: 5 to 157, and an
antisense RNA having a complementary sequence thereto; and dsRNA
having at least one sequence selected from the group consisting of
sequences of SEQ ID NOs: 158 to 310.
2. The composition according to claim 1, wherein the composition
comprises at least one of the followings: siRNA which includes a
sense RNA having at least one sequence selected from the group
consisting of sequences of SEQ ID NOs: 5 to 12, 14 to 19, 21, 23,
24, 26, 28 to 34, 35 to 37, 39 to 41, 43, 45 to 47, 49 to 53, 55 to
60, 62 to 73, 75 to 81, 84 to 87, 89 to 98, 100 to 102, 105 to 116,
118 to 128, 130 to 154, 156 to 157, and an antisense RNA having a
complementary sequence thereto; and dsRNA having at least one
sequence selected from the group consisting of sequences of SEQ ID
NOs: 158 to 165, 167 to 172, 174, 176, 177, 179, 181 to 187, 188 to
190, 192 to 194, 196, 198 to 200, 202 to 206, 208 to 213, 215 to
226, 228 to 234, 237 to 240, 242 to 251, 253 to 255, 258 to 269,
271 to 281, 283 to 307, 309 and 310.
3. The composition according to claim 1, wherein the composition
comprises at least one of the followings: siRNA which includes a
sense RNA having at least one sequence selected from the group
consisting of sequences of SEQ ID NOs: 5 to 28, and an antisense
RNA having a complementary sequence thereto; and dsRNA having at
least one sequence selected from the group consisting of sequences
of SEQ ID NOs: 158 to 181.
4. The composition according to claim 1, wherein the composition
comprises at least one of the followings: siRNA which includes a
sense RNA having at least one sequence selected from the group
consisting of sequences of SEQ ID NOs: 29 to 55, and an antisense
RNA having a complementary sequence thereto; and dsRNA having at
least one sequence selected from the group consisting of sequences
of SEQ ID NOs: 182 to 208.
5. The composition according to claim 1, wherein the composition
comprises at least one of the followings: siRNA which includes a
sense RNA having at least one sequence selected from the group
consisting of sequences of SEQ ID NOs: 56 to 120, and an antisense
RNA having a complementary sequence thereto; and dsRNA having at
least one sequence selected from the group consisting of sequences
of SEQ ID NOs: 209 to 273.
6. The composition according to claim 1, wherein the composition
comprises at least one of the followings: siRNA which includes a
sense RNA having at least one sequence selected from the group
consisting of sequences of SEQ ID NOs: 121 to 157, and an antisense
RNA having a complementary sequence thereto; and dsRNA having at
least one sequence selected from the group consisting of sequences
of SEQ ID NOs: 274 to 310.
7. The composition according to claim 1, wherein the siRNA or dsRNA
is loaded on at least one carrier selected from the group
consisting of liposomes, lipofectamines, dendrimers, micelles,
porous silica particles, amino clay, gold nanoparticles, magnetic
nanoparticles, graphene, oxidized graphene, chitosan, dextran,
pectin, manganese dioxide two-dimensional sheet, PVA, gelatin,
silica, glass particles, protamine, exosome, polyethyleneimine,
N-butyl cyanoacrylate, gel foam, ethanol, nanocrystals, nanotubes,
carbon nanoparticles, hyaluronic acid, iron oxide, polylactic acid,
polybutyl cyanoacrylate, albumin, lipid particles, polyethylene
glycol, poly-L-guluronic alginate, polyglycolic-polyacticpolyactic
acid, polydioxanone, polyglycolic acid-co-caprolactone,
polypropylene and hydrogel.
8. The composition according to claim 7, wherein the carrier is a
porous silica particle characterized in that t when a ratio of
absorbance in the following Equation 1 becomes 1/2 is 20 or more:
A.sub.t/A.sub.0 [Equation 1] wherein A.sub.0 is absorbance of the
porous silica particle measured by placing 5 ml of a suspension
including 1 mg/ml of the porous silica particle into a cylindrical
dialysis membrane having pores with a diameter of 50 kDa; 15 ml of
the same solvent as the suspension is placed outside the dialysis
membrane while being in contact with the dialysis membrane,
followed by horizontal agitation at 60 rpm and 37.degree. C. inside
and outside the dialysis membrane; and A.sub.t is absorbance of the
porous silica particle measured after t hours elapses from the
measurement of A.sub.o.
9. The composition according to claim 8, wherein the t is 40 or
more.
10. The composition according to claim 8, wherein the siRNA
includes a sense RNA having at least one sequence selected from the
group consisting of sequences of SEQ ID NOs: 28, 119 and 136, and
an antisense RNA having a complementary sequence thereto; and the
dsRNA has at least one sequence selected from the group consisting
of sequences of SEQ ID NOs: 181, 272 and 289.
11. The composition according to claim 8, wherein the porous silica
particle has a hydrophilic substituent or a hydrophobic
substituent.
12. The composition according to claim 8, wherein the porous silica
particle has at least one hydrophilic substituent selected from the
group consisting of aldehyde, keto, carbamate, sulfate, sulfonate,
amino, amine, aminoalkyl, silyl, carboxyl, sulfonic acid, thiol,
ammonium, sulfhydryl, phosphate, ester, imide, thioimide, ether,
indene, sulfonyl, methylphosphonate, polyethylene glycol,
substituted or unsubstituted C.sub.1 to C.sub.30 alkyl, substituted
or unsubstituted C3 to C30 cycloalkyl, substituted or unsubstituted
C.sub.6 to C.sub.30 aryl, and C.sub.1 to C.sub.30 ester groups.
13. The composition of claim 8, wherein the porous silica particle
is positively or negatively charged on an outer surface of the
particle or an inside of a pore thereof at neutral pH.
14. The composition of claim 8, wherein the porous silica particle
is positively charged on an outer surface of the particle or an
inside of a pore thereof at neutral pH.
15. The composition of claim 8, wherein the porous silica particle
has an average particle diameter of 100 to 400 nm and a pore
diameter of 4 to 30 nm.
Description
CROSS REFERENCE TO RELATED APPLICATIONS AND CLAIM OF PRIORITY
[0001] This application claims benefit under 35 U.S.C. 119(e), 120,
121, or 365(c), and is a National Stage Entry from International
Application No. PCT/KR2018/008611, filed on Jul. 30, 2018, which
claims priority to the benefit of U.S. Patent Application No.
62/538,034 filed in the US Patent Office on Jul. 28, 2017 and
Korean Patent Application No. 10-2018-0088375 filed in the Korean
Intellectual Property Office on Jul. 30, 2018, the entire contents
of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to a pharmaceutical
composition for preventing or treating liver cancer.
BACKGROUND ART
[0003] Hepatocellular carcinoma (HCC) is the second leading cause
of cancer-related deaths worldwide. HCC is one of few cancers that
have been recently increased in incidence.
[0004] The primary treatment of HCC is surgical resection, and most
of patients are not eligible for curative treatment at the initial
treatment stage. Resection and transdermal ablation involve a
recurrence rate of 70% after 5 years and thus are closely related
to the survival rate.
[0005] Like other cancers, HCC is also characterized by multiple
tumor progression. The damaged liver tissues in the early stage
evolve to small nodular hypercellular lesions called dysplastic
nodules (DNs). Such pre-cancerous lesion develops into small,
well-differentiated hepatocytes with an ambiguous nodular pattern
and then progresses to early hepatocellular carcinoma (eHCC), which
is defined as progressive HCC characterized by an epileptic
appearance and frequent microvascular invasion. Based on current
knowledge of an occurrence of multilevel HCC, high-critical
patients are closely followed up, and diagnostic images show that a
specific lesion with a small size and unknown cause was increased
in number. Ultrasound-guided needle biopsy is performed on such
lesions. The lesion would be subjected to treatment if it is
determined as a cancer by histological diagnosis. However, eHCC
generally exhibits minimal dysplasia and lacks clear invasive or
destructive growth. Therefore, even for hepatopathologists, it is
often difficult to distinguish recurrent nodules, precancerous
lesions and early lesions. Due to such reasons, discovery of
objective molecular markers that standardize histological diagnosis
of eHCC and induce appropriate therapy is eagerly required. In
addition, the discovery of biomarkers related to accurate HCC
diagnosis may facilitate identification of precancerous lesions
possibly progressing to HCC and to determine surgically resectable
lesions, thereby supporting a surgeon to design a surgical range in
HCC patients. Identifying additional molecular markers that predict
possible occurrence of HCC in precancerous lesions may be helpful
for identifying patients at risk for recurrence following surgical
resection.
[0006] The present study is intended to establish a gene selection
strategy to identify potential causative genes by combining
clinicopathological and gene expression data of staged
hepatocarcinoma tissues defined by hepatopathologists. As a result,
10 genes expected to be the cause of early stage HCC could be
selected.
[0007] Clinical and experimental studies have demonstrated that
barriers to procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 and
splicing factor 3b subunit 4 among the 10 presumptive driving genes
could indicate HCC in precancerous lesions, and also could diagnose
eHCC in a large scale of HCC patients, compared to glypican 3,
glutamine synthetase and heat shock protein 70 which are current
HCC diagnostic marker trio.
[0008] In vivo experiments and in vitro tumor formation analysis
demonstrated that target destruction of BANF1, PLOD3 and SF3B4
genes inhibits tumor and metastatic characteristics of HCC cells.
Excessive response of SFB4 could increase slug in p27 and HCC cells
to inhibit epithelial-mesenchymal transition (EMT), which
contributes to transformation and proliferation of malignant cells,
hence interfering with a cell cycle checkpoint and thus causing
over-activation of spliceosome. Further, it could be seen that
production of selective splicing variants inhibiting the growth of
KLF4 tumor was accelerated.
[0009] The results described above suggest that novel HCC
diagnostic markers, that is, BANF1, PLOD3 and SF3B contribute to
early malignant transformation of hepatocytes in formation of
hepatic tumor and are also targets for molecular therapy of liver
malignancy.
SUMMARY
[0010] It is an object of the present invention to provide a
pharmaceutical composition for preventing or treating liver cancer
that knockdown a specific gene highly expressed in early stage
liver cancer cells.
[0011] 1 A pharmaceutical composition for preventing or treating
liver cancer, including: siRNA which includes a sense RNA having at
least one sequence selected from the group consisting of sequences
of SEQ ID NOs: 5 to 157, and an antisense RNA having a
complementary sequence thereto; or
[0012] dsRNA having at least one sequence selected from the group
consisting of sequences of SEQ ID NOs: 158 to 310.
[0013] 2 The composition according to the above 1, wherein the
composition includes:
[0014] siRNA which includes a sense RNA having at least one
sequence selected from the group consisting of sequences of SEQ ID
NOs: 5 to 12, 14 to 19, 21, 23, 24, 26, 28 to 34, 35 to 37, 39 to
41, 43, 45 to 47, 49 to 53, 55 to 60, 62 to 73, 75 to 81, 84 to 87,
89 to 98, 100 to 102, 105 to 116, 118 to 128, 130 to 154, 156 to
157, and an antisense RNA having a complementary sequence thereto;
or
[0015] dsRNA having at least one sequence selected from the group
consisting of sequences of SEQ ID NOs: 158 to 165, 167 to 172, 174,
176, 177, 179, 181 to 187, 188 to 190, 192 to 194, 196, 198 to 200,
202 to 206, 208 to 213, 215 to 226, 228 to 234, 237 to 240, 242 to
251, 253 to 255, 258 to 269, 271 to 281, 283 to 307, 309 and
310.
[0016] 3 The composition according to the above 1, wherein the
composition includes:
[0017] siRNA which includes a sense RNA having at least one
sequence selected from the group consisting of sequences of SEQ ID
NOs: 5 to 28, and an antisense
[0018] RNA having a complementary sequence thereto; or
[0019] dsRNA having at least one sequence selected from the group
consisting of sequences of SEQ ID NOs: 158 to 181.
[0020] 4. The composition according to the above 1, wherein the
composition includes:
[0021] siRNA which includes a sense RNA having at least one
sequence selected from the group consisting of sequences of SEQ ID
NOs: 29 to 55, and an antisense RNA having a complementary sequence
thereto; or
[0022] dsRNA having at least one sequence selected from the group
consisting of sequences of SEQ ID NOs: 182 to 208.
[0023] 5. The composition according to the above 1, wherein the
composition includes:
[0024] siRNA which includes a sense RNA having at least one
sequence selected from the group consisting of sequences of SEQ ID
NOs: 56 to 120, and an antisense RNA having a complementary
sequence thereto; or
[0025] dsRNA having at least one sequence selected from the group
consisting of sequences of SEQ ID NOs: 209 to 273.
[0026] 6. The composition according to the above 1, wherein the
composition includes:
[0027] siRNA which includes a sense RNA having at least one
sequence selected from the group consisting of sequences of SEQ ID
NOs: 121 to 157, and an antisense RNA having a complementary
sequence thereto; or
[0028] dsRNA having at least one sequence selected from the group
consisting of sequences of SEQ ID NOs: 274 to 310.
[0029] 7. The composition according to any one of the above 1 to
6,
[0030] wherein the siRNA or dsRNA is loaded on at least one carrier
selected from the group consisting of liposomes, lipofectamines,
dendrimers, micelles, porous silica particles, amino clay, gold
nanoparticles, magnetic nanoparticles, graphene, oxidized graphene,
chitosan, dextran, pectin, manganese dioxide two-dimensional sheet,
PVA, gelatin, silica, glass particles, protamine, exosome,
polyethyleneimine, N-butyl cyanoacrylate, gel foam, ethanol,
nanocrystals, nanotubes, carbon nanoparticles, hyaluronic acid,
iron oxide, polylactic acid, polybutyl cyanoacrylate, albumin,
lipid particles, polyethylene glycol, poly-L-guluronic alginate,
polyglycolic-polylactic acid, polydioxanone, polyglycolic
acid-co-caprolactone, polypropylene and hydrogel.
[0031] 8. The composition according to the above 7,
[0032] wherein the carrier is a porous silica particle
characterized in that t when a ratio of absorbance in the following
Equation 1 becomes 1/2 is 20 or more:
A.sub.t/A.sub.0 [Equation 1]
[0033] (wherein A.sub.0 is absorbance of the porous silica particle
measured by placing 5 ml of a suspension including 1 mg/ml of the
porous silica particle into a cylindrical dialysis membrane having
pores with a diameter of 50 kDa,
[0034] 15 ml of the same solvent as the suspension is placed
outside the dialysis membrane while being in contact with the
dialysis membrane, followed by horizontal agitation at 60 rpm and
37.degree. C. inside and outside the dialysis membrane, and
[0035] A.sub.t is absorbance of the porous silica particle measured
after t hours elapses from the measurement of A.sub.0).
[0036] 9. The composition according to the above 8, wherein the t
is 40 or more.
[0037] 10. The composition according to the above 8,
[0038] wherein the siRNA includes a sense RNA having at least one
sequence selected from the group consisting of sequences of SEQ ID
NOs: 28, 119 and 136, and an antisense RNA having a complementary
sequence thereto, and
[0039] the dsRNA has at least one sequence selected from the group
consisting of sequences of SEQ ID NOs: 181, 272 and 289.
[0040] 11. The composition according to the above 8,
[0041] wherein the porous silica particle has a hydrophilic
substituent or a hydrophobic substituent.
[0042] 12. The composition according to the above 8,
[0043] wherein the porous silica particle has at least one
hydrophilic substituent selected from the group consisting of
aldehyde, keto, carbamate, sulfate, sulfonate, amino, amine,
aminoalkyl, silyl, carboxyl, sulfonic acid, thiol, ammonium,
sulfhydryl, phosphate, ester, imide, thioimide, ether, indene,
sulfonyl, methylphosphonate, polyethylene glycol, substituted or
unsubstituted C.sub.1 to C.sub.30 alkyl, substituted or
unsubstituted C.sub.3 to C.sub.30 cycloalkyl, substituted or
unsubstituted C.sub.6 to C.sub.30 aryl, and C.sub.1 to C.sub.30
ester groups.
[0044] 13. The composition of claim 8,
[0045] wherein the porous silica particle is positively or
negatively charged on an outer surface of the particle or an inside
of a pore thereof at neutral pH.
[0046] 14. The composition of claim 8,
[0047] wherein the porous silica particle is positively charged on
an outer surface of the particle or an inside of a pore thereof at
neutral pH.
[0048] 15. The composition of claim 8,
[0049] wherein the porous silica particle has an average particle
diameter of 100 to 400 nm and a pore diameter of 4 to 30 nm.
[0050] The pharmaceutical composition of the present invention
provides preventive and therapeutic effects of liver cancer by
specifically knocking down the genes expressed in early stage liver
cancer cells, so as to prevent the development of liver cancer and
inhibit the metastasis and proliferation of liver cancer cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIG. 1 is views illustrating results of measuring expression
levels of indicator factors ("markers") corresponding to siRNAs by
Western blotting, respectively, after in vitro transfection of
siRNAs to Hepa-1c1c7 and SNU-449 cell lines in EXAMPLE 1 by methods
described in EXAMPLE 1-2 or 1-8, wherein each siRNA includes a
sense RNA having a sequence in Table 11 and an antisense RNA having
a complementary sequence to that of the sense RNA.
[0052] FIG. 2 is views illustrating results of analyzing migration
and invasion of markers corresponding to siRNAs by a method
described in EXAMPLE 1-5 and analyzing scratch wound healing
ability of the same by a method described in EXAMPLE 1-6,
respectively, after in vitro transfection of siRNAs to SNU-449 cell
line in EXAMPLE 1-1 by a method described in EXAMPLE 1-2, wherein
each siRNA includes a sense RNA having a sequence in Table 12 and
an antisense RNA having a complementary sequence to the sequence of
the sense RNA.
[0053] A of FIG. 3 is views illustrating results of analyzing
expression levels of markers corresponding to siRNAs and EMT
regulatory proteins by a method described in EXAMPLE 1-9,
respectively, after in vitro transfection of siRNAs to SNU-449 cell
line in EXAMPLE 1 by the method described in EXAMPLE 1-2, wherein
each siRNA includes a sense RNA having a sequence in Table 12 and
an antisense RNA having a complementary sequence to the sequence of
the sense RNA; and B of FIG. 3 is views illustrating the analyzed
results of hepatic tumor sizes and survival rates of mice, after
subcutaneous injection of transfected cells in the above (A) into
athymic nude mice.
[0054] A of FIG. 4 is views illustrating processes of in vivo
transfection of siRNAs by a method described in EXAMPLE 1-8,
ultrasonic images and the number of tumors over time, wherein each
siRNA includes a sense RNA having a sequence in Table 13 and an
antisense RNA having a complementary sequence to the sequence of
the sense RNA; and B of FIG. 4 is a view illustrating results of
analyzing expression inhibitory levels of siRNAs loaded on porous
nanoparticles to indicator genes corresponding to the siRNAs by the
method described in EXAMPLE 1-9.
[0055] FIG. 5 is micrographs illustrating porous silica
particles.
[0056] FIG. 6 is micrographs illustrating porous silica
particles.
[0057] FIG. 7 is micrographs illustrating small pore particles
during production of the porous silica particles.
[0058] FIG. 8 is micrographs illustrating small pore particles.
[0059] FIG. 9 is micrographs demonstrating biodegradability of
porous silica particles.
[0060] FIG. 10 is a view illustrating a tube having a cylindrical
dialysis membrane.
[0061] FIG. 11 is a graph illustrating results of absorbance
reduction of the porous silica particles over time.
[0062] FIG. 12 is a graph and a table illustrating results of
absorbance reduction by particle diameter of the porous silica
particles over time.
[0063] FIG. 13 is a graph and a table illustrating results of
absorbance reduction by pore diameter of the porous silica
particles over time.
[0064] FIG. 14 is a graph illustrating results of absorbance
reduction of the porous silica particles by pH in environments over
time.
[0065] FIG. 15 is a graph illustrating results of absorbance
reduction of the porous silica particles over time.
[0066] FIG. 16 is a view illustrating a tube for identifying
release of bioactive material from the porous silica particles.
[0067] FIG. 17 is a graph illustrating release of bioactive
material loaded on the porous silica particles over time.
[0068] FIG. 18 is micrographs demonstrating siRNA release in mice
by loading the porous silica particles with siRNA.
DETAILED DESCRIPTION
[0069] The terms used in the present invention are defined as
follows.
[0070] "siRNA" refers to a nucleic acid molecule capable of
mediating RNA interference or gene silencing. siRNA can inhibit
expression of a target gene and may be provided as an efficient
gene knockdown method or as a gene therapy method. The siRNA
molecule may have a structure in which a sense strand (a sequence
corresponding to mRNA sequence of the target gene) and an antisense
strand (a complementary sequence to mRNA sequence of the target
gene) are located on sides opposite to each other to form a double
chain. In addition, siRNA molecules may have a single stranded
structure with self-complementary sense and antisense strands. The
siRNA is not limited to a complete pair of double-stranded RNA
modalities that are paired with each other, but may also include
modalities that are not paired due to a mismatch (the corresponding
base is not complementary), a bulge (no base corresponding to one
chain) or the like. The siRNA terminal structure may include blunt
or cohesive terminals as long as it can inhibit expression of a
target gene by RNA interference (RNAi) effects. The cohesive
terminal structure may be a 3'-terminal protruding structure and
5'-terminal protruding structure. Further, siRNA molecules may have
a form in which a short nucleotide sequence (e.g., about 5-15 nt)
is inserted between the self-complementary sense and the antisense
strands. In this case, the siRNA molecule formed by expression of
the nucleotide sequence may form a hairpin structure by
intramolecular hybridization, which in turn forms a stem-and-loop
structure on the whole. This stem-and-loop structure may be
processed in vitro or in vivo to produce siRNA molecules capable of
mediating RNAi.
[0071] "dsRNA" refers to a siRNA precursor molecule that meets a
RISC complex containing DICER enzyme (Ribonuclease III) of a target
cell and is cleaved into siRNA. In this process, RNAi is generated.
dsRNA has a longer sequence by several nucleotides than siRNA and
may have a structure wherein a sense strand (a sequence
corresponding to mRNA sequence of the target gene) and an antisense
strand (a sequence complementary to mRNA sequence of the target
gene) are located on sides opposite to each other to form a double
chain.
[0072] "Nucleic acid" may include any DNA or RNA, for example,
chromosomes, mitochondria, viruses and/or bacterial nucleic acids
present in a tissue sample. One or both strands of a
double-stranded nucleic acid molecule may be included, and further
any fragment or portion of an intact nucleic acid molecule.
[0073] "Gene" refers to any nucleic acid sequence or portion
thereof that has a functional role at the time of protein coding or
transcription, or in the control of other gene expressions. The
gene may include only a portion of the nucleic acid encoding or
expressing any nucleic acid or protein that encodes the functional
protein. The nucleic acid sequence may involve gene abnormality in
exon, intron, an initiation or termination region, a promoter
sequence, another regulatory sequence or a specific sequence
adjacent to the gene.
[0074] As used herein, the term "gene expression" generally refers
to a cellular process in which a polypeptide having biological
activity is produced from a DNA sequence and exhibits biological
activity in the cell. In this sense, the gene expression may
include not only transcription and translation processes but also
post-transcription and post-translation processes that may affect
the biological activity of the gene or gene product. Such processes
may include polypeptide synthesis, transport and post-translational
modification as well as RNA synthesis, processing and transport,
but it is not limited thereto. In the case of a gene which does not
encoding a protein product such as siRNA gene, the term "gene
expression" refers to a process in which a precursor siRNA is
produced from a gene. Normally, the above process is referred to as
transcription, although a transcription product of siRNA gene is
not translated to produce a protein, unlike the transcription
induced by RNA polymerase II on a protein coding gene.
Nevertheless, the formation of mature siRNAs from siRNA genes may
be encompassed by the term "gene expression" as that term is used
herein.
[0075] As used herein, the term "target gene" refers to a gene
targeted for modulation using the method and composition in the
subject matters disclosed herein. Therefore, the target gene
includes a nucleic acid sequence with a specific expression level
down-regulated by siRNA into mRNA or a polypeptide level.
Similarly, the term "target RNA" or "target mRNA" refers to a
transcript of the target gene that is bound to siRNA and induces
modulation of expression in the target gene.
[0076] As used herein, the term "transcription" refers to a
cellular process involving interaction between an
expression-inducible gene, which is RNA of structural information
present in a coding sequence of the gene, and RNA polymerase.
[0077] As used herein, the expression "down-regulation" refers to
considerably decreasing the expression of a specific gene into mRNA
or the expression level into a protein by gene transcription or
gene translation in activated cells, as compared to normal tissue
cells.
[0078] As used herein, the term "treatment" means an approach to
obtain beneficial or desired clinical results. For the purposes of
the present invention, the beneficial or desired clinical results
may include, without limitation, alleviation of symptoms, reduction
in an extent of disease, stabilization (i.e., not worsening) of
disease state, delayed progression of disease or reduction in
progress rate of disease, improvement, temporary mitigation and
alleviation of disease state (partially or wholly), whether or not
it is detectable. Further, the term "treatment" may also refer to
increasing the survival rate compared to that expected survival
when untreated. The treatment refers to both therapeutic treatment
and prophylactic or preventative measures. Such treatments may
include treatments required for disorders that have already
occurred as well as disorders to be prevented.
[0079] As used herein, the term "prevention" means any action to
inhibit or delay development of a related disease. It will be
apparent to those skilled in the art that the composition mentioned
herein may prevent initial symptoms, or related diseases in a case
of administering before symptoms appear.
[0080] Hereinafter, the present invention will be described in
detail.
[0081] The present invention provides a pharmaceutical composition
for preventing or treating liver cancer, that is, hepatocellular
carcinoma (HCC), which includes: siRNA which includes a sense RNA
having at least one sequence selected from the group consisting of
sequences of SEQ ID NOs: 5 to 157, as well as antisense RNA having
a complementary sequence thereto; or
[0082] dsRNA having at least one sequence selected from the group
consisting of sequences of SEQ ID NOs: 158 to 310.
[0083] The siRNA or dsRNA of the present invention may be derived
from animals including humans, such as monkeys, pigs, horses, cows,
sheep, dogs, cats, mice, rabbits, and the like, and is preferably
derived from humans.
[0084] The siRNA or dsRNA of the present invention may be modified
by deletion, substitution or insertion of a functional equivalent
of nucleic acid molecule constituting the siRNA or dsRNA, for
example, a part of the base sequence in the siRNA or dsRNA of the
present invention, however, may also be a concept including
variants which are capable of functionally performing the same
action as the siRNA or dsRNA of the present invention.
[0085] The siRNA or dsRNA of the present invention may be isolated
or prepared using standard molecular biology techniques, such as
chemical synthesis methods or recombinant methods, or may include
commercially available ones. Further, the composition of the
present invention may include not only siRNA or dsRNA itself of the
present invention but also other substances, for example,
compounds, natural products, novel proteins, etc. which are capable
of increasing an expression rate of the siRNA or dsRNA of the
present invention in cells.
[0086] Meanwhile, the siRNA or dsRNA of the present invention may
be provided in a state of being included in a vector for
intracellular expression.
[0087] The siRNA or dsRNA of the present invention may be
introduced into cells by various transformation techniques such as
a complex of DNA and DEAE-dextran, a complex of DNA and a nuclear
protein, a complex of DNA and lipid and the like. To this end, the
siRNA or dsRNA of the present invention may be provided in a form
of being contained in a carrier enabling efficient introduction
into a cell. The carrier is preferably a vector, and both viral
vectors and non-viral vectors are usable. As the viral vector may
include lentivirus, retrovirus, adenovirus, herpes virus and avipox
virus vector, etc., preferably, is a lentivirus vector, but it is
not limited thereto. Lentivirus is one type of retrovirus
characterized by infecting a non-mitotic cell as well as a mitotic
cell due to nucleophilic property of pre-integrated complex (a
virus "shell") that allows active incorporation into nucleopores or
a complete nuclear membrane.
[0088] In addition, the vector containing siRNA or dsRNA of the
present invention preferably includes a selection marker. The
"selection marker" is intended to facilitate selection or screening
of cells into which siRNA or dsRNA of the present invention has
been introduced. The selection marker used in the vector is not
particularly limited as long as it is a gene capable of easily
detecting or determining whether or not the vector was introduced.
However, examples thereof may typically include markers endowing
selectable phenotypes such as drug resistance, auxotrophy,
tolerance to cytotoxic agents, expression of surface protein, etc.,
in particular, green fluorescent protein (GFP), puromycin, neomycin
(Neo), hygromycin (Hyg), histidinol dehydrogenase gene (hisD),
guanine phosphoribosyltransferase (Gpt) or the like. Preferably,
the green fluorescent protein (GFP) and puromycin markers are
used.
[0089] The composition of the present invention may include: siRNA
which includes a sense RNA having at least one sequence selected
from the group consisting of sequences of SEQ ID NOs: 5 to 28 in
Table 1 below, and an antisense RNA having a complementary sequence
thereto; and dsRNA having at least one sequence selected from the
group consisting of sequences of SEQ ID NOs: 158 to 181 in Table 1
below.
[0090] In this regard, siRNA which includes a sense RNA having at
least one sequence selected from the group consisting of sequences
of SEQ ID NOs: 5 to 28 in Table 1 below, and an antisense RNA
having a complementary sequence thereto; or dsRNA having at least
one sequence selected from the group consisting of sequences of SEQ
ID NOs: 158 to 181 in Table 1 below, may target variant 1 sequence
(SEQ ID NO: 1) of human BANF1 gene to inhibit expression of the
human BANF1 gene variant 1 through RNAi, thereby achieving effects
of preventing or treating HCC.
