KR101722948B1 - Double stranded oligo RNA molecule with a targeting ligand and method of preparing the same - Google Patents

Abstract

The present invention relates to a double-stranded oligo RNA structure in which a target-specific ligand is bound to a double-stranded oligo RNA structure in which a polymer compound that improves the transfer of a double-stranded oligo RNA is covalently bonded, And a target-specific double-stranded oligo-RNA delivery technology through the above-described structure. Nanoparticles composed of ligand-bound double-stranded oligo RNA constructs efficiently transfer double-stranded oligonucleotide RNA to the target, which can result in the activity of double-stranded oligonucleotide RNA in target cells at a relatively low concentration of double-stranded oligonucleotide RNA Can prevent the transmission of nonspecific double-stranded oligo RNA to other organs and cells and is a tool for the treatment of various diseases, especially double-stranded oligo RNA for cancer, as well as a new type of double-stranded oligonucleotide RNA delivery system Can be usefully used.

Description

[0001] The present invention relates to a double-stranded oligo RNA construct having a ligand bound thereto, and a method for preparing the same.

The present invention relates to a novel double-stranded oligosaccharide RNA structure having hydrophilic and hydrophobic materials that improve the delivery of double-stranded oligo RNA, which can be useful for the treatment of cancer and infectious diseases, A double-stranded oligosaccharide structure, a double-stranded oligo RNA structure, a double-stranded oligosaccharide RNA structure conjugated with the ligand, a pharmaceutical composition comprising the structure, and a nanoparticle comprising the structure, A method for producing nanoparticles comprising the structure, and a technique for delivering the double-stranded oligo RNA structure having the ligand bound thereto.

RNA interference (RNAi) has been shown to act on sequence-specific mRNAs in a variety of mammalian cells since its discovery (Silence of the transcripts: RNA interference in medicine. J Mol Med (2005) 83: 764-773). When a double-stranded RNA of a long chain is transferred to a cell, the double-stranded RNA is transferred to a small interfering RNA (RNA), which is processed into 21 to 23 base pairs (bp) by endonuclease Dicer , Hereinafter referred to as 'siRNA'). siRNA binds to an RNA-induced silencing complex (RISC), and the antisense strand recognizes and degrades the target mRNA, thereby specifically inhibiting the expression of the target gene in a sequence-specific manner (NUCLEIC-ACID THERAPEUTICS: BASIC PRINCIPLES AND RECENT APPLICATIONS. Reviews Drug Discovery, 2002. 1, 503-514).

According to a study by Bertrand et al., SiRNA is superior to the antisense oligonucleotide (ASO) for the same target gene in inhibiting mRNA expression in vivo / ex vivo ( in vitro and in vivo ) Has long-lasting effects (Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. Biochem. Biophys. Res. Commun. 2002. 296: 1000-1004). In addition, since the action mechanism of siRNA binds complementarily to the target mRNA and regulates the expression of the target gene in a sequence-specific manner, the target that can be applied compared to the conventional antibody-based drug or chemical (small molecule drug) (Progress Towards in Vivo Use of siRNAs. MOLECULAR THERAPY 2006 13 (4): 664-670).

Despite the superior effectiveness of siRNAs and their various uses, in order for siRNA to be developed as a therapeutic agent, the siRNA should be efficiently delivered to the target cell through improvement of the stability of the siRNA and improvement of the cell delivery efficiency (Harnessing in vivo siRNA delivery for drug discovery and therapeutic Development. Drug Discov Today. 2006 Jan; 11 (1-2): 67-73).

Attempts are being made to overcome this problem in part by using nucleic acid-degrading enzyme analogs or using vectors such as viral vectors, liposomes or nanoparticles.

Viral delivery systems such as adenovirus and retroviruses have high transfection efficacy but high immunogenicity and oncogenicity. On the other hand, the non-viral delivery system containing nanoparticles has a lower cell delivery efficiency than the viral delivery system, but has high safety in vivo , target specific delivery, (RNAi) oligonucleotides into cells or tissues, and low cytotoxicity and immune stimulation, thereby effectively inhibiting the expression of target genes (J Clin Invest. 2007 December 3; 117 (12): 3623-4362).

Various nanoparticle delivery systems have been developed for cancer-specific delivery. These nanoparticle systems have been coated with a hydrophilic material to circulate for a long time in the blood, and a positive charge is applied to enhance endocytosis (Active targeting schemes for nanoparticle systems in cancer therapeutics. Advanced Drug Delivery Reviews 60 (2008) 1615-1626). On the other hand, tumor tissues are very strong and have a diffusion-limitation as compared with normal tissue. Such diffusion restriction adversely affects the movement of waste materials such as nutrients, oxygen and carbon dioxide necessary for tumor growth, It overcomes the diffusion limitation by forming blood vessels around the blood through angiogenesis. The blood vessels in tumor tissues formed through angiogenesis have leaky and defective blood vessels with a gap of about 100 nm to 2 μm depending on the type of tumor.

Therefore, the nanoparticles pass through the capillary endothelium of the cancer tissue having a loose vascular structure in comparison with the tissue capillary blood vessels of the normal tissue, thus facilitating access to the tumor interstitium during circulation in the blood, In addition, there is no lymphatic drainage in the tumor tissue, which results in accumulated drug, which is called enhanced permeation and retention (EPR) effect. In this study, we investigated the effect of tumor necrosis factor (TNF) on tumor cell differentiation, .

To overcome the nonspecific in vivo distribution, targeting, and lack of water solubility of therapeutic drugs including anticancer drugs, the size of nanoparticles loaded with therapeutic drug is optimized or the surface is modified to produce Research is under way to improve circulation time. Particularly, polymeric nanoparticles using polymer-drug conjugates and anticancer agents are known as albumin, poly-L-glutamate (PGA), N - (2-hydroxypropyl) ) -methacrylamide the copolymer) and like water-soluble (water-soluble), biodegradability (biodegradable) is connected to the material and the anticancer agents having a characteristic capable tumor specific delivery of anticancer drug-methoxy taksil amide copolymer ((2-hydroxypropyl N) (Clin Cancer Res 2008; 14: 1310-1316). ≪ Desc / Clms Page number 2 > In addition, research on the transfer of anticancer drugs through the formation of polymeric micelles, which are composed of a core composed of an anticancer drug which is usually hydrophobic, and a hydrophilic shell formed by binding an amphiphilic substance to an anticancer drug, is under way (Development of the polymer micelle carrier system for doxorubicin. J Control Release 2001; 74: 295-302).

Therefore, when a hydrophobic substance is further added to a therapeutic drug such as an anticancer drug to enhance the cohesion of the core, micelles can be formed even at a low concentration, and the polymeric micelles Can be formed. A therapeutic drug in which hydrophobic and hydrophilic substances are attached to both ends as biodegradable bonds forms an improved form of polymer micelles, and thus the therapeutic drug can be stably transferred to the cancer tissue as a target organ.

Recently, a technology for transferring double-stranded oligonucleotides has been developed (see Korean Patent Publication No. 2009-0042297), which is a self-assembled nanoparticle formed through a characteristic of a substance bound to a nucleic acid terminal. SAMiRNA is a self-assembled nanoparticle composed of a double-stranded oligo RNA structure with hydrophilic and hydrophobic polymers that improve the transfer of double-stranded oligo RNA. SAMiRNA technology is a technique that improves the transfer of double-stranded oligo RNA into cells have

When the SAMiRNA was applied as a tail vein to a tumor xenograft mouse model with a fluorescent label attached thereto, it was confirmed that the nanoparticles were tumor-specifically transferred by the above-mentioned passive targeting (See Fig. 2).

Active targeting, on the other hand, involves the binding of a targeting moiety to nanoparticles, which involves preferential accumulation of nanoparticles in the target tissue, internalization of the nanoparticles into the target cell (10): 552-8. Epub 2008 Aug 21). In the present study, we investigated the effect of the localization of tumor necrosis factor (TNF) on tumorigenesis.

Active targeting is the binding of targeting moieties such as antibodies or ligands to nanoparticles to induce delivery to improved target cells. Recently, studies are underway to localize siRNA to a desired tissue by binding a targeting moiety to the siRNA.

For example, it has been found that siRNA conjugated with alpha -tocopherol is effectively and stably transferred in vivo to inhibit the expression of a target gene through RNA interference (Kazutaka Nishina et al., The American Society of Gene In addition, cholesterol-conjugated siRNA is superior to CPP (cell penetrating peptide), which is a peptide that promotes penetration, which is mainly used for siRNA delivery, (US-20060014289; Moschos SA et al., Bioconjug. Chem. 18: 1450-1459). It has been reported that the delivery effect is dependent not only on the specificity of the tumor tissue but also on the specificity of the target cell by the binding target moiety.

