Pharmaceutical composition for treating dementia comprising ShRNA
inhibiting S100a9 expression
United States Patent: 8,088,751
Issued: January 3, 2012
Inventors: Suh; Yoo-Hun
(Seoul, KR), Chang; Keun-A (Seoul, KR)
Assignee: SNU R&DB
Foundation (Nakseongdae-dong, Gwanak-gu, Seoul, KR)
Appl. No.: 12/851,273
Filed: August 5, 2010
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Disclosed is a composition for treating
dementia including shRNA to inhibit expression of S100a9. More
particularly, the present disclosure describes a composition for
prevention or treatment of dementia which includes shRNA having a
nucleotide sequence defined by SEQ. ID No. 1 or 2 or a mixture thereof
wherein the nucleotide sequence is complementarily bonded to mRNA of
S100a9 in order to inhibit expression of S100a9, as well as a method for
prevention or treatment of dementia, including administering the foregoing
shRNA into a mammalian cell including a human cell or in vitro established
mammalian cell-line, in order to inhibit expression of S100a9 protein.
Description of the
CLAIM OF PRIORITY
This application claims priority from Korean Patent Application No.
10-2010-50864, filed on May 31, 2010 in the Korean Intellectual Property
Office, the entire disclosure of which is incorporated herein, by
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a pharmaceutical composition for
prevention or treatment of dementia, which includes shRNA containing a
nucleotide sequence of SEQ. ID No. 1 or 2 capable of inhibiting expression
of S100a9, as well as a method for prevention or treatment of dementia by
administering the foregoing shRNA into cells of mammals including, for
example, human beings.
2. Description of the Related Art
It is known that S100a9 is an 5100 family as a calcium-binding protein
associated with inflammation. Increase in activated S100a9 in microglia
cells activates, signal transduction (or signaling) dependent on mitogen-activated
protein kinase (MAPK) cascade, NF-kB or calcium.
Neurodegenerative diseases including cerebral ischemia and Alzheimer's
disease have been known to associate with modified expression or function
of S100 family members and, recently, S100a9 is known to participate in
inflammation of patients with Alzheimer's disease and be considerably
increased in neuritic plaques. However, a pathological mechanism regarding
the foregoing conditions is still not disclosed.
RNA-mediated interference (RNAi) refers to a phenomenon that an RNA
fragment with a size of 21 to 25 nucleotides (nt) is selectively bonded to
mRNA having a complementary sequence and degrades the same in order to
inhibit protein expression.
Since Elbashir research team reported in 2001 that expression of a
particular gene may be selectively inhibited when a short dsRNA with 21
bases (siRNA) is introduced into a cultured mammal cell, applicability of
RNAi in mammalian cells was noticeably increased. At present, gene
expression inhibitory technologies using siRNA are generally used to
understand functions of various genes and are being actively applied to
development of drugs for treating incurable diseases such as cancer,
infectious diseases, etc.
Induction of cell apoptosis in human myelogenous leukemia cells using
siRNA specific to oncogenic genes such as Bcl-2 and c-Raf closely relating
to tumor formation, was reported. It was also disclosed that using siRNA
specific to Bcr-ab1 fused genes massively expressing in chronic
myelogenous leukemia (CML) may remarkably reduce expression of Bcr-abl
Alternatively, approaches for inhibition of viral infection using a
complementary siRNA of CXCR4/CCR5RNA, which is a co-receptor of siRNA or
HIV-1 complementary to HIV RNA, are being actively studied and developed.
In recent years, it was reported that a synthesized siRNA complementary to
hepatitis virus may effectively inhibit gene expression of the hepatitis
Such techniques to inhibit expression of particular genes in animal cells
using siRNA may include, for example, in vitro preparation of siRNA
comprising synthesizing siRNA in vitro and introducing the same into
cells. However, the foregoing method has disadvantages in that
bio-synthesis of siRNA requires high costs and cell introduction of the
synthesized siRNA has relatively low efficacy with regard to cell plasma
infection, in turn entailing insufficient gene inhibition using siRNA
while exhibiting RNAi effects for 2 to 3 days only. In order to overcome
the above problems, a method of introducing a siRNA plasmid vector capable
of expressing siRNA into cells was developed.
