WO2016204515A1 - Stat3 gene specific sirna, double-helical oligo rna structure including sirna, composition containing same, and use thereof - Google Patents

Abstract

The present invention relates to: a STAT3 gene specific siRNA; a highly efficient double-helical oligo RNA structure including the same; a pharmaceutical composition containing the same; and a use thereof and, more particularly, to: a STAT3 gene specific siRNA; a double-helical oligo RNA structure, which includes the same and has a structure in which hydrophilic materials and hydrophobic materials are bound to both ends of the double-helical RNA (siRNA), by using a simple covalent bond or a linker-mediated covalent bond, so as to be effectively transferred into a cell; nanoparticles capable of being produced, in an aqueous solution, by hydrophobic interactions between the double-helical oligo RNA structures; a method for producing the double-helical oligo RNA structure; and a pharmaceutical composition for preventing or treating cancers, especially breast cancer, skin diseases including psoriasis, or inflammatory diseases, containing the double-helical oligo RNA structure. According to the present invention, the STAT3 specific siRNA and the composition for treating cancers, skin diseases, or inflammatory diseases, containing the double-helical oligo RNA structure including the same can achieve a therapeutic effect on cancers, especially breast cancer, skin diseases including psoriasis, or inflammatory diseases by inhibiting the expression of STAT3 with high efficiency without causing side effects, and thus the present invention can be useful in treating breast cancer, skin diseases, or inflammatory diseases for which there currently is no suitable therapeutic agent.

Description

STAT3 gene specific siRNAs, double-stranded oligo RNA constructs comprising such siRNAs, compositions comprising them and uses thereof

The present invention relates to a STAT3 gene specific siRNA, a high efficiency double-stranded oligo RNA construct comprising the same, a pharmaceutical composition comprising the same, and a use thereof, and more particularly, to a STAT3 gene specific siRNA, including the same, and efficiently delivered into a cell. Double-stranded oligo RNA structure, an aqueous solution, in which a hydrophilic material and a hydrophobic material are bonded to both ends of a double-stranded RNA (siRNA) by using simple covalent bonds or linker-mediated covalent bonds. And to nanoparticles that can be produced by hydrophobic interaction of the double helix oligo RNA structures.

The present invention also relates to a method for producing the double helix oligo RNA construct, and a pharmaceutical composition comprising the double helix oligo RNA construct and use thereof.

Techniques for inhibiting gene expression are important tools in the development of therapeutics and target validation for the treatment of diseases. Among these techniques, it has been found that interfering RNA (RNA interference, hereinafter referred to as 'RNAi') acts on sequence specific mRNA in various kinds of mammalian cells since its role was discovered (Silence). of the transcripts: RNA interference in medicine.J Mol Med (2005) 83: 764-773). Once the long-stranded RNA double strand is delivered to the cell, the delivered RNA double strand is small interfering processed into 21 to 23 base pairs (bp) by an endonuclease called Dicer. RNA is converted into RNA, hereinafter called siRNA, and siRNA binds to an RNA-induced silencing complex (RISC), whereby the guide (antisense) strand recognizes and degrades the target mRNA to sequence-express the expression of the target gene. (NUCLEIC-ACID THERAPEUTICS: BASIC PRINCIPLES AND RECENT APPLICATIONS. Nature Reviews Drug Discovery. 2002. 1, 503-514).

According to Bertrand researchers, siRNA against the same target gene is superior to antisense oligonucleotides (ASOs) in inhibiting mRNA expression in vitro and in vivo , and the effect lasts for a long time. (Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo . Biochem. Biophys. Res. Comm. 2002. 296: 1000-1004). In addition, the siRNA action mechanism complementarily binds to the target mRNA and regulates the expression of the target gene in a sequence-specific manner, which is why it is possible to apply a target to an antibody-based drug or a small molecule drug. It has the advantage that it can be expanded (Progress Towards in vivo Use of siRNAs. MOLECULAR THERAPY. 2006 13 (4): 664-670).

Despite the excellent effects of siRNAs and their wide range of uses, siRNAs must be effectively delivered to target cells by improving their stability and improving cell delivery efficiency in order for siRNAs to be developed as therapeutic agents (Harnessing in vivo siRNAs). delivery for drug discovery and therapeutic development.Drug Discov Today. 2006 Jan; 11 (1-2): 67-73).

In order to solve the above problems, some nucleotides or backbones of siRNA are modified to have nuclease resistance or viral vectors, liposomes or nanoparticles, etc., to improve the stability in the body. Research into the use of the carrier is actively attempted.

Delivery systems using viral vectors such as adenoviruses or retroviruses have high transfection efficiency, but high immunogenicity and oncogenicity. On the other hand, non-viral delivery systems containing nanoparticles have lower cell delivery efficiency than viral delivery systems, but have high safety in vivo and target specific delivery. It has the advantage of improved transfer effect such as uptake and internalization of nested RNAi oligonucleotides into cells or tissues, as well as little cytotoxicity and immune stimulation. It is currently being evaluated as a viable delivery method compared to viral delivery systems (Nonviral delivery of synthetic siRNAs in vivo . J Clin Invest. 2007 December 3; 117 (12): 3623-3632).

In the non-viral delivery system, a method of using a nanocarrier is used to form nanoparticles by using various polymers such as liposomes and cationic polymer complexes, and siRNA is added to these nanoparticles, that is, nanocarriers. It has a form to be supported and delivered to the cell. Among the methods using nanocarriers, polymer nanoparticles, polymer micelles, lipoplexes, etc. are mainly used. Among them, lipoplexes are composed of cationic lipids. It interacts with the anionic lipids of the endosome of endosome, induces the destabilizing effect of the endosome and delivers it into cells (Proc. Natl. Acad. Sci. 15; 93 (21): 11493-8, 1996).

In addition, it is possible to induce high efficiency in vivo by connecting chemicals and the like to the end of the siRNA passenger (sense) strand to have enhanced pharmacokinetic characteristics. (Nature 11; 432 (7014): 173-8, 2004). In this case, the stability of the siRNA depends on the nature of the chemical bound to the ends of the siRNA sense (passenger) or antisense (guide) strand. For example, siRNAs in the form of conjugated polymer compounds, such as polyethylene glycol (PEG), form complexes by interacting with the anionic phosphate groups of the siRNAs in the presence of cationic materials, thereby improving improved siRNA stability. Excitation carrier (J Control Release 129 (2): 107-16, 2008). In particular, micelles composed of polymer complexes are extremely uniform in size and spontaneously formed, compared to other systems used as drug delivery vehicles, such as microspheres or nanoparticles. There is an advantage that it is easy to ensure the quality control and reproducibility of the formulation.

In addition, in order to improve the intracellular delivery efficiency of siRNA, a simple covalent or linker-mediated covalent association of a hydrophilic material (e.g., polyethylene glycol, PEG) that is a biocompatible polymer to siRNA Through the siRNA conjugate conjugated to, a technique for securing the stability of the siRNA and efficient cell membrane permeability has been developed (Korean Patent No. 883471). However, the chemical modification of siRNA and the conjugation of polyethylene glycol (PEGylation) alone (PEGylation) still have the disadvantages of low stability in vivo and delivery to the target organ is not smooth. In order to solve this shortcoming, a double-stranded oligo RNA structure in which hydrophilic and hydrophobic materials are coupled to oligonucleotides, especially double-stranded oligo RNA such as siRNA, has been developed. The structure is prepared by SAMiRNA ™ (self assembled) by hydrophobic interaction of hydrophobic materials. self-assembled nanoparticles called micelle inhibitory RNA) (see Korean Patent No. 1224828). SAMiRNA ™ technology yields very small and homogenous nanoparticles compared to conventional delivery technologies. It has the advantage of being able to.

On the other hand, in Korea, one out of four people is dying of cancer (the number one cause of death), and the number is increasing considerably each year due to the development of diagnostic methods and data collection methods, aging population and environmental changes. In addition, global cancer incidence and cancer deaths are on the rise, and prevention, diagnosis, and treatment technologies for cancer conquest are urgent tasks for humankind (BT technology trend report, new drug development trend by major diseases, and Biotechnology Policy Research Center). , 2007, Vol. 72).

Cancer is one of the world's most fatalities, and the development of innovative cancer treatments can reduce the cost of medical treatment and create high added value. The treatment of cancer is divided into surgery, radiation therapy, chemotherapy, and biological therapy. Among these, chemotherapy is a chemical substance that inhibits or kills the proliferation of cancer cells. Even though the anticancer drug is effective, the resistance is lost after a certain period of time, but the development of an anticancer drug that selectively acts on cancer cells and does not develop resistance is urgently needed. 2004. 6 (19). Recently, the development of new anticancer drugs targeting molecular characteristics of cancer by securing molecular genetic information on cancer, and anticancer drugs targeting specific molecular targets that only cancer cells have It is reported that this does not occur. Therefore, it is possible to develop a therapeutic agent that is more effective and has fewer side effects than conventional anticancer drugs through the development of a gene therapeutic agent targeting a characteristic molecular target having only cancer cells.

Psoriasis is a chronic autoimmune inflammatory skin disorder characterized by epidermal hyperproliferation of keratinocytes and endothelial cells, and inflammatory cell accumulation (such as activated T cells) (Griffiths CE, J. Eur. Acad. Dermatol. Venereol. 2003, 17 Suppl 2: 1-5; Creamer JD, et al., Clin.Exp.Dermatol. 1995, 20 (1): 6-9). Psoriasis is characterized by abnormal and rapid growth of the epidermal layer of the skin. The abnormal production of these skin cells generally produces skin cells every 28 to 30 days, but in the case of psoriasis it is replaced every 3 to 5 days. This change is mainly due to the increase of endothelial cells, including dendritic cells, macrophage and T cells, to proliferate immature keratinocytes. These immune cells migrated from cells to envelope cells are TNF-α, interleukin-1β, IL-1β, interleukin-6 (IL-6), and interleukin-36 (IL- 36) and secrete various cytokines such as interleukin-22 (IL-22) and these cytokines have been reported to stimulate the proliferation of keratinocytes (Nestle FO et al., N Engl J Med. 2009; 361). (5): 496-509; Baliwag, Jaymie et al., Cytokine. 2015; 73 (2): 342-350). In particular, dendritic cells connect the innate immune system to the adaptive immune system, and psoriasis-induced T cells migrate to the envelope cells to release interleukin-17 (IL-17) and interferon-gamma (IFN-γ). Secrete and interleukin-23 (IL-23) stimulates the production of interleukin-22 (IL-22) and interleukin-17 (IL-17), which are interleukin-17 (IL-22) 17) may exacerbate psoriasis by stimulating keratinocytes (Ouyang W., Cytokine Growth Factor Rev. 2010; 21 (6): 435-41; Mudigonda P et al., Dermatol Online J. 2012; 18 (10): 1).

Recently, the STAT3 gene has been reported to be critically involved in the development of psoriasis (Stat3 links activated keratinocytes and immunocytes required for development of psoriasis in a novel transgenic mouse model, Nature Medicine.Vol. 11, 2005.Pp. 43-49 Stat3 as a therapeutic target for the treatment of psoriasis: a clinical feasibility study with STA-21, a stat3 inhibitor, J. Invest.Dermatol.Vol. 131. 2011. pp. 108-117). Clinically, the main symptom of psoriasis is a gray or silver flaky patch on the skin that is red and inflamed below.

Common treatments for psoriasis include topical dosing, phototherapy and oral dosing. Topical treatments are therapies including steroids, coal tar, anthraline, vitamin D3 and analogs thereof, retinoids and sunburn, and the like, which have the side effects of skin thinning, stretch marks, burns, irritation and photosensitivity.

Drugs used in oral administration are for the treatment of psoriasis through immunosuppression or inflammatory response. Many psoriasis treatments developed to date do not fundamentally cure psoriasis and provide only temporary relief of symptoms. Because of suppressing the immune system of the human body, if the drug is used for a long time due to the deterioration of the immune function of the human body can cause various infections, including cancer, tuberculosis, and the like.

