therapeutic polymer drug structure, methods for preparing a nanoparticle, double-stranded oligorma s

专利摘要:
STRUCTURE OF THERAPEUTIC POLYMER DRUG, NANOPARTICULA, PHARMACEUTICAL COMPOSITION, OLIGONUCLEOTIDE CONJUGATE AND METHODS OF PREPARATION THEREOF. Providing double-stranded RNA structure in which a polymer compound covalently linked to a double-stranded RNA oligo for the treatment of diseases, particularly cancer, enhancing the release of double-stranded oligo RNA; a target-specific ligand and respective method of target-specific double stranded oligo RNA release technique. Nanoparticle composed of oligo RNA structures linked to the double-stranded linker effectively releasing the double-stranded oligo RNA to a target providing double-stranded oligo RNA activity even when the double-stranded oligo RNA is administered at low concentrations. Furthermore, it prevents the non-specific release of double-stranded oligo RNA into other organs and cells. Furthermore, it refers to a hybrid conjugate, which comprises hydrophilic and hydrophobic materials covalently linked to the ends of an antisense oligonucleotide (ASO), improving the in vivo stability of the ASO; method to prepare hybrid conjugate and nanoparticle composed of the conjugates.
公开号:BR112014014730B1
申请号:R112014014730-2
申请日:2012-12-14
公开日:2021-05-18
发明作者:Jeiwook Chae；Boram Han；Han-Na KIM；Han Oh Park；Pyoung Oh Yoon；Sun Gi Kim；Kwangju JUNG；Taewoo Kwon；Jong Deok Choi；Eun-Jung Jung；Sam Young Lee
申请人:Bioneer Corporation；
IPC主号:

专利说明:

Application field
[001] The present invention relates to a new oligonucleotide structure having attached to it, hydrophilic and hydrophobic materials that improve the release of single-stranded or double-stranded oligonucleotides useful for the treatment of cancer and infectious diseases, and for use of the same.
[002] A first aspect of the present invention relates to a double-stranded oligo RNA structure comprising a target-specific linker attached to a hydrophilic material contained in the framework, a nanoparticle composed of oligo RNA structures linked to the double-stranded linker , a pharmaceutical composition comprising the RNA structure, a pharmaceutical composition comprising a nanoparticle composed of oligo RNA structures linked to the double helix ligand, a method for preparing the structure, a method for preparing a nanoparticle composed of RNA structures, and a technique for releasing the oligo RNA structure linked to the double helix ligand.
[003] A second aspect of the present invention relates to a conjugated antisense oligonucleotide (hereinafter referred to as "ASO") which comprises a hydrophilic and hydrophobic material linked to both ends of the ASO by a single covalent bond or a covalent bond mediated by ligand in order to improve the efficiency of intracellular ASO release, a method for preparing the conjugate, and a technique for releasing a nanoparticle composed of ASO conjugates. State of the Art
[004] Once the role of RNA interference (hereinafter referred to as 'RNAi') has been found, it has been shown that RNAi acts on a sequence-specific mRNA in a variety of mammalian cells (Silence of the transcripts: RNA interference in medicine J Mol Med (2005) 83: 764-773). When a long double-stranded RNA is released into cells, the released RNA is processed by the endonuclease dicer into small, 21-23 base pair (bp) interfering RNA (hereinafter referred to as 'siRNA'). siRNA binds to RISC (RNA-induced silencing complex) and inhibits target gene expression in a sequence-specific mode by the process in which the antisense strand recognizes and degrades the target mRNA (NUCLEIC-ACID THERAPEUTICS: BASIC PRINCIPLES AND RECENT APPLICATIONS Nature Reviews Drug Discovery, 2002. 1, 503-514).
[005] Bertrand et al. reported that siRNA has an excellent inhibitory effect on mRNA expression in vitro and in vivo compared to an antisense oligonucleotide (ASO) for the same target gene and that the effect is long-lasting (Comparison of antisense oligonucleotides and siRNAs in cell culture and in Alive. Biochem. Biophys. Res. Commun. 2002. 296: 1000-1004). Furthermore, due to the fact that siRNA complementary binds to target mRNA to regulate target gene expression in a sequence-specific mode, it can be advantageously used in a wide range of applications compared to conventional drugs or antibody-based chemicals ( drugs small molecule drugs) (Progress Towards in Vivo Use of siRNAs. Molecular Therapy. 2006 13(4):664-670).
[006] siRNA has excellent effects and can be used in a wide range of applications, but for siRNA to be released into cellular therapeutic agents, it is required to improve the stability and efficiency of intracellular siRNA release in order to effectively release siRNA into their target cells (Harnessing in vivo siRNA delivery for drug discovery and therapeutic development. Drug Discov. Today. 2006 Jan;11(1-2):67-73).
[007] In an attempt to satisfy these requirements, nuclease resistant analogues or carriers such as viral vectors, liposomes or nanoparticles have been used.
[008] Viral carriers such as adenoviruses or retroviruses have algae transfection efficacy, but carry the risks of immunogenicity and oncogenicity. However, non-viral carriers including nanoparticles are evaluated for having low intracellular release efficiency compared to viral carriers, but they have advantages, including high in vivo safety, target-specific release, efficient uptake and internalization of RNAi oligonucleotides into cells or tissues, and low cytotoxicity and immune stimulation. Thus, these non-viral carriers are considered to be the most promising delivery method that effectively inhibits target gene expression (Nonviral delivery of synthetic siRNAs in vivo. J. Clin Invest. 2007 December 3; 117(12): 3623-3632 ).
[009] Delivery systems with multiple nanoparticles have been developed for cancer-specific delivery. Such nanoparticle systems are generally designed so that the surface is coated with a hydrophilic material to increase blood circulation time and is positively charged to increase endocytosis (Active targeting schemes for nanoparticle systems in cancer therapeutics. Advanced Drug Delivery Reviews 60 ( 2008) 1615-1626). However, tumor tissue is very rigid and has diffusion limitation, unlike normal tissue, and it overcomes diffusion limitation by forming new blood vessels in the surrounding region by angiogenesis, because this diffusion limitation has adverse effects on the required nutrient migration into the tumor , and waste materials such as oxygen and carbon dioxide. Blood vessels formed in a tumor tissue by angiogenesis have a leaky, defective blood vessel including a lacuna having a size of about 100 nm to 2 m depending on the type of tumor.
[010] Thus, nanoparticles easily pass through the capillary endothelium of cancer tissue having a leaky and defective blood vessel, compared to the structured capillaries of normal tissue, so that they are easily released during their circulations in the blood. Furthermore, the tumor tissue has no lymphatic drainage, and thus a drug is accumulated in it. This mechanism is known as the enhanced permeation and retention effect (EPR). Nanoparticles are easily specifically released into tumor tissue by this effect, and this mechanism is known as passive targeting (Nanoparticles for drug delivery in cancer treatment. Urol Oncol. 2008 Jan-Feb; 26(1):57-64).
[011] To overcome this non-specific in vivo distribution, targeting and lack of water solubility of therapeutic drugs including anti-cancer drugs, studies were conducted to optimize the size of nanoparticles loaded with therapeutic drugs or modify the surface to increase the time of their circulations in the blood. Particularly with respect to polymeric nanoparticles comprising polymer-drug conjugates, studies were conducted to increase tumor-specific release of anticancer drugs by binding anticancer drugs to water-soluble biodegradable materials such as albumin, poly-L-glutamate (PGA) or an N-(2-hydroxypropyl)-methacrylamide copolymer (Therapeutic Nanoparticles for Drug Delivery in Cancer. Clin Cancer Res 2008; 14: 1310-1316). Furthermore, studies have been conducted to bind an amphiphilic material to an anticancer drug to form polymeric micelles consisting of a hydrophobic anticancer drug core and a hydrophilic shell (Development of the polymer micelle carrier system for doxorubicin. J Control Release 2001; 74: 295-302).
[012] Thus, when a hydrophobic material is further linked to a therapeutic drug so that an anticancer drug to increase the cohesive strength of the core, micelles can be formed even at low concentration and polymeric micelles having increased stability due to the hydrophilic material of the shell be formed. A therapeutic drug having hydrophobic and hydrophilic materials bonded to both ends by biodegradable bonding can form polymeric micelles that can stably deliver the therapeutic drug to the target cancer tissue.
[013] Recently, as the technology for releasing double-stranded RNA oligo, the self-assembled SAMiRNA nanoparticle formed technology is based on the characteristics of materials attached to the ends of nucleic acid was developed (Korean Patent Laid-Open Publication No. 2009 -0042297). SAMiRNA is a self-assembled nanoparticle composed of double-stranded oligo RNA structures having attached to it hydrophobic and hydrophilic materials that enhance double-stranded oligo RNA release, and technology to form SAMiRNA may be the technology to improve intracellular oligo release Double-stranded RNA.
[014] It was shown that when fluorescently labeled SAMiRNA was administered to the tail vein of a mouse tumor xenograft model, the nanoparticle was specifically released by the passive targeting mentioned above (see FIG. 2).
[015] However, active targeting uses nanoparticles having a targeting fraction attached to them. The targeting fraction has been reported to cause preferential accumulation of nanoparticles in tissue, something or increase the uptake of nanoparticles into target cells (Does a targeting ligand influence nanoparticle tumor localization or uptake Trends Biotechnol. 2008 Oct; 26(10):552 -8. Epub 2008 Aug 21).
[016] Active targeting means to improve the release of nanoparticles to target cells using a targeting moiety such as an antibody or a ligand, attached to the nanoparticles. In recent years, studies have been conducted to localize siRNAs to the desired tissue using various targeting moieties linked to siRNAs.
[017] For example, it has been shown that an siRNA having α-tocopherol bound thereto was effectively and stably released in vivo and inhibited target gene expression by RNA interference (Kazutaka Nishina et al., The American Society of Gene therapy , 2008, 16(4):734-740). Furthermore, it has been shown that an siRNA having cholesterol attached to it was more effectively released into liver tissue compared to a cell-penetrating peptide (CCP) which is primarily used for siRNA release (US-20060014289; Moschos SA et al., Bioconjug Chem. 18:1450-1459). It has been reported that the releasing effect is caused not only by tumor tissue specificity, but also by the specificity of a cell targeted by the bound targeting fraction.
[018] Active targeting uses materials having the ability to bind to carbohydrates, receptors or antigens, which are specific to or overexpressed on the surface of the target cell (Nanotechnology in cancer therapeutics: bioconjugated nanoparticles for drug delivery. Mol Cancer Ther 2006; 5 (8): 1909-1917). Thus, nanoparticles having an active targeting fraction linked to them in tumor tissue during their blood circulation by passive targeting, and the release of nanoparticles in target cells is increased by the targeting fraction, thus increasing the therapeutic effect of the drug released in the cells . As the targeting moiety, a linker or an antibody is primarily used. It binds to its cell surface receptor with high avidity and specificity and promotes the internalization of nanoparticles by receptor-mediated endocytosis (RME) (Kinetic analysis of receptor-mediated endocytosis (RME) of proteins and peptides: use of RME as a drug delivery system. J Control Release 1996; 39: 191-200).
[019] The cell surface receptor or antigen that is targeted by this ligand or antibody has a characteristic that it is specific to or overexpressed in target cells to facilitate access of the targeting ligand to it, thus increasing the rate of endocytosis. Furthermore, the receptor or antigen releases the nanoparticles having the ligand attached to them in the cells, and is recycled back to the cell surface (Receptor-mediated endocytosis: An overview of a dynamic process. J. Biosci., Oct. 1984, 6(4), pp. 535-542.). Tumor targeting fractions are materials that specifically bind to receptors such as epidermal growth factor or low-density lipoprotein receptor, which are specifically expressed in target cell lines such as folate receptor, which are known to be over-expressed on the surface of various cancer cells (Nanotechnology in cancer therapeutics: bioconjugated nanoparticles for drug delivery. Mol Cancer Ther 2006, 5(8): 1909-1917).
[020] If a targeting moiety, particularly a receptor-specific ligand that enhances receptor-mediated endocytosis (RME) uptake, is bound to SAMiRNA, it can efficiently promote the release of SAMiRNA in target cells, particularly cells carcinogens, and thus SAMiRNA can be released into target cells even at a relatively low concentration and dose so that double-stranded oligo RNA may have high activity and non-specific release of double-stranded oligo RNA in other organs and cells can be inhibited.
[021] Furthermore, SAMiRNA formation technology can be applied not only to double-stranded oligo RNAs, but also to single-stranded oligonucleotides, particularly a single-stranded antisense oligonucleotide (ASO) for therapeutic purposes.
[022] ASO technology is the technology to control the transfer of information from gene to protein by altering mRNA metabolism using RNA or single-stranded DNA. In other words, it is the technology to perform preferential inhibition of expression of the protein of interest using a selected nucleotide sequence that complementary and specifically hybridizes to the protein. Due to the fact that ASO binds to the target gene in a sequence-specific fashion, it does not influence the expression of genes beyond the target gene. Thus, ASO technology can serve as a target tool in analyzing the in vivo function of a specific protein and can even be used as gene therapy against a specific disease (FASEBJ.9,1288-1296,1995).
[023] In recent years, an antagomir which is a new type of single-stranded antisense oligonucleotide has been developed and has been used to inhibit the function of microRNAs in cells. An antagomir or microRNA inhibitor (miRNA inhibitor) which is a short RNA chemically synthesized complementary to the target microRNA is known to inhibit microRNA function. An antagomir preferably has a chemical structure modified such as 2' methoxy or phosphothioate to prevent degradation of the antagomir. Currently, antagomirs that inhibit the functions of miRNAs related to various diseases, including cancer and cardiac and pulmonary fibrosis, are known ("Silencing of microRNAs in vivo with 'antagomirs'" Nature, Dec. 2005, 438(7068): 685-689 ; "MicroRNAs as Therapeutic Targets" New England J. Medicine, 2006, 354 (11): 1194-1195; Meister G. et al., "Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing" RNA, Mar. 2004, 10 (3): 544-550).
[024] Antisense DNA binds to target mRNA to form an RNA/DNA duplex, which is degraded by RNase H (a type of ribonuclease that specifically degrades an mRNA containing an RNA/DNA hybrid duplex formed therein) in vivo. atissens RNA forms an RNA/RNA duplex, and target mRNA degradation is induced by RNase L. RNase L is a ribonuclease that preferentially degrades single-stranded RNA around double-stranded RNA (Pharmacol.Toxicol.32,329-376,1992 ).
[025] However, an ASO comprising the miRNA inhibitor antagomir must be effectively released into the target cells to obtain a desired effect, and the ASO can be degraded by ribonuclease in the blood. Thus, in order to use an ASO for therapeutic purposes, an ASO conjugate must be effectively released across the cell membrane, and the stability of the ASO in vivo must be ensured (Shigeru Kawakami and Mitsuru Hashida, Drug Metab. Pharmacokinet. 22(3) : 142-151, 2007).
[026] Thus, for stability in vivo, most ASOs are in the form of oligodeoxynucleotides (ODNs) obtained by various modifications that provide nuclease resistance. The modification can be the replacement of an -OH group at the 2' carbon position of the sugar moiety of one or more nucleotides with -CH3 (methyl), -OCH3, -NH2, -F (fluorine), -O-2- methoxyethyl, -O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl, -ON-methylacetamido or -O-dimethylamidoxyethyl; the oxygen replacement of the sugar fraction of the nucleotide with sulfur; modifying the bond between the nucleotides into a phosphorothioate, boranephosphonate, or methyl phosphonate bond; or a combination of one or more of them; or modification in the form of PNA (peptide nucleic acid) or LNA (blocked nucleic acid) (see Crooke et al., Ann. Rev. Med. Vol.55: pp 61-65 2004, US5,660,985, US5,958,691, US6 531,584, US5,808,023, US6,326,358, US6,175,001 Braasch DA et al., Bioorg. Med. Chem. Lett. 14:1139-1143, 2003; Chiu YL et al., RNA, 9:1034-1048, 2003; Amarzguioui M. et al., Nucleic Acid Res. 31:589-595, 2003).
[027] To deliver ASOs into target cells, gene delivery techniques that use viruses like adenoviruses or retroviruses, and gene delivery techniques that use non-viral carriers like liposomes, cationic lipids or cationic polymers have been developed. However, viral carriers have problems in terms of safety, because it is not guaranteed that these carriers do not cause abnormalities in the normal functions of host genes after incorporation into the host chromosome, or that they do not activate oncogenes. Also, if the viral gene is continuously expressed even at low levels to cause autoimmune diseases or if the viral carrier causes modified viral infection, ASOs cannot be effectively released.
[028] To overcome such problems, methods to fuse a gene for the non-viral carrier liposome or methods to use cationic lipids or polymers have been studied to overcome the deficiencies of the same. Although these non-viral carriers are less efficient than viral carriers, they have advantages such that they are safe in vivo, cause fewer side effects, and can be produced at low cost (Lehrman S. Nature. 401(6753):517-518 , 1999).
[029] To effectively obtain stable release of ODN molecules including an ASO using non-viral carriers, an effective method of preventing enzymatic and non-enzymatic degradation is required. Thus, methods to chemically modify ASOs to prepare the nuclease-stable ASOs and increase the intracellular uptake of ASOs have been proposed (Shigery Kawakami and Mitsuru Hashida. Drug Metab.Parmacokinet. 22(3): 142-151, 2007).
[030] However, polymers comprising PEG (polyethylene glycol) form compounds having micelle structures spontaneously formed by interaction between them, and these compounds are known as polymer compound micelles (Kataoka K. et al. Macromolecules, 29:8556-8557, 1996 ). These polymer composite micelles have advantages in that they have a very small size compared to other drug delivery systems like microspheres or nanoparticles while their distribution is very uniform, and they are spontaneously formed, making it easy to control the quality of the formulation and guarantee the reproducibility.
[031] In recent years, in order to increase the release efficiency of ASOs, the technology to ensure the stability of ASOs and the efficient permeation of ASOs across the cell membrane by conjugating a hydrophilic material such as the biocompatible polymer PEG to ASOs by a single covalent bond or a linker-mediated covalent bond has been developed (Korean Patent Registry No. 0466254). However, improving the in vivo stability of ASOs and ensuring efficient release of ASOs into the target tissue is difficult to achieve by chemical modification and pequilation alone.
[032] The international application WO2010131916 entitled siRNA Conjugate and Preparation Method Thereof refers to a siRNA-polymer conjugate and a method for preparing the same, and more specifically, to a hybrid conjugate formed by covalently bonding siRNA and a polymeric compound to improve siRNA biostability and a hybrid conjugate preparation method. However, it does not disclose a functional structure according to the present invention, which is directed to the therapeutic polymer-drug structure comprising the linker L attached to the end of the hydrophilic block. and specifically selected from folate, N-acetylgalactosamine (NAG) and mannose.
[033] Patent application CN1911447 entitled Transferrin-Polyethylene Glycol Medicine Molecular Compound and Its Use discloses the composition of the transferrin-polyethanediol-drug molecule for targeted tumor therapy. However, it is silent on the problem of how to effectively deliver RNA to the patient's body, since it is easily degraded in the body.
[034] International application WO2011054939 entitled Compositions and Methods for Inhibiting Expression of KIF10 Genes D4. CN102028955 Novel Targeting Compound for Tumor Gene Interference describes only a dsRNA linked to the linker that is completely different from a linker connected to the polymer-drug therapeutic structure of the present invention. What this document reveals is "the linker can be conjugated to dsRNA" and "it is known that folic acid at the 3' end of the oligonucleotide results in increased cellular uptake of the oligonucleotide". When a ligand is directly attached to the dsRNA and the ligand-dsRNA conjugate is transferred into a cell, the dsRNA becomes very vulnerable to many intracellular enzymes, such as proteases. To avoid this, the aforementioned document teaches that dsRNA requires very complicated modifications.
[035] Patent document CN102028955 entitled Novel Targeting Compound for Tumor Gene Interference discloses a new targeting compound for tumor gene interference, which comprises an aptamer, protamine and small molecule interfering ribonucleic acid. In this structure, protamine acts as a bridge and the aptamer is a target group. That is, this document only teaches that siRNA can be delivered to a specific target molecule via the aptamer and does not reveal a structure with dual functionality.
[036] Patent application US2011112176 entitled Compositions and Methods for Inhibiting Expression of KIF10 Genes discloses a double-stranded ribonucleic acid molecule capable of inhibiting the expression of the KIF10 gene. However, it does not solve the problem of targeting the active principle to specific cells while reducing siRNA degradation in the body.
[037] Patent application JP2011522070 entitled Micellic Assemblies discloses a micelle and a polynucleotide associated with the micelle, the micelle comprising a plurality of block copolymers, each including a hydrophilic block and a hydrophobic block. However, the hydrophilic block with a cationic species is ionic associated with the polynucleotide. This is completely different from the structure in which siRNA is covalently linked between a hydrophilic block and a hydrophobic block.
[038] The international publication WO2010042823 entitled Multifunctional Self-Assembling Polymeric Nanosystems discloses a hydrophilic block and a hydrophobic block for nucleic acid delivery; however, it is encapsulated in a hydrogel shell, which is different from the structure in which siRNA is covalently bonded between a hydrophilic block and a hydrophobic block.
[039] Patent application US2007287681 whose title is siRNA-Hydrophilic Polymer Conjugates for Intracellular Delivery of siRNA and Method Thereof, as well as the publication "Folate Receptor Targeted Delivery of Polyelectrolyte Complex Micelles Prepared from ODN-PEG-Folate Conjugate and Cationic Lipids" , Sun Hwa Kim et al, Biotechnol. Program (20061115), vol. 23, no. 1, pages 232 - 237, discloses a siRNA-hydrophilic polymer conjugate and an ODN-PEG-FOL conjugate, respectively. Since the structures described in these documents do not have a hydrophobic polymer, it is necessary to use a cationic compound to be delivered to the cell. In this case, the cationic compound (eg PEI) can be toxic to the cell, and the complex micelle formed with the cationic compound is not uniform in size, resulting in the problem of low efficiency of delivery of the active ingredient into the cell.
[040] Patent application US2012225129 entitled Conjugates, Particles, Compositions, and Related Methods discloses a nucleic acid covalently linked to a hydrophobic moiety or to a hydrophilic-hydrophobic polymer. Thus, the structure described in that document has reduced tendency to form a structure in which the nucleic acid is located between the hydrophilic and hydrophobic portions, as in the present invention, so that the nucleic acid is stably delivered into a cell.
[041] It results, therefore, that the SAMiRNA technology on nanoparticles obtained by introducing hydrophobic and hydrophilic materials to siRNA to improve the intracellular release of siRNA was developed. However, the application of this technology for the release of ASOs has not yet been reported. Thus, there is a need to develop an ASO delivery system and a method for preparing various chemical modifications in ASOs and conjugating various polymers to the ASOs to protect the ASOs from enzymes to thereby increase their stability, and efficient permeation of the even across the cell membrane. DESCRIPTION OF THE INVENTION
[042] In a first aspect, an objective of the present invention is to provide a therapeutic drug structure, comprising hydrophilic and hydrophobic materials that are biocompatible polymeric compounds linked to both ends of the therapeutic drug by a simple covalent bond or a covalently mediated bond by ligand to increase the efficiency of intracellular delivery of the therapeutic drug and further comprises a ligand bound to the hydrophilic material, a nanoparticle composed of the structure of the therapeutic drug, and a method of preparation thereof.
[043] Another objective of the present invention is to provide a double-stranded oligo RNA structure, comprising hydrophilic and hydrophobic biocompatible polymeric materials linked to both ends of the double-stranded oligo RNA by a single bond or a ligand-mediated covalent bond to increase efficiency intracellular release of double-stranded oligo RNA, and further comprises, linked to the hydrophilic material, a specific ligand for the receptor having the property of improving the internalization of the target cell (particularly cancer cell) by receptor-mediated endocytosis (RME); a nanoparticle composed of the double-stranded oligo RNA structures linked to the ligand; and a pharmaceutical composition comprising the double stranded oligo RNA structure linked to the linker or a nanoparticle composed of the double stranded oligo RNA structures linked to the linker.
[044] Yet another objective of the present invention is to provide methods to prepare a double stranded oligo RNA oligo structure linked to the linker and a nanoparticle comprising the same, and a technique to release a double stranded oligo RNA using the double stranded oligo RNA structure linked to the link .
[045] When a target-specific ligand is attached to a double-stranded oligo RNA structure, a nanoparticle composed of the double-stranded oligo RNA structures attached to the ligand can be effectively released into the target cell. Thus, when a ligand-bound oligo-RNA duplex structure is administered at a relatively low concentration, it may exhibit double-stranded oligo RNA activity in the target cell. Furthermore, due to the fact that the linked ligand can prevent the non-specific release of oligo RNA double helix in other organs and cells, the double helix RNA structure linked to the ligand can be used for the treatment of various diseases and can be effectively used as a new type of double helix oligo RNA delivery system. Particularly, the ligand-bound double-stranded RNA structure can be effectively used for the treatment of diseases, including cancer and infectious diseases.
[046] In a second aspect, an object of the present invention is to provide an ASO-polymer conjugate, comprising hydrophilic and hydrophobic biocompatible polymeric materials linked to both ends of the ASO by a single covalent bond or a ligand-mediated covalent bond to enhance the efficiency of intracellular ASO release, and a method for its preparation.
[047] Another objective of the present invention is to provide a technique to release an ASO using a nanoparticle composed of ASO-polymer conjugates, and a pharmaceutical composition comprising the ASO-polymer conjugate or a nanoparticle composed of ASO-polymer conjugates.
[048] The ASO-polymer conjugate according to the present invention and a nanoparticle composed of the ASO-polymer conjugates can increase the in vivo stability of ASO, making it possible to efficiently release the therapeutic ASO into cells. Furthermore, they can show ASO activity at relatively low concentrations compared to an ASO whose end has not been modified, even in the absence of a transfection agent. Thus, the ASO-polymer conjugate and a nanoparticle composed of the ASO-polymer conjugates can be used for the treatment of various diseases, including cancer and infectious diseases, and can even be very effectively used as a new type of ASO delivery system in basic bioengineering research and medical industries. Brief Description of Figures
[049] FIG. 1 is a schematic view of a nanoparticle (SAMiRNA) composed of double-stranded oligo RNA structures having a linker attached thereto.
[050] FIG. 2 shows tumor-specific release of SAMiRNA. FIG. 2A is a photograph showing the biodistribution of SAMiRNA with time after Cy5.5-labeled SAMiRNA was administered once to the tail vein of a tumor-transplanted mouse at a dose of 5 mg/kg body weight (the portion indicated by the dotted line red is a transplanted portion of the tumor). FIG. 2(B) is an ex vivo photograph of each tissue collected within 48 hours of SAMiRNA administration.
[051] FIG. 3 shows the results of NMR analysis of 1,3,4,6-tetraacetyl-NAG (compound A). 1H NMR (300 MHz, DMSO-D6); δ7.89 ppm (1H, d, J=9.3 Hz), 5.64 ppm (1H, d, J=8.7 Hz), 5.27 ppm (1H, d, J=3.3 Hz) , 5.07ppm (1H, dd, J=11.7, 3.6Hz), 4.22ppm (1H, t, J=6.3Hz), 4.14-3.96ppm (2H,m ), 2.12ppm(3H,s), 2.04ppm(3H,s), 1.99ppm(3H,s), 1.91(3H,s), 1.78ppm(3H,s).
[052] FIG. 4 shows the results of NMR analysis of 3,4,6-triacetyl-1-hexa(ethylene glycol)-N-acetylgalactosamine(NAG) (compound B). 1H NMR (300 MHz, DMSO-D6); δ7.71 ppm (1H, d, J=9.3 Hz), 5.21 ppm (1H, d, J=3.0 Hz), 4.97 ppm (1H, dd, J=11.1.3 .0 Hz), 4.56 ppm (1H, d, J=8.7 Hz), 3.88 ppm (1H, q, J=8.7 Hz), 3.83-3.74ppm (1H, m), 3.62-3.39 ppm (25H, m), 2.10 ppm (3H, s), 2.01 ppm (3H, s), 1.89 ppm (3H, s), 1.77 ppm (3H , s).
[053] FIG. 5 shows the results of NMR analysis of 1-hex(ethylene glycol)-NAG-phosphoramidite) (compound C). (A) 1H NMR (300 MHz, DMSO-D6); δ 7.78 ppm (1H, d, J=9.3 Hz), 5.21 ppm (1H, d, J=3.0 Hz), 4.97 ppm (1H, dd, J=11.1, 3.6 Hz), 4.56 ppm (1H, d, J=8.1 Hz), 3.88 ppm (1H, d, J=9.0 Hz), 3.81-3.41 ppm (30H , m), 2.89 ppm (2H, t, J=5.7 Hz), 2.11 ppm (3H, s), 2.00 ppm (3H, s), 1.89 ppm (3H, s) , 1.77 ppm (3H, s), 1.20-1.12 ppm (12H, m), (B) 31P NMR data (121 MHz, DMSO-D6); δ 147.32 ppm.
[054] FIG. 6 shows a process for making a single-stranded RNA.
[055] FIG. 7 shows a process for preparing a double stranded oligo RNA structure comprising PEG attached to the 5' end of a double stranded oligo RNA and the results of RNA structure analysis. FIG. 7(A) shows a process for attaching PEG to a double-stranded RNA oligo using PEG-phosphoramidite, FIG. 7(B) shows the results of MALDI-TOF MS analysis of a single-stranded (21mer) RNA that was not modified at the 5' end (SEQ ID NO: 1; MW 6662.1), and FIG. 7(C) shows the MALDI-TOF MS results of a single-stranded (21mer) RNA having PEG attached to the 5' end (SEQ ID NO: 1; MW 6662.1).
[056] FIG. 8 shows a process for preparing a mono-NAG-PEG-RNA structure and the results of structure analysis. FIG. 8(A) shows a process for attaching N-acetyl galactosamine (NAG) to PEG-RNA using N-acetyl galactosamine phosphoramidite, FIG. 8(B) shows the results of MALDI-TOF MS analysis of a NAG-PEG-RNA structure (blue, MW 9171.2) comprising N-acetyl galactosamine linked to PEG-RNA (green, MW 8624.1) and shows that the central peak changed by the molecular weight of N-acetyl galactosamine (MW 547).
[057] FIG. 9 shows a process for preparing a triple-NAG-PEG-RNA structure and the results of structure analysis. FIG. 9(A) shows a process for attaching N-acetyl galactosamine to PEG-RNA using phosphoramidite and NAG-phosphoramidite dendrimer, and FIG. 9(B) shows the results of MALDI-TOF MS analysis of PEG-RNA (green, MW 8624.1), and a mono-NAG-PEG structure (blue, MW 9171.2) and a triple-NAG-PEG-RNA structure (red, MW 10630), which comprises N-acetyl galactosamine bound thereto.
[058] FIG. 10 shows a process for preparing a 5' folate-PEG-RNA structure and structure results. FIG. 10(A) shows a process for attaching folate to PEG-RNA by NHS-folate, and FIG. 10(B) shows the results of MALDI-TOF MS analysis of PEG-RNA (green, MW 8624.1) and a folate-PEG-RNA structure (blue, MW 9277.8) and shows that the central peak shifted by molecular weight of folate (MW 615).
[059] FIG. 11 shows the results of analysis of a 5' C24-RNA structure. FIG. 11(A) shows MALDI-TOF MS analysis analysis of a single-stranded RNA complementary to SEQ ID NO: 1 (MW 7349.5), and FIG. 11(B) shows MALDI-TOF MS analysis analysis of a 5' C24-RNA structure complementary to SEQ ID NO: 1 (MW 7830.2).
[060] FIG. 12 shows a process for preparing a 3' CPG-amine-PEG-RNA structure by amine-CPG.
[061] FIG. 12 shows a process for preparing a 3' CPG-amine-PEG-RNA structure by amine-CPG.
[062] FIG. 14 shows a process for preparing a 3' folate-PEG-RNA structure and the results of the structure analysis. FIG. 14(A) shows a process for attaching folate to a 3' amine-PEG-RNA structure by NHS-folate; and FIG. 14(B) shows the results of MALDI-TOF MS analysis of a 3 ‘ folate-PEG-RNA structure (SEQ ID NO: 1; MW 9277.7).
[063] FIG. 15 shows the results of analyzing the physical properties of a nanoparticle (folate-SAMiRNA) composed of 5' folate-RNA-polymer structures. FIG. 15(A) is a graphical diagram showing the size and polydispersed index (PDI) of folate-SAMiRNA, and FIG. 15(B) is a graphical diagram showing the critical micelle concentration of folate-SAMiRNA.
[064] FIG. 16 shows the effect of folate-SAMiRNA in inhibiting target gene expression in a cell line that overexpresses the folate receptor. The level of target gene mRNA in the folate receptor-overexpressing KB tumor lineage was measured by qPCR at 48 hours after treatment with folate-SAMiRNA and SAMiRNA. In FIG. 16, Folate-free in culture medium: folate-free condition; with Folate in culture medium: a condition containing an excessive amount (1 mM) of folate; Con: a test group treated with the SAMiRNA-Con nanoparticle composed of double helix-polymer oligo RNA structures comprising a sequence of SEQ ID NO: 2 (control sequence); SAM: a test group treated with the SAMiRNA-Sur nanoparticle composed of double helix-polymer oligo RNA structures comprising a sequence of SEQ ID NO: 1 (survivin sequence); Folate-SAM: a test group treated with the Folate-SAMiRNA-Sur nanoparticle composed of double-stranded folate-oligo RNA structures comprising a sequence of SEQ ID NO: 1 (survivin sequence) and having a folate ligand attached thereto.
[065] FIG. 17 shows the effect of folate-SAMiRNA in inhibiting target gene expression in tumor tissue. The level of target gene (survivin) mRNA in tumor tissue was measured by qPCR at 48 hours or 72 hours after each of SAMiRNA and folate-SAMiRNA was administered once at a dose of 5 mg/kg body weight to the tail vein of a mouse bearing a tumor composed of the KB tumor cell line that overexpresses folate receptor 48. In FIG. 17, PBS: negative control; SAMiRNA: a group administered with the SAMiRNA-Sur nanoparticle composed of double helix-polymer oligo RNA structures comprising a sequence of SEQ ID NO: 1 (survivin sequence) and having no linker attached thereto; Folate-SAMiRNA: a group administered with the Folate-SAMiRNA-Sur nanoparticle of SEQ ID NO: 1 (survivin sequence) having a folate ligand attached thereto.
[066] FIG. 18 is a schematic view of a nanoparticle comprising an ASO-polymer conjugate.
[067] FIG. 19 shows the MALDI-TOF MS spectrum of each of an ASO and an ASO-polymer conjugate according to the present invention. Four nucleotides at both ends (5' and 3' ends) are modified with 2-OCH3 (methoxy), and 'm' indicates an OCH3 (methoxy) group. FIG. 19(A) shows the MALDI-TOF ASO data (M.W. 5967.9 Da), and FIG. 19(B) shows the MALDI-TOF data of the ASO-polymer conjugate (M.W. 8448 Da).
[068] FIG. 20 shows the results of analyzing the physical properties of a nanoparticle composed of ASO-polymer conjugates. FIG. 20(A) is a graphical diagram showing the size and polydispersity index (PDI) of a nanoparticle composed of ASO-polymer conjugates; and FIG. 20(B) is a graphical diagram showing the critical micelle concentration of the composite nanoparticle of the ASO-polymer conjugates.
[069] FIG. 21 shows the results of analyzing the expression level of mRNA at various treatment concentrations (10, 50 and 100 nM) to assess the effects of an ASO and an ASO-polymer conjugate in inhibiting target gene expression in tumor cells. Scramble: an ASO of SEQ ID NO: 4 (control sequence); Survivin: an ASO of SEQ ID NO: 3 (survivin sequence); ASO: an ASO having no material attached to it; ASO-polymer conjugate: an ASO-polymer conjugate in the form of 3'PEG-ASO-5'lipid). BEST WAY TO CARRY OUT THE INVENTION 1. First aspect of the present invention
[070] In the first aspect of the present invention, the term "antisense strand" means a strand that shows RNAi activity to bind and degrade target mRNA in RISC (RNA-induced silencing complex), and the term "sense strand" means a tape having a sequence complementary to the antisense tape.
[071] As used herein, the term "complementary" or "complementary bond" means two sequences to link to each other to form a double-stranded structure. This includes not only a perfect match between two sequences, but also an incorrect match between two sequences.
[072] The present invention provides a therapeutic drug-polymer structure having a structure of the following formula (1) and comprising a binder attached thereto:

[073] wherein A is a hydrophilic material; B is a hydrophobic material; X and Y are each independently a single covalent bond or a linker-mediated covalent bond; R is a therapeutic drug; and L is a receptor-specific ligand having the property of enhancing target cell internalization by receptor-mediated endocytosis (RME).
[074] Here, the therapeutic drug can be selected from among anticancer drugs, oligo RNA double helices, antiviral drugs, steroidal anti-inflammatory drugs (SAIDs), non-steroidal anti-inflammatory drugs (NSAIDs), antibiotics, antifungal agents, vitamins, hormones, retinoic acid, prostaglandins, prostacyclins, antimetabolic agents, mycotics, choline agonists, adrenaline antagonists, anticonvulsants, antianxiety drugs, tranquilizers, antidepressants, anesthetics, analgesics, anabolic steroids, estrogens, progesterones, immunonucleosides and immunostimulants.
[075] In the present invention, the therapeutic drug is preferably a double stranded RNA oligo or an anticancer drug. If the therapeutic drug is a double-stranded RNA oligo, the hydrophilic material can be attached to the 3' or 5' end of the double-stranded oligo RNA.
[076] In the inventive therapeutic drug-polymer structure comprising a linker attached thereto, a linker may additionally be attached to a specific position (particularly end) of the hydrophilic material attached to the double-stranded RNA oligo or the anticancer drug. The linker can be selected from a target receptor-specific antibody, an aptamer (a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) capable of binding to the target molecule with high affinity and specificity), a peptide, or materials chemicals, including folate (folate is used interchangeably with folic acid, and the term "folate" as used herein refers to folate that is active in nature in the human body), N-Acetylgalactosamine (NAG) and mannose, which has the property of specifically binding to the receptor that enhances target cell uptake by RME. Here, the ligand is a material that effects release in a specific way to the target receptor, and is not limited only by the above-described antibody, aptamer, peptide and chemical materials.
[077] The double-stranded RNA oligo is preferably composed of 19-31 nucleotides. The double stranded RNA oligo that is used in the present invention can be any double stranded RNA oligo for any gene that is used or can be used for gene therapy or research.
[078] The hydrophobic material forms a nanoparticle composed of double-stranded oligo RNA structures by hydrophobic interaction. Among hydrophobic materials, a carbon or cholesterol chain is very suitable for use in preparing the structure of the present invention, because it can be easily ligated in the double-stranded oligo RNA synthesis step.
[079] The hydrophobic material preferably has a molecular weight of 250-1,000. Particularly, the hydrophobic material which is used in the present invention may be a steroid derivative, a glyceride derivative, glycerol ether, polypropylene glycol, a saturated or unsaturated C12-C50 hydrocarbon, diacyl phosphatidylcholine, fatty acid, phospholipid, lipopolyamine or the like , but is not limited to these, and it will be obvious to those skilled in the art that any hydrophobic material suitable for the purpose of the present invention can be used.
[080] Particularly, the steroid derivative can be selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate, and cholestanyl amine, and the glyceride derivative can be selected from mono-, di- and tri-glycerides, where the glyceride fatty acid may be a saturated or unsaturated C12-C50 fatty acid.
[081] Further, the hydrophilic material is preferably a cationic or nonionic polymer having a molecular weight of 2000,000, and more preferably a nonionic polymer having a molecular weight of 1,000-2,000. For example, the hydrophilic material which is used in the present invention is preferably a non-ionic hydrophilic polymer composed such as polyethylene glycol, polyvinyl pyrrolidone or polyoxazoline, but is not limited thereto.
[082] The hydrophilic material may, if necessary, be modified to have a functional group required to bind to the linker. Among hydrophilic materials, particularly polyethylene glycol (PEG) can have various molecular weights and functional groups, have good biocompatibility, do not induce immune response, increase in vivo stability of oligo RNA double helix, and increase RNA release efficiency, and, thus, it is very suitable for preparing the inventive double stranded RNA oligo backbone.
[083] The linker that mediates the covalent bond is not specifically limited, as long as it forms a covalent bond between the hydrophilic material (or the hydrophobic material) and the end of the oligo RNA double helix and provides a bond that can, if necessary, be degraded in a specific environment. Thus, the linker can include any compound that binds the hydrophilic material (or the hydrophobic material) to the double stranded oligo RNA during the structure preparation process.
[084] Furthermore, the covalent bond can be a non-degradable bond or a degradable bond. Here, non-degradable bonds include, among others, an amide bond or a phosphate bond, and degradable bonds include, among others, a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond, or an enzymatically degradable bond.
[085] The present invention provides a double stranded oligo RNA structure conjugated to the linker represented by the following formula (2), which comprises a hydrophilic material attached to the 3' end of the sense strand of a double stranded oligo RNA, a linker attached to the material hydrophilic, and a hydrophobic material attached to the 5' end of the sense tape:

[086] Where A is the hydrophilic material; B is the hydrophobic material; X and Y are each independently a single covalent bond or a linker-mediated covalent bond; S is the sense strand of double-stranded oligo RNA; AS is the antisense strand of double-stranded oligo RNA; and L is the receptor-specific ligand having the property of enhancing target cell internalization by receptor-mediated endocytosis (RME).
[087] A method to prepare the linker-conjugated double-stranded oligo RNA structure represented by formula (2) comprises the steps of:
[088] (1) synthesize a single-stranded RNA on a solid support having a functional group-hydrophilic material attached to it;
[089] (2) covalently attach a hydrophobic material to the 5' end of the single-stranded RNA having the functional group-hydrophilic material attached thereto;
[090] (4) separate the structure of functional group-RNA-polymer and a separately synthesized single-stranded RNA complementary to the solid support;
[091] (5) attaching a linker to the end of the hydrophilic material by the functional group; and
[092] (6) annealing the ligand-bound RNA-polymer structure with the complementary single-stranded RNA to form a double-stranded RNA structure.
[093] In a more preferred embodiment of the present invention, the method may comprise the steps of: (1) attaching a hydrophilic material to a solid support (CPG) having a functional group attached thereto; (2) synthesize a single-stranded RNA on the solid support (CPG) having the functional group-hydrophilic material attached thereto; (3) covalently attach a hydrophobic material to the 5' end of the single-stranded RNA; (4) separate the functional group-RNA-polymer structure and a separately synthesized complementary single-stranded RNA from the solid support (CPG); (5) attaching a linker to the end of the hydrophilic material by the functional group to prepare an RNA-polymer structure having the linker attached thereto; and (6) annealing the linker-bound RNA-polymer structure with the complementary single-stranded RNA to form a double-stranded oligo RNA structure having the linker attached thereto. After step (6), the RNA-polymer structure and the complementary single-stranded RNA can be separated and purified from the reagents by high performance liquid chromatography (HPLC), and then the molecular weight can be measured by a MALDI mass spectrometer -TOF to determine if the desired RNA-polymer structure and RNA were prepared. In the above-described preparation method, the step to synthesize the single-stranded RNA complementary to the single-stranded RNA synthesized in step (3) can be performed before step (1) or in any of steps (1) to (6) .
[094] In another embodiment, the present invention provides a double-stranded oligo RNA structure linked to a linker represented by the following formula (3), which comprises a hydrophilic material attached to the 5' end of the sense strand of a double-stranded oligo RNA, a binder attached to the hydrophilic material, and a hydrophobic material attached to the 3' end of the sense tape:

[095] A method for preparing a linker-linked double-stranded RNA oligo structure represented by formula (3) comprises the steps of:
[096] (1) to synthesize a single-stranded RNA on a solid support having a functional group attached thereto;
[097] (2) covalently attaching a hydrophilic material to the material obtained in step (1);
[098] (3) covalently attaching a binder to the material obtained in step (2);
[099] (4) separating the material obtained in step (3) from the solid support;
[0100] (5) covalently attaching a hydrophobic material to the material resulting from step (4) by the functional group attached to the 3' end; and
[0101] (6) anneal the material resulting from step (5) with a complementary single-stranded RNA to form a double-stranded RNA structure.
[0102] In a more preferred embodiment, the method of preparation may comprise the steps of: (1) synthesizing a single-stranded RNA on a solid support (CPG) having a functional group attached thereto; (2) covalently attach a hydrophilic material to the 5' end of the single-stranded RNA; (3) attach a linker to the hydrophilic material attached to the single-stranded RNA to synthesize a hydrophilic polymer-RNA-functional group structure; (4) separating the functional group-RNA-hydrophilic polymer structure from the solid support (CPG); (5) attaching a hydrophobic material to RNA via the functional group to synthesize an RNA-polymer structure having a linker attached thereto; and (6) annealing the prepared RNA-polymer structure with a complementary single-stranded RNA to prepare a double-stranded RNA-polymer oligo structure.
[0103] After step (5), the RNA can be separated and purified from the reagents by high performance liquid chromatography (HPLC), and then the molecular weight can be measured by a MALDI-TOF mass spectrometer to determine whether the structure of desired RNA-polymer and RNA were prepared. In the above-described preparation method, the step to synthesize the single-stranded RNA complementary to the single-stranded RNA synthesized in step (1) can be performed before step (1) or in any of steps (1) to (6) .
[0104] In another embodiment, the present invention provides a double helix oligo RNA structure linked to a linker represented by the following formula (4), which comprises a hydrophilic or hydrophobic material attached to the 5' end of the sense strand and antisense strand of double RNA propeller:

[0105] Wherein A is a hydrophilic material; B is a hydrophobic material; X and Y are each independently a single covalent bond or a linker-mediated covalent bond; S is the sense strand of a double-stranded RNA oligo; AS is the antisense strand of the double-stranded oligo RNA; and L is a receptor-specific ligand having the property of enhancing target cell internalization by receptor-mediated endocytosis (RME).
[0106] A method to prepare the double-stranded oligo RNA structure represented by formula (4) comprises the steps of:
[0107] (1) synthesize a single-stranded RNA on a solid support;
[0108] (2) covalently attach a hydrophilic material to the 5' end of the single-stranded RNA;
[0109] (3) attach a linker to the hydrophilic material attached to the single-stranded RNA;
[0110] (4) separate the hydrophilic RNA-polymer structure attached to a linker and a complementary hydrophobic RNA-polymer structure separately synthesized from the solid support; and
[0111] (5) anneal the RNA-hydrophilic polymer structure bonded to the binder with the complementary RNA-hydrophobic polymer structure to form a double-stranded structure.
[0112] The preparation method comprises, between steps (1) to (4), a step to synthesize a single-stranded RNA complementary to the single-stranded RNA of step (1), and then covalently bind a hydrophobic material to the synthesized RNA to synthesize a hydrophobic polymer-single-stranded RNA structure.
[0113] The present invention further provides a nanoparticle comprising the double stranded oligo RNA structure having the linker attached to it, and a nanoparticle comprising the polymer-therapeutic drug structure having the linker attached thereto.
[0114] A nanoparticle is formed by interaction between oligo RNA double helix structures linked to the ligand of the present invention. Specifically, a nanoparticle is formed, which has a structure in which a hydrophobic material is located at the center of the nanoparticle, a double-stranded RNA oligo is protected by an outer hydrophilic material, and a binder is located on the surface of the nanoparticle (see FIG. 1). The nanoparticle releases the double-stranded oligo RNA into a cell via the ligand, and thus releases the RNA into a cell with increased efficiency. This nanoparticle can be used to treat diseases. Synthesis of structure and characteristics, intracellular release efficiency and effects of a nanoparticle comprising the structure will be described in further detail in the Examples below.
[0115] The present invention further provides a method of gene therapy that uses a nanoparticle composed of a double-stranded oligo RNA structure linked to the ligand or the therapeutic drug-polymer structures linked to the ligand.
[0116] Specifically the present invention provides a therapeutic method comprising the steps of: preparing a nanoparticle composed of a double stranded oligo RNA structure linked to the ligands; and introduce the nanoparticle into an animal's body.
[0117] The present invention further provides a pharmaceutical composition comprising a pharmaceutically effective amount of a nanoparticle composed of double-stranded oligo RNA structure linked to the ligands.
[0118] For administration, the composition of the present invention may comprise, in addition to the active ingredient described above, at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier that can be used in the present invention must be compatible with the active ingredient of the present invention and may be physiological saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of two or more of these. Furthermore, the composition of the present invention may, if necessary, comprise other conventional additives, including antioxidants, buffers, and bacteriostatic agents. Furthermore, the composition of the present invention can be formulated as injectable forms as aqueous solutions, suspensions, or emulsions with the aid of diluents, dispersants, surfactants, binders and lubricants. Particularly, the composition of the present invention is preferably provided as a lyophilized formulation. For preparing the lyophilized formulations, any conventional method known in the art can be used, and a stabilizer for lyophilization can further be added.
[0119] Furthermore, the composition of the present invention can be formulated into appropriate dosage forms depending on the type of disease or component according to a method known in the art or the method disclosed in Remington's pharmaceutical Science (Mack Publishing Company, Easton PA) .
[0120] The dosage of the pharmaceutical composition of the present invention can be determined by those skilled in the art depending on the conditions of the patient and the severity of the disease. Furthermore, the composition of the present invention may be formulated in the form of powders, tablets, capsules, liquid, injection solutions, ointments, and syrups, and may be provided in unit dosage forms or multiple dosage forms, e.g., ampoules or sealed bottles.
[0121] The pharmaceutical composition of the present invention can be administered orally or parenterally. The pharmaceutical composition according to the present invention can be administered by various routes, including, but not limited to, orally, intravenously, intramuscularly, intraarterially, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, gastrointestinal, sublingual, and local .
[0122] For said clinical administration, the pharmaceutical composition of the present invention can be formulated into appropriate forms using a technique known in the art. The dose of the composition of the present invention may vary depending on various factors such as the patient's body weight, age, sex, health condition and diet, the time and method of administration, excretion rate, and severity of a disease, and may be easily determined by a person skilled in the art. 2. Second aspect of the present invention
[0123] In a second aspect, the present invention provides an ASO-polymer conjugate represented by the following formula 5:

[0124] Where one of A and B is a hydrophilic material, the other is a hydrophobic material, X and Y are each independently a single covalent bond or a ligand-mediated covalent bond, and R is an ASO.
[0125] As used herein, the term "ASO" means to include not only conventional antisense oligonucleotides that are used to inhibit mRNA expression, but even antagomirs that inhibit microRNA functions.
[0126] The hydrophilic material in formula (5) may have a specific target binder attached to it. Target-specific ligand has the property of enhancing target-specific internalization by receptor-mediated endocytosis (RME) and can be selected from target-specific antibodies or aptamers, peptides as receptor-specific ligands, and chemical materials such as folate, N- acetylgalactosamine (NAG), and mannose. Here, the targeting moiety is a material that carries out the release in a target-specific manner, and is not only limited by the above-described antibody, aptamer, peptide and chemical materials.
[0127] In the conjugate of the present invention, the ASO preferably comprises 10-50 oligonucleotides, more preferably 13-25 oligonucleotides.
[0128] To improve stability in vivo, ASO includes oligodeoxynucleotides (ODNs) obtained by various modifications that provide nuclease resistance. The modification may be one or a combination of two or more selected from replacing an -OH group at the 2' carbon position of the sugar moiety of one or more nucleotides with -CH3 (methyl), -OCH3,-NH2, -F (fluorine), -O-2-methoxyethyl, -O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl, -ON-methylacetamido or -O-dimethylamidoxyethyl; the oxygen replacement of the sugar fraction of the nucleotide with sulfur; modification of the bond between the nucleotides into a phosphorothioate, boranephosphonate, or methyl phosphonate bond. Alternatively, the modification can be modification in the form of PNA (peptide nucleic acid) or LNA (blocked nucleic acid).
[0129] An ASO that can be used in the present invention is not specifically limited and can be an ASO for any gene that is used or can be used for gene therapy or research.
[0130] It will be obvious to those skilled in the art that ASOs that are used in the present invention include, among others, an ASO having a perfect pairing with the target mRNA, but still an ASO that wrongly pairs with the target mRNA to inhibit translation of the mRNA.
[0131] The hydrophilic material preferably has a molecular weight of 200-10,000, more preferably 1,000-2,000. Furthermore, the hydrophilic material is preferably a cationic or non-ionic polymer compound.
[0132] For example, the hydrophilic polymer composite that is used in the present invention is preferably a nonionic hydrophilic polymer composite such as PEG (polyethylene), polyvinylpyrrolidone or polyoxazoline, but is not limited thereto.
[0133] The hydrophilic material can, if necessary, be modified to have a functional group required to bond to the other materials. Among hydrophilic materials, particularly polyethylene glycol (PEG) can have various molecular weights and functional groups, have good biocompatibility, do not induce immune response, increase the in vivo stability of ASO, and increase the efficiency of ASO release, and thus this it is very suitable for the preparation of the inventive conjugate.
[0134] Furthermore, the hydrophobic material preferably has a molecular weight of 250-1,000. Particularly, the hydrophobic material which is used in the present invention may preferably be a steroid derivative, a glyceride derivative, glycerol ether, polypropylene glycol, a C12-C50 saturated or unsaturated hydrocarbon, diacyl phosphatidylcholine, fatty acid, phospholipid, lipopolyamine or the like , but is not limited to these, and it will be obvious to those skilled in the art that any hydrophobic material suitable for the purpose of the present invention can be used.
[0135] Particularly, the steroid derivative can be selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate, and cholestanyl amine, and the glyceride derivative can be selected from mono-, di- and triglycerides, where the fatty acid of the glyceride may be a saturated or unsaturated C12-C50 fatty acid.
[0136] The hydrophobic material works to cause a hydrophobic interaction to form a nanoparticle. Among the hydrophobic materials, particularly a carbon or cholesterol chain is very suitable for the preparation of the conjugate of the present invention, because it can be easily attached in the step of preparing an ASO.
[0137] Furthermore, the covalent bond indicated by X or Y may be a non-degradable bond or a degradable bond. Here, non-degradable bonds include, among others, an amide bond or a phosphate bond, and degradable bonds include, among others, a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond, or an enzymatically degradable bond.
[0138] An ASO-polymer conjugate according to the present invention may have a structure in which a hydrophilic material is attached to one of the 5' and 3' ends of an ASO and the hydrophobic material is attached to the other end.
[0139] A method for preparing the ASO-polymer conjugate having the hydrophilic material attached to the 3' end of the ASO may comprise the steps of:
[0140] (a) covalently attaching a hydrophilic material to a solid support;
[0141] (b) synthesize an ASO on the solid support comprising the hydrophilic material;
[0142] (c) covalently attaching a hydrophobic material to the 5' end of the ASO on the solid support; and
[0143] (d) separate and purify the resulting ASO-polymer conjugate from the solid support.
[0144] In a more preferred embodiment, the ASO-polymer conjugate is prepared by a method comprising the steps of: covalently attaching a hydrophilic material to a solid support (controlled pore glass (CPG)); synthesizing an ASO on solid support (CPG), which has the hydrophilic material covalently attached to it, by deblocking, coupling, capping and oxidation; and covalently attaching a hydrophobic material to the 5' end of the ASO. After completion of the preparation of the ASO-polymer conjugate, the ASO-polymer conjugate is separated from the solid support (CPG) treating it with 28%(v/v) ammonia in a water bath at 60°C, and the ASO-conjugate polymer can be separated and purified from the reagents by high performance liquid chromatography (HPLC), after which the molecular weight can be measured by MALDI-TOF mass spectrometer to determine if the desired ASO-polymer conjugate has been prepared.
[0145] In another aspect, a method for preparing an ASO-polymer conjugate comprising a hydrophilic material attached to the 5' end of an ASO comprises the steps of:
[0146] (a) synthesize an ASO on a solid support having a functional group attached thereto;
[0147] (b) covalently attaching a hydrophilic material to the 5' end of the ASO;
[0148] (c) separating the ASO conjugate bound to the hydrophilic material from the solid support; and
[0149] (d) covalently attach a hydrophobic material to the 3' end of the ASO separated from the solid support.
[0150] In a more preferred embodiment, the method of preparation comprises the steps of: synthesizing an ASO on a solid support (CPG) having a functional group attached to it; covalently attaching a hydrophilic material to the 5' end of the ASO; treating the resulting solid with 28 %(v/v) ammonia in a water bath at 60°C to separate the functional group-bound ASO-hydrophilic polymer conjugate from the solid support (CPG); and attaching a hydrophobic material to the ASO via the functional group to form an ASO-polymer conjugate comprising the hydrophilic material and hydrophobic material bonded to both ends of the ASO. When the preparation of the ASO-polymer conjugate has been completed, the ASO-polymer conjugate can be separated and purified from the reagents by high performance liquid chromatography (HPLC), after which the molecular weight can be measured by the MALDI-mass spectrometer. TOF to determine if the desired ASO-polymer conjugate was prepared.
[0151] However, the ASO-polymer conjugate may further comprise a binder attached to the hydrophilic material.
[0152] A method for attaching a linker to the hydrophilic material is determined according to the type of functional group attached to the linker. For example, a phosphoramidite-linker having phosphoramidite as a functional group can be attached to the hydrophilic material in the same way as the ASO synthesis process, and a linker having N-Hydroxysuccinimide (NHS) attached thereto can be attached to the hydrophilic material by an N-Hydroxysuccinimide (NHS) ester linkage.
[0153] A method for preparing an ASO-polymer conjugate comprising a hydrophilic material attached to the 3' end of an ASO comprises the steps of:
[0154] (a) attaching a hydrophilic material to a solid support having a functional group attached thereto;
[0155] (b) synthesize an ASO on the solid support having the functional group-hydrophilic material attached to it;
[0156] (c) covalently attaching a hydrophobic material to the 5' end of the ASO;
[0157] (d) separate an ASO-polymer conjugate, obtained in step (c), from the solid support; and
[0158] (e) attaching a binder to the hydrophilic material of the ASO-polymer conjugate separated from the solid support.
[0159] In a more preferred embodiment, the method of preparation comprises the steps of: attaching a hydrophilic polymer to a solid support having a functional group attached to it; synthesizing an ASO on the solid support (CPG) having the functional group-hydrophilic material attached thereto; covalently attaching a hydrophobic material to the ASO endgroup; separating the resulting functional group-ASO-polymer conjugate from the solid support (CPG); and attaching a linker to the end of the hydrophilic polymer by the functional group, thereby preparing an ASO-polymer conjugate linked to the linker. When the preparation of the binder-bound ASO-polymer conjugate has been completed, the ASO-polymer conjugate can be separated and purified from the reagents by high-performance liquid chromatography (HPLC), after which the molecular weight can be measured by the spectrometer. MALDI-TOF mass to determine if the binder-bound ASO-polymer conjugate was prepared.
[0160] In another aspect, a method for preparing an ASO-polymer conjugate comprising the linker attached to an ASO-polymer conjugate having a hydrophilic material attached to the 5' end of an ASO comprises the steps of:
[0161] (a) to synthesize an ASO on a solid support having a functional group attached thereto;
[0162] (b) covalently attaching a hydrophilic material to the end of the ASO;
[0163] (c) covalently attaching a linker to the ASO-hydrophilic material conjugate;
[0164] (d) separate an ASO-hydrophilic material-linker conjugate, which has the functional group attached to it, from the solid support; and
[0165] (e) covalently attach a hydrophobic material to the 3' end of the ASO of the conjugate separated from the solid conjugate.
[0166] In a more preferred embodiment, the method of preparation comprises the steps of: synthesizing an ASO on a solid support (CPG) having a functional group attached to it; covalently attaching a hydrophilic material to the ASO end group; covalently attaching a linker to the ASO-hydrophilic polymer; separating the functional group-linked ASO-hydrophilic polymer-linker conjugate from the solid support (CPG); and attaching a hydrophobic material to the conjugate separated by the functional group, to thereby synthesize a binder-linked ASO-polymer conjugate having the hydrophobic material attached to the opposite end of the hydrophilic polymer. When the preparation of the binder-bound ASO-polymer conjugate has been completed, the ASO-polymer conjugate can be separated and purified from the reagents by high-performance liquid chromatography (HPLC), after which the molecular weight can be measured by the spectrometer. MALDI-TOF mass to determine if the binder-bound ASO-polymer conjugate was prepared.
[0167] As a result, the ASO-polymer conjugate synthesized in the present invention comprises both hydrophobic and hydrophilic materials, and thus, is amphiphilic in nature. The hydrophilic fraction tends to come out by interaction (as a hydrogen bond) with water molecules in vivo, and the hydrophobic material tends to come in by hydrophobic interaction, and thus a thermodynamically stable nanoparticle is formed. In other words, a nanoparticle is formed in which the hydrophobic material is located in the center of the nanoparticle and the hydrophilic material is located outside the ASO to protect the ASO (see FIG. 18). The nanoparticle formed as described above improves the intracellular release of ASO and can be used for the treatment of diseases. Synthesis of the conjugate and the characteristics, intracellular release efficiency and effects of the conjugate will be described in further detail in the Examples below.
[0168] In addition, the present invention provides a method of gene therapy comprising the steps of: preparing a nanoparticle composed of ASO-polymer conjugates; and releasing ASO in vitro by the nanoparticle. The gene therapy method is not limited to in vitro application.
[0169] The present invention also provides a pharmaceutical composition comprising a pharmaceutically effective amount of the ASO-polymer conjugate or a nanoparticle composed of the ASO-polymer conjugate bound to the binder.
[0170] For administration, the composition of the present invention may comprise, in addition to the active ingredient described above, at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier that can be used in the present invention must be compatible with the active ingredient of the present invention and may be physiological saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of two or more of these. Furthermore, the composition of the present invention may, if necessary, comprise other conventional additives, including antioxidants, buffers, and bacteriostatic agents. Furthermore, the composition of the present invention can be formulated as injectable forms as aqueous solutions, suspensions, or emulsions with the aid of diluents, dispersants, surfactants, binders and lubricants. Particularly, the composition of the present invention is preferably provided as a lyophilized formulation. For preparing the lyophilized formulations, any conventional method known in the art can be used, and a stabilizer for lyophilization can further be added.
[0171] Furthermore, the composition of the present invention can be formulated into appropriate dosage forms depending on the type of disease or component according to a method known in the art or the method disclosed in Remington's pharmaceutical Science (Mack Publishing Company, Easton PA) .
[0172] The dosage of the pharmaceutical composition of the present invention can be determined by those skilled in the art depending on the conditions of the patient and the severity of the disease. Furthermore, the composition of the present invention may be formulated in the form of powders, tablets, capsules, liquid, injection solutions, ointments, and syrups, and may be provided in unit dosage forms or multiple dosage forms, e.g., ampoules or sealed bottles.
[0173] The pharmaceutical composition of the present invention can be administered orally or parenterally. The pharmaceutical composition according to the present invention can be administered by various routes, including, but not limited to, orally, intravenously, intramuscularly, intraarterially, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, gastrointestinal, sublingual, and local . The dose of the composition of the present invention may vary depending on various factors such as the patient's body weight, age, sex, health condition and diet, the time and method of administration, excretion rate, and severity of a disease, and may be easily determined by a person skilled in the art. EXAMPLES
[0174] Hereinafter, the present invention will be described in more detail with reference to the examples. It will be clear to a person of ordinary skill in the art that these examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention. Example 1: Preparation of binder material that can be bonded
[0175] To prepare a double stranded oligo RNA structure having a linker attached to it, a linker material that can be attached to the double stranded oligo RNA structure was prepared.
[0176] Example 1-1: Preparation of reagent 1-hexa(ethylene glycol)- N-acetylgalactosamine-phosphoramidite (compound A, BeC)
[0177] To attach N-acetyl galactosamine (NAG) to a double-stranded oligo RNA structure, 1-hexa(ethylene glycol)-NAG-phosphoramidite was prepared as shown in the following reaction scheme 1. Reaction scheme 1
Example 1-1-1: Preparation of 1,3,4,6-tetraacetyl-NAG(compound A)
The starting material is galactosamine hydrochloride (Sigma Aldrich, USA) (2g, 9.27mmol), acetonitrile (Samjeon, Korea) (31m () and triethylamine (Sigma Aldrich, USA) (15.42m, 111.24 mmol) were mixed with each other and refluxed for 1 hour. The mixture was cooled slowly to room temperature and cooled to 0°C using ice water, and then acetic anhydride (Sigma Aldrich, USA) (8.76m£, 92.70mmol) was added dropwise over 10 minutes. Then the ice water was removed and the remaining material was stirred at room temperature for 24 hours. After completion of the reaction, an aqueous solution of sodium bicarbonate (Samjeon, Korea) was slowly added to the reaction product until the pH was neutral. After the pH became neutral, the reaction solution was stirred at room temperature for 2 hours, and the solid produced was filtered. The filtrate was washed sequentially with ethyl acetate (Samjeon, Korea) (100 ml x 2), distilled water (100 ml x 2) and ethyl acetate (100 ml x 1). The solid was lyophilized to generate 1,3,4,6-tetraacetyl-N-acetyl galactosamine (1.82g, 52.3%) (see FIG. 3). Example 1-1-2: Preparation of 3,4,6-triacetyl-1-hexa(ethylene glycol)-NAG (compound B)
[0179] The 1,3,4,6-tetraacetyl-N-acetyl galactosamine (1.81g, 4.82mmol) prepared in Example 1-1-1, iron III chloride (Sigma Aldrich, USA)(1, 02g, 6.27mmol) and methylene chloride (Samjeon, Korea) (48m£) were mixed with each other and stirred at room temperature for 10 minutes. Then hexa(ethylene glycol)(Sigma Aldrich, USA)(1.58m, 4.82mmol) was added to the mixture, followed by reflux for 2 hours. After completion of the reaction, the reaction solution was filtered through celite (Sigma Aldrich, USA), and the filtrate was washed with methylene chloride (50 ml x 2). The filtrate was concentrated under reduced pressure and added to ethyl acetate (100m) and distilled water (100m), and the aqueous layer was collected. The collected aqueous layer was extracted with methylene chloride (100 ml x 3), and the organic layer was collected, dried with anhydrous magnesium sulfate (Samjeon, Korea) and filtered. The filtrate was concentrated under reduced pressure and dried under vacuum, thus obtaining 3,4,6-triacetyl-1-hexa(ethylene glycol)-N-acetyl galactosamine (2.24g, 74.9%) (see FIG. 4) . Example 1-1-3: Preparation of 1-hex(ethylene glycol)-NAG-phosphoramidite (compound C)
[0180] The compound (2.22g, 3.71mmol) obtained in Example 1-1-2, methylene chloride (37m) and triethylamine (0.94m£, 6.75mmol) were mixed with each the other and stirred at room temperature for 10 minutes. Then, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (Sigma Aldrich, USA) (0.75m£, 3.38mmol) was added to the mixture and stirred for 45 minutes. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and ethyl acetate (100 ml) and distilled water (100 ml) were added thereto. The organic layer was collected, dried with anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography, thus obtaining 1-hexa(ethylene glycol)-N-acetyl galactosamine-phosphoramidite (1.14g, 42.2%) (see FIG. 5). Example 1-2: Preparation of NHS-folate
[0181] To bind folate to a double-stranded oligo RNA structure, NHS-folate was prepared as shown in the following reaction scheme 2:
[0182] Reaction Scheme 2