TABLE-US-00001 TABLE 11 Target sequence 5: GC content: 45.0% 5'-CAA
GAA GCT GGA SEQ ID NO: 5 GGA AAG-3' (SEQ ID Sense strand: NO: 482)
5'-CAA GAA GCU GGA GGA AAG (Position in UU-3' gene sequence: 601)
SEQ ID NO: 326 Antisense strand: 5'- CUU UCC UCC AGC UUC UUG UU-3'
SEQ ID NO: 158 dsRNA: 5'- CAA GAA GCU GGA GGA AAG UU UCU AAA G-3'
Target sequence 6: GC content: 40.9% 5'-GAA AGA TGA AGA SEQ ID NO:
6 CCT CTT CC-3' (SEQ Sense strand: ID NO: 483) 5'-GAA AGA UGA AGA
CCU CUU (Position in CCU U-3' gene sequence: 667) SEQ ID NO: 327
Antisense strand: 5'-GGA AGA GGU CUU CAU CU UCU U-3' SEQ ID NO: 159
dsRNA: 5'- GAA AGA UGA AGA CCU CUU CCU UUC UAA AG-3' Target
sequence 7: GC content: 40.9% 5'-GGA ATG GCT GAA SEQ ID NO: 7 AGA
CAC TT-3' (SEQ Sense strand: ID NO: 484) 5'-GGA AUG GCU GAA AGA CAC
(Position UUU U-3' in gene SEQ ID NO: 328 sequence: 688) Antisense
strand: 5'-AAG UGU CUU UCA GCC AUU CCU U-3' SEQ ID NO: 160 dsRNA:
5'- GGA AUG GCU GAA AGA CAC UUU UUC UAA AG-3' Target sequence 8: GC
content: 55.0% 5'-CCA GTG TTC CCA SEQ ID NO: 8 GTT CCC-3' (SEQ ID
Sense strand: NO: 485) 5'-CCA GUG UUC CCA GUU CCC (Position in
UU-3' gene sequence: 1) SEQ ID NO: 329 Antisense strand: 5'-GGG AAC
UGG GAA CAC UGG UU-3' SEQ ID NO: 161 dsRNA: 5'-CCA GUG UUC CCA GUU
CCC UU UCU AAA G-3' Target sequence 9: GC content: 55.0% 5'-CCA GTC
CAA CTG SEQ ID NO: 9 CGA GGA-3' (SEQ ID Sense strand: NO: 486) 5'-
CCA GUC CAA CUG CGA GGA (Position in UU-3' gene sequence: 19) SEQ
ID NO: 330 Antisense strand: 5'-UCCUCGCAGUUGGACUGG UU-3' SEQ ID NO:
162 dsRNA: 5'- CCAGUCCAACUGCGAGGA UU UCU AAA G-3' Target sequence
10: GC content: 50.0% 5'-CGA CGT GAG TCT SEQ ID NO: 10 GAG TCT-3'
(SEQ ID Sense strand: NO: 487) 5'- CGA CGU GAG UCU GAG (Position in
UCU UU-3' gene sequence: 41) SEQ ID NO: 331 Antisense strand: 5'-
AGACUCAGACUCACGUCG UU-3' SEQ ID NO: 163 dsRNA: 5'-
CGACGUGAGUCUGAGUCU UU UCU AAA G-3' Target sequence 11: GC content:
40.0% 5'-GTC CGT CTT CTA SEQ ID NO: 11 ACT CTT-3' (SEQ ID Sense
strand: NO: 488) 5'- GUC CGU CUU CUA ACU (Position in CUU UU-3'
gene sequence: 116) SEQ ID NO: 332 Antisense strand: 5'-
AAGAGUUAGAAGACGGAC UU-3' SEQ ID NO: 164 dsRNA: 5'-
GUCCGUCUUCUAACUCUU UU UCU AAA G-3' Target sequence 12: GC content:
45.0% 5'-CGT CAA GCC TAA SEQ ID NO: 12 GTC CTT-3' (SEQ ID Sense
strand: NO: 489) 5'- CGUCAAGCCUAAGUCCUU (Position in UU-3' gene
sequence: 149) SEQ ID NO: 333 Antisense strand: 5'-
AAGGACUUAGGCUUGACG UU-3' SEQ ID NO: 165 dsRNA: 5'-
CGUCAAGCCUAAGUCCUU UU UCU AAA G-3' Target sequence 13: GC content:
45.0% 5'-GCA GAG AAA GGA SEQ ID NO: 13 AGT CCT-3' (SEQ ID Sense
strand: NO: 490) 5'- GCAGAGAAAGGAAGUCCU (Position in UU-3' gene
sequence: 185) SEQ ID NO: 334 Antisense strand: 5'-
AGGACUUCCUUUCUCUGC UU-3' SEQ ID NO: 166 dsRNA: 5'-
GCAGAGAAAGGAAGUCCU UU UCU AAA G-3' Target sequence 14: GC content:
50.0% 5'-CGA GAA GCG AGA SEQ ID NO: 14 CCT TAG-3' (SEQ ID Sense
strand: NO: 491) (Position in 5'- CGAGAAGCGAGACCUUAG gene sequence:
234) UU-3' SEQ ID NO: 335 Antisense strand: 5'-CUAAGGUCUCGCUUCUCG
UU-3' SEQ ID NO: 167 dsRNA: 5'- CGAGAAGCGAGACCUUAG UU UCU AAA G-3'
Target sequence 15: GC content: 40.0% 5'-CCT CAA CTC TAT SEQ ID NO:
15 AGC TCT-3' (SEQ ID Sense strand: NO: 492) (Position in 5'-
CCUCAACUCUAUAGCUCU gene sequence: 319) UU-3' SEQ ID NO: 336
Antisense strand: 5'-AGAGCUAUAGAGUUGAGG UU-3' SEQ ID NO: 168 dsRNA:
5'- CCUCAACUCUAUAGCUCU UU UCU AAA G-3' Target sequence 16: GC
content: 45.0% 5'-CTA GTG GCT TGA SEQ ID NO: 16 GGT ATC-3' (SEQ ID
Sense strand: NO: 493) 5'- CUAGUGGCUUGAGGUAUC (Position in UU-3'
gene sequence: 423) SEQ ID NO: 337 Antisense strand: 5'-
GAUACCUCAAGCCACUAG UU-3' SEQ ID NO: 169 dsRNA: 5'-
CUAGUGGCUUGAGGUAUC UU UCU AAA G-3' Target sequence 17: GC content:
40.0% 5'-GGA TTA AGC CTG SEQ ID NO: 17 ATC A AG-3' (SEQ ID Sense
strand: NO: 494) 5'- GGAUUAAGCCUGAUCAAG (Position in UU-3' gene
sequence: 491) SEQ ID NO. 338 Antisense strand:
5'-CUUGAUCAGGCUUAAUCC UU-3' SEQ ID NO: 170 dsRNA: 5'-
GGAUUAAGCCUGAUCAAG UU UCU AAA G-3' Target sequence 18: GC content:
50.0% 5'-GAC TGC TTC GGA SEQ ID NO: 18 TGC CTT-3' (SEQ ID Sense
strand: NO: 495) 5'- GACUGCUUCGGAUGCCUU (Position in UU-3' gene
sequence: 734) SEQ ID NO: 339 Antisense strand:
5'-AAGGCAUCCGAAGCAGUC UU-3' SEQ ID NO: 171 dsRNA: 5'-
GACUGCUUCGGAUGCCUU UU UCU AAA G-3' Target sequence 19: GC content:
45.0% 5'-CCT TCT TGT GAT SEQ ID NO: 19 GCT CTC-3' (SEQ ID Sense
strand: NO: 496) 5'- CCUUCUUGUGAUGCUCUC (Position in UU-3' gene
sequence: 768) SEQ ID NO: 340 Antisense strand: 5'-
GAGAGCAUCACAAGAAGG UU-3' SEQ ID NO: 172 dsRNA: 5'-
CCUUCUUGUGAUGCUCUC UU UCU AAA G-3' Target sequence 20: GC content:
45.0% 5'-CCT CAT CCA GAG SEQ ID NO: 20 TTT GCA-3' (SEQ ID Sense
strand: NO: 497) 5'- CCUCAUCCAGAGUUUGCA (Position in UU-3' gene
sequence: 808) SEQ ID NO: 341 Antisense strand:
5'-UGCAAACUCUGGAUGAGG UU-3' SEQ ID NO: 173 dsRNA: 5'-
CCUCAUCCAGAGUUUGCA UU UCU AAA G-3' Target sequence 21: GC content:
50.0% 5'-CCT GTC CTC TAC SEQ ID NO: 21 GAA GGA-3' (SEQ ID Sense
strand: NO: 498) 5'- CCUGUCCUCUACGAAGGA (Position in UU-3' gene
sequence: 845) SEQ ID NO: 342 Antisense strand: 5'-
UCCUUCGUAGAGGACAGG UU-3' SEQ ID NO: 174 dsRNA: 5'-
CCUGUCCUCUACGAAGGA UU UCU AAA G-3' Target sequence 22: GC content:
36.36% 5'-GAT TGC TAT TGT SEQ ID NO: 22 CGT ACT CA-3' Sense strand:
(SEQ ID NO: 499) 5'-GAUUGCUAUUGUCGUACUCA UU-3' (Position in SEQ ID
NO: 343 gene sequence: 866) Antisense strand: 5'-
UGAGUACGACAAUAGCAAUC UU-3' SEQ ID NO: 175 dsRNA: 5'- sense UU UCU
AAA G-3' Target sequence 23: GC content: 45.0% 5'-GGA TTC TCG CTC
SEQ ID NO: 23 TTG CAT-3' (SEQ ID Sense strand:
NO: 500) 5'- GGAUUCUCGCUCUUGCAU (Position in UU-3' gene sequence:
947) SEQ ID NO: 344 Antisense strand: 5'-AUGCAAGAGCGAGAAUCC UU-3'
SEQ ID NO: 176 dsRNA: 5'- GGAUUCUCGCUCUUGCAU UU UCU AAA G-3' Target
sequence 24: GC content: 45.0% 5'-GGT GAC AGT TAC SEQ ID NO: 24 CAG
CTT-3' (SEQ ID Sense strand: NO: 501) 5'- GGUGACAGUUACCAGCUU
(Position in UU-3' gene sequence: 999) SEQ ID NO: 345 Antisense
strand: 5'-AAGCUGGUAACUGUCACC UU-3' SEQ ID NO: 177 dsRNA: 5'-
GGUGACAGUUACCAGCUU UU UCU AAA G-3' Target sequence 25: GC content:
40.0% 5'-CCT CAC TTT CAA SEQ ID NO: 25 TCC GTT-3' (SEQ ID Sense
strand: NO: 502) 5'- CCUCACUUUCAAUCCGUU (Position in UU-3' gene
sequence: 1054) SEQ ID NO: 346 Antisense strand: 5'-
AACGGAUUGAAAGUGAGG UU-3' SEQ ID NO: 178 dsRNA: 5'-
CCUCACUUUCAAUCCGUU UU UCU AAA G-3' Target sequence 26: GC content:
50.0% 5'-GCA GAA CAG TCA SEQ ID NO: 26 CTG TCC-3' (SEQ ID Sense
strand: NO: 503) 5'- GCAGAACAGUCACUGUCC (Position in UU-3' gene
sequence: 1096) SEQ ID NO: 347 Antisense strand:
5'-GGACAGUGACUGUUCUGC UU-3' SEQ ID NO: 179 dsRNA: 5'-
GCAGAACAGUCACUGUCC UU UCU AAA G-3' Target sequence 27: GC content:
36.36% 5'-GAT CAA TAA AGT SEQ ID NO: 27 CAG TGG CT-3' (SEQ Sense
strand: ID NO: 504) 5'-GAUCAAUAAAGUCAGUGGCU UU-3' (Position in SEQ
ID NO: 348 gene sequence: 1128) Antisense strand:
5'-AGCCACUGACUUUAUUGAUC UU-3' SEQ ID NO: 180 dsRNA: 5'-
GAUCAAUAAAGUCAGUGGCU UU UCU AAA G-3' Target sequence 28: GC
content: 47.83% 5'-AAG AAG CTG GAG SEQ ID NO: 28 GAA AGG GGT -3'
Sense strand: (SEQ IDNQ 505) 5'-AAGAAGCUGGAGGAAAGGGGU UU-3' SEQ ID
NO: 349 Antisense strand: 5'-CCCCUUUCCUCCGCUUCUU UU-3' SEQ ID NO:
181 dsRNA: 5'- AAGAAGCUGGAGGAAAGGGGU UU UCU AAA G-3'
[0091] The composition of the present invention may include: siRNA
which includes a sense RNA having at least one sequence selected
from the group consisting of sequences of SEQ ID NOs: 29 to 55 in
Table 2 below, and an antisense RNA having a complementary sequence
thereto; or dsRNA having at least one sequence selected from the
group consisting of sequences of SEQ ID NOs: 182 to 208 in Table 2
below.
[0092] In this regard, siRNA which includes a sense RNA having at
least one sequence selected from the group consisting of sequences
of SEQ ID NOs: 29 to 55 in Table 2 below, and an antisense RNA
having a complementary sequence thereto; or dsRNA having at least
one sequence selected from the group consisting of sequences of SEQ
ID NOs: 182 to 208 in Table 2 below, may target variant 2 sequence
(SEQ ID NO: 2) of human BANF1 gene to inhibit expression of the
human BANF1 gene variant 2 through RNAi, thereby achieving effects
of preventing or treating HCC.
TABLE-US-00002 TABLE 2 Target sequence 29: GC content: 40.9% 5'-ATG
AC A ACC TCC SEQ ID NO: 29 CAA AAG CA-3' (SEQ Sense strand: ID NO:
506) 5'-AUGACAACCUCCCAAAAGCA (Position in UU-3' gene sequence: 452)
SEQ ID NO: 350 Antisense strand: 5'-UGCUUUUGGGAGGUUGUCAU UU-3' SEQ
ID NO: 182 dsRNA: 5'-AUGACAACCUCCCAAAAGCA UU UCU AAA G-3' Target
sequence 30: GC content: 40.9% 5'-CCG AGA CTT CGT SEQ ID NO: 30 GGC
AGA-3' (SEQ ID Sense strand: NO: 507) 5'- CCGAGACUUCGUGGCAGA
(Position in UU-3' gene sequence: 472) SEQ ID NO: 351 Antisense
strand: 5'-UCUGCCACGAAGUCUCGG UU-3' SEQ ID NO: 183 dsRNA 5'-
CCGAGACUUCGUGGCAGA UU UCU AAA G-3' Target sequence 31: GC content:
52.94% 5'-AGC CTG GCT GGG SEQ ID NO: 31 ATT-3' (SEQ ID NO: Sense
strand: 508) (Position 5'- AGCCUGGCUGGGAUU in gene UU-3' sequence:
515) SEQ ID NO: 352 Antisense strand: 5'- AAUCCCAGCCAGGCU UU-3' SEQ
ID NO: 184 dsRNA: 5'- AGCCUGGCUGGGAUU UU UCU AAA G-3' Target
sequence 32:5'- GC content: 42.1% CAA GAA GCT GGA SEQ ID NO: 32 GGA
AA-3' Sense strand: (SEQ ID NO: 509) 5'- CAAGAAGCUGGAGGAAA
(Position UU-3' in gene SEQ ID NO: 353 sequence: 544) Antisense
strand: 5'- UUUCCUCCAGCUUCUUG UU-3' SEQ ID NO: 185 dsRNA: 5'-
CAAGAAGCUGGAGGAAA UU UCU AAA G-3' Target sequence 33: GC content:
40.9% 5'-CCA GTT TCT GGT SEQ ID NO: 33 GCT AAA GA-3' (SEQ Sense
strand: ID NO: 510) 5'-CCAGUUUCUGGUGCUAAAGA (Position in UU-3' gene
sequence: 592) SEQ ID NO: 354 Antisense strand:
5'-UCUUUAGCACCAGAAACUGG UU-3' SEQ ID NO: 186 dsRNA: 5'-
CCAGUUUCUGGUGCUAAAGA UU UCU AAA G-3' Target sequence 34: GC
content: 40.0% 5'-AAG ATG AAG ACC SEQ ID NO: 34 TCT TCC-3' (SEQ ID
Sense strand: NO: 511) 5'- AAGAUGAAGACCUCUUCC (Position in UU-3'
gene sequence: 612) SEQ ID NO: 355 Antisense strand:
5'GGAAGAGGUCUUCAUCUU UU-3' SEQ ID NO 187 dsRNA: 5'-
AAGAUGAAGACCUCUUCC UU UCU AAA G-3' Target sequence 35: GC content:
52.38% 5'-GGA CTG CTT CGG SEQ ID NO: 35 ATG CCT T-3' (SEQ ID Sense
strand:5'- NO: 512) GGACUGCUUCGGAUGCCUU UU-3' (Position in SEQ ID
NO: 356 gene sequence: 676) Antisense strand:
5'-AAGGCAUCCGAAGCAGUCC UU-3' SEQ ID NO: 188 dsRNA: 5'-
GGACUGCUUCGGAUGCCUU UU UCU AAA G-3' Target sequence 36: GC content:
52.38% 5'-AGT GGT GCG ACG SEQ ID NO: 36 CCT TCT T-3' (SEQ ID Sense
strand: NO: 513) 5'-AGUGGUGCGACGCCUUCUU (Position in UU-3' gene
sequence: 698) SEQ ID NO: 357 Antisense strand:
5'-AAGAAGGCGUCGCACCACU UU-3' SEQ ID NO: 189 dsRNA: 5'-
AGUGGUGCGACGCCUUCUU UU UU UCU AAA G-3' Target sequence 37: GC
content: 52.38% 5'-CTC TCT GGG AAG SEQ ID NO: 37 CTC TCA AT-3'
Sense strand: (SEQ ID NO: 514) 5'-AGUGGUGCGACGCCUUCUU (Position in
UU-3' gene sequence: 724) SEQ ID NO: 358 Antisense strand:
5'-AAGAAGGCGUCGCACCACU UU-3' SEQ ID NO: 190 dsRNA: 5'-
AGUGGUGCGACGCCUUCUU UU UCU AAA G-3' Target sequence 38:5'- GC
content: 40.9% TTG CTA TTG TCG SEQ ID NO: 38 TAC TCA CC-3' Sense
strand: (SEQ ID 5'-UUGCUAUUGUCGUACUCACC NO: 515) UU-3' (Position in
SEQ ID NO: 359 gene sequence: 811) Antisense strand:
5'-GGUGAGUACGACAAUAGCAA UU-3' SEQ ID NO: 191 dsRNA: 5'-
UUGCUAUUGUCGUACUCACC UU UCU AAA G-3' Target sequence 39: GC
content: 45.0% 5'-GAT TCT CGC TCT SEQ ID NO: 39 TGC ATG-3' (SEQ ID
Sense strand: NO: 516) 5'- GAUUCUCGCUCUUGCAUG (Position in UU-3'
gene sequence: 891) SEQ ID NO: 360 Antisense strand:
5'-CAUGCAAGAGCGAGAAUC UU-3' SEQ ID NO: 192 dsRNA: 5'-
GAUUCUCGCUCUUGCAUG UU UCU AAA G-3' Target sequence 40: GC content:
45.45% 5'-CAG TTC CCT GGT SEQ ID NO: 40 GAC AGT TA-3' (SEQ Sense
strand: ID NO: 517) 5'-CAGUUCCCUGGUGACAGUUA (Position in UU-3' gene
sequence: 933) SEQ ID NO: 361 Antisense strand:5'-
UAACUGUCACCAGGGAACUG UU-3' SEQ ID NO: 193 dsRNA: 5'-
CAGUUCCCUGGUGACAGUUA UU UCU AAA G-3' Target sequence 41: GC
content: 45.0% 5'-CCA GCT TTC CTG SEQ ID NO: 41 AAT GGA-3' (SEQ ID
Sense strand: NO: 518) 5'- CCAGCUUUCCUGAAUGGA (Position in UU-3'
gene sequence: 953) SEQ ID NO: 362 Antisense strand: 5'-
UCCAUUCAGGAAAGCUGG UU-3' SEQ ID NO: 194 dsRNA: 5'-
CCAGCUUUCCUGAAUGGA UU UCU AAA G-3' Target sequence 42: GC content:
36.36% 5'-CTC ACT TTC AAT SEQ ID NO: 42 CCG TTT GA-3' (SEQ ID Sense
strand: NO: 519) 5'-CUCACUUUCAAUCCGUUUGA (Position in UU-3' gene
sequence: 998) SEQ ID NO: 363 Antisense strand:
5'-UCAAACGGAUUGAAAGGAG UU-3' SEQ ID NO: 195 dsRNA: 5'-
CUCACUUUCAAUCCGUUUGA UU UCU AAA G-3' Target sequence 43: GC
content: 45.45% 5'-CAG AAC AGT CAC SEQ ID NO: 43 TGT CCT TG-3' (SEQ
ID Sense strand: NO: 520) 5'-CAGAACAGUCACUGUCCUUG (Position in
UU-3' gene sequence: 1039) SEQ ID NO: 364 Antisense strand:
5'-CAAGGACAGUGACUGUUCUG UU-3' SEQ ID NO 196 dsRNA: 5'-
CAGAACAGUCACUGUCCUUG UU UCU AAA G-3' Target sequence 44: GC
content: 55.0% 5'-CAC CAG TCC AAC SEQ ID NO: 44 TGC GAG-3' (SEQ ID
Sense strand: NO: 521) 5'- CACCAGUCCAACUGCGAG (Position in UU-3'
gene sequence: 8) SEQ ID NO: 365 Antisense strand:
5'-CUCGCAGUUGGACUGGUG UU-3' SEQ ID NO: 197 dsRNA: 5'-
CACCAGUCCAACUGCGAG UU UCU AAA G-3' Target sequence 45:5'- GC
content: 50.0% TGC GAC GTG AGT SEQ ID NO: 45 CTG AGT CT-3' (SEQ ID
Sense strand: NO: 522) (Position in 5'-UGCGACGUGAGUCUGAGUCU gene
sequence: 39) UU-3' SEQ ID NO: 366 Antisense strand:
5'-AGACUCAGACUCACGUCGCA UU-3' SEQ ID NO: 198 dsRNA: 5'-
UGCGACGUGAGUCUGAGUCU UU UCU AAA G-3' Target sequence 46:5'- GC
content: 45.0% CCT CCG AAA ACC SEQ ID NO: 46 GTA CTT-3' (SEQ ID
Sense strand: NO: 523) (Position in 5'- CCUCCGAAAACCGUACUU gene
sequence: 63) UU-3' SEQ ID NO: 367 Antisense strand:
5'-AAGUACGGUUUUCGGAGG UU-3' SEQ ID NO: 199 dsRNA: 5'-
CCUCCGAAAACCGUACUU UU UCU AAA G-3' Target sequence 47: GC content:
45.45%
5'-CCT TGT CCG TCT TCT SEQ ID NO: 47 AAC TC-3' (SEQ ID NO: Sense
strand: 524) 5'-CCUUGUCCGUCUUCUAACUC (Position in gene UU-3'
sequence: 113) SEQ ID NO: 368 Antisense strand:
5'-GAGUUAGAAGACGGACAAGG UU-3' SEQ ID NO: 200 dsRNA: 5'-
CCUUGUCCGUCUUCUAACUC UU UCU AAA G-3' Target sequence 48: GC
content: 52.38% 5'-CCA GGT CCG TCA SEQ ID NO: 48 AGC CTA A-3' (SEQ
ID Sense strand: NO: 525) 5'-CCAGGUCCGUCAAGCCUAA (Position in UU-3'
gene sequence: 142) SEQ ID NO. 369 Antisense strand:
5'-UUAGGCUUGACGGACCUGG UU-3' SEQ ID NO: 201 dsRNA: 5'-
CCAGGUCCGUCAAGCCUAA UU UCU AAA G-3' Target sequence 49: GC content:
45.0% 5'-GCA GCA GAG AAA SEQ ID NO 49 GGA AGT-3' (SEQ ID Sense
strand: NO: 526) 5'- GCAGCAGAGAAAGGAAGU (Position in UU-3' gene
sequence: 182) SEQ ID NO: 370 Antisense strand:
5'-UACUUCCUUUCUCUGCUGC UU-3' SEQ ID NO: 202 dsRNA: 5'-
GCAGCAGAGAAAGGAAGU UU UCU AAA G-3' Target sequence 50: GC content:
45.0% 5'-CCT ATC TCC CTC SEQ ID NO: 50 AGA ACT-3' (SEQ ID Sense
strand: NO: 527) 5'- CCUAUCUCCCUCAGAACU (Position in UU-3' gene
sequence: 214) SEQ ID NO: 371 Antisense strand:
5'-AGUUCUGAGGGAGAUAGG UU-3' SEQ ID NO: 203 dsRNA: 5'-
CCUAUCUCCCUCAGAACU UU UCU AAA G-3' Target sequence 51: GC content:
45.45% 5'-GAG AAG CGA GAC SEQ ID NO: 51 CTT AGA AG-3' (SEQ Sense
strand: ID NO: 528) 5'-GAGAAGCGAGACCUUAGAAG (Position in UU-3' gene
sequence: 236) SEQ ID NO: 372 Antisense strand:
5'-CUUCUAAGGUCUCGCUUCUC UU-3' SEQ ID NO: 204 dsRNA: 5'-
GAGAAGCGAGACCUUAGAAG UU UCU AAA G-3' Target sequence 52: GC
content: 40.9% 5'-GCC TCA ACT CTA SEQ ID NO: 52 TAG CTC TA-3' (SEQ
ID Sense strand: NO: 529) 5'-GCCUCAACUCUAUAGCUCUA (Position in
UU-3' gene sequence: 319) SEQ ID NO: 373 Antisense strand:
5'-UAGAGCUAUAGAGUUGAGGC UU-3' SEQ ID NO: 205 dsRNA: 5'-
GCCUCAACUCUAUAGCUCUA UU UCU AAA G-3' Target sequence 53: GC
content: 40.0% 5'-CCA ACG TGG AAT SEQ ID NO: 53 GTT TCT-3' (SEQ ID
Sense strand: NO: 530) 5'- CCAACGUGGAAUGUUUCU (Position in UU-3'
gene sequence: 354) SEQ ID NO: 374 Antisense strand:
5'-AGAAACAUUCCACGUUGG UU-3' SEQ ID NO 206 dsRNA: 5'-
CCAACGUGGAAUGUUUCU UU UCU AAA G-3' Target sequence 54: GC content:
41.67% 5'-GAA GCG GAA GTG SEQ ID NO: 54 GAA GAA AGT T-3' Sense
strand: (SEQ ID NO: 531) 5'-GAAGCGGAAGUGGAAGAAAGUU (Position in
gene UU-3' sequence: 401) SEQ ID NO: 375 Antisense strand:
5'-AACUUUCUUCCACUUCCGCUUC UU-3' SEQ ID NO: 207 dsRNA:
5'-GAAGCGGAAGUGGAAGAAAGUU UU UCU AAA G-3' Target sequence 55: GC
content: 40.9% 5'-CTA GTG GCT TGA SEQ ID NO: 55 GAT TAA GC-3' (SEQ
Sense strand: ID NO: 532) 5'-CUAGUGGCUUGAGAUUAAGC (Position in
UU-3' gene sequence: 423) SEQ ID NO: 376 Antisense strand:
5'-GCUUAAUCUCAAGCCACUAG UU-3' SEQ ID NO: 208 dsRNA: 5'-
CUAGUGGCUUGAGAUUAAGC UU UCU AAA G-3'
[0093] The composition of the present invention may include: siRNA
which includes a sense RNA having at least one sequence selected
from the group consisting of sequences of SEQ ID NOs: 56 to 120 in
Table 3 below, and an antisense RNA having a complementary sequence
thereto; or dsRNA having at least one sequence selected from the
group consisting of sequences of SEQ ID NOs: 209 to 273 in Table 3
below.
[0094] In this regard, siRNA which includes a sense RNA having at
least one sequence selected from the group consisting of sequences
of SEQ ID NOs: 56 to 120 in Table 3 below, and an antisense RNA
having a complementary sequence thereto; or dsRNA having at least
one sequence selected from the group consisting of sequences of SEQ
ID NOs: 209 to 273 in Table 3 below, may target a sequence of human
PLOD3 gene (SEQ ID NO: 3) to inhibit expression of the human PLOD3
gene through RNAi, thereby achieving effects of preventing or
treating HCC.
TABLE-US-00003 TABLE 3 Target sequence 56: 5'- GC content: 50.0%
CCA GAG AAG CTG SEQ ID NO: 56 Sense strand: 5'- CTG GTG AT-3' (SEQ
CCAGAGAAGCUGCUGGUGAU UU-3' ID NO: 533) (Position in SEQ ID NO: 377
Antisense strand: 5'- gene sequence: 562) AUCACCAGCAGCUUCUCUGG
UU-3' SEQ ID NO: 209 dsRNA: 5'-CCAGAGAAGCUGCUGGUGAU UU UCU AAA G-3'
Target sequence 57: 5'- GC content: 55.0% CCA CAG CTG AAA SEQ ID
NO: 57 Sense strand: 5'-CCACAGCUGAAACCGAGG CCG AGG-3' (SEQ ID UU-3'
NO: 534) (Position in SEQ ID NO: 378 Antisense strand: 5'- gene
sequence: 590) CCUCGGUUUCAGCUGUGG UU-3' SEQ ID NO: 210 dsRNA:
5'-CCACAGCUGAAACCGAGG UU UCU AAA G-3' Target sequence 58: 5'- GC
content: 50.0% CTC TGC GGA GTT SEQ ID NO: 58 Sense strand:
5'-CUCUGCGGAGUUCUUC UU- CTT C-3' (SEQ ID NO: 3' 535) (Position in
gene SEQ ID NO: 379 Antisense strand: 5'-GAAGAACUCCGCAGAG sequence:
627) UU-3' SEQ ID NO: 211 dsRNA: 5'-CUCUGCGGAGUUCUUC UU UCU AAA
G-3' Target sequence 59: 5'- GC content: 50.0% AAC TAC ACT GTG SEQ
ID NO: 59 Sense strand: 5'-AACUACACUGUGCGGACC CGG ACC-3' (SEQ ID
UU-3' NO: 536) (Position in SEQ ID NO: 380 Antisense strand: 5'-
gene sequence: 643) GGUCCGCACAGUGUAGUU UU-3' SEQ ID NO: 212 dsRNA:
5'-AACUACACUGUGCGGACC UU UCU AAA G-3' Target sequence 60: 5'- GC
content: 50.0% GTG ATG TGG CTC SEQ ID NO: 60 Sense strand:
5'-GUGAUGUGGCUCGAACAG GAA CAG-3' (SEQ ID UU-3' NO: 537) (Position
in SEQ ID NO: 381 Antisense strand:5'- gene sequence: 689)
CUGUUCGAGCCACAUCAC UU-3' SEQ ID NO: 213 dsRNA:
5'-GUGAUGUGGCUCGAACAG UU UCU AAA G-3' Target sequence 61: 5'- GC
content: 36.36% GGT TAA AGA AGG SEQ ID NO: 61 Sense strand: 5'- AAA
TGG AG-3' (SEQ GGUUAAAGAAGGAAAUGGAG UU-3' ID NO: 538) (Position in
SEQ ID NO: 382 Antisense strand: 5'- gene sequence: 731)
