Patent Description:
In order to be effectively delivered into a cell, the double-stranded oligo RNA structure may have a structure in which hydrophilic materials and hydrophobic materials are conjugated to both ends of a double-stranded RNA by a simple covalent bond or a linker-mediated covalent bond and may be converted into a nanoparticle form in an aqueous solution by a hydrophobic interaction of the double-stranded oligo RNA structures.

In addition, the present invention relates to a pharmaceutical composition containing the double-stranded oligo RNA structure, a method of preparing the double-stranded oligo RNA structure, and a technology of delivering a double-stranded oligo RNA using the double-stranded oligo RNA structure.

Since a role of an RNA interference (hereinafter, referred to as 'RNAi') had been recognized, it was found that the RNA interference sequence-specifically acts on mRNA in various kinds of mammalian cells (<NPL>).

When a long-chain double-stranded RNA is delivered into a cell, the delivered double-stranded RNA is converted into a small interfering RNA (hereinafter, referred to as 'siRNA') which is processed to <NUM> to <NUM> base pairs (bp) by Dicer endonuclease. siRNA has a short-chain double-stranded RNA having <NUM> to <NUM> bases and is coupled to an RNA-induced silencing complex (RISC), whereby a guide (antisense) strand recognizes and degrades a target mRNA to sequence-specifically inhibit expression of a target gene (<NPL>).

The long-chain double-stranded RNA delivered from the outside has a problem of eliciting a non-sequence-specific immune stimulation through interferon expression in a mammal cell; however, it was found that the problem may be overcome by a short-stranded siRNA (<NPL>).

It is known that a chemically synthesized siRNA has a double strand of about <NUM> to <NUM> base pairs and consists of a <NUM>-nt(nucleotide) overhang structure at <NUM>' end, and in order that the double-stranded siRNA expresses an activity, the structure may consist of <NUM>'-hydroxyl groups (OH) and <NUM>'-phosphate groups (PO<NUM>) (<NPL>; <NPL>).

It is known that a commercialized and synthesized siRNA has a structure in which hydroxyl groups are present at both ends, and when the synthesized siRNA is delivered into a cell, siRNA <NUM>' end is phosphorylated by a phosphorylation enzyme (kinase) to express functions of siRNA (<NPL>).

In addition, since siRNA is complementarily coupled to a target mRNA to sequence-specifically regulate an expression of the target gene, a mechanism of the siRNA has an advantage that a target to be capable of being applied may be remarkably increased as compared to the existing antibody-based medical product or chemical material (small molecular drug) (<NPL>).

In order to develop the siRNA as a therapeutic agent despite of excellent effect and variously usable range of the siRNA, the siRNA is required to be effectively delivered into a target cell by improving stability of the siRNA and a cell delivery efficiency (<NPL>).

In addition, the siRNA still has a non-specific innate immune stimulation, such that <NUM>-methoxy-, <NUM>-fluoro-substituents have been developed to overcome the non-specific innate immune stimulation.

Since the siRNA is not capable of passing through a hydrophobic phospholipid bilayer of a cell due to negative charges thereof, it is difficult to be delivered into the cell through a simple diffusion.

In order to increase siRNA delivery efficiency in vivo or in vitro, various kinds of cell delivery materials have been developed. Liposomes, cationic surfactants, and the like, are commonly used, and the use of carrier, that is, a fusion method of a gene with liposome or a method of using lipid or a polymer having cations has been known, or a method of chemically modifying siRNA or a method of using conjugate has been known (<NPL>).

Since the siRNA is not capable of passing through a hydrophobic phospholipid bilayer of a cell due to negative charges thereof, it is difficult to be delivered into the cell through a simple diffusion, such that in order to overcome the difficulty, methylphosphonate or peptide nucleic acid (PNA) is used in a basic binding structure of the siRNA. In addition, a carrier is used, for example, a fusion method of a gene with liposome or a method of using lipid or a polymer having cations is used (<NPL>).

Among them, as a method of using a nanocarrier, a method of using various polymers such as liposome, cationic polymer complex, and the like, is to carry siRNA on a nanocarrier by formation of nanoparticles to deliver siRNA. Among the methods of using nanocarriers, a method of using polymeric nanoparticle, polymer micelle, lipoplex, or the like, is mainly used, wherein the lipoplex consists of cationic lipid to interact with anionic lipid of endosome of a cell, thereby eliciting a destabilization effect of the endosome to deliver the siRNA into a cell (<NPL>).

In addition, it is known that chemical materials, and the like, are connected to end portions of a siRNA passenger (sense strand) to provide increased pharmacokinetics characteristics and high efficacy may be induced in vivo (<NPL>). Here, stability of the siRNA may vary depending on properties of the chemical materials bound to ends of the siRNA sense (passenger) or antisense (guide) strand. For example, a siRNA having a polymer compound such as polyethylene glycol (PEG) conjugated thereto interacts with an anionic phosphate group of siRNA in the presence of cationic materials to form a complex, thereby being a siRNA carrier having an improved stability (<NPL>). In particular, micelle consisting of polymer complexes has an extremely small size, significantly uniform distribution, and is spontaneously form, thereby being easy to manage quality of formulation and secure reproducibility, as compared to other systems used as a drug delivery vehicle, such as microsphere, nanoparticle, and the like.

