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WO2019022586A2 - Pharmaceutical composition for preventing or treating liver cancer - Google Patents

Pharmaceutical composition for preventing or treating liver cancer Download PDF

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Publication number
WO2019022586A2
WO2019022586A2 PCT/KR2018/008611 KR2018008611W WO2019022586A2 WO 2019022586 A2 WO2019022586 A2 WO 2019022586A2 KR 2018008611 W KR2018008611 W KR 2018008611W WO 2019022586 A2 WO2019022586 A2 WO 2019022586A2
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Prior art keywords
seq
group
dsrna
sequence
porous silica
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PCT/KR2018/008611
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French (fr)
Korean (ko)
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WO2019022586A9 (en
WO2019022586A3 (en
Inventor
원철희
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주식회사 레모넥스
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Priority to EP18838318.6A priority Critical patent/EP3689355A4/en
Priority to JP2020504312A priority patent/JP6967810B2/en
Priority to US16/634,675 priority patent/US20210093654A1/en
Priority to CN202310211626.6A priority patent/CN116898870A/en
Priority to AU2018306411A priority patent/AU2018306411B2/en
Priority to CN201880062438.9A priority patent/CN111132682B/en
Priority to CN202310211629.XA priority patent/CN116236501A/en
Application filed by 주식회사 레모넥스 filed Critical 주식회사 레모넥스
Priority claimed from KR1020180088375A external-priority patent/KR20190013635A/en
Publication of WO2019022586A2 publication Critical patent/WO2019022586A2/en
Publication of WO2019022586A3 publication Critical patent/WO2019022586A3/en
Publication of WO2019022586A9 publication Critical patent/WO2019022586A9/en
Priority to US17/536,880 priority patent/US12076336B2/en
Priority to AU2022201756A priority patent/AU2022201756A1/en
Priority to AU2022201755A priority patent/AU2022201755A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a pharmaceutical composition for preventing or treating liver cancer.
  • HCC Hepatocellular carcinoma
  • the primary treatment for hepatocellular carcinoma is surgical resection, and few patients are eligible for curative treatment at the initial treatment stage.
  • Resection and percutaneous ablation have a recurrence rate of 70% after 5 years and are closely related to the survival rate.
  • HCC is characterized by multiple tumor progression.
  • the damaged liver tissue evolves in the early stages to small nodular hypercellular lesions called dysplastic nodules (DNs).
  • DNs small nodular hypercellular lesions
  • eHCC early hepatocarcinoma
  • eHCC early hepatocarcinoma
  • liver cancer diagnostic markers BANF1, PLOD3 and SF3B contribute to early malignant transformation of hepatocytes in liver tumor formation and are promising targets for molecular therapy of liver malignancy.
  • siRNA comprising a sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 5 to 157 and an antisense RNA consisting of the sequence complementary thereto; or
  • a dsRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 158 to 310;
  • a prophylactic or therapeutic agent for liver cancer comprising at least one sequence selected from the group consisting of SEQ ID NOs: 234, 237 to 240, 242 to 251, 253 to 255, 258 to 269, 271 to 281, 283 to 307, 309 and 310 A pharmaceutical composition.
  • siRNA consisting of a sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 5 to 28 and an antisense RNA comprising a sequence complementary thereto;
  • a dsRNA comprising at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 158 to 181;
  • siRNA consisting of a sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 29 to 55 and an antisense RNA comprising a sequence complementary thereto; or
  • a dsRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 182 to 208;
  • siRNA consisting of a sense RNA consisting of at least one sequence selected from the group consisting of SEQ ID NOS: 56-120 and an antisense RNA consisting of a sequence complementary thereto; or
  • a dsRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 209 to 273.
  • composition comprising siRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of SEQ ID NOS: 121 to 157 and antisense RNA having a sequence complementary thereto; or
  • a pharmaceutical composition for preventing or treating liver cancer comprising a dsRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 274 to 310.
  • the siRNA or dsRNA may be selected from the group consisting of liposomes, lipofectamines, dendrimers, micelles, porous silica particles, amino clay, gold nanoparticles, magnetic nanoparticles, graphene, oxidized graphene, chitosan, dextran, pectin, , Gelatin, silica, glass particles, protamine, exosome, polyethyleneimine, N-butylcyanoacrylate, gel foam, gelatin, ethanol, nanocrystals, nanotubes, carbon nanoparticles, hyaluronic acid, iron oxide, polylactic acid, poly Polyglycolic acid, polydioxanone, polyglycolic acid-co-caprolactone, polypropylene and polyglycolic acid, and mixtures thereof.
  • the carrier is supported on at least one support selected from the group consisting of hydrogels.
  • the support is porous silica particles having a t of 20 or more, wherein the ratio of absorbance of the following formula (1) is 1/2:
  • a 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles in a cylindrical permeable membrane having pores having a diameter of 50 kDa,
  • the pH of the suspension is 7.4 and A t is the absorbance of the porous silica particles measured after passage of time from the measurement of A 0 ).
  • composition of claim 8 wherein t is greater than or equal to 40.
  • the siRNA comprises at least one sequence selected from the group consisting of SEQ ID NOS: 28, 119, and 136, and an antisense RNA comprising a sequence complementary thereto,
  • the dsRNA comprises at least one sequence selected from the group consisting of SEQ ID NOS: 181, 272, and 289.