CUCCAUUUCCUUCUUUAACC UU-3' SEQ ID NO: 214 dsRNA:
5'-GGUUAAAGAAGGAAAUGGAG UU UCU AAA G-3' Target sequence 62: 5'- GC
content: 36.36% GGA GGA TAT GAT SEQ ID NO: 62 Sense strand: 5'- CAT
CAT GT-3' (SEQ GGAGGAUAUGAUCAUCAUGU UU-3' ID NO: 539) (Position in
SEQ ID NO: 383 Antisense strand: 5'- gene sequence: 765)
ACAUGAUGAUCAUAUCCUCC UU-3' SEQ ID NO: 215 dsRNA:
5'-GGAGGAUAUGAUCAUCAUGU UU UCU AAA G-3' Target sequence 63: 5'- GC
content: 45.0% GGA TAG CTA CGA SEQ ID NO: 63 Sense strand:
5'-GGAUAGCUACGACGUGAU CGT GAT-3' (SEQ ID UU-3' NO: 540) (Position
in SEQ ID NO: 384 Antisense strand: 5'- gene sequence: 789)
AUCACGUCGUAGCUAUCC UU-3' SEQ ID NO: 216 dsRNA:
5'-GGAUAGCUACGACGUGAU UU UCU AAA G-3' Target sequence 64: 5'- GC
content: 45.0% CAC AGA GCT GCT SEQ ID NO: 64 Sense strand:
5'-CACAGAGCUGCUGAAGAA GAA GAA-3' (SEQ ID UU-3' NO: 541) (Position
in SEQ ID NO: 385 Antisense strand: 5'- gene sequence: 822)
UUCUUCAGCAGCUCUGUG UU-3' SEQ ID NO: 217 dsRNA:
5'-CACAGAGCUGCUGAAGAA UU UCU AAA G-3' Target sequence 65: 5'- GC
content: 45.0% TGC TCT TCT CTG SEQ ID NO: 65 Sense strand:
5'-UGCUCUUCUCUGCAGAGA CAG AGA-3' (SEQ ID UU-3' NO: 542) (Position
in SEQ ID NO: 386 Antisense strand: 5'- gene sequence: 863)
UCUCUGCAGAGAAGAGCA UU-3' SEQ ID NO: 218 dsRNA:
5'-UGCUCUUCUCUGCAGAGA UU UCU AAA G-3' Target sequence 66: 5'- GC
content: 47.62% GCT TCC TCA ATT SEQ ID NO: 66 Sense strand:
5'-GCUUCCUCAAUUCUGGUGG CTG GTG G-3' (SEQ ID UU-3' NO: 543)
(Position in SEQ ID NO: 387 Antisense strand: 5'- gene sequence:
941) CCACCAGAAUUGAGGAAGC UU-3' SEQ ID NO: 219 dsRNA:
5'-GCUUCCUCAAUUCUGGUGG UU UCU AAA G-3' Target sequence 67: 5'- GC
content: 40.9% ATT CAT CGG TTT SEQ ID NO: 67 Sense strand: 5'- TGC
CAC CA-3' (SEQ AUUCAUCGGUUUUGCCACCA UU-3' ID NO: 544) (Position in
SEQ ID NO: 388 Antisense strand: 5'- gene sequence: 950)
UGGUGGCAAAACCGAUGAAU UU-3' SEQ ID NO: 220 dsRNA:
5'-AUUCAUCGGUUUUGCCACCA UU UCU AAA G-3' Target sequence 68: 5'- GC
content: 38.1% AGT GGA AGT ACA SEQ ID NO: 68 Sense strand:
5'-AGUGGAAGUACAAGGAUGA AGG ATG A-3' (SEQ UU-3' ID NO: 545)
(Position in SEQ ID NO: 389 Antisense strand: 5'- gene sequence:
1001) UCAUCCUUGUACUUCCACU UU-3' SEQ ID NO: 221 dsRNA:
5'-AGUGGAAGUACAAGGAUGA UU UCU AAA G-3' Target sequence 69: 5'- GC
content: 40.0% CAG CCT TAA TCT SEQ ID NO: 69 Sense strand:
5'-CAGCCUUAAUCUGGAUCA GGA TCA-3' (SEQ ID UU-3' NO: 546) (Position
in SEQ ID NO: 390 Antisense strand: 5'- gene sequence: 1080)
UGAUCCAGAUUAAGGCUG UU-3' SEQ ID NO: 222 dsRNA:
5'-CAGCCUUAAUCUGGAUCA UU UCU AAA G-3' Target sequence 70: 5'- GC
content: 45.45% GTC TCG GAT CTT SEQ ID NO: 70 Sense strand: 5'- TCA
GAA CC-3' (SEQ GUCUCGGAUCUUUCAGAACC UU-3' ID NO: 547) (Position in
SEQ ID NO: 391 Antisense strand: 5'- gene sequence: 1101)
GGUUCUGAAAGAUCCGAGAC UU-3' SEQ ID NO: 223 dsRNA:
5'-GUCUCGGAUCUUUCAGAACC UU UCU AAA G-3' Target sequence 71: 5'- GC
content: 40.0% GGC TTT AGA TGA SEQ ID NO: 71 Sense strand:
5'-GGCUUUAGAUGAAGUGGU AGT GGT-3' (SEQ ID UU-3' NO: 548) (Position
in SEQ ID NO: 392 Antisense strand: 5'- gene sequence: 1128)
ACCACUUCAUCUAAAGCC UU-3' SEQ ID NO: 224 dsRNA:
5'-GGCUUUAGAUGAAGUGGU UU UCU AAA G-3' Target sequence 72: 5'- GC
content: 45.0% GTT TGA TCG GAA SEQ ID NO: 72 Sense strand:
5'-GUUUGAUCGGAACCGUGU CCG TGT-3' (SEQ ID UU-3' NO: 549) (Position
in SEQ ID NO: 393 Antisense strand: 5'- gene sequence: 1152)
ACACGGUUCCGAUCAAAC UU-3' SEQ ID NO: 225 dsRNA:
5'-GUUUGAUCGGAACCGUGU UU UCU AAA G-3' Target sequence 73: 5'- GC
content: 45.0% TTG TGG TCC ATG SEQ ID NO: 73 Sense strand:
5'-UUGUGGUCCAUGGAAACG GAA ACG-3' (SEQ ID UU-3' NO: 550) (Position
in SEQ ID NO: 394 Antisense strand: 5'- gene sequence: 1205)
CGUUUCCAUGGACCACAA UU-3' SEQ ID NO: 226 dsRNA:
5'-UUGUGGUCCAUGGAAACG UU UCU AAA G-3' Target sequence 74: 5'- GC
content: 47.62% CCA CTA AGC TGC SEQ ID NO: 74 Sense strand:
5'-CCACUAAGCUGCAGCUCAA AGC TCA A-3' (SEQ ID UU-3' NO: 551)
(Position in SEQ ID NO: 395 Antisense strand: 5'- gene sequence:
1223) UUGAGCUGCAGCUUAGUGG UU-3' SEQ ID NO: 227 dsRNA:
5'-CCACUAAGCUGCAGCUCAA UU UCU AAA G-3' Target sequence 75: 5'- GC
content: 52.38% CCA ATG GCT GGA SEQ ID NO: 75 Sense strand:
5'-CCAAUGGCUGGACUCCUGA CTC CTG A-3' (SEQ ID UU-3' NO: 552)
(Position in SEQ ID NO: 396 Antisense strand: 5'- gene sequence:
1265) TCAGGAGTCCAGCCATTGG UU-3' SEQ ID NO: 228 dsRNA:
5'-CCAAUGGCUGGACUCCUGA UU UCU AAA G-3' Target sequence 76: 5'- GC
content: 52.38% GCT GTG GCT TCT SEQ ID NO: 76 Sense strand:
5'-GCUGUGGCUUCUGCAACCA GCA ACC A-3' (SEQ ID UU-3' NO: 553)
(Position in SEQ ID NO: 397 Antisense strand: 5'- gene sequence:
1289) UGGUUGCAGAAGCCACAGC UU-3' SEQ ID NO: 229 dsRNA:
5'-GCUGUGGCUUCUGCAACCA UU UCU AAA G-3' Target sequence 77: 5'- GC
content: 45.0% GTG TTT GTG GAA SEQ ID NO: 77 Sense strand:
5'-GUGUUUGUGGAACAGCCU CAG CCT-3' (SEQ ID UU-3' NO: 554) (Position
in SEQ ID NO: 398 Antisense strand: 5'- gene sequence: 1360)
AGGCUGUUCCACAAACAC UU-3' SEQ ID NO: 230 dsRNA:
5'-GUGUUUGUGGAACAGCCU UU UCU AAA G-3' Target sequence 78: 5'- GC
content: 47.62% GCT GCT ACT CCT SEQ ID NO: 78 Sense strand:
5'-GCUGCUACUCCUGGACUAU GGA CTA T-3' (SEQ ID UU-3' NO: 555)
(Position in SEQ ID NO: 399 Antisense strand: 5'- gene sequence:
1407) AUAGUCCAGGAGUAGCAGC UU-3' SEQ ID NO: 231 dsRNA:
5'-GCUGCUACUCCUGGACUAU UU UCU AAA G-3' Target sequence 79: 5'- GC
content: 45.45% TTC CTG CAC AAC SEQ ID NO: 79 Sense strand: 5'- AAC
GAG GT-3' (SEQ UUCCUGCACAACAACGAGGU UU-3' ID NO: 556) (Position in
SEQ ID NO: 400 Antisense strand: 5'- gene sequence: 1447)
ACCUCGUUGUUGUGCAGGAA UU-3' SEQ ID NO: 232 dsRNA:
5'-UUCCUGCACAACAACGAGGU UU UCU AAA G-3' Target sequence 80: 5'- GC
content: 55.0% CCA CAT CGC TGA SEQ ID NO: 80 Sense strand:
5'-CCACAUCGCUGACUCCUG CTC CTG-3' (SEQ ID UU-3' NO: 557) (Position
in SEQ ID NO: 401 Antisense strand: 5'- gene sequence: 1479)
CAGGAGUCAGCGAUGUGG UU-3' SEQ ID NO: 233 dsRNA:
5'-CCACAUCGCUGACUCCUG UU UCU AAA G-3' Target sequence 81: 5'- GC
content: 50.0% AGC TCC AGG ACC SEQ ID NO: 81 Sense strand: 5'- ACT
TCT CA-3' (SEQ AGCUCCAGGACCACUUCUCA UU-3' ID NO: 558) (Position in
SEQ ID NO: 402 Antisense strand: 5'- gene sequence: 1502)
TGAGAAGTGGTCCTGGAGCT UU-3' SEQ ID NO: 234 dsRNA:
5'-AGCUCCAGGACCACUUCUCA UU UCU AAA G-3' Target sequence 82: 5'- GC
content: 50.0% ATG GCC ATG GAC SEQ ID NO: 82 Sense strand:
5'-AUGGCCAUGGACCUGUGU CTG TGT-3' (SEQ ID UU-3' NO: 559) (Position
in SEQ ID NO: 403 Antisense strand: 5'- gene sequence: 1576)
ACACAGGUCCAUGGCCAU UU-3' SEQ ID NO: 235 dsRNA:
5'-AUGGCCAUGGACCUGUGU UU UCU AAA G-3' Target sequence 83: 5'- GC
content: 40.9% CGA GTG TGA GTT SEQ ID NO: 83 Sense strand: 5'- CTA
CTT CA-3' (SEQ CGAGUGUGAGUUCUACUUCA UU-3' ID NO: 560) (Position in
SEQ ID NO: 404 Antisense strand:5'- gene sequence: 1605)
UGAAGUAGAACUCACACUCG UU-3' SEQ ID NO: 236 dsRNA:
5'-CGAGUGUGAGUUCUACUUCA UU UCU AAA G-3' Target sequence 84: 5'- GC
content: 55.0% GCT GTC CTC ACC SEQ ID NO: 84 Sense strand:
5'-GCUGUCCUCACCAACCUG AAC CTG-3' (SEQ ID UU-3' NO: 561) (Position
in SEQ ID NO: 405 Antisense strand: 5'- gene sequence: 1639)
CAGGUUGGUGAGGACAGC UU-3' SEQ ID NO: 237 dsRNA:
5'-GCUGUCCUCACCAACCUG UU UCU AAA G-3' Target sequence 85: 5'- GC
content: 45.0% CTG CGT ATC CTC SEQ ID NO: 85 Sense strand:
5'-CUGCGUAUCCUCAUUGAG ATT GAG-3' (SEQ ID UU-3' NO: 562) (Position
in SEQ ID NO: 406 Antisense strand: 5'- gene sequence: 1663)
CUCAAUGAGGAUACGCAG UU-3' SEQ ID NO: 238 dsRNA:
5'-CUGCGUAUCCUCAUUGAG UU UCU AAA G-3' Target sequence 86: 5'- GC
content: 45.0% GAG AAC AGG AAG SEQ ID NO: 86 Sense strand:
5'-GAGAACAGGAAGGUGAUC GTG ATC-3' (SEQ ID UU-3' NO: 563) (Position
in SEQ ID NO: 407 Antisense strand: 5'- gene sequence: 1681)
GAUCACCUUCCUGUUCUC UU-3' SEQ ID NO: 239 dsRNA:
5'-GAGAACAGGAAGGUGAUC UU UCU AAA G-3'
Target sequence 87: 5'- GC content: 45.0% CAA GCT GTG GTC SEQ ID
NO: 87 Sense strand: 5'-CAAGCUGUGGUCCAACUU CAA CTT-3' (SEQ ID UU-3'
NO: 564) (Position in SEQ ID NO: 408 Antisense strand: 5'- gene
sequence: 1722) AAGUUGGACCACAGCUUG UU-3' SEQ ID NO: 240 dsRNA:
5'-CAAGCUGUGGUCCAACUU UU UCU AAA G-3' Target sequence 88: 5'- GC
content: 55.0% GAG GAC TAC GTG SEQ ID NO: 88 Sense strand:
5'-GAGGACUACGUGGAGCUG GAG CTG-3' (SEQ ID UU-3' NO: 565) (Position
in SEQ ID NO: 409 Antisense strand: 5'- gene sequence: 1780)
CAGCUCCACGUAGUCCUC UU-3' SEQ ID NO: 241 dsRNA:
5'-GAGGACUACGUGGAGCUG UU UCU AAA G-3' Target sequence 89: 5'- GC
content: 40.9% GTG TGT GGA ATG SEQ ID NO: 89 Sense strand: 5'- TAC
CAT AC-3' (SEQ GUGUGUGGAAUGUACCAUAC UU-3' ID NO: 566) (Position in
SEQ ID NO: 410 Antisense strand: 5'- gene sequence: 1817)
GUAUGGUACAUUCCACACAC UU-3' SEQ ID NO: 242 dsRNA:
5'-GUGUGUGGAAUGUACCAUAC UU UCU AAA G-3' Target sequence 90: 5'- GC
content: 52.38% AGA GGG ATG TGT SEQ ID NO: 90 Sense strand:
5'-AGAGGGAUGUGUUCUCGGG TCT CGG G-3' (SEQ ID UU-3' NO: 567)
(Position in SEQ ID NO: 411 Antisense strand: 5'- gene sequence:
1888) CCCGAGAACACAUCCCUCU UU-3' SEQ ID NO: 243 dsRNA:
5'-AGAGGGAUGUGUUCUCGGG UU UCU AAA G-3' Target sequence 91: 5'- GC
content: 40.9% CCT TCT GTA AGA SEQ ID NO: 91 Sense strand: 5'- GCT
TTC GA-3' (SEQ CCUUCUGUAAGAGCUUUCGA UU-3' ID NO: 568) (Position in
SEQ ID NO: 412 Antisense strand: 5'- gene sequence: 1931)
UCGAAAGCUCUUACAGAAGG UU-3' SEQ ID NO: 244 dsRNA:
5'-CCUUCUGUAAGAGCUUUCGA UU UCU AAA G-3' Target sequence 92: 5'- GC
content: 45.45% ACA AGG GCA TCT SEQ ID NO: 92 Sense strand: 5'- TCC
TCC AT-3' (SEQ ACAAGGGCAUCUUCCUCCAU UU-3' ID NO: 569) (Position in
SEQ ID NO: 413 Antisense strand: 5'- gene sequence: 1952)
AUGGAGGAAGAUGCCCUUGU UU-3' SEQ ID NO: 245 dsRNA:
5'-ACAAGGGCAUCUUCCUCCAU UU UCU AAA G-3' Target sequence 93: 5'- GC
content: 40.0% CTG AGC AAT CAG SEQ ID NO: 93 Sense strand:
5'-CUGAGCAAUCAGCAUGAA CAT GAA-3' (SEQ ID UU-3' NO: 570) (Position
in SEQ ID NO: 414 Antisense strand: 5'- gene sequence: 1972)
UUCAUGCUGAUUGCUCAG UU-3' SEQ ID NO: 246 dsRNA:
5'-CUGAGCAAUCAGCAUGAAUU UCU AAA G-3' Target sequence 94: 5'- GC
content: 45.0% CCA CTT CCA GAT SEQ ID NO: 94 Sense strand:
5'-CCACUUCCAGAUACGACA ACG ACA-3' (SEQ ID UU-3' NO: 571) (Position
in SEQ ID NO: 415 Antisense strand: 5'- gene sequence: 2006)
UGUCGUAUCUGGAAGUGG UU-3' SEQ ID NO: 247 dsRNA:
5'-CCACUUCCAGAUACGACA UU UCU AAA G-3' Target sequence 95: 5'- GC
content: 47.62% ACC TCT GGC AGA SEQ ID NO: 95 Sense strand:
5'-ACCUCUGGCAGAUCUUCGA TCT TCG A-3' (SEQ ID UU-3' NO: 572)
(Position in SEQ ID NO: 416 Antisense strand: 5'- gene sequence:
2042) UCGAAGAUCUGCCAGAGGU UU-3' SEQ ID NO: 248 dsRNA:
5'-ACCUCUGGCAGAUCUUCGA UU UCU AAA G-3' Target sequence 96: 5'- GC
content: 55.0% CGT CGA CTG GAA SEQ ID NO: 96 Sense strand:
5'-CGUCGACUGGAAGGAGCA GGA GCA-3' (SEQ ID UU-3' NO: 573) (Position
in SEQ ID NO: 417 Antisense strand: 5'- gene sequence: 2067)
UGCUCCUUCCAGUCGACG UU-3' SEQ ID NO: 249 dsRNA:
5'-CGUCGACUGGAAGGAGCA UU UCU AAA G-3' Target sequence 97: 5'- GC
content: 40.0% GTA CAT CCA CGA SEQ ID NO: 97 Sense strand:
5'-GUACAUCCACGAGAACUA GAA CTA-3' (SEQ ID UU-3' NO: 574) (Position
in SEQ ID NO: 418 Antisense strand: 5'- gene sequence: 2085)
UAGUUCUCGUGGAUGUAC UU-3' SEQ ID NO: 250 dsRNA:
5'-GUACAUCCACGAGAACUA UU UCU AAA G-3' Target sequence 985'- GC
content: 50.0% AAG GAA TCG TGG SEQ ID NO: 98 Sense strand: 5'- AGC
AGC CA-3' (SEQ AAGGAAUCGUGGAGCAGCCA UU-3' ID NO: 575) (Position in
SEQ ID NO: 419 Antisense strand: 5'- gene sequence: 2123)
UGGCUGCUCCACGAUUCCUU UU-3' SEQ ID NO: 251 dsRNA:
5'-AAGGAAUCGUGGAGCAGCCA UU UCU AAA G-3' Target sequence 995'- GC
content: 40.0% CTG CTG TCA GAA SEQ ID NO: 99 Sense strand:
5'-CUGCUGUCAGAACAAAUG CAA ATG-3' (SEQ ID UU-3' NO: 576) (Position
in SEQ ID NO: 420 Antisense strand: 5'- gene sequence: 2167)
CAUUUGUUCUGACAGCAG UU-3' SEQ ID NO: 252 dsRNA:
5'-CUGCUGUCAGAACAAAUG UU UCU AAA G-3' Target sequence 100: 5'- GC
content: 50.0% TGT GAT GAG CTG SEQ ID NO: 100 Sense strand: 5'- GTG
GCA GA-3' (SEQ UGUGAUGAGCUGGUGGCAGA UU-3' ID NO: 577) (Position in
SEQ ID NO: 421 Antisense strand: 5'- gene sequence: 2185)
UCUGCCACCAGCUCAUCACA UU-3' SEQ ID NO: 253 dsRNA:
5'-UGUGAUGAGCUGGUGGCAGA UU UCU AAA G-3' Target sequence 101: 5'- GC
content: 45.0% GCA TGA GGA TTC SEQ ID NO: 101 Sense strand:
5'-GCAUGAGGAUUCAAGGCU AAG GCT-3' (SEQ ID UU-3' NO: 578) (Position
in SEQ ID NO: 422 Antisense strand: 5'- gene sequence: 2238)
AGCCUUGAAUCCUCAUGC UU-3' SEQ ID NO: 254 dsRNA:
5'-GCAUGAGGAUUCAAGGCU UU UCU AAA G-3' Target sequence 102: 5'- GC
content: 47.62% CTG GAG GCT ACG SEQ ID NO: 102 Sense strand: 5'-
AGA ATG T-3' (SEQ ID CUGGAGGCUACGAGAAUGU UU-3' NO: 579) (Position
in SEQ ID NO: 423 Antisense strand: 5'- gene sequence: 2256)
ACAUUCUCGUAGCCUCCAG UU-3' SEQ ID NO: 255 dsRNA:
5'-CUGGAGGCUACGAGAAUGU UU UCU AAA G-3' Target sequence 103: 5'- GC
content: 45.0% TGG ACA TCC ACA SEQ ID NO: 103 Sense strand:
5'-UGGACAUCCACAUGAAGC TGA AGC-3' (SEQ ID UU-3' NO: 580) (Position
in SEQ ID NO: 424 Antisense strand: 5'- gene sequence: 2285)
GCUUCAUGUGGAUGUCCA UU-3' SEQ ID NO: 256 dsRNA:
5'-UGGACAUCCACAUGAAGC UU UCU AAA G-3' Target sequence 104: 5'- GC
content: 54.54% TAC GAG GAC CAG SEQ ID NO: 104 Sense strand: 5'-
TGG CTG CA-3' (SEQ UACGAGGACCAGUGGCUGCA UU-3' ID NO: 581) (Position
in SEQ ID NO: 425 Antisense strand: 5'- gene sequence: 2311)
TGCAGCCACTGGTCCTCGTA UU-3' SEQ ID NO: 257 dsRNA:
5'-UACGAGGACCAGUGGCUGCA UU UCU AAA G-3' Target sequence 105: 5'- GC
content: 47.62% CAT GAC CGA GAG SEQ ID NO: 105 Sense strand: 5'-
CCT GTT T-3' (SEQ ID CAUGACCGAGAGCCUGUUU UU-3' NO: 582) (Position
in SEQ ID NO: 426 Antisense strand:5'- gene sequence: 2355)
AAACAGGCUCUCGGUCAUG UU-3' SEQ ID NO: 258 dsRNA:
5'-CAUGACCGAGAGCCUGUUU UU UCU AAA G-3' Target sequence 106: 5'- GC
content: 40.9% GTG ATG AAC TTT SEQ ID NO: 106 Sense strand: 5'- GTG
GTT CG-3' 3' GUGAUGAACUUUGUGGUUCG UU-3' (SEQ ID NO: 583) SEQ ID NO:
427 Antisense strand: 5'- (Position in gene CGAACCACAAAGUUCAUCAC
UU-3' sequence: 2401) SEQ ID NO: 259 dsRNA: 5'-GUGAUGAACUUUGUGGUUCG
UU UCU AAA G-3' Target sequence 107: 5'- GC content: 55.0% AGA CGA
GCA GCC SEQ ID NO: 107 Sense strand: 5'-AGACGAGCAGCCGUCUCU GTC
TCT-3' (SEQ ID UU-3' NO: 584) (Position in SEQ ID NO: 428 Antisense
strand: 5'- gene sequence: 2429) AGAGACGGCUGCUCGUCU UU-3' SEQ ID
NO: 260 dsRNA: 5'-AGACGAGCAGCCGUCUCU UU UCU AAA G-3' Target
sequence 108: 5'- GC content: 50.0% GAC TCA TCC ACC SEQ ID NO: 108
Sense strand: 5'- TTC ACC CT-3' (SEQ GACUCAUCCACCUUCACCCU UU-3' ID
NO: 585) (Position in SEQ ID NO: 429 Antisense strand: 5'- gene
sequence: 2461) AGGGUGAAGGUGGAUGAGUC UU-3' SEQ ID NO: 261 dsRNA:
5'- GACUCAUCCACCUUCACCCUUU UCU AAA G-3' Target sequence 109: 5'- GC
content: 50.0% TTC CTG CGC TAC SEQ ID NO: 109 Sense strand: 5'- GAC
TGT GT-3' (SEQ UUCCUGCGCUACGACUGUGU UU-3' ID NO: 586) (Position in
SEQ ID NO: 430 Antisense strand: 5'- gene sequence: 2533)
ACACAGUCGUAGCGCAGGAA UU-3' SEQ ID NO: 262 dsRNA:
5'-UUCCUGCGCUACGACUGUGU UU UCU AAA G-3' Target sequence 110: 5'- GC
content: 45.45% CAC ACG CTA CAT SEQ ID NO: 110 Sense strand: 5'-
CAT GGT GT-3' (SEQ CACACGCUACAUCAUGGUGU UU-3' ID NO: 587) (Position
in SEQ ID NO: 431 Antisense strand: 5'- gene sequence: 2637)
ACACCAUGAUGUAGCGUGUG UU-3' SEQ ID NO: 263 dsRNA:
5'-CACACGCUACAUCAUGGUGU UU UCU AAA G-3' Target sequence 111: 5'- GC
content: 40.9% TGC CAT TGT GCC SEQ ID NO: 111 Sense strand: 5'- TTT
TTA GG-3' (SEQ UGCCAUUGUGCCUUUUUAGG UU-3' ID NO: 588) (Position in
SEQ ID NO: 432 Antisense strand: 5'- gene sequence: 2701)
CCUAAAAAGGCACAAUGGCA UU-3' SEQ ID NO: 264 dsRNA:
5'-UGCCAUUGUGCCUUUUUAGG UU UCU AAA G-3' Target sequence 112: 5'- GC
content: 40.9% CAC TTC CTG AGT SEQ ID NO: 112 Sense strand: 5'- TCA
TGT TC-3' (SEQ CACUUCCUGAGUUCAUGUUC UU-3' ID NO: 589) (Position in
SEQ ID NO: 433 Antisense strand: 5'- gene sequence: 2769)
GAACAUGAACUCAGGAAGUG UU-3' SEQ ID NO: 265 dsRNA:
5'-CACUUCCUGAGUUCAUGUUC UU UCU AAA G-3' Target sequence 113: 5'- GC
content: 40.9% CCT GAA CTG AAT SEQ ID NO: 113 Sense strand: 5'- ATG
TCA CC-3' (SEQ CCUGAACUGAAUAUGUCACC UU-3' ID NO: 590) (Position in
SEQ ID NO: 434 Antisense strand: 5'- gene sequence: 2796)
GGUGACAUAUUCAGUUCAGG UU-3' SEQ ID NO: 266 dsRNA:
5'-CCUGAACUGAAUAUGUCACC UU UCU AAA G-3' Target sequence 114: 5'- GC
content: 40.9% CGC AGT CTC ACT SEQ ID NO: 114 Sense strand: 5'- CTG
AAT AAA-3' CGCAGUCUCACUCUGAAUAAA UU-3' (SEQ ID NO: 591) SEQ ID NO:
435 Antisense strand: 5'- (Position in gene UUUAUUCAGAGUGAGACUGCG
UU-3' sequence: 2937) SEQ ID NO: 267 dsRNA: 5'-
CGCAGUCUCACUCUGAAUAAAUU UCU AAA G-3' Target sequence 115: 5'- GC
content: 38.06% GGA CAG TTT GTA SEQ ID NO: 115 Sense strand: 5'-
AGT CTT G-3' (SEQ ID GGACAGUUUGUAAGUCUUG UU-3' NO: 592) (Position
in SEQ ID NO: 436 Antisense strand: 5'- gene sequence: 2958)
CAAGACUUACAAACUGUCC UU-3' SEQ ID NO: 268 dsRNA:
5'-GGACAGUUUGUAAGUCUUG UU UCU AAA G-3' Target sequence 116: 5'- GC
content: 50.0% TCA CTT CCC CTG SEQ ID NO: 116 Sense strand: 5'- TCC
AGG TT-3' (SEQ UCACUUCCCCUGUCCAGGUU UU-3' ID NO: 593) (Position in
SEQ ID NO: 437 Antisense strand: 5'- gene sequence: 121)
AACCUGGACAGGGGAAGUGA UU-3' SEQ ID NO: 269 dsRNA:
5'-UCACUUCCCCUGUCCAGGUU UU UCU AAA G-3' Target sequence 117: 5'- GC
content: 40.9% TCA GCT TCC ACA SEQ ID NO: 117 Sense strand: 5'- TGT
GTC AA-3' (SEQ UCAGCUUCCACAUGUGUCAA UU-3' ID NO: 594) (Position in
SEQ ID NO: 438 Antisense strand: 5'- gene sequence: 141)
UUGACACAUGUGGAAGCUGA UU-3' SEQ ID NO: 270 dsRNA:
5'-UCAGCUUCCACAUGUGUCAA UU UCU AAA G-3' Target sequence 118: 5'- GC
content: 47.62% GAC AAT CCT CGC SEQ ID NO: 118 Sense strand:
5'-
CTT GTC T-3' (SEQ ID GACAAUCCUCGCCUUGUCU UU-3' NO: 595) (Position
in SEQ ID NO: 439 Antisense strand: 5'- gene sequence: 241)
AGACAAGGCGAGGAUUGUC UU-3' SEQ ID NO: 271 dsRNA:
5'-GACAAUCCUCGCCUUGUCU UU UCU AAA G-3' Target sequence 119: 5'- GC
content: 45.45% CAT CTG GAG CTT SEQ ID NO: 119 Sense strand: 5'-
TCT GTA GC-3' (SEQ GCAUCUGGAGCUUUCUGUA UU-3' ID NO: 596) (Position
in SEQ ID NO: 440 Antisense strand: 5'- gene sequence: 270)
UACAGAAAGCUCCAGAUGC UU-3' SEQ ID NO: 272 dsRNA:
5'-GCAUCUGGAGCUUUCUGUA UU UCU AAA G-3' Target sequence 120: 5'- GC
content: 55.0% GAG ATC CCA GGA SEQ ID NO: 120 Sense strand:
5'-GAGAUCCCAGGAUCCUGG TCC TGG-3' (SEQ ID UU-3' NO: 597) (Position
in SEQ ID NO: 441 Antisense strand: 5'- gene sequence: 414)
CCAGGAUCCUGGGAUCUC UU-3' SEQ ID NO: 273 dsRNA:
5'-GAGAUCCCAGGAUCCUGG UU UCU AAA G-3'
[0095] The composition of the present invention may include: siRNA
which includes a sense RNA having at least one sequence selected
from the group consisting of sequences of SEQ ID NOs: 121 to 157 in
Table 4 below, and an antisense RNA having a complementary sequence
thereto; or dsRNA having at least one sequence selected from the
group consisting of sequences of SEQ ID NOs: 274 to 310 in Table 4
below.
[0096] In this regard, siRNA which includes a sense RNA having at
least one sequence selected from the group consisting of sequences
of SEQ ID NOs: 121 to 157 in Table 4 below, and an antisense RNA
having a complementary sequence thereto; or dsRNA having at least
one sequence selected from the group consisting of sequences of SEQ
ID NOs: 274 to 310 in Table 4 below, may target a sequence of human
SF3B4 gene (SEQ ID NO: 4) to inhibit expression of the human SF3B4
gene through RNAi, thereby achieving effects of preventing or
treating HCC.