Recently, in order to improve an intracellular delivery efficiency of siRNA, technology of using a siRNA conjugate in which hydrophilic material which is a biocompatible polymer (for example, polyethylene glycol (PEG)) is conjugated to the siRNA by a simple covalent bond or a linker-mediated covalent bond, to thereby secure stability of siRNA and have effective cell membrane permeability was developed (see <CIT>).

However, the chemical modification of siRNA and the conjugation with the polyethylene glycol (PEG) (PEGylation) still has disadvantages that stability in vivo is low and delivery into a target organ is not smooth. In chemical modification of siRNA, a bond to RISC without modification at <NUM>' end of an antisense (guide) strand recognizing a target mRNA is significantly important to initiation of RNAi mechanism. In a case of a sense (passenger) strand, through the existing research, functions of siRNA are confirmed even in a case where the conjugates are bound to both of ends, such that the sense (passenger) strand is utilized for a conjugate bond (<NPL>).

In a case of a double-stranded oligo RNA structure in which the hydrophilic materials and the hydrophobic materials are bound to the double-stranded oligo RNA, self-assembling nanoparticles are formed by a hydrophobic interaction of the hydrophobic materials, wherein the self-assembling nanoparticle is referred to as 'SAMiRNA' (<CIT>).

The technology of forming the self-assembling nanoparticles (SAMiRNA) by binding the hydrophobic materials and the hydrophilic materials to an end of the double-stranded oligo RNA has a possibility of RNA strand bias of a double-stranded oligo RNA, that is, RNAi functions may be inhibited depending on a position where the hydrophilic materials and the hydrophobic materials are bound to the end. Therefore, a technology of delivering a double-stranded oligo RNA capable of effectively permeating a cell membrane without inhibiting the functions of the double-stranded oligo RNA through optimization of the double-stranded oligo RNA structure is inevitably required to be developed.

<CIT> discloses an siRNA conjugate and a method for its preparation, specifically a hybrid conjugate formed by covalently bonding siRNA and a polymeric compound for improving the in vivo stability of siRNA.

<CIT> discloses a siRNA-polymer conjugate, which is a hybrid conjugate formed by covalently bonding siRNA and a polymeric compound for improving the in vivo stability of siRNA.

<CIT> discloses a duplex oligonucleotide complex comprising a sense strand that ranges in size from about <NUM> to about <NUM> nucleotides; an antisense strand that ranges in size from about <NUM> to about <NUM> nucleotides, wherein the antisense strand has significant levels of complementarity to both the sense strand and a target gene, and wherein the sense strand and the antisense strand form a duplex; a conjugate moiety that facilitates cellular delivery; and a linker molecule that is from about <NUM> to about <NUM> atoms in length and attaches the conjugate moiety to said sense strand; wherein between about <NUM>% to about <NUM>% of the nucleotides of the sense strand and between about <NUM>% to about <NUM>% of the nucleotides of the antisense strand are chemically modified nucleotides.

<NPL> discloses a human biochemical system that recapitulates siRNA-mediated target RNA degradation. By using affinitytagged siRNAs, it is demonstrated that a single-stranded siRNA resides in the RNA-induced silencing complex together with elF2C1 and/or elF2C2.

<NPL> discloses the role of ATP in the RNA interference (RNAi) pathway and it is shown that two ATP-dependent steps exist and that the RNAi reaction comprises at least four sequential steps.

An object of the present invention is to provide a structure a self-assembling nanoparticle (SAMiRNA) with a maximized efficacy in vivo. The SAMiRNA of the present invention means a nanoparticle formed by a hydrophobic interaction among hydrophobic materials in double-stranded oligo RNA structures in which hydrophilic materials and hydrophobic materials are bound to ends of a double-stranded oligo RNA.

An object of the present invention is to find out that a RNAi function varies depending on positions of the hydrophilic materials and the hydrophobic materials bound to the double-stranded oligo RNA structure, and thus, is to provide a technology of delivering the SAMiRNA in which RNAi functions are maximized by optimization of the double-stranded oligo RNA structure forming the SAMiRNA.

The double-stranded oligo RNA structure according to the present invention is a double-stranded oligo RNA structure comprising hydrophilic and hydrophobic material bound to a sense strand of the double-stranded oligo RNA, wherein the hydrophobic material is bound to <NUM>' end of a sense strand of the double-stranded oligo RNA and the hydrophilic material is bound to <NUM>' end thereof as shown in the following formula <NUM>:.

wherein A is a hydrophobic material, B is a hydrophilic material, X and Y are each a simple covalent bond or a linker-mediated covalent bond independently of each other, S is a sense strand of the double-stranded oligo RNA, and pAS is an antisense strand of the double-stranded oligo RNA, in which a phosphate group is bound to <NUM>' end portion of the double-stranded oligo RNA.

The double-stranded oligo RNA structure according to the present invention has a form in which the hydrophilic materials and the hydrophobic materials helping delivery into a cell are bound to the RNA by a simple covalent bond or a linker-mediated covalent bond to be capable of being self-assembled as the nanoparicle (SAMiRNA) in an aqueous solution, which may be used as an RNA inhibitor with a high efficacy significantly usable for treatment of cancer and infectious diseases, and the like, and may also be used as a pharmaceutical composition containing the double-stranded oligo RNA structure for treatment of diseases. In particular, an RNA inhibiting activity with a high efficacy may be provided even at a low concentration of the SAMiRNA dosage and thus could be used as a therapeutic agent of cancer and infectious diseases, and the like.