The term atopy is a term that has the meaning "weird" or "inappropriate" in origin. Atopic dermatitis is a relatively common chronic inflammatory skin disease that occurs most often in infancy and childhood, and improves and worsens.It can be diagnosed with three characteristics: individual or family history of atopy, severe itching, and eczema. May be exacerbated by seasonal and climate change, irritation and allergies. The etiology of atopic dermatitis is not yet clear, but studies to date indicate a lack of functionalities due to hypersensitivity reactions with increased IgE antibodies, or due to undifferentiated differentiation of T lymphocytes resulting from decreased cellular mediated immune function, Blockage of adrenergic receptors present in the skin is the cause of the onset. Therefore, atopic dermatitis is considered to be a genetic disease involving immunological abnormalities. Recently, STAT3 genes have been reported to influence the development of atopic dermatitis (Diminished allergic disease in patients with STAT3 mutations reveals a role for STAT3 signaling in mast cell degranulation, J. Allergy and Clinical Immunology, Vol. 132, 2013. Pp. 1388-1396).

For the treatment and management of atopic dermatitis, it is common for most dermatologists to prescribe steroid hormones, that is, topical corticosteroids, to improve the inflammatory response along with moisturizers that keep the skin's surface moisturized. However, when the topical corticosteroids are used for a long time, there are problems that cause various skin side effects such as skin atrophy, vasodilation, pigmentation and swelling ancestors. In addition, patients with severe atopic dermatitis may be given phototherapy, such as appropriate UV treatment, interferon gamma, immunosuppressants such as cyclosporin, and immunoglobulin intravenous injection. Therefore, there is a need to develop a raw material or pharmaceuticals for treating atopic dermatitis having an anti-inflammatory effect without showing such side effects.

Allergic dermatitis is a recurrent eczema lesion accompanied by a chronic itching. The rash tends to appear on the face, neck, elbows, knees, and other flexures, and may worsen and spread throughout the body. The number of patients with allergic dermatitis increases year by year, depending on the environment in which various allergens are increased, dietary changes, and the like, and the symptoms tend to be severe. Treatment of allergic dermatitis is mainly drug therapy, and corticosteroids, immunosuppressants, and antihistamines are used. These drugs may only relieve symptoms or settle inflammation, but are only temporary. In addition, corticosteroids and immunosuppressants are known to have side effects of infection and poisoning.

Therefore, current therapeutic agents for allergic dermatitis may not be sufficiently satisfactory in their effectiveness and alleviation of side effects.

In addition, rheumatoid arthritis known as STAT3-mediated inflammatory disease, degenerative arthritis (Korean Publication No. 10-2014-0032925), osteoporosis, hyperimmunoglobulinemia, anemia, nephritis, chronic thyroiditis, Crohn's disease, pancreatitis, etc. (Korean Publication No. 10-1416149) And the like can be the object of prevention and treatment according to the present invention.

Since RNA interference is known to inhibit gene expression with high specificity and efficiency, siRNAs targeting various genes have been studied as a therapeutic agent for cancer. These genes include cancer cells such as oncogenes, anti-apoptotic molecules, telomerases, growth factor receptor genes, and signaling molecules. Inhibiting the expression of genes necessary for survival or inducing apoptosis is the main direction (RNA interference in cancer. Biomolecular Engineering. 2006; 23: 17-34).

STAT3 (signal transducer and activator of transcription 3) is a transcription factor that promotes transcription by delivering extracellular extracellular growth factor and cytokine signals to the nucleus. When tyrosine residues of the transactivation domain are phosphorylated and activated in the state, they enter the nucleus (STAT3 inhibitors for cancer therapy: Have all roads been explored- Jak-Stat. 2013; 1; 2 (1): e22882). Phosphorylated STAT3 (p-STAT3) binds to the DNA of the nucleus and induces the expression of a broad range of target genes involved in tumorigenesis, such as proliferation and differentiation of cells. It is constantly active in about 70% (Role of STAT3 in cancer metastasis and translational advances. BioMed research international. 2013; 2013: 421821).

However, transcription factors such as STAT3 were considered to be undruggable in the field of traditional synthetic drug development because of the tertiary structure of the protein, which makes it difficult to find a target that can inhibit activity (Transcription Factor STAT3 as a Novel Molecular Target for Cancer Prevention. Cancers. 2014; 16; 6 (2): 926-57). Therefore, the market demand for siRNA therapeutics and their delivery technology that can inhibit the expression of STAT3 is very high.

Accordingly, the present inventors have made diligent efforts to solve the above problems, and as a result, siRNA specific to the STAT3 gene can specifically inhibit the activity of the STAT3 gene, and a double-stranded oligo RNA structure comprising the same and a drug containing the same The present invention was completed by confirming that the anticancer effect, the skin disease and the inflammatory disease of the pharmaceutical composition are very excellent.

Summary of the Invention

An object of the present invention is to solve the above problems, a novel siRNA that can inhibit its expression with a very high efficiency specific to STAT3 and a double-stranded oligo RNA structure comprising the same, and such a double-stranded oligo RNA structure It is to provide a method of manufacturing.

Another object of the present invention is to provide a pharmaceutical composition comprising the STAT3-specific siRNA or a double-stranded oligo RNA structure comprising such siRNA as an active ingredient.

Still another object of the present invention is to provide a method for preventing or treating cancer, skin disease or inflammatory disease using the STAT3-specific siRNA or a double-stranded oligo RNA structure comprising such siRNA.

In order to achieve the above object, the present invention provides a STAT3 specific siRNA comprising a sense strand comprising any sequence selected from SEQ ID NO: 1 to SEQ ID NO: 200 and an antisense strand comprising a sequence complementary thereto. to provide.

“STAT3 specific siRNA” in the present invention means siRNA specific to a gene encoding a STAT3 protein.

In addition, in the sense strand comprising the sequence according to SEQ ID NO: 1 to SEQ ID NO: 200 or an antisense strand complementary thereto, as long as the specificity for STAT3 is maintained, one or more bases include a sequence substituted, deleted, or inserted. It is apparent to those skilled in the art that STAT3 specific siRNAs including sense strands and antisense strands are also included in the scope of the present invention.

SEQ ID NO: 1 to SEQ ID NO: 200 is the sense strand sequence of STAT3 specific siRNA.

In the present invention, the sense strand or the anti-sense strand of the siRNA is not limited to the number of nucleotides if it can perform the function of siRNA, preferably it may be characterized in that consisting of 19 to 31 nucleotides.

In the present invention, the siRNA preferably comprises a sense strand of STAT3 specific siRNA according to SEQ ID NO: 12, 17, 42, 101, 108, 114, 119, 137, 141, 166, 170, 171 It may be characterized by, more preferably comprising a sense strand of STAT3 specific siRNA according to SEQ ID NO: 17, 42, 101, 119, 137, most preferably SEQ ID NO: 42, 101 , 137 may be characterized in that it comprises a sense strand of the STAT3-specific siRNA.

STAT3 specific siRNA provided in the present invention has a base sequence designed to complementarily bind to the mRNA encoding the gene, it is characterized in that it can effectively suppress the expression of the gene. In addition, the siRNA may include an overhang, which is a structure including one or two or more unpaired nucleotides at the 3 ′ end of the siRNA.

In the present invention, the siRNA may be characterized in that it comprises a variety of modification (modification) for imparting nuclease resistance resistance and non-specific immune response to improve the stability in vivo. Modifications of the siRNAs have been described in which the —OH group at the 2 ′ carbon position of the sugar structure in one or more nucleotides is —CH ₃ (methyl), —OCH ₃ (methoxy), —NH ₂ , —F (fluorine), —O-2-methicone. Methoxyethyl -O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl, -ON-methylacetamido or -O- Modification by substitution with dimethylamidooxyethyl; Modification in which oxygen in a sugar structure in nucleotides is replaced with sulfur; Or a combination of one or more modifications selected from nucleotide-linked phosphorothioate or boranophosphate, methyl phosphonate bonds, peptide nucleic acid (PNA), Modifications in the form of locked nucleic acid (LNA) or unlocked nucleic acid (UNA) are also possible (Ann. Rev. Med. 55, 61-65 2004; US 5,660,985; US 5,958,691; US 6,531,584; US 5,808,023; US 6,326,358 ; US 6,175,001; Bioorg.Med. Chem. Lett. 14: 1139-1143, 2003; RNA, 9: 1034-1048, 2003; Nucleic Acid Res. 31: 589-595, 2003; Nucleic Acids Research, 38 (17) 5761-5773, 2010; Nucleic Acids Research, 39 (5) 1823-1832, 2011).

STAT3 specific siRNA provided by the present invention not only inhibits the expression of the gene of interest, but also significantly inhibits the expression of the protein of interest. In addition, since it is known to improve the sensitivity of radiation or chemotherapy, which is a typical combination with cancer-specific RNAi used in the treatment of cancer (The Potential RNAi-based Combination Therapeutics. Arch Pharm Res 34 (1). ): 1-2, 2011), it will be apparent to those skilled in the art that the STAT3 specific siRNA of the present invention can be used in combination with existing radiation or chemotherapy.

In another aspect of the present invention, there is provided a conjugate in which a hydrophilic substance and a hydrophobic substance are conjugated to both ends of an siRNA for efficient delivery and stability of breast cancer associated genes, particularly STAT3 specific siRNA, in vivo.

In the case of siRNA conjugates in which a hydrophilic material and a hydrophobic material are bound to siRNA, self-assembled nanoparticles are formed by hydrophobic interaction of the hydrophobic material (see Korean Patent Registration No. 1224828). And not only the stability in the body is extremely excellent, but also the particle size is very uniform through the improvement of the structure is easy to QC (Quality control), there is an advantage that the manufacturing process as a drug is simple (see WO2015002511).

As one preferred embodiment, the double-stranded oligo RNA structure comprising the STAT3 specific siRNA according to the present invention preferably comprises a structure as shown in the following structural formula (1).

Structural Formula 1

In structural formula (1), A is a hydrophilic monomer, B is a hydrophobic substance, J is a m-hydrophilic monomer, or a linker connecting m hydrophilic monomers and oligonucleotides to each other, and n is a hydrophilic monomer. The number of repetitions of the repeated hydrophilic material blocks, X and Y are each independently a simple covalent bond or a linker mediated covalent bond, R is a STAT3 specific siRNA, and m is an integer from 1 to 10, preferably 6, n is an integer of 1-10, Preferably it is 4.

As long as STAT3 specificity is maintained, the STAT3-specific siRNA may not only not only match perfect sequences but also some sequences when the antisense strand of the siRNA is 100% nucleotide complementary to the binding site of the STAT3 gene. If not, that is, there is a mismatch. Such siRNA is preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, most preferably 100% relative to a portion of the mRNA sequence of the STAT3 gene It may consist of a sequence having a homology of.

Such siRNAs may be duplexed and may also include single molecule polynucleotides, but are not limited to antisense oligonucleotides or microRNAs (miRNAs).

More preferably, the double-stranded oligo RNA structure comprising STAT3-specific siRNA according to the present invention has the structure of the following structural formula (2).

Structural Formula 2

A, B, J, m, n, X and Y in the formula (2) is the same as the definition in the formula (1), S is the sense strand of STAT3-specific siRNA, AS is antisense of STAT3-specific siRNA Means strand.

More preferably, the double-stranded oligo RNA construct comprising STAT3-specific siRNA has the structure of formula (3) below.

Structural Formula 3

One to three phosphate groups may be bonded to the 5 ′ end of the antisense strand of the double-stranded oligo RNA structure comprising the STAT3-specific siRNAs in the formulas (1) to (3), It is apparent to those skilled in the art that shRNA may be used instead of siRNA.

The hydrophilic material monomers (A) in the structural formulas (1) to (3) can be used without limitation as long as they meet the object of the present invention among the monomers of the nonionic hydrophilic polymer, preferably in Table 1 The monomers selected from the compounds (1) to (3) described, more preferably the monomers of compound (1) can be used, and in compound (1) G is preferably selected from CH ₂ , O, S and NH And more preferably O is used.

