U.S. patent application number 16/781766 was filed with the patent office on 2020-07-30 for multi-conjugate of sirna and preparing method thereof.
The applicant listed for this patent is KIP Co. Ltd.. Invention is credited to Soo Hyeon Lee, Hye Jung Mok, Tae Gwan Park.
Application Number | 20200239892 16/781766 |
Document ID | 20200239892 / US20200239892 |
Family ID | 1000004752180 |
Filed Date | 2020-07-30 |
Patent Application | download [pdf] |
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United States Patent
Application |
20200239892 |
Kind Code |
A1 |
Park; Tae Gwan ; et
al. |
July 30, 2020 |
MULTI-CONJUGATE OF SIRNA AND PREPARING METHOD THEREOF
Abstract
The present invention relates to a multi-conjugate of small
interfering RNA (siRNA) and a preparing method of the same, more
precisely a multi-conjugate of siRNA prepared by direct binding of
double stranded sense/antisense siRNA monomers or indirect covalent
bonding mediated by a cross-linking agent or a polymer, and a
preparing method of the same. The preparing method of a siRNA
multi-conjugate of the present invention is characterized by simple
and efficient reaction and thereby the prepared siRNA
multi-conjugate of the present invention has high molecular weight
multiple times the conventional siRNA, so that it has high negative
charge density, suggesting that it has excellent ionic interaction
with a cationic gene carrier and high gene delivery efficiency.
Inventors: |
Park; Tae Gwan; (US)
; Mok; Hye Jung; (Daejeon, KR) ; Lee; Soo
Hyeon; (Daejeon, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KIP Co. Ltd. |
Daejeon |
|
KR |
|
|
Family ID: |
1000004752180 |
Appl. No.: |
16/781766 |
Filed: |
February 4, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16040795 |
Jul 20, 2018 |
10597659 |
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16781766 |
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15483528 |
Apr 10, 2017 |
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16040795 |
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14312530 |
Jun 23, 2014 |
9644209 |
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15483528 |
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14048951 |
Oct 8, 2013 |
9255269 |
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14312530 |
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12514306 |
May 8, 2009 |
8580946 |
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PCT/KR2009/002252 |
Apr 29, 2009 |
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14048951 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/1136 20130101;
C12N 15/113 20130101; C12N 2310/51 20130101; C12N 2310/14 20130101;
C12N 15/111 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; C12N 15/11 20060101 C12N015/11 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 13, 2009 |
KR |
10-2009-0021705 |
Claims
1. A multi-conjugate of siRNA having the following structure of
[Structure Formula I] or [Structure Formula II] prepared by direct
or indirect covalent bonding of double-stranded sense/antisense
siRNA monomers mediated by a cross-linking agent or a polymer:
(--X-A-).sub.n [Structural Formula I] (Wherein, X is
double-stranded siRNA monomer; A is either presented or not
presented, a cross-linking agent or a polymer; and n is the number
of double-stranded siRNA monomer), x-A-(X-A-).sub.n-x' [Structural
Formula II] (Wherein, X is double-stranded siRNA monomer; x or x'
is single-stranded siRNA monomer A is either presented or not
presented, a cross-linking agent or a polymer; and n is the number
of double-stranded siRNA monomer).
2. The siRNA multi-conjugate according to claim 1, wherein the
double-stranded sense/antisense siRNA monomer is composed of 15-29
nucleotides.
3. The siRNA multi-conjugate according to claim 1, wherein the
number n indicating the number of the double-stranded
sense/antisense siRNA monomer is one of 1-100.
4. A preparing method of a multi-conjugate of siRNA having the
structure of [Structural Formula I] or [Structural Formula II]
containing the step of direct or indirect covalent bonding of
double-stranded sense/antisense siRNA monomers mediated by a
cross-linking agent or a polymer (--X-A-).sub.n [Structural Formula
I] (Wherein, X is double-stranded siRNA monomer; A is either
presented or not presented, a cross-linking agent or a polymer; and
n is the number of double-stranded siRNA monomer),
x-A-(X-A-).sub.n-x' [Structural Formula II] (Wherein, X is
double-stranded siRNA monomer; x or x' is single-stranded siRNA
monomer A is either presented or not presented, a cross-linking
agent or a polymer; and n is the number of double-stranded siRNA
monomer).
5. The preparing method of a siRNA multi-conjugate according to
claim 4, wherein the method is composed of the following steps: 1)
preparing yXy by complementary hydrogen bonding of single-stranded
sense/antisense siRNA monomers (yx+x'y) having substitutions with
same functional groups at the ends; and 2) inducing covalent bond
of the yXy mediated by a cross-linking agent or a polymer: (Herein,
X is double-stranded siRNA monomer; x and x' are single-stranded
sense/antisense siRNA monomers; and y is the functional group
introduced in the end).
6. The preparing method of a siRNA multi-conjugate according to
claim 4, wherein the method is composed of the following steps: 1)
preparing dimers of xyyx and x'yyx' by covalent bonding of
single-stranded sense/antisense siRNA monomers (yx and x'y) having
substitutions with same functional groups at the ends; and 2)
inducing complementary hydrogen bond between the dimers xyyx and
x'yyx': (Herein, X is double-stranded siRNA monomer; x and x' are
single-stranded sense/antisense siRNA monomers; and y is the
functional group introduced in the end).
7. The preparing method of a siRNA multi-conjugate according to
claim 4, wherein the method is composed of the following steps: 1)
preparing yXz by complementary hydrogen bonding of single-stranded
sense/antisense siRNA monomers (yx+x'z) having substitutions with
different functional groups at the ends; and 2) inducing covalent
bond of the yXz mediated by a cross-linking agent or a polymer:
(Herein, X is double-stranded siRNA monomer; x and x' are
single-stranded sense/antisense siRNA monomers; and y and z are the
functional groups introduced in the ends).
8. The preparing method of a siRNA multi-conjugate according to
claim 4, wherein the method is composed of the following steps: 1)
preparing dimers of xyzx and x'yzx' by covalent bonding of
single-stranded sense/antisense siRNA monomers (yx and xz, x'y and
x'z) having substitutions with different functional groups at the
ends; and 2) inducing complementary hydrogen bond between the
dimers xyzx and x'yzx': (Herein, X is double-stranded siRNA
monomer; x and x' are single-stranded sense/antisense siRNA
monomers; and y and z are the functional groups introduced in the
ends).
9. The preparing method of a siRNA multi-conjugate according to
claim 4, wherein the covalent bond is selected from the group
consisting of non-cleavable bonds (amide bond and urethane bond),
acid cleavable bonds (ester bond, hydrazone bond and acethal bond),
reductant cleavable bond (disulfide bond), bio-cleavable bonds and
enzyme cleavable bonds.
10. The preparing method of a siRNA multi-conjugate according to
claim 4, wherein the single stranded sense/antisense siRNA monomer
has the substitution with a functional group at 3'end or at
5'end.