  • porous silica particle has a hydrophilic substituent or a hydrophobic substituent.
  • the porous silica particles may be in the form of particles of an aliphatic group having an aldehyde group, a keto group, a carbamate group, a sulfate group, a sulfonate group, an amino group, an amine group, an aminoalkyl group, a silyl group, a carboxyl group, a sulfonic acid group, a thiol group, , A substituted or unsubstituted C 1 to C 30 alkyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aryl group, A substituted or unsubstituted C 6 to C 30 aryl group, and a C 1 to C 30 ester group, in the presence of at least one hydrophilic substituent selected from the group consisting of a substituted or unsubsti
  • porous silica particles have a positive or negative charge at an external surface or pore interior at neutral pH.
  • porous silica particles are positively charged at neutral pH at the outer surface and inside the pores.
  • porous silica particles have an average diameter of 100 to 400 nm and a pore diameter of 4 to 30 nm.
  • the pharmaceutical composition of the present invention provides a preventive and therapeutic effect of liver cancer by specifically knocking down the genes expressed in early liver cancer cells to prevent the development of liver cancer and inhibiting the metastasis and proliferation of liver cancer cells.
  • Fig. 1 shows siRNAs comprising the sense RNA having the sequence of Table 11 and the antisense RNA consisting of the complementary sequence to the Hepa-1c1c7 and SNU-449 cell strains of Example 1, , And then the amount of expression of corresponding indicator factors of each siRNA was measured by Western blotting.
  • FIG. 2 shows the results of in vitro transfection of the SNU-449 cell line of Example 1-1 with siRNAs consisting of sense RNA having the sequence of Table 12 and antisense RNA of the complementary sequence according to the method of Example 1-2 , Migration and invasion responses of the corresponding surface factors of each siRNA were analyzed according to the methods of Examples 1-5, and the results of analysis of the scratch wound healing ability according to the method of Example 1-6 are shown.
  • FIG. 3 (A) shows the results of in vitro transfection of SNU-449 cell line of Example 1 with siRNAs consisting of sense RNA having the sequence of Table 12 and antisense RNA consisting of the complementary sequence according to the method of Example 1-2
  • FIG. 3 (B) is a graph showing the results of analysis of the expression level of the EMT regulatory proteins and the expression level of the corresponding indicator factors of the respective siRNAs according to the method of Example 1-9, In a subcutaneous tissue of athymic nude mice, and then analyzed the size of the liver tumor and the survival rate of the mouse.
  • FIG. 4 (A) shows siRNAs consisting of sense RNA having the sequence of Table 13 and antisense RNA consisting of the complementary sequence thereof in vivo according to the method of Example 1-8, and the process, ultrasound image
  • FIG. 4 (B) is a graph showing the results of analysis of inhibitory levels of the expression levels of the respective indicator genes of the siRNAs carried on the porous nanoparticles by the method of Example 1-9.
  • 5 is a photomicrograph of a porous silica particle.
  • FIG. 7 is a microphotograph of a small pore particle during the production of porous silica particles.
  • 10 is a tube having a cylindrical permeable membrane.
  • 11 is a graph showing the results of absorbance reduction with time of the porous silica particles.
  • FIG. 12 is a graph showing the results of absorbance reduction by particle size of porous silica particles over time.
  • 13 is a graph showing the results of absorbance reduction of pore diameters with time of porous silica particles.
  • 15 is a graph showing the results of absorbance reduction with time of the porous silica particles.
  • 16 is a tube for confirming release of physiologically active substance from porous silica particles.
  • 17 is a graph showing the release of the physiologically active substance carried on the porous silica particles with time.
  • 18 is a photomicrograph showing the release of siRNA in mice by loading siRNA onto porous silica particles.
  • siRNA means a nucleic acid molecule capable of mediating RNA interference or gene silencing. Since siRNA can inhibit expression of a target gene, it is provided as an efficient gene knockdown method or as a gene therapy method.
  • the siRNA molecule may have a structure in which the sense strand (the corresponding sequence corresponding to the mRNA sequence of the target gene) and the antisense strand (the sequence complementary to the mRNA sequence of the target gene) are located on opposite sides to form a double strand.
  • siRNA molecules may have a single stranded structure with self-complementary sense and antisense strands.
  • the siRNA is not limited to a complete pair of double-stranded RNA portions that are paired with each other, but is paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) May be included.
  • the siRNA terminal structure is capable of blunt or cohesive termini as long as it can inhibit the expression of the target gene by RNA interference (RNAi) effect.
  • RNAi RNA interference
  • the sticky end structure can be a 3'-end protruding structure and a 5'-end protruding structure.
  • siRNA molecules may have a form in which a short nucleotide sequence (e.g., about 5-15 nt) is inserted between the self-complementary sense and antisense strands, in which case the siRNA molecule formed by the expression of the nucleotide sequence To form a hairpin structure, which in turn forms a stem-and-loop structure.
  • This stem-and-loop structure is processed in vitro or in vivo to produce siRNA molecules that are capable of mediating RNAi.
  • the "dsRNA” is a siRNA precursor molecule that meets the RISC complex containing the DICER enzyme of the target cell (Ribonuclease III) and is cleaved into siRNA, which in turn generates RNAi.
  • the dsRNA has a sequence that is several nucleotides longer than the siRNA, and the sense strand (the corresponding sequence corresponding to the mRNA sequence of the target gene) and the antisense strand (the sequence complementary to the mRNA sequence of the target gene) As shown in FIG.