The hydrophilic material monomer A and the linker J included in each of the hydrophilic material blocks may be the same as or different from each of the hydrophilic material blocks independently. That is, if three hydrophilic material blocks are used (n = 3), the first block contains the hydrophilic monomer according to compound (1), the second block contains the hydrophilic monomer according to compound (2), and the third block contains Other hydrophilic material monomers may be used for every hydrophilic material block, such as a hydrophilic material monomer according to compound (3), and any hydrophilic material monomer selected from compounds (1) to (3) may be used for every hydrophilic material block. One hydrophilic monomer may equally be used. Similarly, the linker that mediates the binding of the hydrophilic material monomer may also use the same linker for each hydrophilic material block, or different linkers for each hydrophilic material block.

The hydrophobic material (B) in the structural formulas (1) to (3) serves to form nanoparticles composed of the oligonucleotide structure according to the structural formula (1) through hydrophobic interaction.

In the present invention, the hydrophobic material preferably has a molecular weight of 250 to 1,000, steroid derivatives, glyceride derivatives, glycerol ether, polypropylene glycol, C ₁₂ to C ₅₀ unsaturated or saturated hydrocarbons, diacylphosphatidylcholine, fatty acids, phospholipids, lipopolyamines, and the like may be used, but are not limited thereto. Any hydrophobic material can be used as long as it can be understood by those skilled in the art to which the present invention pertains.

In the present invention, the steroid derivative (steroid) may be selected from the group consisting of cholesterol, cholestanol, cholic acid, chosteryl formate, cotestanyl formate and colistanylamine, the glyceride derivative is Mono-, di- and tri-glycerides and the like, wherein the fatty acid of the glyceride is preferably C ₁₂ to C ₅₀ unsaturated or saturated fatty acid.

Particularly, among the hydrophobic substances, saturated or unsaturated hydrocarbons or cholesterols are preferable in that they have the advantage of being easily bonded in the synthesis step of the oligonucleotide structure according to the present invention.

The hydrophobic material is bound to the distal end of the hydrophilic material, and may be bonded at any position of the sense strand or the antisense strand of the siRNA.

The hydrophilic or hydrophobic material and the STAT3-specific siRNA in the structural formulas (1) to (3) according to the present invention are bound by simple covalent bonds or linker-mediated covalent bonds (X or Y). The linker that mediates the covalent bond is not particularly limited as long as it provides covalent bond at the end of the STAT3-specific siRNA with a hydrophilic substance or a hydrophobic substance, and can be decomposed in a specific environment as necessary. Thus, the linker can be used any compound that binds to activate the STAT3-specific siRNA and / or hydrophilic material (or hydrophobic material) during the preparation of the double helix oligo RNA structure according to the present invention.

In the present invention, the covalent bond may be any of non-degradable bonds or degradable bonds. In this case, the non-degradable bonds include amide bonds or phosphorylation bonds, and the degradable bonds include disulfide bonds, acid decomposable bonds, ester bonds, anhydride bonds, biodegradable bonds or enzymatic bonds, but are not limited thereto. .

In addition, STAT3 specific siRNAs represented by R (or S and AS) in the above formulas (1) to (3) can be used without limitation as long as all siRNAs have properties that can specifically bind to STAT3, Preferably, the present invention may be characterized by consisting of a sense strand comprising any one sequence selected from SEQ ID NO: 1 to SEQ ID NO: 200 and an antisense strand comprising a complementary sequence thereto.

In the present invention, the siRNA preferably comprises a sense strand of STAT3 specific siRNA according to SEQ ID NO: 12, 17, 42, 101, 108, 114, 119, 137, 141, 166, 170, 171, More preferably the sense strand of the STAT3 specific siRNA according to SEQ ID NO: 17, 42, 101, 119, 137, most preferably the sense strand of the STAT3 specific siRNA according to SEQ ID NO: 42, 101, 137 It may be characterized in that it comprises a.

Tumor tissue, on the other hand, is very robust and has diffusion-limitation compared to normal tissue, which can adversely affect the movement of waste products such as nutrients, oxygen and carbon dioxide, which are necessary for tumor growth. Angiogenesis overcomes diffusion limitations by forming blood vessels around. Blood vessels in tumor tissue formed through angiogenesis have a leaky and defective blood vessel having a gap of about 100 nm to 2 um depending on the type of tumor. As a result, nanoparticles pass through the capillary endothelium of cancerous tissues, which contain loose blood vessel structures, compared to the organized capillaries of normal tissues, thereby facilitating access to tumor interstitium during blood circulation. In addition, there is no lymphatic drainage in tumor tissues, which results in the accumulation of drugs. This is called the enhanced permeation and retention (EPR) effect. The delivery of nanoparticles to tumor tissue specificity by this effect is called passive targeting (Nanoparticles for drug delivery in cancer treatment.Urol. Oncol. 2008 Jan-Feb; 26 (1): 57-64 ). Active targeting is a case where a targeting moiety is bound to a nanoparticle, which promotes the accumulation of nanoparticles in the target tissue or internalization of the nanoparticles into the target cell. Does a targeting ligand influence nanoparticle tumor localization or uptake Trends Biotechnol. 2008 Oct; 26 (10): 552-8. Epub 2008 Aug 21). Active targeting uses target cell surface specific or overexpressed carbohydrates, receptors, and antigens (targeting moieties) that have the ability to bind to antigens (Nanotechnology in cancer). therapeutics: bioconjugated nanoparticles for drug delivery.Mol Cancer Ther 2006; 5 (8): 1909-1917).

Therefore, if the double-stranded oligo RNA structure comprising the STAT3-specific siRNA according to the present invention and the nanoparticles formed therefrom are provided with a targeting moiety, the target moiety can be efficiently promoted to a target cell, and at a relatively low concentration, It can be delivered to target cells and exhibit high target gene expression regulating function, and can prevent delivery of non-specific STAT3 specific siRNA to other organs and cells.

Accordingly, the present invention provides a receptor (L), particularly a receptor for enhancing internalization of a target cell through receptor-mediated endocytosis (RME), in a construct according to Structural Formulas (1) to (3). Provides a double-stranded oligo RNA, structure is further coupled to a ligand (ligand) having a specific binding properties, the form of the ligand is bound to the double-stranded oligo RNA structure according to the formula (1) is represented by the following formula (4) Have the same structure.

Structural Formula 4

(Li-Z)-(A _m -J) _n -XRYB

In the above formula (4), A, B, J, m, n, X and Y are the same as the definition in the formula (1) to (3), L is receptor-mediated endocytosis, RME) refers to a ligand having a property of specifically binding to a receptor that promotes target cell internalization, i means an integer of 1 to 5, preferably an integer of 1 to 3, Z is It is a linker that mediates the binding of a ligand to a hydrophilic monomer in a simple covalent or hydrophilic block of materials.

The ligand in the above formula (4) is preferably a target receptor-specific antibody or aptamer having the characteristics of receptor-mediated endocytosis (RME) that promotes internalization specifically to the target cell. , Peptides; Or folic acid (Folate, in general, folate and folic acid are used interchangeably with each other, folic acid in the present invention means folate that is active in the natural state or human body), N-acetyl galactosamine (N-acetyl Galactosamine, NAG Hexamine (hexoamine), glucose (glucose), mannose, and other chemicals, such as sugar or carbohydrate (carbohydrate), such as, but is not limited to this, preferably glucose.

In still another aspect of the present invention, the present invention provides a method for preparing a double-stranded oligo RNA construct comprising the STAT3 specific siRNA.

The process for preparing a double helix oligo RNA construct comprising a STAT3 specific siRNA according to the invention is, for example,

(1) bonding the hydrophilic material on a solid support basis;

(2) synthesizing a single RNA strand based on the solid support to which the hydrophilic material is bound;

(3) covalently binding a hydrophobic substance to the 5 'end of the RNA single strand;

(4) synthesizing an RNA single strand of a sequence complementary to the sequence of the RNA single strand;

(5) separating and purifying the RNA-polymer structure and the RNA single strand from the solid support when the synthesis is completed;

(6) preparing a double-stranded oligo RNA structure by annealing RNA single strands of sequences complementary to the prepared RNA-polymer construct;

It may be made, including.

In the present invention, the solid support is preferably Controlled Pore Glass (CPG), but is not limited thereto. In the case of CPG, the diameter is preferably 40 to 180 μm, and preferably has a pore size of 500 μs to 3000 μm. Do. After the step (5), when the preparation is completed, the purified RNA-polymer construct and RNA single strand can be determined by measuring the molecular weight with a MALDI-TOF mass spectrometer to prepare the desired RNA-polymer construct and RNA single strand. In the preparation method, the step (4) of synthesizing the RNA single strand of the sequence complementary to the sequence of the RNA single strand synthesized in step (2) is performed before (1) or (1) to (5) It can be done during a process.

In addition, the RNA single strand comprising the RNA single strand and the complementary sequence synthesized in step (2) may be a manufacturing method characterized in that used in the form of a phosphate group bonded to the 5 'end.

The present invention also provides a method for preparing a double-stranded oligo RNA structure in which ligand is additionally bound to a double-stranded oligo RNA structure comprising a STAT3-specific siRNA.

Methods for preparing oligo RNA constructs comprising STAT3 specific siRNAs with bound ligands are, for example,

(1) binding a hydrophilic material to the solid support to which the functional group is bound;

(2) synthesizing a single RNA strand based on the solid support to which the functional group-hydrophilic material is bound;

(3) synthesizing by covalently attaching a hydrophobic material to the 5 'end of the RNA single strand;

(4) synthesizing an RNA single strand of a sequence complementary to the sequence of the RNA single strand;

(5) separating the RNA single strand of the functional group-RNA-polymer structure and the complementary sequence from the solid support when synthesis is complete;

(6) binding a ligand to a hydrophilic material terminal using the functional group to prepare a ligand-RNA-polymer structure single strand;

(7) preparing a ligand-double-stranded oligo RNA structure by annealing RNA single strands of sequences complementary to the prepared ligand-RNA-polymer construct;

It may be made, including.

After the step (6), when the preparation is completed, the ligand-RNA-polymer structure and the RNA single strand of the complementary sequence is separated and purified, and then the desired ligand-RNA-polymer structure and complement by measuring the molecular weight with a MALDI-TOF mass spectrometer. You can check whether the RNA is prepared. Ligand-double-stranded oligo RNA structures can be prepared by annealing RNA single strands of sequences complementary to the prepared ligand-RNA-polymer constructs. In the preparation method, the step (4) of synthesizing the RNA single strand of the sequence complementary to the sequence of the RNA single strand synthesized in step (3) is an independent synthesis process before (1) or (1) to ( 6) may be performed during any of the steps.

In another aspect of the invention, there is provided a nanoparticle comprising a double helix oligo RNA construct comprising a STAT3 specific siRNA.

As described above, the double-stranded oligo RNA structure containing STAT3-specific siRNA is amphiphilic containing both hydrophobic and hydrophilic substances, and the hydrophilic portion is formed by interacting with hydrogen molecules and water molecules in the body. They have an affinity and are directed outwards, and hydrophobic materials are directed inward through hydrophobic interactions between them, forming thermodynamically stable nanoparticles. That is, the hydrophobic material is located in the center of the nanoparticles, and the hydrophilic material is located in the outward direction of the STAT3 specific siRNA to form a nanoparticle that protects the STAT3 specific siRNA. The thus formed nanoparticles enhance the intracellular delivery of STAT3-specific siRNA and the siRNA efficacy.

Nanoparticles according to the invention may be formed of a double-stranded oligo RNA structure comprising siRNA having the same sequence, or may be composed of a double-stranded oligo RNA structure comprising siRNA comprising different sequences In addition, siRNAs comprising different sequences in the present invention may be different target genes, for example, STAT3 specific siRNAs, and may include cases in which the sequences are different while having the same target gene specificity.