In particular, the double-stranded oligo RNA structure according to the present invention may use the SAMiRNA which is a nanoparticular synthetic oligo RNA inhibitor and may maximize an efficacy in gene-specifically inhibiting RNA through a binding of the hydrophilic materials and the hydrophobic materials and <NUM>' phosphorylation of an antisense strand in the SAMiRNA so as to have an optimized activity in vivo.

The present invention provides a double-stranded oligo RNA structure comprising hydrophilic and hydrophobic material bound to a sense strand of the double-stranded oligo RNA, wherein the hydrophobic material is bound to <NUM>' end of a sense strand of the double-stranded oligo RNA and the hydrophilic material is bound to <NUM>' end thereof as shown in the following formula <NUM>:.

It is preferred that the double-stranded oligo RNA strand consists of <NUM> to <NUM> nucleotides in the double-stranded oligo RNA structure of the present invention. As the double-stranded oligo RNA usable in the present invention, a double-stranded oligo RNA to any gene used for gene therapy or gene study or having a possibility therefore may also be adopted.

The double-stranded oligo RNA has various modifications for providing resistance of nuclease and decreasing a non-specific immune stimulation in order to improve stability in vivo, wherein the modification may be one or two or more combinations selected from modification in which -OH group at <NUM>' carbon in a sugar structure in one or more nucleotides is substituted with -CH<NUM>(methyl), - OCH<NUM>(methoxy), -NH<NUM>, -F(fluorine), -O-<NUM>-methoxyethyl-O-propyl, -O-<NUM>-methylthioethyl, -O-<NUM>-aminopropyl, -O-<NUM>-dimethylaminopropyl, -O-N-methylacetamido or -O-dimethylamidooxyethyl; modification in which oxygen in a sugar structure in nucleotides is substituted with sulfur; and modification to phosphorothioate or boranophosphophate, methyl phosphonate bindings from bindings among nucleotides, or may be modification to peptide nucleic acid (PNA), locked nucleic acid (LNA) or unlocked nucleic acid (UNA) (see <NPL>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT> <NPL>; <NPL>; <NPL>, <NPL>, <NPL>).

The hydrophobic material serves to generate a hydrophobic interaction to form the nanoparticle (SAMiRNA) consisting of the double-stranded oligo RAN structures. Particularly, among the hydrophobic materials, carbon chain or cholesterol has an easy binding ability in synthesis of the double-stranded oligo RNA structure, which is significantly appropriate for preparation of the double-stranded oligo RNA structure of the present invention.

In addition, it is preferred that the hydrophobic material has a molecular weight of <NUM> to <NUM>,<NUM>.

In particular, the hydrophobic material may include a steroid derivative, a glyceride derivative, glycerol ether, polypropylene glycol, unsaturated or saturated hydrocarbons containing <NUM> to <NUM> carbon atoms in a case of hydrocarbon chain, diacylphosphatidylcholine, fatty acid, phospholipid, lipopolyamine, and the like, as an example; but is not limited thereto. It is apparent to those skilled in the art that any hydrophobic materials are capable of being used as long as a material is to meet objects of the present invention.

In particular, the steroid derivative may be selected from a group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate, and cholestanyl amine, and the glyceride derivative may be selected from mono-, di-and tri-glyceride, and the like, wherein a fatty acid of the glyceride is a C<NUM> to C<NUM> unsaturated or saturated fatty acid.

In addition, the hydrophilic material is preferably a cationic or non-ionic polymer material having a molecular weight of <NUM> to <NUM>,<NUM>, more preferably, a non-ionic polymer material having a molecular weight of <NUM>,<NUM> to <NUM>,<NUM>. For example, non-ionic hydrophilic polymer compounds such as polyethylene glycol, polyvinyl pyrrolidone, polyoxazoline, and the like, are preferably used as the hydrophilic polymer compound, but are not necessarily limited thereto.

The hydrophilic material may be modified by having functional groups required for binding with other materials such as a target specific ligand, and the like, as needed. Among the hydrophilic materials, in particular, polyethylene glycol (PEG) is significantly appropriate for preparing the double-stranded oligo RNA structure of the present invention since various molecular weights and functional groups may be introduced thereinto, affinity in vivo is excellent, an immune stimulation is not induced, bio-compatibility is excellent, stability of the double-stranded oligo RNA in vivo is increased, and a delivery efficiency is increased.

In addition, a linker mediating the covalent bond is covalently bound to the hydrophilic material (or a hydrophobic material) at an end of the double-stranded oligo RNA, but is not particularly limited as long as the bond which is degraded in a specific environment is provided as needed. Therefore, any compound for the binding to activate the oligo RNA and/or the hydrophilic material (or hydrophobic material) in preparation of the double-stranded oligo RNA structure may be used as the linker.

The covalent bond may be any one of a non-degradable bond or a degradable bond. Here, examples of the non-degradable bond may include an amide bond or a phosphorylation bond, and examples of the degradable bond may include a disulfide bond, an acid degradable bond, an ester bond, an anhydride bond, a biodegradable bond or an enzymatically degradable bond, and the like, but the present invention is not necessarily limited thereto. The invention relates to a method of preparing a double-stranded oligo RNA structure as defined above comprising:.

More preferably, the method may include:.

After the step (<NUM>) or (<NUM>') above, when the preparation is completed, the reactant may be purified by high performance liquid chromatography (HPLC) and a molecular weight thereof may be measured by MALDI-TOF mass spectrometry to confirm whether or not a desired double-stranded oligo RNA and a double-stranded oligo RNA structure are prepared.