  • Nucleic acid is meant to include any DNA or RNA, such as chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in a tissue sample. Includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of the intact nucleic acid molecule.
  • Gene means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression.
  • the gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein.
  • the nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.
  • gene expression generally refers to a cellular process in which a biologically active polypeptide is produced from a DNA sequence and exhibits biological activity in the cell.
  • gene expression includes post-transcriptional and post-transcriptional processes that not only involve transcription and translation processes, but can also affect the biological activity of the gene or gene product. Such procedures include, but are not limited to, RNA synthesis, processing and transport as well as polyp peptide synthesis, transport and post-translational modification of the polypeptide.
  • gene expression refers to a process in which a precursor siRNA is produced from a gene.
  • this process is referred to as transcription, although the transcription product of the siRNA gene is not translated to produce a protein, unlike the transcription induced by RNA polymerase II on the protein coding gene. Nevertheless, the generation of mature siRNAs from siRNA genes is encompassed by the term " gene expression " as that term is used herein
  • target gene refers to a gene that is targeted for modulation using methods and compositions of the subject matter disclosed herein.
  • the target gene comprises a nucleic acid sequence whose expression level is down-regulated by the siRNA to an mRNA or polypeptide level.
  • target RNA or " target mRNA” refers to a transcript of a target gene that will bind siRNA and induce modulation of expression of the target gene.
  • transcription refers to a cellular process involving the interaction of an RNA polymerase with a gene that induces expression as RNA of structural information present in the coding sequence of the gene.
  • down-regulation refers to the expression of a specific gene into mRNA or the expression level of a protein by an intracellular transcription or gene translation in an activated cell, .
  • Treatment means an approach to obtaining beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of the disease state, (Either partially or totally), detectable or undetected, whether or not an improvement or temporary relief or reduction
  • treatment may mean increasing the survival rate compared to the expected survival rate when not receiving treatment. Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include treatments required for disorders that have already occurred as well as disorders to be prevented.
  • compositions herein may prevent early onset symptoms, or related disorders when administered prior to appearance.
  • the present invention provides a siRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 5 to 157 and an antisense RNA comprising a sequence complementary thereto; or
  • a dsRNA comprising at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 158 to 310.
  • the present invention also provides a pharmaceutical composition for preventing or treating liver cancer.
  • siRNA or dsRNA of the present invention can be used in an animal including humans, such as monkeys, pigs, horses, cows, sheeps, dogs, cats, Mice, rabbits, and the like, and preferably may be derived from humans.
  • the siRNA or dsRNA of the present invention may be modified by deletion, substitution or insertion of a functional equivalent of the nucleic acid molecule constituting it, for example, a part of the base sequence of the siRNA or dsRNA of the present invention , And variants capable of functionally functioning with the siRNA or dsRNA of the present invention.
  • siRNA or dsRNA of the present invention can be isolated or prepared using standard molecular biology techniques, such as chemical synthesis methods or recombinant methods, or commercially available.
  • the composition of the present invention includes siRNA or dsRNA itself of the present invention, as well as other substances capable of increasing the expression rate of the siRNA or dsRNA of the present invention in a cell, for example, a compound, a natural product, .
  • siRNA or dsRNA of the present invention can be provided as a vector for intracellular expression.
  • the siRNA or dsRNA of the present invention can be introduced into cells using various transformation techniques such as a complex of DNA and DEAE-dextran, a complex of DNA and nuclear protein, a complex of DNA and lipid, etc.
  • the dsRNA may be in a form that is contained within a carrier that enables efficient introduction into the cell.
  • the carrier is preferably a vector, and both viral vectors and non-viral vectors are usable.
  • the viral vector for example, lentivirus, retrovirus, adenovirus, herpes virus and avipox virus vector can be used, Preferably a lentiviral vector, but is not limited thereto.
  • Lentiviruses are a type of retrovirus that is not only a mitotic cell but also a mitotic cell due to the nucleophilicity of a nucleopore or a pre-integrated complex (a virus "shell") that allows active incorporation into a complete nuclear membrane There is a feature that can be made.
  • the vector comprising siRNA or dsRNA of the present invention preferably further comprises a selection marker.
  • the " selection marker" is intended to facilitate screening of cells into which siRNA or dsRNA of the present invention has been introduced.
  • the selectable marker used in the vector is not particularly limited as long as it is a gene capable of easily detecting or measuring the introduction of a vector.
  • the selectable marker is selected from the group consisting of resistance to drugs, tolerance to cytotoxic agents, Such as GFP (green fluorescent protein), puromycin, neomycin (Neo), hygromycin (Hyg), histidine dehydrogenase Histidinol dehydrogenase gene hisD) and guanine phosphosribosyltransferase (Gpt).
  • GFP green fluorescent protein
  • puromycin marker can be used.
  • the present invention relates to a composition
  • a composition comprising siRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 5 to 28 of Table 1 and antisense RNA comprising the sequence complementary thereto; Or at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 158 to 181 in Table 1 below.
  • SiRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 5 to 28 of Table 1 and antisense RNA consisting of the complementary sequence thereof; Or at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 158 to 181 in Table 1 targets the variant 1 sequence (SEQ ID NO: 1) of the human BANF1 gene, and the expression of the human BANF1 gene variant 1 RNAi, thereby preventing or treating liver cancer.