Active targeting utilizes a substance capable of binding to carbohydrates, receptors, and antigens that are specific or over-expressed on a target cell surface (Nanotechnology in cancer therapeutics: bioconjugated nanoparticles for drug delivery. Mol Cancer Ther 2006; 5 (8): 1909-1917). Thus, nanoparticles bound with an active targeting moiety are accumulated in the tumor tissue during circulation in the blood by passive targeting, and the targeting moiety increases the intracellular delivery of the nanoparticles, The therapeutic effect of the drug delivered to the cell is improved. The target moiety is mainly a ligand or an antibody that binds to the cell surface receptor with high affinity and specificity to induce receptor-mediated endocytosis (RME) Mediated endocytosis (RME) of proteins and peptides: use of RME as a drug delivery system. J Control Release 1996; 39: 191 200).

Receptors or antigens on the cell surface that are targets of such ligands or antibodies are specific or overexpressed in the target cells, so that the targeting ligand should be easy to access and the rate of endocytosis is high. Receptor-mediated endocytosis: An overview of a dynamic process. J. Biosci., Vol. 6, p. Number 4, October 1984, pp. 535-542.). A targeting moiety is a receptor that is specifically expressed in a target cell line such as an epidermal growth factor or a low-density lipoprotein receptor, or a receptor that is overexpressed on the cell surface of various cancer species (Molocer Cancer Ther 2006; 5 (8): 1909-1917), which specifically binds to tumor-specific receptors, such as folate receptors known to be involved in tumorigenesis (Nanotechnology in cancer therapeutics: bioconjugated nanoparticles for drug delivery. .

The targeting moieties to SAMiRNA, particularly receptor-specific ligands having receptor-mediated endocytosis (RME) properties, are effectively combined to facilitate delivery to target cells, particularly cancer cells, Can also be transferred to the target cell, which can exhibit high double-stranded oligo RNA activity and inhibit the transfer of nonspecific double-stranded oligo RNA to other organs and cells.

Korean Patent Laid-Open Publication No. 2009-0042297: siRNA conjugate and preparation method thereof. Date of publication Nov. 24, 2010, Applicant: Bioneer

It is an object of the present invention to provide a therapeutic drug in which both hydrophilic and hydrophobic substances, which are biocompatible polymer compounds, are bound to both ends of a therapeutic drug by simple covalent bonding or linker-mediated covalent bonding in order to improve the intracellular delivery efficiency of the therapeutic drug A therapeutic drug structure in which a ligand is bound to a hydrophilic substance, and nanoparticles comprising the structure.

Another object of the present invention is to provide a method for improving the intracellular delivery efficiency of a double-stranded oligo RNA, wherein a hydrophilic and hydrophobic substance, which is a biocompatible polymer compound, is attached to a terminal of a double-stranded oligo RNA with a simple covalent bond or a linker- Binding double-stranded oligonucleotide construct, a receptor-mediated endocytosis (RME) -specific ligand-binding double-stranded RNA that enhances cellular internalization of the target cell specifically to the hydrophilic material A pharmaceutical composition comprising nanoparticles composed of an oligosaccharide structure, an oligosaccharide structure, a nanoparticle composed of the double-stranded oligosaccharide structure to which the ligand is bound, and a double-stranded oligosaccharide structure to which the ligand is bonded or a double-stranded oligosaccharide structure to which the ligand is bonded .

Another object of the present invention is to provide a double-stranded oligo RNA construct having the ligand bound thereto, a method for producing nanoparticles containing the same, and a double-stranded oligo RNA delivery technology using the double-stranded oligosaccharide RNA structure bound to the ligand .

In the present invention, the antisense strand is a strand that exhibits RNAi activity that is complementary to a target mRNA in RISC (RNA-induced silencing complex) to decompose a target mRNA, and a sense strand is a strand having a sequence complementary to an antisense strand do.

In the present invention, the term complementary or complementary binding means that two sequences are combined to form a double-stranded structure. It is not necessarily a perfect match, and mismatches are included in some sequences. .

The present invention provides a therapeutic drug-polymer structure to which a ligand having a structure represented by the following formula (1) is bonded.

A ligand-bound therapeutic drug-polymer structure having a structure represented by the following formula (1);

L-A-X-R-Y-B (1)

In the formula (1), A is a hydrophilic substance and B is a hydrophobic substance; X and Y are independently a simple covalent bond or a linker-mediated covalent bond; R is a therapeutic drug; L means a ligand that specifically binds to a receptor that promotes target cell internalization through receptor-mediated endocytosis (RME).

At this time, the therapeutic drug is selected from the group consisting of anticancer drugs, double helix oligo RNA, antiviral drugs, steroidal antiinflammatory drugs (SAID), NSAIDs, antibiotics, An antidepressant, an anesthetic, an analgesic, an analgesic, an anabolic steroid, an estrogen, a progesterone, a glycosaminoglycan, a polynucleotide, an immunosuppressant, an antineoplasic agent, And immunostimulating agents.

The therapeutic drug in the present invention is preferably a double-stranded oligo RNA or an anticancer agent. In this case, when the therapeutic drug is a double-stranded oligo RNA, the hydrophilic substance may be bonded at any position of the 3 'or 5' end of the double-stranded oligo RNA.

In the ligand-binding therapeutic drug-polymer structure of the present invention, a double-stranded oligo RNA or a hydrophilic substance bound to an anticancer agent may be additionally provided with a ligand at a specific position, particularly at the terminal. The ligand may be a target receptor-specific antibody or an aptamer having an RME characteristic that promotes internalization of a target cell specifically (a characteristic of being able to bind to aptamer target molecule with high affinity and specificity) (DNA, RNA or modified nucleic acid)}, peptide or folate (generally, folate and folic acid are used intermingled with each other, and folate in the present invention is a natural state or a folate activated form in the human body and it means), N - may be selected from acetyl galactosamine (N -Acetylgalactosamine, chemicals including NAG), mannose (mannose). In this case, the ligand is a substance that allows the target receptor-specific delivery, and is not limited to the antibody, the aptamer, the peptide, and the chemical substance.

Preferably, the double-stranded oligo RNA is comprised of 19 to 31 nucleotides. Double helical oligo RNAs that can be used in the present invention may be selected from double helical oligo RNAs for any genes that are used or likely to be used for gene therapy or research.

The hydrophobic material causes a hydrophobic interaction to form nanoparticles composed of a double helix oligo RNA structure. Among the hydrophobic substances, carbon chains and cholesterol are particularly suitable for the production of the structure of the present invention because they can be easily conjugated in the step of synthesizing double-stranded oligo RNA.

The hydrophobic substance preferably has a molecular weight of 250 to 1,000. Particularly, the hydrophobic substance may be a steroid derivative, a glyceride derivative, a glycerol ether, a polypropylene glycol, a C ₁₂ to C ₅₀ unsaturated or saturated hydrocarbons, diacyl phosphatidylcholine, fatty acids, phospholipids, lipopolyamines and the like may be used, but the present invention is not limited thereto, It is obvious to those skilled in the art that any hydrophobic material can be used as long as it conforms to the present invention.

In particular, the steroid derivative may be selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cortestanyl morpholate, and cholestanyl amine, wherein the glyceride derivative is selected from the group consisting of mono-, - and tri-glycerides, wherein the fatty acid of the glyceride is characterized by a C ₁₂ to C ₅₀ unsaturated or saturated fatty acid.

In addition, the hydrophilic material is preferably a cationic or nonionic polymer having a molecular weight of 200 to 10,000, more preferably 1,000 to 2,000. For example, the hydrophilic substance is preferably a nonionic hydrophilic polymer compound such as polyethylene glycol, polyvinylpyrrolidone, or polyoxazoline, but is not limited thereto.

The hydrophilic substance may be modified so as to have a functional group required for binding with the ligand, if necessary. Among the hydrophilic substances, polyethylene glycol (PEG) can introduce various molecular weights and functional groups, has good bio-compatibility such as good affinity in vivo and does not induce an immune response, and double helix Oligonucleotides, oligonucleotides, oligonucleotides, oligonucleotides, oligonucleotides, oligonucleotides, oligonucleotides, oligonucleotides, and oligonucleotides.

The linker that mediates the covalent bond is particularly limited as long as it provides a bond capable of covalently bonding at the end of a residue derived from a hydrophilic substance (or a hydrophobic substance) and a double-stranded oligo RNA, no. Thus, the linker may include any compound that binds to activate double-stranded oligo RNA and / or hydrophilic (or hydrophobic) materials during fabrication of the structure.

The covalent bond may be either a non-cleavable bond or a cleavable bond. At this time, the non-degradable bond has an amide bond or a phosphorylated bond, and the degradable bond includes a disulfide bond, an acid-decomposable bond, an ester bond, an anhydride bond, a biodegradable bond or an enzyme degradable bond.