TABLE-US-00004 TABLE 4 Target sequence 121: 5'- GC content: 40.9%
AAT CAG GAT GCC SEQ ID NO: 121 Sense strand: 5'- ACT GTG TA-3' (SEQ
ID AAUCAGGAUGCCACUGUGUA UU-3' NO: 598) (Position in SEQ ID NO: 442
Antisense strand: 5'- gene sequence: 521) UACACAGUGGCAUCCUGAUU
UU-3' SEQ ID NO: 274 dsRNA: 5'-AAUCAGGAUGCCACUGUGUA UU UCU AAA G-3'
Target sequence 122: 5'- GC content: 40.9% CTG GAT GAG AAG SEQ ID
NO: 122 Sense strand: 5'- GTT AGT GA-3' (SEQ ID
CUGGAUGAGAAGGUUAGUGA UU-3' NO: 599) (Position in SEQ ID NO: 443
Antisense strand: 5'- gene sequence: 551) UCACUAACCUUCUCAUCCAG
UU-3' SEQ ID NO: 275 dsRNA: 5'-CUGGAUGAGAAGGUUAGUGA UU UCU AAA G-3'
Target sequence 123: 5'- GC content: 45.45% TGT GGG AAC TGT SEQ ID
NO: 123 Sense strand: 5'- TTC TCC AG-3' (SEQ ID
UGUGGGAACUGUUUCUCCAG UU-3' NO: 600) (Position in SEQ ID NO: 444
Antisense strand: 5'- gene sequence: 579) CUGGAGAAACAGUUCCCACA
UU-3' SEQ ID NO: 276 dsRNA: 5'-UGUGGGAACUGUUUCUCCAG UU UCU AAA G-3'
Target sequence 124: 5'- GC content: 45.0% CTG GAC CAG TAG SEQ ID
NO: 124 Sense strand: 5'- TCA ACA-3' (SEQ ID CUGGACCAGUAGUCAACA
UU-3' NO: 601) (Position in SEQ ID NO: 445 Antisense strand: 5'-
gene sequence: 599) UGUUGACUACUGGUCCAG UU-3' SEQ ID NO: 277 dsRNA:
5'-CUGGACCAGUAGUCAACA UU UCU AAA G-3' Target sequence 125: 5'- GC
content: 42.86% CCA AAG GAT AGA SEQ ID NO: 125 Sense strand: 5'-
GTC ACT G-3' (SEQ ID CCAAAGGAUAGAGUCACUG UU-3' NO: 602) (Position
in SEQ ID NO: 446 Antisense strand: 5'- gene sequence: 626)
CAGUGACUCUAUCCUUUGG UU-3' SEQ ID NO: 278 dsRNA:
5'-CCAAAGGAUAGAGUCACUG UU UCU AAA G-3' Target sequence 126: 5'- GC
content: 47.83% CAG CAC CAA GGC SEQ ID NO: 126 Sense strand: 5'-
TAT GGC TTT-3' (SEQ CAGCACCAAGGCUAUGGCUUU UU-3' ID NO: 603)
(Position in SEQ ID NO: 447 Antisense strand: 5'- gene sequence:
647) AAAGCCAUAGCCUUGGUGCUG UU-3' SEQ ID NO: 279 dsRNA: 5'-
CAGCACCAAGGCUAUGGCUUU UU UCU AAA G-3' Target sequence 127: 5'- GC
content: 40.9% GTG GAA TTC TTG SEQ ID NO: 127 Sense strand: 5'- AGT
GAG GA-3' (SEQ GUGGAAUUCUUGAGUGAGGA UU-3' ID NO: 604) (Position in
SEQ ID NO: 448 Antisense strand: 5'- gene sequence: 668)
UCCUCACUCAAGAAUUCCAC UU-3' SEQ ID NO: 280 dsRNA:
5'-GUGGAAUUCUUGAGUGAGGA UU UCU AAA G-3' Target sequence 128: 5'- GC
content: 36.36% GCT GAC TAT GCC SEQ ID NO: 128 Sense strand: 5'-
ATT AAG AT-3' (SEQ ID GCUGACUAUGCCAUUAAGAU UU-3' NO: 605) (Position
in SEQ ID NO: 449 Antisense strand: 5'- gene sequence: 692)
AUCUUAAUGGCAUAGUCAGC UU-3' SEQ ID NO: 281 dsRNA:
5'-GCUGACUAUGCCAUUAAGAU UU UCU AAA G-3' Target sequence 129: 5'- GC
content: 33.33% ACA TGA TCA AAC SEQ ID NO: 129 Sense strand: 5'-
TCT ATG G-3' (SEQ ID ACAUGAUCAAACUCUAUGG UU-3' NO: 606) (Position
in SEQ ID NO: 450 Antisense strand: 5'- gene sequence: 717)
CCAUAGAGUUUGAUCAUGU UU-3' SEQ ID NO: 282 dsRNA:
5'-ACAUGAUCAAACUCUAUGG UU UCU AAA G-3' Target sequence 130: 5'- GC
content: 45.0% GGT GAA CAA AGC SEQ ID NO: 130 Sense strand: 5'- ATC
AGC-3' (SEQ ID GGUGAACAAAGCAUCAGC UU-3' NO: 607) (Position in SEQ
ID NO: 451 Antisense strand: 5'- gene sequence: 748)
GCUGAUGCUUUGUUCACC UU-3' SEQ ID NO: 283 dsRNA:
5'-GGUGAACAAAGCAUCAGC UU UCU AAA G-3' Target sequence 131: 5'- GC
content: 40.0% CCT GAG ATT GAT SEQ ID NO: 131 Sense strand: 5'- GAG
AAG-3' (SEQ ID CCUGAGAUUGAUGAGAAG UU-3' NO: 608) (Position in SEQ
ID NO: 452 Antisense strand: 5'- gene sequence: 818)
CUUCUCAUCAAUCUCAGG UU-3' SEQ ID NO: 284 dsRNA:
5'-CCUGAGAUUGAUGAGAAG UU UCU AAA G-3' Target sequence 132: 5'- GC
content: 42.1% GGT CAT CTT ACA SEQ ID NO: 132 Sense strand:
5'-GGUCAUCUUACAAACCC AAC CC-3' (SEQ ID NO: UU-3' 609) (Position in
gene SEQ ID NO: 453 Antisense strand: 5'- sequence: 865)
GGGUUUGUAAGAUGACC UU-3' SEQ ID NO: 285 dsRNA: 5'-GGUCAUCUUACAAACCC
UU UCU AAA G-3' Target sequence 133: 5'- GC content: 55.0% CCT GAC
ACA GGC SEQ ID NO: 133 Sense strand: 5'-CCUGACACAGGCAACUCC AAC
TCC-3' (SEQ ID UU-3' NO: 610) (Position in SEQ ID NO: 454 Antisense
strand: 5'- gene sequence: 899) GGAGUUGCCUGUGUCAGG UU-3' SEQ ID NO:
286 dsRNA: 5'-CCUGACACAGGCAACUCC UU UCU AAA G-3' Target sequence
134: 5'- GC content: 40.9% GCT TCA TTT GAT SEQ ID NO: 134 Sense
strand: 5'- GCT TCG GA-3' (SEQ ID GCUUCAUUUGAUGCUUCGGA UU-3' NO:
611) (Position in SEQ ID NO: 455 Antisense strand: 5'- gene
sequence: 941) UCCGAAGCAUCAAAUGAAGC UU-3' SEQ ID NO: 287 dsRNA:
5'-GCUUCAUUUGAUGCUUCGGA UU UCU AAA G-3' Target sequence 135: 5'- GC
content: 40.9% TGC AGC AAT TGA SEQ ID NO: 135 Sense strand: 5'- AGC
CAT GA-3' (SEQ UGCAGCAAUUGAAGCCAUGA UU-3' ID NO: 612) (Position in
SEQ ID NO: 456 Antisense strand: 5'- gene sequence: 961)
UCAUGGCUUCAAUUGCUGCA UU-3' SEQ ID NO: 288 dsRNA:
5'-UGCAGCAAUUGAAGCCAUGA UU UCU AAA G-3' Target sequence 136: 5'- GC
content: 47.62% GCA GTA CCT CTG SEQ ID NO: 136 Sense strand: 5'-
TAA CCG T-3' (SEQ ID GCAGUACCUCUGUAACCGU UU-3' NO: 613) (Position
in SEQ ID NO: 457 Antisense strand: 5'- gene sequence: 985)
ACGGUUACAGAGGUACUGC UU-3' SEQ ID NO: 289 dsRNA:
5'-GCAGUACCUCUGUAACCGU UU UCU AAA G-3' Target sequence 137: 5'- GC
content: 40.9% CAC CGT ATC TTA SEQ ID NO: 137 Sense strand: 5'- TGC
CTT CA-3' (SEQ ID CACCGUAUCUUAUGCCUUCA UU-3' NO: 614) (Position in
SEQ ID NO: 458 Antisense strand: 5'- gene sequence: 1009)
UGAAGGCAUAAGAUACGGUG UU-3' SEQ ID NO: 290 dsRNA:
5'-CACCGUAUCUUAUGCCUUCA UU UCU AAA G-3' Target sequence 138: 5'- GC
content: 50.0% GAA CGA CTT CTG SEQ ID NO: 138 Sense strand: 5'- GCA
GCT CA-3' (SEQ GAACGACUUCUGGCAGCUCA UU-3' ID NO: 615) (Position in
SEQ ID NO: 459 Antisense strand: 5'- gene sequence: 1067)
UGAGCUGCCAGAAGUCGUUC UU-3' SEQ ID NO: 291 dsRNA:
5'-GAACGACUUCUGGCAGCUCA UU UCU AAA G-3' Target sequence 139: 5'- GC
content: 45.45% CCT CAT CAG CTG SEQ ID NO: 139 Sense strand: 5'-
TTT GCA GA-3' (SEQ ID CCUCAUCAGCUGUUUGCAGA UU-3' NO: 616) (Position
in SEQ ID NO: 460 Antisense strand: 5'- gene sequence: 1112)
UCUGCAAACAGCUGAUGAGG UU-3' SEQ ID NO: 292 dsRNA:
5'-CCUCAUCAGCUGUUUGCAGA UU UCU AAA G-3' Target sequence 140: 5'- GC
content: 45.45% TGG TCA TGG ACA SEQ ID NO: 140 Sense strand: 5'-
CTC ACA TC-3' (SEQ ID UGGUCAUGGACACUCACAUC UU-3' NO: 617) (Position
in SEQ ID NO: 461 Antisense strand: 5'- gene sequence: 1351)
GAUGUGAGUGUCCAUGACCA UU-3' SEQ ID NO: 293 dsRNA:
5'-UGGUCAUGGACACUCACAUC UU UCU AAA G-3' Target sequence 141: 5'- GC
content: 45.0% GAT GTC TCA GAT SEQ ID NO: 141 Sense strand: 5'- GCA
GCT-3' (SEQ ID GAUGUCUCAGAUGCAGCU UU-3' NO: 618) (Position in SEQ
ID NO: 462 Antisense strand: 5'- gene sequence: 1408)
AGCUGCAUCUGAGACAUC UU-3' SEQ ID NO: 294 dsRNA:
5'-GAUGUCUCAGAUGCAGCU UU UCU AAA G-3' Target sequence 142: 5'- GC
content: 45.0% CCT CAT GGC TTA SEQ ID NO: 142 Sense strand: 5'- GGA
CAT-3' (SEQ ID CCUCAUGGCUUAGGACAU UU-3' NO: 619) (Position in SEQ
ID NO: 463 Antisense strand: 5'- gene sequence: 1439)
AUGUCCUAAGCCAUGAGG UU-3' SEQ ID NO: 295 dsRNA:
5'-CCUCAUGGCUUAGGACAU UU UCU AAA G-3' Target sequence 143: 5'- GC
content: 40.9% TCA CAT TTT CCT TCC SEQ ID NO: 143 Sense strand: 5'-
TCC TG-3' (SEQ ID NO: UCACAUUUUCCUUCCUCCUG UU-3' 620) (Position in
gene SEQ ID NO: 464 Antisense strand: 5'- sequence: 1771)
CAGGAGGAAGGAAAAUGUGA UU-3' SEQ ID NO: 296 dsRNA:
5'-UCACAUUUUCCUUCCUCCUG UU UCU AAA G-3' Target sequence 144: 5'- GC
content: 45.45% CCT TGG ACC AAT SEQ ID NO: 144 Sense strand: 5'-
CAG AGA TG-3' (SEQ CCUUGGACCAAUCAGAGAUG UU-3' ID NO: 621) (Position
in SEQ ID NO: 465 Antisense strand: 5'- gene sequence: 1818)
CAUCUCUGAUUGGUCCAAGG UU-3' SEQ ID NO: 297 dsRNA:
5'-CCUUGGACCAAUCAGAGAUG UU UCU AAA G-3' Target sequence 145: 5'- GC
content: 40.9% GGC AAA GGT ACT SEQ ID NO: 145 Sense strand: 5'- AAT
CCC TT-3' (SEQ ID GGCAAAGGUACUAAUCCCUU UU-3' NO: 622) (Position in
SEQ ID NO: 466 Antisense strand: 5'- gene sequence: 1852)
AAGGGAUUAGUACCUUUGCC UU-3' SEQ ID NO: 298 dsRNA:
5'-GGCAAAGGUACUAAUCCCUU UU UCU AAA G-3' Target sequence 146: 5'- GC
content: 40.0% TTC CAC AGG AGG SEQ ID NO: 146 Sense strand: 5'- TAT
TTC-3' (SEQ ID UUCCACAGGAGGUAUUUC UU-3' NO: 623) (Position in SEQ
ID NO: 467 Antisense strand: 5'- gene sequence: 1911)
GAAAUACCUCCUGUGGAA UU-3' SEQ ID NO: 299 dsRNA:
5'-UUCCACAGGAGGUAUUUC UU UCU AAA G-3' Target sequence 147: 5'- GC
content: 40.0% GGT CCT GAG TAT SEQ ID NO: 147 Sense strand: 5'- TTT
GCA-3' (SEQ ID GGUCCUGAGUAUUUUGCA UU-3' NO: 624) (Position in SEQ
ID NO: 468 Antisense strand: 5'- gene sequence: 1940)
UGCAAAAUACUCAGGACC UU-3' SEQ ID NO: 300 dsRNA:
5'-GGUCCUGAGUAUUUUGCA UU UCU AAA G-3' Target sequence 148: 5'- GC
content: 42.86% CCA AAT CTG CAA SEQ ID NO: 148 Sense strand: 5'-
GAA GGC T-3' (SEQ ID CCAAAUCUGCAAGAAGGCU UU-3' NO: 625) (Position
in SEQ ID NO: 469 Antisense strand: 5'- gene sequence: 18)
AGCCUUCUUGCAGAUUUGG UU-3' SEQ ID NO: 301 dsRNA:
5'-CCAAAUCUGCAAGAAGGCU UU UCU AAA G-3' Target sequence 149: 5'- GC
content: 43.48% GGA ACT CTT CAG SEQ ID NO: 149 Sense strand: 5'-
CAC ATC CTT-3' (SEQ GGAACUCUUCAGCACAUCCUU UU-3' ID NO: 626)
(Position in SEQ ID NO: 470 Antisense strand: 5'- gene sequence:
95) AAGGAUGUGCUGAAGAGUUCC UU-3' SEQ ID NO: 302 dsRNA: 5'-
GGAACUCUUCAGCACAUCCUU UU UCU AAA G-3' Target sequence 150: 5'- GC
content: 40.9% CTC TGG ACA ACA SEQ ID NO: 150 Sense strand: 5'- GAA
GAA GA-3' (SEQ CUCUGGACAACAGAAGAAGA UU-3' ID NO: 627) (Position in
SEQ ID NO: 471 Antisense strand: 5'- gene sequence: 116)
UCUUCUUCUGUUGUCCAGAG UU-3' SEQ ID NO: 303 dsRNA:
5'-CUCUGGACAACAGAAGAAGA UU UCU AAA G-3' Target sequence 151: 5'- GC
content: 40.0% TGA GAG CAG TGT SEQ ID NO: 151 Sense strand: 5'- GAT
TCT-3' (SEQ ID UGAGAGCAGUGUGAUUCU UU-3' NO: 628) (Position in SEQ
ID NO: 472 Antisense strand: 5'- gene sequence: 201)
AGAAUCACACUGCUCUCA UU-3' SEQ ID NO: 304 dsRNA:
5'-UGAGAGCAGUGUGAUUCU UU UCU AAA G-3'
Target sequence 152: 5'- GC content: 42.1% CAA GTC TAG CAG SEQ ID
NO: 152 Sense strand: 5'-CAAGUCUAGCAGUGCAU TGC AT-3' (SEQ ID NO:
UU-3' 629) (Position in gene SEQ ID NO: 473 Antisense strand: 5'-
sequence: 221) AUGCACUGCUAGACUUG UU-3' SEQ ID NO: 305 dsRNA:
5'-CAAGUCUAGCAGUGCAU UU UCU AAA G-3' Target sequence 153: 5'- GC
content: 42.86% CTC GCT AAG ACA SEQ ID NO: 153 Sense strand: 5'-
ACT AGC A-3' (SEQ ID CUCGCUAAGACAACUAGCA UU-3' NO: 630) (Position
in SEQ ID NO: 474 Antisense strand: 5'- gene sequence: 270)
UGCUAGUUGUCUUAGCGAGA UU-3' SEQ ID NO: 306 dsRNA:
5'-CUCGCUAAGACAACUAGCA UU UCU AAA G-3' Target sequence 154: 5'- GC
content: 45.45% CAG GTT AAG TTT SEQ ID NO: 154 Sense strand: 5'-
CGG AGG CT-3' (SEQ CAGGUUAAGUUUCGGAGGCU UU-3' ID NO: 631) (Position
in SEQ ID NO: 475 Antisense strand: 5'- gene sequence: 331)
AGCCUCCGAAACUUAACCUG UU-3' SEQ ID NO: 307 dsRNA:
5'-CAGGUUAAGUUUCGGAGGCU UU UCU AAA G-3' Target sequence 155: 5'- GC
content: 54.54% GCT TCC AGG CAC SEQ ID NO: 155 Sense strand: 5'-
CTC CTC TT-3' (SEQ ID GCUUCCAGGCACCUCCUCUU UU-3' NO: 632) (Position
in SEQ ID NO: 476 Antisense strand: 5'- gene sequence: 369)
AAGAGGAGGUGCCUGGAAGC UU-3' SEQ ID NO: 308 dsRNA:
5'-GCUUCCAGGCACCUCCUCUU UU UCU AAA G-3' Target sequence 156: 5'- GC
content: 50.0% GAA GTG GAA GTC SEQ ID NO: 156 Sense strand: 5'- GTG
CTG AG-3' (SEQ GAAGUGGAAGUCGUGCUGAG UU-3' ID NO: 633) (Position in
SEQ ID NO: 477 Antisense strand: 5'- gene sequence: 427)
CUCAGCACGACUUCCACUUC UU-3' SEQ ID NO: 309 dsRNA:
5'-GAAGUGGAAGUCGUGCUGAG UU UCU AAA G-3' Target sequence 157: 5'- GC
content: 50.0% GAT CTC TTT CGC SEQ ID NO: 157 Sense strand: 5'- CAT
GGC TG-3' (SEQ ID GAUCUCUUUCGCCAUGGCUG UU-3' NO: 634) (Position in
SEQ ID NO: 478 Antisense strand: 5'- gene sequence: 481)
CAGCCAUGGCGAAAGAGAUC UU-3' SEQ ID NO: 310 dsRNA:
5'-GAUCUCUUUCGCCAUGGCUG UU UCU AAA G-3'
[0097] The siRNA or dsRNA of the present invention may be one
capable of being loaded on a carrier while carrying RNA molecules
depending on types of the carrier, which is not particularly
limited as long as it is known in the art, and may include, but is
not limited to, at least one selected from the group consisting of,
for example, liposomes, lipofectamines, dendrimers, micelles,
porous silica particles, amino clay, gold nanoparticles, magnetic
nanoparticles, graphene, oxidized graphene, chitosan, dextran,
pectin, manganese dioxide two-dimensional sheet, PVA, gelatin,
silica, glass particles, protamine, exosome, polyethyleneimine,
N-butyl cyanoacrylate, gel foam, ethanol, nanocrystals, nanotubes,
carbon nanoparticles, hyaluronic acid, iron oxide, polylactic acid,
polybutyl cyanoacrylate, albumin, lipid particles, polyethylene
glycol, poly-L-guluronic alginate, polyglycolic-polylactic acid,
polydioxanone, polyglycolic acid-co-caprolactone, polypropylene and
hydrogel, preferably, porous silica particles having advantages
such as high RNA retention, sustained release, biodegradability,
etc.
[0098] The siRNA or dsRNA of the present invention may be loaded on
porous silica particles, wherein the particles are particles of
silica material (SiO.sub.2) and have a nano-sized particle
diameter.
[0099] The porous silica particles may be porous particles having
nano-sized pores and may carry physiologically active substances
("bioactive materials") such as siRNA or dsRNA of the present
invention on the surfaces of the particles and/or insides of the
pores.
[0100] The porous silica particles are biodegradable particles and,
when the particles loaded with the bioactive material and is
administered in a body, may release the bioactive material while
being biodegraded in the body. That is, biodegradation of the
porous silica particles results in release of the bioactive
material. In this case, the porous silica particles according to
the present invention may be slowly degraded in the body so that
the loaded bioactive material can have sustained release
properties.
[0101] For example, t when a ratio of absorbance in the following
Equation 1 becomes 1/2 may be 20 or more.
A.sub.t/A.sub.0 [Equation 1]
[0102] (wherein A.sub.0 is absorbance of the porous silica
particles measured by placing 5 ml of a suspension including 1
mg/ml of the porous silica particles into a cylindrical dialysis
membrane having pores with a diameter of 50 kDa,
[0103] 15 ml of the same solvent as the suspension is placed
outside the dialysis membrane while being in contact with the
dialysis membrane, followed by horizontal agitation at 60 rpm and
37.degree. C. inside and outside the dialysis membrane,
[0104] pH of the suspension is 7.4, and
[0105] A.sub.t is absorbance of the porous silica particles
measured after t hours elapses from the measurement of
A.sub.0).
[0106] Equation 1 indicates how fast the porous silica particles
are degraded under environments similar to the body.
[0107] In Equation 1, the absorbance Ao and At may be measured, for
example, after placing the porous silica particles and the
suspension in a cylindrical dialysis membrane, and further placing
the same suspension on the outside of the dialysis membrane.
[0108] The particles are biodegradable and may be slowly degraded
in the suspension, wherein the diameter of 50 kDa corresponds to
about 5 nm, the biodegraded particles can pass through a 50 kDa
dialysis membrane, this cylindrical dialysis membrane is under
horizontal agitation at 60 rpm such that the suspension is evenly
admixed, and the degraded particles may come out of the dialysis
membrane.
[0109] The absorbance in Equation 1 may be measured, for example,
under an environment in which the suspension outside the dialysis
membrane is replaced with a new suspension. The suspension may be
one that is constantly replaced, one that is replaced at a constant
period wherein the constant period may be periodic or irregular.
For example, the replacement may be performed within a range of 1
hour to 1 week, in particular, at 1-, 2-, 3-, 6-, 12-, 24-hours
intervals, or 2-, 3-, 4-, 7-days interval, etc., 2 0 but it is not
limited thereto.
[0110] A ratio of absorbance of 1/2 means that, after t hours, the
absorbance becomes half of the initial absorbance, therefore, means
that approximately half of the porous silica particles have been
degraded.
[0111] The suspension may be a buffer solution and, for example, at
least one selected from the group consisting of phosphate buffered
saline (PBS) and simulated body fluid (SBF), and more specifically,
PBS.
[0112] t when the ratio of absorbance in Equation 1 becomes 1/2 is
20 or more or 24 or more, for example, t may be 20 to 120,
specifically, 20 to 96, 20 to 72, 30 to 70, 40 to 70, 50 to 65,
etc. within the above range, but it is not limited thereto.
[0113] The particles are characterized in that t when the ratio of
absorbance in Equation 1 becomes 1/5 may be, for example, 70 to
140, specifically, 80 to 140, 80 to 120, 80 to 110, 70 to 140, 70
to 120, 70 to 110, etc. within the above range, but it is not
limited thereto.
[0114] The particles are characterized in that t when the ratio of
absorbance in Equation 1 becomes 1/20 may be, for example, 130 to
220, specifically, 130 to 200, 140 to 200, 140 to 180, 150 to 180,
etc. within the above range, but it is not limited thereto.
[0115] The particles are characterized in that t when the measured
absorbance becomes 0.01 or less may be, for example, 250 or more,
specifically, 300 or more, 350 or more, 400 or more, 500 or more,
1000 or more, etc. within the above range while having an upper
limit of 2000, but it is not limited thereto.
[0116] The particles are characterized in that the absorbance ratio
in Equation 1 has high positive correlation with t, specifically,
Pearson correlation coefficient may be 0.8 or more, for example,
0.9 or more, 0.95 or more, etc.
[0117] t in Equation 1 means how fast the porous silica particles
are degraded under environments similar to the body, for example,
may be controlled by adjusting the surface area, particle diameter,
pore diameter, substituent on the surface of the porous silica
particle and/or inside the pore, compactness of the surface,
etc.
[0118] More particularly, t may be reduced by increasing the
surface area of the particle or may be increased by reducing the
surface area thereof. The surface area may be regulated by
adjusting the diameter of the particles and/or the diameter of the
pores. In addition, placing a substituent on the surface of the
particle and/or the inside of the pore may reduce direct exposure
of the porous silica particles to the environment (such as a
solvent), thereby increasing t. Further, loading the bioactive
material on the porous silica particles and increasing affinity
between the bioactive material and the porous silica particles may
reduce direct exposure of the porous silica particles to the
environment, thereby increasing t. In addition, the surface may be
made more densely in the preparation of the particles so as to
increase t. In the above, various examples of adjusting tin
Equation 1 have been described, but it is not limited thereto.
[0119] The porous silica particles may be, for example, spherical
particles, but it is not limited thereto.
[0120] The average diameter of the porous silica particles may be,
for example, 100 to 1000 nm, specifically, 100 to 800 nm, 100 to
500 nm, 100 to 400 nm, 100 to 300 nm, 100 to 200 nm, etc. within
the above range, but it is not limited thereto.
[0121] The average pore diameter of the particles may be, for
example, 1 to 100 nm, specifically, 4 to 100 nm, 4 to 50 nm, 4 to
30 nm, 10 to 30 nm, etc. within the above range, but it is not
limited thereto. Due to the large pore diameter, the particles may
carry a large amount of the bioactive material and/or the bioactive
material having a large size.
[0122] The porous silica particles may have a BET surface area of,
for example, 200 to 700 m.sup.2/g, specifically, 200 to 700
m.sup.2/g, 200 to 650 m.sup.2/g, 250 to 650 m.sup.2/g, 300 to 700
m.sup.2/g, 300 to 650 m.sup.2/g, 300 to 600 m.sup.2/g, 300 to 550
m.sup.2/g, 300 to 500 m.sup.2/g, 300 to 450 m.sup.2/g, etc. within
the above range, but it is not limited thereto.
[0123] The porous silica particles may have a volume per gram (g)
of, for example, 0.7 to 2.2 ml, specifically, 0.7 to 2.0 ml, 0.8 to
2.2 ml, 0.8 to 2.0 ml, 0.9 to 2.0 ml, 1.0 to 2.0 ml, etc. within
the above range, but it is not limited thereto. If the volume per
gram (g) is too small, a degradation rate may be too high. Further,
it may be difficult to manufacture excessively large particles or
the particles may not have a complete shape.
[0124] The porous silica particles may have a hydrophilic
substituent and/or a hydrophobic substituent on an outer surface
thereof and/or inside the pore. For example, only hydrophilic
substituents or only hydrophobic substituents may be present on
both the surface of the particle and the inside of the pore,
hydrophilic substituents or hydrophobic substituents may be present
on either the surface of the particle or the inside of the pore,
otherwise, a hydrophilic substituent may be present on the surface
of the particle while a hydrophobic substituent may be present
inside of the pore, and vice versa.
[0125] Release of the bioactive material loaded on the porous
silica particles is mainly conducted by degradation of the
particles. Specifically, interaction of the porous silica particles
with respect to the release environment of the bioactive material
is controlled by adjusting the substituent, thereby regulating a
degradation rate of the particles thus to control a release rate of
the bioactive material. Further, the bioactive material may be
diffused and released from the particles wherein adjusting the
substituent may regulate a binding force of the bioactive material
with the particles, thereby controlling the release of the
bioactive material.
[0126] Further, for improvement of the binding force between the
particles and a poorly soluble (hydrophobic) bioactive material, an
additional process may be further included so that a hydrophobic
substituent is present inside the pore while a hydrophilic
substituent is present on the surface of the particle, in
consideration of easiness in use and formulation of the composition
according to the present invention.
[0127] The hydrophilic substituent may include, for example,
aldehyde, keto, carbamate, sulfate, sulfonate, amino, amine,
aminoalkyl, silyl, carboxyl, sulfonic acid, thiol, ammonium,
sulfhydryl, phosphate, ester, imide, thioimide, ether, indene,
sulfonyl, methylphosphonate, polyethylene glycol, substituted or
unsubstituted C.sub.1to C.sub.30 alkyl, substituted or
unsubstituted C.sub.3 to C.sub.30 cycloalkyl, substituted or
unsubstituted C.sub.6 to C.sub.30 aryl, and C.sub.1 to C.sub.30
ester groups, etc., while the hydrophobic substituent may include,
for example, substituted or unsubstituted C.sub.1to C.sub.30 alkyl,
substituted or unsubstituted C.sub.3 to C.sub.30 cycloalkyl,
substituted or unsubstituted C.sub.6 to C.sub.30 aryl, C.sub.2 to
C.sub.30 heteroaryl, halogen, C.sub.1 to C.sub.30 ester, and
halogen-containing groups, etc.
[0128] The "substituted" functional group in the "substituted or
unsubstituted" mentioned above may include at least one selected
from the group consisting of aldehyde, keto, carbamate, sulfate,
sulfonate, amino, amine, aminoalkyl, silyl, carboxyl, sulfonic
acid, thiol, ammonium, sulfhydryl, phosphate, ester, imide,
thioimide, ether, indene, sulfonyl, methylphosphonate and
polyethylene glycol.
[0129] Further, the porous silica particles may be positively
and/or negatively charged on the outer surface thereof and/ or the
inside of the pore. For example, both the surface of the particle
and the inside of the pore may be positively or negatively charged.
Alternatively, only the surface of the particle or the inside of
the pore may be positively or negatively charged. Otherwise, the
surface of the particle may be positively charged while the inside
of the pore may be negatively charged, and vice versa.
[0130] The charging may be performed, for example, by the presence
of a cationic substituent or an anionic substituent.
[0131] The cationic substituent may include, for example, an amino
group or other nitrogen-containing group as a basic group, while
the anionic substituent may include, for example, a carboxyl group
(--COOH), a sulfonic acid group (--SO.sub.3H), or a thiol group
(--SH), etc., but it is not limited thereto.
[0132] Similarly, due to charging as described above, interaction
between the porous silica particles with respect to the environment
for releasing the bioactive material is controlled by adjusting the
substituent so that a degradation rate of the particles may be
regulated thus to control a release rate of the bioactive material.
Further, the bioactive material may be diffused and released from
the particles wherein adjusting the substituent may regulate a
binding force of the bioactive material with the particles, thereby
controlling the release of the bioactive material.
[0133] Other than the above substituents, the porous silica
particles may further include another substituent, which is present
on the surface of the particle and/or the inside of the pore, in
order to carry a bioactive material, transfer the bioactive
material to a target cell, carry a material used for other purposes
or bind other additional substituents, etc., wherein the
substituent may further include an antibody, a ligand, a cell
permeable peptide, an aptamer, etc. coupled thereto.
[0134] The above-mentioned substituents, charges, coupled
substances, etc. present on the surface of the particle and/or the
inside of the pore may be added thereto, for example, by surface
modification.
[0135] The surface modification may be performed, for example, by
reacting a compound having a substituent to be introduced, with the
particles. In this regard, the compound may include, for example,
alkoxysilane having a Cl to C10 alkoxy group, but it is not limited
thereto. The alkoxysilane may have at least one alkoxy group,
specifically, 1 to 3 alkoxy groups, and may have a substituent to
be introduced into a site in which the alkoxy group is not bonded
or a substituent substituted with the alkoxy group.
[0136] The porous silica particles may be prepared by, for example,
a small pore particle preparation and pore expansion process. If
necessary, the particles may be prepared through further
calcination, and surface modification processes, etc. If the
particles are subjected to both the calcination and the surface
modification processes, the particles may be surface-modified after
the calcinations.
[0137] The small pore particles may be, for example, particles
having an average pore diameter of 1 to 5 nm.
[0138] The small pore particles may be obtained by adding a
surfactant and a silica precursor to a solvent and then agitating
and homogenizing the solution.
[0139] Water and/or organic solvents may be used as the solvent,
and the organic solvent used herein may include, for example:
ethers such as 1,4-dioxane (particularly cyclic ethers);
halogenated hydrocarbons such as chloroform, methylene chloride,
carbon tetrachloride, 1,2-dichloroethane, dichloroethylene,
trichloroethylene, perchloroethylene, dichloropropane, amyl
chloride, 1,2-dibromoethane, etc.; ketones such as acetone,
methylisobutylketone, y-butyrolactone,
1,3-dimethyl-imidazolidinone, methylethylketone, cyclohexanone,
cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc.; carbon-based
aromatics such as benzene, toluene, xylene, tetramethylbenzene,
etc.; alkyl amides such as N,N-dimethylformamide,
N,N-dibutylformamide, N,N-dimethylacetamide, N-methylpyrrolidone,
etc.; alcohols such as methanol, ethanol, propanol, butanol, etc.;
glycol ethers (CELLOSOLVE) such as ethyleneglycol monoethylether,
ethyleneglycol monomethylether, ethyleneglycol monobutylether,
diethyleneglycol monoethylether, diethyleneglycol monomethylether,
diethyleneglycol monobutylether, propyleneglycol monomethylether,
propyleneglycol monoethylether, dipropyleneglycol diethylether,
triethyleneglycol monoethylether, etc.; and dimethylacetamide
(DMAc), N,N-diethylacetamide, dimethylformamide (DMF),
diethylformamide (DEF), N,N-dimethylacetamide (DMAc),
N-methylpyrrolidone (NMP), N-ethylpyrrolidone (NEP),
1,3-dimethyl-2-imidazolidinone, N,N-dimethylmethoxyacetamide,
dimethyl sulfoxide, pyridine, dimethyl sulfone, hexamethyl
phosphoamide, tetramethylurea, N-methylcarrolactam,
tetrahydrofuran, m-dioxane, p-dioxane, 1,2-dimethoxyethan and the
like. Specifically, alcohol, more specifically, methanol may be
used, but it is not limited thereto.
[0140] When using a mixed solvent of water and an organic solvent
as the solvent, a ratio of water and an organic solvent may be used
in a volume ratio of, for example, 1:0.7 to 1.5, e.g., 1:0.8 to
1.3, but it is not limited thereto.
[0141] The surfactant may be, for example, cetyltrimethylammonium
bromide (CTAB), hexadecyltrimethylammonium bromide (TMABr),
hexadecyltrimethylpyridinium chloride (TMPrCl), tetramethylammonium
chloride (TMACl), and the like, and specifically, CTAB may be
used.
[0142] The surfactant may be added in an amount of, for example, 1
to 10 g per liter of solvent, specifically, 1 to 8 g, 2 to 8 g, 3
to 8 g, etc. within the above range, but it is not limited
thereto.
[0143] The silica precursor may be added after the agitation with
addition of the surfactant to the solvent. The silica precursor may
be, for example, tetramethyl orthosilicate (TMOS), but it is not
limited thereto.
[0144] The agitation may be performed, for example, for 10 to 30
minutes, but it is not limited thereto.
[0145] The silica precursor may be added thereto, for example, in
an amount of 0.5 to 5 ml per liter of solvent, specifically, 0.5 to
4 ml, 0.5 to 3 ml, 0.5 to 2 ml, 1 to 2 ml, etc. within the above
range, but it is not limited thereto. Rather, if necessary, sodium
hydroxide as a catalyst may be further used, wherein the catalyst
may be added while agitating after adding the surfactant to the
solvent and before adding the silica precursor to the solvent.
[0146] Sodium hydroxide may be used in an amount of, for example,
0.5 to 8 ml per liter of solvent, specifically, 0.5 to 5 ml, 0.5 to
4 ml, 1 to 4 ml, 1 to 3 ml, 2 to 3 ml, etc. within the above range,
based on 1 M aqueous sodium hydroxide solution, but is not limited
thereto.
[0147] After the addition of the silica precursor, the solution may
be reacted with agitation. The agitation may be performed, for
example, for 2 to 15 hours, specifically, 3 to 15 hours, 4 to 15
hours, 4 to 13 hours, 5 to 12 hours, 6 to 12 hours, 6 to 10 hours,
etc. within the above range, but it is not limited thereto. If an
agitating time (reaction time) is too short, nucleation may be
insufficient.
[0148] After the agitation, the solution may be aged. Aging may be
performed, for example, for 8 to 24 hours, specifically, 8 to 20
hours, 8 to 18 hours, 8 to 16 hours, 8 to 14 hours, 10 to 16 hours,
10 to 14 hours, etc. within the above range, but it is not limited
thereto.
[0149] Thereafter, the reaction product may be washed and dried to
obtain porous silica particles and, if necessary, unreacted
material may be isolated before washing, which may be performed,
for example, by separating the supernatant through
centrifugation.
[0150] The centrifugation may be implemented, for example, at 6,000
to 10,000 rpm, for example, for 3 to 60 minutes, specifically, 3 to
30 minutes, 5 to 30 minutes, etc. within the above range, but it is
not limited thereto.
[0151] The washing may be carried out with water and/or an organic
solvent. In particular, since different substances are soluble in
different solvents, respectively, water and the organic solvent may
be used once or several times by turns. Alternatively, water and/or
the organic solvent may be used alone for washing once or several
times. Such several times may include, for example, two or more,
ten or less, specifically, three or more and ten or less, four or
more and eight or less, four or more and six or less, etc.
[0152] The organic solvent used herein may include, for example:
ethers such as 1,4-dioxane (particularly cyclic ethers);
halogenated hydrocarbons such as chloroform, methylene chloride,
carbon tetrachloride, 1,2-dichloroethane, dichloroethylene,
trichloroethylene, perchloroethylene, dichloropropane, amyl
chloride, 1,2-dibromoethane, etc.; ketones such as acetone,
methylisobutylketone, y-butyrolactone,
1,3-dimethyl-imidazolidinone, methylethylketone, cyclohexanone,
cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc.; carbon-based
aromatics such as benzene, toluene, xylene, tetramethylbenzene,
etc.; alkyl amides such as N,N-dimethylformamide,
N,N-dibutylformamide, N,N-dimethylacetamide, N-methylpyrrolidone,
etc.; alcohols such as methanol, ethanol, propanol, butanol, etc.;
glycol ethers (CELLOSOLVE) such as ethyleneglycol monoethylether,
ethyleneglycol monomethylether, ethyleneglycol monobutylether,
diethyleneglycol monoethylether, diethyleneglycol monomethylether,
diethyleneglycol monobutylether, propyleneglycol monomethylether,
propyleneglycol monoethylether, dipropyleneglycol diethylether,
triethyleneglycol monoethylether, etc.; and dimethylacetamide
(DMAc), N,N-diethylacetamide, dimethylformamide (DMF),
diethylformamide (DEF), N,N-dimethylacetamide (DMAc),
N-methylpyrrolidone (NMP), N-ethylpyrrolidone (NEP),
1,3-dimethyl-2-imidazolidinone, N,N-dimethylmethoxyacetamide,
dimethyl sulfoxide, pyridine, dimethyl sulfone, hexamethyl
phosphoamide, tetramethylurea, N-methylcarrolactam,
tetrahydrofuran, m-dioxane, p-dioxane, 1,2-dimethoxyethane, etc.,
and, specifically, alcohol and, more specifically, ethanol may be
used, but it is not limited thereto.
[0153] The washing may be performed under centrifugation, for
example, at 6,000 to 10,000 rpm, for example, for 3 to 60 minutes,
specifically, 3 to 30 minutes, 5 to 30 minutes, etc. within the
above range, but it is not limited thereto.
[0154] The washing may be performed by filtering particles with a
filter without centrifugation. The filter may include pores with a
diameter of less than or equal to the diameter of the porous silica
particles. If the reaction solution is filtered through such a
filter, only particles remain on the filter and may be washed by
pouring water and/or an organic solvent over the filter.
[0155] For washing, water and the organic solvent may be used once
or several times by turns. Alternatively, the washing may be
performed once or several times even with water or the organic
solvent alone. The several times may include, for example, two or
more and ten or less, specifically, three or more and ten or less,
four or more and eight or less, four or more and six or less and
the like.
[0156] The drying may be performed, for example, at 20 to
100.degree. C., but it is not limited thereto. Alternatively, the
drying may be performed in a vacuum state.
[0157] Thereafter, the pores of the obtained porous silica
particles may be expanded using, for example, a pore swelling
agent.
[0158] The pore swelling agent used herein may include, for
example, trimethylbenzene, triethylbenzene, tripropylbenzene,
tributylbenzene, tripentylbenzene, trihexylbenzene, toluene,
benzene, etc. and, specifically, trimethylbenzene may be used, but
it is not limited thereto.
[0159] Alternatively, the pore swelling agent used herein may be,
for example, N,N-dimethylhexadecylamine (DMHA), but it is not
limited thereto.
[0160] Pore expansion described above may be performed, for
example, by mixing porous silica particles in a solvent with a pore
swelling agent, and heating and reacting the mixture.
[0161] The solvent used herein may be, for example, water and/or an
organic solvent. The organic solvent used herein may include, for
example: ethers such as 1,4-dioxane (particularly cyclic ethers);
halogenated hydrocarbons such as chloroform, methylene chloride,
carbon tetrachloride, 1,2-dichloroethane, dichloroethylene,
trichloroethylene, perchloroethylene, dichloropropane, amyl
chloride, 1,2-dibromoethane, etc.; ketones such as acetone,
methylisobutylketone, cyclohexanone, etc.; carbon-based aromatics
such as benzene, toluene, xylene, etc.; alkyl amides such as
N,N-dimethylformamide, N,N-dibutylformamide, N,N-dimethylacetamide,
N-methylpyrrolidone, etc.; alcohols such as methanol, ethanol,
propanol, butanol, etc.; and, specifically, alcohol and, more
specifically, ethanol may be used, but it is not limited
thereto.
[0162] The porous silica particles may be added in a ratio of, for
example, 10 to 200 g per liter of solvent, specifically, 10 to 150
g, 10 to 100 g, 30 to 100 g, 40 to 100 g, 50 to 100 g, 50 to 80 g,
60 to 80 g, etc. within the above range, but it is not limited
thereto.
[0163] The porous silica particles may be evenly dispersed in a
solvent, for example, the porous silica particles may be added to
the solvent and ultrasonically dispersed therein. In the case of
using a mixed solvent, the second solvent may be added after the
porous silica particles are dispersed in the first solvent.
[0164] The pore swelling agent may be added in an amount of, for
example, 10 to 200 parts by volume (vol. parts), specifically, 100
to 150 vol. parts, 10 to 100 vol. parts, 10 to 80 vol. parts, 30 to
80 vol. parts, 30 to 70 vol. parts based on 100 vol. parts of
solvent within the above range, but it is not limited thereto.
[0165] The reaction may be performed, for example, at 120 to
180.degree. C., specifically, 120 to 170.degree. C., 120 to
160.degree. C., 120 to 150.degree. C., 130 to 180.degree. C., 130
to 170.degree. C., 130 to 160.degree. C., 130 to 150.degree. C.,
etc. within the above range, but it is not limited thereto.
[0166] The reaction may be performed, for example, for 24 to 96
hours, 2 0 specifically, 30 to 96 hours, 30 to 80 hours, 30 to 72
hours, 24 to 80 hours, 24 to 72 hours, 36 to 96 hours, 36 to 80
hours, 36 to 72 hours, 36 to 66 hours, 36 to 60 hours, 48 to 96
hours, 48 to 88 hours, 48 to 80 hours, 48 to 72 hours, etc. within
the above range, but it is not limited thereto.