  • Target sequence 5 5'-CAA GAA GCT GGA GGA AAG-3 '(Position in gene sequence: 601) GC content: 45.0% SEQ ID NO: 5 Sense strand: 5'-CAA GAA GCU GGA GGA AAG UU-3 ' SEQ ID NO: 326 Antisense strand: 5'- CUU UCC UCC AGC UUC UUG UU-3 ' SEQ ID NO: 158 dsRNA: 5'-CAA GAA GCU GGA GGA AAG UU UCU AAA G-3 ' Target sequence 6: 5'-GAA AGA TGA AGA CCT CTT-3 '(Position in gene sequence: 667) GC content: 40.9% SEQ ID NO: 6 Sense strand: 5'-GAA AGA UGA AGA CCU CUU CCU U-3 ' SEQ ID NO: 327 Antisense strand: 5'-GGA AGA GGU CUU CAU CU UCU U-3 ' SEQ ID NO:
  • the present invention is a siRNA comprising a sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 29 to 55 of Table 2 and an antisense RNA comprising a sequence complementary thereto; Or at least one sequence selected from the group consisting of SEQ ID NOS: 182 to 208 in Table 1 below.
  • siRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 29 to 55 of Table 2 and antisense RNAs comprising the sequence complementary thereto; Or at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 182 to 208 shown in Table 2 below targets the variant 2 sequence (SEQ ID NO: 2) of the human BANF1 gene and the expression of the human BANF1 gene variant 2 RNAi, thereby preventing or treating liver cancer.
  • Target sequence 29 5'-ATG ACA ACC TCC CAA AAGCA-3 '(Position in gene sequence: 452) GC content: 40.9% SEQ ID NO: 29 Sense strand: 5'- AUGACAACCUCCCAAAAGCA UU-3 ' SEQ ID NO: 350 Antisense strand: 5'- UGCUUUUGGGAGGUUGUCUCUU UU-3 ' SEQ ID NO: 182 dsRNA: 5'- AUGACAACCUCCCAAAAGCA UU UCU AAA G-3 ' Target sequence 30: 5'-CCG AGA CTT CGT GGC AGA-3 '(Position in gene sequence: 472) GC content: 40.9% SEQ ID NO: 30 Sense strand: 5'- CCGAGACUUCGUGGCAGA UU-3 ' SEQ ID NO: 351 Antisense strand: 5'-UCUGCCACGAAGUCUCGG UU-3 ' SEQ ID NO: 183 dsRNA: 5'-CCGAGA
  • the present invention is a siRNA comprising a sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 56-120 of Table 3 and an antisense RNA consisting of the sequence complementary thereto; Or at least one sequence selected from the group consisting of SEQ ID NOS: 209 to 273 of Table 3 below.
  • SiRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of SEQ ID NOS: 56-120 of Table 3 and antisense RNA consisting of the sequence complementary thereto; Or at least one sequence selected from the group consisting of SEQ ID NOS: 209 to 273 of Table 3, targets the sequence of the human PLOD3 gene (SEQ ID NO: 3) and inhibits the expression of the human PLOD3 gene through RNAi Thereby preventing or treating liver cancer.
  • Target sequence 56 5'-CCA GAG AAG CTG CTG GTG AT-3 '(Position in gene sequence: 562) GC content: 50.0% SEQ ID NO: 56 Sense strand: 5'- CCAGAGAAGCUGCUGGUGAU UU-3 ' SEQ ID NO: 377 Antisense strand: 5'- AUCACCAGCAGCUUCUCUGG UU-3 ' SEQ ID NO: 209 dsRNA: 5'- CCAGAGAAGCUGCUGGUGAU UU UCU AAA G-3 ' Target sequence 57: 5'-CCA CAG CTG AAA CCG AGG-3 '(Position in gene sequence: 590) GC content: 55.0% SEQ ID NO: 57 Sense strand: 5'- CCACAGCUGAAACCGAGG UU-3 ' SEQ ID NO: 378 Antisense strand: 5'- CCUCGGUUUCAGCUGUGG UU-3 ' SEQ ID NO: 210 dsRNA: 5'-
  • the present invention is a siRNA comprising a sense RNA consisting of at least one sequence selected from the group consisting of SEQ ID NOs: 121 to 157 shown in Table 4 below and an antisense RNA comprising a sequence complementary thereto; Or at least one sequence selected from the group consisting of SEQ ID NOS: 274 to 310 shown in Table 4 below.
  • siRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of SEQ ID NOs: 121 to 157 of Table 4 and antisense RNA consisting of the sequence complementary thereto; Or at least one sequence selected from the group consisting of SEQ ID NOS: 274 to 310 shown in Table 4 below, targets the sequence of the human SF3B4 gene (SEQ ID NO: 4) and inhibits the expression of the human SF3B4 gene through RNAi Thereby preventing or treating liver cancer.