11. The preparing method of a siRNA multi-conjugate according to
claim 4, wherein the polymer is one or more non-ionic hydrophilic
polymers selected from the group consisting of PEG, Pluronic,
polyvinylpyrolidone and polyoxazolin; or one or more biocleavable
polyester polymers selected from the group consisting of
poly-L-lactic acid, poly-glycolic acid, poly-D-lactic-co-glycolic
acid, poly-L-lactic-co-glycolic acid, poly-D,L-lactic-co-glycolic
acid, polycaprolactone, polyvalerolactone, polyhydroxybutyrate and
polyhydroxyvalerate.
12. The preparing method of a siRNA multi-conjugate according to
claim 4, wherein the cross-linking agent has the molecular weight
of 1.00-10000 and is one or more compounds selected from the group
consisting of DTME (Dithio-bis-maleimidoethane), BM(PEG).sub.2
(1,8-Bis-maleimidodiethyleneglycol), maleimide, NHS
(N-hydroxysuccinimide), vinylsulfone, iodoacetyl, nitrophenyl
azide, isocyanate, pyridyldisulfide, hydrazide and hydroxyphenyl
azide.
13. The preparing method of a siRNA multi-conjugate according to
claim 4, wherein a cell selective ligand is additionally added to
the end of the siRNA multi-conjugate.
14. The preparing method of a siRNA multi-conjugate according to
claim 13, wherein the ligand is one or more compounds selected from
the group consisting of cell specific antibody, cell selective
peptide, cell growth factor, folic acid, galactose, mannose, RGD,
and transferrin.
15. The preparing method of a siRNA multi-conjugate according to
claim 4, wherein the step of activating the said functional group
is additionally included.
16. An ionic complex formed by ionic interaction between the siRNA
multi-conjugate of claim 1; and one or more cationic gene carriers
selected from the group consisting of cationic peptides, cationic
lipids and cationic polymers.
17. A method for treating cancer containing the step of
administering the ionic complex of claim 16 to a subject.
18. A method for treating angiogenesis related disease containing
the step of administering the ionic complex of claim 16 to a
subject.
19. A use of the ionic complex of claim 16 for the production of an
anticancer agent.
20. A use of the ionic complex of claim 16 for the production of a
therapeutic agent for angiogenesis related disease.
Description
BACKGROUND
Technical Field
[0001] The present invention relates to a multi-conjugate of siRNA
(small interfering RNA) and a preparing method of the same, more
precisely a multi-conjugate of siRNA prepared by direct or indirect
covalent bonding of double-stranded sense/antisense siRNA monomers
mediated by a cross-linking agent or a polymer.
Background Art
[0002] SiRNA (small interfering RNA) is a short double-stranded RNA
composed of 19-22 nucleic acids, which targets mRNA (messenger RNA)
of a gene whose nucleotide sequence is identical with its sense
strand in order to suppress expression of the gene by decomposing
the target gene (Elbashir, S. M., Harborth, J., Lendeckel, W.,
Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of
21-nucleotide RNAs mediate RNA interference in cultured mammalian
cells. Nature 411, 494-8).
[0003] SiRNA is capable of inhibiting gene expression even with 10
times less amount than the required amount of conventional
antisense oligonucleotide, suggesting that it has excellent gene
selectivity indicating that it is highly capable of inhibiting a
target gene alone. However, siRNA is so unstable in vivo that it is
easily decomposed within a short period of time and it is anionic
which makes cell membrane transmission difficult, resulting in low
intracellular delivery efficiency.
[0004] To increase siRNA delivery efficiency, a nano-sized
ion-complex is generally used which is prepared by ionic bonding of
siRNA and diverse functional cationic polymers, lipids or cationic
peptides. However, the conventional siRNA has the molecular weight
of about 15,000 and has a very stiff structure of double strand.
So, it is very difficult to prepare a stable siRNA/cationic gene
carrier complex (Gary, D. J., Puri, N., and Won, Y. Y. (2007)
Polymer-based siRNA delivery: perspectives on the fundamental and
phenomenological distinctions from polymer-based DNA delivery. J
Control Release 121, 64-73).
[0005] Therefore, studies have been actively undergoing to prepare
a stable siRNA complex with a gene carrier. It has been attempted
to increase charge density of each element of an siRNA or a
cationic carrier to increase stability of an ionic complex. As an
example, it has been attempted to induce strong ionic interaction
by increasing molecular weight of cationic polymer or lipid or by
introducing a strong cationic group into a cationic carrier.
However, even if the said method can increase gene delivery
efficiency, it also increases non-specific cytotoxicity owing to
the strong cations of a gene carrier, making the clinical
application difficult. So, a new approach has recently been made to
modify siRNA itself to produce a stable complex with the
conventional gene carrier.
[0006] According to recent reports, 4-8 additional nucleotides
(deoxythymine, deoxyadenine) are added to sense strand in order to
increase the molecular weight of siRNA, resulting in complementary
base pairing in siRNA complex (Bolcato-Bellemin, A. L., Bonnet, M.
E., Creusat, G., Erbacher, P., and Behr, J. P. (2007) Sticky
overhangs enhance siRNA-mediated gene silencing. Proc Natl Acad Sci
USA 104, 16050-5). However, at this time, complementary bindings of
4-8 nucleotides are so unstable that it cannot be confirmed by
electrophoresis.
[0007] Thus, the present inventors continued study to increase
stability and delivery efficiency of siRNA. As a result, the
present inventors completed this invention by confirming that a
multi-conjugate of siRNA prepared by direct or indirect covalent
bonding of double-stranded sense/antisense siRNA monomers mediated
by a cross-linking agent or a polymer has excellent gene delivery
efficiency owing to strong ionic bond with a cationic gene carrier
and does not induce severe immune response, compared with the
conventional siRNA, so that it can be effectively used for gene
therapy.
SUMMARY
Technical Problem
[0008] It is an object of the present invention to provide a
multi-conjugate of siRNA having higher gene inhibition efficiency
than the conventional siRNA.
[0009] It is another object of the present invention to provide a
simple but highly efficient preparing method of a multi-conjugate
of siRNA that is capable of binding stably with a gene carrier
owing to the high negative-charge density.
Technical Solution
[0010] To achieve the above objects, the present invention provides
a multi-conjugate of siRNA having the following structure of
[Structural Formula I] or [Structure Formula II] prepared by direct
or indirect covalent bonding of double-stranded sense/antisense
siRNA monomers mediated by a cross-linking agent or a polymer:
(--X-A-).sub.n [Structural Formula I]
[0011] (wherein,
[0012] X is double-stranded siRNA monomer;
[0013] A is either presented or not presented, a cross-linking
agent or a polymer; and n is the number of double-stranded siRNA
monomer),
x-A-(X-A-).sub.n-x' [Structural Formula II]
[0014] (wherein,
[0015] X is double-stranded siRNA monomer;
[0016] x or x' is single-stranded siRNA monomer
[0017] A is either presented or not presented, a cross-linking
agent or a polymer; and
[0018] n is the number of double-stranded siRNA monomer).