A double-stranded oligo-RNA having a ligand in which a hydrophilic substance is bonded to the 3 'end of the double stranded oligo RNA sense strand, a ligand is bonded to the hydrophilic substance, and a hydrophobic substance is bonded to the 5' A method for providing an RNA structure and producing the same

(Wherein A is a hydrophilic substance, B is a hydrophobic substance, X and Y are independently a simple covalent bond or linker-mediated covalent bond, S is a sense strand of a double-stranded oligo RNA, AS is a Is an antisense strand of double-stranded oligo RNA; L is a ligand with specific binding properties to receptors that promote target cell internalization via receptor-mediated endocytosis (RME) .)

(1) synthesizing a single strand of RNA based on a solid support to which a functional group-hydrophilic substance is bound;

(2) covalently bonding a hydrophobic substance to the 5 'terminus of the RNA single strand bound with the functional group-hydrophilic substance;

(4) separating the functional single-stranded RNA-polymer structure and a separately synthesized single strand of complementary sequence RNA from the solid support;

(5) binding the ligand to the end of the hydrophilic material through the functional group;

(6) RNA double strand is formed through annealing to single strand of the complementary sequence of the RNA-polymer structure to which the ligand is bound;

Linked oligonucleotide construct comprising a double-stranded oligonucleotide.

More specific preferred examples include (1) binding a hydrophilic substance to a solid support (CPG) to which a functional group is bonded; (2) synthesizing a single strand of RNA in a solid support (CPG) to which a functional group-hydrophilic substance is bound; (3) covalently bonding a hydrophobic substance to the 5 'end of the single strand of RNA; (4) separating the functional group-RNA-polymer structure from the solid support (CPG) and a single strand of the complementary sequence separately synthesized when the synthesis is completed; (5) preparing an RNA-polymer structure having a ligand bound to the end of the hydrophilic substance using the functional group; (6) The RNA single strand of the sequence complementary to the prepared ligand-bound RNA-polymer structure may be prepared by annealing to prepare a double-stranded oligo RNA structure having a ligand bound thereto. After the step (6), when the preparation is completed, single strands of the RNA of the reactants, the RNA-polymer structure and the complementary sequence are separated and purified using High Performance Liquid Chromatography (HPLC) The molecular weight can be measured with a mass spectrometer to confirm that the desired RNA-polymer structure and RNA have been prepared. The step of synthesizing a single strand of RNA having a sequence complementary to that of the single strand of RNA synthesized in the above step (3) may be performed before the step (1) or during any one of steps (1) to (6) It can be done.

In yet another aspect,

As shown in the following formula (3), a double-stranded oligo-RNA oligonucleotide comprising a double-stranded oligo RNA sense strand conjugated with a hydrophilic substance, a ligand to a hydrophilic substance and a hydrophobic substance at the 3'- RNA structure, and a method for producing the same

(1) synthesizing a single strand of RNA based on a solid support to which a functional group is bonded;

(2) covalently bonding a hydrophilic material to the material obtained in step (1);

(3) covalently bonding a ligand to the material obtained in step (2);

(4) separating the material obtained in step (3) from the solid support;

(5) covalently bonding the material obtained in step (4) to a hydrophobic material through a functional group bound to the 3 'terminus;

(6) annealing a single strand of complementary sequence RNA with the material obtained in step (5) to form double strands;

Linked oligonucleotide construct comprising a double-stranded oligonucleotide.

More specific preferred examples include (1) synthesizing a single strand of RNA based on a solid support (CPG) to which a functional group is bonded; (2) covalently bonding a hydrophilic substance to the 5 'end of the single strand of RNA; (3) synthesizing a functional group-RNA-hydrophilic polymer structure in which a ligand is bound to a single-stranded hydrophilic substance of the RNA; (4) separating the ligand-bound functional group-RNA-hydrophilic polymer structure from the solid support (CPG) upon completion of synthesis; (5) synthesizing an RNA-polymer structure to which a ligand is bound by binding a hydrophobic substance through a functional group; And (6) the RNA single strand of the sequence complementary to the prepared RNA-polymer structure may be prepared by annealing to prepare a double-stranded oligo-RNA-polymer structure.

After the step (5), when the preparation is completed, the reaction product is purified using High Performance Liquid Chromatography (HPLC), and the molecular weight is measured by a MALDI-TOF mass spectrometer to obtain an RNA- And whether the RNA was prepared. The step of synthesizing an RNA single strand having a sequence complementary to that of the RNA single strand synthesized in the above step (1) may be performed before (1) or during any of (1) to (6) It can be done.

In another embodiment, a process for producing a double-stranded oligo RNA structure in which a double-stranded oligo RNA sense strand and a ligand in which a hydrophilic substance or a hydrophobic substance is bound to the 5'-end of the antisense strand, silver

X and Y are independently a simple covalent bond or a linker-mediated covalent bond; S is a sense strand of a double-stranded oligo RNA, and AS is a hydrophobic substance, Is an antisense strand of double-stranded oligo RNA; L is a ligand with specific binding properties to receptors that promote target cell internalization through receptor-mediated endocytosis (RME) it means.)

(1) synthesizing a single strand of RNA based on a solid support;

(2) covalently bonding a hydrophilic substance to the 5 'end of the single strand of RNA;

(3) binding a ligand to a single strand of the RNA with a hydrophilic material;

(4) separating a single strand of the RNA-hydrophilic polymer structure having the ligand from the solid support and the RNA-hydrophobic polymer structure of the separately synthesized complementary sequence from the solid support;

(5) annealing the RNA-hydrophobic polymer structure having a complementary sequence with the ligand-bound RNA-hydrophilic polymer structure to form double strands;

A method for preparing a double-stranded oligo RNA structure,

In the middle of the steps (1) to (4), a single strand of RNA consisting of a single strand and a complementary sequence of RNA of step (1) is synthesized and then a hydrophobic substance is covalently bonded to form an RNA- hydrophobic polymer structure And a step of synthesizing a single strand.

In addition, the present invention provides nanoparticles composed of the double-stranded oligo RNA structure to which the ligand is bound, and nanoparticles composed of the therapeutic drug-polymer structure to which the ligand is bound.

The nanoparticles are formed by the interaction between the double-stranded oligo RNA constructs to which the ligand of the present invention is bound. The structure of nanoparticles is described in detail. The hydrophobic substance is located at the center of the nanoparticles. The double helix RNA is protected by the external hydrophilic substance, and forms a nanoparticle in which the ligand is located on the surface of the nanoparticle 1). The nanoparticles have intracellular delivery of double-stranded oligo RNA through the ligand, which has an improved cell transfer efficiency and can be applied for the treatment of diseases. More specific structures of the structure and characteristics of the nanoparticles composed of the structure, cell transfer efficiency and effect will be described in more detail in the following examples.

The present invention also provides a method for gene therapy using nanoparticles composed of a double-stranded oligo RNA structure or a ligand-conjugated therapeutic drug-polymer structure to which the ligand is bound.

Specifically, the present invention provides a therapeutic method comprising the steps of preparing nanoparticles composed of a double-stranded oligo RNA structure to which the ligand is bound, and injecting the nanoparticles into the body of an animal.

The present invention also provides a pharmaceutical composition comprising a pharmaceutically effective amount of a nanoparticle composed of the double-stranded oligo RNA construct to which the ligand is bound.

The composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. The pharmaceutically acceptable carrier should be compatible with the active ingredient of the present invention and may be prepared by mixing and using at least one of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, And other conventional additives such as antioxidants, buffers, bacteriostats and the like may be added as necessary. In addition, a diluent, a dispersant, a surfactant, a binder, and a lubricant may be additionally added to prepare a formulation for injection, such as an aqueous solution, a suspension or an emulsion. Further, it can be advantageously formulated according to the individual diseases or according to each disease, using methods disclosed in the art or by methods disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

The pharmaceutical compositions of the present invention can be routinely determined by one of ordinary skill in the art based on the patient's symptoms and the severity of the disease. It may also be formulated into various forms such as powders, tablets, capsules, liquids, injections, ointments, syrups and the like, and may be provided in unit-dose or multi-dose containers such as sealed ampoules and bottles.

The pharmaceutical composition of the present invention can be administered orally or parenterally. The route of administration of the pharmaceutical composition according to the present invention may be, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intradermal, intracardiac, transdermal, subcutaneous, intraperitoneal, , Sublingually or topically.

For such clinical administration, the pharmaceutical composition of the present invention can be formulated into a suitable formulation using known techniques. The dosage of the composition of the present invention may vary depending on the patient's body weight, age, sex, health condition, diet, administration time, method, excretion rate, severity of disease, and the like, You can decide.