In addition, double-stranded oligo RNA constructs comprising other cancer specific target gene specific siRNAs in addition to STAT3-specific siRNAs may also be included in the nanoparticles according to the invention.

In still another aspect, the present invention provides a method for preventing or treating cancer, skin disease or inflammatory disease comprising STAT3-specific siRNA, a double-stranded oligo RNA structure comprising the same, and / or nanoparticles comprising the double-stranded oligo RNA structure. It provides a composition for.

STAT3-specific siRNA according to the present invention, a double-stranded oligo RNA structure comprising the same and / or a composition comprising nanoparticles consisting of the double-stranded oligo RNA structure as an active ingredient induces the proliferation of cancer cells and the death of cancer cells, or keratin It induces proliferation and death of forming cells and thus induces the activity of abnormal immune cells, thereby preventing or treating cancer, skin disease or inflammatory disease. Therefore, the STAT3-specific siRNA and the composition comprising the same according to the present invention can be used for various cancers, including gastric cancer, lung cancer, pancreatic cancer, colorectal cancer, liver cancer, prostate cancer, ovarian cancer and kidney cancer, including breast cancer in which the gene is reported to be overactivated. It is effective in preventing or treating skin diseases and inflammatory diseases including cancer or psoriasis.

In particular, in the present invention, the composition for preventing or treating cancer comprising the double-stranded oligo RNA structure comprises any one sequence selected from SEQ ID NO: 1 to SEQ ID NO: 200, preferably SEQ ID NO: 12, 17, 42, 101, Any one selected from the sequences according to 108, 114, 119, 137, 141, 166, 170, 171, more preferably the sequences according to SEQ ID NOs: 17, 42, 101, 119, 137, most preferably And a double stranded oligo RNA construct comprising a STAT3 specific siRNA comprising a sense strand comprising a sequence according to SEQ ID NOs: 42, 101 and 137 and an antisense strand comprising a sequence complementary thereto. .

In addition, siRNA specific to cancer, skin disease or inflammatory disease specific target genes other than STAT3 or a double-stranded oligo RNA construct comprising the same may be additionally included in the composition according to the present invention.

Along with the double-stranded oligo RNA construct comprising STAT3-specific siRNA as described above, the cancer further includes all of the double-stranded oligo RNA construct comprising siRNA specific to cancer, skin disease or inflammatory disease specific target gene other than STAT3. When used together with a composition for the prevention or treatment of skin diseases or inflammatory diseases, synergistic effects can be achieved, such as combination therapy, which is often used to treat cancer, skin diseases or inflammatory diseases.

Cancers that can be prevented or treated by the composition according to the present invention may exemplify breast cancer, stomach cancer, colon cancer, pancreatic cancer, prostate cancer, liver cancer, ovarian cancer, kidney cancer and lung cancer, but are not limited thereto. Psoriasis, atopic dermatitis, ringworm and allergic skin diseases can be exemplified, but not limited thereto. Inflammatory diseases may include rheumatoid arthritis, degenerative arthritis, hyperimmunoglobulinemia, chronic thyroiditis, Crohn's disease, and pancreatitis. It is not limited to this

The composition of the present invention may be prepared by including at least one pharmaceutically acceptable carrier in addition to the above-mentioned effective ingredient. Pharmaceutically acceptable carriers must be compatible with the active ingredients of the present invention and include saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these ingredients. It can mix and use, and if needed, other conventional additives, such as antioxidant, buffer, and bacteriostatic agent, can be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions and the like. In particular, it is desirable to formulate and provide a formulation in the form of a lyophilized form. Methods commonly known in the art may be used for the preparation of lyophilized formulations, and stabilizers for lyophilization may be added. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's pharmaceutical Science, Mack Publishing Company, Easton PA.

The content and method of administration of the active ingredient and the like contained in the composition of the present invention can be determined by a person of ordinary skill in the art based on the symptoms and severity of the disease of a typical patient. It may also be formulated in various forms, such as powders, tablets, capsules, solutions, injections, ointments, syrups, and the like, and may also be provided in unit-dose or multi-dose containers, such as sealed ampoules and bottles.

The composition of the present invention can be administered orally or parenterally. Routes of administration of the compositions according to the invention include, but are not limited to, for example, bronchial, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal Sublingual, or topical administration is possible. The dosage of the composition according to the present invention may vary in the range depending on the weight, age, sex, health condition, diet, time of administration, method, excretion rate or severity of the disease, etc. of the patient, and is easily available to those skilled in the art. Can decide. In addition, the compositions of the present invention can be formulated into suitable formulations using known techniques for clinical administration.

In the present invention, STAT3-specific siRNA according to the present invention, a double-stranded oligo RNA structure comprising the siRNA, a composition or a nanoparticle comprising the same for use in the manufacture of a medicament for the prevention or treatment of cancer, skin diseases or inflammatory diseases To provide.

The present invention also provides a method for preventing and treating cancer, skin disease or inflammatory disease comprising administering a double-stranded oligo RNA structure, a composition or a nanoparticle comprising the same to a patient in need thereof. to provide.

1 is a schematic diagram of nanoparticles composed of a double helix oligo RNA structure according to the present invention.

2 is a double-stranded oligo RNA structure comprising an siRNA comprising a sequence of SEQ ID NO: 12, 17, 42, 101, 108, 114, 116, 119, 137, 141, 170 or 171 according to the present invention as a sense strand It is a measurement graph of the size (size) and polydispersity index (PDI) of the nanoparticles made.

Figure 3 is a graph of the expression inhibition of the identified target gene after transforming the siRNA having the sense strand of SEQ ID NO: 1 to 200 of the present invention in the MCF-7 cell line at a concentration of 5 nM.

Figure 4 is a graph of the expression inhibition of the identified target genes after transforming the siRNA having the sense strand of SEQ ID NO: 1 to 200 of the present invention in the MCF-7 cell line at a concentration of 1 nM.

5 shows the sequence of SEQ ID NOs: 12, 17, 42, 55, 91, 95, 101, 108, 111, 114, 116, 119, 137, 139, 141, 152, 166, 168, 170 or 171 of the present invention. The siRNA having the sense strand is transformed into the MDA-MB-231 cell line at a concentration of 0.2 nM, 1 nM or 5 nM, and then a graph of the target gene expression inhibition confirmed.

FIG. 6 shows siRNA having a sense strand having a sequence of SEQ ID NOs. 12, 17, 42, 101, 108, 114, 116, 119, 137, 141, 170 or 171 of the present invention as 1.6 pM, 8 pM, 40 pM, It is a graph of the target gene expression inhibition confirmed after transforming the MCF-7 cell line at a concentration of 200 pM, 1 nM or 5 nM.

7 is 1.6 pM, 8 pM, 40 pM, siRNA having a sense strand of the sequence SEQ ID NO: 12, 17, 42, 101, 108, 114, 116, 119, 137, 141, 170 or 171 of the present invention, After transforming MDA-MB-231 cell line at a concentration of 200 pM, 1 nM or 5 nM, it is a graph of confirmed target gene expression inhibition.

Figure 8 is an analysis of the IC50 (inhibition concentration 50%) of siRNA having a sense strand of the sequence of SEQ ID NO: 12, 17, 42, 101, 108, 114, 116, 119, 137, 141, 170 or 171 of the present invention As a graph, A is the MCF-7 cell line and B is the MDA-MB-231 cell line.

Figure 9 shows the target gene expression after transforming the siRNA having the sense strand of SEQ ID NO: 17, 42, 101, 119, 137 of the present invention in a 5 nM or 50 nM MDA-MB-231 cell line Inhibition amount and thus cell viability reduction amount graph, A is the target gene expression inhibition amount, B is the cell viability reduction amount.

10 is a nano-contained double-stranded RNA structure comprising an siRNA having a sense strand of SEQ ID NO: 12, 17, 42, 101, 108, 114, 116, 119, 137, 141, 170 or 171 of the present invention When the particles were treated with the MDA-MB-231 cell line at a concentration of 100 nM or 200 nM, it is a graph showing the amount of inhibition of expression of the target gene.

Figure 11 is a ligand (at a concentration of 100 nM, 200 nM or 500 nM of nanoparticles consisting of a double-stranded oligo RNA structure comprising all of the siRNA comprising the sense strand of SEQ ID NO: 42, 101, 137 according to the present invention) Inhibition amount of the target gene according to the presence or absence of glucose) is a graph analyzed in the MDA-MB-231 cell line.

Figure 12 is a ligand to the same nanoparticles (SAMiRNA-STAT3-Combo) consisting of a double-stranded oligo RNA structure comprising all of the siRNA comprising the sequence of SEQ ID NO: 42, 101, 137 as the sense strand according to the present invention It is a graph measuring the size and polydispersity index (PDI) of glucose-containing nanoparticles (Glucose-SAMiRNA-STAT3-Combo).

Figure 13 is a microvenous administration of mouse breast cancer xenograft model of the nanoparticles (SAMiRNA-STAT3-Combo) containing all of the double-stranded RNA structure comprising a siRNA having the sense strand of SEQ ID NO: 42, 101, 137 of the present invention In order to confirm the tumor growth inhibitory effect, after administration of the nanoparticle microvenous, it is a graph confirming the size of the tumor. PBS, PBS (excipient) administration control group; SAMiRNA-CONT, nanoparticle 5 mg / kg body weight administration control comprising siRNA having the sense strand of SEQ ID NO: 201; Experimental group administered with 1 mg / kg body weight or 5 mg / kg body weight of nanoparticles (SAMiRNA-CONT-Combo) containing all of the siRNAs having the sense strand of SAMiRNA-STAT3, SEQ ID NOs: 42, 101, and 137 it means. To confirm statistical significance, Two-way ANOVA with Bonferroni postests was used with GraphPad PRISM5 software (La Jolla, Calif.), And p <0.05 was determined as a significant difference between experimental groups. #, p <0.05 versus PBS; ###, p <0.001 versus PBS; *, p <0.05 versus SAMiRNA-CONT (5 mpk); **, p <0.01 versus SAMiRNA-CONT (5 mpk); ***, p <0.001 versus SAMiRNA-CONT (5 mpk)

Figure 14 includes all of the double-stranded RNA structure comprising a siRNA having a sense strand of SEQ ID NO: 42, 101, 137 of the present invention, nanoparticles containing glucose as a ligand (Glucose-SAMiRNA-STAT3-Combo) In order to confirm the tumor growth inhibitory effect of the mouse breast cancer xenograft model of the intravenous administration, the graph of the tumor size after the nanoparticle microvenous administration. PBS, PBS (excipient) administration control group; Glucose-SAMiRNA-CONT, nanoparticle 5 mg / kg body weight administration control comprising siRNA having the sense strand of SEQ ID NO: 201; 1 mg / kg body weight or 5 mg / kg body weight of nanoparticles (Glucose-SAMiRNA-CONT-Combo) containing all of the siRNAs having the sense strand of Glucose-SAMiRNA-STAT3, SEQ ID NOs: 42, 101, and 137 Means the experimental group administered. To confirm statistical significance, Two-way ANOVA with Bonferroni postests was used with GraphPad PRISM5 software (La Jolla, Calif.), And p <0.05 was determined as a significant difference between experimental groups. ###, p <0.001 versus PBS; ‡‡, p <0.01 versus Glucose-SAMiRNA-CONT (5 mpk); ‡‡‡, p <0.001 versus Glucose-SAMiRNA-CONT (5 mpk)

15 and 16 are the same as the nanoparticles (SAMiRNA-STAT3-Combo, FIG. 15) including all the double-stranded RNA structures including siRNAs having the sense strands of SEQ ID NOs: 42, 101, and 137 of the present invention. In order to confirm the tumor growth inhibitory effect of the mouse breast cancer xenograft model in which the glucose-attached nanoparticles (Glucose-SAMiRNA-STAT3-Combo, FIG. 16) are attached to the particles, the tumors after the administration of the microparticles of the nanoparticles, Is a graph confirming the weight (angle A of FIGS. 15 and 16) and the percentage of tumor weight to weight ratio (angle B of FIGS. 15 and 16). PBS, PBS (excipient) administration control group; SAMiRNA-CONT, nanoparticle 5 mg / kg body weight administration control comprising siRNA having the sense strand of SEQ ID NO: 201; Experimental group for administration of 1 mg / kg body weight or 5 mg / kg body weight of nanoparticles (SAMiRNA-CONT-Combo) including all siRNAs having a sense strand of the sequence of SAMiRNA-STAT3, SEQ ID NOs: 42, 101, and 137; Glucose-SAMiRNA-CONT, nanoparticle 5 mg / kg body weight administration control comprising siRNA having the sense strand of SEQ ID NO: 201; 1 mg / kg body weight or 5 mg / kg body weight of nanoparticles (Glucose-SAMiRNA-CONT-Combo) containing all of the siRNAs having the sense strand of Glucose-SAMiRNA-STAT3, SEQ ID NOs: 42, 101, and 137 Means the experimental group administered. In order to confirm statistical significance, One-way ANOVA and Dunnett's Multiple comparison test was performed using GraphPad PRISM5 software (La Jolla, CA), and p <0.05 was determined as a significant difference between the experimental groups. ##, p <0.01 versus PBS; *, p <0.05 versus SAMiRNA-CONT (5 mpk); **, p <0.01 versus SAMiRNA-CONT (5 mpk); ‡, p <0.05 versus Glucose-SAMiRNA-CONT (5 mpk).