[0019] The present invention also provides a preparing method of a
multi-conjugate of siRNA having the structure of [Structural
Formula I] or [Structural Formula II] containing the step of direct
or indirect covalent bonding of double-stranded sense/antisense
siRNA monomers mediated by a cross-linking agent or a polymer.
[0020] The present invention further provides an ionic complex
formed by ionic interaction between the said siRNA multi-conjugate
and a cationic gene carrier selected from the group consisting of
cationic peptides, cationic lipids and cationic polymers.
[0021] The present invention also provides a method for treating
cancer or angiogenesis related disease containing the step of
administering the said ionic complex to a subject.
[0022] In addition, the present invention provides a use of the
said ionic complex for the production of an anticancer agent or a
therapeutic agent for angiogenesis related disease.
[0023] Hereinafter, the present invention is described in
detail.
[0024] The present invention provides a multi-conjugate of siRNA
having the following structure of [Structural Formula I] or
[Structure Formula II] prepared by direct or indirect covalent
bonding of double-stranded sense/antisense siRNA monomers mediated
by a cross-linking agent or a polymer:
(--X-A-).sub.n [Structural Formula I]
[0025] (wherein,
[0026] X is double-stranded siRNA monomer;
[0027] A is either presented or not presented, a cross-linking
agent or a polymer; and n is the number of double-stranded siRNA
monomer),
x-A-(X-A-).sub.n-x' [Structural Formula II]
[0028] (wherein,
[0029] X is double-stranded siRNA monomer;
[0030] x or x' is single-stranded siRNA monomer
[0031] A is either presented or not presented, a cross-linking
agent or a polymer; and
[0032] n is the number of double-stranded siRNA monomer).
[0033] In this invention, the said double-stranded sense/antisense
siRNA monomer preferably has 15-50 nucleotides and more preferably
has 15-29 nucleotides, but not always limited thereto.
[0034] In this invention, the number of the said double-stranded
sense/antisense siRNA monomer is preferably 1-150 and more
preferably 1-100, but not always limited thereto.
[0035] The multi-conjugate of siRNA of the present invention is
preferably prepared by the following two methods, but not always
limited thereto:
[0036] 1) The first method: single-stranded sense siRNA and
antisense siRNA each having a functional group at terminal are
reacted respectively in the presence of a cross-linking agent or a
polymer to produce single-stranded sense and antisense siRNA
dimers. Each dimer is complementarily annealed in aqueous solution
(see FIGS. 1B and 1D).
[0037] Particularly, sense strand siRNA and antisense strand siRNA
each having sulfhydryl group substituted at terminal are reacted to
produce dimers by cleavable or non-cleavable covalent bond using
DIME (Dithio-bis-maleimidoethane) or BM(PEG).sub.2
(1,8-bis(maleimido)diethylene glycol), the cross-linking agent
reactable with sulfhydryl group. Then, equal amount of each dimer
is loaded in PBS to induce complementary binding via hydrogen bond,
resulting in the preparation of a siRNA multi-conjugate.
[0038] 2) The second method: double-stranded siRNA (prepared by
complementary hydrogen bond) having a functional group substituted
at terminal proceeds to covalent bond mediated by a cross-linking
agent or a polymer, resulting in the preparation of a siRNA
multi-conjugate (see FIGS. 1A and 1C).
[0039] Particularly, single-stranded siRNA introduced with
sulfhydryl group at terminal proceeds to complementary hydrogen
bond, and the resultant product proceeds to covalent bond based on
oxidation in the presence of a cross-linking agent or DMSO,
resulting in the preparation of a siRNA multi-conjugate.
[0040] In the production of the said multi-conjugate of siRNA, the
molecular weight of siRNA oligo-strand is preferably selected in
the range from 10,000-50,000, but not always limited thereto. The
siRNA for the present invention is not limited and any one that is
being used for treatment or study can be accepted, for example any
siRNA that is being used for gene therapy or study or is expected
to be used in near future is selected, which is exemplified by
c-myc, c-myb, c-fos, c-jun, bcl-2 or VEGF, VEGF-B, VEGF-C, VEGF-D,
PIGF, etc.
[0041] Hydroxy group (--OH) at terminal of the said siRNA can be
substituted with a functional group such as sulfhydryl group
(--SH), carboxyl group (--COOH) or amine group (--NH.sub.2).
[0042] The substitution can be performed at 3'end or 5'end, and is
preferably occurred at 3' ends of both sense and antisense are
substituted with such functional group, but not always limited
thereto.
[0043] The polymer herein can be a non-ionic hydrophilic polymer
such as polyethyleneglycol (PEG), polyvinylpyrolidone and
polyoxazolin, or a hydrophobic polymer such as PLGA and PLA,
etc.
[0044] The cross-linking agent herein has the molecular weight of
100-10000, which is exemplified by DTME
(Dithio-bis-maleimidoethane), BM(PEG).sub.2
(1,8-Bis-maleimidodiethyleneglycol), maleimide, NHS
(N-hydroxysuccinimide), vinylsulfone, iodoacetyl, nitrophenyl
azide, isocyanate, pyridyldisulfide, hydrazide or hydroxyphenyl
azide, but not always limited thereto.
[0045] Either the cross-linking agent having external
stimulus-mediated cleavable bonds or the cross-linking agent having
non-cleavable bonds can be used herein, for example, the
cross-linking agent of the present invention can have non-cleavable
bond such as amide bond and urethane bond, and cleavable bond such
as acid cleavable bond (covalent bond of ester, hydrazone, acethal,
etc) and reductant cleavable bond (disulfide bond), etc. And
additionally, any cross-linking agent available for drug
modification can be used without limitation.
[0046] The said multi-conjugate of siRNA can include cell selective
ligand at the end.
[0047] The ligand can be selected from the group consisting of cell
specific antibody, cell selective peptide, cell growth factor,
folic acid, galactose, mannose, RGD, and transferrin. Such ligand
can be introduced at the end of the multi-conjugate by disulfide
bond, amide bond, or ester bond, etc.
[0048] The multi-conjugate of siRNA of the present invention can
form an ionic complex by ionic interaction with a cationic gene
carrier (cationic lipid, cationic polymer, cationic peptide,
etc).
[0049] The cationic peptide is KALA (cationic fusogenic peptide),
polylysine, polyglutamic acid or protamine. The KALA preferably has
the peptide sequence of WEAKLAKALAKALAKHLAKALAKALAACEA (SEQ. ID.
NO: 1), but not always limited thereto.
[0050] The cationic lipid is dioleyl phosphatidylethanolamine or
cholesterol dioleyl phosphatidylcholine.
[0051] The cationic polymer is polyethyleneimine, polyamine or
polyvinylamine.