[0183] The starting material folic acid (Sigma Aldrich, USA)(3g, 6.8 mmol), dimethyl sulfoxide (Sigma Aldrich, USA)(60m£), N-hydroxysuccinimide (Sigma Aldrich, USA)(0, 86g, 7.5mmol) and 1,3-dicyclohexylcarbodiimide (Sigma Aldrich, USA)(1.54g, 7.5mmol) were mixed with each other and stirred at room temperature for 18 hours. After completion of the reaction, the reaction mixture was added dropwise to 950ml of a 3:5 mixture of ethyl acetate:n-hexane (Samjeon, Korea) for 10 minutes, and the solid produced NHS-folate (3.79g) was filtered (Robert J. Lee and Philip S. Low (1994) J. Biological Chemistry. 269: 31983204). Example 1-3: Peptide Preparation
[0184] Due to the fact that peptide compounds include α-amino acid, a linkage relationship between a peptide derivative having this structure and the amine functional group of PEG has been realized. In this linkage reaction, the amine functional groups present in PEG and the peptide derivative interact with each other during the linkage reaction with the carboxylic acid of the peptide, and for this reason, a process to replace the amine functional group of the peptide compound with a protecting group was required prior to the binding reaction. The amine group of the peptide compound was replaced with a 9-fluorenylmethyloxycarbonyl (Fmoc) or tert-butyloxycarbonyl (t-BOC) protecting group to remove its reactivity with the carboxylic acid of the peptide, thus preparing a peptide compound capable of binding to PEG . Example 2: Preparation of double-helix oligoRNA structure having a linker attached to the 5' end
[0185] The linker material prepared in Example 1 can be constructed in the phosphoramidite form like the NAG-phosphoramidite of Example 1-1 so that it can be attached to the PEG end by a general chemical oligo synthesis process (consisting of in unlocking, coupling, capping and oxidizing). Alternatively, this can be constructed in the NHS-linker form like the NHS-folate from Example 1-2 so that it can be linked to amine-linked PEG via an ester linkage, to thereby synthesize a PEG-RNA structure having the linker. attached to the 5' end of the RNA. The synthesized PEG-RNA structure having the linker attached to the 5' end was annealed with a complementary 5' C24-RNA structure having a hydrophobic group attached thereto, to thereby synthesize the double stranded oligo RNA structure having the linker attached to the 5' end . Example 2-1: Preparation of double helix RNA oligo
[0186] In the following examples, a double-stranded anti-survivin RNA oligo was used to inhibit survivin. Survivin is a protein that is commonly expressed in most tumors or mutant cell lines to date and is expected to be an important target in anti-cancer therapy (Survivin: a new target for anti-cancer therapy. Cancer Treat Rev. 2009 Nov; 35(7):553-62). The survivin double stranded RNA oligo according to the present invention consists of a sense strand set forth in SEQ ID NO: 1 and an antisense strand complementary thereto, and a double stranded oligo nucleotide which is used as a control of an established sense strand in SEQ ID NO: 2 and an antisense strand complementary thereto. The double stranded RNA oligo used in this example consists of the following nucleotide sequence. (SEQ ID NO: 1) 5'-AAG GAG AUC AAC AUU UUC A-3' (SEQ ID NO: 2) 5'-CUU ACG CUG AGU ACU UCG A-3'
[0187] To synthesize double-stranded oligo RNA, single-stranded RNA was synthesized by linking nucleotides by phosphodiester bonds of the RNA backbone using β-cyanoethyl phosphoramidite protected by tert-butyldimethylsilyl (Polymer support oligonucleotide synthesis: use of β-cyanoethyl-N ,N-dialkylamine-/N-morpholine phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucleic Acids Res. 1984 Jun 11;12(11):4539-57).
[0188] In the synthesis process, a cycle that consists of unlocking, coupling, capping and oxidizing was repeated a solid support having a nucleoside attached to it, thus obtaining a desired RNA sequence. The process to synthesize single-stranded RNA was performed using an RNA synthesizer (384 Synthesizer, BIONEER, Korea) (see FIG. 6). Example 2-2: Preparation of 5' PEG-RNA structure
[0189] The RNA synthesized in Example 2-1 was reacted with a PEG phosphoramidite reagent according to a general RNA synthesis procedure, thus preparing a 5' PEG-RNA structure (see FIG. 7). Example 2-3:SynthesisofPEG-RNAhavinglinkerattachedtothe5'end Example 2-3-1:Preparation ofNAG-PEG-RNAstructureusingN-acetylgalactosamine(NAG)phosphoramidite
[0190] The 5' PEG-RNA structure synthesized in Example 2-2 was reacted with the NAG phosphoramidite reagent (synthesized in Example 1-1) according to a general RNA synthesis procedure to bind N-acetyl galactosamine (NAG) to the PEG-RNA backbone by a phosphodiester bond. The N-acetyl galactosamine linker can provide one or more N-acetyl galactosamine molecules to PEG using a dendrimer linker. Example 2-3-1-1: Preparation of mono NAG-PEG-RNA structure
[0191] The PEG-RNA structure synthesized in Example 2-2 was reacted with the NAG phosphoramidite reagent (synthesized in Example 1-1) according to a general RNA synthesis procedure to attach N-acetyl galactosamine (NAG) to the structure PEG-RNA by a phosphodiester linkage, thus synthesizing a 5' mono-NAG-PEG RNA structure (see FIG. 8). Example 2-3-1-2: Preparation of NAG-PEG-RNA Triple Structure
[0192] The PEG-RNA structure synthesized in Example 2-2 was reacted with a phosphoramidite dendrimer reagent (Trebler Phosphoramidite, Glen research, USA), and then reacted with the NAG phosphoramidite reagent (synthesized in Example 1-1) according to a general RNA synthesis process to link three NAGs to the PEG-RNA backbone via phosphodiester bonds, to thereby synthesize a 5' triple-NAG-PEG-RNA backbone (see FIG. 9). Example 2-3-2: Preparation of Folate-PEG-RNA Structure
[0193] The PEG-RNA structure synthesized in Example 2-2 was reacted with an amine phosphoramidite reagent according to a general RNA synthesis procedure to link an amine group to the PEG-RNA structure via a phosphodiester bond, to thereby synthesize an amine-PEG-RNA structure. The synthesized amine-PEG-RNA structure was linked with NHS-folate (synthesized in Example 1-2) via an ester bond to synthesize a 5' folate-PEG-RNA structure (see FIG. 10). Example 2-3-3: Preparation of peptide-PEG-RNA structure by amine and peptide compound modification
[0194] The linkage reaction between the carboxyl group of the protected peptide compound prepared in Example 1-3 and the amine group of PEG of the amine-PEG-RNA structure prepared in Example 2-3-2 was carried out using BOP (benzotriazol-1 -yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate) (Sigma Aldrich, USA) and HOBT (1-hydroxybenzotriazole) (Sigma Aldrich, USA). After the ligation reaction, the reaction product was treated with piperidine (Sigma Aldrich, USA) to remove the protecting group, thus preparing a 5' peptide-PEG-oligo RNA double helix structure. Example 2-4: Preparation of 5' C24-RNA structure
[0195] An RNA structure complementary to the RNA sequence of the 5' linker-PEG-oligo double-stranded RNA structure of Example 2-3 was synthesized by the RNA synthesis method of Example 2-1, and then treated with a reagent C24 tetradocosane containing a disulfide bond according to a general RNA synthesis procedure to link C24 to the RNA backbone by a phosphodiester bond, thereby synthesizing a 5' C24-RNA backbone (see FIG. 11). Example: 2-5: Recovery and annealing
[0196] Each of the single-stranded RNA synthesized in Examples 21, 2-2, 2-3 and 2-4 was treated with 28% (v/v) ammonia in a water bath at 60°C to separate the synthesized RNAs and RNA structures from CPGs, followed by deprotection. The unprotected 5' linker-PEG-RNA double-stranded structures and 5' C24-oligo RNA double-stranded structures were treated with a 10:3:4 (v/v/v) mixture of N-methylpyrrolidone, triethylamine and triethylamine trihydrofluoride in an oven at 70°C to remove 2'TBDMS (tert-butyldimethylsilyl).
[0197] The RNAs were separated from the reagents by high performance liquid chromatography (HPLC) to separate the RNAs, and the molecular weights of RNAs were measured by MALDI-TOF MS (SHIMADZU, Japan) to determine if the RNAs were consistent with the desired nucleotide sequences and double stranded RNA oligo structures.
[0198] Next, to prepare a double-stranded oligo RNA structure having a linker attached to it, the PEG-RNA sense RNA attached to the linker and antisense RNA were mixed with each other in the same amount, and the mixture was added to 1X buffer of annealing (30 mM HEPES, 100 mM Potassium Acetate, 2 mM Magnesium Acetate, pH 7.0 to 7.5) and allowed to react in a water bath at a constant temperature at 90°C for 3 minutes, then left to react react at 37°C, thereby preparing a desired double stranded oligo RNA structure having a linker attached to the 5' end of each strand. The prepared double stranded RNA oligo having the linker attached to the 5' end was subjected to electrophoresis to confirm annealing of the strands. Example 3: Preparation of double-helix oligoRNA structure having a linker attached to the 3' end
[0199] Amine-CPG was synthesized in 3' amine-PEG-RNA using a PEG phosphoramidite reagent, and then linked with NHS-linker like NHS-folate from Example 1-2 via an ester bond, to thereby synthesize a PEG-structure double-stranded RNA oligo having a linker attached to the 3' end. The synthesized double-stranded PEG-oligo RNA backbone having a linker attached to the 3' end was ligated with the hydrophobic material C24 to form PEG-RNA-C24 having a linker attached to the 3' end. PEG-RNA-C24 was annealed with a complementary RNA to synthesize the double stranded oligo RNA structure having a linker attached to the 3' end. Example 3-1: Preparation of 3' amine-PEG-RNA structure
[0200] Amine-CPG was treated with a polyethylene glycol phosphoramidite reagent according to a general RNA synthesis procedure to synthesize 3' CPG-amine-PEG. The 3' CPG-amine-PEG was synthesized in a 3' CPG-amine-PEG-RNA structure having a desired RNA sequence according to the RNA synthesis procedure of Example 2-1 (see FIG. 12). Example3-2:Preparation of 3‘amine-PEGRNA-C24 structure
[0201] The 3' CPG-amine-PEG-RNA structure synthesized in Example 31 was treated with a C24 tetradocosane reagent containing a disulfide bond according to a general RNA synthesis procedure to link C24 to RNA via a phosphodiester bond, to thus synthesizing a 3' amine-PEG RNA-C24 structure (see FIG. 13). Example3-3:Preparation of C24-PEGRNA structure having linker attached to the 3’ end
[0202] The 3' amine-PEG-RNA-C24 structure synthesized in Example 32 was treated with 28% ammonia in a water bath at 60°C to separate the synthesized double-stranded 3' amine-PEG-oligo RNA structure and the 3' amine-PEG-RNA-C24 structure of CPG, followed by deprotection. The deprotected 3' amine-PEG-RNA-C24 structure is treated with a 10:3:4 (v/v/v) mixture of N-methylpyrrolidone, triethylamine and triethylamine trihydrofluoride in an oven at 70°C to remove 2' TBDMS ( tert-butyldimethylsilyl). The separated 3' amine-PEG-RNA-C24 structure was linked with a material linker such as NHS-linker by an ester bond, to thereby synthesize a PEG-RNA-C24 structure having a linker attached to the 3' end. Example 3-3-1: Preparation of 3' Folate-PEG-RNA Structure
[0203] The amine-PEG-RNA-C24 structure synthesized in Example 3-2 was linked with NHS-folate (synthesized in Example 1-2) by an ester linkage to synthesize a 3' folate-PEG-RNA-C24 structure ( FIG. 14). Example 3-4: Preparation of complementary RNA structure
[0204] A single-stranded RNA complementary to the 3' linker-PEG-RNA-C24 structure sequence of Example 3-3 was synthesized using the RNA synthesis method of Example 2-1. The synthesized single-stranded RNAs were treated with 28% ammonia in a water bath at 60°C to separate the synthesized RNAs from the CPG, followed by deprotection. Unprotected RNAs were treated with a 10:3:4 (v/v/v) mixture of N-methylpyrrolidone, triethylamine and triethylamine trihydrofluoride in an oven at 70°C to remove 2‘ TBDMS (2‘ tert-butyldimethylsilyl). Example 3-5: Annealing
[0205] The RNA and 3' linker-PEG RNA-C24 reaction products were separated from the reagents by high-performance liquid chromatography (HPLC; LC-20A Prominence, SHIMADZU, Japan), and the molecular weights of the materials separated were measured by MALDI TOF-MS (SHIMADZU, Japan) to determine if they were consistent with the desired nucleotide sequence and 3' linker-PEG RNA-C24 structure.
[0206] Next, to prepare double-stranded oligo RNA structure having a linker attached to it, the PEG-RNA sense RNA attached to the linker and antisense RNA were mixed with each other in the same amount, and the mixture was added to 1X buffer of annealing (30 mM HEPES, 100 mM Potassium Acetate, 2 mM Magnesium Acetate, pH 7.0 to 7.5) and allowed to react in a constant temperature water bath at 90°C for 3 minutes, then allowed to react at 37°C, a desired double stranded oligo RNA structure was thus prepared having a linker attached to the 3' end of each of the strands. The prepared double stranded RNA oligo having the linker attached to the 3' end was subjected to electrophoresis to confirm annealing of the strands. Example 4: Formation of nanoparticles composed of double-stranded oligoRNA structures having ligand attached to it
[0207] The double stranded oligo RNA structures having the linker at the 5' end, and the double stranded oligo RNA structures having the linker at the 3' end, synthesized in Examples 2 and 3, form a nanoparticle (ie micelle) composed of double-stranded oligo RNA structures attached to the linker by hydrophobic interactions between the hydrophobic material attached to the ends of double-stranded oligo RNA (see FIG. 1). Critical micelle size and concentration (CMC) measurements and transmission electron microscopy (TEM) analysis for a nanoparticle composed of the 5' folate-linker-oligo double-stranded RNA synthesized in Example 2 were measured to confirm nanoparticle formation . Example4-1:Measurement of particle size in a particle composed of double-stranded 5‘folate-oligoRNA structures
[0208] Nanoparticle size was measured by measuring zeta potential. Specifically, the 5' double-stranded folate-oligo RNA structures were dissolved in 1.5 mDP DPBS (Dulbecco's phosphate buffered saline) at a concentration of 50 µg/m^, and then homogenized with a sonicator (Wiseclean, DAIHAN, Korea) (700 W; amplitude: 20%). The size of homogenized nanoparticles was measured with a Zetasizer (Nano-ZS, MALVERN, GB) under the following conditions: refractive index: 1.459, absorption index: 0.001, solvent temperature PBS (phosphate buffered saline: 25°C, viscosity at that temperature: 1.0200, and refractive index: 1.335. Each measurement consisted of 20 readings and was repeated three times.
[0209] It was shown that nanoparticles (folate-SAMiRNA) composed of folate-bound double-stranded oligo RNA structures had a size of about 100-200 nm. A lower polydispersion index (PDI) value indicates a more even distribution of particles. The PDI value of folate-SAMiRNA was measured to be less than 0.4, suggesting that nanoparticles having a relatively uniform size were formed. The size of nanoparticles composed of such structures has been shown to be appropriate for absorption into cells by endocytosis (Nanotoxicology: nanoparticles reconstruct lipids. Nat Nanotechnol. 2009 Feb;4(2):84-5) (see FIG. 15(A) ). Example4-2:Measurement of ice-critical concentration of nanoparticles composed of double-stranded oligoRNA structures
[0210] An amphiphilic material containing both an oleophilic group and a hydrophilic group on the molecule can act as a surfactant. When a surfactant is dissolved in an aqueous solution, the hydrophobic fractions come in to avoid contact with the water, and the hydrophilic fractions come out, thus forming a micelle. The concentration at which the micelle is first formed is defined as the critical micelle concentration (CMC). One method for measuring CMC using a fluorescent dye is based on a rapid change in the slope of the fluorescent intensity graph of a fluorescent dye before and after micelle formation.
[0211] For the measurement of critical micelle concentration of nanoparticles composed of the folate-linked double-stranded oligo RNA structures, 0.04 mM DPH (1,6-Diphenyl-1,3,5-hexatriene, Sigma Aldrich, USA) how a fluorescent dye was prepared. 1 nmol/μ^' of the 5' folate-oligo double-stranded RNA synthesized in Example 2 was diluted with DPBS serially from 0.0977 /m a to 50 g/m^, thus preparing 180 ^ each of samples of the 5' double-stranded folate-oligo RNA structure. To the preparation sample, 20 µl each of 0.04 mM DPH in methanol and methanol alone as a control was added and shaken well. Then, homogenization using a sonicator (Wiseclean, DAIHAN, Korea) was carried out in the same manner as described in Example 4-1 (700 W; amplitude: 20%). Each of the homogenized samples was allowed to react at room temperature under a condition protected from light for about 24 hours, and the fluorescence intensities (excitation: 355 nm, emission: 428 nm, top reading) were measured. Because the measured fluorescence intensities were used to determine the relative fluorescence intensity, the relative fluorescence intensity ([fluorescence intensity of sample containing DPH]-[fluorescence intensity of sample containing methanol alone]) at the same concentration was calculated and graphically displayed on the Y-axis as a function of the log value of the concentration of 5' folate-oligo double-stranded RNA structures (X-axis) (see FIG. 15(B)).
[0212] The fluorescence intensities measured at various concentrations increase as the concentration increases, and the point at which the concentration increases rapidly is the CMC concentration. Thus, the low concentration regions where the fluorescence did not increase and the high concentration region where the fluorescence intensity increased were divided into several points to draw trend lines, and the X axis value where the two trend lines crossed with each other was determined according to the CMC concentration (FIG. 15(B)). The measured CMC of the double-stranded folate-oligo RNA structure was very low (1.33 μg/m^), suggesting that Folate-SAMiRNA can easily form micelles at a very low concentration. Example4-3:Observation of double-stranded oligoRNA by transmission electron microscope (TEM)
[0213] The morphology of nanoparticles formed from the double-stranded folate-oligo RNA structures was observed by a transmission electron microscope (TEM).
[0214] Specifically, double-stranded folate-oligo RNA structures were dissolved in DPBS (Dulbecco's phosphate buffered saline) at a concentration of 100 µg/m^, and then homogenized with a sonicator (Wiseclean, DAIHAN, Korea) ( 700 W; amplitude: 20%). The nanoparticles formed from the double-stranded folate-oligo RNA structures were observed by negative staining with a material having high electron density. The nanoparticles observed by the transmission electron microscope (TEM) had a size similar to the size of the nanoparticle measured in Example 4-1, suggesting that the nanoparticles were easily formed. Example 5: In vitro release of double-stranded RNA linker-ligand structures
[0215] To assess whether nanoparticles (folate-SAMiRNA) composed of the 5' double-stranded folate-oligo RNA structures synthesized in Example 2 show improved effects of double-stranded oligo RNA in vitro, the KB cell lineage that overexpresses the receptor for folate was grown in the presence or absence of folate without transfection. As a result, it was shown that the bound ligand improved the efficiency of intracellular SAMiRNA release and that SAMiRNA had the effect of inhibiting target gene expression. Example 5-1: Tumor cell lineage culture
[0216] The oral epithelial carcinoma (KB) cell line purchased from the American type Culture Collection (ATCC) was cultured in folate-free RPMI-1640 medium (Gibco, USA) supplemented with 10%(v/v) FBS, 100 units/m^ penicillin and 100 ^g/m^ streptomycin, under the conditions of 37°C and 5%(v/v) CO 2 . Example 5-2: Tumor cell lineage transfection with folate-SAMiRNA
[0217] Tumor cells (1.3 x 105 cells/well) cultured in Example 5-1 were cultured in a folate-free RPMI-1640 medium in a 6-well plate for 18 hours under the conditions described in Example 5- 1, and then the medium was removed and the same amount of Opti-MEM medium was added to each well.
[0218] Nanoparticles (SAMiRNA-Sur) composed of double-stranded oligo RNA structures comprising a sequence of SEQ ID NO: 1 that inhibits the expression of the target gene survivin, nanoparticles (SAMiRNA-Con) composed of double-stranded oligo RNA structures- polymer comprising a control sequence of SEQ ID NO: 2, and nanoparticles (Folate-SAMiRNA-Sur) composed of double-stranded oligo RNA linker-linked folate structures comprising a sequence of SEQ ID NO: 1, were dissolved in DPBS in a concentration of 50 µ/mm according to the same method as described in Example 4-1, and were homogenized by sonication, thus obtaining homogenized nanoparticles composed of each of the structures.
[0219] To form a condition where an excessive amount of folate in Opti-MEM medium, folate was further added to the medium to form a condition containing 1 mM folate and a condition to which folate was not further added. Then, cells were treated with 200 nM of each sample and cultured under conditions of 37°C and 5%(v/v) CO2 for 48 hours. Example 5-3:Relative quantitative analysis of survivin RNA
[0220] Total RNA was extracted from the transfected cell line of Example 5-2 and synthesized into cDNA, and then the relative expression level of survivin was quantified in real-time PCR according to the method described in the Korean Patent Laid-Open Publication No. 2009-0042297.
[0221] SAMiRNA-Con is a test group treated with nanoparticles composed of double-stranded oligo RNA structures comprising a control sequence of SEQ ID NO: 2, and SAMiRNA-Sur is a test group treated with nanoparticles composed of oligo RNA structures double helix comprising a sequence of SEQ ID NO: 1 (survivin sequence). Folate-SAMiRNA-Sur is a test group treated with nanoparticles composed of double-stranded folate-oligo RNA structures comprising a sequence of SEQ ID NO: 1 (survivin sequence).
[0222] The degree of inhibition of target mRNA expression was the target gene expression level in the test group treated with each of SAMiRNA-Sur and Folate-SAMiRNA-Sur relative to the target gene expression level in the test group treated with SAMiRNA-Con and was determined by comparative quantification (see FIG. 16).
[0223] When an excess amount was present in the medium, it can be seen that the folate receptor in the KB cell line was saturated with an excessive amount of folate, and thus the effect of promoting intracellular uptake by the folate ligand bound to SAMiRNA was masked, suggesting that the folate ligand influences the efficiency of intracellular SAMiRNA release to play a critical role in inhibiting target gene mRNA expression.
[0224] In the case of the group treated with SAMiRNA-Sur, there was no significant difference in the inhibition of target gene expression between the presence and absence of folate, but in the case of the group treated with folate-SAMiRNA-Sur, the inhibition of expression of the target gene was about twice as high in the absence of folate than in the presence of folate. In other words, when an excessive amount of folate was present, the change in the inhibitory effect of target gene expression by the linked folate ligand was not observed, but when folate was absent, the increase in the inhibitory effect of target gene expression by the ligand bound folate was observed.