  • Target sequence 121 5'-AAT CAG GAT GCC ACT GTG TA-3 '(Position in gene sequence: 521) GC content: 40.9% SEQ ID NO: 121 Sense strand: 5'- AAUCAGGAUGCCACUGUGUA UU-3 ' SEQ ID NO: 442 Antisense strand: 5'- UACACAGUGGCAUCCUGAUU UU-3 ' SEQ ID NO: 274 dsRNA: 5'- AAUCAGGAUGCCACUGUGUA UU UCU AAA G-3 ' Target sequence 122: 5'-CTG GAT GAG AAG GTT AGT GA-3 '(Position in gene sequence: 551) GC content: 40.9% SEQ ID NO: 122 Sense strand: 5'- CUGGAUGAGAAGGUUAGUGA UU-3 ' SEQ ID NO: 443 Antisense strand: 5'- UCACUAACCUUCUCAUCCAG UU-3 ' SEQ ID NO: 275 d
  • the siRNA or dsRNA of the present invention may be one that is carried on a carrier and is capable of carrying RNA molecules in the kind of the carrier.
  • the siRNA or dsRNA is not particularly limited as long as it is known in the art.
  • the siRNA or dsRNA of the present invention may be carried on a porous silica particle, and the particle is a particle of silica (SiO 2 ) and has a nano-sized particle size.
  • the porous silica particles are porous particles having nano-sized pores and can carry physiologically active substances such as siRNA or dsRNA of the present invention on the surface and / or pores thereof.
  • the porous silica particles are biodegradable particles, and when physiologically active substances are supported on the body, they can be biodegraded in the body and release physiologically active substances. That is, biodegradation of the porous silica particles results in the release of the physiologically active substance.
  • the porous silica particles according to the present invention may be slowly decomposed in the body, so that the supported physiologically active substance may have sustained release properties. For example, t at which the ratio of the absorbance of the following formula (1) is 1/2 is 20 or more:
  • a 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles in a cylindrical permeable membrane having pores having a diameter of 50 kDa,
  • the pH of the suspension was 7.4,
  • a t is the absorbance of the porous silica particles measured after passage of time t from the measurement of A 0 ).
  • Equation (1) means that the porous silica particles are decomposed at a certain rate in an environment similar to the inside of the body.
  • the absorbances A 0 and A t in Equation (1) may be measured by, for example, placing the porous silica particles and suspension in a cylindrical permeable membrane and putting the same suspension in the outside of the permeable membrane.
  • the porous silica particles are biodegradable and can be slowly decomposed in the suspension.
  • the porous silica particles having a diameter of 50 kDa corresponding to about 5 nm can pass through the permeable membrane having a diameter of 50 kDa and the cylindrical permeable membrane can be permeated under a condition of 60 rpm Therefore, the suspension may be uniformly mixed, and the decomposed porous silica particles may come out of the permeable membrane.
  • the absorbance in Equation (1) may be measured under an environment in which, for example, the suspension outside the permeable membrane is replaced with a new suspension.
  • the suspension may be constantly being replaced, replaced at regular intervals, and the period may be a periodic or irregular period. For example, in the range of 1 hour to 1 week, an interval of 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 7 Day interval, etc., but is not limited thereto
  • the absorbance becomes half of the initial absorbance after t time, meaning that about half of the porous silica particles are decomposed.
  • the suspension may be a buffer solution, and may be at least one selected from the group consisting of, for example, PBS (phosphate buffered saline) and SBF (simulated body fluid), and more specifically, PBS.
  • PBS phosphate buffered saline
  • SBF simulated body fluid
  • T may be 20 or more, for example, t may be 20 to 120, for example, 20 to 96, 20 to 72, 30 to 70, 40 to 70, 50 to 65, and the like.
  • the porous silica particles may have a ratio t of 1/5 of the absorbance of the formula (1), for example, 70 to 140, and may be, for example, in the range of 80 to 140, 80 to 120, 80 to 110, To 140, 70 to 120, 70 to 110, and the like.
  • the porous silica particles may have a ratio t of 1/20 of the absorbance of the formula (1), for example, 130 to 220, and for example, within the range of 130 to 200, 140 to 200, 140 to 180, 150 To 180, and the like.
  • the porous silica particles may have a t of, for example, not less than 250, such as not less than 300, not less than 350, not less than 400, not less than 500, not less than 1000, and the like, , But is not limited thereto.
  • the ratio of the absorbance of Equation 1 to t has a high positive correlation.
  • the Pearson correlation coefficient may be 0.8 or more, for example, 0.9 or more and 0.95 or more.
  • T represents the degree of decomposition of the porous silica particles at a certain rate in an environment similar to that of the body. This means that the surface area, the particle size, the pore diameter, the surface of the porous silica particles, and / The substituent, the degree of compactness of the surface, and the like.
  • the surface area of a particle can be increased to decrease t, or the surface area can be decreased to increase t.
  • the surface area can be controlled by controlling the diameter of the particles and the diameter of the pores. It is also possible to increase the t by placing substituents in the surface and / or pores to reduce the direct exposure of the porous silica particles to the environment (solvent, etc.). Also, it is possible to increase the affinity between the physiologically active substance and the porous silica particles by supporting the physiologically active substance on the porous silica particles, and to reduce the direct exposure of the porous silica silica particles to the environment, thereby increasing t. It is also possible to increase the t by fabricating the surface more densely during the production of the particles. While various examples have been described above for adjusting t in Equation 1, the present invention is not limited thereto.
  • the porous silica particles may be, for example, spherical particles, but are not limited thereto.
  • the average diameter of the porous silica particles may be, for example, 100 nm to 1000 nm, and may be within the above range, for example, 100 nm to 800 nm, 100 nm to 500 nm, 100 nm to 400 nm, 100 nm to 300 nm, , But is not limited thereto.