Especially, an siRNA plasmid vector expressing a short hairpin RNA (shRNA),
wherein sense and anti-sense sequences of siRNA target sequence are
located from a promoter of RNA polymerase III by interposing a loop having
5 to 9 bases, is characterized in that the shRNA expressed after
introduction thereof into cells is transformed into siRNA by an siRNA
processing enzyme (that is, Dicer or RNase III) and the transformed siRNA
can selectively inhibit expression of specific genes.
SUMMARY OF THE INVENTION
In order to solve conventional problems described above, an object of the
present invention is to provide a pharmaceutical composition for
prevention and treatment of dementia, including shRNA with a specific
sequence of RNA inhibiting expression of S100a9.
Another object of the present invention is to provide a method of
inhibiting expression of S100a9 protein by administering the foregoing
shRNA into mammalian cells including human cells, or in vitro established
A still further object of the present invention is to a method for
prevention or treatment of dementia by administering the foregoing shRNA
to a mammal such as the human.
In order to accomplish the foregoing purposes, the present invention
provides a pharmaceutical composition for prevention or treatment of
dementia, which includes shRNA having a nucleotide sequence defined by
SEQ. ID No. 1 or 2.
The present invention also provides a method of inhibiting expression of
S100a9 protein by administering the foregoing shRNA into cells.
The present invention further provides a method for prevention or
treatment of dementia by administering the foregoing shRNA to a mammal
such as the human.
According to the present invention, shRNA having a nucleotide sequence
defined by SEQ. ID 1 or 2 efficiently inhibits expression of S100a9,
thereby preventing or treating dementia.
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, preferred embodiments of the present invention will be
described in more detail through the following examples, in conjunction
with accompanying drawings.
According to an exemplary embodiment of the present invention, there is
provided a composition for treating dementia, comprising shRNA to inhibit
expression of S100a9. More particularly, described herein are: a
pharmaceutical composition for prevention or treatment of dementia, which
comprises shRNA having a nucleotide sequence defined by SEQ. ID No. 1 or 2
wherein the nucleotide sequence is complementarily bonded to mRNA of
S100a9 in order to inhibit expression of S100a9; a method of inhibiting
expression of S100a9 protein, comprising administering the foregoing shRNA
to cells (mammalian cells including the human cells or in vitro
established mammalian cells); and a method for prevention or treatment of
dementia by administering the shRNA to mammalian cells such as the human
The present invention will be described in detail below.
The expression "shRNA" refers to a short double-stranded chain wherein a
loop is cut into the chain by a dicer and the chain, like siRNA, reacts
with RICS so as to express RNAi phenomenon. RNA consists of a stem-loop
structure, wherein a long RNA having 19 to 29 nucleotides produces a pair
of bases at both sides of the loop site having 5 to 10 nucleotides, thus
forming the double-stranded stem. In general, shRNA undergoes in vivo
transcription by Po1 III promoter and is synthesized, followed by cutting
the synthesized shRNA loop using a dicer, and reacting the cut chains with
RISC, like siRNA.
The inventive shRNA comprises an anti-sense base sequence defined by SEQ.
ID No. 1 or 2, wherein S100a9 expression may be efficiently inhibited by
such a sequence. The base sequences defined by SEQ. ID Nos. 1 and 2 are
shown in TABLE 1
-- see Original Patent.
According to one embodiment of the present invention, in order to reduce
S100a9 expression, a lentivirus vector encoding the anti-sense base
sequence defined by SEQ. ID No. 1 or 2 is provided. Such a system enables
more stable and continuous expression of siRNA for a long period of time.