[0052] To investigate target gene inhibition efficiency of the
multi-conjugate of siRNA of the present invention, the inventors
mixed siRNA with the gene carrier Linear PEI to prepare an ionic
complex, which was then treated to cancer cells expressing GFP
stably. Then, GFP level was measured using fluorophotometer. As a
result, the multi-conjugate of siRNA of the present invention
demonstrated higher gene delivery efficiency using a cationic gene
carrier than conventional siRNA, suggesting that the
multi-conjugate of siRNA of the present invention had excellent
target gene inhibition efficiency (see FIG. 4).
[0053] To investigate binding force with a cationic gene carrier
and stability of the siRNA multi-conjugate of the present
invention, the inventors produced an ionic complex by mixing the
gene carrier Linear PEI and siRNA, followed by observation of shape
and size using Atomic Force Microscopy (AFM). As a result, compared
with conventional siRNA, the siRNA multi-conjugate of the present
invention demonstrated excellent binding force with a cationic
polymer and had smaller size which favored production of even and
small nano particles (see FIG. 5).
[0054] To investigate the amount of a cationic polymer conjugated
to the siRNA multi-conjugate of the present invention, gel
retardation assay was performed. As a result, compared with
conventional siRNA, the siRNA multi-conjugate of the present
invention demonstrated higher charge density, which favored
production of an ionic complex by binding a cationic polymer even
at a low concentration (see FIG. 6).
[0055] The present inventors also investigated gene inhibition
efficiency of the siRNA multi-conjugate of the present invention.
To do so, the inventors produced an ionic complex by mixing the
gene carrier Linear PEI with siRNA, which was treated to cancer
cells. Then, VEGF level was measured by ELISA. As a result,
compared with conventional siRNA, the siRNA multi-conjugate of the
present invention formed an ionic complex with a cationic polymer
more stably and evenly, suggesting that the multi-conjugate of
siRNA of the present invention had excellent gene delivery
efficiency and excellent selective inhibition of a target gene (see
FIG. 7).
[0056] The present inventors also investigated gene inhibition
efficiency of the siRNA multi-conjugate of the present invention
according to the molecular weight. First, siRNA was separated over
the size by gel separation method. Each separated siRNA was mixed
with Linear PEI to produce an ionic complex. Then, the complex was
treated to cancer cells, followed by quantification of VEGF by
ELISA. As a result, as the molecular weight of the siRNA
multi-conjugate of the present invention was increased, charge
density was increased, and accordingly the efficiency of gene
delivery using a cationic polymer was also increased (see FIG.
8).
[0057] The present inventors also investigated immune response
inducing ability of the siRNA multi-conjugate of the present
invention. The present inventors produced an ionic complex by
mixing the cationic gene carrier Linear PEI, jet PEI or DOTAP with
siRNA, which was treated to human PBMC. Then, Interferon-.alpha.
(INF-.alpha.) was quantified by ELISA. As a result, there was no
significant induction of INF-.alpha. by the siRNA multi-conjugate
of the present invention (see FIG. 9A).
[0058] To investigate immune response inducing capacity of the
siRNA multi-conjugate of the present invention, the inventors
produced an ionic complex by mixing the cationic gene carrier
Linear PEI with siRNA, which was intravenously injected to ICR
mouse at 7 weeks. Blood was taken from the heart of the mouse,
followed by quantification of blood siRNA by ELISA. As a result, in
this animal model, there was no significant induction of
INF-.alpha. by the siRNA multi-conjugate of the present invention,
compared with conventional siRNA (see FIG. 9B).
[0059] Therefore, the siRNA multi-conjugate of the present
invention was confirmed to have higher molecular weight, higher
negative charge density, higher stability, and higher ionic
strength with a cationic gene carrier than conventional siRNA,
resulting in high gene delivery efficiency. The siRNA
multi-conjugate of the present invention can be simply produced by
using the therapeutic gene siRNA alone, which favors
biocompatibility. Moreover, even as multi-conjugate form, the siRNA
of the present invention maintains gene inhibition activity but has
increased gene delivery efficiency, suggesting that it has higher
intracellular introduction efficiency and thus higher gene
inhibition effect than conventional siRNA.
[0060] VEGF binds to VEGF receptor existing on vascular endothelial
cell surface and is then functioning to accelerate growth and
migration of endothelial cell and angiogenesis. In particular,
tumor growth and metastasis are closely related to angiogenesis.
So, inhibition of angiogenesis can be a new approach to treat
cancer by inhibiting the growth of tumor. The siRNA multi-conjugate
of the present invention can inhibit VEGF significantly, so that it
can be effectively used for treating cancer.
[0061] The present invention also provides a preparing method of a
multi-conjugate of siRNA having the structure of [Structure Formula
I] or [Structure Formula II] containing the step of direct or
indirect covalent bonding of double-stranded sense/antisense siRNA
monomers mediated by a cross-linking agent or a polymer.
[0062] In a preferred embodiment of the present invention, the
preparing method of the multi-conjugate of siRNA is composed of the
following steps: [0063] 1) preparing yXy by complementary hydrogen
bonding of single-stranded sense/antisense siRNA monomers (yx+x'y)
having substitutions with same functional groups at the ends; and
[0064] 2) inducing covalent bond of the yXy mediated by a
cross-linking agent or a polymer (Herein, X is double-stranded
siRNA monomer, x and x' are single-stranded sense/antisense siRNA
monomers, and y is the functional group introduced in the
end.).
[0065] In another preferred embodiment of the present invention,
the preparing method of the multi-conjugate of siRNA is composed of
the following steps: [0066] 1) preparing dimers of xyyx and x'yyx'
by covalent bonding of single-stranded sense/antisense siRNA
monomers (yx and x'y) having substitutions with same functional
groups at the ends; and [0067] 2) inducing complementary hydrogen
bond between the dimers xyyx and x'yyx' (Herein, X is
double-stranded siRNA monomer, x and x' are single-stranded
sense/antisense siRNA monomers, and y is the functional group
introduced in the end.).
[0068] In another preferred embodiment of the present invention,
the preparing method of the multi-conjugate of siRNA is composed of
the following steps: [0069] 1) preparing yXz by complementary
hydrogen bonding of single-stranded sense/antisense siRNA monomers
(yx+x'z) having substitutions with different functional groups at
the ends; and [0070] 2) inducing covalent bond of the yXz mediated
by a cross-linking agent or a polymer (Herein, X is double-stranded
siRNA monomer, x and x' are single-stranded sense/antisense siRNA
monomers, and y and z are the functional groups introduced in the
ends.).
[0071] In another preferred embodiment of the present invention,
the preparing method of the multi-conjugate of siRNA is composed of
the following steps: [0072] 1) preparing dimers of xyzx and x'yzx'
by covalent bonding of single-stranded sense/antisense siRNA
monomers (yx and xz, x'y and x'z) having substitutions with
different functional groups at the ends; and [0073] 2) inducing
complementary hydrogen bond between the dimers xyzx and x'yzx'
(Herein, X is double-stranded siRNA monomer, x and x' are
single-stranded sense/antisense siRNA monomers, and y and z are the
functional groups introduced in the ends.).