In the preparation method thereof, the synthesizing of the RNA single strand of a complementary sequence to a sequence of the RNA single strand synthesized in the step (<NUM>) or (<NUM>'), which is an independent synthesis process, may be performed before the step (<NUM>) or the step (<NUM>') or may be performed during any one step of the steps (<NUM>) to (<NUM>) or (<NUM>') to (<NUM>').

The present invention relates to a method of preparing a double-stranded oligo RNA structure of claim <NUM> comprising:.

The functional group of the present invention is not limited as long as objects of the present invention may be achieved, preferably, may be selected from amine, thiol, carboxyl, aldehyde, biotin, and the like.

In addition, the preparation method of the double-stranded oligo RNA structure may include: forming an RNA single strand based on a solid support containing the hydrophobic material bound thereto, covalently binding a hydrophilic material thereto to thereby prepare an RNA polymer structure and forming the double-stranded oligo RNA structure by annealing the RNA single strand and an RNA single strand of a complementary sequence thereto.

Meanwhile, in the double-stranded oligo RNA structure containing the ligand bound thereto of the present invention, a target specific ligand may be additionally provided with a specific position, in particular, an end, of the hydrophilic material bound to the double-stranded oligo RNA structure. The targeting moiety may be specifically bound to a receptor promoting internalization of a target cell, through receptor-mediated endocytosis (RME). The materials may be a target specific antibody, aptamer, peptide; or chemical materials including folate, N-acetyl galactosamine (NAG) and mannose, and the like, which are selected from a receptor specific ligand. Here, the targeting moiety may be any material as long as the material is specifically bound to the target receptor to perform the delivery, and thus, is not limited to the antibody, aptamer, peptide, and chemical materials.

The method of preparing the double-stranded oligo RNA structure containing a ligand bound thereto may include:.

After the step (<NUM>‴) above, when the preparation is completed, the double-stranded oligo RNA structure containing the reactant and the ligand bound thereto and the RNA single strand of a complementary sequence thereto may be separated and purified by high performance liquid chromatography (HPLC) and molecular weights thereof may be measured by MALDI-TOF mass spectrometry to confirm whether or not a desired double-stranded oligo RNA structure containing the ligand bound thereto and RNA complementary thereto are prepared. In the preparation method, the synthesizing of the RNA single strand of a complementary sequence to a sequence of the RNA single strand synthesized in the step (<NUM>‴), which is an independent synthesis process, may be performed before the step (<NUM>‴) or may be performed during any one step of the steps (<NUM>‴) to (<NUM>‴).

In addition, the present invention provides a nanoparticle (SAMiRNA) comprising a double-stranded oligo RNA structure as defined above or a double-stranded oligo RNA structure containing a ligand bound thereto as defined above containing the ligand bound thereto. The double-stranded oligo RNA structure is amphipathic containing both of hydrophobic materials and hydrophilic materials, wherein the hydrophilic materials have affinity through an interaction such as hydrogen bond, and the like, with water molecules present in the body to be toward the outside and the hydrophobic materials are toward the inside by a hydrophobic interaction therebetween, thereby forming a thermodynamically stable nanoparticle (SAMiRNA). That is, the hydrophobic material is positioned in the center of the nanoparticle and the hydrophilic material is positioned in an outside direction of the double-stranded oligo RNA, thereby forming the nanoparticle protecting the double-stranded oligo RNA (see <FIG>). The formed nanoparticle may improve the intracellular delivery
of the double-stranded oligo RNA and the function thereof, and may be utilized for a purpose of treating diseases. More specific synthesis of the structure and characteristics, intracellular delivery efficiency and effects of the nanoparticle (SAMiRNA) consisting of the double-stranded oligo RNA structure will be described by the following Examples in more detail.

In addition, the present invention provides an in vitro method of controlling the expression of gene by using a double-stranded oligo RNA structure according to any one of claims <NUM> to <NUM>, a double-stranded oligo RNA structure containing a ligand bound thereto according to claim <NUM>, or a nanoparticle according to claim <NUM>.

Specifically, described is a treatment method including: preparing nanoparticle (SAMiRNA) consisting of the double-stranded oligo RNA structure and administrating the nanoparticle (SAMiRNA) to the body of an animal.

In addition, the present invention provides a pharmaceutical composition comprising a double-stranded oligo RNA structure as defined above, a double-stranded oligo RNA structure containing a ligand bound thereto as defined above or a nanoparticle as defined above.

The composition of the present invention may be prepared by additionally containing at least one kind of pharmaceutically acceptable carrier in addition to the above-described effective components for administration. The pharmaceutically acceptable carrier is required to be compatible with the effective components of the present invention. One component selected from saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and other components or a combination of two or more components thereof may be used, and other conventional additives such as antioxidant, buffer, fungistat, and the like, may be added thereto as needed. In addition, the composition may be prepared as a formulation for injection, such as an aqueous solution, suspension, emulsion, and the like, by additionally adding diluent, dispersant, surfactant, binder and lubricant thereto. In particular, it is preferred that the composition is prepared as a lyophilized formulation. To prepare the lyophilized formulation, any method which is generally known in the corresponding art of the present invention may be used, wherein a stabilizer for lyophlization may be added thereto.

In addition, appropriate methods in the art or a method disclosed in <NPL> may be preferably used for formulation depending on each disease or component.