Figure 17 shows the expression of the identified target gene after transforming the siRNA having a sense strand of the SEQ ID NO: 42 of the present invention into a HaCaT cell line at a concentration of 50, 100, 200, 500 nM or 1 μM Inhibition graph.

Detailed description of the invention and preferred Embodiment

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is obvious to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1. Target Sequence Design of STAT3 and Preparation of siRNA

200 kinds of target sequences (sense strands) capable of binding to the mRNA sequence (NM_213662) of the STAT3 gene were designed, and siRNAs of antisense strands, which were complementary sequences of the target sequences, were prepared. First, the target sequence sequence to which siRNA can bind in the mRNA sequence of the genes was designed using the gene design program (Turbo si-Designer) developed by Bioneer Corporation. The siRNA for the STAT3 gene of the present invention is a double stranded structure consisting of a sense strand consisting of 19 nucleotides and an antisense strand complementary thereto. In addition, siCONT (SEQ ID NO: 201), a siRNA having a sequence that does not inhibit expression of any gene, was prepared. The siRNA was prepared by linking phosphodiester bonds forming an RNA backbone structure using β-cyanoethyl phosphoramidite (Nucleic Acids Research, 12: 4539-4557, 1984). Specifically, using a RNA synthesizer (384 Synthesizer, BIONEER, Korea), a series of processes consisting of deblocking, coupling, oxidation, and capping on a solid support to which nucleotides are attached Was repeated to obtain a reaction containing RNA of the desired length. The reaction was isolated and purified by HPLC LC918 (Japan Analytical Industry, Japan) equipped with Daisogel C18 (Daiso, Japan) column and matched with target sequence using MALDI-TOF mass spectrometer (Shimadzu, Japan). Confirmed. Then, the desired double-stranded siRNA (SEQ ID NOS: 1-201) was prepared by combining the sense and antisense RNA strands (see Table 2).

Example 2 Synthesis of Double Helix Oligo RNA Constructs

The double-stranded oligo RNA structure prepared in the present invention has a structure as shown in the following structural formula (5).

Structural Formula 5

In the above formula (5), S is the sense strand of the siRNA, AS is the antisense strand of the siRNA, PO ₄ is a phosphate group, ethylene glycol is a hydrophilic monomer (monomer) is a repeating frequency of A of the formula (3), hydrophilic monomer m is as 6 hexaethylene glycol (hexa ethylene glycol) linker (J) a phosphoric acid group (PO ₃ ^-) by being coupled through the repetition number (n) of hydrophilic material block 4, C ₂₄ is a hydrophobic material (B) Tetradocosane, which contains disulfide bonds, and 5 'and 3' refer to the terminal orientation of the double helix oligo RNA, and glucose is a ligand that enhances receptor mediated intracellularization (RME).

The sense strand of siRNA in the above formula (5) is β-cyanoethyl in the manner mentioned above using DMT-hexaethylin glycol-CPG prepared according to the method described in Example 1 of the existing patent (WO2015002511) as a support. By using phosphamidite to connect phosphodiester bonds forming an RNA framework, oligo RNA-hydrophilic substance structures including a sense strand having hexaethylene glycol bonded to the 3 'terminal are synthesized. Tetradocoic acid containing disulfide bonds was bound to the 5 'end to prepare a sense strand of a desired RNA-polymer construct. In the case of the antisense strand to be annealed with the strand, the antisense strand of the sequence complementary to the sense strand was prepared through the reaction mentioned above.

After the synthesis was completed, deprotection was performed by separating the synthesized RNA single-strand and RNA-polymer constructs from CPG by treating 28% (v / v) ammonia in a 60 ° C water bath. The protective residue was removed through the reaction. RNA single-stranded and RNA-polymerized structures with no protective residues were added in a volume ratio of N-methylpyrrolidon, triethylamine and triethylaminetrihydrofluoride in an oven at 70 ° C. 2 'TBDMS (tert-butyldimethylsilyl) was removed by treatment at a ratio of 10: 3: 4.

The reaction was isolated and purified by HPLC LC918 (Japan Analytical Industry, Japan) equipped with Daisogel C18 (Daiso, Japan) column and matched with target sequence using MALDI-TOF mass spectrometer (Shimadzu, Japan). Confirmed. Subsequently, the same amount of the sense strand and the antisense strand were mixed in the same manner to prepare each double-stranded oligo RNA structure, 1 × annealing buffer (30 mM HEPES, 100 mM potassium acetate), 2 mM magnesium acetate, pH 7.0 -7.5), reacted for 3 minutes in a 90 ° C constant temperature water bath and then again at 37 ° C, SEQ ID NOs: 12, 17, 42, 101, 108, 114, 116, 119, 137, 141, 170, 171 and Double-stranded oligo RNA constructs comprising siRNA having the sense strand of siRNA 201 (hereinafter SAMiRNA-STAT3 # 12, 17, 42, 101, 108, 114, 116, 119, 137, 141, 170, 171 and SAMiRNA- Each called CONT). It was confirmed that the prepared double helix oligo RNA structure was annealed through electrophoresis.

Example 3 Preparation and Size and Polydispersity Index of Nanoparticles (SAMiRNA) Consisting of Double Helix Oligo RNA Structure

The double-stranded oligo RNA structure synthesized in Example 2 forms a micelle (micelle) as nanoparticles by hydrophobic interaction between hydrophobic materials bound to the ends of the double-stranded oligo RNA (see FIG. 1).

The formation of nanoparticles composed of the corresponding SAMiRNA was confirmed by analyzing the size (diameter, d.nm) and polydispersity index (hereinafter, PDI (polydispersity index)) of the nanoparticles consisting of SAMiRNA-STAT3 and SAMiRNA-CONT.

Example 3-1. Preparation of Cellular Nanoparticles

50 μg of SAMiRNA-STAT3 # 12, 17, 42, 101, 108, 114, 116, 119, 137, 141, 170, 171 and SAMiRNA-CONT synthesized in Example 2 were added to 1.5 ml Phosphate Buffered Saline (PBS). After dissolving at a concentration of / ㎖, to prepare a nanoparticle powder by lyophilization for 48 hours at -75 ℃, 5mTorr conditions, dissolved in 1.5 ㎖ PBS solvent to prepare homogenized nanoparticles were used for cell experiments.

Example 3-2. Preparation of Nanoparticles for Animal Experiments

After dissolving the SAMiRNA-CONT synthesized in Example 2 at a concentration of 250 ㎍ / ㎖ in 1 ml PBS (Phosphate Buffered Saline) to prepare a nanoparticle powder by lyophilization for 6 days at -75 ℃, 5mTorr conditions , Dissolved in 0.2 mL PBS solvent to prepare homogenized nanoparticles were used in animal experiments.

The same amount of SAMiRNA-STAT3 # 42, # 101, # 137 synthesized in Example 2 was mixed and dissolved in 1 ml PBS (Phosphate Buffered Saline) at a concentration of 250 μg / ml, and then, at -75 ° C. and 5 mTorr conditions. Nanoparticle powders were prepared by daily freeze-drying, and then dissolved in 0.2 ml PBS, a solvent, to prepare homogenized nanoparticles and used in animal experiments.

The prepared SAMiRNA-CONT homogenizer and SAMiRNA-STAT3 homogenizer were diluted 1/25 in PBS and used for cell transformation confirmation experiments of animal nanoparticles at a concentration of 50 μg / ml.

Example 3-3. Nanoparticle Size and PDI Measurement

The size and dispersion of the nanoparticles were measured by a dynamic light-scattering method. The homogenized nanoparticles prepared in Examples 3-1 and 3-2 were measured for size and dispersion with a dynamic light scattering meter (Nano-ZS, MALVERN, UK), the refractive index of the material (Refractive index) is 1.459, the absorption rate (Absorption index) was 0.000, the temperature of the solvent PBS 25 ℃ and the resulting viscosity (viscosity) was measured by entering the value of 1.0200 and the refractive index of 1.335. One measurement consisted of a size measurement consisting of 15 repetitions, which were repeated six times.

As a result, the average cell size of the nanoparticles was 98.7 ± 26.4 (mean ± SD, n = 6) and the polydispersity index value of 0.289 ± 0.064 (mean ± SD, n = 6). ), For animal experimental nanoparticles, the average 79.4 ± 6.3 (mean ± SD, n = 6) d.nm size and 0.265 ± 0.007 (mean ± SD, n = 6) polydispersity index values (see FIG. 12). ) Confirmed.

As the value of the polydispersity index was lower, the particles were evenly distributed, and the nanoparticles of the present invention were found to have a very uniform size.

Example 4 Confirmation of Inhibition of Target Gene Expression Using siRNA in Human Breast Cancer Cell Line, MCF-7

Human breast cancer cell line, MCF-7, was transformed using siRNA having a sense strand of SEQ ID Nos. 1 to 201 prepared in Example 1, and the expression pattern of the target gene was analyzed in the transformed cell line.

Example 4-1. Culture of Human Breast Cancer Cell Line, MCF-7

Human breast cancer cell line, MCF-7, obtained from the American Type Culture Collection (ATCC) was cultured using RPMI 1640 (Roswell Park Memorial Institute 1640) culture medium (GIBCO / Invitrogen, USA, 10% (v / v) fetal bovine serum. , 100 units / ml penicillin and 100 μg / ml streptomycin) were incubated at 37 ° C. under 5% (v / v) CO ₂ .

Example 4-2. Transfection of STAT3 siRNA in Human Breast Cancer Cell Line, MCF-7

MCF-7 cell line cultured in Example 4-1 in a 12-well plate at 37 ℃, 5% (v / v) CO ₂ in RPMI 1640 culture medium for 18 hours at a cell number of 1 × 10 ⁵ per well After culturing under conditions, 500 μl of Opti-MEM medium (GIBCO, USA) was dispensed per well after removing the medium.

2 μl of Lipofectamine RNAi Max (Invitrogen, USA) and 248 μl of Opti-MEM medium were mixed to prepare a mixed solution, reacted for 5 minutes at room temperature, and then the respective sequences prepared in Example 1 above. 1 μl or 5 μl of siRNA (1 pmole / μl concentration) having the sense strand No. 1 to 201 was added to Opti-MEM medium to prepare a siRNA solution having a concentration of 4 nM or 20 nM in the final 250 μl amount. . The lipofectamine RNAi Max (Lipofectamine ™ RNAi Max) mixture and the siRNA solution were mixed and reacted for 15 minutes at room temperature to prepare a solution for transfection.