[0074] In the preparing method of a multi-conjugate of siRNA of the
present invention, siRNA oligo strand is preferably selected from
those having the molecular weight of 10,000-50,000. The siRNA
herein is not limited and any siRNA that is used for treatment or
study can be accepted, for example any siRNA that has been used for
gene therapy or study or is expected to be used for that purpose
can be accepted, which is exemplified by c-myc, c-myb, c-fos,
c-jun, bcl-2 or VEGF, VEGF-B, VEGF-C, VEGF-D, PIGF, etc.
[0075] In the preparing method of a multi-conjugate of siRNA of the
present invention, the covalent bond is preferably selected from
the group consisting of non-cleavable bonds (amide bond and
urethane bond), acid cleavable bonds (ester bond, hydrazone bond
and acethal bond), reductant cleavable bond (disulfide bond),
bio-cleavable bonds and enzyme cleavable bonds, but not always
limited thereto.
[0076] In the preparing method of a multi-conjugate of siRNA of the
present invention, hydroxyl group (--OH) at the end of the
single-stranded sense/antisense siRNA monomer is preferably
substituted with one of the functional groups selected from the
group consisting of sulfhydryl group (--SH), carboxyl group
(--COOH) and amine group (--NH.sub.2), but not always limited
thereto.
[0077] The substitution is preferably performed at 3' end or 5'
end, and it is preferred that sense and antisense are substituted
with functional groups at 3' ends, but not always limited
thereto.
[0078] In the preparing method of a multi-conjugate of siRNA of the
present invention, the polymer used as a mediator for covalent bond
is preferably one or more non-ionic hydrophilic polymers selected
from the group consisting of PEG, Pluronic, polyvinylpyrolidone and
polyoxazolin; or one or more biocleavable polyester polymers
selected from the group consisting of poly-L-lactic acid,
poly-glycolic acid, poly-D-lactic-co-glycolic acid,
poly-L-lactic-co-glycolic acid, poly-D,L-lactic-co-glycolic acid,
polycaprolactone, polyvalerolactone, polyhydroxybutyrate and
polyhydroxyvalerate, but not always limited thereto.
[0079] In the preparing method of a multi-conjugate of siRNA of the
present invention, the cross-linking agent is one or more compounds
having the molecular weight of 100-10,000, which is preferably
selected from the group consisting of DIME
(Dithio-bis-maleimidoethane), BM(PEG).sub.2
(1,8-Bis-maleimidodiethyleneglycol), maleimide, NHS
(N-hydroxysuccinimide), vinylsulfone, iodoacetyl, nitrophenyl
azide, isocyanate, pyridyldisulfide, hydrazide and hydroxyphenyl
azide, but not always limits thereto.
[0080] In the preparing method of a multi-conjugate of siRNA of the
present invention, the cell selective ligand is preferably added to
the end of the said siRNA multi-conjugate, but not always limited
thereto.
[0081] The said ligand is one or more compounds preferably selected
from the group consisting of cell specific antibody, cell selective
peptide, cell growth factor, folic acid, galactose, mannose, RGD
and transferrin, but not always limited thereto.
[0082] In the preparing method of a multi-conjugate of siRNA of the
present invention, the step of activating the functional group of
the said siRNA can be additionally included, but not always limited
thereto.
[0083] The material used for the activation of the functional group
of the said siRNA is one or more compounds preferably selected from
the group consisting of 1-ethyl-3,3-dimethylaminopropyl
carbodiimide, imidazole, N-hydroxysuccinimide,
dichlorohexylcarbodiimide, N-.beta.-Maleimidopropionic acid,
N-.beta.-maleimidopropyl succimimide ester and N-Succinimidyl
3-(2-pyridyldithio)propionate, but not always limited thereto.
[0084] In the preparing method of a multi-conjugate of siRNA of the
present invention, the preparation reaction is not limited, but
generally performed at room temperature for about 24-48 hours.
Ratio of each reactant added to the reaction is not limited and the
length of siRNA multi-conjugate can be regulated by adjusting molar
ratio (%) of a cross-linking agent to siRNA.
[0085] The aqueous solution used for complementary binding in this
invention is PBS, Tris buffer or HEPES buffer, and more preferably
the buffer having salt concentration of at least 100 mM.
[0086] The present invention also provides an ionic complex
facilitating cellular transduction, which is formed by ionic
interaction between the said siRNA multi-conjugate and a cationic
gene carrier selected from the group consisting of cationic
peptides, cationic lipids and cationic polymers.
[0087] An ionic complex can be prepared by ionic interaction
between the siRNA multi-conjugate of the present invention and a
cationic gene carrier. The said ionic complex is a small complex
formed by interaction between an anionic gene and a polymer having
counter ions (ex, cationic material), which is preferably 100-200
nm in size, but not always limited thereto.
[0088] In this invention, the cationic peptide is preferably
selected from the group consisting of KALA (cationic fusogenic
peptide), polylysine, polyglutamic acid and protamine, but not
always limited thereto.
[0089] In this invention, the cationic lipid is preferably selected
from the group consisting of dioleyl phosphatidylethanolamine and
cholesterol dioleyl phosphatidyl choline, but not always limited
thereto.
[0090] In this invention, the cationic polymer is preferably
selected from the group consisting of polyethylenimine, polyamines
and polyvinylamine, but not always limited thereto.
[0091] In the preparation of the ionic complex, the siRNA
multi-conjugate of the present invention is diluted in PBS, to
which a cationic substance is added. Then, the mixture stands at
room temperature, resulting in the preparation of an ionic complex
in aqueous solution. At this time, the content of the cationic
substance is regulated by adjusting the ratio of positive charge of
the cationic substance to negative charge of the siRNA to 1:1
(+/-=1/1).about.100:1.
[0092] The present invention also provides a method for treating
cancer or angiogenesis related disease containing the step of
administering the said ionic complex to a subject.
[0093] In addition, the present invention provides a use of the
said ionic complex for the production of an anticancer agent or a
therapeutic agent for angiogenesis related disease.
[0094] The term "subject" herein indicates human or animals
including monkey, dog, goat, pig and mouse that have cancer or
angiogenesis related disease that can be improved by the
administration of the ionic complex of the present invention.
[0095] The term "administration" herein indicates providing a
required amount of the ionic complex of the present invention to a
subject through a proper method.
[0096] The term "treatment" herein indicates all behavior performed
to improve or relieving symptoms of cancer or angiogenesis related
disease by administering the ionic complex of the present
invention.