[0225] Thus, it can be observed that nanoparticles composed of the oligo RNA structure double helix linked to the ligands show improved intracellular release efficiency and increased inhibition of the target gene in cells in which the receptor ligand is overexpressed. Example 6: In vivo release of ligand-bound oligoRNA double-stranded structure
[0226] To assess whether nanoparticles (folate-SAMiRNA) composed of 5' double-stranded folate-oligo RNA structures synthesized in Example 2 improve the effect of double-stranded oligo RNA under in vivo conditions, the nanoparticles were administered to a mouse having a tumor composed of KB cell lineage overexpressing the folate receptor, and the effects of released folate-SAMiRNA and SAMiRNA in inhibiting target gene expression in tumor tissue were evaluated. Example 6-1: Preparation of KB xenograft model
[0227] The KB cell line grown in Example 5-1 was injected subcutaneously into each of the 5-week-old nude mice (BALB/C nude) at a density of 1 X 106 cells. After injection, tumor growth was observed by measuring the long axis and short axis lengths of the tumor at 2-day intervals, and it was shown that the tumor grew to a volume of about 150-200 mm3 at 2 weeks after injection. Example 6-2: Ligand-attached double-stranded oligoRNA structures and administration of ligand-attached double-stranded oligoRNA structures
[0228] For administration in the KB xenograft model prepared in Example 6-1, the 5' folate-oligo double-stranded RNA structures comprising the sequence of SEQ ID NO: 1, synthesized in Example 2, and double-stranded RNA structures having no linker attached thereto, they were homogenized in the same manner as described in Example 4-1, thus obtaining homogenized nanoparticles composed of double-stranded oligo RNA structures. The homogenized nanoparticles were administered once into the tail vein of the KB xenograft models (n=4) at a dose of 5 mg/kg body weight), and tumor tissue was collected at 48 or 72 hours after administration. Total RNA was extracted from the tumor tissue collected into cDNA, and then the relative expression level of survivin mRNA was quantified in real-time PCR according to the method described in Korean Patent Laid-Open Publication No. 2009-0042297 (see FIG. . 17).
[0229] In FIG. 17, PBS is a test group administered with an isolated solvent as a negative control, SAMiRNA is a test group administered with nanoparticles composed of double-stranded oligo RNA structures having no ligand attached to it, and Folate-SAMiRNA is a group of test administered with nanoparticles composed of the double-stranded 5' folate-oligo RNA structures.
[0230] The inhibitory effect of SAMiRNA target gene expression was greater at 72 hours after administration than at 48 hours after administration. The inhibition of target gene expression in the group administered with the structure linked to the ligand (Folate-SAMiRNA) was 160% at 48 hours after administration and 120% at 72 hours after administration.
[0231] Thus, it can be seen that the double-stranded RNA structure having the folate ligand attached to it is rapidly released into the target tumor tissue in vivo that overexpresses the folate receptor, so that the double-stranded oligo RNA effect is improved, and that the effect is maintained over time. Example 7: Preparation of ASO-polymer conjugate
[0232] In the examples of the present invention, a survivin ASO was used to inhibit survival (Biol. Proced. Online 2004; 6(1): 250-256). Survivin is a protein that is commonly expressed in most tumors or mutant cell lines tested to date and is expected to be an important target in cancer therapy (Abbrosini G. etal.Nat.Med,3(8):917- 921,1997).
[0233] The ASOs used in the following examples are a specific sequence for survivin set forth in SEQ ID NO:3 and a control sequence set forth in SEQ ID NO:4.
[0234] (SEQ ID NO: 3) survivin ASO (ISIS 23722), 5-TGTGCTATTCTGTGAATT-
[0235] (SEQ ID NO: 4) scrambled sequence (ISIS 28598), 5-TAAGCTGTTCTATGTGTT-
[0236] ASO sequences were synthesized by nucleotide ligation through the phosphodiester linkages of the DNA backbone using β-cyanoethyl phosphoramidite (Shina et al. NucleicAcidsResearch,12:4539-4557,1984).
[0237] In the ASO synthesis process, a cycle that consists of unlocking, coupling, capping and oxidizing was repeated a solid support bearing (CPG) having a nucleoside attached to it, thus obtaining a desired RNA sequence.
[0238] Specifically, in an unlocking step which is the first step, a CPG having a nucleoside attached to it is treated with 3% trichloroacetic acid (TCA) to remove DMT (4,4'-dimethoxytrityl). In a coupling step which is the next step, the nucleotide chains are linked to each other by the linkage reaction between the 5'-hydroxyl group formed on the CPG in the previous step and a phosphoramidite nucleoside monomer having a desired sequence. In a capping step which is the third step, an unreacted 5'-hydroxyl group in the coupling step is blocked to eliminate the formation of a nucleotide chain having a desired nucleotide sequence in the coupling step of the next cycle. In the capping step, the unreacted 5'-hydroxyl group is acetylated by treating it with acetic anhydride and N-methylimidazole. In an oxidation step which is the final step, the phosphitetriester bond between a 5'-hydroxyl group and phosphoramidite, formed in the coupling step, is converted to a phosphodiester bond. In this oxidation step, the phosphitetriester bond is treated with 0.02 M oxidation solution (0.02 M-I2 in THF/pyridine/H2O) to convert phosphate to phosphate. A series of processes for synthesizing ASO was carried out using a DNA synthesizer (384 Synthesizer, BIONEER, Korea).
[0239] In a process to synthesize an ASO-polymer conjugate (3'PEG-ASO-5'lipid), an ASO was synthesized by deblocking, coupling, capping and oxidation of a 3' PEG-CPG support having the hydrophilic material PEG at the 3' end, and C24 tetradocosan hydrophobic material containing a disulfide bond was attached to the 5' end, thereby preparing a desired ASO-polymer conjugate (see Korean Patent Laid-Open Publication No. 2009-0042297).
[0240] Still, to prepare a 3'-PEG-ASO-5'lipid linker, PEG was linked to a CPG having a functional group such as an amine group attached to it, using a PEG phosphoramidite reagent by a process that consists of unlocking , coupling, capping, and oxidizing and the C24 tetradocosan hydrophobic material containing a disulfide bond was attached to the 5' end, thus preparing a 3'-functional group-PEG-ASO-5'-lipid to which a desired linker can be attached. Upon completion of the synthesis, the reaction product was treated with 28% (v/v) ammonia in a water bath at 60°C to separate the ASO-polymer conjugate, to which a binder can be attached, from the CPG. Then, a linker was attached to the conjugate by the functional group, thus preparing an ASO-polymer conjugate (3'-PEG-ASO—5'lipid linker).
[0241] However, to synthesize an ASO-polymer conjugate (3'lipid-ASO-5'PEG), an ASO was synthesized in a CPG having a functional group as an amine group by a process that consists of unlocking, coupling, cap and oxidize, and PEG was attached to the 5' end using PEG phosphoramidite, thus preparing a functional group-ASO-hydrophilic polymer conjugate. After completion of the synthesis of functional group-ASO-hydrophilic polymer conjugate, the reaction product was treated with 28%(v/v) ammonia in a water bath at 60°C to separate the functional group-ASO-hydrophilic polymer conjugate from the CPG, and then a hydrophobic material was attached to the conjugate via the functional group, thereby preparing an ASO-polymer conjugate having desired hydrophilic and hydrophobic materials attached thereto. Then, the ASO-polymer conjugate was separated and purified from the reagents by high performance liquid chromatography (HPLC) (LC-20A Prominence, SHIMADZU, Japan), and the molecular weights of the ASO and the ASO-polymer conjugate were measured by MALDI TOF-MS (SHIMADZU, Japan) to determine if the nucleotide sequence to be synthesized has been obtained. Example 8: Synthesis of ASO-Phosphothioate Modified Polymer Conjugate
[0242] The ASO used in this Example was obtained by replacing the phosphate group of the DNA structure with a phosphothioate group to obtain S-oligos and linking the S-oligos by phosphothioate bonds.
[0243] Specifically, an ASO comprising S-oligos was synthesized by a process that consists of unlocking, coupling, capping and oxidizing in which the oxidation step was performed by treatment with 0.1 M sulfurization reagent in place of 0.02 M of oxidation solution. By this ASO synthesis process, phosphothioate-modified ASOs having sequences of SEQ ID NOS: 3 and 4 were synthesized (Shina etal.NucleicAcidsResearch,12:4539-4557,1984). The remaining synthesis procedures used in this Example were similar to those used in Example 7.
[0244] To synthesize an ASO in which 4 nucleotides at both ends (5' and 3' ends) were modified with 2-OCH3 (methoxy), a nucleoside in the modified region in the form of 2'-OCH3-DNA was synthesized using 2'-OCH3-DNA-cyanoethyl phosphoramidite [rA(Bz),rC(Ac),rG(ibu),rU] to replace the DNA backbone with phosphothiolate, and then, as described above, the DNA backbone was synthesized by ligating S-oligos via phosphothiolate bonds.
[0245] To prepare an ASO-polymer conjugate modified by phosphothiolate, an ASO conjugate modified with phosphothiolate was synthesized on a 3'PEG-CPG support by a cycle that consists of unlocking, coupling, capping and oxidizing as known in the art (see Patent Korean Laid-Open Publication No. 2009-0042297), and then the hydrophobic material tetradocosane C24 containing a disulfide bond was attached to the 5' end, thereby preparing an ASO-polymer conjugate modified by phosphothiolate.
[0246] Furthermore, to attach a linker to the end of the hydrophilic material of the ASO-polymer conjugate modified by phosphothiolate, the functional group to which a linker can be attached was attached to the 3' CPG, and then a hydrophilic material was attached to the CPG, and the above-mentioned reaction was carried out, thereby preparing an ASO-polymer conjugate to which a linker can be attached.
[0247] After the completion of the synthesis of the ASO-polymer conjugate, the reaction product was treated with 28%(v/v) ammonia in a water bath at 60°C to separate the ASO and the ASO-polymer conjugate from the CPG. Then, the ASO-polymer conjugate was separated and purified from the reagents by high performance liquid chromatography (HPLC) (LC-20A Prominence, SHIMADZU, Japan), and the molecular weights of the ASO and the ASO-polymer conjugate were measured by MALDI TOF-MS (SHIMADZU, Japan) to determine if the nucleotide sequence to be synthesized has been obtained (see FIG. 19). Example 9: Evaluation of the stability of ASO-polymer conjugate under mimetic conditions in vivo
[0248] To assess whether the stability of the ASO-polymer conjugates, synthesized and separated in Examples 7 and 8, was increased compared to the original ASO having the polymer attached to it, the following experiment was performed. Specifically, an ASO having polymer bound thereto and the ASO-polymer conjugate were incubated in 30 and 50%(v/v) medium containing FBS (fetal bovine serum), which mimics in vivo conditions, for 0, 3, 5 , 7 and 10 days, and then the degradation of the ASO-polymer conjugate was analyzed in comparison with that of the original ASO by electrophoresis or polymerase chain reaction (PCR).
[0249] As a result, the ASO-polymer conjugate was stable regardless of the FBS concentration, even though PEG separated from the ASO with the passage of time, whereas the stability of ASO having no polymer attached to it began to drop after day 3. Example 10: Analysis of physical properties of a particle composed of ASO-polymer conjugates
[0250] The ASO-polymer conjugates form a nanoparticle composed of the ASO-polymer conjugates by interaction between the hydrophobic materials attached to the ends of the ASOs (see FIG. 18). The size and critical micelle concentration (CMC) of the composite nanoparticles of the ASO-polymer conjugates was measured by measuring the zeta potential. Example 10-1: Measurement of composite particles of ASO-polymer conjugates
[0251] The size of the nanoparticles composed of the ASO-polymer conjugates, prepared in Example 8 and comprising a sequence of SEQ ID NO: 3, was measured by measuring the zeta potential. Specifically, 50 µg of the ASO-polymer conjugates were dissolved in 1 ml of DPBS (Dulbecco's Phosphate Buffered Saline), and then homogenized with a sonicator (Wiseclean, DAIHAN, Korea) (700 W; amplitude: 20%). The size of the homogenized nanoparticles was measured with a zeta potential measuring device (Nano-ZS, MALVERN, GB) under the following conditions: refractive index: 1.454, absorption index: 0.001, temperature of water as a solvent: 25° Ç. Each measurement consisted of 20 size readings and was repeated three times.
[0252] It can be observed that the nanoparticles formed by the ASO-polymer conjugates had a size of 100-200 nm and a polydispersity index (PDI) of less than 0.4 (see FIG. 20(A)). A lower polydispersion index (PDI) value indicates a more even distribution of particles. Thus, it can be seen that the nanoparticles formed from the ASO-polymer conjugates have a relatively uniform size, which is suitable for absorption into cells by endocytosis (Kenneth A. Dawson et al. nature nanotechnology 4:84-85, 2009). Example 10-2:Measurement of myelocritical concentration of ASO-polymer conjugates
[0253] An amphiphilic material containing both an oleophilic group and a hydrophilic group on the molecule can act as a surfactant. When a surfactant is dissolved in an aqueous solution, the hydrophobic fractions come in to avoid contact with the water, and the hydrophilic fractions come out, thus forming a micelle. The concentration at which the micelle is first formed is defined as the critical micelle concentration (CMC). One method for measuring CMC using a fluorescent dye is based on a rapid change in the slope of the fluorescent intensity graph of a fluorescent dye before and after micelle formation.
[0254] For the measurement of critical micelle concentration of nanoparticles composed of ASO-polymer conjugates, 0.04 mM DPH (1,6-Diphenil-1,3,5-hexatriene, SIGMA, USA) as a fluorescent dye was prepared . 1 nmolM of the ASO-polymer conjugates comprising a sequence of SEQ ID NO: 3 were serially diluted with DPBS from 0.0977 /mm to 50 g/m,, thereby preparing 180 of each of the ASO-polymer conjugate samples. To the prepared sample, 20 µl each of 0.04 mM DPH in methanol and methanol alone as a control was added and the well shaken. Then homogenization using a sonicator (Wiseclean, DAIHAN, Korea) was carried out in the same manner as described in Example 10-1 (700 W; amplitude: 20%). Each of the homogenized samples was allowed to react at room temperature under a condition protected from light for about 24 hours, and the fluorescence intensities (excitation: 355 nm, emission: 428 nm, top reading) were measured. Because the fluorescence intensities are used to determine the relative fluorescence intensity, the relative fluorescence intensity ([fluorescence intensity of sample containing DPH]-[fluorescence intensity of sample containing methanol alone]) at the same concentration was calculated and graphically displayed on the Y-axis as a function of the log value of the concentration of ASO-polymer conjugates (X-axis) (see FIG. 20(B)).
[0255] The fluorescence intensities measured at various concentrations increase as the concentration increases, and the point at which the concentration increases rapidly is the CMC concentration. Thus, the low concentration regions where the fluorescence did not increase and the high concentration region where the fluorescence intensity increased were divided into several points to draw trend lines, and the X axis value where the two trend lines crossed with each other was determined according to the CMC concentration. The measured CMC of the ASO-polymer conjugates was very low (1.56 μg/mβ), suggesting that nanoparticles formed from the ASO-polymer conjugates can easily form micelles even at a very low concentration. Example 11: Inhibition of target gene expression in tumor cell lines by ASO-polymer conjugate and transfection agent
[0256] Each of ASO having the polymer conjugated thereto, and the ASO-polymer conjugate, prepared in Example 8, was transfected into a human colorectal cancer cell line (SW480) as a tumor cell line, and the expression patterns of survivin in the transfected tumor cell line were analyzed. Example 11-1: Tumor cell lineage culture
[0257] The colorectal cancer cell line (SW480) obtained from the American type Culture Collection (ATCC) was grown in a growth medium (L-15 medium from Leibovitz, GIBCO/Invitorgen; USA), supplemented with 10%(v/ v) FBS, 100 units/ml penicillin and 100 µl/ml streptomycin, under conditions of 37°C and 5%(v/v) carbon dioxide (CO 2 ). Example 11-2: Tumor cell line transfection with ASO-polymer conjugate
[0258] Each of ASO having polymer conjugated thereto, and the ASO-polymer conjugate, prepared in Example 8, was transfected into a human colorectal cancer cell line (SW480) as a tumor cell line, and the expression patterns of survivin in the transfected tumor cell line were analyzed.
[0259] The tumor cell line grown in Example 11-1 was grown in a growth medium (L-15 medium from Leibovitzs, GIBCO/Invitorgen; USA) in a 6-well plate at a density of 1.3 x 105 cells for 18 hours under the conditions described in Example 11-1, and then the medium was removed, and 800 µl of Opti-MEM medium (modified Eagle's Minimal Essential Medium, GIBCO/Invitorgen; USA) was added to each well.
[0260] Meanwhile, 2 µl Lipofectamine™ 2000 and 198 µl Opti-MEM medium were mixed with each other and allowed to react at room temperature for 5 minutes. The reaction product was treated with 25 pmol/^ of each of the ASO-polymer conjugates, prepared mps Examples 7 and 8, to final concentrations of 10, 50 and 100 nM, and was then allowed to react at room temperature for 20 minutes, thus preparing the transfection solutions.
[0261] Then, 200 µl of each of the transfection solutions was added to each well containing the tumor cell line and Opti-MEM, and then the cells were cultured for 6 hours, followed by removal of the Opti-MEM medium. Then, 2.5 µg of growth medium (L-15 medium from Leibovitz, GIBCO/Invitorgen; USA) was added to each well, and then the cells were cultured for 24 hours under the conditions of 37°C and 5°C. %(v/v) carbon dioxide (CO2). Example 11-3:Relative quantitative analysis of survivin RNA
[0262] Total RNA was extracted from the transfected cell line of Example 11-2 and synthesized into cDNA, and then the mRNA level of the survivin gene was comparatively quantified in real-time PCR according to the method described in Korean Patent Laid- Open Publication No. 2009-0042297 (see FIG. 21).
[0263] To analyze the inhibitory effects of ASO target gene expression having in the polymer conjugated to it the ASO-polymer conjugate, the cells were transfected with each of ASO and the ASO-polymer conjugate along with the transfection reagent, and then the mRNA expression levels of the survivin gene in the cells were analyzed. As a result, it was demonstrated that the inhibition of survivin gene expression by the ASO-polymer conjugate was similar to that by the ASO having the polymer conjugated to it, suggesting that the conjugated polymer does not interfere with the mechanism of action of ASO. Example 12: Inhibition of target gene expression in tumor cell lineage by isolated ASO-polymer conjugate
[0264] Each of the ASO-polymer conjugates prepared in Examples 7 and 8 was transfected into a human colorectal cancer cell line (SW480) as a tumor cell line, and the expression patterns in the transfected tumor cell line were analyzed. Example 12-1: Tumor cell lineage culture
[0265] The colorectal cancer cell line (SW480) obtained from the American type Culture Collection (ATCC) was grown in a growth medium (L-15 medium from Leibovitz, GIBCO/Invitorgen; USA), supplemented with 10%(v/ v) FBS, 100 units/ml penicillin and 100 µl/ml streptomycin, under conditions of 37°C and 5%(v/v) carbon dioxide (CO 2 ). Example 12-2: Tumor cell line transfection with ASO-polymer conjugate
[0266] The tumor cell line grown in Example 12-1 was grown in a growth medium (M-15 medium from Leibovitz, GIBCO/Invitorgen; USA) in a 6-well plate at a density of 1.3 x 105 cells per 18 hours under the conditions described in Example 5-1, then the medium was removed, and 800 µl of Opti-MEM medium was added to each well.
[0267] 100^ of Opti-MEM medium and 5, 10 or 100^ (500 nM, 1 μM or 10 μM) of each of the ASO-polymer conjugates (1 nmol/^) prepared in Examples 1 and 2 were mixed with each other and homogenized with a sonicator (Wiseclean, DAIHAN, Korea) in the same manner as described in Example 4-1 (700 W; amplitude: 20%), thus preparing transfection solutions containing homogenized nanoparticles formed from the ASO-hydrophobic material conjugates.
[0268] Then, 100µ of each of the transfection solutions was added to each well containing the tumor cell line and Opti-MEM, and the cells were cultured for 24 hours, after which 1 ml of 20% of medium growth containing FBS (M-15 medium from Leibovitz, GIBCO/Invitorgen; USA) was added to this. Then, cells were further cultured for 24 hours under conditions of 37°C and 5%(v/v) carbon dioxide (CO2). Thus, cells were cultured for a total of 48 hours after treatment with the ASO-polymer conjugate. Example 12-3:Relative quantitative analysis of survivin RNA
[0269] Total RNA was extracted from the transfected cell line of Example 12-2 and synthesized into cDNA, and then the mRNA levels of the survivin gene were comparatively quantified in real-time PCR according to the method described in Korean Patent Publication No. . 2009-0042297.
[0270] The inhibition of mRNA expression of the survivin gene was compared between the ASO having the polymer conjugated to it and the ASO-polymer conjugate under a condition containing no transfection reagent. As a result, it can be seen that ASO-polymer inhibited target gene expression at a relatively low concentration compared to unconjugated ASO under a condition containing no transfection reagent.
[0271] Thus, it can be observed that the ASO-polymer conjugates synthesized in the present invention or nanoparticles composed of the ASO-polymer conjugates are released into cells even under a condition not containing any transfection reagent so that ASO inhibits the expression of the target gene . INDUSTRIAL APPLICABILITY
[0272] As described above, the new oligonucleotide structure according to the present invention and a pharmaceutical composition comprising the same can be used for the treatment of cancer and infectious diseases very efficiently and usefully.
[0273] Although the present invention has been described in detail with reference to specific features, it will be apparent to those skilled in the art that this description is for a preferred embodiment only and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