The present invention relates to the combination of hydrophilic and hydrophobic materials that facilitate the delivery of double helix oligo RNA, which can be useful for the treatment of cancer and infectious diseases, using simple covalent or linker-mediated covalent bonds Ligand-double-stranded oligonucleotide construct conjugated with a ligand having a receptor-mediated endocytosis (RME) characteristic that specifically promotes internalization of cancer cells specifically to a target-specific double-stranded oligonucleotide RNA structure, And a technique for transferring double helix oligo RNA specifically to a receptor expressed on a cell surface through a double helical oligo RNA structure having the ligand bound thereto. When the target specific ligand is bound to the double helix oligo RNA structure, the nanoparticle composed of the duplex helical oligo RNA structure to which the ligand is bound can be efficiently transferred to the target cell. Therefore, Even when the oligonucleotide construct is administered, the double-stranded oligonucleotide RNA can be expressed in the target cell. In addition, ligand binding inhibits the transfer of nonspecific double-stranded oligo RNA to other organs and cells, thus being a tool for the treatment of double-stranded oligo RNA for various diseases, as well as a new type of double-stranded oligonucleotide RNA delivery system Can be usefully used.

Figure 1 is a schematic diagram of a nanoparticle (SAMiRNA) formed by a double-stranded oligo RNA construct with ligands bound thereto
Figure 2 shows tumor specific delivery of SAMiRNA. (A) SAMiRNA labeled with Cy5.5 in tumor-transplanted mice was administered to tail vein at a dose of 5 mg / kg body weight, followed by the in vivo distribution of SAMiRNA biodistribution (red dotted line is the site of tumor implantation); (B) ex vivo photograph of SAMiRNA obtained after 48 hours from the administration of SAMiRNA.
FIG. 3 is a diagram showing NMR analysis results of 1,3,4,6-tetraacetyl- N -acetylgalactosamine (1,3,4,6-Tetraacetyl-NAG) (Compound A). ^{(1 H NMR (300 MHz,} DMSO-D6); δ7.89 ppm (1H, d, J = 9.3 Hz), 5.64 ppm (1H, d, J = 8.7 Hz), 5.27 ppm (1H, d, J = 3.3 Hz), 4.17-3.96 ppm (2H, m), 2.12 ppm (3H, s), 5.07 ppm (1H, dd, J = 2.04 ppm (3H, s), 1.99 ppm (3H, s), 1.91 (3H, s), 1.78 ppm (3H, s)
Of acetyl galactosamine (3,4,6-Triacetyl-1-hexa (ethylene glycol) -NAG) ( compound B) - 4 3,4,6- triacetyl-1-hexa (ethylene glycol) - N NMR analysis. Fig. ^{1 H NMR (300 MHz, DMSO} -D 6); d, J = 11.1, 3.0 Hz), 4.56 ppm (1 H, d, J = 3.0 Hz), 4.97 ppm (1H, J = 8.7 Hz), 3.88 ppm (1H, q, J = 8.7Hz), 3.83-3.74ppm (1H, m), 3.62-3.39ppm (25H, m), 2.10ppm (3H, 3H, s), 1.89 ppm (3H, s), 1.77 ppm (3H, s)
5 is a diagram showing NMR analysis results of 1-hexa (ethylene glycol) -N -acetylgalactosamine-phosphoamidite (1-Hexa (Ethylene Glycol) -NAG-Phosphoramidite) (Compound C). ^{(A) 1 H NMR (300} MHz, DMSO-D 6); (1H, d, J = 3.0 Hz), 4.97 ppm (1H, dd, J = 11.1, 3.6 Hz), 4.56 ppm = 8.1 Hz), 3.88 ppm (1H, d, J = 9.0 Hz), 3.81-3.41 ppm (30H, m), 2.89 ppm (2H, t, J = 5.7 Hz), 2.11 ppm ppm (3H, s), 1.89 ppm (3H, s), 1.77 ppm (3H, s), 1.20-1.12 ppm (12H, m), (B) 31 P NMR data (121 MHz, DMSO-D 6); [delta] 147.32 ppm.
6 is a diagram showing a process for producing a single strand of RNA;
FIG. 7 shows a schematic diagram of a double-stranded oligo RNA construct having a double-stranded oligo RNA having a 5'end at its 5'-end, and (A) PEG-phosphoramidite (B) a result showing the result of mass analysis (MALDI-TOF MS) of a single-stranded RNA (21mer) having no 5'terminal modification (SEQ ID NO: 1, MW 6662.1) (MALDI-TOF MS) of single-stranded RNA (21mer) to which PEG is attached at the 5 'end (SEQ ID NO: 1, MW 8662.1).
FIG. 8 shows the results of the reaction formula for the preparation of the mono-NAG-PEG-RNA structure and the result of the preparation of (A) N -acetylgalactosamine-phosphoramidite to N -acetylgalactosamine (MALDI-TOF MS) of NAG-PEG-RNA (blue, MW 9171.2) structure with N -acetylgalactosamine bound to PEG-RNA (green, MW 8624.1) The results show that the central peak moves by the molecular weight (MW 547) of N -acetylgalactosamine.
FIG. 9 shows the results of the reaction scheme and the result of the preparation of the triple-NAG-PEG-RNA structure, (A) using dendrimer phosphoramidite and N -acetylgalactosamine-phosphoramidite the PEG-RNA N - the process of combining the lactose samin go acetyl, (B) PEG-RNA ( green, MW 8624.1) , and N - the mono-NAG-PEG structure Lactobacillus samin combined go acetyl (blue, MW 9171.2), and (MALDI-TOF MS) results of a triple-NAG-PEG-RNA construct (red, MW 10630).
FIG. 10 shows a reaction scheme for preparing a 5'-Folate-PEG-RNA structure and (A) a process for binding folate to PEG-RNA through NHS-Folate, (B) (MALDI-TOF MS) of the folate-PEG-RNA structure (blue, MW 9277.8) conjugated with folate and the central peak shifted by the molecular weight of the folate (MW 615) As a result of confirming the drawing.
11 is a 5 'produced a result of ₂₄ C -RNA structure, (A) SEQ ID No. 1 and mass spectrometry of single-stranded RNA of complementary sequence (MALDI-TOF MS) results figure (MW 7349.5), (B) SEQ ID NO: Mass analysis (MALDI-TOF MS) of the single stranded 5 'C ₂₄ -RNA structure of sequence 1 and complementary sequence (MW 7830.2).
12 is a diagram showing a process for producing a 3 'CPG-amine-PEG-RNA structure through an amine-CPG;
13 is a diagram showing a process for producing a 3 'CPG-amine-PEG-RNA-C ₂₄ structure through an amine-CPG;
FIG. 14 shows a reaction scheme for preparing a 3 'Folate-PEG-RNA structure and (A) a process for binding folate to a 3' amine-PEG-RNA structure via NHS-Folate, (MALDI-TOF MS) of the 3 'Folate-PEG-RNA structure (SEQ ID NO: 1, MW 9277.7).
15 is a graph showing the results of physical property analysis of a 5 'Folate-RNA-polymer structure nanoparticle (Folate-SAMiRNA), (A) a Folate-SAMiRNA size and polydisperse index (PDI) ) Critical Micelle Concentration of Folate-SAMiRNA
16 shows the effect of Folate-SAMiRNA on the inhibition of target gene expression inhibition in a folate receptor overexpressing cell line. In the case of folate receptor (Folate Receptor) overexpressing KB tumor cells, Folate-SAMiRNA and SAMiRNA with or without folate were treated under conditions that did not contain folate and excessive folate The amount of mRNA of the target gene in the tumor cell line was measured by qPCR at 48 hours after the treatment. (Without folate in culture medium, conditions without folate with folate in culture medium, condition with excess (1 mM) folate), double-stranded oligo RNA-polymer of SEQ ID NO: 2 SAM, an experimental group treated with SAMiRNA-Con, a nanoparticle composed of a structure, and Folate-SAM, an experimental group treated with SAMiRNA-Sur, a nanoparticle composed of a double-stranded oligo RNA-polymer structure consisting of SEQ ID NO: 1 Folate ligand-bound Folate-Folate-SAMiRNA-Sur nanoparticle composed of a double-stranded oligo-RNA structure (SEQ ID NO: 1)
FIG. 17 is a graph showing the inhibitory effect of Folate-SAMiRNA on tumor gene expression in tumor tissues. FIG. SAMiRNA and Folate-SAMiRNA were administered to rats with tumors composed of KB tumor cells, a cell line overexpressing folate receptor (Folate receptor), in an amount of 5 mg / Kg body weight, followed by 48 hours or 72 hours At one point, the amount of target gene (survivin) mRNA in tumor tissue is measured by qPCR. SAMiRNA-Sur-administered Folate-SAMiRNA, which is a nanoparticle composed of a double-stranded oligo RNA-polymer structure consisting of PBS, negative control SAMiRNA and a ligand-free survivin sequence, SEQ ID NO: 1, folate ligand SAMiRNA-Sur, which is a nanoparticle composed of SEQ ID NO: 1,

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood, however, that these embodiments are provided for purposes of illustration only and are not to be construed as limiting the scope of the present invention as defined by the appended claims. will be.