[0167] By adjusting the time and the temperature within the above
ranges, respectively, the reaction may be performed sufficiently
without being too much. For example, when the reaction temperature
is lower, the reaction time may be increased, otherwise, when the
reaction temperature is lower, the reaction time may be shortened.
If the reaction is not sufficient, pore expansion may not be
sufficient. On the other hand, if the reaction proceeds
excessively, the particles may collapse due to the expansion of the
pores.
[0168] The reaction may be performed, for example, while gradually
increasing the temperature. Specifically, the reaction may be
performed while gradually increasing the temperature at a rate of
0.5 to 15.degree. C./min from the room temperature, specifically, 1
to 15.degree. C./min, 3 to 15.degree. C./min, 3 to 12.degree.
C./min, 3 to 10.degree. C./min, etc. within the above range, but it
is not limited thereto.
[0169] After the reaction, the reaction solution may be cooled
slowly, for example, cooled by lowering the temperature step by
step. Specifically, the reaction solution may be cooled by
gradually decreasing the temperature at a rate of 0.5 to 20.degree.
C./min to room temperature, specifically, 1 to 20.degree. C./min, 3
to 20.degree. C./min, 3 to 12.degree. C./min, 3 to 10.degree.
C./min, etc. within the above range, but it is not limited
thereto.
[0170] After cooling, the reaction product may be washed and dried
to obtain porous silica particles having expanded pores. If
necessary, unreacted material may be isolated prior to washing, for
example, by centrifugation to separate a supernatant.
[0171] The centrifugation may be performed, for example, at 6,000
to 10,000 rpm for 3 to 60 minutes, specifically, 3 to 30 minutes, 5
to 30 minutes, etc. within the above range, but it is not limited
thereto.
[0172] The washing may be carried out with water and/or an organic
solvent. In particular, since different substances are soluble in
different solvents respectively, water and the organic solvent may
be used once or several times by turns. Alternatively, water and/or
the organic solvent may be used alone for washing once or several
times. Such several times may include, for example, two or more,
ten or less, specifically, three times, 4 times, 5 times, 6 times,
7 times, 8 times, etc.
[0173] The organic solvent used herein may include, for example:
ethers such as 1,4-dioxane (particularly cyclic ethers);
halogenated hydrocarbons such as chloroform, methylene chloride,
carbon tetrachloride, 1,2-dichloroethane, dichloroethylene,
trichloroethylene, perchloroethylene, dichloropropane, amyl
chloride, 1,2-dibromoethane, etc.; ketones such as acetone,
methylisobutylketone, cyclohexanone, etc.; carbon-based aromatics
such as benzene, toluene, xylene, etc.; alkyl amides such as
N,N-dimethylformamide, N,N-dibutylformamide, N,N-dimethylacetamide,
N-methylpyrrolidone, etc.; alcohols such as methanol, ethanol,
propanol, butanol, etc.; specifically, alcohol, more specifically,
ethanol may be used, but it is not limited thereto.
[0174] The washing may be carried out under centrifugation, for
example at 6,000 to 10,000 rpm, for example, for 3 to 60 minutes,
specifically, 3 to 30 minutes, 5 to 30 minutes, etc. within the
above range, but it is not limited thereto.
[0175] The washing may be performed by filtering particles with a
filter without centrifugation. The filter may have pores with a
diameter of less than or equal to the diameter of the porous silica
particles. If the reaction solution is filtered through such a
filter, only particles remain on the filter and may be washed by
pouring water and/or an organic solvent over the filter.
[0176] For washing, water and the organic solvent may be used once
or several times by turns. Alternatively, the washing may be
performed once or several times even with water or the organic
solvent alone. The several times may include, for example, two or
more and ten or less, specifically, three or more and ten or less,
four or more and eight or less, four or more and six or less and
the like.
[0177] The drying may be performed, for example, at 20 to
100.degree. C., but it is not limited thereto. Alternatively, the
drying may be performed in a vacuum state.
[0178] Thereafter, the pores of the obtained porous silica
particles may be subjected to calcinations, which is a process of
heating the particles to have a denser structure on the surface
thereof and the inside of the pore, and removing organic materials
filling the pores. For example, the calcinations may be performed
at 400 to 700.degree. C. for 3 to 8 hours, specifically, at 500 to
600.degree. C. for 4 to 5 hours, but it is not limited thereto.
[0179] Then, the obtained porous silica particles may be modified
on the surface and/or the inside of the pore as described
above.
[0180] The surface modification may be performed on the surface of
the particle and/or the inside of the pore. The surface of the
particle and the inside of the pore may be surface-modified in the
same manner or differently.
[0181] The particles may be charged or have hydrophilic and/or
hydrophobic properties through surface modification. The surface
modification may be performed, for example, by reacting a compound
having a hydrophilic, hydrophobic, cationic or anionic substituent
to be introduced, with the particles. In this regard, the compound
may include, for example, alkoxysilane having a C1 to C10 alkoxy
group, but it is not limited thereto. The alkoxysilane may have at
least one alkoxy group, specifically, 1 to 3 alkoxy groups, and may
have a substituent to be introduced into a site in which the alkoxy
group is not bonded or a substituent substituted with the alkoxy
group.
[0182] When the alkoxysilane is reacted with the porous silicon
particles, an alkoxysilane can be bonded to the surface of the
porous silica particle and/or the inside of pore through a covalent
bond between a silicon atom and an oxygen atom. Further, since the
alkoxysilane has a substituent to be introduced, this substituent
may be introduced into the surface of the porous silica particle
and/or the inside of the pore.
[0183] The above reaction may be performed by reacting the porous
silica particles dispersed in a solvent with alkoxysilane.
[0184] Water and/or organic solvents may be used as the solvent,
and the organic solvent used herein may include, for example:
ethers such as 1,4-dioxane (particularly cyclic ethers);
halogenated hydrocarbons such as chloroform, methylene chloride,
carbon tetrachloride, 1,2-dichloroethane, dichloroethylene,
trichloroethylene, perchloroethylene, dichloropropane, amyl
chloride, 1,2-dibromoethane, etc.; ketones such as acetone,
methylisobutylketone, y-butyrolactone,
1,3-dimethyl-imidazolidinone, methylethylketone, cyclohexanone,
cyclopentanone, 4-hydroxy-4-methyl-2-pentanone, etc.; carbon-based
aromatics such as benzene, toluene, xylene, tetramethylbenzene,
etc.; alkyl amides such as N,N-dimethylformamide,
N,N-dibutylformamide, N,N-dimethylacetamide, N-methylpyrrolidone,
etc.; alcohols such as methanol, ethanol, propanol, butanol, etc.;
glycol ethers (CELLOSOLVE) such as ethyleneglycol monoethylether,
ethyleneglycol monomethylether, ethyleneglycol monobutylether,
diethyleneglycol monoethylether, diethyleneglycol monomethylether,
diethyleneglycol monobutylether, propyleneglycol monomethylether,
propyleneglycol monoethylether, dipropyleneglycol diethylether,
triethyleneglycol monoethylether, etc.; and dimethylacetamide
(DMAc), N,N-diethylacetamide, dimethylformamide (DMF),
diethylformamide (DEF), N,N-dimethylacetamide (DMAc),
N-methylpyrrolidone (NMP), N-ethylpyrrolidone (NEP),
1,3-dimethyl-2-imidazolidinone, N,N-dimethylmethoxyacetamide,
dimethyl sulfoxide, pyridine, dimethyl sulfone, hexamethyl
phosphoamide, tetramethylurea, N-methylcarrolactam,
tetrahydrofuran, m-dioxane, p-dioxane, 1,2-dimethoxyethan and the
like. Specifically, alcohol, more specifically, methanol may be
used, but it is not limited thereto.
[0185] The positively charging may be performed by reacting the
particles with, for example, alkoxysilane having a basic group,
that is, a nitrogen-containing group such as amino, aminoalkyl,
etc. Specifically, N-[3-(trimethoxysilyl)propyl]ethylenediamine,
N1-(3-trimethoxysilylpropy)diethylenetriamine,
(3-aminopropyl)trimethoxysilane,
N-[3-(trimethoxysily0propyl]aniline, trimethoxy
[3-(methylamino)propyl]silane,
3-(2-aminoethylamino)propyldimethoxymethylsilane, etc. may be used,
but it is not limited thereto.
[0186] The negatively charging may be performed by reacting the
particles with, for example, alkoxysilane having an acidic group
such as carboxyl, sulfonic acid, thiol, etc. Specifically,
(3-mercaptopropyl)trimethoxysilane may be used, but it is not
limited thereto.
[0187] The hydrophilic property may be obtained by reacting the
particles with, for example, alkoxysilane having a hydrophilic
group such as hydroxyl, carboxyl, amino, carbonyl, sulfhydryl,
phosphate, thiol, ammonium, ester, imide, thioimide, keto, ether,
indene, sulfonyl, polyethyleneglycol, etc. Specifically,
N-[3-(trimethoxysilyl) propyl]ethylenediamine,
N1-(3-trimethoxysilylpropyl)diethylenetriamine,
(3-aminopropyl)trimethoxysilane,
(3-mercaptopropyl)trimethoxysilane, trimethoxy
[3-(methylamino)propyl]silane,
3-(2-aminoethylamino)propyldimethoxymethylsilane, etc. may be used,
but it is not limited thereto.
[0188] The hydrophobic property may be obtained by reacting the
particles with, for example, alkoxysilane having a hydrophobic
substituent such as substituted or unsubstituted C.sub.1 C.sub.30
alkyl, substituted or unsubstituted C.sub.3 to C.sub.30 cycloalkyl,
substituted or unsubstituted C.sub.6 to C.sub.30 aryl, substituted
or unsubstituted C.sub.2 to C.sub.30 heteroaryl, halogen, C.sub.1
to C.sub.30 ester, and halogen-containing groups, etc.
Specifically, trimethoxy(octadecyl)silane,
trimethoxy-n-octylsilane, trimethoxy(propyl)silane,
isobutyl(trimethoxy)silane, trimethoxy(7-octen-1-yl)silane,
trimethoxy(2-phenylethyl)silane, vinyltrimethoxysilane,
cyanomethyl, 3-[(trimethoxysilyl)propyl]trithiocarbonate and
(3-bromopropyl)trimethoxysilane, etc. may be used, but it is not
limited thereto.
[0189] For improvement of the binding force between the particles
and a poorly soluble (hydrophobic) bioactive material through
surface modification, an additional process may be further included
so that a hydrophobic substituent is present inside the pore while
a hydrophilic substituent is present on the surface of the
particle, in consideration of easiness in use and formulation of
the composition according to the present invention. Further, a
substituent for binding another material other than the bioactive
material to the surface of the particle may be further
provided.
[0190] Further, the surface modification may be performed in
combination. For example, two or more surface modification may be
performed on the outer surface of the particle or inside the pore.
As a specific example, a compound containing a carboxyl group may
be bonded to an amide-introduced silica particle through amide bond
to change positively charged particles to have different surface
characteristics, but it is not limited thereto.
[0191] The reaction of the porous silica particles with
alkoxysilane may be performed, for example, under heating.
[0192] The heating may be performed at 80 to 180.degree. C., for
example, in a range of 80 to 160.degree. C., 80 to 150.degree. C.,
100 to 160.degree. C., 100 to 150.degree. C., 110 to 150.degree.
C., etc., but it is not limited thereto.
[0193] The reaction of the particles with alkoxysilane may be
implemented for 4 to 20 hours, for example, in a range of 4 to 18
hours, 4 to 16 hours, 6 to 18 hours, 6 to 16 hours, 8 to 18 hours,
8 to 16 hours, 8 to 14 hours, 10 to 14 hours, etc., but it is not
limited thereto.
[0194] A reaction temperature, time, and the amount of the compound
used for surface modification may be selected depending on a
desired extent of surface modification. Further, varying reaction
conditions depending on hydrophilicity, hydrophobicity and a charge
level of the bioactive material may regulate hydrophilicity,
hydrophobicity and charge level of the silica particles, thereby
controlling the release rate of the bioactive material. For
example, if the bioactive material has strong negative charge at
neutral pH, the reaction temperature may be increased, the reaction
time may be extended, or an amount of the compound to be treated
may also be increased so that the porous silica particles have
strong positive charge, but it is not limited thereto.
[0195] Further, the porous silica particles may be manufactured by,
for example, preparation of small pore particles, expansion of
pores, surface modification, modification of inside of the pore and
the like.
[0196] Preparation of the small pore particles and pore expansion
may be performed by the processes described above, and the washing
and drying processes may be performed after the preparation of the
small pore particles and after the pore expansion.
[0197] If necessary, isolation of the unreacted material may be
preceded by washing, for example, conducted by separating the
supernatant through centrifugation.
[0198] The centrifugation may be performed at, for example, 6,000
to 10,000 rpm, for example, for 3 to 60 minutes, specifically, 3 to
30 minutes, 5 to 30 minutes, etc. within the above range, but it is
not limited thereto.
[0199] The washing after the preparation of the small pore
particles may be performed by any method under conditions within
the above-illustrated range, but it is not limited thereto.
[0200] The washing after the pore expansion may be performed under
more relaxed conditions than the above illustrative embodiments.
For example, washing may be carried out three times or less, but it
is not limited thereto.
[0201] The surface of the particle and/or the inside of the pore
may be modified by the above-described method, wherein the
modification may be performed in an order of the surface of the
particle and then the inside of the pore, and particle washing may
be further performed between the above two processes.
[0202] When the washing is carried out in more relaxed conditions
after the preparation of small pore particles and pore expansion,
the pores are filled with a reaction solution such as a surfactant
used in the particle preparation and the pore expansion, such that
the inside of the pore is not modified during surface modification,
instead, only the surface of the particle may be modified. After
then, washing the particles may remove the reaction solution in the
pores.
[0203] Particle washing between surface modification and
modification of the inside of the pore may be performed with water
and/or an organic solvent. In particular, since different
substances are soluble in different solvents respectively, water
and the organic solvent may be used once or several times by turns.
Alternatively, water and/or the organic solvent may be used alone
for washing once or several times. Such several times may include,
for example, two or more, ten or less, specifically, three or more
and ten or less, four or more and eight or less, four or more and
six or less, etc.
[0204] The washing may be performed under centrifugation, for
example, at 6,000 to 10,000 rpm, for example, for 3 to 60 minutes,
specifically, 3 to 30 minutes, 5 to 30 minutes, etc. within the
above range, but it is not limited thereto.
[0205] The washing may be performed by filtering particles with a
filter without centrifugation. The filter may include pores with a
diameter of less than or equal to the diameter of the porous silica
particles. If the reaction solution is filtered through such a
filter, only particles remain on the filter and may be washed by
pouring water and/or an organic solvent over the filter.
[0206] For washing, water and the organic solvent may be used once
or several times by turns. Alternatively, the washing may be
performed once or several times even with water or the organic
solvent alone. The several times may include, for example, two or
more and ten or less, specifically, three or more and ten or less,
four or more and eight or less, four or more and six or less and
the like. [22 6] The drying may be performed, for example, at 20 to
100.degree. C., but it is not limited thereto. Alternatively, the
drying may be performed in a vacuum state.
[0207] The bioactive material such as siRNA or dsRNA of the present
invention may be loaded on the surface of the particle and/or the
inside of the pore.
[0208] The loading may be implemented, for example, by mixing the
porous silica particles and the bioactive material in a
solvent.
[0209] Water and/or organic solvents may be used as the solvent,
and the organic solvent used herein may include, for example:
ethers such as 1,4-dioxane (particularly cyclic ethers);
halogenated hydrocarbons such as chloroform, methylene chloride,
carbon tetrachloride, 1,2-dichloroethane, dichloroethylene,
trichloroethylene, perchloroethylene, dichloropropane, amyl
chloride, 1,2-dibromoethane, etc.; ketones such as acetone,
methylisobutylketone, cyclohexanone, etc.; carbon-based aromatics
such as benzene, toluene, xylene, etc.; alkyl amides such as
N,N-dimethylformamide, N,N-dibutylformamide, N,N-dimethylacetamide,
N-methylpyrrolidone, etc.; alcohols such as methanol, ethanol,
propanol, butanol, etc. and the like.
[0210] Further, a phosphate buffered saline solution (PBS),
simulated body fluid (SBF), borate-buffered saline, tris-buffered
saline may be used as the solvent.
[0211] A ratio of the porous silica particles and the bioactive
material is not particularly limited and, for example, the weight
ratio may be 1:0.05 to 0.8, specifically, 1:0.05 to 0.7, 1:0.05 to
0.6, 1:0.1 to 0.8, 1:0.1 to 0.6, 1:0.2 to 0.8, 1:0.2 to 0.6, etc.
within the above range.
[0212] The bioactive material such as siRNA or dsRNA of the present
invention loaded on the porous silica particles may be gradually
released over an extended period of time. Such slow release may be
continuous or non-continuous, or linear or non-linear, and may vary
due to the characteristics of the porous silica particles and/or
interaction thereof with the bioactive material.
[0213] The bioactive material loaded on the porous silica particles
is released while the porous silica particles are biodegraded. More
particularly, the porous silica particles according to the present
invention may be slowly degraded to release the loaded bioactive
material in a sustained manner. Such release may be controlled by,
for example, adjusting the surface area, particle diameter, pore
diameter, substituents on the surface of the particle and/or the
inside of pore, compactness of the porous silica particles, and the
like, but it is not limited thereto.
[0214] In addition, the bioactive material loaded on the particles
may be released while being separated from the porous silica
particles and diffused, which is affected by the relationship
between the porous silica particles, the bioactive material and the
bioactive material releasing environment. Therefore, adjusting
these conditions may control the release of bioactive material. For
example, the release of bioactive material may be controlled by
strengthening or weakening the binding force of the porous silica
particles with the bioactive material by surface modification.
[0215] More particularly, if the loaded bioactive material is
poorly water-soluble (hydrophobic), the surface of the particle
and/or the inside of the pore may have a hydrophobic substituent to
increase the binding force between the particles and the bioactive
material, whereby the bioactive material may be released in a
sustained manner. This may be achieved by, for example, surface
modification of the particles with alkoxysilane having a
hydrophobic substituent.
[0216] As used herein, "poorly soluble" means being insoluble
(practically insoluble) or only slightly soluble (with respect to
water), which is a terminology defined in "pharmaceutical Science"
18.sup.th Edition (U.S.P, Remington, Mack Publishing Company).
[0217] The poorly water-soluble bioactive material may have, for
example, water solubility of less than 10 g/L, specifically less
than 5 g/L, more specifically less than 1 g/L at 1 atmosphere and
25.degree. C., but it is not limited thereto.
[0218] When the loaded bioactive material is water-soluble
(hydrophilic), the surface of the particle and/or the inside of the
pore may have a hydrophilic substituent to increase the binding
force between the porous silica particles and the bioactive
material, whereby the bioactive materials may be released in a
sustained manner.
[0219] This may be achieved by, for example, surface modification
of the porous silica particles with alkoxysilane having a
hydrophilic substituent.
[0220] The water-soluble bioactive material may have, for example,
water solubility of 10 g/L or more at 1 atmosphere and 25.degree.
C., but it is not limited thereto.
[0221] When the loaded bioactive material is charged, the surface
of the particle and/or the inside of the pore may be charged with
the opposite charge thus to increase the binding force between the
porous silica particles and the bioactive material, whereby the
bioactive material may be released in a sustained manner. This may
be achieved by, for example, surface modification of the porous
silica particles with alkoxysilane having an acidic group or a
basic group.
[0222] Specifically, if the bioactive material is positively
charged at neutral pH, the surface of the particle and/or the
inside of the pore may be negatively charged at neutral pH thus to
increase the binding force between the porous silica particles and
the bioactive material, whereby the bioactive material may be
released in a sustained manner. This may be achieved by, for
example, surface modification of the porous silica particles with
alkoxysilane having an acidic group such as a carboxyl group
(-COOH), sulfonic acid group (--SO.sub.3H), etc.
[0223] Further, if the bioactive material is negatively charged at
neutral pH, the surface of the particle and/or the inside of the
pore may be positively charged thus to increase the binding force
between the porous silica particles and the bioactive material,
whereby the bioactive material may be release in a sustained
manner. This may be achieved by, for example, surface modification
of the porous silica particles with alkoxysilane having a basic
group such as an amino group, nitrogen-containing group, etc.
[0224] The loaded bioactive material may be released for a period
of, for example, 7 days to 1 year or more depending on the type of
treatment required, release environment, and porous silica
particles to be used, etc.
[0225] Since the porous silica particles are biodegradable and may
be degraded by 100%, the bioactive material loaded thereon can be
released by 100%.
[0226] The pharmaceutical composition for preventing or treating
liver cancer, which includes siRNA or dsRNA of the present
invention, may further include pharmaceutically acceptable carrier
and may be formulated together with the same. As used herein, the
term "pharmaceutically acceptable carrier" refers to a carrier or
diluent that does not irritate an organism and does not inhibit
biological activities and properties of the administered compound.
The pharmaceutically acceptable carrier in a composition formulated
in a liquid solution is sterile and physiologically compatible, and
may include saline, sterile water, Ringer's solution, buffered
saline, albumin injectable solution, dextrose solution,
maltodextrin solution, glycerol, ethanol, and a combination of one
or more of these components. Further, if necessary, other
conventional additives such as antioxidants, buffers and
bacteriostatic agents may also be added thereto. In addition,
diluents, dispersants, surfactants, binders and lubricants may also
be added so as to formulate the composition into injectable
formulations such as aqueous solution, suspension, emulsion, etc.,
pills, capsules, granules or tablets and the like.
[0227] The composition of the present invention is applicable in
any type of formulation that contains the siRNA or dsRNA of the
present invention as an active ingredient, and may be prepared in
oral or parenteral formulations. Such pharmaceutical formulations
of the invention may include any one suitable for oral, rectal,
nasal, topical (including the cheek and sublingual), subcutaneous,
vaginal or parenteral (intramuscular, subcutaneous) administration,
or otherwise, may be suitable for administration through inhalation
or insufflation.
[0228] The composition of the present invention may be administered
in a pharmaceutically effective amount. An effective dose level may
be determined in consideration of the type of disease, severity,
activity of the drug, sensitivity to the drug, administration time,
administration route and rate of release, duration of treatment,
factors including concurrent drug use, and other factors well known
in the medical field. The composition of the present invention may
be administered as a separate therapeutic agent or in combination
with other therapeutic agents, may be administered sequentially or
simultaneously with conventional therapeutic agents, and may be
administered in single or multiple doses. Taking all of the above
factors into consideration, it is important to administer a minimum
amount that can obtain maximum effects without side effects, which
can be easily determined by those skilled in the art.
[0229] Dosage of the composition of the present invention may vary
greatly depending on a weight, age, gender and/or health condition
of a patient, diet, administration time, method of administration,
excretion rate and severity of the disease. Specifically, an
appropriate dosage may depend on the amount of drug accumulated in
the body and/or specific efficacy of the siRNA or dsRNA of the
present invention to be used. In general, the dosage may be
estimated based on EC50 determined to be effective in in vivo
animal models as well as in vitro. For example, the dosage may
range from 0.01 .mu.g to 1 g per kg of body weight, and the
composition may be administered once or several times per unit
period, in daily, weekly, monthly or yearly unit periods.
Otherwise, the composition may be continuously administered for a
long period of time via an infusion pump. The number of repeated
doses is determined in consideration of a retention time of drug
remaining in the body, a concentration of drug in the body and the
like. Even after the treatment in the course of the disease
treatment, the composition may be administered for preventing
relapse.
[0230] The composition of the present invention may further include
at least one active ingredient having the same or similar function
in relation to treatment of liver cancer or a compound which
maintains/increases solubility and/or absorbency of the active
ingredient. Further, chemotherapeutic agents, anti-inflammatory
agents, antiviral agents and/or immune-modulators, etc. may be
optionally included.
[0231] In addition, the composition of the present invention may be
formulated by any conventional method known in the art to provide
rapid, sustained or delayed release of the active ingredient after
the administration thereof to a mammal. The formulation may be in a
form of powders, granules, tablets, emulsions, syrups, aerosols,
soft or hard gelatin capsules, sterile injectable solutions,
sterile powders.
[0232] Hereinafter, the present invention will be described in
detail with reference to the following examples.
EXAMPLE 1--Experimental Materials and Methods
[0233] 1. Cell Culture
[0234] Human liver cancer cell line (SNU-449) and murine Hepa-1c1c7
liver cancer cell line were obtained from Korean Cell Line Bank
(Seoul, Korea). All of the cell lines were cultured in EMEM
(American Type Culture Collection, Manassas, Va.), RPMI-1640 or
DMEM medium (Lonza, Walkersville, Md.) which is supplemented with
10% fetal bovine serum (FBS, Lonza) and 100 units/mL
penicillin-streptomycin
[0235] (Invitrogen, Carlsbad, Calif.), in a humidified incubator at
37.degree. C. under 5% CO.sub.2 condition.
[0236] 2. Synthesis and Transfection of siRNA and dsRNA
[0237] The siRNA and dsRNA used in this experiment were synthesized
by
[0238] Lemonex (Seoul, Korea). Further, human BANF1, PLOD3 and
SF3B4 expression plasmids subcloning gene ORF sequences
(BANF1:NM_003860, PLOD3: NM_001084, SF3B4: NM_005850) in pcDNA3.1
+/C-(K)-DYK plasmid, respectively, were purchased from Genscrip.TM.
(Piscataway, N.J., USA). Transfection was performed using
Lipofectamine RNAiMAX or Lipofectamine 2000 reagent (Invitrogen)
according to the manufacturer's manual.
[0239] 3. Extraction of RNA and DNA, and Execution of RT-PCR and
qRT-PCR
[0240] Total RNA was isolated from frozen tissue and cells using
trizol reagent (Invitrogen). 1 .mu.g of total RNA was
reverse-transcribed with cDNA by Tetro cDNA synthesis kit (Bioline,
London, UK) according to the manufacturer's manual. RT-PCR
reactions were conducted with nTaq DNA polymerase (Enzynomics,
Taejon, Korea), followed by detection using ethidium bromide in a
Gel Doc XR imaging system (Bio-Rad, Hercules, Calif.). qRT-PCR was
performed by SensiFAST SYBR No-ROX Kit (Bioline) and was monitored
in real time by iQ.TM.-5 (Bio-Rad). An average Ct (threshold cycle)
acquired from three replicate experiments was used for the
calculation. Normalized gene expression was determined using a
relative quantification method. The results were expressed as an
average value of three replicate experiments. Genomic DNA from
tissues and cells was isolated using DNAzol reagent (Invitrogen)
according to the manual. For analysis of replication number
variation, the SF3B4 genomic DNA region was amplified from 20 pairs
(non-tumor and tumor) HCC tissues using a primer set from exon-1 to
intron-1 according to the genome sequence of Genbank accession No.
NC_000001.11. qRT-PCR was performed as described above, and
glyceraldehyde-3-phosphate dehydrogenase was used as an endogenous
loading control. The primer sequences used for RT-PCR and qRT-PCR
are shown in Table 5 below.
TABLE-US-00005 TABLE 5 Accession Gene No. Primer Nucleotide
sequence SEQ ID NO BANF1 NM_003860 Forward 5'- SEQ ID NO:
GAACCGTTACGGGAACTGA 316 A-3' Reverse 5'- SEQ ID NO:
CCCGGAAGAGGTCTTCATC 317 T-3' PLOD3 NM_001084 Forward 5'- SEQ ID NO:
CAGCTCCAGGACCACTTCT 318 C-3' Reverse 5'- SEQ ID NO:
GAGCGGGCGTAGTACTCAT 319 C-3' SF3B4 NM_005850 Forward 5'- SEQ ID NO:
CTCAGATGCAGCTTGCACA 320 C-3' Reverse 5'- SEQ ID NO:
GGAGGGCCAGTGTATCCAT- 321 3' GAPDH NM_002046 Forward 5'- SEQ ID NO:
ACCAGGTGGTCTCCTCTGA 322 C-3' Reverse 5'- SEQ ID NO:
TGCTGTAGCCAAATTCGTTG- 323 3'
[0241] 4. Cell Growth and Proliferation Assay
[0242] Cell lines were seeded in 12-well plates at 30% confluence
for cell growth assay. After transfection or inhibitor treatment,
cells were incubated with 0.5 mg/mL of MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for
1 h at 37.degree. C. every 24 hours. The formazan crystal was
dissolved with dimethyl sulfoxide (DMSO), and absorbance was read
at 570 nm using VICTOR3.TM. multilabel plate reader (PerkinElmer,
Boston, Mass.).
[0243] Cell lines were seeded in 24-well plates at 30% confluence
for cell proliferation assay. After transfection, cells were
treated with 5-bromo-2'-deoxyuridine (BrdU) reagent for 2 h and
fixed at room temperature for 30 min. Cells were incubated with
anti-BrdU antibody for 1 h at room temperature. Unbound antibody
was removed by washing buffer. Horseradish peroxidase-conjugated
secondary antibody was added to each well. The substrate solution
was added and the reaction was stopped with stop solution after 30
min. The final product was quantified at 490 nm by VICTOR3.TM.
multilabel plate reader (PerkinElmer).
[0244] 5. Cell Motility and Invasion Assay
[0245] For in vitro cell motility and invasion assays, Transwell
plates and cell culture inserts (BD Biosciences) were used. For the
coating of the invasion assay, Matrigel (BD Biosciences) was
diluted to 0.3 mg/ml concentration with coating buffer (0.01 M
Tris, 0.7% NaCl, pH 8.0) and 100 .mu.l Matrigel was coated onto the
upper compartment of the cell culture insert. After incubation for
1 h at 37.degree. C., the cell culture insert was ready for
seeding. After si-SF3B4 transfection, cells were appropriately
seeded (0.5.times.10.sup.5 cells/well for the motility assay,
1.times.10.sup.5 cells/well for the invasion assay) into the cell
culture insert with serum-free medium in the presence of 5% FBS as
chemoattractant. After incubation for 6 h (migration assay) or 12 h
(invasion assay) at 37.degree. C., migrated or invaded cells were
stained using Diff-Quik staining kit (Sysmex, Japan). Cells were
photographed using an Axiovert 200 inverted microscope (Zeiss,
Jena, Germany) at .times.200 magnification. Cells were enumerated
in three random fields of view.
[0246] 6. Wound Healing Assay
[0247] Transfected cells were seeded in wells of a 6-well plate. At
100% confluence, a scratch was made on a uniform layer using a
micropipette tip. Photographs of the same area of the wound were
taken after 0 and 24 h with IX70 fluorescence inverted microscope
(Olympus, Tokyo, Japan).
[0248] 7. Mouse Liver Cancer Model
[0249] For xenograft tumorigenesis assay, 1.times.10.sup.7 cells of
transfected cells were mixed with 0.2 ml PBS (pH 7.4) and 30% (v/v)
Matrigel matrix (BD Biosciences). Cell suspensions were
subcutaneously injected in 6-week-old male Balb/c-nude mice. Mice
were examined twice per week for tumor formation at the injection
sites. Tumor volumes were calculated using: 0.5.times.length
(L).times.width (W.sup.2). Each experimental group consisted of 10
mice and tumor growth was quantified by measuring tumor sizes in
three orthogonal direction using calipers. Results are expressed as
mean tumor volumes and 95% confidence interval. The H-ras12V
activated homozygous transgenic mice were kindly provided by Dr.
Dae-Yeoul Yu (Laboratory of Human Genomics, Korea Research
Institute of Bioscience and Biotechnology, Daej eon, Korea).
Transgenic mice were H-ras12V activated. Male mice spontaneously
developed HCC beginning at 15-weeks-of-age. We surgically obtained
the non-tumor region and HCC mass from five mice (35-weeks-old) and
pick out three pairs of HCC tissue by pathological scoring.
Diethylnitrosamine (DEN) was used to induce HCC.
[0250] 8. Porous Silica Particles (Mesoporous Nanoparticles)
Transfection
[0251] siRNA specific to BANF1, PLOD3, SF3B4 was loaded in 80 .mu.l
of 3 nmol InViVojection.TM. RNAi-nano reagent (the porous silica
nanoparticles in EXAMPLE 1-12(1)-2)-(ii), Cat. No. DHMSN-vivoRNA;
Lemonex Inc., Seoul, Korea) and prepared in 200 .mu.l of PBS. A
mixture of siRNA or dsRNA and nanoparticles was intravenously
injected into H-ras transgenic HCC mouse model through tail vein
every week from week 9 to week 23. Sonograms (ultrasonic
photographs) were taken at 17, 19, and 21 weeks by an ultrasonic
machine (Affiniti 50, Philips, Seoul, Korea).
[0252] 9. Western Blotting Analysis
[0253] Cells were dissolved in a protein extraction buffer (50 mM
HEPES, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 mM
Na.sub.2P.sub.2O.sub.7, 1 mM Na.sub.3VO.sub.4, 100.times.Halt
protease inhibitor cocktail). Lysate containing the same amount of
protein was separated by SDS-PAGE and transferred onto a
polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The blots were
blocked with 5% skim milk and incubated along with each antibody
(Table 6).
TABLE-US-00006 TABLE 6 Protein Manufacturer Catalog No. Dilution
BANF1 Santa Cruz sc-33787 1:200 PLOD3 Proteintech 11027-1-AP 1:1000
SF3B4 Abcam ab157117 1:1000 E-cadherin BD Biosciences 610404 1:1000
N-cadherin BD Biosciences 610920 1:1000 Fibronectin Santa Cruz
sc-9068 1:1000 Snail Abcam ab78105 1:1000 Slug Cell Signaling #9585
1:500 GAPDH Santa Cruz sc-32233 1:1000
[0254] 10. Statistical Analysis
[0255] Survival curves were plotted using the Kaplan-Meier product
limit method, and significant differences between survival curves
were determined using the log-rank test. All experiments were
performed at least three times, and all samples were analyzed in
triplicate. Results are presented as mean .+-.standard deviation
(SD) or standard error of the mean (SEM). The statistical
significance of the difference between experimental groups was
assessed by paired or unpaired student's t-tests using Graphpad.TM.