权利要求:
Claims (15)
[0001]
1. THERAPEUTIC DRUG-POLYMER STRUCTURE having a structure of the following formula (1) and comprising a linker attached thereto:
[0002]
2. THERAPEUTIC DRUG-POLYMER STRUCTURE, according to claim 1, characterized in that the double-stranded oligo RNA comprises modification comprising the replacement of an -OH group at the 2' carbon position of the sugar moiety of one or more nucleotides with -CH3 (methyl), -OCH3, -NH2, -F (fluorine), -O-2-methoxyethyl, -O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, -O-3 -dimethylaminopropyl, -ON-methylacetamido or -O-dimethylamidoxyethyl; the replacement of oxygen in the sugar fraction of the nucleotide with sulfur; the modification of the bond between the nucleotides in one or a combination of two or more selected from the group consisting of a phosphorothioate, boranephosphonate and methyl phosphonate bond, or is modified in the form of PNA (peptide nucleic acid) or LNA (locked nucleic acid) .
[0003]
3. THERAPEUTIC DRUG-POLYMER STRUCTURE, according to claim 1, characterized in that the hydrophobic material has a molecular weight of 250-1,000.
[0004]
4. THERAPEUTIC DRUG-POLYMER STRUCTURE, according to claim 3, characterized in that the hydrophobic material is selected from the group consisting of a steroid derivative, a glyceride derivative, glycerol ether, polypropylene glycol, a C12-hydrocarbon unsaturated or saturated C50, diacyl phosphatidylcholine, fatty acid, phospholipid, and lipopolyamine.
[0005]
5. THERAPEUTIC DRUG-POLYMER STRUCTURE, according to claim 4, characterized in that the steroid derivative is selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate, and cholestanyl amine.
[0006]
6. THERAPEUTIC DRUG-POLYMER STRUCTURE, according to claim 4, characterized in that the glyceride derivative is selected from among mono-, di- and tri-glycerides.
[0007]
7. THERAPEUTIC DRUG-POLYMER STRUCTURE, according to claim 1, characterized in that the hydrophilic material has a molecular weight of 200-10,000.
[0008]
8. THERAPEUTIC DRUG-POLYMER STRUCTURE, according to claim 7, characterized in that the hydrophilic material is selected from the group consisting of polyethylene glycol, polyvinyl pyrrolidone, and polyoxazoline.
[0009]
9. THERAPEUTIC DRUG-POLYMER STRUCTURE, according to claim 1, characterized in that the covalent bond is either a non-degradable bond or a degradable bond.
[0010]
10. THERAPEUTIC DRUG-POLYMER STRUCTURE according to claim 9, characterized in that the non-degradable bond is either an amide bond or a phosphate bond wherein the degradable bonds are selected from the group consisting of a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond, or an enzymatically degradable bond.
[0011]
11. METHOD FOR PREPARING A DOUBLE-HELIX OLIGO RNA STRUCTURE CONNECTED TO A LINKER as defined in claim 1, characterized in that it comprises the steps of: (1) synthesizing a single-stranded RNA on a solid support having a functional group-hydrophilic material linked to this; (2) covalently attaching a hydrophobic material to the 5' end of the single-stranded RNA having the functional group-hydrophilic material attached thereto; (3) separate the functional group-RNA-polymer structure and a complementary single-stranded RNA separately synthesized from the solid support; (4) covalently attaching a linker to the end of the hydrophilic material by the functional group; and (5) annealing the ligand-bound RNA-polymer structure with the complementary single-stranded RNA to form a double-stranded RNA structure, where the ligand is selected from target-specific antibodies, aptamers, peptides, and receptor-specific chemical materials.
[0012]
12. METHOD FOR PREPARING A DOUBLE HELIX OLIGO RNA STRUCTURE as defined in claim 1, characterized in that it comprises the steps of: (1) synthesizing a single-stranded RNA on a solid support; (2) covalently attach a hydrophilic material to the 5' end of the single-stranded RNA; (3) attaching a linker to the hydrophilic material attached to the single-stranded RNA; (4) separate the linker-bound, hydrophilic RNA-polymer structure and a complementary hydrophobic RNA-polymer structure separately synthesized from the solid support; and (5) annealing the linker-bound, hydrophilic RNA-polymer structure with the complementary hydrophobic RNA-polymer structure to form a double-stranded structure, the method of preparation comprising, between steps (1) to (4) , a step of synthesizing a single-stranded RNA complementary to the single-stranded RNA of step (1), and then covalently attaching a hydrophobic material to the synthesized single-stranded RNA to synthesize a hydrophobic single-stranded RNA-polymer structure, and where the linker is selected among target specific antibodies, aptamers, peptides, receptor specific chemical materials.
[0013]
13. METHOD FOR PREPARING A DOUBLE HELIX OLIGO RNA STRUCTURE ATTACHED TO A LINKER as defined in claim 1, characterized in that it comprises the steps of: (1) synthesizing a single stranded RNA on a solid support having a functional group attached to This one; (2) covalently attaching a hydrophilic material to the material obtained in step (1); (3) covalently attaching a binder to the material obtained in step (2); (4) separating the material obtained in step (3) from the solid support; (5) covalently attaching a hydrophobic material to the material resulting from step (4) by the functional group attached to the 3' end; and (6) annealing the material resulting from step (5) with a complementary single-stranded RNA to form a double-stranded RNA structure, and where the linker is selected from target-specific antibodies, aptamers, peptides, receptor-specific chemical materials.
[0014]
14. NANOPARTICLE characterized in that it comprises a therapeutic drug-polymer structure, according to any one of claims 1 to 10.
[0015]
15. PHARMACEUTICAL COMPOSITION characterized by comprising a therapeutic drug-polymer structure, according to any one of claims 1 to 10 or the nanoparticle of claim 14.