After dispensing 500 μl of the transfection solution into each well of the tumor cell line of the 12-well plate in which Opti-MEM was dispensed, and incubating for 6 hours, the Opti-MEM medium was removed and 1 ml of RPMI 1640 culture medium was dispensed per well. Incubated for 24 hours at 37 ° C. and 5% (v / v) CO ₂ conditions.

Example 4-3. Quantitative Analysis of Target Gene mRNA

After cDNA was prepared by extracting total RNA from the cell line transfected in Example 4-2, mRNA expression of the target gene was quantified relative to each other using real-time PCR.

Example 4-3-1. RNA Isolation and cDNA Preparation from Transfected Cells

Using RNA extraction kit (AccuPrep Cell total RNA extraction kit, BIONEER, Korea), the total RNA is extracted from the cell line transfected in Example 4-2, the extracted RNA is RNA reverse transcriptase (AccuPower RocketScript Cycle RT Premix) / dT20, Bioneer, Korea), to prepare a cDNA in the following manner. Specifically, 1 μg of RNA extracted per tube was added to AccuPower RocketScript Cycle RT Premix / dT20 (Bioneer, South Korea) contained in 0.25 ml Eppendorf tubes and treated with DEPC (diethyl pyrocarbonate) to achieve a total volume of 20 μl. Distilled water was added. This was a gene amplifier (MyGenie ™ 96 Gradient Thermal Block, BIONEER, Korea), hybridizing RNA and primer for 30 seconds at 37 ° C, preparing cDNA at 48 ° C for 4 minutes (cDNA synthesis), and then at 55 ° C. After repeating the denaturation process for 30 seconds 12 times, the amplification reaction was terminated by inactivating the enzyme at 95 ° C. for 5 minutes.

Example 4-3-2. Relative Quantitative Analysis of Target Gene mRNA

Using the cDNA prepared in Example 4-3-1 as a template, the relative amount of STAT3 gene mRNA was quantified by real-time PCR in the following manner. The cDNA prepared in Example 4-3-1 was diluted 5 times with distilled water, 3 μl of diluted cDNA and 25 μl of 2 × GreenStar ™ PCR master mix (BIONEER, Korea), 16 μl of distilled water, STAT3 qPCR primer (F , 10 pmole / μl of each of the mixture, BIONEER, Korea, see Table 3) 6 μl of the mixture was placed in a 96-well PCR plate. On the other hand, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), a housekeeping gene (hereinafter referred to as HK gene), was used as a standard gene to compare and correct the expression level of target gene mRNA. The 96-well plate containing the mixed solution was subjected to the following reaction using an Exicycler ™ 96 Real-Time Quantitative Thermal Block (BIONEER, Korea). After 15 minutes of reaction at 95 ° C to remove enzyme activation and secondary structure of cDNA, denaturing at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 30 seconds, SYBR Four procedures of the SYBR green scan were repeated 42 times, followed by a final extension at 72 ° C. for 3 minutes, then maintaining the temperature at 55 ° C. for 1 minute, and melting from 55 ° C. to 95 ° C. curve). After the completion of PCR, the obtained Ct (threshold cycle) value of each target gene was obtained by calculating the Ct value of the target gene corrected through the GAPDH gene, and then treated with siRNA (siCONT) of the control sequence that does not cause gene expression inhibition. The difference of ΔCt value was calculated | required using the experimental group as a control. Using the ΔCt value and the formula 2 (−ΔCt) × 100, the expression level of the target gene of the cells treated with STAT3-specific siRNA was quantified (see FIG. 3, 5 nM concentration siRNA treatment; see FIG. 4, 1 nM). Concentration siRNA treatment). In order to select high efficiency siRNAs, 20 siRNAs having a high mRNA expression level for a target gene were reduced at 1 nM and 5 nM concentrations (SEQ ID NOs: 12, 17, 42, 55, 91, 95, 101, 108, 111, 114, 116, 119, 137, 139, 141, 152, 166, 168, 170, 171 with sense strands).

Example 5 Expression Inhibition of Target Genes Using siRNA in Human Breast Cancer Cell Line, MDA-MB-231.

Prepared in Example 1, and selected from SEQ ID NOs: 12, 17, 42, 55, 91, 95, 101, 108, 111, 114, 116, 119, 137, 139, 141, 152 Another human breast cancer cell line, MDA-MB-231, was transformed using siRNA having sense strands 166, 168, 170, and 171, and the expression pattern of the target gene was analyzed in the transformed cell line.

Example 5-1. Culture of Human Breast Cancer Cell Line, MDA-MB-231

Human breast cancer cell line, MDA-MB-231, obtained from the American Type Culture Collection (ATCC) was cultured under the same conditions as in Example 4-1.

Example 5-2. Transfection of STAT3 siRNA in Human Breast Cancer Cell Line, MDA-MB-231

The MDA-MB-231 cell line cultured in Example 5-1 was used in a 12-well plate at 37 ° C., 5% (v / v) CO 2, in an RPMI 1640 culture medium for 18 hours at a cell number of 0.8 × 10 5 per well. After culturing under conditions, 500 μl of Opti-MEM medium (GIBCO, USA) was dispensed per well after removing the medium.

2 μl of Lipofectamine RNAi Max (Invitrogen, USA) and 248 μl of Opti-MEM medium were mixed to prepare a mixed solution, reacted for 5 minutes at room temperature, and prepared in Example 1 above. SEQ ID NO: 12, 17, 42, 55, 91, 95, 101, 108, 111, 114, 116, 119, 137, 139, 141, 152, 166, 168, 170, 171 selected from 4-3-2. 0.2 μl, 1 μl or 5 μl of siRNA having a sense strand (1 pmole / μl concentration) was added to the Opti-MEM medium to prepare a siRNA solution having a concentration of 0.8 nM, 4 nM or 20 nM in the final 250 μl amount. . The lipofectamine RNAi Max (Lipofectamine ™ RNAi Max) mixture and the siRNA solution were mixed and reacted for 15 minutes at room temperature to prepare a solution for transfection.

After dispensing 500 μl of the transfection solution per tumor cell line of each well of Opti-MEM-dispensed 12-well plate and incubating for 6 hours, the Opti-MEM medium was removed and 1 ml of RPMI 1640 culture medium was dispensed per well. Incubated for 24 hours at 37 ° C. and 5% (v / v) CO ₂ conditions.

Example 5-3. Quantitative Analysis of Target Gene mRNA

Quantitative analysis was performed in the same manner as in Example 4-3 (see FIG. 5). Based on the results, 12 siRNAs with high efficiency at all siRNA concentrations of 0.2 nM, 1 nM and 5 nM were selected (SEQ ID NOs: 12, 17, 42, 101, 108, 114, 116, 119, 137, 141, 170, 171 sequence having sense strand).

Example 6. Measurement of IC ₅₀ (inhibition concentration 50%) in human breast cancer cell lines (MCF-7, MDA-MB-231)

The siRNA prepared in Example 1 and having the sense strand of the sequence of SEQ ID NOs 12, 17, 42, 101, 108, 114, 116, 119, 137, 141, 170 and 171 selected in Example 5-3 The human breast cancer cell line (MCF-7, MDA-MB-231) was transformed, and the exact efficiency of siRNA was analyzed by confirming the IC ₅₀ value by analyzing the expression level of the target gene in the transformed cell line.

Example 6-1. Culture of Human Breast Cancer Cell Lines (MCF-7, MDA-MB-231)

Human breast cancer cell lines (MCF-7, MDA-MB-231) obtained from the American Type Culture Collection (ATCC) were cultured under the same conditions as in Example 4-1.

Example 6-2. Transfection of Target siRNA in Human Breast Cancer Cell Lines (MCF-7, MDA-MB-231)

The MCF-7 and MDA-MB-231 cell lines cultured in Examples 4-1 and 5-1 above were RPMI 1640 for 18 hours in a 12-well plate at 37 ° C. and 5% (v / v) CO ₂ . After incubation in culture medium, 500 μl of Opti-MEM medium (GIBCO, USA) was dispensed per well after removing the medium.

2 μl of Lipofectamine RNAi Max (Invitrogen, USA) and 248 μl of Opti-MEM medium were mixed to prepare a mixed solution, reacted for 5 minutes at room temperature, and prepared in Example 1 above. 0.0016 μl, 0.008 μl, 0.04 μl, 0.2 μl, siRNA having the sense strand of SEQ ID NO: 12, 17, 42, 101, 108, 114, 116, 119, 137, 141, 170 or 171 selected from 5-3 Μl or 5 μl were added to Opti-MEM medium to prepare siRNA solutions with concentrations of 6.4 pM, 32 pM, 0.16 nM, 0.8 nM, 4 nM or 20 nM in amounts of the final 250 μl. The lipofectamine RNAi Max (Lipofectamine ™ RNAi Max) mixture and the siRNA solution were mixed and reacted for 15 minutes at room temperature to prepare a solution for transfection.

After dispensing 500 μl of the transfection solution into each well of Opti-MEM-dispersed tumor cell lines and incubating for 6 hours, the Opti-MEM medium was removed and 1 ml of RPMI 1640 culture medium was dispensed. Incubation was performed under conditions of 37 ° C. and 5% (v / v) CO _{2 for} a period of time.

Example 6-3. Quantitative Analysis of Target Gene mRNA

After extracting the entire RNA from the cell line transfected in Example 6-2 to prepare a cDNA, using the real-time PCR (real-time PCR) the mRNA expression amount of the target gene in the same manner as in Example 4-3 Relative quantification (see Figure 6, MCF-7 cell line; see Figure 7, MDA-MB-231 cell line).

Example 6-4. IC ₅₀ analysis

SoftMax Pro program (Molecular Devices, USA) was used to confirm the IC ₅₀ value based on the results identified in Example 6-3. Nonlinear regression analysis was performed by converting the% expression level of the target gene into the% inhibition rate by siRNA, graphing the log of the concentration of the siRNA, and substituting it into the 4-parameter logistics model. The IC ₅₀ value was confirmed through (see FIG. 8). IC ₅₀ values in two breast cancer cells clearly identified the efficacy of each siRNA (see Table 4, SEQ ID NOs: 17, 42, 101, 119, 137, five sequences were selected).

Example 7. Expression inhibition and cell growth inhibition of target genes by STAT3-specific siRNA

A breast cancer cell line, MDA-MB-231, was transformed at a concentration of 5, 50 nM of an siRNA having a sense strand of SEQ ID NOs: 17, 42, 101, 119, and 137, which are the highly efficient siRNA selected in Example 6-4. After the expression of the target gene expression inhibition and cell growth inhibition was confirmed.

Example 7-1. Transfection of Target siRNA in Human Liver Cancer Cell Lines

The MDA-MB-231 cell line cultured in Example 5-1 was RPMI for 18 hours at a cell number of 0.6 × 10 ⁵ per well in a 12-well plate under conditions of 5% (v / v) CO ₂ at 37 ° C. After incubation in -1640 culture medium, 500 μl of Opti-MEM medium (GIBCO, USA) was dispensed per well after the medium was removed. Meanwhile, 2 μl of Lipofectamine RNAi Max (Invitrogen, USA) and 248 μl of Opti-MEM medium were mixed to prepare a mixed solution, reacted for 5 minutes at room temperature, and prepared in Example 1, 5 μl or 50 μl of siRNA (1 pmole / μl) having the sense strand of SEQ ID NO: 17, 42, 101, 119, 137 selected in Example 6-3 was added to the Opti-MEM medium so that the total amount was 250 μl. SiRNA solutions having concentrations of 20 nM or 200 nM, respectively, were prepared. The lipofectamine RNAi Max (Lipofectamine ™ RNAi Max) mixture and the siRNA solution were mixed and reacted for 15 minutes at room temperature to prepare a solution for transfection.

Thereafter, 500 μl of the solution for transfection was dispensed into each well of the tumor cell line to which Opti-MEM was dispensed, and cultured for 6 hours, and then the Opti-MEM medium was removed. Here, 1 ml of RPMI 1640 culture medium was dispensed and then incubated at 37 ° C. for 5 hours under 5% (v / v) CO ₂ .