A process of preparing shRNA and introducing the same into a cell or an
animal may depend on cell-biological performances of target gene products
and/or purposes of experiments, and all of siRNAs or shRNAs in association
with S100a9 gene should not always inhibit expression of proteins with
physiologically important effects. For instance, among five types of
shRNAs (shRNA90, shRNA168, shRNA204, shRNA255 and shRNA280) regarding
S100a9, shRNA255 (SEQ. ID No. 1) and shRNA280 (SEQ. ID No. 2) only have
efficiently inhibited S100a9 expression, as described below.
In the present description, "dementia" refers to all brain and nervous
system diseases developed by expression of S100a9 gene and means
complicated clinical syndromes wherein a brain undergoes organic damage or
degradation, in turn causing deterioration in cognitive functions such as
intelligence, learning, language, etc., as well as deterioration in
advanced mental functions. Such diseases may include, although are not
restricted to, neuro-degenerative disorders such as: Alzheimer's disease;
Parkinson's disease; senile dementia; prion disease; Lewy body dementia;
Huntington's disease; Creutzfeldt-Jakob disease, and the like.
Furthermore, the present invention may provide a method of inhibiting
expression of S100a9 protein in mammalian cells such as human cells or in
vitro established mammalian cell-lines, comprising administering the
foregoing shRNA into the cells, as well as a method for prevention or
treatment of dementia comprising administering the foregoing shRNA to
The administering process of the shRNA to the cell is to introduce a
lentivirus vector containing the inventive shRNA sequence into the cell
and may include any conventional methods used by persons skilled in the
art. The foregoing cell may be a mammalian cell such as the human cell.
In the present description, "gene" may refer to an encoded nucleic acid
molecule relative to a specific protein or, occasionally, mean a
functional or structural RNA molecule.
In the present description, "vector" refers to a nucleic acid molecule
transportable to another nucleic acid linked thereto. "Lentivirus vector"
means a vector extracted from lentivirus (that is, specific
shared-nucleotide sequence in lentivirus).
Preferred embodiments of the present invention will be described by the
following examples. However, these examples are provided for illustrative
purposes but are not construed to restrict the scope of the present
invention as defined by the appended claims.
Experimental Example 1
Using a trizol reagent (invitrogen), Total RNA was extracted from brain
tissues or cells. The RNA was bound with an oligo-primer (dT) and, using a
reverse transcriptase (RT) (AccuPower RT premix, Pioneer), cDNA was
prepared. The prepared single-stranded cDNA was used as a target of
polymerase chain reaction (PCR) and an annealing temperature was adjusted
according to properties of the primer. More particularly, a temperature
control cycle comprising 95.degree. C. for 1 minute, 58.degree. C. for 1
minute and 72.degree. C. for 1 minute was executed 30-40 times.
The used primer was S100a9 forward, 5'-CAGCATAACCACCATCATCG-3' (SEQ ID NO:
3) reverse, 5'-GTCCTGGTTTGTGTCCAGGT-3' (SEQ ID NO: 4) actin forward,
5'-CCAGATCATGTTTGAGACCT-3' (SEQ ID NO: 5) reverse,
5'-GTTGCCAATAGTGATGACCT-3' (SEQ ID NO: 6). Electrophoresis of PCR product
was conducted in 1.2% Agarose gel. From a band obtained by staining the
product with Ethidium Bromide (EtBr), homeostatic mRNA level was scanned
using a densitometer and the obtained result was compensated using actin.
Experimental Example 2
The following antibodies were used in the present experiment: Anti-APP
C-terminal polyclonal antibody (C9) (Chemicon, California), 6E10 (Chemicon),
CD11b (Chemicon), IL-1.beta. (R&D), TNF-.alpha. (R&D), iNOS (Santa Cruz),
anti-S100a9 (R&D), anti-GAPDH (Santa Cruz), anti-tubulin (Santa Cruz),
Neprilysin (alpha diagnostic).