[0097] The cancer herein is preferably selected from the group
consisting of breast cancer, colorectal cancer, rectal cancer,
non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), renal
cell cancer, prostate cancer, liver cancer, pancreatic cancer,
soft-tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma, head
and neck cancer, melanoma, ovarian cancer, mesothelioma and
multiple myeloma, but not always limited thereto.
[0098] The angiogenesis related disease herein is selected from the
group consisting of hemangioma, angiofibroma, arthritis, diabetic
retinosis, retrolental fibroplasia, neovascular glaucoma,
neovascular corneal disease, macular degeneration, macular
dystrophy, pterygium, retinal degeneration, retrolental
fibroplasia, trachoma, psoriasis, capillarectasia, granuloma
pyogenicum, dermatitis seborrheica and acne, but not always limited
thereto.
[0099] The siRNA multi-conjugate of the present invention is
prepared by siRNA alone, so that it has excellent biocompatibility
and excellent stability and improved binding capacity with various
gene carriers without reducing gene inhibition activity of siRNA,
suggesting that the siRNA multi-conjugate of the present invention
has higher gene inhibition activity than conventional siRNA.
[0100] In this invention, an ionic complex prepared by mixing a
siRNA multi-conjugate and the representative gene carrier Linear
PEI was treated to cancer cells. As a result, significant VEGF
inhibition effect was demonstrated, compared with conventional
siRNA. That is, the siRNA multi-conjugate of the present invention
forms a very stable and regular ionic complex with a cationic
polymer and therefore the conjugate demonstrates excellent gene
delivery effect and target gene inhibition effect.
[0101] Therefore, the ionic complex of the present invention can be
used for diverse gene therapy, because the siRNA multi-conjugate
can include diverse siRNA, in particular for gene therapy for
cancer or angiogenesis related disease.
Advantageous Effect
[0102] The preparing method of a multi-conjugate of siRNA of the
present invention is characterized by simple reaction process and
high efficiency. Therefore, the siRNA multi-conjugate prepared by
the above method has the molecular weight of multiple times the
conventional siRNA, and thus has higher negative charge density,
indicating that the siRNA multi-conjugate of the present invention
has significantly high gene delivery efficiency resulted from
excellent ionic interaction with a cationic gene carrier.
BRIEF DESCRIPTION OF DRAWINGS
[0103] The application of the preferred embodiments of the present
invention is best understood with reference to the accompanying
drawings, wherein:
[0104] FIG. 1 is a diagram illustrating the preparing method of a
multi-conjugate of siRNA. FIG. 1A: Double-stranded siRNA having
functional groups introduced in both ends is prepared by
complementary base-pairing via hydrogen bonding of single-stranded
sense siRNA and single-stranded antisense siRNA having same
functional group substituted at one end. Then, a multi-conjugate of
siRNA is prepared by covalent bonding using a cross-linking agent
or a polymer. FIG. 1B: Dimer form of sense strand and antisense
strand siRNA is first prepared by covalent bonding single-stranded
sense siRNA and single-stranded antisense siRNA having same
functional group substituted at one end using a cross-linking agent
or a polymer. Then, a multi-conjugate of siRNA is prepared by
complementary hydrogen bonding of each oligonucleotide. FIG. 1C
Double-stranded siRNA having different functional groups introduced
in both ends is prepared by complementary base-pairing via hydrogen
bonding of single-stranded sense siRNA and single-stranded
antisense siRNA having different functional group at each end.
Then, a multi-conjugate of siRNA is prepared by direct covalent
bonding or covalent bonding mediated by a cross-linking agent. FIG.
1D Dimer form of sense strand and antisense strand siRNA is first
prepared by direct covalent bonding of single-stranded sense siRNA
and single-stranded antisense siRNA having different functional
group at each end or covalent bonding mediated by a cross-linking
agent. Then, a multi-conjugate of siRNA is prepared by
complementary base-pairing via hydrogen bonding of each
oligonucleotide.
[0105] FIG. 2 illustrates the result of electrophoresis of the
multi-conjugate of siRNA. Double-stranded siRNA is prepared by
hydrogen bonding between sense strand siRNA and antisense strand
siRNA both having the substitution with sulfhydryl group at 3'end.
Then, FIG. 2A a multi-conjugate of siRNA is prepared by using the
cross-linking agent DIME or FIG. 2B a multi-conjugate of siRNA is
prepared by using the cross-linking agent BM(PEG).sub.2 (by the
method of FIG. 1A).
[0106] FIG. 3A illustrates the result of electrophoresis of the
multi-conjugate of siRNA prepared by using double-stranded siRNA
having the substitution with sulfhydryl group at 3' ends of both
sense and antisense siRNA. FIG. 3B illustrates the result of
electrophoresis of the multi-conjugate of siRNA prepared by
complementary binding of dimers produced from sense and antisense
siRNA having substitution with sulfhydryl group at 3'end in the
presence of a cross-linking agent. FIG. 3C and FIG. 3D illustrate
observation of dimer by electrophoresis after preparing the dimer
by using sense and antisense siRNA of the dimer in the presence of
a cross-linking agent that is capable of linking sense siRNA and
antisense siRNA having substitution with sulfhydryl group at 3'end
by cleavable covalent bond which is disulfide bond FIG. 3C or by
non-cleavable covalent bond FIG. 3D.
[0107] FIG. 4 illustrates that a complex is prepared with the siRNA
multi-conjugate to GFP of FIG. 1A and FIG. 1B and the conventional
siRNA using the cationic gene carrier linear PEI at NP ratio 20.
Then, the complex is treated to the cancer cell line MDA-MB-435
expressing GFP stably, followed by quantification of GFP
inhibition.
[0108] FIG. 5 illustrates that an ionic complex is prepared from
the siRNA multi-conjugate (prepared by the method of FIG. 1B) and
the conventional siRNA (Naked) using the cationic gene carrier
linear PEI at NP ratio 2 and 10, and then the size and shape are
observed by atomic force microscopy (AFM).
[0109] FIG. 6 illustrates that ion complexes are prepared by using
the siRNA multi-conjugate (prepared by the method of FIG. 1B) and
the conventional siRNA (Naked) using the cationic gene carrier
linear PEI over different NP ratios, followed by electrophoresis to
observe the ionic complex formation.
[0110] FIG. 7 illustrates that a siRNA multi-conjugate linked by
cleavable disulfide bond or non-cleavable covalent bond is prepared
by the method of FIG. 1B using siRNA inhibiting VEGF (Vascular
endothelial growth factor), followed by comparison of gene
inhibition efficiency with the conventional siRNA (Naked). Linear
PEI is used as a gene carrier. Gene inhibition efficiency is
measured over the concentration of siRNA FIG. 7A and over the NP
ratio FIG. 7B by ELISA, by which VEGF level is quantified.
[0111] FIG. 7C illustrates the result of quantification of mRNA by
RT-PCR to investigate gene inhibition efficiency.