7.0 software. Statistical significance was determined for
p<0.05. Chi-square test (2-sided) was used to determine
associations between parameters
[0256] 11. Preparation of Porous Silica Particles (Mesoporous
Nanoparticles)
[0257] (1) Preparation of Particle 1
[0258] 1) Preparation of Small Pore Particles
[0259] A 2 L round bottom flask was charged with 960 ml of
distilled water (DW) and 810 ml of MeOH. 7.88 g of CTAB was added
to the flask and 4.52 ml of 1 M NaOH was rapidly added with
agitating. The mixture was agitated for 10 minutes to give a
homogeneously mixed solution, and 2.6 ml of TMOS was added thereto.
The mixture was homogenously mixed under agitation for 6 hours and
then aged for 24 hours.
[0260] Then, the reaction solution was centrifuged at 8000 rpm and
25.degree. C. for 10 minutes to remove the supernatant. During
centrifugation at 8000 rpm and 25.degree. C., the product was
washed five times with ethanol and distilled water by turns.
[0261] Thereafter, the resultant product was dried in an oven at
70.degree. C. to obtain 1.5 g of powdery small pore silica
particles (average pore diameter: 2 nm, particle diameter: 200
nm).
[0262] 2) Pore Expansion
[0263] 1.5 g of small pore silica particle powders were added to 10
ml of ethanol, followed by ultrasonic dispersion.
[0264] Further, 10 ml of water and 10 ml of trimethyl benzene (TMB)
were added thereto, followed by ultrasonic dispersion.
[0265] Thereafter, the dispersion was placed in an autoclave and
reacted at 160.degree. C. for 48 hours.
[0266] The reaction started at 25.degree. C. and proceeded with
heating at a rate of 10.degree. C./min, followed by cooling down at
a rate of 1 to 10.degree. C./min in an autoclave.
[0267] The cooled reaction solution was centrifuged at 8000 rpm and
25.degree. C. for 10 minutes to remove the supernatant. During
centrifugation at 8000 rpm and 25.degree. C. for 10 minutes, the
product was washed five times with ethanol and distilled water by
turns.
[0268] Thereafter, the resultant product was dried in an oven at
70.degree. C. to obtain powdery porous silica particles (pore
diameter: 10 to 15 nm, particle size: 200 nm).
[0269] 3) Calcinations
[0270] The porous silica particles prepared in the above 2) were
placed in a glass vial and heated at 550.degree. C. for 5 hours.
After the completion of the reaction, the particles were gradually
cooled to room temperature, thereby preparing particles.
[0271] (2) Preparation of Particle 2
[0272] Porous silica particles were prepared in the same manner as
in EXAMPLE 1-11(1), except that the reaction conditions at the time
of pore expansion were changed to 140.degree. C. and 72 hours.
[0273] (3) Preparation of Particle 3 (10 L Scale)
[0274] Porous silica particles were prepared in the same manner as
in EXAMPLE 1-11(1), except that 5-fold larger vessels were used and
all the materials were used in a 5-fold capacity.
[0275] (4) Preparation of Particle 4 (Particle Diameter: 300
nm)
[0276] Porous silica particles were prepared in the same manner as
in EXAMPLE 1-11(1), except that 920 ml of distilled water and 850
ml of methanol were used in the preparation of small pore
particles.
[0277] (5) Preparation of Particle 5 (Particle Diameter: 500
nm)
[0278] Porous silica particles were prepared in the same manner as
in EXAMPLE 1-11(1), except that 800 ml of distilled water, 1010 ml
of methanol and 10.6 g of CTAB were used in the preparation of
small pore particles.
[0279] (6) Preparation of Particle 6 (Particle Diameter: 1000
nm)
[0280] Porous silica particles were prepared in the same manner as
in EXAMPLE 1-11(1), except that 620 ml of distilled water, 1380 ml
of methanol and 7.88 g of CTAB were used in the preparation of
small pore particles.
[0281] (7) Preparation of Particle 7 (Pore Diameter: 4 nm)
[0282] Porous silica particles were prepared in the same manner as
in EXAMPLE 1-11(1), except that 2.5 ml of TMB was used at the time
of pore expansion.
[0283] (8) Production of Particle 8 (Pore Diameter: 7 nm)
[0284] Porous silica particles were prepared in the same manner as
in EXAMPLE 1-11(1), except that 4.5 ml of TMB was used at the time
of pore expansion.
[0285] (9) Preparation of Particle 9 (Pore Diameter: 17 nm)
[0286] Porous silica particles were prepared in the same manner as
in EXAMPLE 1-11(1), except that 11 ml of TMB was used at the time
of pore expansion.
[0287] (10) Preparation of Particle 10 (Pore Diameter: 23 nm)
[0288] Porous silica particles were prepared in the same manner as
in EXAMPLE 1-11(1), except that 12.5 ml of TMB was used at the time
of pore expansion.
[0289] (11) Preparation of Particle 11 (Dual Modification)
[0290] 1) Preparation of Small Pore Particles
[0291] Small pore particles were prepared in the same manner as in
EXAMPLE 1-11(1).
[0292] 2) Pore Expansion
[0293] The small pore particles were reacted with TMB in the same
manner as in EXAMPLE 1-11(1)-2), cooled, and centrifuged to remove
the supernatant. Thereafter, the mixture was centrifuged under the
same conditions as in EXAMPLE 1-11(1)-2), washed three times with
ethanol and distilled water by turns, and then dried under the same
conditions as in EXAMPLE 1-11(1)-2), thereby obtaining powdery
silica particles (pore diameter: 10 to 15 nm, particle diameter:
200 nm).
[0294] 3) Surface Modification
[0295] 0.8 to 1 g of porous silica particles having expanded pores
were dispersed in 50 ml of toluene, and then 5 ml of
(3-aminopropyl)triethoxysilane was added thereto, followed by
heating at 120.degree. C. for 12 hours under reflux. The product
was subjected to the washing and drying processes described above
and then dispersed along with 1 ml of triethyleneglycol (PEG3,
2-[2-(2-methoxyethoxy)ethoxy]acetic acid), 100 mg of EDC
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 200 mg of
N-Hydroxysuccinimide (NHS) in 30 ml of PBS. The dispersion was
subjected to reaction with agitating at room temperature for 12
hours. Then, the resultant product was subjected to the
above-described washing and drying processes.
[0296] Since the reaction solution in the previous step remained in
the pores, the insides of the pores were not modified.
[0297] 4) Washing Inside of Pore
[0298] 800 mg of the surface-modified particle powders were
dissolved in 40 ml of 2 M HCl/ethanol and refluxed with vigorous
agitating for 12 hours.
[0299] Then, the cooled reaction solution was centrifuged at 8000
rpm for 10 minutes to remove the supernatant. During centrifugation
at 8000 rpm and 25.degree. C. for 10 minutes, the product was
washed five times with ethanol and distilled water by turns.
[0300] Thereafter, the resultant product was dried in an oven at
70.degree. C. to obtain powdery porous silica particles.
[0301] 5) Modification of Inside of Pore
[0302] (i) A propyl group was introduced into the pores in the same
manner as in EXAMPLE 1-12(2)-1) described below.
[0303] (ii) An octyl group was introduced into the pores in the
same manner as in EXAMPLE 1-12(2)-2) described below.
[0304] 12. Surface Modification of Porous Silica Particles
[0305] (1) Positive Charging
[0306] 1) Particles Having a Particle Diameter of 300 nm
[0307] The porous silica particles in EXAMPLE 1-11(4) were reacted
with (3-aminopropyl)triethoxysilane (APTES) thus to be positively
charged.
[0308] More particularly, 100 mg of porous silica particles were
dispersed in 10 ml of toluene in a 100 ml round bottom flask
provided with a bath sonicator. Then, 1 ml of APTES was added
thereto, and the mixture was reacted for 12 hours with agitation at
400 rpm and 130.degree. C.
[0309] After the reaction, the mixture was slowly cooled to room
temperature, centrifuged at 8000 rpm for 10 minutes to remove the
supernatant. During centrifugation at 8000 rpm and 25.degree. C.
for 10 minutes, the product was washed five times with ethanol and
distilled water by turns.
[0310] Thereafter, the resultant product was dried in an oven at
70.degree. C. to obtain powdery porous silica particles having an
amino group on the surfaces of the particles and the insides of
pores.
[0311] 2) Particles Having a Particle Diameter of 200 nm
[0312] (i) The porous silica particles in EXAMPLE 1-11(1) were
reacted with (3-aminopropyl)triethoxysilane (APTES) thus to be
positively charged, and then were modified in the same manner as in
EXAMPLE 1-12(1)-1), except that 0.4 ml of APTES was added and the
reaction time was changed to 3 hours.
[0313] (ii) The porous silica particles in EXAMPLE 1-11(9) were
reacted with (3-aminopropyl)triethoxysilane(APTES) thus to be
positively charged, and then were modified in the same manner as in
EXAMPLE 1-12(1)-1), except that the particles prepared in EXAMPLE
1-11(9) were used.
[0314] (iii) The porous silica particles in EXAMPLE 1-11(10) were
reacted with (3-aminopropyl)triethoxysilane(APTES) thus to be
positively charged, and then were modified in the same manner as in
EXAMPLE 1-12(1)-1), except that the particles prepared in EXAMPLE
1-11(10) were used.
[0315] (2) Introduction of Hydrophobic Group
[0316] 1) Propyl Group
[0317] The porous silica particles in EXAMPLE 1-11(1) were reacted
with trimethoxy(propyl)silane to introduce a propyl group into the
surfaces of the particles and the insides of the pores, and then
were modified in the same manner as in EXAMPLE 1-12(1), except that
0.35 ml of trimethoxy(propyl)silane was added instead of APTES and
the reaction was conducted for 12 hours.
[0318] 2) Octyl Group
[0319] The porous silica particles of EXAMPLE 1-11(1) were reacted
with trimethoxy-n-octylsilane to introduce a propyl group into the
surfaces of the particles and the insides of the pores, and then
were modified in the same manner as in EXAMPLE 1-12(1), except that
0.5 ml of trimethoxy-n-octylsilane was added instead of APTES and
the reaction was conducted for 12 hours.
[0320] (3) Negative Charging
[0321] 1) Carboxyl Group
[0322] The porous silica particles in EXAMPLE 1-11(1) were reacted
with succinic anhydride thus to be negatively charged, and then
were modified in the same manner as in EXAMPLE 1-12(1)-1), except
that dimethyl sulfoxide (DMSO) was used instead of toluene, 80 mg
of succinic anhydride was added instead of APTES, followed by
agitation at room temperature for 24 hours to conduct the reaction,
and DMSO was used instead of distilled water at the time of
washing.
[0323] 2) Thiol Group
[0324] Modification was implemented in the same manner as in
EXAMPLE 1-12(1)-1), except that 1.1 ml of MPTES was used instead of
APTES.
[0325] 3) Sulfonic Acid Group
[0326] 100 mg of the porous silica nanoparticles in EXAMPLE
1-12(3)-2) were dispersed in 1 ml of 1 M aqueous sulfuric acid
solution and 20 ml of 30% aqueous hydrogen peroxide, followed by
agitation at room temperature to induce oxidative reaction thus to
oxidize a thiol group into a sulfonic acid group. Thereafter, the
product was washed and dried in the same manner as in EXAMPLE
1-12(1)-1).
[0327] 13. Loading of siRNA or dsRNA on Porous Silica Particles
[0328] 21 base pair duplex siRNA targeting green fluorescent
protein (GFP) synthesized by Bionia Co. Ltd., on request was
purchased from the same
TABLE-US-00007 (Sequence: sense; 5'-GGCUACGUCCAGGAGCGCACC-3', (SEQ
ID NO: 324) antisense; 5'-UGCGCUCCUGGACGUAGCCUU-3' (SEQ ID NO:
325)).
[0329] After mixing 10 .mu.g of the porous silica particles in
EXAMPLE 1-12(1)-2)-(ii) and 50 pmol of siRNA were mixed in
1.times.PBS condition and allowed to be loaded at room temperature
for 30 minutes.
EXAMPLE 2--Analysis of Inhibitory Rate of Indicator Gene Expression
by siRNA or dsRNA of the Present Invention
[0330] According to the experimental procedures in EXAMPLE 1-1 to
3, indicator genes of siRNA and dsRNA of the present invention
(BANF1 variant 1, BANF1 variant 2, PLOD3, and SF3B4) were analyzed,
and the results are shown in Tables 7 to 10 below.
[0331] Referring to Tables 7 to 10 below, it can be seen that all
the siRNAs and dsRNAs of the present invention could inhibited the
expression of the indicator genes at high inhibitory rates.
TABLE-US-00008 TABLE 7 Verification of validity of siRNA, dsRNA for
inhibition of human BANF1, transcript variant 1, mRNA (Gene Bank
number: NM_003860.3) expression Base SEQ ID NO Expression
inhibitory rate (%) 5 87.73 6 79.64 7 82.3 8 76.21 9 89.6 10 83.42
11 73.18 12 85.44 13 69.57 14 77.3 15 82.92 16 91.38 17 84.11 18
88.36 19 87.83 20 67.72 21 82.29 22 63.23 23 76.24 24 87.7 25 62.57
26 72.92 27 65.58 28 72.91
TABLE-US-00009 TABLE 8 Verification of validity of siRNA, dsRNA for
inhibition of human BANF1, transcript variant 2, mRNA (Gene Bank
number: NM_001143985.1) expression Base SEQ ID NO Expression
inhibitory rate (%) 29 92.55 30 91.49 31 86.44 32 77.1 33 73.82 34
76.6 35 88.33 36 82.53 37 81.64 38 68.4 39 79.72 40 91.6 41 87.37
42 53.77 43 86.39 44 68.63 45 83.22 46 78.16 47 73.48 48 68.3 49
85.27 50 88.74 51 92.32 52 74.8 53 84.31 54 64.9 55 74.72
TABLE-US-00010 TABLE 9 Verification of validity of siRNA, dsRNA for
inhibition of human PLOD3 gene sequence (Gene Bank number:
NM_001084.4) expression Base SEQ ID NO Expression inhibitory rate
(%) 56 87.62 57 78.13 58 92.72 59 83.49 60 86.8 61 64.29 62 73.33
63 85.83 64 82.68 65 76.92 66 91.64 67 85.77 68 79.1 69 82.4 70
84.63 71 89.26 72 76.4 73 76.8 74 68.27 75 77.44 76 86.26 77 84.3
78 81.52 79 79.35 80 76.63 81 85.32 82 62.72 83 64.3 84 77.13 85
83.78 86 86.71 87 82.33 88 68.46 89 74.88 90 72.7 91 83.69 92 85.3
93 76.62 94 82.11 95 83.46 96 71.25 97 72.73 98 87.6 99 69.91 100
81.38 101 78.27 102 74.8 103 69.76 104 62.45 105 87.22 106 82.7 107
74.5 108 86.25 109 83.7 110 74.13 111 76.29 112 73.52 113 82.86 114
73.52 115 73.71 116 82.55 117 69.72 118 77.8 119 86.49 120 71.3
TABLE-US-00011 TABLE 10 Verification of validity of siRNA, dsRNA
for inhibition of Human SF3B4 gene sequence (Gene Bank number:
NM_005850.4) expression Base SEQ ID NO Expression inhibitory rate
(%) 121 83.71 122 81.83 123 87.62 124 86.39 125 78.64 126 82.7 127
84.25 128 74.11 129 63.36 130 76.25 131 74.32 132 92.19 133 84.72
134 81.3 135 83.4 136 88.63 137 78.25 138 85.1 139 81.68 140 83.57
141 78.33 142 72.45 143 76.72 144 81.36 145 83.2 146 72.41 147
73.64 148 86.77 149 84.8 150 82.56 151 73.12 152 78.66 153 82.5 154
76.63 155 62.95 156 89.6 157 77.2
EXAMPLE 3--Identification of Excellent RNA Delivery by Porous
Silica Particles
[0332] With respect to Hepa-lcic7 and SNU-449 cell lines in EXAMPLE
1, siRNAs, each of which includes a sense RNA having a sequence
shown in Table 11 below and an antisense RNA having a complementary
sequence thereto, were subjected to in vitro transfection by the
methods described in EXAMPLE 1-2 or 1-8, respectively. Then,
expression levels of the corresponding markers of the above siRNAs
were measured by Western blotting, and the results are shown in
FIG. 1.
[0333] Referring to FIG. 1, when the siRNAs shown in Table 11 were
transfected, it can be seen that the markers were effectively
inhibited. Specifically, when siRNA was loaded on the porous silica
particles and then transfected, the expression inhibitory rate was
demonstrated to be higher.
TABLE-US-00012 TABLE 111 Sense RNA Name in SEQ ID NO sequence FIG.
1 Target gene SEQ ID NO: 311 5'- Banf1 Mouse BANF1 gene
CCUCAGCGUUUCAAU CUUUUU-3' SEQ ID NO: 312 5'- Plod3 Mouse PLOD3 gene
CGACUGCAGAAUCUC CUCUUU-3' SEQ ID NO: 313 5'- Sf3b4 Mouse SF3B4 gene
CUGCUUUACGAUACU UUCAUU-3' SEQ ID NO: 314 5'- Control --
CCUACGCCACCAAUU UCGU-3' SEQ ID NO: 28 5'- BANF1 Human BANF1 gene
AAGAAGCUGGAGGAA AGGGGUUU-3' SEQ ID NO: 119 5'- PLOD3 Human PLOD3
gene GCAUCUGGAGCUUUC UGUA UU-3' SEQ ID NO: 136 5'- SF3B4 Human SF3B
gene GCAGUACCUCUGUAA CCGU UU-3'
EXAMPLE 4--Identification of Liver Cancer Cell Metastatic Potential
Inhibition by siRNA or dsRNA of the Present Invention
[0334] 1 Cell Motility and Invasion Assay and Wound Healing
Assay
[0335] With respect to SNU-449 cell line in EXAMPLE 1-1, siRNAs,
each of which includes a sense RNA having a sequence shown in Table
12 below and an antisense RNA having a complementary sequence
thereto, were subjected to in vitro transfection by the method
described in EXAMPLE 1-2. Migration and invasion of markers
corresponding to the above siRNAs were analyzed by the method in
EXAMPLE 1-5, while a scratch wound healing ability was analyzed by
the method in EXAMPLE 1-6, and the analyzed results are shown in
FIG. 2.
TABLE-US-00013 TABLE 12 SEQ ID NO Sense RNA sequence Name in FIG.
Target gene SEQ ID NO: 314 5'- Control -- CCUACGCCACCAAUUUC GU-3'
SEQ ID NO: 28 5'- BANF1 Human BANF1 AAGAAGCUGGAGGAAAG gene
GGGUUU-3' SEQ ID NO: 119 5'- PLOD3 Human PLOD3 GCAUCUGGAGCUUUCUG
gene UA UU-3' SEQ ID NO: 136 5'- SF3B4 Human SF3B4
GCAGUACCUCUGUAACC gene GU UU-3'
[0336] Referring to A of FIG. 2, it can be seen that, as compared
to the control group, the cells with the markers, which were
knockdown by transfection of siRNAs listed in Table 12, exhibited
significantly reduced migration and invasion. Referring to B of
FIG. 2, it was observed that, as compared to the control group,
wound-healing ability was considerably decreased. These results
demonstrate that the siRNA or dsRNA of the present invention can
inhibit metastatic potential of liver cancer cells, while reducing
malignant progression.
[0337] 2. Identification of EMT Regulatory Proteins Inhibition
[0338] In order to identify whether siRNA or dsRNA of the present
invention can inhibit indicator actors for expression of
N-cadherin, Fibronectin, Snail and Slug, which are representative
epithelial-mesenchymal transition (EMT) regulatory proteins in
relation to the metastasis of liver cancer cells, which in turn can
inhibit metastasis of liver cancer, siRNAs, each of which includes
a sense RNA having the sequence shown in Table 12 above and an
antisense RNA having the complementary sequence thereto, were
subjected to in vitro transfection to the SNU-449 cell line in
EXAMPLE 1 by the method described in EXAMPLE 1-2. Then, expression
levels of the markers corresponding to the siRNAs and expression
levels of the EMT regulatory proteins above were analyzed by the
method in EXAMPLE 1-9, and the analyzed results are shown in A of
FIG. 3.
[0339] Referring to A of FIG. 3, it can be seen that, as compared
to the control group, the cells with the markers, which were
knockdown by transfection of siRNAs listed in Table 12, exhibited
that the expression levels of N-cadherin, Fibronectin, Snail and
Slug, which are epithelial-mesenchymal transition (EMT) regulatory
proteins, as well as the expression levels of the markers are
inhibited. These results demonstrate that the siRNA or dsRNA of the
present invention may selectively inhibit the expression of the
corresponding markers, thereby inhibiting metastatic potential of
liver cancer cells.
EXAMPLE 5--Identification of Tumor Growth Inhibition by siRNA or
dsRNA of the Present Invention
[0340] siRNAs, each of which includes a sense RNA having the
sequence shown in Table 12 above and an antisense RNA having the
complementary sequence thereto, were subjected to in vitro
transfection to the SNU-449 cell line in EXAMPLE 1 by the method
described in EXAMPLE 1-2. Thereafter, athymic nude mice were
subjected to subcutaneous injection of the transfected cells,
followed by analyzing sizes of hepatic tumors and survival rates of
the mice, and the analyzed results are shown in B of FIG. 3.
[0341] Referring to the left image of B of FIG. 3, it can be seen
that most of the experimental groups have a significantly smaller
hepatic tumor size than the control group. Therefore, it is
understood that knockdown of the markers by the siRNA or dsRNA of
the present invention may reduce overall tumor growth rate and
decrease the average tumor volume.
[0342] Referring to the right image of B of FIG. 3, it can be seen
that the tumor-free survival rates of the experimental groups are
significantly higher than that of the control group. Specifically,
when 50 days elapses after the subcutaneous injection of the
transfected cells, the control group exhibited tumors in 6 mice of
10 mice whereas the experimental group exhibited tumors in 1 to 2
mice only among 10 mice, thereby indicating that the siRNA or dsRNA
of the present invention may effectively inhibit the growth of
hepatic tumors.
EXAMPLE 6--Identification of liver cancer prevention efficacy of
siRNA or dsRNA of the present invention
[0343] siRNAs, each of which includes a sense RNA having a sequence
shown in Table 13 below and an antisense RNA having a complementary
sequence thereto, were subjected to in vivo transfection by the
method described in EXAMPLE 1-8, and processes thereof, ultrasonic
images and the number of tumors over time are shown in A of FIG. 4.
Further, expression inhibitory levels for the indicator genes by
the siRNAs loaded on the porous nanoparticles were analyzed by the
method in EXAMPLE 1-9 and shown in B of FIG. 4.
[0344] Referring to A of FIG. 4, in the case of the control group
injected with only porous nanoparticles, multiple large hepatic
tumors were found in 3 to 4 mice 17 weeks after the injection
whereas the experimental group injected with porous nanoparticles
loaded with siRNA exhibited that only a relatively small hepatic
tumor was found in 2 to 4 mice 19 weeks after the injection.
Further, as shown in B of FIG.
[0345] 4, it can be seen that, as compared to the control group,
the experimental group could significantly reduce the expression
levels of the indicator gens in vivo as a result of Western
blotting. These results demonstrate that the siRNA or dsRNA of the
present invention may effectively inhibit the expression of the
markers in vivo, and exert excellent effects in inhibition of
hepatic tumor formation and prevention of liver cancer.
TABLE-US-00014 TABLE 13 SEQ Sense RNA Name in ID NO sequence FIG. 1
Target gene SEQ ID 5'- Banfl Mouse BANF1 NO: 311 CCUCAGCGUUUCAAUCU
gene UUUU-3' SEQ ID 5'- Plod3 Mouse PLOD3 NO: 312 CGACUGCAGAAUCUCCU
gene CUUU-3' SEQ ID 5'- 5f3b4 Mouse 5F3B4 NO: 313 CUGCUUUACGAUACUUU
gene CAUU-3'
EXAMPLE 7--Identification of Formation of Porous Silica Particles
and Pore Expansion
[0346] The small pore particles and the prepared porous silica
particles in EXAMPLE 1-11(1) to (3) were observed with a microscope
to identify whether the small pore particles were uniformly formed
and the pores were sufficiently expanded thus to uniformly form the
porous silica particle (FIGS. 5 to 8).
[0347] FIG. 5 is photographs illustrating the porous silica
particles in EXAMPLE 1-11(1) and FIG. 6 is photographs illustrating
the porous silica particles in EXAMPLE 1-11(2), demonstrating that
the porous silica particles in a spherical shape with sufficiently
expanded pores were uniformly formed. Further, FIG. 7 is
photographs illustrating the small pore particles in EXAMPLE
1-11(1), and FIG. 8 is comparative photographs illustrating the
small pore particles in EXAMPLEs 1-11(1) and 1-11(3), demonstrating
that spherical small pore particles were uniformly formed.
EXAMPLE 8--Identification of Biodegradability of Porous Silica
articles
[0348] In order to identify the biodegradability of the porous
silica particles in EXAMPLE 1-11(1), the degree of biodegradation
at 37.degree. C. under SBF (pH 7.4) was observed with a microscope
at 0 hours, 120 hours and 360 hours, and the observed results are
shown in FIG. 9.
[0349] Referring to FIG. 9, it can be seen that the porous silica
particles were almost completely degraded after 360 hours elapse
from the observation.
EXAMPLE 9--Measurement of Absorbance Ratio of Porous Silica
Particles
[0350] 1. Measurement Method
[0351] The absorbance ratio according to the following Equation 1
was measured:
A.sub.t/A.sub.0 [Equation 1]
[0352] (wherein A.sub.0 is absorbance of the porous silica
particles measured by placing 5 ml of a suspension including 1
mg/ml of the porous silica particles into a cylindrical dialysis
membrane having pores with a diameter of 50 kDa,
[0353] 15 ml of the same solvent as the suspension is placed
outside the dialysis membrane while being in contact with the
dialysis membrane, followed by horizontal agitation at 60 rpm and
37.degree. C. inside and outside the dialysis membrane, and
[0354] At is absorbance of the porous silica particles measured
after t hours elapses from the measurement of A.sub.0).
[0355] Specifically, 5 mg of the porous silica particle powders
were dissolved in 5 ml of SBF (pH 7.4). Thereafter, 5 ml of the
porous silica particle solution was placed in a dialysis membrane
having a pore diameter of 50 kDa shown in FIG. 10. 15 ml of SBF was
added to an outer membrane, and SBF of the outer membrane was
replaced every 12 hours. Degradation of the porous silica particles
was performed while horizontally agitating at 37.degree. C. and 60
rpm.
[0356] Thereafter, the absorbance was measured by UV-vis
spectroscopy and analyzed at .lamda.=640 nm. [431]
[0357] 2. Absorbance Ratio Measurement Results
[0358] The absorbance ratio of the porous silica particles in
EXAMPLE 1-11(1) was 2 5 measured according to the above method, and
the results are shown in FIG. 11.
[0359] Referring to FIG. 11, it can be seen that t when the
absorbance ratio becomes 1/2 was about 58 hours, demonstrating very
slow degradation.
[0360] 3. Measurement Results by Particle Size
[0361] The absorbance of the porous silica particles in each of
EXAMPLEs 1-11(1), (5) and (6) was measured according to Equation 1
above and the results are shown in FIG. 12 (using SBF as a
suspension and a solvent).
[0362] Referring to FIG. 12, it can be seen that t is decreased as
the particle diameter is increased.
[0363] 4. Measurement Results to Pore Diameter Average Diameter
[0364] The absorbance of the porous silica particles in each of
EXAMPLEs 1-11(1) and (9), and the small pore silica particles in
EXAMPLE 1-11(1) as a control group was measured according to the
above Equation 1 (using SBF as suspension and solvent).
[0365] Referring to FIG. 13, it can be seen that the porous silica
particles in the examples have significantly larger t than the
control.
[0366] 5. Measurement Results by pH
[0367] The absorbance of the porous silica particles in EXAMPLE
1-11(4) was measured by pH. The absorbance was measured in SBF and
in Tris at pH 2, 5 and 7.4, respectively, and the results are shown
in FIG. 14.
[0368] Referring to FIG. 14, there was a difference in t by pH, but
t when all absorbance ratios became 1/2 was 24 or more.
[0369] 6. Charging
[0370] The absorbance of the porous silica particles in EXAMPLE
1-12(1)-1) was measured, and the results are shown in FIG. 15
(using Tris (pH 7.4) as a suspension and a solvent).
[0371] Referring to FIG. 15, even the positively charged particles
showed that t when the absorbance ratio of the absorbance became
1/2 was 24 or more.
EXAMPLE 10--Release of siRNA or dsRNA Loaded on Porous Silica
Particles
[0372] 10 .mu.l of the porous silica particles loaded with the
siRNA in EXAMPLE 1-13 was resuspended in SBF (pH 7.4, 37.degree.
C.) and placed in a dialysis membrane having a pore diameter of 20
kDa (the tube in FIG. 16). Then, the dialysis tube was immersed in
1.5 ml of SBF. The release of siRNA was performed while
horizontally agitating at 37.degree. C. and 60 rpm.
[0373] The release solvent was recovered at 0.5, 1, 2, 4, 8, 12, 24
hours prior to 24 hours and then, every 24 hours, 0.5 ml of the
release solvent was recovered for fluorescence measurement and SBF
was added thereto.
[0374] The fluorescence intensity of the siRNA was measured at a
wavelength of 670 nm (.lamda..sub.ex=647 nm) to determine the
degree of siRNA release, and the results are shown in FIG. 17.
[0375] Referring to FIG. 17, it can be seen that the time at which
50% of the siRNA was released was about 40 hours or more.
EXAMPLE 11--Identification of Target Delivery of siRNA or dsRNA
Loaded on Porous Silica Particles
[0376] In order to verify whether the siRNAs of the present
invention can play a role of a transporter in a desired level for
study of siRNA delivery in animal level, tumor inhibitory rates due
to the release of bioactive material in mice (rats) were
confirmed.
[0377] Specifically, Balb/c nude male mice (5 weeks old) were
purchased from Orient Bio Inc., and 3 million HeLa cells (cervical
cancer cells) were dispersed in sterilized 1.times.PBS to
proliferate Xenograft tumors subcutaneously injected into the mice.
When 70 mm.sup.3 size of solidified tumors were observed, PBS,
FITC-porous silica particles (porous silica particles in EXAMPLE
1-12(1)-2)-(ii)), and FITC-porous silica particles loaded with
siRNA (porous silica particles in EXAMPLE 1-12(1)-2)-(ii)) were
injected into tumors in the mice, respectively. Then, fluorescence
intensities and distribution thereof were measured immediately
before, immediately after, and 48 hours after the administration,
by means of FOBI fluorescence in vivo imaging system (Neo science,
Korea).
[0378] FITC labeling were implemented by: dispersing 50 mg of
silica particles in 1 ml of dimethyl sulfoxide (DMSO); adding 25
.mu.g (10 .mu.l) of FITC-NHS (N-hydroxysuccinimide) solution (2.5
mg/mL) thereto; reacting the mixture at room temperature for 18
hours while shielding light with aluminum foil; purifying the
reaction product through centrifugation (8500 rpm, 10 minutes);
discarding the supernatant while collecting settled particles; and
evenly dispersing the particles in ethanol, wherein the above
processes were repeated three and four times with ethanol and
distilled water to purify until FITC color is invisible in the
supernatant.
[0379] Referring to FIG. 18 demonstrating results of the above
experiments, the control group refers to administration of PBS
alone, Cy5-siRNA refers to administration of siRNA in EXAMPLE 1-13
alone, FITC-DDV refers to administration of FITC-labeled porous
silica particles alone, and the complex refers to administration of
FITC-labeled porous silica particles loaded with the siRNA in
EXAMPLE 1-13. Referring to this figure, it can be seen that the
siRNA loaded on the particles and delivered into the body has a
longer duration of activity and stays longer at the injected site,
thereby exhibiting strong fluorescence even after 48 hours.
[0380] A sequence listing electronically submitted with the present
application on Jan. 28, 2020 as an ASCII text file named
20200128_Q25720LC02_TU_SEQ, created on Jan. 28, 2020 and having a
size of 124,000 bytes, is incorporated herein by reference in its
entirety.