Example 7-2. Quantitative Analysis of Target Gene mRNA

Quantitative analysis was performed in the same manner as in Example 4-3 (see FIGS. 9 and A).

Example 7-3. Confirmation of cell growth inhibition

The cells transformed in Example 7-1 were washed twice with 500 μl of DPBS per well, and 500 μl of TrypLETM Express with Phenol Red (1 ×, Gibco, USA) per well was used at 37 ° C. for 5% (v / v) The cells were suspended by treatment for 2 minutes under conditions of CO ₂ . Stop the reaction by adding 500 μl RPMI 1640 culture medium per well, mix 100 μl with the same amount of 2x Trypan Blue Solution, and take 10 μl of LUNATM Automated Cell Counter (Logos Biosystems, USA). ) Was used to measure the number of cells. The measurement was performed three times (see Fig. 9, B).

As a result of confirming the cell proliferation inhibitory effect through the cell number, it was confirmed that the cell viability decreased in a concentration-dependent manner, in particular, siRNA having the sense strand of SEQ ID NO: 42, 101, 137 was treated with 50 nM. The experimental group was found to have about 40% inhibition in vitro .

Example 8 Confirmation of Inhibition of Target Gene Expression Using Nanoparticles (SAMiRNA-STAT3) Consisting of Double Helix Oligo RNA Structures Containing STAT3-Specific siRNA in Human Breast Cancer Cell Line, MDA-MB-231

SiRNA prepared in Example 3-1 and having the sense strand as SEQ ID NOs: 12, 17, 42, 101, 108, 114, 119, 137, 141, 166, 170, and 171 selected in Example 5-3 Of the target gene in the human breast cancer cell line, MDA-MB-231 using a nanoparticle comprising (SAMiRNA-STAT3 # 12, 17, 42, 101, 108, 114, 119, 137, 141, 166, 170, 171). Expression patterns were analyzed.

Example 8-1. Culture of Human Breast Cancer Cell Line, MDA-MB-231

Human breast cancer cell line, MDA-MB-231, obtained from the American Type Culture Collection (ATCC) was cultured under the same conditions as in Example 5-1.

Example 8-2. Treatment of SAMiRNA-STAT3 in Human Breast Cancer Cell Line, MDA-MB-231

MDA-MB-231 cell line incubated in Example 5-1 in a 12-well plate at 37 ℃, 5% (v / v) CO in RPMI 1640 culture medium for 18 hours at a cell number of 0.6 × 10 ⁵ per well _The culture was carried out under the conditions of ₂ .

In order to prepare a SAMiRNA treatment solution, 26.6 μl or 53.2 μl of each was prepared in Example 3-1 and 50 μg selected in Example 5-3 in each 973.4 μl or 946.8 μl of OPTI-MEM medium. SAMiRNA treatment of 100 nM or 200 nM by mixing SAMiRNA-STAT3 # 12, 17, 42, 101, 108, 114, 119, 137, 141, 166, 170, 171 or SAMiRNA-CONT at a concentration of /3.3 (13.3 uM) Liquid was prepared.

After removing the medium, 1 ml of 100 nM or 200 nM SAMiRNA treatment solution per well was treated twice a day for 2 days, and then cultured under 37 ° C. and 5% (v / v) CO ₂ conditions.

Example 8-3. Quantitative Analysis of Target Gene mRNA

Quantitative analysis was performed in the same manner as in Example 4-3 (see FIG. 10).

Example 9 According to the presence or absence of Glucose Ligand of Nanoparticles (SAMiRNA-STAT3-Combo) consisting of a double-stranded oligo RNA construct containing all STAT3-specific siRNAs for animal experiments in human breast cancer cell line, MDA-MB-231 Comparison of expression inhibition of target genes

Prepared in Example 3-2, the same as the animal experimental nanoparticles (SAMiRNA-STAT3-Combo) containing all of the siRNA having a sense strand of SEQ ID NO: 42, 101, 137 selected in Example 7-3 nano Inhibition of target gene expression by nanoparticles (Glucose-SAMiRNA-STAT3-Combo) with glucose as the ligand in the particles was compared and analyzed in human breast cancer cell line, MDA-MB-231.

Example 9-1. Culture of Human Breast Cancer Cell Line, MDA-MB-231

Human breast cancer cell line, MDA-MB-231, obtained from the American Type Culture Collection (ATCC) was cultured under the same conditions as in Example 5-1.

Example 9-2. Treatment of Nanoparticles in Human Breast Cancer Cell Line, MDA-MB-231

MDA-MB-231 cell line incubated in Example 5-1 in a 12-well plate at 37 ℃, 5% (v / v) CO in RPMI 1640 culture medium for 18 hours at a cell number of 0.6 × 10 ⁵ per well _The culture was carried out under the conditions of ₂ .

50 μg diluted after preparation in Example 3-2 of 26.6 μl, 53.2 μl or 133.0 μl each in 973.4 μl, 946.8 μl or 867.0 μl of OPTI-MEM medium to prepare a SAMiRNA treatment solution. SAMiRNA-treated solutions of 100 nM, 200 nM or 500 nM were prepared by mixing SAMiRNA-STAT3-Combo or Glucose-SAMiRNA-STAT3-Combo at a concentration of / ml (13.3 uM).

After removing the medium, 1 ml of 100 nM, 200 nM, or 500 nM of SAMiRNA treatment solution per well was treated twice a day for 2 days and incubated under 37 ° C. and 5% (v / v) CO ₂ conditions.

Example 9-3. Quantitative Analysis of Target Gene mRNA

Quantitative analysis was performed in the same manner as in Example 4-3 (see FIG. 11). After confirming that the inhibitory expression of the target gene was equally distributed by the F-test of the two groups for variance, and performing t-test on the two equally distributed families, p <0.05 was obtained at the 500 nM treatment concentration. Significantly, Glucose-SAMiRNA-STAT3-Combo nanoparticles were found to inhibit the expression of target genes more strongly than SAMiRNA-STAT3-Combo.

Example 10 Confirmation of Tumor Growth Inhibition Effect of Nanoparticles Consisting of Double Helix Oligo RNA Structure Containing STAT3-Specific siRNA in Animal Tumor Model

Prepared in Example 3-2, the same as the animal experimental nanoparticles (SAMiRNA-STAT3-Combo) containing all siRNA having a sense strand of SEQ ID NO: 42, 101, 137 selected in Example 7-3 Tumor growth inhibitory effect in mouse breast cancer xenograft model by glucose nanoparticles (Glucose-SAMiRNA-STAT3-Combo) as a ligand to the nanoparticles was confirmed.

Example 10-1. Construction of mouse breast cancer xenograft model

Subcutaneous injection of the human breast cancer cell line, MDA-MB-231, 3 × 10 ⁶ cells cultured in Example 5-1 into the left flank of female nude mice (Balb / c nude mice) , The tumor size was measured at intervals of 2 days to confirm the engraftment and growth of tumor cells to prepare a model.

Example 10-2. Confirmation of weight change by nanoparticles consisting of a double-stranded oligo RNA structure containing STAT3-specific siRNA

Nanoparticles having glucose as a ligand to the same nanoparticles as animal experimental nanoparticles (SAMiRNA-STAT3-Combo) containing all of the siRNA having a sense strand of SEQ ID NO: 42, 101, 137 prepared in Example 3-2 In order to confirm the weight change of the experimental animals by microvenous administration of the particle (Glucose-SAMiRNA-STAT3-Combo), the body weight was measured at two-day intervals (see Table 5, SAMiRNA-STAT3-Combo-administered group; Table 6). See Glucose-SAMiRNA-STAT3-Combo administration group). As a result, no significant weight gain or weight loss was observed, indicating that there were no external factors that could affect the interpretation of the results.

Example 10-3. Confirmation of tumor growth inhibition by nanoparticles consisting of double helix oligo RNA constructs containing STAT3-specific siRNA

Prepared in Example 3-2, the same as the animal experimental nanoparticles (SAMiRNA-STAT3-Combo) containing all of the siRNA having a sense strand of SEQ ID NO: 42, 101, 137 selected in Example 7-3 nano Tumor growth inhibitory effect in mouse breast cancer xenograft model by nanoparticles (Glucose-SAMiRNA-STAT3-Combo) having glucose as ligand in the particles was confirmed.

Example 10-3-1. Experimental grouping of mouse breast cancer xenograft model

When the average tumor size of the mouse breast cancer model produced in Example 8-1 was 140 mm 3, the experimental groups were organized in groups of 6 per group according to the tumor size.

Example 10-3-2. Administration of nanoparticles consisting of a double helix oligo RNA construct comprising STAT3-specific siRNA

Prepared in Example 3-2, the same as the animal experimental nanoparticles (SAMiRNA-STAT3-Combo) containing all of the siRNA having a sense strand of SEQ ID NO: 42, 101, 137 selected in Example 7-3 nano In a concentration of 1 mg / kg body weight or 5 mg / kg body weight, nanoparticles (Glucose-SAMiRNA-STAT3-Combo) containing glucose as a ligand in the particles were used, and 100 µl of the dose was injected into 1 ml syringe (0.25 mm x 8 mm, 31 Gauge). , BD328820, USA) was administered intravenously to the experimental animal group configured in Example 10-3-1 once a day for 2 weeks, for a total of 14 consecutive weeks, and all the following administration groups were blind. ).

Administration group: PBS, PBS (excipient) administration control group; SAMiRNA-CONT, nanoparticle 5 mg / kg body weight administration control comprising siRNA having the sense strand of SEQ ID NO: 201; Experimental group for administration of 1 mg / kg body weight or 5 mg / kg body weight of nanoparticles (SAMiRNA-CONT-Combo) including all siRNAs having a sense strand of the sequence of SAMiRNA-STAT3, SEQ ID NOs: 42, 101, and 137; Glucose-SAMiRNA-CONT, nanoparticle 5 mg / kg body weight administration control comprising siRNA having the sense strand of SEQ ID NO: 201; Administration of 1 mg / kg body weight or 5 mg / kg body weight of nanoparticles (Glucose-SAMiRNA-CONT-Combo) containing all siRNAs having the sense strand of Glucose-SAMiRNA-STAT3, SEQ ID NOs: 42, 101, and 137 Experimental group

Example 10-3-3. Confirmation of effect by measuring tumor size

Tumor size was measured at 2, 4, 6, 8, 10, 12, and 14 days after administration during the 14-day continuous administration period of Example 10-3-2 (see FIGS. 13 and 14). On the 14th day of the last administration day, 30% of SAMiRNA-STAT3 (1mg / kg body weight) and 37% of SAMiRNA-STAT3 (5mg / kg body weight) were compared with PBS, an excipient control group. In comparison with the negative control SAMiRNA-CONT, SAMiRNA-STAT3 (1 mg / kg body weight) was 24% and SAMiRNA-STAT3 (5mg / kg body weight) was 31%. In addition, compared to PBS, an excipient control group, Glucose-SAMiRNA-STAT3 (1 mg / kg body weight) was 9%, Glucose-SAMiRNA-STAT3 (5 mg / kg body weight) was 46%, and a negative control Glucose-SAMiRNA-CONT was used. In contrast, Glucose-SAMiRNA-STAT3 (1mg / kg body weight) was -15% and Glucose-SAMiRNA-STAT3 (5mg / kg body weight) was 32%. Tumor growth inhibitory effect was not shown, but Glucose-SAMiRNA-STAT3 (5mg / kg body weight) showed significant tumor growth inhibition effect.