[0112] FIG. 8A illustrates that a siRNA multi-conjugate is prepared
by the method of FIG. 1B and each siRNA is separated, followed by
electrophoresis. FIG. 8B illustrates that gene inhibition
efficiency of each separated multi-conjugate is compared by
quantifying VEGF by ELISA.
[0113] FIG. 9 illustrates the IFN-alpha induction by the siRNA
multi-conjugate prepared by the method of FIG. 1B. FIG. 9A
illustrates that an ionic complex is prepared by using the
conventional siRNA or the siRNA multi-conjugate linked by cleavable
or non-cleavable covalent bond and a cationic gene carrier (Linear
PEI, Jet-PEI or DOTAP). The ionic complex is introduced into PBMC
(Peripheral Blood Mononuclear Cell) extracted from human blood,
followed by quantification of IFN-alpha released from the cell by
ELISA. FIG. 9B illustrates that an ionic complex is prepared by
using the conventional siRNA or the siRNA multi-conjugate linked by
cleavable or non-cleavable covalent bond and the cationic gene
carrier Linear PEI, which is intravenously injected into ICR mouse,
followed by quantification of IFN-alpha released from blood by
ELISA.
DETAILED DESCRIPTION
Mode for Invention
[0114] Practical and presently preferred embodiments of the present
invention are illustrative as shown in the following Examples.
[0115] However, it will be appreciated that those skilled in the
art, on consideration of this disclosure, may make modifications
and improvements within the spirit and scope of the present
invention.
Example 1: Preparation of Double-Stranded siRNA Via Hydrogen Bond
of Sense Strand siRNA and Antisense Strand siRNA Having
Substitution with Same Functional Group at their Ends and
Preparation of Multi-Conjugate of siRNA Using Cross-Linking
Agent
[0116] 100 nmol of sense or antisense strand siRNA having
substitution with sulfhydryl group at 3' end was dissolved in 260
.mu.l of 1.times.PBS, which stood at 37.degree. C. for 1 hour,
resulting in double-stranded siRNA. To reduce sulfhydryl group at
both ends of the prepared double-stranded siRNA, 22 .mu.l of
25.times.PBS, 260 .mu.l of 2M DTT (dithiothreitol) solution and 4
.mu.l of 5N NaOH solution (to adjust pH) were added thereto,
followed by reaction for 12 hours. Upon completion of the reaction,
remaining DTT was eliminated by dialysis and the solution was
concentrated to 1 nmol/.mu.l. 25.times.PBS was added to adjust the
final concentration to 5.times.PBS. The cross-linking agent DTME or
BM(PEG).sub.2 was added at the concentration of half the
concentration of thiol group, followed by reaction at room
temperature for 24 hours. Upon completion of the reaction,
remaining foreign materials such as cross-linking agent, etc, were
eliminated by dialysis and the solution was concentrated to make
the final concentration to 1.about.2 .mu.g/.mu.l to prepare a siRNA
multi-conjugate (see FIG. 1A). The prepared siRNA was confirmed by
electrophoresis. (see FIG. 2)
[0117] A multi-conjugate was prepared by direct covalent bonding of
double-stranded siRNA mediated by oxidation without using a
cross-linking agent. Double-stranded siRNA having substitution with
thiol group at 3' ends of sense and antisense strands was treated
with DTT by the same manner as described above, followed by
dialysis and concentration to make the final concentration of the
solution to 1 nmol/.mu.l. DMSO and diamide were added to the above
solution to oxidize sulfhydryl, resulting in the formation of
disulfide bond. The prepared double-stranded siRNA multi-conjugate
was confirmed by electrophoresis (see FIG. 3A).
Example 2: Preparation of Dimer of Each Sense Strand siRNA and
Antisense Strand siRNA Having the Substitution with Same Functional
Group at the End Using Cross-Linking Agent and Preparation of siRNA
Multi-Conjugate Via Hydrogen Bond
[0118] 100 nmol of sense or antisense strand siRNA having the
substitution with sulfhydryl group at 3' end was dissolved in 260
.mu.l of DEPC (Diethyl pyrocarbonate) treated deionized water, to
which 22 .mu.l of 25.times.PBS was added. 260 .mu.l of 2M DTT
(dithiothreitol) was added thereto and then 4 .mu.l of 5N NaOH was
added to adjust pH, followed by reaction for 12 hours. Upon
completion of the reaction, remaining DTT was eliminated by
dialysis and the solution was concentrated. As a result, sense or
antisense strand siRNA having the final concentration of 1
nmol/.mu.l was prepared. 25.times.PBS was added to adjust the final
concentration to 5.times.PBS. The cross-linking agent DIME or
BM(PEG).sub.2 was added thereto at the concentration of half the
concentration of thiol group, followed by reaction at room
temperature for hours. Upon completion of the reaction, foreign
materials such as cross-linking agent, etc, were eliminated by
dialysis, and the solution was concentrated to prepare the dimer
form of sense or antisense siRNA having the final concentration of
1.about.2 .mu.g/.mu.l (see FIG. 1B). The dimer prepared by
cleavable disulfide bond (see FIG. 3C) or non-cleavable covalent
bond (see FIG. 3D) were confirmed by electrophoresis. Equal amount
of sense and antisense dimers stood in PBS at 37.degree. C. for 1
hour to induce hydrogen bond. As a result, a siRNA multi-conjugate
was prepared and confirmed by electrophoresis (see FIG. 3B).
Example 3: Preparation of Double-Stranded siRNA by Hydrogen Bonding
of Sense Strand siRNA and Antisense Strand siRNA Having Different
Functional Groups at their Ends and Preparation of siRNA Conjugate
Using Cross-Linking Agent
[0119] Sense strand and antisense strand siRNA having respectively
amine group and sulfhydryl group at 3'end were prepared. 100 nmol
of each sense and antisense siRNA was dissolved in 260 .mu.l of
PBS, which stood at 37.degree. C. for 1 hour, resulting in the
preparation of double-stranded siRNA. DTT was treated thereto in
order to prepare single-stranded siRNA having sulfhydryl group
substituted at the end, followed by dialysis and concentration to
make the final concentration of 1 nmol/.mu.l. The cross-linking
agent sulfo-SMCC
(sulfosuccinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxylate)
was added to the prepared double stranded siRNA, followed by
reaction for 24 hours to prepare a multi-conjugate of siRNA. Upon
completion of the reaction, remaining foreign materials such as
cross-linking agent, etc, were eliminated by dialysis and the
solution was concentrated to make the final concentration to
1.about.2 .mu.g/.mu.l (see FIG. 1C).