Sequence CWU 1
1
6341270DNAArtificial SequenceHuman BANF1 gene variant1 1atgacaacct
cccaaaagca ccgagacttc gtggcagagc ccatggggga gaagccagtg 60gggagcctgg
ctgggattgg tgaagtcctg ggcaagaagc tggaggaaag gggttttgac
120aaggcctatg ttgtccttgg ccagtttctg gtgctaaaga aagatgaaga
cctcttccgg 180gaatggctga aagacacttg tggcgccaac gccaagcagt
cccgggactg cttcggatgc 240cttcgagagt ggtgcgacgc cttcttgtga
2702270DNAArtificial SequenceHuman BANF1 gene variant2 2atgacaacct
cccaaaagca ccgagacttc gtggcagagc ccatggggga gaagccagtg 60gggagcctgg
ctgggattgg tgaagtcctg ggcaagaagc tggaggaaag gggttttgac
120aaggcctatg ttgtccttgg ccagtttctg gtgctaaaga aagatgaaga
cctcttccgg 180gaatggctga aagacacttg tggcgccaac gccaagcagt
cccgggactg cttcggatgc 240cttcgagagt ggtgcgacgc cttcttgtga
27032217DNAArtificial SequenceHuman PLOD3 gene 3atgacctcct
cggggcctgg accccggttc ctgctgctgc tgccgctgct gctgccccct 60gcggcctcag
cctccgaccg gccccggggc cgagacccgg tcaacccaga gaagctgctg
120gtgatcactg tggccacagc tgaaaccgag gggtacctgc gtttcctgcg
ctctgcggag 180ttcttcaact acactgtgcg gaccctgggc ctgggagagg
agtggcgagg gggtgatgtg 240gctcgaacag ttggtggagg acagaaggtc
cggtggttaa agaaggaaat ggagaaatac 300gctgaccggg aggatatgat
catcatgttt gtggatagct acgacgtgat tctggccggc 360agccccacag
agctgctgaa gaagttcgtc cagagtggca gccgcctgct cttctctgca
420gagagcttct gctggcccga gtgggggctg gcggagcagt accctgaggt
gggcacgggg 480aagcgcttcc tcaattctgg tggattcatc ggttttgcca
ccaccatcca ccaaatcgtg 540cgccagtgga agtacaagga tgatgacgac
gaccagctgt tctacacacg gctctacctg 600gacccaggac tgagggagaa
actcagcctt aatctggatc ataagtctcg gatctttcag 660aacctcaacg
gggctttaga tgaagtggtt ttaaagtttg atcggaaccg tgtgcgtatc
720cggaacgtgg cctacgacac gctccccatt gtggtccatg gaaacggtcc
cactaagctg 780cagctcaact acctgggaaa ctacgtcccc aatggctgga
ctcctgaggg aggctgtggc 840ttctgcaacc aggaccggag gacactcccg
ggggggcagc ctcccccccg ggtgtttctg 900gccgtgtttg tggaacagcc
tactccgttt ctgccccgct tcctgcagcg gctgctactc 960ctggactatc
cccccgacag ggtcaccctt ttcctgcaca acaacgaggt cttccatgaa
1020ccccacatcg ctgactcctg gccgcagctc caggaccact tctcagctgt
gaagctcgtg 1080gggccggagg aggctctgag cccaggcgag gccagggaca
tggccatgga cctgtgtcgg 1140caggaccccg agtgtgagtt ctacttcagc
ctggacgccg acgctgtcct caccaacctg 1200cagaccctgc gtatcctcat
tgaggagaac aggaaggtga tcgcccccat gctgtcccgc 1260cacggcaagc
tgtggtccaa cttctggggc gccctgagcc ccgatgagta ctacgcccgc
1320tccgaggact acgtggagct ggtgcagcgg aagcgagtgg gtgtgtggaa
tgtaccatac 1380atctcccagg cctatgtgat ccggggtgat accctgcgga
tggagctgcc ccagagggat 1440gtgttctcgg gcagtgacac agacccggac
atggccttct gtaagagctt tcgagacaag 1500ggcatcttcc tccatctgag
caatcagcat gaatttggcc ggctcctggc cacttccaga 1560tacgacacgg
agcacctgca ccccgacctc tggcagatct tcgacaaccc cgtcgactgg
1620aaggagcagt acatccacga gaactacagc cgggccctgg aaggggaagg
aatcgtggag 1680cagccatgcc cggacgtgta ctggttccca ctgctgtcag
aacaaatgtg tgatgagctg 1740gtggcagaga tggagcacta cggccagtgg
tcaggcggcc ggcatgagga ttcaaggctg 1800gctggaggct acgagaatgt
gcccaccgtg gacatccaca tgaagcaggt ggggtacgag 1860gaccagtggc
tgcagctgct gcggacgtat gtgggcccca tgaccgagag cctgtttccc
1920ggttaccaca ccaaggcgcg ggcggtgatg aactttgtgg ttcgctaccg
gccagacgag 1980cagccgtctc tgcggccaca ccacgactca tccaccttca
ccctcaacgt tgccctcaac 2040cacaagggcc tggactatga gggaggtggc
tgccgcttcc tgcgctacga ctgtgtgatc 2100tcctccccga ggaagggctg
ggcactcctg caccccggcc gcctcaccca ctaccacgag 2160gggctgccaa
cgacctgggg cacacgctac atcatggtgt cctttgtcga cccctga
221741275DNAArtificial SequenceHuman SF3B4 gene 4atggctgccg
ggccgatctc cgagcggaat caggatgcca ctgtgtacgt ggggggcctg 60gatgagaagg
ttagtgaacc gctgctgtgg gaactgtttc tccaggctgg accagtagtc
120aacacccaca tgccaaagga tagagtcact ggccagcacc aaggctatgg
ctttgtggaa 180ttcttgagtg aggaagatgc tgactatgcc attaagatca
tgaacatgat caaactctat 240gggaagccaa tacgggtgaa caaagcatca
gctcacaaca aaaacctgga tgtaggggcc 300aacattttca ttgggaacct
ggaccctgag attgatgaga agttgcttta tgatactttc 360agcgcctttg
gggtcatctt acaaaccccc aaaattatgc gggaccctga cacaggcaac
420tccaaaggtt atgcctttat taattttgct tcatttgatg cttcggatgc
agcaattgaa 480gccatgaatg ggcagtacct ctgtaaccgt cctatcaccg
tatcttatgc cttcaagaag 540gactccaagg gtgagcgcca tggctcagca
gccgaacgac ttctggcagc tcagaacccg 600ctctcccagg ctgatcgccc
tcatcagctg tttgcagatg cacctcctcc accctctgct 660cccaatcctg
tggtatcatc attggggtct gggcttcctc caccaggcat gcctcctcct
720ggctccttcc cacccccagt gccacctcct ggagccctcc cacctgggat
acccccagcc 780atgcccccac cacctatgcc tcctggggct gcaggacatg
gccccccatc ggcaggaacc 840ccaggggcag gacatcctgg tcatggacac
tcacatcctc acccattccc accgggtggg 900atgccccatc cagggatgtc
tcagatgcag cttgcacacc atggccctca tggcttagga 960catccccacg
ctggaccccc aggctctggg ggccagccac cgccccgacc accacctgga
1020atgcctcatc ctggacctcc tccaatgggc atgccccccc gagggcctcc
attcggatct 1080cccatgggtc acccaggtcc tatgcctccg catggtatgc
gtggacctcc tccactgatg 1140cccccccatg gatacactgg ccctccacga
cccccaccct atggctacca gcgggggcct 1200ctccctccac ccagacccac
tccccggcca ccagttcccc ctcgaggccc acttcgaggc 1260cctctccctc agtaa
1275520RNAArtificial SequencesiRNA sense strand 5caagaagcug
gaggaaaguu 20622RNAArtificial SequencesiRNA sense strand
6gaaagaugaa gaccucuucc uu 22722RNAArtificial SequencesiRNA sense
strand 7ggaauggcug aaagacacuu uu 22820RNAArtificial SequencesiRNA
sense strand 8ccaguguucc caguucccuu 20920RNAArtificial
SequencesiRNA sense strand 9ccaguccaac ugcgaggauu
201020RNAArtificial SequencesiRNA sense strand 10cgacgugagu
cugagucuuu 201120RNAArtificial SequencesiRNA sense strand
11guccgucuuc uaacucuuuu 201220RNAArtificial SequencesiRNA sense
strand 12cgucaagccu aaguccuuuu 201320RNAArtificial SequencesiRNA
sense strand 13gcagagaaag gaaguccuuu 201420RNAArtificial
SequencesiRNA sense strand 14cgagaagcga gaccuuaguu
201520RNAArtificial SequencesiRNA sense strand 15ccucaacucu
auagcucuuu 201620RNAArtificial SequencesiRNA sense strand
16cuaguggcuu gagguaucuu 201720RNAArtificial SequencesiRNA sense
strand 17ggauuaagcc ugaucaaguu 201820RNAArtificial SequencesiRNA
sense strand 18gacugcuucg gaugccuuuu 201920RNAArtificial
SequencesiRNA sense strand 19ccuucuugug augcucucuu
202020RNAArtificial SequencesiRNA sense strand 20ccucauccag
aguuugcauu 202120RNAArtificial SequencesiRNA sense strand
21ccuguccucu acgaaggauu 202222RNAArtificial SequencesiRNA sense
strand 22gauugcuauu gucguacuca uu 222320RNAArtificial SequencesiRNA
sense strand 23ggauucucgc ucuugcauuu 202420RNAArtificial
SequencesiRNA sense strand 24ggugacaguu accagcuuuu
202520RNAArtificial SequencesiRNA sense strand 25ccucacuuuc
aauccguuuu 202620RNAArtificial SequencesiRNA sense strand
26gcagaacagu cacuguccuu 202722RNAArtificial SequencesiRNA sense
strand 27gaucaauaaa gucaguggcu uu 222823RNAArtificial SequencesiRNA
sense strand 28aagaagcugg aggaaagggg uuu 232922RNAArtificial
SequencesiRNA sense strand 29augacaaccu cccaaaagca uu
223020RNAArtificial SequencesiRNA sense strand 30ccgagacuuc
guggcagauu 203117RNAArtificial SequencesiRNA sense strand
31agccuggcug ggauuuu 173219RNAArtificial SequencesiRNA sense strand
32caagaagcug gaggaaauu 193322RNAArtificial SequencesiRNA sense
strand 33ccaguuucug gugcuaaaga uu 223420RNAArtificial SequencesiRNA
sense strand 34aagaugaaga ccucuuccuu 203521RNAArtificial
SequencesiRNA sense strand 35ggacugcuuc ggaugccuuu u
213621RNAArtificial SequencesiRNA sense strand 36aguggugcga
cgccuucuuu u 213721RNAArtificial SequencesiRNA sense strand
37aguggugcga cgccuucuuu u 213822RNAArtificial SequencesiRNA sense
strand 38uugcuauugu cguacucacc uu 223920RNAArtificial SequencesiRNA
sense strand 39gauucucgcu cuugcauguu 204022RNAArtificial
SequencesiRNA sense strand 40caguucccug gugacaguua uu
224120RNAArtificial SequencesiRNA sense strand 41ccagcuuucc
ugaauggauu 204222RNAArtificial SequencesiRNA sense strand
42cucacuuuca auccguuuga uu 224322RNAArtificial SequencesiRNA sense
strand 43cagaacaguc acuguccuug uu 224420RNAArtificial SequencesiRNA
sense strand 44caccagucca acugcgaguu 204522RNAArtificial
SequencesiRNA sense strand 45ugcgacguga gucugagucu uu
224620RNAArtificial SequencesiRNA sense strand 46ccuccgaaaa
ccguacuuuu 204722RNAArtificial SequencesiRNA sense strand
47ccuuguccgu cuucuaacuc uu 224821RNAArtificial SequencesiRNA sense
strand 48ccagguccgu caagccuaau u 214920RNAArtificial SequencesiRNA
sense strand 49gcagcagaga aaggaaguuu 205020RNAArtificial
SequencesiRNA sense strand 50ccuaucuccc ucagaacuuu
205122RNAArtificial SequencesiRNA sense strand 51gagaagcgag
accuuagaag uu 225222RNAArtificial SequencesiRNA sense strand
52gccucaacuc uauagcucua uu 225320RNAArtificial SequencesiRNA sense
strand 53ccaacgugga auguuucuuu 205424RNAArtificial SequencesiRNA
sense strand 54gaagcggaag uggaagaaag uuuu 245522RNAArtificial
SequencesiRNA sense strand 55cuaguggcuu gagauuaagc uu
225622RNAArtificial SequencesiRNA sense strand 56ccagagaagc
ugcuggugau uu 225720RNAArtificial SequencesiRNA sense strand
57ccacagcuga aaccgagguu 205818RNAArtificial SequencesiRNA sense
strand 58cucugcggag uucuucuu 185920RNAArtificial SequencesiRNA
sense strand 59aacuacacug ugcggaccuu 206020RNAArtificial
SequencesiRNA sense strand 60gugauguggc ucgaacaguu
206122RNAArtificial SequencesiRNA sense strand 61gguuaaagaa
ggaaauggag uu 226222RNAArtificial SequencesiRNA sense strand
62ggaggauaug aucaucaugu uu 226320RNAArtificial SequencesiRNA sense
strand 63ggauagcuac gacgugauuu 206420RNAArtificial SequencesiRNA
sense strand 64cacagagcug cugaagaauu 206520RNAArtificial
SequencesiRNA sense strand 65ugcucuucuc ugcagagauu
206621RNAArtificial SequencesiRNA sense strand 66gcuuccucaa
uucugguggu u 216722RNAArtificial SequencesiRNA sense strand
67auucaucggu uuugccacca uu 226821RNAArtificial SequencesiRNA sense
strand 68aguggaagua caaggaugau u 216920RNAArtificial SequencesiRNA
sense strand 69cagccuuaau cuggaucauu 207022RNAArtificial
SequencesiRNA sense strand 70gucucggauc uuucagaacc uu
227120RNAArtificial SequencesiRNA sense strand 71ggcuuuagau
gaagugguuu 207220RNAArtificial SequencesiRNA sense strand
72guuugaucgg aaccguguuu 207320RNAArtificial SequencesiRNA sense
strand 73uuguggucca uggaaacguu 207421RNAArtificial SequencesiRNA
sense strand 74ccacuaagcu gcagcucaau u 217521RNAArtificial
SequencesiRNA sense strand 75ccaauggcug gacuccugau u
217621RNAArtificial SequencesiRNA sense strand 76gcuguggcuu
cugcaaccau u 217720RNAArtificial SequencesiRNA sense strand
77guguuugugg aacagccuuu 207821RNAArtificial SequencesiRNA sense
strand 78gcugcuacuc cuggacuauu u 217922RNAArtificial SequencesiRNA
sense strand 79uuccugcaca acaacgaggu uu 228020RNAArtificial
SequencesiRNA sense strand 80ccacaucgcu gacuccuguu
208122RNAArtificial SequencesiRNA sense strand 81agcuccagga
ccacuucuca uu 228220RNAArtificial SequencesiRNA sense strand
82auggccaugg accuguguuu 208322RNAArtificial SequencesiRNA sense
strand 83cgagugugag uucuacuuca uu 228420RNAArtificial SequencesiRNA
sense strand 84gcuguccuca ccaaccuguu 208520RNAArtificial
SequencesiRNA sense strand 85cugcguaucc ucauugaguu
208620RNAArtificial SequencesiRNA sense strand 86gagaacagga
aggugaucuu 208720RNAArtificial SequencesiRNA sense strand
87caagcugugg uccaacuuuu 208820RNAArtificial SequencesiRNA sense
strand 88gaggacuacg uggagcuguu 208922RNAArtificial SequencesiRNA
sense strand 89guguguggaa uguaccauac uu 229021RNAArtificial
SequencesiRNA sense strand 90agagggaugu guucucgggu u
219122RNAArtificial SequencesiRNA sense strand 91ccuucuguaa
gagcuuucga uu 229222RNAArtificial SequencesiRNA sense strand
92acaagggcau cuuccuccau uu 229320RNAArtificial SequencesiRNA sense
strand 93cugagcaauc agcaugaauu 209420RNAArtificial SequencesiRNA
sense strand 94ccacuuccag auacgacauu 209521RNAArtificial
SequencesiRNA sense strand 95accucuggca gaucuucgau u
219620RNAArtificial SequencesiRNA sense strand 96cgucgacugg
aaggagcauu 209720RNAArtificial SequencesiRNA sense strand
97guacauccac gagaacuauu 209822RNAArtificial SequencesiRNA sense
strand 98aaggaaucgu ggagcagcca uu 229920RNAArtificial SequencesiRNA
sense strand 99cugcugucag aacaaauguu 2010022RNAArtificial
SequencesiRNA sense strand 100ugugaugagc ugguggcaga uu
2210120RNAArtificial SequencesiRNA sense strand 101gcaugaggau
ucaaggcuuu 2010221RNAArtificial SequencesiRNA sense strand
102cuggaggcua cgagaauguu u 2110320RNAArtificial SequencesiRNA sense
strand 103uggacaucca caugaagcuu 2010422RNAArtificial SequencesiRNA
sense strand 104uacgaggacc aguggcugca uu 2210521RNAArtificial
SequencesiRNA sense strand 105caugaccgag agccuguuuu u
2110622RNAArtificial SequencesiRNA sense strand 106gugaugaacu
uugugguucg uu 2210720RNAArtificial SequencesiRNA sense strand
107agacgagcag ccgucucuuu 2010822RNAArtificial SequencesiRNA sense
strand 108gacucaucca ccuucacccu uu 2210922RNAArtificial
SequencesiRNA sense strand 109uuccugcgcu acgacugugu uu
2211022RNAArtificial SequencesiRNA sense strand 110cacacgcuac
aucauggugu uu 2211122RNAArtificial SequencesiRNA sense strand
111ugccauugug ccuuuuuagg uu 2211222RNAArtificial
SequencesiRNA sense strand 112cacuuccuga guucauguuc uu
2211322RNAArtificial SequencesiRNA sense strand 113ccugaacuga
auaugucacc uu 2211423RNAArtificial SequencesiRNA sense strand
114cgcagucuca cucugaauaa auu 2311521RNAArtificial SequencesiRNA
sense strand 115ggacaguuug uaagucuugu u 2111622RNAArtificial
SequencesiRNA sense strand 116ucacuucccc uguccagguu uu
2211722RNAArtificial SequencesiRNA sense strand 117ucagcuucca
caugugucaa uu 2211821RNAArtificial SequencesiRNA sense strand
118gacaauccuc gccuugucuu u 2111921RNAArtificial SequencesiRNA sense
strand 119gcaucuggag cuuucuguau u 2112020RNAArtificial
SequencesiRNA sense strand 120gagaucccag gauccugguu
2012122RNAArtificial SequencesiRNA sense strand 121aaucaggaug
ccacugugua uu 2212222RNAArtificial SequencesiRNA sense strand
122cuggaugaga agguuaguga uu 2212322RNAArtificial SequencesiRNA
sense strand 123ugugggaacu guuucuccag uu 2212420RNAArtificial
SequencesiRNA sense strand 124cuggaccagu agucaacauu
2012521RNAArtificial SequencesiRNA sense strand 125ccaaaggaua
gagucacugu u 2112623RNAArtificial SequencesiRNA sense strand
126cagcaccaag gcuauggcuu uuu 2312722RNAArtificial SequencesiRNA
sense strand 127guggaauucu ugagugagga uu 2212822RNAArtificial
SequencesiRNA sense strand 128gcugacuaug ccauuaagau uu
2212921RNAArtificial SequencesiRNA sense strand 129acaugaucaa
acucuauggu u 2113020RNAArtificial SequencesiRNA sense strand
130ggugaacaaa gcaucagcuu 2013120RNAArtificial SequencesiRNA sense
strand 131ccugagauug augagaaguu 2013219RNAArtificial SequencesiRNA
sense strand 132ggucaucuua caaacccuu 1913320RNAArtificial
SequencesiRNA sense strand 133ccugacacag gcaacuccuu
2013422RNAArtificial SequencesiRNA sense strand 134gcuucauuug
augcuucgga uu 2213522RNAArtificial SequencesiRNA sense strand
135ugcagcaauu gaagccauga uu 2213621RNAArtificial SequencesiRNA
sense strand 136gcaguaccuc uguaaccguu u 2113722RNAArtificial
SequencesiRNA sense strand 137caccguaucu uaugccuuca uu
2213822RNAArtificial SequencesiRNA sense strand 138gaacgacuuc
uggcagcuca uu 2213922RNAArtificial SequencesiRNA sense strand
139ccucaucagc uguuugcaga uu 2214022RNAArtificial SequencesiRNA
sense strand 140uggucaugga cacucacauc uu 2214120RNAArtificial
SequencesiRNA sense strand 141gaugucucag augcagcuuu
2014220RNAArtificial SequencesiRNA sense strand 142ccucauggcu
uaggacauuu 2014322RNAArtificial SequencesiRNA sense strand
143ucacauuuuc cuuccuccug uu 2214422RNAArtificial SequencesiRNA
sense strand 144ccuuggacca aucagagaug uu 2214522RNAArtificial
SequencesiRNA sense strand 145ggcaaaggua cuaaucccuu uu
2214620RNAArtificial SequencesiRNA sense strand 146uuccacagga
gguauuucuu 2014720RNAArtificial SequencesiRNA sense strand
147gguccugagu auuuugcauu 2014821RNAArtificial SequencesiRNA sense
strand 148ccaaaucugc aagaaggcuu u 2114923RNAArtificial
SequencesiRNA sense strand 149ggaacucuuc agcacauccu uuu
2315022RNAArtificial SequencesiRNA sense strand 150cucuggacaa
cagaagaaga uu 2215120RNAArtificial SequencesiRNA sense strand
151ugagagcagu gugauucuuu 2015219RNAArtificial SequencesiRNA sense
strand 152caagucuagc agugcauuu 1915321RNAArtificial SequencesiRNA
sense strand 153cucgcuaaga caacuagcau u 2115422RNAArtificial
SequencesiRNA sense strand 154cagguuaagu uucggaggcu uu
2215522RNAArtificial SequencesiRNA sense strand 155gcuuccaggc
accuccucuu uu 2215622RNAArtificial SequencesiRNA sense strand
156gaaguggaag ucgugcugag uu 2215722RNAArtificial SequencesiRNA
sense strand 157gaucucuuuc gccauggcug uu 2215827RNAArtificial
SequencedsRNA 158caagaagcug gaggaaaguu ucuaaag 2715929RNAArtificial
SequencedsRNA 159gaaagaugaa gaccucuucc uuucuaaag
2916029RNAArtificial SequencedsRNA 160ggaauggcug aaagacacuu
uuucuaaag 2916127RNAArtificial SequencedsRNA 161ccaguguucc
caguucccuu ucuaaag 2716227RNAArtificial SequencedsRNA 162ccaguccaac
ugcgaggauu ucuaaag 2716327RNAArtificial SequencedsRNA 163cgacgugagu
cugagucuuu ucuaaag 2716427RNAArtificial SequencedsRNA 164guccgucuuc
uaacucuuuu ucuaaag 2716527RNAArtificial SequencedsRNA 165cgucaagccu
aaguccuuuu ucuaaag 2716627RNAArtificial SequencedsRNA 166gcagagaaag
gaaguccuuu ucuaaag 2716727RNAArtificial SequencedsRNA 167cgagaagcga
gaccuuaguu ucuaaag 2716827RNAArtificial SequencedsRNA 168ccucaacucu
auagcucuuu ucuaaag 2716927RNAArtificial SequencedsRNA 169cuaguggcuu
gagguaucuu ucuaaag 2717027RNAArtificial SequencedsRNA 170ggauuaagcc
ugaucaaguu ucuaaag 2717127RNAArtificial SequencedsRNA 171gacugcuucg
gaugccuuuu ucuaaag 2717227RNAArtificial SequencedsRNA 172ccuucuugug
augcucucuu ucuaaag 2717327RNAArtificial SequencedsRNA 173ccucauccag
aguuugcauu ucuaaag 2717427RNAArtificial SequencedsRNA 174ccuguccucu
acgaaggauu ucuaaag 2717529RNAArtificial SequencedsRNA 175gauugcuauu
gucguacuca uuucuaaag 2917627RNAArtificial SequencedsRNA
176ggauucucgc ucuugcauuu ucuaaag 2717727RNAArtificial SequencedsRNA
177ggugacaguu accagcuuuu ucuaaag 2717827RNAArtificial SequencedsRNA
178ccucacuuuc aauccguuuu ucuaaag 2717927RNAArtificial SequencedsRNA
179gcagaacagu cacuguccuu ucuaaag 2718029RNAArtificial SequencedsRNA
180gaucaauaaa gucaguggcu uuucuaaag 2918130RNAArtificial
SequencedsRNA 181aagaagcugg aggaaagggg uuuucuaaag
3018229RNAArtificial SequencedsRNA 182augacaaccu cccaaaagca
uuucuaaag 2918327RNAArtificial SequencedsRNA 183ccgagacuuc
guggcagauu ucuaaag 2718424RNAArtificial SequencedsRNA 184agccuggcug
ggauuuuucu aaag 2418526RNAArtificial SequencedsRNA 185caagaagcug
gaggaaauuu cuaaag 2618629RNAArtificial SequencedsRNA 186ccaguuucug
gugcuaaaga uuucuaaag 2918727RNAArtificial SequencedsRNA
187aagaugaaga ccucuuccuu ucuaaag 2718828RNAArtificial SequencedsRNA
188ggacugcuuc ggaugccuuu uucuaaag 2818930RNAArtificial
SequencedsRNA 189aguggugcga cgccuucuuu uuuucuaaag
3019028RNAArtificial SequencedsRNA 190aguggugcga cgccuucuuu
uucuaaag 2819129RNAArtificial SequencedsRNA 191uugcuauugu
cguacucacc uuucuaaag 2919227RNAArtificial SequencedsRNA
192gauucucgcu cuugcauguu ucuaaag 2719329RNAArtificial SequencedsRNA
193caguucccug gugacaguua uuucuaaag 2919427RNAArtificial
SequencedsRNA 194ccagcuuucc ugaauggauu ucuaaag 2719529RNAArtificial
SequencedsRNA 195cucacuuuca auccguuuga uuucuaaag
2919629RNAArtificial SequencedsRNA 196cagaacaguc acuguccuug
uuucuaaag 2919727RNAArtificial SequencedsRNA 197caccagucca
acugcgaguu ucuaaag 2719829RNAArtificial SequencedsRNA 198ugcgacguga
gucugagucu uuucuaaag 2919927RNAArtificial SequencedsRNA
199ccuccgaaaa ccguacuuuu ucuaaag 2720029RNAArtificial SequencedsRNA
200ccuuguccgu cuucuaacuc uuucuaaag 2920128RNAArtificial
SequencedsRNA 201ccagguccgu caagccuaau uucuaaag
2820227RNAArtificial SequencedsRNA 202gcagcagaga aaggaaguuu ucuaaag
2720327RNAArtificial SequencedsRNA 203ccuaucuccc ucagaacuuu ucuaaag
2720429RNAArtificial SequencedsRNA 204gagaagcgag accuuagaag
uuucuaaag 2920529RNAArtificial SequencedsRNA 205gccucaacuc
uauagcucua uuucuaaag 2920627RNAArtificial SequencedsRNA
206ccaacgugga auguuucuuu ucuaaag 2720731RNAArtificial SequencedsRNA
207gaagcggaag uggaagaaag uuuuucuaaa g 3120829RNAArtificial
SequencedsRNA 208cuaguggcuu gagauuaagc uuucuaaag
2920929RNAArtificial SequencedsRNA 209ccagagaagc ugcuggugau
uuucuaaag 2921027RNAArtificial SequencedsRNA 210ccacagcuga
aaccgagguu ucuaaag 2721125RNAArtificial SequencedsRNA 211cucugcggag
uucuucuuuc uaaag 2521227RNAArtificial SequencedsRNA 212aacuacacug
ugcggaccuu ucuaaag 2721327RNAArtificial SequencedsRNA 213gugauguggc
ucgaacaguu ucuaaag 2721429RNAArtificial SequencedsRNA 214gguuaaagaa
ggaaauggag uuucuaaag 2921529RNAArtificial SequencedsRNA
215ggaggauaug aucaucaugu uuucuaaag 2921627RNAArtificial
SequencedsRNA 216ggauagcuac gacgugauuu ucuaaag 2721727RNAArtificial
SequencedsRNA 217cacagagcug cugaagaauu ucuaaag 2721827RNAArtificial
SequencedsRNA 218ugcucuucuc ugcagagauu ucuaaag 2721928RNAArtificial
SequencedsRNA 219gcuuccucaa uucugguggu uucuaaag
2822029RNAArtificial SequencedsRNA 220auucaucggu uuugccacca
uuucuaaag 2922128RNAArtificial SequencedsRNA 221aguggaagua
caaggaugau uucuaaag 2822227RNAArtificial SequencedsRNA
222cagccuuaau cuggaucauu ucuaaag 2722329RNAArtificial SequencedsRNA
223gucucggauc uuucagaacc uuucuaaag 2922427RNAArtificial
SequencedsRNA 224ggcuuuagau gaagugguuu ucuaaag 2722527RNAArtificial
SequencedsRNA 225guuugaucgg aaccguguuu ucuaaag 2722627RNAArtificial
SequencedsRNA 226uuguggucca uggaaacguu ucuaaag 2722728RNAArtificial
SequencedsRNA 227ccacuaagcu gcagcucaau uucuaaag
2822828RNAArtificial SequencedsRNA 228ccaauggcug gacuccugau
uucuaaag 2822928RNAArtificial SequencedsRNA 229gcuguggcuu
cugcaaccau uucuaaag 2823027RNAArtificial SequencedsRNA
230guguuugugg aacagccuuu ucuaaag 2723128RNAArtificial SequencedsRNA
231gcugcuacuc cuggacuauu uucuaaag 2823229RNAArtificial
SequencedsRNA 232uuccugcaca acaacgaggu uuucuaaag
2923327RNAArtificial SequencedsRNA 233ccacaucgcu gacuccuguu ucuaaag
2723429RNAArtificial SequencedsRNA 234agcuccagga ccacuucuca
uuucuaaag 2923527RNAArtificial SequencedsRNA 235auggccaugg
accuguguuu ucuaaag 2723629RNAArtificial SequencedsRNA 236cgagugugag
uucuacuuca uuucuaaag 2923727RNAArtificial SequencedsRNA
237gcuguccuca ccaaccuguu ucuaaag 2723827RNAArtificial SequencedsRNA
238cugcguaucc ucauugaguu ucuaaag 2723927RNAArtificial SequencedsRNA
239gagaacagga aggugaucuu ucuaaag 2724027RNAArtificial SequencedsRNA
240caagcugugg uccaacuuuu ucuaaag 2724127RNAArtificial SequencedsRNA
241gaggacuacg uggagcuguu ucuaaag 2724229RNAArtificial SequencedsRNA
242guguguggaa uguaccauac uuucuaaag 2924328RNAArtificial
SequencedsRNA 243agagggaugu guucucgggu uucuaaag
2824429RNAArtificial SequencedsRNA 244ccuucuguaa gagcuuucga
uuucuaaag 2924529RNAArtificial SequencedsRNA 245acaagggcau
cuuccuccau uuucuaaag 2924627RNAArtificial SequencedsRNA
246cugagcaauc agcaugaauu ucuaaag 2724727RNAArtificial SequencedsRNA
247ccacuuccag auacgacauu ucuaaag 2724828RNAArtificial SequencedsRNA
248accucuggca gaucuucgau uucuaaag 2824927RNAArtificial
SequencedsRNA 249cgucgacugg aaggagcauu ucuaaag 2725027RNAArtificial
SequencedsRNA 250guacauccac gagaacuauu ucuaaag 2725129RNAArtificial
SequencedsRNA 251aaggaaucgu ggagcagcca uuucuaaag
2925227RNAArtificial SequencedsRNA 252cugcugucag aacaaauguu ucuaaag
2725329RNAArtificial SequencedsRNA 253ugugaugagc ugguggcaga
uuucuaaag 2925427RNAArtificial SequencedsRNA 254gcaugaggau
ucaaggcuuu ucuaaag 2725528RNAArtificial SequencedsRNA 255cuggaggcua
cgagaauguu uucuaaag 2825627RNAArtificial SequencedsRNA
256uggacaucca caugaagcuu ucuaaag 2725729RNAArtificial SequencedsRNA
257uacgaggacc aguggcugca uuucuaaag 2925828RNAArtificial
SequencedsRNA 258caugaccgag agccuguuuu uucuaaag
2825929RNAArtificial SequencedsRNA 259gugaugaacu uugugguucg
uuucuaaag 2926027RNAArtificial SequencedsRNA 260agacgagcag
ccgucucuuu ucuaaag 2726129RNAArtificial SequencedsRNA 261gacucaucca
ccuucacccu uuucuaaag 2926229RNAArtificial SequencedsRNA
262uuccugcgcu acgacugugu uuucuaaag 2926329RNAArtificial
SequencedsRNA 263cacacgcuac aucauggugu uuucuaaag
2926429RNAArtificial SequencedsRNA 264ugccauugug ccuuuuuagg
uuucuaaag 2926529RNAArtificial SequencedsRNA 265cacuuccuga
guucauguuc uuucuaaag 2926629RNAArtificial SequencedsRNA
266ccugaacuga auaugucacc uuucuaaag 2926730RNAArtificial
SequencedsRNA 267cgcagucuca cucugaauaa auuucuaaag
3026828RNAArtificial SequencedsRNA 268ggacaguuug uaagucuugu
uucuaaag 2826929RNAArtificial SequencedsRNA 269ucacuucccc
uguccagguu uuucuaaag 2927029RNAArtificial SequencedsRNA
270ucagcuucca caugugucaa uuucuaaag 2927128RNAArtificial
SequencedsRNA 271gacaauccuc gccuugucuu uucuaaag
2827228RNAArtificial SequencedsRNA 272gcaucuggag cuuucuguau
uucuaaag 2827327RNAArtificial SequencedsRNA 273gagaucccag
gauccugguu ucuaaag 2727429RNAArtificial SequencedsRNA
274aaucaggaug ccacugugua uuucuaaag 2927529RNAArtificial
SequencedsRNA 275cuggaugaga agguuaguga uuucuaaag
2927629RNAArtificial SequencedsRNA 276ugugggaacu guuucuccag
uuucuaaag 2927727RNAArtificial SequencedsRNA 277cuggaccagu
agucaacauu ucuaaag 2727828RNAArtificial SequencedsRNA 278ccaaaggaua
gagucacugu uucuaaag 2827930RNAArtificial SequencedsRNA
279cagcaccaag gcuauggcuu uuuucuaaag 3028029RNAArtificial
SequencedsRNA 280guggaauucu ugagugagga uuucuaaag
2928129RNAArtificial SequencedsRNA 281gcugacuaug ccauuaagau
uuucuaaag 2928228RNAArtificial SequencedsRNA 282acaugaucaa
acucuauggu uucuaaag 2828327RNAArtificial SequencedsRNA
283ggugaacaaa gcaucagcuu ucuaaag 2728427RNAArtificial SequencedsRNA
284ccugagauug augagaaguu ucuaaag 2728526RNAArtificial SequencedsRNA
285ggucaucuua caaacccuuu cuaaag 2628627RNAArtificial SequencedsRNA
286ccugacacag gcaacuccuu ucuaaag 2728729RNAArtificial SequencedsRNA
287gcuucauuug augcuucgga uuucuaaag 2928829RNAArtificial
SequencedsRNA 288ugcagcaauu gaagccauga uuucuaaag
2928928RNAArtificial SequencedsRNA 289gcaguaccuc uguaaccguu
uucuaaag 2829029RNAArtificial SequencedsRNA 290caccguaucu
uaugccuuca uuucuaaag 2929129RNAArtificial SequencedsRNA
291gaacgacuuc uggcagcuca uuucuaaag 2929229RNAArtificial
SequencedsRNA 292ccucaucagc uguuugcaga uuucuaaag
2929329RNAArtificial SequencedsRNA 293uggucaugga cacucacauc
uuucuaaag 2929427RNAArtificial SequencedsRNA 294gaugucucag
augcagcuuu ucuaaag 2729527RNAArtificial SequencedsRNA 295ccucauggcu
uaggacauuu ucuaaag 2729629RNAArtificial SequencedsRNA 296ucacauuuuc
cuuccuccug uuucuaaag 2929729RNAArtificial SequencedsRNA