Experimental Example 3
Western Blot Analysis
After a human brain cell with dementia and a transformed mouse brain cell
were dissolved in an RIPA buffer containing a protease inhibitor (Roche),
the prepared mixture was precipitated in a centrifuge at a desired speed
for 10 minutes and the supernatant was collected to conduct protein assay
(or determine protein concentration). For electrophoresis, each protein
with a constant concentration (30 ug to 60 ug) was boiled together with a
sample buffer, followed by conducting SDS-PAGE under denaturing
conditions. The protein was transferred to a PVDF membrane (Amersham
Pharmacia) and blocked using a 5% skimmed milk solution, followed by
washing with 0.05% Tween20-TBS three times. Using an antibody associated
with a desired protein, the product was incubated for 2 hours. Then, after
binding the incubated product with a HRP-polymer secondary antibody (Amersham
Pharmacia), the same was detected using an enhanced-chemiluminescent
detection system (Amersham Pharmacia). Loading a standard marker during
electrophoresis, a size of the detected band was determined, thus
verifying gene expression.
Experimental Example 4
Measurement of Luciferase Activity
48 hours after CT, wtAPP and/or sweAPP contained in pcDNA vector together
with fe65 were transfected with SHSY5Y cells, a human S100a9 promoter
contained in pgL3 vector (Dr. Claus Kerkhoff, Muenstet Univ., Germany) was
dissolved using a reporter lysis buffer contained in a Luciferase assay
system (Promega, WI, USA), Luciferase activity was determined using a
Biocouter M1500 luminometer (Lumac, GE Groningen, Netherlands). Protein
assay was executed using a Bradford protein assay reagent (Bio-Rad) while
Luciferase activity was standardized into a protein assay value.
Experimental example 5
Mice brains and human AD brains in 10% neutral buffered formalin for 48 h
were dehydrated and embedded in paraffin. Before immunostaining, slides
were deparaffinized in xylene and then dehydrated through graded alcohols
to water. The fluorescent immunohistochemistry was performed with
appropriate primary antibodies at 4uC for O/N and visualized using
Cy3-conjugated or FITC-conjugated secondary antibody (Jackson, West Grove,
Pa.). DAPI counter staining was performed. Images were collected using the
LSM 510 program on a Zeiss confocal microscope (Carl Zeiss MicroImaging,
Inc.). For the non-fluorecence labeling, Immunohistochemistry was
performed using a Vectastain avidin-biotin complex (ABC) elite kit.
Reaction product was detected using 3,3-diaminobenzidinetetrahydrochloride
(DAB). Photomicrographs were acquired with a, color digital camera DFC280
(Leica) attached to a microscope (BX-51; Olympus).
Experimental Example 6
Statistical assay comprised analysis of variance (ANOVA) executed using a
statistical package program (SPSS) (statistical package social science,
version 14.0, Chicago, Ill.) and significance test conducted by Duncan's
multiple rage test with p<0.05 in order to determine significance of test
Determination of S100a9 Protein Derived from Alzheimer's Disease
From each of a normal mouse aged 11 months and a mouse with dementia,
APPV717I-CT100 Tg, a brain was removed and whole RNA was extracted from a
hippocampus site of the brain using trizol (invitgen). Double-stranded
cDNA was synthesized from mRNA and hybridized to CodeLink Twinchip.TM.
Mouse-20K (Amersham Bioscience). All microarray experiments were conducted
by Digital Genomics (Seoul, South Korea). A scanned DNA chip was
standardized by conventional processes such as image plot, histogram, box
plot, RNA degradation plot, scatter plot, MA plot, etc. through a computer
program. Genes obtained according to the foregoing procedures were
subjected to SAM analysis in order to investigate a genetic origin or
functions of gene and, according to clustering of genes showing noticeable
fold change or similar genes in terms of functions, genes specifically
expressed in the dementia mouse rather than the normal mouse were
In order to extract gene candidate groups associated with Alzheimer's
disease, whole RNAs in hippocampus sites of the CT-Tg mouse and the same
aged control mouse were subjected to microarray assay and verified by
RT-PCR and Western blotting.