Example 1. Preparation of a bondable ligand material

A ligand material capable of binding to a double stranded oligo RNA structure was prepared to prepare a double stranded oligo RNA structure having a ligand bound thereto.

Example 1-1. 1-hexa (ethylene glycol) - N -Acetylgalactosamine-phosphoamidite (1-Hexa (Ethylene Glycol) - N -Acetyl Galactosamine-Phosphoramidite) Reagents (Compound A, B, C)

N-acetyl-galactosamine (N -Acetyl Galactosamine, NAG) the double-helix oligonucleotide as shown below to bind to RNA structure Scheme I 1- hexa (ethylene glycol) - N-acetyl-galactosamine-phosphoramidite Deet (1-Hexa (Ethylene Glycol) -NAG-Phosphoramidite).

Scheme I

Example 1-1-1. Preparation of 1,3,4,6-tetraacetyl-NAG (1,3,4,6-Tetraacetyl-NAG) (Compound A)

Starting material Galactosamine hydrochloride (Sigma Aldrich, USA) (2 g, 9.27 mmol), acetonitrile (Samseon, Korea) (31 mL), triethylamine (Sigma Aldrich, USA) 15.42 ml, 111.24 mmol) were mixed and refluxed for 1 hour. The mixture was cooled slowly to room temperature, cooled to 0 캜 with ice water, and acetic anhydride (Sigma Aldrich, USA) (8.76 mL, 92.70 mmol) was added dropwise over 10 minutes. After the addition was finished, the ice was removed and stirred at room temperature for 24 hours. After the reaction was completed, the mixture was slowly added to the aqueous solution with saturated aqueous sodium bicarbonate (Samjeon, Korea) until the pH became neutral. After the pH became neutral, the mixture was stirred at room temperature for 2 hours. The resultant solid was filtered and the filtrate was washed with ethyl acetate (100 mL 2), distilled water (100 mL 2), ethyl acetate (100 ml x 1). The solid was vacuum-dried to obtain 1,3,4,6-tetraacetyl- N -acetylgalactosamine (1.82 g, 52.3%) (see FIG. 3).

Example 1-1-2. Triacetyl-1-hexa (ethylene glycol) - N -Acetylgalactosamine (3,4,6-Triacetyl-1-hexa (ethylene glycol) -NAG) (Compound B)

Tetraacetyl- N -acetylgalactosamine (1.81 g, 4.82 mmol) obtained in Example 1-1-1 and iron Ⅲ chloride (Sigma Aldrich, USA) (1.02 g , 6.27 mmol) and methylene chloride (Samjeon, Korea) (48 ml) were mixed and stirred at room temperature. After stirring for 10 minutes, hexa (ethylene glycol) (Hexa (ethylene glycol), Sigma Aldrich, USA) (1.58 ml, 4.82 mmol) was added thereto and refluxed for 2 hours. When the reaction was completed, it was filtered using Celite (Sigma Aldrich, USA) and the filtrate was washed with methylene chloride (50 ml x 2). The filtrate was concentrated under reduced pressure, ethyl acetate (100 ml) and distilled water (100 ml) were added, and the water layer was extracted. The obtained water layer was extracted with methylene chloride (100 ml × 3), and the organic layer was collected and dried over anhydrous magnesium sulfate (Magnesium sulfate anhydride, Samseon, Korea) and filtered. The filtrate was concentrated under reduced pressure and vacuum dried to obtain 3,4,6-triacetyl-1-hexa (ethylene glycol) -N -acetylgalactosamine (2.24 g, 74.9%) (see FIG. 4).

Example 1-1-3, 1-hexa (ethylene glycol) - N -Acetylgalactosamine-1-Hexa (Ethylene Glycol) -NAG-Phosphoramidite) (Compound C)

Methylene chloride (37 ml) and triethylamine (0.94 ml, 6.75 mmol) obtained in Example 1-1-2 (2.22 g, 3.71 mmol) were mixed and stirred at room temperature. After stirring for 10 minutes, 2-cyanoethyl N, N - diisopropyl-chloro-phosphoramidite Deet (2-Cyanoethyl N, N -diisopropylchlorophosphoramidite, Sigma Aldrich, USA) (0.75 ㎖, 3.38 mmol) by putting 45 minutes Lt; / RTI > After completion of the reaction, the reaction mixture was concentrated under reduced pressure, ethyl acetate (100 ml) and distilled water (100 ml) were added, and the organic layer was extracted. The obtained organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure and separated by a column to obtain 1-hexa (ethylene glycol) -N -acetylgalactosamine-phosphoramidite (1.14 g, 42.2%) (see FIG.

Examples 1-2. Production of NHS-Folate

NHS-Folate was prepared as shown in Scheme II below to bind the folate to the double stranded RNA structure.

Reaction Scheme II

The starting material, folic acid (Folic acid, Sigma Aldrich, USA) (3 g, 6.8 mmol) , dimethyl sulfonic Foxy cargo (Dimethyl sulfoxide, Sigma Aldrich, USA) (60 ㎖), N - hydroxy-succinimide (N -Hydroxysuccinimide, (Sigma Aldrich, USA) (0.86 g, 7.5 mmol) and 1,3-dicyclohexylcarbodiimide (Sigma Aldrich, USA) (1.54 g, 7.5 mmol) were mixed and stirred at room temperature for 18 hours . When the reaction was completed, the reaction mixture was added dropwise to a solution (950 ml) of ethyl acetate: n-hexane (n-hexane, Samseon, Korea) at a ratio of 3: 5 over 10 minutes to obtain 3.79 g of solid NHS-Folate (Robert J. Lee and Philip S. Low (1994) J. Biological Chemistry. 269: 3198-3204).

Examples 1-3. Production of Peptides

Since the peptide compounds contain an alpha-amino acid, the binding reaction between the peptide derivative having such a structure and the amine functional group existing in PEG was carried out. As a precondition for this coupling reaction, the amine functional groups present in PEG and peptide derivatives require a first step of substituting the amine functional groups present in the peptide compound with a protecting group, since they interact with each other in the binding reaction with the carboxylic acid present in the peptide . The amine group present in the peptide compound is replaced by a protecting group of 9-fluorenylmethyloxycarbonyl (Fmoc) or tert-butyloxycarbonyl (t-BOC) A peptide compound having a form capable of binding to PEG by removing reactivity with a carboxylic acid was prepared.

Example 2. Preparation of double-stranded oligo RNA construct with ligand bound at the 5 'end

The bindable ligand material prepared in Example 1 was formed in the form of phosphoramidite as in NAG-phosphoramidite of Example 1-1, and was subjected to a general chemical oligo synthesis process (blocking removal (a process consisting of deblocking, coupling, capping and oxidation), or in the form of an NHS-ligand like the NHS-Folate of Example 1-2, the amine group may be combined and then coupled in the form of a PEG-RNA ligand bound at the 5 'end through an ester reaction with an amine PEG. The PEG-RNA structure with the ligand bound to the synthesized 5'-end was annealed with a 5'C ₂₄ -RNA structure in which a hydrophobic residue of a complementary sequence was bound to form a double-stranded oligo RNA structure was synthesized.

Example  2-1. Double spiral up RNA Manufacturing

In the following examples, a sustained double-stranded oligo RNA was used to suppress survivin. Survivin is a protein commonly expressed in most neoplastic tumors or transgenic cell lines tested so far and is expected to be an important target in chemotherapy (Survivin: a new target for anti-cancer therapy. Cancer Treat Rev. 2009 Nov; 35 (7): 553-62). The double stranded oligo RNA of the present invention is composed of the sense strand described in SEQ ID NO: 1 and the antisense strand complementary thereto, and the double stranded oligo RNA used as a control is a sense strand described in SEQ ID NO: Strand and an antisense strand complementary thereto. The double stranded oligo RNA used in this example is composed of the following base sequences.

(SEQ ID NO: 1) 5'-AAG GAG AUC AAC AUU UUC A-3 '

(SEQ ID NO: 2) 5'-CUU ACG CUG AGU ACU UCG A-3 '

The double-stranded oligonucleotide was prepared by ligating phosphodiester bonds forming a RNA backbone structure using? -Cyanoethyl phosphoramidite (? -Cyanoethyl phosphoramidite) (Polymer support oligonucleotide synthesis XVIII: use of β-cyanoethyl- N, N- dialkylamino- / N- morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product Nucleic Acids Res. 11; 12 (11): 4539-57).