The pharmaceutical composition of the present invention may be determined based on symptoms of the general patient and severity of the disease by a general expert in the art. In addition, the composition may be formulated with various types such as powder, tablet, capsule, solution, injection, ointment, syrup, and the like, and may be provided as a unit-dosage or a multi-dosage container, for example, a sealed ampoule, bottle, and the like.

The pharmaceutical composition of the present invention may be orally or parenterally administered. Examples of an administration route of the pharmaceutical composition according to the present invention may include oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal, sublingual or topical administration, but the present invention is not limited thereto.

For the clinical administration as described above, the pharmaceutical composition of the present invention may be prepared as an appropriate formulation by known technology. The dosage of the composition may have various ranges thereof depending on weight, age, gender, health condition, diet, administration time, method, excretion rate the severity of disease, and the like, of a patient, and may be easily determined by a general expert in the art.

Described is a method of regulating gene expression in vivo or in vitro, using the double-stranded oligo RNA structure. Further described is a method of regulating gene expression in vivo or in vitro, using the nanoparticle containing the double-stranded oligo RNA structure.

Hereinafter, the present invention will be described in detail with reference to the following Examples.

Hereinafter, in order to inhibit Survivin, a double-stranded oligo RNA to Survivin was used. The Survivin, which is protein commonly expressed in most neoplastic tumors or transformed cell lines tested until now, is expected as an important target in cancer treatment (<NPL>).

The double-stranded oligo RNA to Survivin of the present invention consists of a sense strand of SEQ ID NO: <NUM> and an antisense strand of a complementary sequence thereto, and a double-stranded oligo RNA used as a control group consists of a sense strand of SEQ ID NO: <NUM> and an antisense strand of a complementary sequence thereto. Sequence of the double-stranded oligo RNA used in the present Examples is as follows:.

In the double-stranded oligo RNA, the double-stranded oligo RNA single strand was synthesized by a method of using β-cyanoethylphosphoramidite to connect phosphodiester bonds configuring an RNA framework (Polymer support oligonucleotide synthesis XVIII: use of beta-cyanoethyl-N,N-dialkylamino- /N-morpholinophosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product.

A desired sequence of the RNA single strand is obtained by starting the synthesis process on the solid support (CPG) containing nucleoside bound thereto and repeating a cycle including deblocking, coupling, capping, and oxidation. The RNA <NUM> Synthesizer (BIONEER, Korea) was used for a series of the corresponding synthesis process of the double-stranded oligo RNA.

By comparison between the double-stranded oligo RNA configuring the double-stranded oligo RNA structure and the double-stranded oligo RNA depending on a binding side of a polymer material in view of an efficacy of inhibiting expression of a target gene, the double-stranded oligo RNA structures having the following structures were prepared for optimization of the double-stranded oligo RNA structure.

The double-stranded oligo RNA structures prepared in the present invention have structures shown in the following Table <NUM>, respectively.

In Table <NUM>, S is a sense strand of the double-stranded oligo RNA; AS is an antisense strand of the double-stranded oligo RNA; PO<NUM> is a phosphate group; PEG is a hydrophilic material: polyethylene glycol; C<NUM> is a hydrophobic material: tetradocosane containing a bisulfide bond; and <NUM>' and <NUM>' mean directionality of an end of the double-stranded oligo RNA.

The synthesis of the sense strand of the double-stranded oligo RNA structure was performed using β-cyanoethylphosphoramidite to connect phosphodiester bonds configuring an RNA framework as described above and polyethylene glycol (PEG) was additionally bound to <NUM>' end portion thereof, thereby preparing a sense strand of SAMiRNALP of the double-stranded oligo RNA structure.

In a case of an antisense strand performing an annealing with the sense strand of the double-stranded oligo RNA structure, the synthesis of the antisense strand was performed using β-cyanoethylphosphoramidite to connect phosphodiester bonds configuring an RNA framework as described above, then tetradocosane (C<NUM>) containing a bisulfide bond which is the hydrophobic material, was additionally bound to <NUM>' end portion thereof, thereby preparing an antisense strand of SAMiRNALP.

A desired sense strand of S-SAMiRNALP was prepared by performing the reaction having <NUM>' polyethylene glycol (PEG)-CPG prepared by Example <NUM> of the related art document (<CIT>) as a support, to synthesize a double-stranded oligo RNA-hydrophilic material structure having a sense strand containing PEG bound to <NUM>' end portion thereof, and binding tetradocosane(C<NUM>) containing a bisulfide bond to <NUM>' end. In a case of an antisense strand annealing with the sense strand of the S-SAMiRNALP, the antisense strand of a complementary sequence to the sense strand was prepared by a method of using β-cyanoethylphosphoramidite to connect phosphodiester bonds configuring an RNA framework as described above.

A sense strand of AS-SAMiRNALP was prepared by a method of using β-cyanoethylphosphoramidite to connect phosphodiester bonds configuring an RNA framework as described above.

A desired antisense strand performing an annealing with the sense strand of the AS-SAMiRNALP was prepared by performing the reaction having <NUM>' PEG-CPG prepared by Example <NUM> of the related art document (<CIT>) as a support, to synthesize a double-stranded oligo RNA-hydrophilic polymer structure having an antisense strand containing PEG bound to <NUM>' end portion thereof, and binding tetradocosane(C<NUM>) containing a bisulfide bond to <NUM>' end.

In order to maximize an effect of the double-stranded oligo RNA, S-SAMiRNALP-PO<NUM> has a sense strand containing a structure of hydrophilic materials and hydrophobic materials bound thereto and an antisense strand containing a phosphate group bound to <NUM>' end portion thereof.