Example 10-3-4. Tumor Weighing to Determine Effectiveness

At 24 hours after the last dose on day 14, the animals were sacrificed to determine tumor weight and tumor weight to tumor ratio (%) (see FIGS. 15 and 16). SAMiRNA-STAT3 (1mg / kg body weight) was 30%, 27% and SAMiRNA-STAT3 (5mg / kg body weight) was 43% and 43% compared to PBS, an excipient control group. SAMiRNA-STAT3 (1 mg / kg body weight) was 32% and 30%, and SAMiRNA-STAT3 (5 mg / kg body weight) was 45% and 46%, compared to SAMiRNA-CONT, a negative control. In addition, Glucose-SAMiRNA-STAT3 (1mg / kg body weight) was -10%, -18%, and Glucose-SAMiRNA-STAT3 (5mg / kg body weight) was 40% and 39% compared to PBS, an excipient control group. The anticancer effect of Glucose-SAMiRNA-STAT3 (1mg / kg body weight) was not shown. However, Glucose-SAMiRNA-STAT3 (5mg / kg body weight) showed a significant anticancer effect. Glucose-SAMiRNA-STAT3 (1mg / kg body weight) was -30% and -40%, and Glucose-SAMiRNA-STAT3 (5mg / kg body weight) was 30% and 28%, respectively. Tumor growth inhibitory effect was not shown by (1mg / kg body weight), but anti-cancer effect was shown in Glucose-SAMiRNA-STAT3 (5mg / kg body weight).

Example 10-4. Regulations on handling, care and disposal of laboratory animals

All methods for handling, managing, and treating laboratory animals were performed in accordance with the approval and regulations of the Committee on Animal Research at Bioneer Corporation (AEC-20081229-0004). The experimental animals were visually observed the health status, such as daily activity, fecal status, and confirmed the changes in dietary intake and weight.

Example 11 Confirmation of Inhibition of Target Gene Expression Using Nanoparticles (SAMiRNA-STAT3) Consisting of Double Helix Oligo RNA Constructs Containing STAT3-Specific siRNA in Human Epidermal Keratin-forming Cell Line (Keratinocyte Cell Line), HaCaT

Target in the human keratin-forming cell line, HaCaT, using nanoparticles (SAMiRNA-STAT3 # 42) prepared from Example 3-1 and containing siRNA having the sense strand of SEQ ID NO: 42 selected in Example 5-3 The expression patterns of the genes were analyzed.

Example 11-1. Human Epidermal Keratin-forming Cell Line (Keratinocyte Cell Line), Culture of HaCaT

HaCaT, a human keratinocytes cell line obtained from the American Type Culture Collection (ATCC), is a DMEM (Dulbecco's modified Eagle's medium) culture medium (GIBCO / Invitrogen, USA, 10% (v / v) fetal bovine serum, penicillin 100 units / ml and streptomycin 100 μg / ml) at 37 ° C. under 5% (v / v) CO ₂ .

Example 11-2. Treatment of SAMiRNA-STAT3 in the Human Epidermal Keratin-forming Cell Line (Keratinous Cell Line), HaCaT

HaCaT cell lines cultured in Example 11-1 were incubated in 12-well plates at 37 ° C., 5% (v / v) CO ₂ in DMEM culture medium for 18 hours at a cell number of 0.6 × 10 5 per well.

Prepared in Example 3-1 of 2.8, 5.6, 11.2, 28.1 μl or 56.1 μl each in 997, 994, 989, 972 μl or 944 μl of OPTI-MEM medium to prepare a SAMiRNA treatment solution. SAMiRNA treatments of 50, 100, 200, 500 nM or 1 μM were prepared by mixing SAMiRNA-STAT3 # 42 or SAMiRNA-CONT at 250 μg / ml (17.8 μM) concentrations selected in Example 5-3.

After removing the medium, 1 ml of 50, 100, 200, 500 nM or 1 μM SAMiRNA treatment solution per well was treated twice a day for 2 days and incubated under 37 ° C. and 5% (v / v) CO ₂ conditions. .

Example 11-3. Quantitative Analysis of Target Gene mRNA

Quantitative analysis was performed in the same manner as in Example 4-3.

As a result, it was confirmed that the expression level of the target gene in the human epidermal keratin-forming cell line decreases with the concentration of siRNA (FIG. 17).

STAT3 specific siRNA according to the present invention, a composition for the treatment of cancer, skin disease or inflammatory disease comprising a double-stranded oligo RNA structure comprising the same by inhibiting the expression of STAT3 with high efficiency without side effects, including cancer, in particular breast cancer, psoriasis Since it can have a therapeutic effect on skin diseases or inflammatory diseases, it can be very useful for the treatment of breast cancer, skin diseases or inflammatory diseases which do not currently have an appropriate therapeutic agent.

Electronic file attached.

Claims (35)

STAT3 specific siRNA comprising a sense strand comprising any one sequence selected from SEQ ID NO: 1 to SEQ ID NO: 200 and an antisense strand comprising a sequence complementary thereto. The method of claim 1, STAT3 specific siRNA, characterized in that the sense strand or antisense strand of the siRNA consists of 19 to 31 nucleotides. The method of claim 1, The siRNA includes a sense strand and a complementary sequence thereof, including any one sequence selected from the group consisting of SEQ ID NOs: 12, 17, 42, 101, 108, 114, 119, 137, 141, 166, 170, and 171. STAT3-specific siRNA comprising an antisense strand. The method of claim 1, STAT3 specific siRNA, characterized in that the sense strand or antisense strand of the siRNA comprises one or more chemical modifications. The method of claim 4, wherein The chemical modification is The —OH group at the 2 ′ carbon position of the sugar structure in the nucleotides is —CH ₃ (methyl), —OCH ₃ (methoxy), —NH ₂ , —F (fluorine), —O-2-methoxyethyl —O-propyl ( propyl), -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl, -ON-methylacetamido or -O-dimethylamidooxyethyl Modification by substitution; Modification in which oxygen in a sugar structure in nucleotides is replaced with sulfur; Modification of nucleotide bonds to phosphorothioate or boranophosphate, methyl phosphonate bonds; Modification to peptide nucleic acid (PNA), locked nucleic acid (LNA) or unlocked nucleic acid (UNA) form; STAT3 specific siRNA, characterized in that at least one modification selected from the group consisting of. The method of claim 1, STAT3-specific siRNA, characterized in that one or more phosphate groups are bonded to the 5 'end of the antisense strand of the siRNA. A double helix oligo RNA structure comprising the structure of formula (1) below. Structural Formula 1 (A _m -J) _n -XRYB In structural formula (1), A is a hydrophilic monomer, B is a hydrophobic substance, J is a m-hydrophilic monomer, or a linker connecting m hydrophilic monomers and oligonucleotides to each other, and n is a hydrophilic monomer. The number of repetitions of the repeated hydrophilic block, X and Y are each independently a simple covalent bond or a linker mediated covalent bond, R is a STAT3-specific siRNA, m is an integer from 1 to 10, n is 1 To an integer of 10. The method of claim 7, wherein Wherein R is a double-stranded oligo RNA structure, characterized in that consisting of the sense strand and antisense strand of siRNA according to claim 7. The method of claim 8, The 5 'end of the sense strand and the antisense strand is connected to X, and the 3' end is connected to Y double helix oligo RNA structure. The method of claim 7, wherein The STAT3-specific siRNA is a double helix oligo RNA structure, characterized in that siRNA according to any one of claims 1 to 6. The method of claim 7, wherein The hydrophilic substance monomer is a double-stranded oligo RNA structure, characterized in that selected from the group consisting of ethylene glycol (EG), polyvinylpyrrolidone and polyoxazoline. The method of claim 7, wherein The double-stranded oligo RNA structure, characterized in that the molecular weight of the hydrophobic material is 250 to 1,000. The method of claim 12, The hydrophobic material may be a steroid derivative, a glyceride derivative, a glycerol ether, a polypropylene glycol, a C ₁₂ to C ₅₀ unsaturated or saturated hydrocarbon, diacylphosphatidylcholine ( A double-stranded oligo RNA structure, characterized in that selected from the group consisting of diacylphosphatidylcholine, fatty acids, phospholipids and lipopolyamines. The method of claim 13, The steroid derivative is a double-stranded oligo RNA structure, characterized in that selected from the group consisting of cholesterol, colistanol, cholic acid, cholesteryl formate, cotestanyl formate and colistanylamine. The method of claim 13, The glyceride derivative is a double helix oligo RNA structure, characterized in that selected from mono-, di- and tri- glycerides The method of claim 7, wherein The covalent bond represented by X and Y is a double-stranded oligo RNA structure, characterized in that the non-degradable bond or degradable bond. The method of claim 16, The non-degradable bond is a double-stranded oligo RNA structure, characterized in that the amide bond or phosphorylation bond. The method of claim 16, The degradable bond is a double-stranded oligo RNA structure, characterized in that disulfide bonds, acid-degradable bonds, ester bonds, anhydride bonds, biodegradable bonds or enzyme-degradable bonds. The method of claim 7, wherein The RNA construct may further include a ligand having a specific binding property to a receptor that promotes target cell internalization through receptor-mediated endocytosis (RME). A double helix oligo RNA structure characterized by. The method of claim 19, The ligand is selected from the group consisting of target receptor specific antibodies or aptamers, peptides, folate, N-acetyl Galactosamine (NAG), glucose and mannose A double helix oligo RNA structure characterized by. 21. A method for preparing a double helix oligo RNA structure according to any one of claims 7 to 20, comprising the following steps: (1) bonding the hydrophilic material on a solid support basis; (2) synthesizing a single RNA strand based on the solid support to which the hydrophilic material is bound; (3) covalently binding a hydrophobic substance to the 5 'end of the RNA single strand; (4) synthesizing an RNA single strand of a sequence complementary to the sequence of the RNA single strand; (5) separating and purifying the RNA-polymer structure and the RNA single strand from the solid support when the synthesis is completed; And (6) preparing a double-stranded oligo RNA structure by annealing RNA single strand of the sequence complementary to the prepared RNA-polymer structure. 22. The method of claim 21, wherein the solid support is Controlled Pore Glass (CPG). The method of claim 21, wherein the RNA single strand comprising the RNA single strand and the complementary sequence has a form in which a phosphate group is bound to a 5 ′ end. 22. The method of claim 21, wherein the solid support is further coupled to a functional group. A nanoparticle comprising a double helix oligo RNA structure according to any one of claims 7 to 20. The method of claim 25, The nanoparticles are nanoparticles, characterized in that the double-stranded oligo RNA structure comprising a siRNA comprising a different sequence is mixed. A pharmaceutical composition for preventing or treating cancer, skin disease or inflammatory disease comprising the siRNA of claim 1, the double-stranded oligo RNA structure of claim 7, or the nanoparticles of claim 24 as an active ingredient. The method of claim 27, The cancer is a pharmaceutical composition, characterized in that selected from the group consisting of breast cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, prostate cancer, breast cancer, ovarian cancer, kidney cancer and lung cancer. The method of claim 27, The skin disease is a pharmaceutical composition, characterized in that selected from the group consisting of psoriasis, atopic dermatitis and allergic skin diseases. The method of claim 27, The skin disease is a pharmaceutical composition, characterized in that selected from the group consisting of psoriasis, atopic dermatitis and allergic skin diseases. A formulation in freeze dried form comprising the pharmaceutical composition of claim 27. The siRNA according to any one of claims 1 to 6, the double-stranded oligo RNA structure according to any one of claims 7 to 20, the nanoparticles according to any one of claims 24 to 25, Or a method for preventing or treating cancer, skin disease or inflammatory disease, which is administered to a subject in need thereof. 33. The method of claim 32, The cancer is selected from the group consisting of breast cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, prostate cancer, ovarian cancer, kidney cancer and lung cancer, the method of preventing or treating cancer, skin disease or inflammatory disease. 33. The method of claim 32, The skin disease is selected from the group consisting of psoriasis, atopic dermatitis and allergic skin diseases, the method of preventing or treating cancer, skin diseases or inflammatory diseases. 33. The method of claim 32, The inflammatory disease is selected from the group consisting of rheumatoid arthritis, degenerative arthritis, osteoporosis, hyperimmunoglobulinemia, nephritis, chronic thyroiditis, Crohn's disease and pancreatitis.

Images