  • the porous silica particles may have an average pore diameter of, for example, 1 nm to 100 nm, and may be within the above range, for example, 4 nm to 100 nm, 4 nm to 50 nm, 4 nm to 30 nm, 10 nm to 30 nm, It is not. It is possible to carry a large amount of the physiologically active substance and to carry the physiologically active substance having a large size.
  • the porous silica particles may have a BET surface area of, for example, 200 m 2 / g to 700 m 2 / g.
  • a BET surface area of, for example, 200 m 2 / g to 700m 2 / g.
  • 200m 2 / g to 650m 2 / g 250m 2 / g to 650m 2 / g
  • 300m 2 / g to 700m 2 / g 300m 2 / g to 650 m 2 / g
  • 300 m 2 / g to 550 m 2 / g 300 m 2 / g to 500 m 2 / g, 300 m 2 / g to 450 m 2 /
  • the porous silica particles may have a volume per gram of, for example, 0.7 ml to 2.2 ml. For example, it may be within the range of 0.7 ml to 2.0 ml, 0.8 ml to 2.2 ml, 0.8 ml to 2.0 ml, 0.9 ml to 2.0 ml, 1.0 ml to 2.0 ml, and the like. If the volume per gram is too small, the rate of decomposition may become too high, and excessively large particles may be difficult to manufacture or may not have a perfect shape.
  • the porous silica particles may have a hydrophilic substituent and / or a hydrophobic substituent on the outer surface and / or inside the pore.
  • a hydrophilic substituent and / or a hydrophobic substituent on the outer surface and / or inside the pore.
  • hydrophilic substituents may be present on both the surface and the pores, only hydrophobic substituents may be present, hydrophilic substituents may be present only on the surface or pores, hydrophobic substituents may be present, hydrophilic substituents may be present on the surface, Or vice versa, and vice versa.
  • the release of the physiologically active substance carried on the porous silica particles is mainly performed by the decomposition of the particles, and the interaction of the porous silica particles with respect to the release environment of the physiologically active substance is controlled by the control of the substituent,
  • the rate of release of the physiologically active substance can be controlled by adjusting the rate of the physiologically active substance, and the physiologically active substance can be diffused and released from the particles. Can be adjusted.
  • a hydrophobic substituent is present inside the pore, and a hydrophilic substituent may be present on the surface of the particle in view of ease of use and formulation Do.
  • the hydrophilic substituent may be, for example, an aldehyde group, a keto group, a carbamate group, a sulfate group, a sulfonate group, an amino group, an amine group, an aminoalkyl group, a silyl group, a carboxyl group, a sulfonic acid group, a thiol group, A substituted or unsubstituted C 1 to C 30 alkyl group, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C1 to C30 alkyl group, An unsubstituted C 3 to C 30 cycloalkyl group, a substituted or unsubstituted C 6 to C 30 aryl group, and a C 1 to C 30 ester group, and the hydrophobic substituent includes, for example, a substituted or unsub
  • the 'substituted' functional group in the 'substituted or unsubstituted' may be an aldehyde group, a keto group, a carbamate group, a sulfate group, a sulfonate group, an amino group, an amine group, an aminoalkyl group, a silyl group, a carboxyl group, At least one member selected from the group consisting of an alkyl group, an alkoxy group, an alkoxy group, an ammonium group, a sulfhydryl group, a phosphate group, an ester group, an imide group, a thioimide group, a keto group, an ether group, an indene group, a sulfonyl group, a methylphosphonate group and a polyethylene glycol group But is not limited thereto.
  • porous silica particles may be those in which the outer surface and / or the inside of the pores are positively charged and / or negatively charged.
  • both the surface and the pores can be positively charged, negatively charged, only positively charged on the surface or pore, negatively charged, surface can be positively charged, negatively charged inside the pore , And vice versa.
  • the charging may be performed, for example, by the presence of a cationic substituent or an anionic substituent.
  • the cationic substituent may be, for example, an amino group or other nitrogen-containing group as a basic group, and the anionic substituent may be, for example, a carboxyl group (-COOH), a sulfonic acid group (-SO 3 H) SH), and the like.
  • the interaction of the porous silica particles with respect to the release environment of the physiologically active substance is controlled by controlling the substituent, so that the rate of decomposition of the particles themselves can be controlled to control the release rate of the physiologically active substance, May be diffused and released from the particles.
  • the substituent By controlling the substituent, the binding force of the physiologically active substance to the particles can be controlled and the release of the physiologically active substance can be controlled.
  • porous silica particles may be coated on the surface and / or inside of the pores by carrying a physiologically active substance, transferring a physiologically active substance to a target cell, carrying a substance for other purposes, And may further include an antibody, a ligand, a cell permeable peptide, an umbilical cord or the like coupled thereto.
  • Substituents, charges, bonding substances, etc. in the surface and / or pores described above can be added, for example, by surface modification.
  • the surface modification can be performed, for example, by reacting a compound having a substituent to be introduced with a particle, and the compound may be, for example, an alkoxysilane having a C1 to C10 alkoxy group, but is not limited thereto.
  • the alkoxysilane is one having at least one of the above-mentioned alkoxy groups and may have, for example, 1 to 3 substituents, and may have a substituent to be introduced or a substituted substituent at a site where an alkoxy group is not bonded.