A sense strand of S-SAMiRNALP-PO<NUM> was prepared by the same method as the sense strand of S-SAMiRNALP, and an antisense strand performing an annealing of the S-SAMiRNALP-PO<NUM> containing a phosphate group bound to <NUM>' end was prepared by performing the reaction using β-cyanoethylphosphoramidite to connect phosphodiester bonds configuring an RNA framework as described above and then, using chemical phosphorylation reagent (CPR) which is [<NUM>-(<NUM>,<NUM>'Dimethoxytrityloxy)-<NUM>,<NUM>-dicarboxyethyl]propyl-(<NUM>-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite to bind a phosphate group to <NUM>' end (see <FIG>). Otherwise, the antisense strand of S-SAMiRNALP-PO<NUM> containing a phosphate group bound thereto was prepared by using a method comprising collecting the RNA single strand from CPG and treating a phosphorylation enzyme (kinase) to bind the phosphate group to <NUM>' end.

When the synthesis was completed, the synthesized RNA single strand and the RNA-polymer structure were separated from CPG with <NUM> %(v/v) ammonia in a water bath at <NUM> and protecting moiety was removed by deprotection reaction. The RNA single strand and the RNA-polymer structure from which the protecting moiety was removed were treated with N-methylpyrrolidone, triethylamine and triethylaminetrihydrofluoride at a volume ratio of <NUM>:<NUM>:<NUM>, in an oven at <NUM>, to remove <NUM>' TBDMS(tertbutyldimethylsilyl).

In addition, in order to bind a ligand to the end portion of the hydrophilic polymer of the double-stranded oligo RNA structure, functional groups capable of binding the ligand was bound to <NUM>' CPG, the hydrophilic polymer was bound thereto, and the reaction was performed, thereby preparing the sense strand of the SAMiRNALP to which the ligand is capable of being bound. In more detail, <NUM>' amine-PEG-RNA was synthesized using a PEG phosphoramidite reagent in amine-CPG containing functional groups such as amine groups, and the like, bound thereto and a hydrophobic material such as C<NUM> was bound to the <NUM>' amine-PEG-RNA oligo to synthesize <NUM>' amine-PEG-RNA-C<NUM>, followed by treatment with <NUM> %(v/v) ammonia in a water bath at <NUM> to separate the synthesized RNA single strand and the RNA-polymer structure from CPG and the protecting moiety was removed by deprotection reaction. The RNA single strand and the RNA-polymer structure from which the protecting moiety was removed were treated with N-methylpyrrolidone, triethylamine and triethylaminetrihydrofluoride at a volume ratio of <NUM>:<NUM>:<NUM>, in an oven at <NUM>, to remove <NUM>' TBDMS(tert-butyldimethylsilyl). The collected <NUM>' amine-PEG-RNA-C<NUM> therefrom was subjected to an ester reaction with a bindable ligand material consisting of N-Hydroxysuccinimide ligand to synthesize a <NUM>' ligand-bound PEG-RNA-C<NUM> oligo. The RNA single strand, the RNA-polymer structure, and the RNA-polymer structure containing the ligand bound thereto were separated from the reactants by HPLC, and molecular weights thereof were measured by MALDI-TOF-MS (SHIMADZU, Japan) to confirm whether or not sequence and the RNA-polymer structure correspond to those to be synthesized.

Then, in order to prepare each double-stranded oligo RNA structure, the sense strand and the antisense strand in an equivalent amount were mixed to each other and put into 1X annealing buffer (<NUM> HEPES, <NUM> potassium acetate, <NUM> magnesium acetate, pH <NUM> to <NUM>), followed by reaction in a constant temperature water bath at <NUM> for <NUM> minutes and then reaction at <NUM>, thereby preparing the desired SAMiRNALP and the desired S-SAMiRNALP-PO<NUM> containing a phosphate group bound thereto, respectively. Annealing of the prepared SAMiRNALP was confirmed by electrophoresis.

S-SAMiRNALP and S-SAMiRNALP-PO<NUM> forms a nanoparticle, that is, micelle, by a hydrophobic interaction between the hydrophobic materials bound to the end of the double-stranded oligo RNA (see <FIG>).

Formation of the nanoparticle (SAMiRNA) consisting of the corresponding SAMiRNALP was confirmed by analysis of nanoparticle size, critical micelle concentration (CMC) and Transmission Electron Microscope (TEM) of the S-SAMiRNALP prepared by Example <NUM> above.

A size of the nanoparticle was measured by zeta-potential measurement. In detail, the SAMiRNALP was dissolved into <NUM> mℓ of Dulbecco's phosphate buffered saline (DPBS) so as to have a concentration of <NUM> µg/mℓ and then treated by a ultrasonic homogenizer (Wiseclean, DAIHAN, Korea) so that a size of nanoparticle is homogenized (<NUM> W; amplitude: <NUM> %). A size of the homogenized nanoparticle was measured by zeta-potential measurement (Nano-ZS, MALVERN, England) under conditions in which a refractive index to the material is <NUM>, an absorption index is <NUM>, a temperature of a solvent: PBS is <NUM> and the corresponding viscosity and refractive index are <NUM> and <NUM>, respectively. Once measurement was conducted by a size measurement including <NUM> times repeats and then repeated three times.