类似技术:

---

公开号 | 公开日 | 专利标题

---

BR112014014730B1|2021-05-18|therapeutic polymer drug structure, methods for preparing a nanoparticle, double-stranded oligorma structure, and pharmaceutical composition

---

US8779114B2|2014-07-15|siRNA conjugate and preparation method thereof

---

KR101752812B1|2017-06-30|High-efficiency nanoparticle-type double-helical oligo-rna structure and method for prep aring same

---

US9707250B2|2017-07-18|Hydrophobically modified antisense oligonucleotides comprising a triple alkyl chain

---

BR112016000160B1|2021-06-08|improved nanoparticle-type oligonucleotide structure having high efficiency and method for preparing the same

---

JP2016531563A|2016-10-13|Liver cancer-related gene-specific siRNA, double helical oligo RNA structure containing such siRNA, and cancer preventive or therapeutic composition containing the same

---

KR20130068032A|2013-06-25|'stable antisense oligonucleotide conjugate and its manufacturing method'

---

KR101722948B1|2017-04-04|Double stranded oligo RNA molecule with a targeting ligand and method of preparing the same

---

WO2021113851A2|2021-06-10|Peptide docking vehicle for targeted nucleic acid delivery

---

Kozuch2018|Fluorescently Labeled siRNAs and their Theranostic Applications in Cancer Gene Therapy

同族专利:

---

公开号 | 公开日

---

US20140371432A1|2014-12-18|

---

BR112014014730A2|2017-06-13|

---

PL2796150T3|2021-08-30|

---

CA2859127C|2017-04-25|

---

JP5952423B2|2016-07-13|

---

CN104244987A|2014-12-24|

---

EP3733848A1|2020-11-04|

---

RU2599449C1|2016-10-10|

---

JP2015506919A|2015-03-05|

---

JP2016216468A|2016-12-22|

---

EP2796150B1|2021-02-24|

---

WO2013089522A1|2013-06-20|

---

AU2012353058B2|2015-05-21|

---

EP2796150A4|2015-07-01|

---

CA2859127A1|2013-06-20|

---

EP2796150A1|2014-10-29|

---

AU2012353058A1|2014-07-03|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US6005087A|1995-06-06|1999-12-21|Isis Pharmaceuticals, Inc.|2'-modified oligonucleotides|

---

US5660985A|1990-06-11|1997-08-26|Nexstar Pharmaceuticals, Inc.|High affinity nucleic acid ligands containing modified nucleotides|

---

US5386023A|1990-07-27|1995-01-31|Isis Pharmaceuticals|Backbone modified oligonucleotide analogs and preparation thereof through reductive coupling|

---

US6277967B1|1998-07-14|2001-08-21|Isis Pharmaceuticals, Inc.|Carbohydrate or 2′-modified oligonucleotides having alternating internucleoside linkages|

---

US6175001B1|1998-10-16|2001-01-16|The Scripps Research Institute|Functionalized pyrimidine nucleosides and nucleotides and DNA's incorporating same|

---

KR100466254B1|2002-02-25|2005-01-14|한국과학기술원|Conjugates Comprising Oligonucleotide And Hydrophilic Polymers for Gene Transfer, Hybrid Polyion Complex Micelles Self-assembled from Said Conjugates and Their Manufacturing Method|

---

US8969543B2|2003-04-03|2015-03-03|Bioneer Corporation|SiRNA-hydrophilic polymer conjugates for intracellular delivery of siRNA and method thereof|

---

KR100663880B1|2004-02-12|2007-01-03|한국과학기술연구원|Compositions of polymer-lipid mixtures, their structures and preparation method thereof|

---

JP4635046B2|2004-04-20|2011-02-23|マリナ・バイオテック・インコーポレーテッド|Methods and compositions for enhancing delivery of double stranded RNA or double stranded hybrid nucleic acids for modulating gene expression in mammalian cells|

---

RU2434942C2|2004-09-28|2011-11-27|Кварк Фармасьютикалс, Инк.|Oligiribonucleotides and methods of their application for treatment of acute renal failure and other diseases|

---

CN1911447B|2006-06-30|2010-05-12|复旦大学|Transferrin-polyethylene glycol medicine molecular compound, and used of medicine prepared by the same|

---

AU2007286545A1|2006-08-24|2008-02-28|Alcon Research, Ltd.|RNAi-mediated inhibition of gremlin for treatment of IOP-related conditions|

---

KR100802278B1|2006-12-26|2008-02-11|재단법인서울대학교산학협력재단|A supercritical nanoparticle process with vitamin k using molecular association theory|

---

MY149171A|2007-05-16|2013-07-31|Mat Malta Advanced Technologies Ltd|Treatment and prevention of influenza|

---

KR20090070846A|2007-12-27|2009-07-01|재단법인서울대학교산학협력재단|A supercritical nanoparticle process with ceramide using molecular association theory|

---

MX2010012239A|2008-05-13|2011-05-24|Univ Washington|Micellic assemblies.|

---

AU2009302217A1|2008-10-09|2010-04-15|Northeastern University|Multifunctional self-assembling polymeric nanosystems|

---

KR101224828B1|2009-05-14|2013-01-22|바이오니아|SiRNA conjugate and preparing method thereof|

---

AR078921A1|2009-11-09|2011-12-14|Hoffmann La Roche|COMPOSITIONS AND METHODS TO INHIBIT THE EXPRESSION OF GENES OF THE QUINESINAS SUPERFAMILY, KIF10|

---

CN102028955A|2010-08-09|2011-04-27|华中科技大学同济医学院附属同济医院|Novel targeting compound for tumor gene interference|

---

CA2808901A1|2010-08-20|2012-02-23|Cerulean Pharma Inc.|Conjugates, particles, compositions, and related methods|

---

RU2599449C1|2011-12-15|2016-10-10|Байонир Корпорейшн|New oligonucleotides conjugates and use thereof|

---

CA2860527C|2012-01-05|2019-04-09|Bioneer Corporation|High-efficiency nanoparticle-type double-helical oligo-rna structure and method for preparing same|RU2599449C1|2011-12-15|2016-10-10|Байонир Корпорейшн|New oligonucleotides conjugates and use thereof|

---

CA2860527C|2012-01-05|2019-04-09|Bioneer Corporation|High-efficiency nanoparticle-type double-helical oligo-rna structure and method for preparing same|

---

CN105705638B|2013-07-05|2019-11-26|柏业公司|What is improved has dynamical nanoparticle form oligonucleotide structure and preparation method thereof|

---

EP3591053A1|2013-07-05|2020-01-08|Bioneer Corporation|Respiratory disease-related gene specific sirna, double-helical oligo rna structure containing sirna, compositon containing same for preventing or treating respiratory disease|

---

KR101715228B1|2013-07-05|2017-03-13|바이오니아|Dengue virus-specific sirna, double helix oligo-rna structure comprising sirna, and composition for suppressing proliferation of dengue virus comprising rna structure|

---

KR20150006742A|2013-07-09|2015-01-19|바이오니아|Liver cancer related genes-specific siRNA, double-stranded oligo RNA molecules comprising the siRNA, and composition for the prevention or treatment of cancer comprising the same|

---

EP3128008A4|2014-04-04|2018-03-28|Bioneer Corporation|Novel double-stranded oligo rna and pharmaceutical composition comprising same for preventing or treating fibrosis or respiratory diseases|

---

CN106795200B|2014-10-10|2020-06-19|豪夫迈·罗氏有限公司|Galnac phosphoramidites, nucleic acid conjugates thereof, and uses thereof|

---

US11186839B2|2016-11-21|2021-11-30|Tribiotica Llc|Methods for preventing titration of bimolecular templated assembly reactions by structurally-determined differential hybridizations|

---

JP2019535281A|2016-11-21|2019-12-12|トリビオティカ・エルエルシー|Method for directed folding assembly or dimerization of proteins by templated assembly reaction|

---

CA3057292A1|2017-03-22|2018-09-27|The Regents Of The University Of California|Modified oligonucleotides and therapeutic uses thereof|

---

US20200369703A1|2017-12-26|2020-11-26|Guangzhou Ribobio Co., Ltd.|Modified oligonucleotides and compound that can be used for synthesizing same|

---

KR102141124B1|2018-01-30|2020-08-04|바이오니아|Double-stranded Oligonucleotide Complex comprising Double― stranded miRNA and Uses thereof|

---

EP3820453A1|2018-07-10|2021-05-19|The Regents of The University of California|Biomolecule coated particles and films and uses thereof|

---

WO2021197371A1|2020-03-31|2021-10-07|南京金斯瑞生物科技有限公司|Biological conjugate containing rigid linker|

法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

---

2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: A61K 47/00 (2006.01), C12N 15/11 (2006.01), A61K 9 |

---

2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

---

2019-04-24| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

---

2020-05-19| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

---

2020-09-01| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

---

2021-03-02| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

---

2021-05-18| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 14/12/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

---

申请号 | 申请日 | 专利标题

---

KR1020110135162A|KR20130068032A|2011-12-15|2011-12-15|'stable antisense oligonucleotide conjugate and its manufacturing method'|

---

KR10-2011-0135162|2011-12-15|

---

KR1020120001710A|KR101722948B1|2012-01-05|2012-01-05|Double stranded oligo RNA molecule with a targeting ligand and method of preparing the same|

---

KR10-2012-0001710|2012-01-05|

---

PCT/KR2012/010967|WO2013089522A1|2011-12-15|2012-12-14|Novel oligonucleotide conjugates and use thereof|

---