The synthesis process starts from a nucleoside bonded solid support (CPG) phase and repeats cycles consisting of deblocking, coupling, capping and oxidation , And the RNA of the desired sequence is obtained. The synthesis of the corresponding single strand of RNA was carried out using an RNA synthesizer (384 Synthesizer, BIONEER, Korea) (see FIG. 6).

Example 2-2. Preparation of 5 'PEG-RNA construct

The RNA synthesized in Example 2-1 was treated with a PEG phosphoramidite reagent to prepare a 5'PEG-RNA structure through a general RNA synthesis process (see FIG. 7).

Examples 2-3. Synthesis of PEG-RNA with Ligand Bound at the 5 '

Example 2-3-1. N - Preparation of NAG-PEG-RNA structure using NAG phosphoramidite

The N -acetylgalactosamine phosphoramidite reagent synthesized in Example 1-1 was added to the 5'PEG-RNA structure synthesized in Example 2-2 to synthesize N -acetyl Galactosamine was phosphodiester bonded to the PEG-RNA construct. The N - acetylgalactosamine ligand can bind a single molecule or more molecules of N - acetylgalactosamine to PEG using a dendrimer linker.

Example 2-3-1-1. Preparation of mono NAG-PEG-RNA structure

Using acetyl galactosamine phosphoramidite Deet (NAG phosphoramidite) reagent through the normal RNA synthesis N - - Example 2-2 synthesized in Example 1-1, the synthesized PEG-RNA structure in the N-acetyl galactosyl go The 5'mono-NAG-PEG RNA structure was synthesized by phosphodiester-binding to the PEG-RNA structure (see FIG. 8).

Example 2-3-1-2. Preparation of triple NAG-PEG-RNA structure

The PEG-RNA structure synthesized in Example 2-2 was synthesized with a Dendrimer Phosphoramidite (Trebler Phosphoramidte, Glen Research, USA) reagent, and then the NAG phosphoramidite reagent synthesized in Example 1-1 was used Through the general RNA synthesis process, a 5-lipo-NAG-PEG-RNA structure was synthesized by phosphodiester linkage of 3 NAGs to the PEG-RNA construct (see FIG. 9).

Example 2-3-2. Preparation of Folate-Polyethylene Glycol-RNA (Folate-PEG-RNA) Structure

PEG-RNA structure synthesized in Example 2-2 was subjected to general RNA synthesis using an amine phosphoramidite reagent to convert an amine group into a PEG-RNA structure by phosphodiester linkage to form an amine-PEG-RNA structure Were synthesized. A 5'-Folate-PEG-RNA structure was synthesized through an ester reaction of the NHS-Folate synthesized in Example 1-2 in the synthesized amine-PEG-RNA structure (see FIG. 10).

Example 2-3-3. Amine modification and preparation of peptide-PEG-RNA constructs through peptide compounds

Binding reaction of the protected peptide compound prepared in Example 1-3 and the carboxyl group of the peptide to the amine group of PEG of the amine-PEG-RNA structure of Example 2-3-2 was performed by using BOP (Benzotriazole-1-yl-oxy-tris - (dimethylamino) -phosphonium hexafluorophosphate, Sigma Aldrich, USA) and HOBT (1-Hydroxybenzotriazole, Sigma Aldrich, USA). After the binding reaction proceeded, the 5 'peptide-PEG-double helix oligo RNA construct was prepared by treating with piperidine (Sigma Aldrich, USA) to remove the protecting group.

Examples 2-4. 5'C ₂₄ Preparation of RNA Structures

The RNA structure of the sequence complementary to the PEG-double-stranded oligo RNA structure sequence to which the 5 'ligand of Example 2-3 was ligated was synthesized through the RNA synthesis method of Example 2-1, and the carbon containing the disulfide bond C ₂₄ was bound to the RNA structure by phosphodiester bond through a general RNA synthesis process using several 24 tetradocosane reagents to synthesize a 5'C ₂₄ -RNA structure (see FIG. 11).

Examples 2-5. Recovery reaction and annealing

Single strands of RNA synthesized in Examples 2-1, 2-2, 2-3, and 2-4 were synthesized by treating 28% (v / v) ammonia in a water bath at 60 ° C The RNA and RNA constructs were detached from the CPG and the protection residues were removed via a deprotection reaction. The PEG-double helix oligo RNA structure and the 5'C ₂₄ -double helix oligo RNA structure in which the ligand is bonded at the 5 'terminal from which the protective residue has been removed are reacted with N -methylpyrrolidone ( N- methylpyrrolidone) Triethylamine and triethylamine trihydrofluoride were treated at a volume ratio of 10: 3: 4 to remove 2'TBDMS (tert-butyldimethylsilyl).

The reaction products were separated and purified by high performance liquid chromatography (HPLC), and their molecular weights were measured with a MALDI-TOF mass spectrometer (MALDI-TOF MS, SHIMADZU, Japan) It was confirmed that it was helical and matched with the RNA structure.

Then, the ligand-bound PEG-RNA sense RNA and the antisense RNA were mixed in the same volume to prepare a double-stranded oligo RNA structure with each ligand bound thereto, and the mixture was diluted with 1X annealing buffer (30 mM HEPES, 100 mM potassium acetate, acetate, pH 7.0 to 7.5), reacted in a constant temperature water bath at 90 ° C. for 3 minutes, and then reacted at 37 ° C. to prepare a double-stranded oligo RNA structure having a ligand bound to each 5'-end of each desired double strand. The double-stranded oligo RNA structure with the ligand bound to the prepared 5 'end was annealed through electrophoresis.

Example 3. Preparation of double-stranded oligo RNA construct with 3 ' terminal ligand bound

The amine-CPG was synthesized in the form of 3 'amine-PEG-RNA using PEG phosphoramidite reagent and then synthesized in NHS-ligand form like NHS-Folate of Example 1-2 A PEG-double helix oligo RNA structure with a ligand bound at the 3 'end can be synthesized through an ester reaction with the ligand material capable of binding. The PEG-duplex oligosaccharide structure having the ligand bound to the synthesized 3 'end is bound to a hydrophobic substance C ₂₄ to form a ligand-bound PEG-RNA-C ₂₄ at the 3' end, which is a complementary sequence RNA is annealed and synthesized as a double stranded oligo RNA structure with a ligand bound at the 3 'end.

Example 3-1. Preparation of 3 'amine-PEG-RNA structure

Amine-CPG was synthesized as a 3'CPG-amine-PEG through a general RNA synthesis process using a polyethylene glycol phosphoramidite reagent. 3'CPG-amine-PEG was synthesized as a 3'CPG-amine-PEG-RNA structure having the desired sequence RNA through the RNA synthesis process of Example 2-1 (see FIG. 12).

Example 3-2. 3 'amine-PEG RNA-C ₂₄ Fabrication of Structures

The embodiment 3'CPG-amine-PEG-RNA structure synthesized in Example 3-1 is a C ₂₄ to RNA through the normal RNA synthesis using a carbon number of 24-tetra-docosane acid (tetradocosane) reagents that contain a disulfide bond Phosphodiester bond to synthesize a structure (see Fig. 13).

Example 3-3. PEG RNA-C < RTI ID = 0.0 > ₂₄ Fabrication of Structures

The 3 'amine-PEG-RNA-C ₂₄ structure synthesized in Example 3-2 was synthesized by treating 28% ammonia in a water bath at 60 ° C. -PEG-double helix oligo RNA structure and 3 'amine-PEG-RNA-C ₂₄ structure were removed from the CPG and the protection residues were removed through a deprotection reaction. The residues removed are protected 3 'amine (amine) -PEG-RNA-C 24 N structure in the oven 70 ℃-methylpyrrolidone (N -methylpyrolidon), triethyl amine (triethylamine) and triethylaminetrihydrofluoride dihydro-fluoro Triethylamine trihydrofluoride was treated at a volume ratio of 10: 3: 4 to remove 2'TBDMS (tert-butyldimethylsilyl). Separating the 3 'amine (amine) -PEG-RNA-C 24 structure form NHS- ligand binding consisting of ligand available materials and ester (ester) via the reaction 3, the PEG-RNA-C ₂₄ structure the ligand is coupled to the terminal .

Example 3-3-1. Preparation of 3'Folate-PEG-RNA Structure

The 3 'amine-PEG-RNA-C ₂₄ structure synthesized in Example 3-2 was reacted with the NHS-Folate synthesized in Example 1-2 to form 3'Folate-PEG-RNA- C ₂₄ structure was synthesized (see Fig. 14).