It was confirmed that the nanoparticle consisting of S-SAMiRNALP had a size of about <NUM> and PDI of about <NUM> (see <FIG>). As the PDI is low, the corresponding particles become uniformly distributed, and thus, it could be appreciated that the nanoparticle consisting of S-SAMiRNALP has a relatively uniform size. It was confirmed that the size of the nanoparticle consisting of the structure has an appropriate size to be intaken in the cell through endocytosis (Nanotoxicology: nanoparticles reconstruct lipids.

An amphiphile containing both of hydrophobic materials and hydrophilic materials in a single molecule may be a surfactant, wherein when the surfactant is dissolved into an aqueous solution, the hydrophobic groups thereof move toward the center portion to prevent water contact and the hydrophilic groups thereof moves toward the outside to form micelle. Here, a concentration at which the micelle is initially formed is referred to as a critical micelle concentration (CMC). A method of measuring CMC using fluorescent pigment is based on property of the fluorescent pigment in which the slope of the graph curve of the fluorescence intensity is rapidly changed before/after the micelle is formed.

In order to measure CMC of the nanoparticle consisting of S-SAMiRNALP, <NUM> DPH (<NUM>,<NUM>-Diphenyl-<NUM>,<NUM>,<NUM>-hexatriene, SIGMA, USA) was prepared as the fluorescent pigment. <NUM> nmole/µℓ of S-SAMiRNALP was diluted from a concentration of <NUM> µg/mℓ to the maximum of <NUM> µg/mℓ with DPBS for each step to prepare S-SAMiRNALP samples having the total volume of <NUM> µℓ. <NUM> µℓ of <NUM> DPH and methanol which is a solvent of DPH for a control group were added to the prepared samples, respectively and mixed well, and treated by a ultrasonic homogenizer (Wiseclean, DAIHAN, Korea) according to the same method as Example <NUM>-<NUM> so that a size of the nanoparticle is homogenized (<NUM> W; amplitude: <NUM> %). The homogenized samples were reacted at room temperature without light for about <NUM> hours, and fluorescent values (excitation: <NUM>, emission: <NUM>, top read) were measured. In order to confirm relative fluorescent values among the measured fluorescent values, the fluorescent value of the sample containing DPH and the fluorescent value of the sample containing the only methanol (Y axis) were measured and shown as a graph with respect to log value of the treated concentration of S-SAMiRNALP (X axis) (see <FIG>).

The fluorescent value measured for each concentration was rapidly increased while moving from a low concentration section to a high concentration section, wherein the concentration at the rapidly increased point is CMC concentration. Therefore, when drawing trend lines by dividing the low concentration section in which the fluorescent amount is not increased and the high concentration section in which the fluorescent amount is increased into several sections, an X value in an intersection of the two trend lines is CMC concentration. It was observed that the measured CMC of the nanoparticle consisting of S-SAMiRNALP is <NUM> µg/mℓ, which is significantly low, and thus, it was confirmed that the micelle may be formed by the nanoparticle consisting of S-SAMiRNALP even at a significantly low concentration.

The nanoparticle consisting of S-SAMiRNALP was observed by TEM in order to confirm the shape thereof.

In detail, the SAMiRNALP was dissolved into DPBS so as to have the final concentration of <NUM> µg/mℓ and treated by a ultrasonic homoginezer (Wiseclean, DAIHAN, Korea) so that a size of nanoparticle is homogenized (<NUM> W; amplitude: <NUM> %). The nanoparticle consisting of S-SAMiRNALP was observed with a material having a high electron density through a negative staining method (see <FIG>).

It was confirmed that the nanoparticle observed by TEM is well formed with a size similar to that of the nanoparticle measured in Example <NUM>-<NUM>.

Expression of Survivin gene of a transfected tumor cell line was analyzed using each nanoparticle consisting of SAMiRNALP, S-SAMiRNALP, or AS-SAMiRNALP prepared by Example <NUM> above.

<NUM>% (v/v) fetal bovine serum, <NUM> units/mℓ of penicillin and <NUM>µg/mℓ of streptomycin were added to an EMEM culture medium (ATCC-formulated eagle's minimum essential medium) containing HeLa acquired from American type Culture Collection (ATCC) and cultured at <NUM> and <NUM> % (v/v) CO<NUM>.

The tumor cell line (<NUM> X <NUM><NUM>) cultured in Example <NUM>-<NUM> above were cultured in a <NUM>-well plate in the EMEM culture medium for <NUM> hours under the same condition as Example <NUM>-<NUM> above, the medium was removed, and the equivalent amount of Opti-MEM medium per each well was deposited.

<NUM>µℓ of the Opti-MEM medium and each <NUM> µg/mℓ of the SAMiRNALP, S-SAMiRNALP and AS-SAMiRNALP were added to DPBS, and then treated by a ultrasonic homoginezer (Wiseclean, DAIHAN, Korea) according to the same method as Example <NUM>-<NUM> to thereby homogenize (<NUM> W; amplitude: <NUM>) each nanoparticle consisting of SAMiRNALP, S-SAMiRNALP and AS-SAMiRNALP, thereby preparing solutions. Then, each well of the tumor cell line in which the Opti-MEM is deposited was treated with a transfection solution at a concentration of <NUM> and <NUM>, and cultured at <NUM> and <NUM> % (v/v) CO<NUM> for the total of <NUM> hours.

The total RNA was extracted from the transfected cell line in Example <NUM>-<NUM> above, cDNA was synthesized, and an expression amount of mRNA of Survivin was relatively quantitative analyzed by real-time PCR according to a method disclosed in <CIT> (see <FIG>).