Example 4: Preparation of Dimer of Each Sense Strand siRNA and
Antisense Strand siRNA Having the Substitution with Different
Functional Groups at their Ends Using Cross-Linking Agent and
Preparation of siRNA Multi-Conjugate Via Hydrogen Bond
[0120] Sense and antisense siRNA each having amine group and
sulfhydryl group at 3' end were linked to make double-stranded
siRNA using the cross-linking agent sulfo-SMCC. Single-stranded
siRNA having the substitution with sulfhydryl group at the end was
treated with DTT, followed by dialysis and concentration until the
final concentration reached 1 nmol/.mu.l. Single-stranded siRNA
having the substitution with amine group at the end was dissolved
in DEPC treated distilled water at the concentration of 1
nmol/.mu.l. Each solution containing sense and antisense having
respectively amine group and sulfhydryl group was treated with
sulfo-SMCC to prepare sense or antisense dimer. The prepared sense
or antisense dimer was mixed in PBS, which stood at 37.degree. C.
for 1 hour, resulting in the preparation of a double-stranded siRNA
multi-conjugate (see FIG. 1D).
Experimental Example 1: Measurement of GFP Expression
[0121] An ionic complex was prepared from the siRNA multi-conjugate
(prepared by the method of FIG. 1A) linked by cleavable disulfide
bond or non-cleavable covalent bond using the siRNA inhibiting GFP
gene, the conventional siRNA, a cross linking agent and linear PEI
(polyethyleneimine). The prepared ionic complex was treated to the
cancer cell line MDA-MB-435 expressing GFP stably for 5 hours. 48
hours later, GFP expression was quantified with a
fluorophotometer.
[0122] As a result, the siRNA multi-conjugate of the present
invention demonstrated excellent gene delivery efficiency using a
cationic gene carrier and excellent target gene inhibition
activity, compared with the conventional siRNA (see FIG. 4).
Experimental Example 2: Measurement of Binding Strength with
Cationic Gene Carrier and Stability
[0123] To confirm whether the siRNA multi-conjugate (prepared by
the method of FIG. 1B) had excellent binding strength with a
cationic gene carrier and could form a stable ionic complex,
compared with the conventional siRNA, the present inventors mixed
the representative gene carrier Linear PEI and each siRNA to
produce each ionic complex. And then, shape and size of each
complex were observed by AFM.
[0124] As a result, the siRNA multi-conjugate of the present
invention demonstrated excellent binding strength with a cationic
polymer and was capable of forming small but even nano particles,
compared with the conventional siRNA (see FIG. 5).
[0125] To investigate the amount of the cationic polymer binding to
each siRNA, gel retardation assay was performed.
[0126] As a result, the siRNA multi-conjugate of the present
invention had higher charge density than the conventional siRNA,
suggesting that the siRNA multi-conjugate of the present invention
can form an ionic complex by binding with a cationic polymer even
at a low concentration (see FIG. 6).
Experimental Example 3: Investigation of Gene Inhibition Efficiency
Using VEGF
[0127] An ionic complex was prepared from the siRNA multi-conjugate
(prepared by the method of FIG. 1B) linked by cleavable disulfide
bond or non-cleavable covalent bond using the siRNA inhibiting VEGF
gene, the conventional siRNA, and linear PEI (polyethyleneimine).
The prepared ionic complex was treated to PC3 cancer cells for 5
hours. Then VEGF secreted for 21 hours was quantified by ELISA. The
experiment was performed over the siRNA concentration (0, 18, 45,
and 90 nM) and over the NP ratio (0, 10, 15, and 20), which was the
ratio of amine in the cationic carrier to phosphate of nucleotide.
To investigate weather intracellular mRNA was reduced selectively,
each ionic complex was treated to cancer cells for 5 hours. 18
hours later, RNA was extracted, followed by PCR to measure the
level of intracellular VEGF mRNA.
[0128] As a result, the siRNA multi-conjugate of the present
invention could form a stable and even ionic complex with a
cationic polymer, compared with the conventional siRNA, and
demonstrated excellent gene delivery efficiency and target gene
inhibition activity (see FIG. 7).
Experimental Example 4: Investigation of VEGF Inhibition
Efficiency
[0129] Electrophoresis was performed with the VEGF siRNA
multi-conjugate prepared by the method of FIG. 1B, and siRNA was
sorted over the size by gel separation method, followed by
electrophoresis to confirm thereof. Each siRNA was mixed with
Linear PEI to form a complex. 90 nM of the siRNA conjugate was
treated to PC3 cell, and then VEGF gene inhibition effect was
measured.
[0130] As a result, as molecular weight of the siRNA
multi-conjugate increased, charge density was increased, suggesting
the improvement of gene delivery efficiency using a cationic
polymer (see FIG. 8).
Experimental Example 5: Induction of Immune Response by siRNA
Multi-Conjugate
[0131] To investigate immune response induction capacity of the
siRNA multi-conjugate prepared by the method of FIG. 1B, peripheral
blood mononuclear cells (PBMC) were separated from human blood by
using Fisher lymphocyte separation medium. An ionic complex was
prepared from the conventional VEGF siRNA or the siRNA
multi-conjugate linked by cleavable disulfide bond or non-cleavable
covalent bond by using the cationic gene carrier linear PEI, jet
PEI or DOTAP. The siRNA complex was treated to PBMC at the final
concentration of 360 nM for 24 hours. INF-alpha level in the
supernatant was measured by ELISA.
[0132] As a result, the siRNA multi-conjugate of the present
invention did not induce INF-alpha significantly, compared with the
conventional siRNA. In particular, the siRNA multi-conjugate
prepared by disulfide bond demonstrated similar INF-alpha induction
to the conventional siRNA (see FIG. 9A).
[0133] To confirm whether the prepared siRNA multi-conjugate could
induce INF-alpha secretion in mouse, 40 .mu.l of the conventional
siRNA or the siRNA multi-conjugate prepared by cleavable disulfide
bond or non-cleavable covalent bond was mixed with the cationic
gene carrier linear PEI to form an ionic complex, which was
injected intravenously into ICR mouse at 7 weeks. After 6 hours of
the treatment, blood was taken from the heart of the mouse,
followed by ELISA to quantify blood siRNA.
[0134] As a result, compared with the conventional siRNA, the siRNA
multi-conjugate of the present invention did not induce INF-alpha
secretion significantly in the animal model (see FIG. 9B).
INDUSTRIAL APPLICABILITY
[0135] The siRNA multi-conjugate of the present invention can be
applied in medicinal field including gene therapy owing to the
improved gene delivery efficiency and thereby further contributes
to the advancement of national industry by realizing diverse
applications thereof in related fields.
[0136] Those skilled in the art will appreciate that the
conceptions and specific embodiments disclosed in the foregoing
description may be readily utilized as a basis for modifying or
designing other embodiments for carrying out the same purposes of
the present invention. Those skilled in the art will also
appreciate that such equivalent embodiments do not depart from the
spirit and scope of the invention as set forth in the appended
claims.
Sequence CWU 1
1
1130PRTArtificial Sequencecationic fusogenic peptide 1Trp Glu Ala
Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Ala Lys His1 5 10 15Leu Ala
Lys Ala Leu Ala Lys Ala Leu Ala Ala Cys Glu Ala 20 25 30
* * * * *