Example 3-4. Preparation of complementary sequence RNA constructs

The RNA single strand of the sequence complementary to the ligand-bound PEG-RNA-C ₂₄ sequence at the 3 'end of Example 3-3 was synthesized through the RNA synthesis method of Example 2-1. The synthesized RNA single strands were treated with 28% ammonia in a water bath at 60 ° C to remove the synthesized RNA from the CPG and deprotection reaction to remove the protecting moiety. The residue is protected RNA is removed from the N 70 ℃ oven-methylpyrrolidone (N -methylpyrolidon), triethyl amine (triethylamine) and triethylaminetrihydrofluoride hydro fluoride (triethylamine trihydrofluoride) is 10: volume of 4: 3 To remove 2'TBDMS (2'tert-butyldimethylsilyl).

Examples 3-5. Annealing

RNA and a 3 'ligand-bound PEG RNA-C ₂₄ structure were separated and purified by high performance liquid chromatography (HPLC) (LC-20A Prominence, SHIMADZU, Japan) The molecular weight was measured with an analyzer (MALDI TOF-MS, SHIMADZU, Japan), and it was confirmed whether the base sequence to be synthesized and the PEG RNA-C ₂₄ structure to which the ligand was bonded at the 3 'end were compatible.

Then, the ligand-bound PEG-RNA sense RNA and the antisense RNA were mixed in the same volume to prepare a double-stranded oligo RNA structure with each ligand bound thereto, and the mixture was diluted with 1X annealing buffer (30 mM HEPES, 100 mM potassium acetate, acetate, pH 7.0 to 7.5), reacted in a constant temperature water bath at 90 ° C for 3 minutes, and then reacted at 37 ° C to prepare a double-stranded oligo RNA structure in which a ligand was bound to each 3 'end of each desired double strand. The double - stranded oligo RNA structure with the ligand bound to the 3 'end was annealed by electrophoresis.

Example 4. Nanoparticle Formation of a Ligand-Bound Double-stranded Oligo RNA Structure

The double-stranded oligo RNA structure having the ligand bound to the 5 'end synthesized in Examples 2 and 3 and the double-stranded oligo RNA structure having the ligand bound to the 3' end are hydrophobic mutual interactions between the hydrophobic substances bonded to the ends of the double- Thereby forming a nanoparticle, that is, a micelle composed of a double-stranded oligo RNA structure having a ligand bound thereto (see FIG. 1). The nanoparticle size and critical micelle concentration (CMC) and transmission electron microscope (TEM) analysis of the 5'-Folate-ligand-double helix oligo RNA structure synthesized in Example 2 .

Example 4-1. Measurement of Particle Size of 5'Folate-Double Helical Oligo RNA Structure

The size of the nanoparticles was measured through a zeta-potential measurement. Specifically, the 5'Folate-double helix oligo RNA structure was dissolved in 1.5 ml DPBS (Dulbecco's phosphate buffered saline) at a concentration of 50 μg / ml, and the size of the nanoparticles was measured with an ultrasonic disperser (Wiseclean, DAIHAN, Was homogenized (700 W; amplitude 20%). The size of the homogenized nanoparticles was measured with a zeta potential meter (Nano-ZS, MALVERN, UK). The refractive index for the material was 1.459 and the absorption index was 0.001. salian) at 25 캜 and a viscosity of 1.0200 and a refractive index of 1.335 were measured. One measurement consisted of a size measurement consisting of 20 iterations, which was repeated three times.

It was confirmed that the nanoparticle (Folate-SAMiRNA) comprising the double-stranded oligo RNA structure conjugated with folate had a size of about 100 to 200 nm. The PDI value of Folate-SAMiRNA was measured to be less than 0.4, and it was found that polydisperse index (PDI) was relatively uniform. Could know. The size of the nanoparticles composed of the structure was found to be a proper size for ingesting into the cells through endocytosis (Nanotoxicology: nanoparticles reconstruct lipids. Nat Nanotechnol. 2009 Feb; 4 (2): 84-5).

Example 4-2. Determination of critical micelle concentration of double helix oligo RNA construct

An amphiphile containing a hydrophilic group and a hydrophilic group in a single molecule can be a surfactant. When the surfactant is dissolved in an aqueous solution, the hydrophobic part is collected to the center to avoid contact with water, and the hydrophilic part is directed to the outside Thereby forming micelles. At this time, the concentration at which the micelle is first formed is referred to as a critical micelle concentration (CMC). The method of measuring CMC using fluorescent dyes is based on the property that the slope of the curve of the fluorescence intensity curve of the fluorescent dye rapidly changes before and after the micelle formation.

To measure the critical micelle concentration of nanoparticles (Folate-SAMiRNA) composed of folate-bound double-stranded oligo RNA constructs, 0.04 mM DPH (1,6-Diphenyl-1,3,5-hexatriene, Sigma Aldrich, USA). 1 nmole / ㎕ of 5'Folate-double-stranded oligosaccharide structure synthesized in Example 2 was diluted stepwise with DPBS at a concentration of 0.0977 / / ml to a maximum concentration of 50 / / ㎖, and a total of 180 의 of 5'Folate- Prepare a sample of oligo RNA construct. 20 μl each of 0.04 mM DPH as a control and DPH as a solvent for methanol were added to the prepared samples and mixed well. The mixture was thoroughly mixed with the nanoparticles through an ultrasonic disperser (Wiseclean, DAIHAN, Korea) The size was homogenized (700 W; amplitude 20%). The homogenized sample was reacted for 24 hours at room temperature under light, and fluorescence (excitation: 355 nm, emission: 428 nm, top read) was measured. Since the measured fluorescence values identify the relative fluorescence values, the relative fluorescence values (fluorescence values of samples containing DPH - fluorescence values of samples containing only methanol) at the same concentration were obtained (Y axis) (X-axis) of the " Folate-double-stranded oligo RNA structure " concentration (see Fig. 2 (B)).

Fluorescence values measured at each concentration are rapidly increased while moving from a low-concentration section to a high-concentration section. The concentration of such a rapidly increasing point becomes CMC concentration. Therefore, the low-concentration section in which the fluorescence amount does not increase and the high-concentration section in which the fluorescence amount is increased are divided into several points, the trend line is drawn, and the X value of the intersection point where the two trend lines meet becomes the CMC concentration (FIG. 14, - It was observed that CMC of double stranded oligonucleotide construct was very low at 1.33 ㎍ / ㎖, and it was confirmed that Folate-SAMiRNA can form micelle well even at very low concentration.

Example 4-3. Transmission electron microscopy of double helix oligo RNA structure

The morphology of the nanoparticles composed of the folate-double helix oligo RNA structure was observed through a transmission electron microscope (TEM).

Specifically, the folate-double-stranded oligosaccharide structure was dissolved in DPBS (Dulbecco's Phosphate-Buffered Saline) to a final concentration of 100 ng / ml, and the size of the nanoparticles was homogenized with an ultrasonic disperser (Wiseclean, DAIHAN, W; amplitude 20%). Nanoparticles composed of folate-double helix oligo RNA constructs were observed through negative staining with a high electron density material. It was confirmed that the nanoparticles observed through a transmission electron microscope (TEM) were well formed to a size similar to that of the nanoparticles measured in Example 4-1.

Example 5. Construction of double-stranded oligo RNA constructs conjugated with ligands in vitro  delivery

In order to confirm the efficacy of the improved double-stranded oligo RNA in vitro of the 5 'folate-double helical oligo RNA structure synthesized in Example 2 (Folate-SAMiRNA), the folate- receptors are overexpressed by treating the KB cell line in the absence of transfection material depending on whether folate is present or not, thereby improving the efficiency of SAMiRNA delivery and inhibiting the expression of the target gene according to the ligand bound thereto The effect was confirmed.

Example 5-1. Culture of tumor cell lines

The human oral carcinoma cell (KB) strain obtained from the American Type Culture Collection (ATCC) was inoculated into RPMI-1640 culture medium (Gibco, USA) without folate (10% (v / v) 100 units / ml of serum, penicillin, and 100 占 퐂 / ml of streptomycin were added and cultured at 37 占 폚 and 5% (v / v) CO ₂ .

Example 5-2. Transformation of tumor cell line through Folate-SAMiRNA

The tumor cell line (1.3 × 10 ⁵ / well) cultured in Example 5-1 was cultured on a 6-well plate for 18 hours in RPMI-1640 culture medium without folate under condition 5-1 of the above example After the culture, the medium was removed, and the same amount of Opti-MEM medium per well was dispensed.

(SAMiRNA-Sur) comprising a double-stranded oligo RNA structure comprising SEQ ID NO: 1 which is a double-stranded oligo RNA sequence which inhibits the expression of survivin as a target gene, a double-stranded oligonucleotide comprising SEQ ID NO: 2 of the control sequence (Folate-SAMiRNA-Sur) composed of a nanoparticle (SAMiRNA-Con) composed of a helical oligo-RNA polymer structure and a double helical oligo RNA structure having a folate ligand binding to SEQ ID NO: 4-1 were added to DPBS at a concentration of 50 μg / ml in the same manner, and the nanoparticles composed of each structure were prepared by homogenization through sonication.