As a result, S100a9 was selected as a gene closely associated with the
above disease. As shown in FIG. 1A (see Original Patent), mRNA level of
S100a9 was derived from hippocampus and cortex sites in the brain of the
CT-Ta mouse. From results of western blotting and immunohistochemical
assay, it was also identified that S100a9 protein is increased in the
hippocampus and cortex sites in the brain of the CT-Tg mouse, compared to
the control (FIG. 1 (see Original Patent)). As shown in FIG. 1C (see Original Patent),
a Swedish type APP over-expressive Tg2576 mouse exhibited considerable
increase in S100a9 at the hippocampus and cortex sites of the brain of the
above mouse, compared to the control.
For both CT-Tg and Tg2576 mice, in order to examine physiological
significance of S100a9, a patient with human Alzheimer's disease and the
same-aged control were subjected to assay of S100a9 levels. Brain tissue
of a normal person who was 69 to 87 years old, as well as paraffin-fixed
brain tissue and lyophilized brain tissue of an Alzheimer's disease
patient were obtained from Netherlands Brain Bank (NBB). According to
neuro-pathological diagnosis, the brain tissues of the Alzheimer's disease
patient were in Braak & Braak stage V or VI while the normal brain tissue
was in Braak & Braak stage 0 or 1. For immunohistochemical staining, the
hippocampus part was cut into coronal sections with 4 mm. The lyophilized
brain tissue was used for western blotting. As shown in FIG. 10 (see Original Patent),
analysis results demonstrated that the brain of the patient with
Alzheimer's disease and a whole fused product thereof exhibit increase in
expression of S100a9, compared to the control.
Determination of Expression of Amyloid Beta or Amyloid Precursor Protein
in BV2 Cell and Microglia Cell
It was identified that S100a9 expression is increased in the brain with
Alzheimer's disease as well as CT-Tg and Tg2576 mice, all of which produce
amyloid beta or amyloid precursor protein (CT) in large quantities. This
relates closely to excessive production of amyloid beta and/or amyloid
precursor protein. As to the brain of the CT-Tg mouse used for a gene
chip, S100a9 was highly expressed in CD11b-positive microglia (FIG. 2A (see Original Patent)).
In order to determine whether Alzheimer's disease is associated with a
pathological mechanism of S100a9, endogenous expression of S100a9 in BV2
cell as a microglia cell-line was derived. The mouse BV2 cell was
incubated with a DMEM medium containing 5% fetal bovine serum (FBS,
Hyclone) and 1% antibiotic (100 U/ml/100 .mu.g/ml) (Life Technology) in a
5% CO.sub.2 cell incubator at 37.degree. C. After transfection with CT50
or CT99 in murine BV2 microglia cells, gene expression of S100a9 was
detected by RT-PCR. As a result, it was found that the mRNA level of
S100a9 is considerably increased 48 hours and 72 hours after transfection
with CT50 or CT99, especially, 48 hours after transfection (FIG. 2B).
Amyloid beta or amyloid precursor protein can be induced by controlling
promoter activity of S100a9. Possibility of such induction was
investigated according to promoter activity assay. 48 hours after
transfection, transfection effects of CT50 and CT99 upon S100a9 promoter
activity were determined (1.5860.32 by CT50, 2.0960.32 by CT99; FIG. 2C).
Effects of normal APP (wtAPP) and Swedish type APP (sweAPP) upon S100a9
promoter activity were also determined (1.4360.41 by wtAPP, 1.3560.20 by
sweAPP) and, in particular, CT99 considerably increased S100a9 promoter
activity (FIG. 2C). Subsequently, amyloid beta and amyloid precursor
protein peptides were treated at different concentrations (1, 10 mM of CT
or 2, 20 mM of A.beta.) for 48 hours, in turn inducing
concentration-dependent mRNA level of S100a9 (FIG. 2D). 10 mM amyloid
precursor protein significantly increased the mRNA level of S100a9.