  • the porous silica particles may be prepared by, for example, preparing fine pore particles and a pore expansion process, and may be manufactured by further performing a calcination process, a surface modification process or the like, if necessary. When both the calcination and the surface modification process are performed, the surface modification may be performed after the calcination.
  • the particles of the small pores may be, for example, particles having an average pore diameter of 1 nm to 5 nm.
  • the small pore particles can be obtained by adding a surfactant and a silica precursor to a solvent and stirring and homogenizing the same.
  • the solvent can be water and / or organic solvent
  • the organic solvent includes, for example, ethers (especially cyclic ethers) such as 1,4-dioxane; Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone,?
  • water and the organic solvent may be used in a volume ratio of 1: 0.7 to 1.5, for example, 1: 0.8 to 1.3 by volume, It is not.
  • the surfactant may be, for example, CTAB (cetyltrimethylammonium bromide), hexadecyltrimethylammonium bromide (TMABr), hexadecyltrimethylpyridinium chloride (TMPrCl), tetramethylammonium chloride (TMACl), and the like.
  • CTAB cetyltrimethylammonium bromide
  • TMABr hexadecyltrimethylammonium bromide
  • TMPrCl hexadecyltrimethylpyridinium chloride
  • TMACl tetramethylammonium chloride
  • the surfactant may be added in an amount of, for example, 1 g to 10 g per liter of solvent, for example, in the range of 1 g to 8 g, 2 g to 8 g, 3 g to 8 g, and the like.
  • the silica precursor may be added after stirring and adding a surfactant to the solvent.
  • the silica precursor may be, for example, TMOS (Tetramethyl orthosilicate), but is not limited thereto.
  • the stirring may be carried out, for example, for 10 minutes to 30 minutes, but is not limited thereto.
  • the silica precursor may be added, for example, in an amount of 0.5 ml to 5 ml per liter of solvent, for example, 0.5 ml to 4 ml, 0.5 ml to 3 ml, 0.5 ml to 2 ml, 1 ml to 2 ml, But may be further added with sodium hydroxide as a catalyst as needed, which may be added with stirring before addition of the surfactant to the solvent, followed by addition of the silica precursor.
  • the sodium hydroxide may be, for example, 0.5 ml to 8 ml per liter of solvent, for example 1 ml of sodium hydroxide solution, 0.5 ml to 5 ml, 0.5 ml to 4 ml, 1 ml to 4 ml, 1 ml to 3 ml, But is not limited thereto.
  • the solution can be reacted with stirring.
  • the stirring may be carried out for example for 2 hours to 15 hours, for example, within the above range for 3 hours to 15 hours, 4 hours to 15 hours, 4 hours to 13 hours, 5 hours to 12 hours, 6 hours to 12 hours , 6 hours to 10 hours, and the like, but is not limited thereto. If the stirring time (reaction time) is too short, nucleation may be insufficient.
  • the solution may be aged.
  • the aging can be carried out, for example, for 8 hours to 24 hours, for example within the above range, 8 to 20 hours, 8 to 18 hours, 8 to 16 hours, 8 to 14 hours, 10 to 16 hours , 10 hours to 14 hours, and the like, but is not limited thereto.
  • reaction product may be washed and dried to obtain porous silica particles, and if necessary, separation of the unreacted material may be preceded by washing, for example, by separating the supernatant liquid by centrifugation.
  • the centrifugation can be performed at, for example, 6,000 to 10,000 rpm, for example, for 3 minutes to 60 minutes, for example, 3 minutes to 30 minutes, 3 minutes to 30 minutes, 5 minutes To 30 minutes, and the like, but the present invention is not limited thereto.
  • the washing may be performed using water and / or an organic solvent. Specifically, since the materials soluble in each solvent are different, water and an organic solvent may be used once or several times alternately. Alternatively, water or an organic solvent may be used once or several times It can be washed several times. The number of times may be, for example, 2 or more, 10 or less, for example, 3 or more and 10 or less, 4 or more or 8 or less, 4 or more or 6 or less, and the like.
  • the organic solvent includes, for example, ethers such as 1,4-dioxane (particularly, cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone,?
  • ethers such as 1,4-dioxane (particularly, cyclic ethers)
  • Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane
  • Acetone methyl isobutyl ketone
  • the washing may be carried out under centrifugation, for example, at a speed of 6,000 to 10,000 rpm, for example, for 3 minutes to 60 minutes, for example 3 minutes to 30 minutes, Min to 30 min, 5 min to 30 min, and the like, but the present invention is not limited thereto.
  • the washing may be performed by filtering the particles with a filter without centrifugation.
  • the filter may have pores smaller than the diameter of the porous silica particles.
  • the water and the organic solvent may be used once or several times at the time of the washing, and the water or the organic solvent may be washed once or several times.
  • the number of times may be, for example, 2 or more, 10 or less, for example, 3 or more and 10 or less, 4 or more or 8 or less, 4 or more or 6 or less, and the like.
  • the drying may be performed at, for example, 20 ° C to 100 ° C, but is not limited thereto, and may be performed in a vacuum state.
  • the pores of the obtained porous silica particles are expanded, which can be carried out using a pore-expanding agent.
  • pore-expanding agent for example, trimethylbenzene, triethylbenzene, tripropylbenzene, tributylbenzene, tripentylbenzene, trihexylbenzene, toluene, benzene and the like can be used. Specifically, trimethylbenzene can be used. But is not limited to.
  • the pore-expanding agent may be, for example, N, N-dimethylhexadecylamine (DMHA), but is not limited thereto.