The nanoparticle consisting of AS-SAMiRNALP, which is a case in which the structure was bound to the only antisense strand bound to the target mRNA in the double-stranded oligo RNA mechanism, shows a low inhibiting efficacy as compared to the existing nanoparticle consisting of SAMiRNALP. The nanoparticle consisting of S-SAMiRNALP, which is a case in which the structure was bound to the only sense strand being contrary to the above case of nanoparticle consisting of AS-SAMiRNALP, shows a high inhibiting efficacy even at a low concentration as compared to the existing nanoparticle consisting of SAMiRNALP (see <FIG>).

Sur584 means SAMiRNALP having double-stranded oligo RNA sequence (SEQ ID NO: <NUM>) specific to the target gene Survivin according to each structures of SAMiRNALP, and CON means SAMiRNALP including control group sequence (SEQ ID NO: <NUM>) not affecting expression of the target gene. A degree of inhibiting expression of mRNA of the target gene was calculated with the expression amount of the target gene of a sample treated with Sur584 with respect to the expression amount of the target gene of a sample treated with CON by Comparative Quantitation.

The nanoparticle consisting of S-SAMiRNALP which is the optimized structure was well delivered into the cell without a transfection material and thus, an effect of inhibiting expression of mRNA of the target gene was observed, which was confirmed to have an increase effect of the double-stranded oligo RNA by about three times or more (nanoparticle groups consisting of SAMiRNALP - <NUM>% inhibition vs. nanoparticle groups consisting of S-SAMiRNALP - <NUM> % inhibition) when being treated with <NUM>, as compared to the existing nanoparticle consisting of SAMiRNAL. In addition, it was observed that the Experimental group of the nanoparticle consisting of AS-SAMiRNALP in which all structures are bound to the antisense strand which is a negative control group has a low effect of inhibiting expression of mRNA of the target gene even at a high concentration.

Each of S-SAMiRNALP of SEQ ID NO: <NUM> and S-SAMiRNALP-PO<NUM> of SEQ ID NO: <NUM> prepared by Example <NUM> above were transfected into Hela by a transfection material and expression of Survivin of the transfected tumor cell line was analyzed.

The tumor cell lines (<NUM> X <NUM><NUM>) cultured in Example <NUM>-<NUM> above were cultured in a <NUM>-well plate in the EMEM culture medium for <NUM> hours under the same condition as Example <NUM>-<NUM> above, the medium was removed, and 800mℓ of Opti-MEM medium per each well was deposited.

Meanwhile, <NUM>µℓ of Lipofectamine™ RNAiMax (Invitrogen, USA) and <NUM>µℓ of Opti-MEM medium were mixed and reacted at room temperature for <NUM> minutes, and treated with each of the S-SAMiRNALP and S-SAMiRNALP-PO<NUM>(25pmole/µℓ) prepared by Example <NUM> above so as to have the final concentration of <NUM>, <NUM> and <NUM>, respectively, followed by reaction at room temperature for <NUM> minutes, thereby preparing solution.

Then, <NUM>µℓ of each transfection solution was deposited into each well of the tumor cell line containing Opti-MEM deposited therein and cultured for <NUM> hours, and Opti-MEM medium was removed. Here, <NUM> mℓ of EMEM culture medium was deposited thereto and cultured at <NUM> and <NUM> % (v/v) CO<NUM> for <NUM> hours.

The total RNA was extracted from the transfected cell line in Example <NUM>-<NUM> above, cDNA was synthesized, and an expression amount of mRNA of Survivin gene was analyzed based on relative quantification by real-time PCR according to a method disclosed in <CIT>.

In order to analyze an effect of inhibiting expression of the target gene by S-SAMiRNALP and S-SAMiRNALP-PO<NUM>, the transformation was conducted with the transfection material, and the degree of expression of mRNA of Survivin gene was measured, and thus, it could be confirmed that the cases treated with S-SAMiRNALP and S-SAMiRNALP-PO<NUM> show an effect of inhibiting expression of the target gene similar to that of the naked double-stranded oligo RNA in which nothing is bound to the end of the double-stranded oligo RNA. S-SAMiRNALP-PO<NUM> showed relatively high efficacy of inhibiting expression as compared to S-SAMiRNALP, and in particular, high effect of inhibiting expression of the target gene was shown at a low concentration of <NUM> (see <FIG>). It was observed that the RNA structure containing S-SAMiRNALP polymer bound thereto does not inhibit mechanism of RNAi, and in particular, in S-SAMiRNALP-PO<NUM>, an effect of double-stranded oligo RNA is increased by the additionally bound phosphate group.

Claim 1:
A double-stranded oligo RNA structure comprising hydrophilic and hydrophobic material bound to a sense strand of the double-stranded oligo RNA, wherein the hydrophobic material is bound to <NUM>' end of a sense strand of the double-stranded oligo RNA and the hydrophilic material is bound to <NUM>' end thereof as shown in the following formula <NUM>:

        A-X-<NUM>' S3' -Y-B pAS     Formula <NUM>

wherein A is a hydrophobic material, B is a hydrophilic material, X and Y are each a simple covalent bond or a linker-mediated covalent bond independently of each other, S is a sense strand of the double-stranded oligo RNA, and pAS is an antisense strand of the double-stranded oligo RNA, in which a phosphate group is bound to <NUM>' end portion of the double-stranded oligo RNA.