In order to form an excessive amount of folate in the Opti-MEM medium, additional folate is added to form a condition containing the final 1 mM folate and no additional folate. The samples were then treated at a concentration of 200 nM and cultured for 48 hours at 37 ° C and 5% (v / v) CO ₂ .

Example 5-3. Relative quantitative analysis of mRNA of survivin gene

The total RNA was extracted from the transfected cell line in Example 5-2 to synthesize cDNA, and then the amount of mRNA expression of survivin was measured using real-time PCR (Real-time PCR) Relative quantification was carried out according to the method of Publication No. 2009-0042297.

SAMiRNA-Con is an experimental group treated with a nanoparticle composed of a double-stranded oligo RNA structure of SEQ ID NO: 2, which is a control sequence, and SAMiRNA-Sur is an experimental group of a nanoparticle composed of a double stranded oligo RNA structure of SEQ ID NO: 1 . Folate-SAMiRNA-Sur is an experimental group treated with a folate-double-stranded oligo RNA structure in which the folate ligand of SEQ ID NO: 1, which is a survivin sequence, is bound.

The degree of target mRNA expression inhibition was calculated by relative quantitation relative to the amount of target gene expression of SAMiRNA-Sur and Folate-SAMiRNA-Sur-treated samples for SAMiRNA-Con-treated samples 16).

In the presence of excessive folate in the medium, the Folate Receptor of the KB cell line was saturated with excessive folate and inhibited the promotion of cell internalization by the folate ligand bound to SAMiRNA And it is expected that it will play a crucial role in inhibiting the mRNA expression of the target gene by affecting the cell delivery efficiency of SAMiRNA.

In the case of treatment with SAMiRNA-Sur, there was no significant difference in the presence or absence of folate and the expression inhibition effect of the target gene. However, in the case of treatment with Folate-SAMIRNA-Sur, in the absence of folate It was confirmed that the inhibition of the target gene expression was improved about twice as much as that in the case where folate was present in an excessive amount. That is, in the presence of an excessive amount of folate, there was no change in the effect of inhibiting the expression of the target gene due to folate ligand binding. However, in the absence of folate, the folate ligand Binding to the target gene was inhibited.

Therefore, it was confirmed that the nanoparticles composed of a double-stranded oligo RNA structure having a ligand bound thereto have improved transmission efficiency and an inhibitory effect on target gene expression in cells overexpressing the ligand receptor.

Example 6. Construction of a double-stranded oligo RNA construct conjugated with a ligand in vivo in vivo ) relay

In order to confirm the efficacy of the double-stranded oligo RNA in vivo conditions of the 5 'folate-double helix RNA structure synthesized in Example 2 (Folate-SAMiRNA), the folate receptor (Folate-SAMiRNA) and SAMiRNA were administered to rats having a tumor consisting of a KB cell line, which is a cell line overexpressing the Folate receptor, to confirm the inhibitory effect of the target gene in the tumor tissue.

Example 6-1. Preparation of KB xenograft model

KB cells cultured in Example 5-1 were subcutaneously injected into subcutaneous tissues of 5-week old nude mice (BALB / C nu) at 1 × 10 ⁶ cells / well. Tumor growth was observed by measuring the lengths of the major axis and minor axis at 2-day intervals after administration, and it was confirmed that the tumors grew at about 150 to 200 mm ³ at a point 2 weeks after administration.

Example 6-2. Administration of a double-stranded oligo RNA construct with ligand binding and a double-stranded oligo RNA construct with ligand binding

The double-stranded oligo RNA structure without ligand binding to the 5'Folate-double-stranded oligo-RNA structure and SEQ ID NO: 1 synthesized in Example 2 was added to the KB xenograft model prepared in Example 6-1, The nanoparticles composed of the double-stranded oligo RNA structure were homogenized through an ultrasonic dispersing machine. Homogenized nanoparticles were administered to the KB xenograft model (n = 4) via tail vein at a dose of 5 mg / kg body weight (n = 4) and at 48 or 72 hours after administration And the tumor tissues were collected. The collected tumor tissues were subjected to real-time PCR to synthesize cDNA by extracting total RNA, and the expression amount of survivin mRNA was relatively quantified by the method of Korean Patent Laid-Open Publication No. 2009-0042297 (See FIG. 17).

SAM is an experimental group to which nanoparticles composed of a double-stranded oligo RNA structure without ligand binding are administered. Folate-SAMiRNA is a group of folate-ligand binding Of the 5'-Folate-double helix oligo RNA construct.

The inhibition of target gene expression by SAMiRNA was prominent at 72 hours after the lapse of 48 hours. When the expression level of target gene was inhibited at 48 hours, the ligand-bound structure Folate-SAMiRNA) showed an improved efficiency of 160% and showed an improvement of 120% even after 72 hours.

Thus, the double-stranded oligo RNA construct with the folate ligand bound was rapidly transferred in vivo to tumor tissue over-expressing the target folate receptor (Folate receptor), resulting in an improved double-stranded oligo RNA effect, Can be maintained even after a lapse of time.

Claims (20)

A structure in which a double-stranded oligo RNA binds to a hydrophobic substance and a hydrophilic substance, and a ligand is bound to the hydrophilic substance as in the structure of the following formula (1), wherein the ligand is a peptide, folate, or N-acetylgalactosamine Structure.
LAXRYB Equation (1)
(Wherein A is a hydrophilic substance, B is a hydrophobic substance, X and Y are independently a simple covalent bond or a linker-mediated covalent bond, R is a double helical oligo RNA, L is a ligand, Refers to a ligand that binds specifically to receptors on the surface and facilitates cellular internalization of the construct through receptor-mediated endocytosis (RME).) delete delete delete The method according to claim 1,
Wherein said double helix oligo RNA is comprised of 19 to 31 nucleotides. The method according to claim 1,
Wherein the hydrophobic substance has a molecular weight of 250 to 1,000. The method according to claim 6,
The hydrophobic material may be selected from the group consisting of steroids, glycerides, glycerol ethers, polypropylene glycols, C ₁₂ to C ₅₀ unsaturated or saturated hydrocarbons, diacyl phosphatidylcholine A fatty acid, a phospholipid, a lipopolyamine, and the like. 8. The method of claim 7,
Wherein the steroid is selected from the group of cholesterol, cholestanol, cholic acid, cholestearyl formate, cortestanyl morpholate and cholestanyl amine. 8. The method of claim 7,
Wherein the glyceride is selected from the group consisting of mono-, di-, and tri-glycerides. The method according to claim 1,
Wherein the hydrophilic material has a molecular weight of 200 to 10,000. 11. The method of claim 10,
Wherein the hydrophilic material is selected from the group consisting of polyethylene glycol, polyvinylpyrolidone, and polyoxalzoline. The method according to claim 1,
Wherein said covalent bond is a non-degradable or degradable bond. 13. The method of claim 12,
Wherein the non-degradable bond is an amide bond or a phosphorylated bond. 13. The method of claim 12,
Wherein said degradable linkage is selected from the group of disulfide bonds, acid degradable bonds, ester bonds, anhydride bonds, biodegradable bonds, and enzymatically degradable bonds. delete A structure wherein a double-stranded oligo RNA binds to a hydrophobic substance and a hydrophilic substance and a ligand is bonded to the hydrophilic substance as shown in the following formula (4), wherein the ligand is a ligand that is a peptide, folic acid, or N-acetylgalactosamine As a method of manufacturing,

X and Y are independently a simple covalent bond or a linker-mediated covalent bond; S is a sense strand of a double-stranded oligo RNA, and AS is a hydrophobic substance, Is an antisense strand of double helix oligo RNA; L is a ligand that binds specifically to a cell surface receptor with a ligand to promote cell internalization of the construct through receptor-mediated endocytosis (RME) .)
(1) synthesizing a single strand of RNA in a solid support;
(2) covalently bonding a hydrophilic substance to the 5 'end of the single strand of RNA;
(3) binding a ligand to a hydrophilic substance bound to the single strand of RNA;
(4) separating a single-stranded RNA-hydrophilic polymer structure having a ligand bound thereto from a solid support and a RNA-hydrophobic polymer structure having a separately synthesized complementary sequence;
(5) annealing the RNA-hydrophobic polymer structure having a complementary sequence with the ligand-bound RNA-hydrophilic polymer structure to form double strands; And
In the middle of steps (1) to (4), a single strand of RNA consisting of a single strand of RNA and a complementary sequence of step (1) is synthesized and then a hydrophobic substance is covalently bonded to form an RNA- RTI ID = 0.0 > 1, < / RTI > comprising the step of synthesizing a single strand of the structure. delete A nanoparticle comprising the structure of claim 1. delete delete

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