297ccuuggacca aucagagaug uuucuaaag 2929829RNAArtificial
SequencedsRNA 298ggcaaaggua cuaaucccuu uuucuaaag
2929927RNAArtificial SequencedsRNA 299uuccacagga gguauuucuu ucuaaag
2730027RNAArtificial SequencedsRNA 300gguccugagu auuuugcauu ucuaaag
2730128RNAArtificial SequencedsRNA 301ccaaaucugc aagaaggcuu
uucuaaag 2830230RNAArtificial SequencedsRNA 302ggaacucuuc
agcacauccu uuuucuaaag 3030329RNAArtificial SequencedsRNA
303cucuggacaa cagaagaaga uuucuaaag 2930427RNAArtificial
SequencedsRNA 304ugagagcagu gugauucuuu ucuaaag 2730526RNAArtificial
SequencedsRNA 305caagucuagc agugcauuuu cuaaag 2630628RNAArtificial
SequencedsRNA 306cucgcuaaga caacuagcau uucuaaag
2830729RNAArtificial SequencedsRNA 307cagguuaagu uucggaggcu
uuucuaaag 2930829RNAArtificial SequencedsRNA 308gcuuccaggc
accuccucuu uuucuaaag 2930929RNAArtificial SequencedsRNA
309gaaguggaag ucgugcugag uuucuaaag 2931029RNAArtificial
SequencedsRNA 310gaucucuuuc gccauggcug uuucuaaag
2931121RNAArtificial SequenceMouse siRNA sense strand 311ccucagcguu
ucaaucuuuu u 2131221RNAArtificial SequenceMouse siRNA sense strand
312cgacugcaga aucuccucuu u 2131321RNAArtificial SequenceMouse siRNA
sense strand 313cugcuuuacg auacuuucau u 2131419RNAArtificial
SequenceControl siRNA sense strand 314ccuacgccac caauuucgu
1931519RNAArtificial SequenceControl siRNA antisense strand
315acgaaauugg uggcguagg 1931620DNAArtificial SequenceHuman BANF1
primer forward strand 316gaaccgttac gggaactgaa 2031720DNAArtificial
SequenceHuman BANF1 primer reverse strand 317cccggaagag gtcttcatct
2031820DNAArtificial SequenceHuman PLOD3 primer forward strand
318cagctccagg accacttctc 2031920DNAArtificial SequenceHuman PLOD3
primer reverse strand 319gagcgggcgt agtactcatc 2032020DNAArtificial
SequenceHuman SF3B4 primer forward strand 320ctcagatgca gcttgcacac
2032119DNAArtificial SequenceHuman SF3B4 primer reverse strand
321ggagggccag tgtatccat 1932220DNAArtificial SequenceHuman GAPDH
primer forward strand 322accaggtggt ctcctctgac 2032320DNAArtificial
SequenceHuman GAPDH primer reverse strand 323tgctgtagcc aaattcgttg
2032421RNAArtificial SequenceGFP siRNA sense strand 324ggcuacgucc
aggagcgcac c 2132521RNAArtificial SequenceGFP siRNA antisense
strand 325ugcgcuccug gacguagccu u 2132620RNAArtificial
SequencesiRNA antisense strand 326cuuuccucca gcuucuuguu
2032721RNAArtificial SequencesiRNA antisense strand 327ggaagagguc
uucaucuucu u 2132822RNAArtificial SequencesiRNA antisense strand
328aagugucuuu cagccauucc uu 2232920RNAArtificial SequencesiRNA
antisense strand 329gggaacuggg aacacugguu 2033020RNAArtificial
SequencesiRNA antisense strand 330uccucgcagu uggacugguu
2033120RNAArtificial SequencesiRNA antisense strand 331agacucagac
ucacgucguu 2033220RNAArtificial SequencesiRNA antisense strand
332aagaguuaga agacggacuu 2033320RNAArtificial SequencesiRNA
antisense strand 333aaggacuuag gcuugacguu 2033420RNAArtificial
SequencesiRNA antisense strand 334aggacuuccu uucucugcuu
2033520RNAArtificial SequencesiRNA antisense strand 335cuaaggucuc
gcuucucguu 2033620RNAArtificial SequencesiRNA antisense strand
336agagcuauag aguugagguu 2033720RNAArtificial SequencesiRNA
antisense strand 337gauaccucaa gccacuaguu 2033820RNAArtificial
SequencesiRNA antisense strand 338cuugaucagg cuuaauccuu
2033920RNAArtificial SequencesiRNA antisense strand 339aaggcauccg
aagcagucuu 2034020RNAArtificial SequencesiRNA antisense strand
340gagagcauca caagaagguu 2034120RNAArtificial SequencesiRNA
antisense strand 341ugcaaacucu ggaugagguu 2034220RNAArtificial
SequencesiRNA antisense strand 342uccuucguag aggacagguu
2034322RNAArtificial SequencesiRNA antisense strand 343ugaguacgac
aauagcaauc uu 2234420RNAArtificial SequencesiRNA antisense strand
344augcaagagc gagaauccuu 2034520RNAArtificial SequencesiRNA
antisense strand 345aagcugguaa cugucaccuu 2034620RNAArtificial
SequencesiRNA antisense strand 346aacggauuga aagugagguu
2034720RNAArtificial SequencesiRNA antisense strand 347ggacagugac
uguucugcuu 2034822RNAArtificial SequencesiRNA antisense strand
348agccacugac uuuauugauc uu 2234921RNAArtificial SequencesiRNA
antisense strand 349ccccuuuccu ccgcuucuuu u 2135022RNAArtificial
SequencesiRNA antisense strand 350ugcuuuuggg agguugucau uu
2235120RNAArtificial SequencesiRNA antisense strand 351ucugccacga
agucucgguu 2035217RNAArtificial SequencesiRNA antisense strand
352aaucccagcc aggcuuu 1735319RNAArtificial SequencesiRNA antisense
strand 353uuuccuccag cuucuuguu 1935422RNAArtificial SequencesiRNA
antisense strand 354ucuuuagcac cagaaacugg uu 2235520RNAArtificial
SequencesiRNA antisense strand 355ggaagagguc uucaucuuuu
2035621RNAArtificial SequencesiRNA antisense strand 356aaggcauccg
aagcaguccu u 2135721RNAArtificial SequencesiRNA antisense strand
357aagaaggcgu cgcaccacuu u 2135821RNAArtificial SequencesiRNA
antisense strand 358aagaaggcgu cgcaccacuu u 2135922RNAArtificial
SequencesiRNA antisense strand 359ggugaguacg acaauagcaa uu
2236020RNAArtificial SequencesiRNA antisense strand 360caugcaagag
cgagaaucuu 2036122RNAArtificial SequencesiRNA antisense strand
361uaacugucac cagggaacug uu 2236220RNAArtificial SequencesiRNA
antisense strand 362uccauucagg aaagcugguu 2036322RNAArtificial
SequencesiRNA antisense strand 363ucaaacggau ugaaagugag uu
2236422RNAArtificial SequencesiRNA antisense strand 364caaggacagu
gacuguucug uu 2236520RNAArtificial SequencesiRNA antisense strand
365cucgcaguug gacugguguu 2036622RNAArtificial SequencesiRNA
antisense strand 366agacucagac ucacgucgca uu 2236720RNAArtificial
SequencesiRNA antisense strand 367aaguacgguu uucggagguu
2036822RNAArtificial SequencesiRNA antisense strand 368gaguuagaag
acggacaagg uu 2236921RNAArtificial SequencesiRNA antisense strand
369uuaggcuuga cggaccuggu u 2137021RNAArtificial SequencesiRNA
antisense strand 370uacuuccuuu cucugcugcu u 2137120RNAArtificial
SequencesiRNA antisense strand 371aguucugagg gagauagguu
2037222RNAArtificial SequencesiRNA antisense strand 372cuucuaaggu
cucgcuucuc uu 2237322RNAArtificial SequencesiRNA antisense strand
373uagagcuaua gaguugaggc uu 2237420RNAArtificial SequencesiRNA
antisense strand 374agaaacauuc cacguugguu 2037524RNAArtificial
SequencesiRNA antisense strand 375aacuuucuuc cacuuccgcu ucuu
2437622RNAArtificial SequencesiRNA antisense strand 376gcuuaaucuc
aagccacuag uu 2237722RNAArtificial SequencesiRNA antisense strand
377aucaccagca gcuucucugg uu 2237820RNAArtificial SequencesiRNA
antisense strand 378ccucgguuuc agcugugguu 2037918RNAArtificial
SequencesiRNA antisense strand 379gaagaacucc gcagaguu
1838020RNAArtificial SequencesiRNA antisense strand 380gguccgcaca
guguaguuuu 2038120RNAArtificial SequencesiRNA antisense strand
381cuguucgagc cacaucacuu 2038222RNAArtificial SequencesiRNA
antisense strand 382cuccauuucc uucuuuaacc uu 2238322RNAArtificial
SequencesiRNA antisense strand 383acaugaugau cauauccucc uu
2238420RNAArtificial SequencesiRNA antisense strand 384aucacgucgu
agcuauccuu 2038520RNAArtificial SequencesiRNA antisense strand
385uucuucagca gcucuguguu 2038620RNAArtificial SequencesiRNA
antisense strand 386ucucugcaga gaagagcauu 2038721RNAArtificial
SequencesiRNA antisense strand 387ccaccagaau ugaggaagcu u
2138822RNAArtificial SequencesiRNA antisense strand 388ugguggcaaa
accgaugaau uu 2238921RNAArtificial SequencesiRNA antisense strand
389ucauccuugu acuuccacuu u 2139020RNAArtificial SequencesiRNA
antisense strand 390ugauccagau uaaggcuguu 2039122RNAArtificial
SequencesiRNA antisense strand 391gguucugaaa gauccgagac uu
2239220RNAArtificial SequencesiRNA antisense strand 392accacuucau
cuaaagccuu 2039320RNAArtificial SequencesiRNA antisense strand
393acacgguucc gaucaaacuu 2039420RNAArtificial SequencesiRNA
antisense strand 394cguuuccaug gaccacaauu 2039521RNAArtificial
SequencesiRNA antisense strand 395uugagcugca gcuuaguggu u
2139621DNAArtificial SequencesiRNA antisense strand 396tcaggagtcc
agccattggu u 2139721RNAArtificial SequencesiRNA antisense strand
397ugguugcaga agccacagcu u 2139820RNAArtificial SequencesiRNA
antisense strand 398aggcuguucc acaaacacuu 2039921RNAArtificial
SequencesiRNA antisense strand 399auaguccagg aguagcagcu u
2140022RNAArtificial SequencesiRNA antisense strand 400accucguugu
ugugcaggaa uu 2240120RNAArtificial SequencesiRNA antisense strand
401caggagucag cgaugugguu 2040222DNAArtificial SequencesiRNA
antisense strand 402tgagaagtgg tcctggagct uu 2240320RNAArtificial
SequencesiRNA antisense strand 403acacaggucc auggccauuu
2040422RNAArtificial SequencesiRNA antisense strand 404ugaaguagaa
cucacacucg uu 2240520RNAArtificial SequencesiRNA antisense strand
405cagguuggug aggacagcuu 2040620RNAArtificial SequencesiRNA
antisense strand 406cucaaugagg auacgcaguu 2040720RNAArtificial
SequencesiRNA antisense strand 407gaucaccuuc cuguucucuu
2040820RNAArtificial SequencesiRNA antisense strand 408aaguuggacc
acagcuuguu 2040920RNAArtificial SequencesiRNA antisense strand
409cagcuccacg
uaguccucuu 2041022RNAArtificial SequencesiRNA antisense strand
410guaugguaca uuccacacac uu 2241121RNAArtificial SequencesiRNA
antisense strand 411cccgagaaca caucccucuu u 2141222RNAArtificial
SequencesiRNA antisense strand 412ucgaaagcuc uuacagaagg uu
2241322RNAArtificial SequencesiRNA antisense strand 413auggaggaag
augcccuugu uu 2241420RNAArtificial SequencesiRNA antisense strand
414uucaugcuga uugcucaguu 2041520RNAArtificial SequencesiRNA
antisense strand 415ugucguaucu ggaagugguu 2041621RNAArtificial
SequencesiRNA antisense strand 416ucgaagaucu gccagagguu u
2141720RNAArtificial SequencesiRNA antisense strand 417ugcuccuucc
agucgacguu 2041820RNAArtificial SequencesiRNA antisense strand
418uaguucucgu ggauguacuu 2041922RNAArtificial SequencesiRNA
antisense strand 419uggcugcucc acgauuccuu uu 2242020RNAArtificial
SequencesiRNA antisense strand 420cauuuguucu gacagcaguu
2042122RNAArtificial SequencesiRNA antisense strand 421ucugccacca
gcucaucaca uu 2242220RNAArtificial SequencesiRNA antisense strand
422agccuugaau ccucaugcuu 2042321RNAArtificial SequencesiRNA
antisense strand 423acauucucgu agccuccagu u 2142420RNAArtificial
SequencesiRNA antisense strand 424gcuucaugug gauguccauu
2042522DNAArtificial SequencesiRNA antisense strand 425tgcagccact
ggtcctcgta uu 2242621RNAArtificial SequencesiRNA antisense strand
426aaacaggcuc ucggucaugu u 2142722RNAArtificial SequencesiRNA
antisense strand 427cgaaccacaa aguucaucac uu 2242820RNAArtificial
SequencesiRNA antisense strand 428agagacggcu gcucgucuuu
2042922RNAArtificial SequencesiRNA antisense strand 429agggugaagg
uggaugaguc uu 2243022RNAArtificial SequencesiRNA antisense strand
430acacagucgu agcgcaggaa uu 2243122RNAArtificial SequencesiRNA
antisense strand 431acaccaugau guagcgugug uu 2243222RNAArtificial
SequencesiRNA antisense strand 432ccuaaaaagg cacaauggca uu
2243322RNAArtificial SequencesiRNA antisense strand 433gaacaugaac
ucaggaagug uu 2243422RNAArtificial SequencesiRNA antisense strand
434ggugacauau ucaguucagg uu 2243523RNAArtificial SequencesiRNA
antisense strand 435uuuauucaga gugagacugc guu 2343621RNAArtificial
SequencesiRNA antisense strand 436caagacuuac aaacuguccu u
2143722RNAArtificial SequencesiRNA antisense strand 437aaccuggaca
ggggaaguga uu 2243822RNAArtificial SequencesiRNA antisense strand
438uugacacaug uggaagcuga uu 2243921RNAArtificial SequencesiRNA
antisense strand 439agacaaggcg aggauugucu u 2144021RNAArtificial
SequencesiRNA antisense strand 440uacagaaagc uccagaugcu u
2144120RNAArtificial SequencesiRNA antisense strand 441ccaggauccu
gggaucucuu 2044222RNAArtificial SequencesiRNA antisense strand
442uacacagugg cauccugauu uu 2244322RNAArtificial SequencesiRNA
antisense strand 443ucacuaaccu ucucauccag uu 2244422RNAArtificial
SequencesiRNA antisense strand 444cuggagaaac aguucccaca uu
2244520RNAArtificial SequencesiRNA antisense strand 445uguugacuac
ugguccaguu 2044621RNAArtificial SequencesiRNA antisense strand
446cagugacucu auccuuuggu u 2144723RNAArtificial SequencesiRNA
antisense strand 447aaagccauag ccuuggugcu guu 2344822RNAArtificial
SequencesiRNA antisense strand 448uccucacuca agaauuccac uu
2244922RNAArtificial SequencesiRNA antisense strand 449aucuuaaugg
cauagucagc uu 2245021RNAArtificial SequencesiRNA antisense strand
450ccauagaguu ugaucauguu u 2145120RNAArtificial SequencesiRNA
antisense strand 451gcugaugcuu uguucaccuu 2045220RNAArtificial
SequencesiRNA antisense strand 452cuucucauca aucucagguu
2045319RNAArtificial SequencesiRNA antisense strand 453ggguuuguaa
gaugaccuu 1945420RNAArtificial SequencesiRNA antisense strand
454ggaguugccu gugucagguu 2045522RNAArtificial SequencesiRNA
antisense strand 455uccgaagcau caaaugaagc uu 2245622RNAArtificial
SequencesiRNA antisense strand 456ucauggcuuc aauugcugca uu
2245721RNAArtificial SequencesiRNA antisense strand 457acgguuacag
agguacugcu u 2145822RNAArtificial SequencesiRNA antisense strand
458ugaaggcaua agauacggug uu 2245922RNAArtificial SequencesiRNA
antisense strand 459ugagcugcca gaagucguuc uu 2246022RNAArtificial
SequencesiRNA antisense strand 460ucugcaaaca gcugaugagg uu
2246122RNAArtificial SequencesiRNA antisense strand 461gaugugagug
uccaugacca uu 2246220RNAArtificial SequencesiRNA antisense strand
462agcugcaucu gagacaucuu 2046320RNAArtificial SequencesiRNA
antisense strand 463auguccuaag ccaugagguu 2046422RNAArtificial
SequencesiRNA antisense strand 464caggaggaag gaaaauguga uu
2246522RNAArtificial SequencesiRNA antisense strand 465caucucugau
ugguccaagg uu 2246622RNAArtificial SequencesiRNA antisense strand
466aagggauuag uaccuuugcc uu 2246720RNAArtificial SequencesiRNA
antisense strand 467gaaauaccuc cuguggaauu 2046820RNAArtificial
SequencesiRNA antisense strand 468ugcaaaauac ucaggaccuu
2046921RNAArtificial SequencesiRNA antisense strand 469agccuucuug
cagauuuggu u 2147023RNAArtificial SequencesiRNA antisense strand
470aaggaugugc ugaagaguuc cuu 2347122RNAArtificial SequencesiRNA
antisense strand 471ucuucuucug uuguccagag uu 2247220RNAArtificial
SequencesiRNA antisense strand 472agaaucacac ugcucucauu
2047319RNAArtificial SequencesiRNA antisense strand 473augcacugcu
agacuuguu 1947422RNAArtificial SequencesiRNA antisense strand
474ugcuaguugu cuuagcgaga uu 2247522RNAArtificial SequencesiRNA
antisense strand 475agccuccgaa acuuaaccug uu 2247622RNAArtificial
SequencesiRNA antisense strand 476aagaggaggu gccuggaagc uu
2247722RNAArtificial SequencesiRNA antisense strand 477cucagcacga
cuuccacuuc uu 2247822RNAArtificial SequencesiRNA antisense strand
478cagccauggc gaaagagauc uu 2247919RNAArtificial SequenceMouse
siRNA antisense strand 479aaagauugaa acgcugagg 1948019RNAArtificial
SequenceMouse siRNA antisense strand 480agaggagauu cugcagucg
1948119RNAArtificial SequenceMouse siRNA antisense strand
481ugaaaguauc guaaagcag 1948218DNAArtificial SequenceHuman BANF1
gene variant1 482caagaagctg gaggaaag 1848320DNAArtificial
SequenceHuman BANF1 gene variant1 483gaaagatgaa gacctcttcc
2048420DNAArtificial SequenceHuman BANF1 gene variant1
484ggaatggctg aaagacactt 2048518DNAArtificial SequenceHuman BANF1
gene variant1 485ccagtgttcc cagttccc 1848618DNAArtificial
SequenceHuman BANF1 gene variant1 486ccagtccaac tgcgagga
1848718DNAArtificial SequenceHuman BANF1 gene variant1
487cgacgtgagt ctgagtct 1848818DNAArtificial SequenceHuman BANF1
gene variant1 488gtccgtcttc taactctt 1848918DNAArtificial
SequenceHuman BANF1 gene variant1 489cgtcaagcct aagtcctt
1849018DNAArtificial SequenceHuman BANF1 gene variant1
490gcagagaaag gaagtcct 1849118DNAArtificial SequenceHuman BANF1
gene variant1 491cgagaagcga gaccttag 1849218DNAArtificial
SequenceHuman BANF1 gene variant1 492cctcaactct atagctct
1849318DNAArtificial SequenceHuman BANF1 gene variant1
493ctagtggctt gaggtatc 1849418DNAArtificial SequenceHuman BANF1
gene variant1 494ggattaagcc tgatcaag 1849518DNAArtificial
SequenceHuman BANF1 gene variant1 495gactgcttcg gatgcctt
1849618DNAArtificial SequenceHuman BANF1 gene variant1
496ccttcttgtg atgctctc 1849718DNAArtificial SequenceHuman BANF1
gene variant1 497cctcatccag agtttgca 1849818DNAArtificial
SequenceHuman BANF1 gene variant1 498cctgtcctct acgaagga
1849920DNAArtificial SequenceHuman BANF1 gene variant1
499gattgctatt gtcgtactca 2050018DNAArtificial SequenceHuman BANF1
gene variant1 500ggattctcgc tcttgcat 1850118DNAArtificial
SequenceHuman BANF1 gene variant1 501ggtgacagtt accagctt
1850218DNAArtificial SequenceHuman BANF1 gene variant1
502cctcactttc aatccgtt 1850318DNAArtificial SequenceHuman BANF1
gene variant1 503gcagaacagt cactgtcc 1850420DNAArtificial
SequenceHuman BANF1 gene variant1 504gatcaataaa gtcagtggct
2050521DNAArtificial SequenceHuman BANF1 gene variant1
505aagaagctgg aggaaagggg t 2150620DNAArtificial SequenceHuman BANF1
gene variant2 506atgacaacct cccaaaagca 2050718DNAArtificial
SequenceHuman BANF1 gene variant2 507ccgagacttc gtggcaga
1850815DNAArtificial SequenceHuman BANF1 gene variant2
508agcctggctg ggatt 1550917DNAArtificial SequenceHuman BANF1 gene
variant2 509caagaagctg gaggaaa 1751020DNAArtificial SequenceHuman
BANF1 gene variant2 510ccagtttctg gtgctaaaga 2051118DNAArtificial
SequenceHuman BANF1 gene variant2 511aagatgaaga cctcttcc
1851219DNAArtificial SequenceHuman BANF1 gene variant2
512ggactgcttc ggatgcctt 1951319DNAArtificial SequenceHuman BANF1
gene variant2 513agtggtgcga cgccttctt 1951420DNAArtificial
SequenceHuman BANF1 gene variant2 514ctctctggga agctctcaat
2051520DNAArtificial SequenceHuman BANF1 gene variant2
515ttgctattgt cgtactcacc 2051618DNAArtificial SequenceHuman BANF1
gene variant2 516gattctcgct cttgcatg 1851720DNAArtificial
SequenceHuman BANF1 gene variant2 517cagttccctg gtgacagtta
2051818DNAArtificial SequenceHuman BANF1 gene variant2
518ccagctttcc tgaatgga 1851920DNAArtificial SequenceHuman BANF1
gene variant2 519ctcactttca atccgtttga 2052020DNAArtificial
SequenceHuman BANF1 gene variant2 520cagaacagtc actgtccttg
2052118DNAArtificial SequenceHuman BANF1 gene variant2
521caccagtcca actgcgag 1852220DNAArtificial SequenceHuman BANF1
gene variant2 522tgcgacgtga gtctgagtct 2052318DNAArtificial
SequenceHuman BANF1 gene variant2 523cctccgaaaa ccgtactt
1852420DNAArtificial SequenceHuman BANF1 gene variant2
524ccttgtccgt cttctaactc 2052519DNAArtificial SequenceHuman BANF1
gene variant2 525ccaggtccgt caagcctaa 1952618DNAArtificial
SequenceHuman BANF1 gene variant2 526gcagcagaga aaggaagt
1852718DNAArtificial SequenceHuman BANF1 gene variant2
527cctatctccc tcagaact 1852820DNAArtificial SequenceHuman BANF1
gene variant2 528gagaagcgag accttagaag 2052920DNAArtificial
SequenceHuman BANF1 gene variant2 529gcctcaactc tatagctcta
2053018DNAArtificial SequenceHuman BANF1 gene variant2
530ccaacgtgga atgtttct 1853122DNAArtificial SequenceHuman BANF1
gene variant2 531gaagcggaag tggaagaaag tt 2253220DNAArtificial
SequenceHuman BANF1 gene variant2 532ctagtggctt gagattaagc
2053320DNAArtificial SequenceHuman PLOD3 gene 533ccagagaagc
tgctggtgat 2053418DNAArtificial SequenceHuman PLOD3 gene
534ccacagctga aaccgagg 1853516DNAArtificial SequenceHuman PLOD3
gene
535ctctgcggag ttcttc 1653618DNAArtificial SequenceHuman PLOD3 gene
536aactacactg tgcggacc 1853718DNAArtificial SequenceHuman PLOD3
gene 537gtgatgtggc tcgaacag 1853820DNAArtificial SequenceHuman
PLOD3 gene 538ggttaaagaa ggaaatggag 2053920DNAArtificial
SequenceHuman PLOD3 gene 539ggaggatatg atcatcatgt
2054018DNAArtificial SequenceHuman PLOD3 gene 540ggatagctac
gacgtgat 1854118DNAArtificial SequenceHuman PLOD3 gene
541cacagagctg ctgaagaa 1854218DNAArtificial SequenceHuman PLOD3
gene 542tgctcttctc tgcagaga 1854319DNAArtificial SequenceHuman
PLOD3 gene 543gcttcctcaa ttctggtgg 1954420DNAArtificial
SequenceHuman PLOD3 gene 544attcatcggt tttgccacca
2054519DNAArtificial SequenceHuman PLOD3 gene 545agtggaagta
caaggatga 1954618DNAArtificial SequenceHuman PLOD3 gene
546cagccttaat ctggatca 1854720DNAArtificial SequenceHuman PLOD3
gene 547gtctcggatc tttcagaacc 2054818DNAArtificial SequenceHuman
PLOD3 gene 548ggctttagat gaagtggt 1854918DNAArtificial
SequenceHuman PLOD3 gene 549gtttgatcgg aaccgtgt
1855018DNAArtificial SequenceHuman PLOD3 gene 550ttgtggtcca
tggaaacg 1855119DNAArtificial SequenceHuman PLOD3 gene
551ccactaagct gcagctcaa 1955219DNAArtificial SequenceHuman PLOD3
gene 552ccaatggctg gactcctga 1955319DNAArtificial SequenceHuman
PLOD3 gene 553gctgtggctt ctgcaacca 1955418DNAArtificial
SequenceHuman PLOD3 gene 554gtgtttgtgg aacagcct
1855519DNAArtificial SequenceHuman PLOD3 gene 555gctgctactc
ctggactat 1955620DNAArtificial SequenceHuman PLOD3 gene
556ttcctgcaca acaacgaggt 2055718DNAArtificial SequenceHuman PLOD3
gene 557ccacatcgct gactcctg 1855820DNAArtificial SequenceHuman
PLOD3 gene 558agctccagga ccacttctca 2055918DNAArtificial
SequenceHuman PLOD3 gene 559atggccatgg acctgtgt
1856020DNAArtificial SequenceHuman PLOD3 gene 560cgagtgtgag
ttctacttca 2056118DNAArtificial SequenceHuman PLOD3 gene
561gctgtcctca ccaacctg 1856218DNAArtificial SequenceHuman PLOD3
gene 562ctgcgtatcc tcattgag 1856318DNAArtificial SequenceHuman
PLOD3 gene 563gagaacagga aggtgatc 1856418DNAArtificial
SequenceHuman PLOD3 gene 564caagctgtgg tccaactt
1856518DNAArtificial SequenceHuman PLOD3 gene 565gaggactacg
tggagctg 1856620DNAArtificial SequenceHuman PLOD3 gene
566gtgtgtggaa tgtaccatac 2056719DNAArtificial SequenceHuman PLOD3
gene 567agagggatgt gttctcggg 1956820DNAArtificial SequenceHuman
PLOD3 gene 568ccttctgtaa gagctttcga 2056920DNAArtificial
SequenceHuman PLOD3 gene 569acaagggcat cttcctccat
2057018DNAArtificial SequenceHuman PLOD3 gene 570ctgagcaatc
agcatgaa 1857118DNAArtificial SequenceHuman PLOD3 gene
571ccacttccag atacgaca 1857219DNAArtificial SequenceHuman PLOD3
gene 572acctctggca gatcttcga 1957318DNAArtificial SequenceHuman
PLOD3 gene 573cgtcgactgg aaggagca 1857418DNAArtificial
SequenceHuman PLOD3 gene 574gtacatccac gagaacta
1857520DNAArtificial SequenceHuman PLOD3 gene 575aaggaatcgt
ggagcagcca 2057618DNAArtificial SequenceHuman PLOD3 gene
576ctgctgtcag aacaaatg 1857720DNAArtificial SequenceHuman PLOD3
gene 577tgtgatgagc tggtggcaga 2057818DNAArtificial SequenceHuman
PLOD3 gene 578gcatgaggat tcaaggct 1857919DNAArtificial
SequenceHuman PLOD3 gene 579ctggaggcta cgagaatgt
1958018DNAArtificial SequenceHuman PLOD3 gene 580tggacatcca
catgaagc 1858120DNAArtificial SequenceHuman PLOD3 gene
581tacgaggacc agtggctgca 2058219DNAArtificial SequenceHuman PLOD3
gene 582catgaccgag agcctgttt 1958320DNAArtificial SequenceHuman
PLOD3 gene 583gtgatgaact ttgtggttcg 2058418DNAArtificial
SequenceHuman PLOD3 gene 584agacgagcag ccgtctct
1858520DNAArtificial SequenceHuman PLOD3 gene 585gactcatcca
ccttcaccct 2058620DNAArtificial SequenceHuman PLOD3 gene
586ttcctgcgct acgactgtgt 2058720DNAArtificial SequenceHuman PLOD3
gene 587cacacgctac atcatggtgt 2058820DNAArtificial SequenceHuman
PLOD3 gene 588tgccattgtg cctttttagg 2058920DNAArtificial
SequenceHuman PLOD3 gene 589cacttcctga gttcatgttc
2059020DNAArtificial SequenceHuman PLOD3 gene 590cctgaactga
atatgtcacc 2059121DNAArtificial SequenceHuman PLOD3 gene
591cgcagtctca ctctgaataa a 2159219DNAArtificial SequenceHuman PLOD3
gene 592ggacagtttg taagtcttg 1959320DNAArtificial SequenceHuman
PLOD3 gene 593tcacttcccc tgtccaggtt 2059420DNAArtificial
SequenceHuman PLOD3 gene 594tcagcttcca catgtgtcaa
2059519DNAArtificial SequenceHuman PLOD3 gene 595gacaatcctc
gccttgtct 1959620DNAArtificial SequenceHuman PLOD3 gene
596catctggagc tttctgtagc 2059718DNAArtificial SequenceHuman PLOD3
gene 597gagatcccag gatcctgg 1859820DNAArtificial SequenceHuman
SF3B4 gene 598aatcaggatg ccactgtgta 2059920DNAArtificial
SequenceHuman SF3B4 gene 599ctggatgaga aggttagtga
2060020DNAArtificial SequenceHuman SF3B4 gene 600tgtgggaact
gtttctccag 2060118DNAArtificial SequenceHuman SF3B4 gene
601ctggaccagt agtcaaca 1860219DNAArtificial SequenceHuman SF3B4
gene 602ccaaaggata gagtcactg 1960321DNAArtificial SequenceHuman
SF3B4 gene 603cagcaccaag gctatggctt t 2160420DNAArtificial
SequenceHuman SF3B4 gene 604gtggaattct tgagtgagga
2060520DNAArtificial SequenceHuman SF3B4 gene 605gctgactatg
ccattaagat 2060619DNAArtificial SequenceHuman SF3B4 gene
606acatgatcaa actctatgg 1960718DNAArtificial SequenceHuman SF3B4
gene 607ggtgaacaaa gcatcagc 1860818DNAArtificial SequenceHuman
SF3B4 gene 608cctgagattg atgagaag 1860917DNAArtificial
SequenceHuman SF3B4 gene 609ggtcatctta caaaccc 1761018DNAArtificial
SequenceHuman SF3B4 gene 610cctgacacag gcaactcc
1861120DNAArtificial SequenceHuman SF3B4 gene 611gcttcatttg
atgcttcgga 2061220DNAArtificial SequenceHuman SF3B4 gene
612tgcagcaatt gaagccatga 2061319DNAArtificial SequenceHuman SF3B4
gene 613gcagtacctc tgtaaccgt 1961420DNAArtificial SequenceHuman
SF3B4 gene 614caccgtatct tatgccttca 2061520DNAArtificial
SequenceHuman SF3B4 gene 615gaacgacttc tggcagctca
2061620DNAArtificial SequenceHuman SF3B4 gene 616cctcatcagc
tgtttgcaga 2061720DNAArtificial SequenceHuman SF3B4 gene
617tggtcatgga cactcacatc 2061818DNAArtificial SequenceHuman SF3B4
gene 618gatgtctcag atgcagct 1861918DNAArtificial SequenceHuman
SF3B4 gene 619cctcatggct taggacat 1862020DNAArtificial
SequenceHuman SF3B4 gene 620tcacattttc cttcctcctg
2062120DNAArtificial SequenceHuman SF3B4 gene 621ccttggacca
atcagagatg 2062220DNAArtificial SequenceHuman SF3B4 gene
622ggcaaaggta ctaatccctt 2062318DNAArtificial SequenceHuman SF3B4
gene 623ttccacagga ggtatttc 1862418DNAArtificial SequenceHuman
SF3B4 gene 624ggtcctgagt attttgca 1862519DNAArtificial
SequenceHuman SF3B4 gene 625ccaaatctgc aagaaggct
1962621DNAArtificial SequenceHuman SF3B4 gene 626ggaactcttc
agcacatcct t 2162720DNAArtificial SequenceHuman SF3B4 gene
627ctctggacaa cagaagaaga 2062818DNAArtificial SequenceHuman SF3B4
gene 628tgagagcagt gtgattct 1862917DNAArtificial SequenceHuman
SF3B4 gene 629caagtctagc agtgcat 1763019DNAArtificial SequenceHuman
SF3B4 gene 630ctcgctaaga caactagca 1963120DNAArtificial
SequenceHuman SF3B4 gene 631caggttaagt ttcggaggct
2063220DNAArtificial SequenceHuman SF3B4 gene 632gcttccaggc
acctcctctt 2063320DNAArtificial SequenceHuman SF3B4 gene
633gaagtggaag tcgtgctgag 2063420DNAArtificial SequenceHuman SF3B4
gene 634gatctctttc gccatggctg 20
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