[ratio=2.1560.29, p=0.0048 versus NC (negative control), student's t-test]
(FIG. 2E). Immunocytochemical analysis also demonstrated that S100a9 is
dose-dependently derived by amyloid beta or amyloid precursor protein
Evaluation of Effects of S100a9 Derived from Amyloid Beta or Amyloid
Protein Precursor Upon Increase in Calcium Content of Cell
48 hours after treatment using amyloid precursor protein or amyloid beta
peptides at different concentrations, BV2 cells cultured in a cover glass
coated with 10 mg/ml polyethyleneimine (PEI) were washed twice using a
Hank's solution (Gibco) and incubated using 10 .mu.M Fluo-3/AM at
37.degree. C. for 20 minutes. After mounting the cells, the cells were
subjected to excitation with an Argon ion laser (wavelength (.lamda.)=488
nm) using a laser-scanning confocal microscope and fluorescence
measurement at .lamda.>515 nm. Change of calcium content [Ca.sup.2+].sub.i
in cells was observed and compared with the control. Using 0.1, 1 and 10
mM amyloid precursor protein peptides or 1 and 10mM amyloid beta peptides,
BV2 cells were treated for 48 hours and [Ca.sup.2+].sub.i levels thereof
were evaluated by a Fluo3/AM method.
[Ca.sup.2+].sub.i level was dose-dependently increased and such a degree
of increase was calculated relative to the control. 10 mM amyloid
precursor protein significantly increased [Ca.sup.2+].sub.i level. In
order to determine whether S100a9 may increase [Ca.sup.2+].sub.i level,
S100a9 siRNA (si-S100a9) was treated using BV2 cells already treated with
amyloid precursor protein peptides.
As a result, S100a9 gene knockdown caused decrease in S100a9 expression
(FIG. 3D) and significantly reduced increase in [Ca.sup.2+].sub.i level by
amyloid precursor protein (from ratio=12.6360.65 to ratio=1.4960.45,
p=0.00016 versus si-CTL/CT 10 mM, Student's t-test). si-CTL did not
influence [Ca.sup.2+].sub.i level (FIGS. 3A and 3B). [Ca.sup.2+].sub.i
level obtained by amyloid beta treatment and S100a9 gene knockdown during
combination of si-S100a9 significantly reduced increase in
[Ca.sup.2+].sub.i level by 10 mM amyloid beta (from ratio=3.3160.58 to
ratio=1.0260.20, p=0.0086 versus si-CTL/Ab 10 mM, Student's t-test).
Determination of Increase in Proinflammatory Cytokines by Treatment Using
Amyloid Beta or Amyloid Precursor Protein
According to the present invention, it was found that treatment using
amyloid beta or amyloid precursor protein derives significant and
dose-dependent increase in IL-1.beta. and TNF-.alpha. as proinflammatory
cytokines (FIG. 30). In order to test efficacy of S100a9 induction for
production of proinflammatory cytokines, si-S100a9 was used for silencing
S100a9. IL-113 and TNF-.alpha. levels derived from amyloid precursor
protein were reduced to 60% by si-CTL and S100a9 expression was also
reduced (FIG. 3D).
Nitrogen oxide (NO) generation was measured on the basis of NO.sub.2
increase/decrease in a cultured solution. After treating BV2 cells
incubated in a 96-well plate using amyloid precursor protein peptides,
increase in NO content of the cultured solution was measured using a
Griess reagent (G4410, Sigma). After admixing the cultured solution with
the same amount of Griess reagent and measuring light absorption for 5
minutes at 540 nm in a spectrophotometer, the obtained results were
analyzed, compared to those of the control. According to the results, it
was found that iNOs level increased by the amyloid precursor protein was
reduced to 70% by si-S100a9 (FIG. 3D). As shown in FIG. 3E, silencing of
S100a9 gene significantly inhibited release of amyloid precursor
protein-derived NO from 21.2962.78 (mM) to 3.1960.88 (mM).
Claim 1 of 6 Claims
1. A pharmaceutical composition for
treatment of dementia, comprising an shRNA sequence defined by SEQ ID NO:
1 or SEQ ID NO: 2.
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