  • DMHA N, N-dimethylhexadecylamine
  • the pore expansion can be performed, for example, by mixing the porous silica particles in a solvent with a pore-expanding agent, and heating and reacting.
  • the solvent can be, for example, water and / or organic solvents
  • the organic solvent can be, for example, ethers (especially cyclic ethers) such as 1,4-dioxane
  • Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane
  • Ketones such as acetone, methyl isobutyl ketone, and cyclohexanone
  • Carbon-based aromatic compounds such as benzene, toluene and xylene
  • Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone
  • Alcohols such as methanol, ethanol, propanol and butano
  • the porous silica particles may be present in an amount of from 10 g to 200 g per liter of solvent, for example from 10 g to 150 g, 10 g to 100 g, 30 g to 100 g, 40 g to 100 g, 50 g to 100 g, 50 g to 80 g, But the present invention is not limited thereto.
  • the porous silica particles may be uniformly dispersed in a solvent, for example, the porous silica particles may be added to a solvent and ultrasonically dispersed.
  • the second solvent may be added after dispersing the porous silica particles in the first solvent.
  • the pore-expanding agent may be, for example, 10 to 200 parts of skin to 100 parts of skin, 10 to 150 parts of skin, 10 to 100 parts of skin, 10 to 80 parts of skin, 30 to 80 parts of skin, 70 parts skin, and the like, but the present invention is not limited thereto.
  • the reaction can be carried out, for example, at 120 ° C to 180 ° C.
  • 120 ° C to 180 ° C For example, within the above-mentioned range, it is possible to use a temperature of 120 to 170 ° C, 120 to 160 ° C, 120 to 150 ° C, 130 to 180 ° C, 130 to 170 ° C, 130 to 160 ° C, 130 to 150 ° C, But is not limited thereto.
  • the reaction can be carried out, for example, for 24 hours to 96 hours.
  • 24 hours to 96 hours For example, within the above range from 30 hours to 96 hours, 30 hours to 96 hours, 30 hours to 80 hours, 30 hours to 72 hours, 24 hours to 80 hours, 24 hours to 72 hours, 36 hours to 96 hours, 36 48 hours to 48 hours, 48 hours to 80 hours, 48 hours to 72 hours, and the like, for example, from 1 hour to 80 hours, 36 hours to 72 hours, 36 hours to 66 hours, 36 hours to 60 hours, But is not limited thereto.
  • the reaction time may be increased when the reaction temperature is lowered, or the reaction time may be shortened when the reaction temperature is lowered. If the reaction is insufficient, the expansion of the pores may not be sufficient, and if the reaction proceeds excessively, the particles may collapse due to over-expansion of the pores.
  • the reaction can be carried out, for example, by raising the temperature stepwise. Specifically, it can be performed by raising the temperature from room temperature to the temperature stepwise at a rate of 0.5 ° C / min to 15 ° C / min, for example, within a range of 1 ° C / min to 15 ° C / Min to 15 ° C / min, 3 ° C / min to 12 ° C / min, 3 ° C / min to 10 ° C / min, and the like.
  • the reaction solution may be gradually cooled, for example, it may be cooled stepwise. Specifically, it may be performed by gradually warming the temperature to room temperature at a rate of 0.5 ° C / minute to 20 ° C / minute.
  • the temperature may be 1 ° C / minute to 20 ° C / 20 ° C / min, 3 ° C / min to 12 ° C / min, 3 ° C / min to 10 ° C / min, and the like.
  • reaction product may be washed and dried to obtain pore-expanded porous silica particles, and if necessary, separation of the unreacted material may be preceded by separation of the supernatant by, for example, centrifugation .
  • the centrifugation can be performed at, for example, 6,000 to 10,000 rpm, for example, for 3 minutes to 60 minutes, for example, 3 minutes to 30 minutes, 3 minutes to 30 minutes, 5 minutes To 30 minutes, and the like, but the present invention is not limited thereto.
  • the washing may be performed using water and / or an organic solvent.
  • water and an organic solvent may be used once or several times alternately.
  • water or an organic solvent may be used once or several times It can be washed several times.
  • the number of times may be, for example, two times or more and ten times or less, for example, three times, four times, five times, six times, seven times, eight times, and the like.
  • the organic solvent includes, for example, ethers such as 1,4-dioxane (particularly, cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone, and cyclohexanone; Carbon-based aromatic compounds such as benzene, toluene and xylene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Etc., and specifically ethanol, more specifically ethanol, may be used, but the present invention
  • the washing may be carried out under centrifugation, for example, at a speed of 6,000 to 10,000 rpm, for example, for 3 minutes to 60 minutes, for example 3 minutes to 30 minutes, Min to 30 min, 5 min to 30 min, and the like, but the present invention is not limited thereto.
  • the washing may be performed by filtering the particles with a filter without centrifugation.
  • the filter may have pores smaller than the diameter of the porous silica particles.
  • the water and the organic solvent may be used once or several times at the time of the washing, and the water or the organic solvent may be washed once or several times.
  • the number of times may be, for example, 2 or more, 10 or less, for example, 3 or more and 10 or less, 4 or more or 8 or less, 4 or more or 6 or less, and the like.
  • the drying may be performed at, for example, 20 ° C to 100 ° C, but is not limited thereto, and may be performed in a vacuum state.
  • the obtained particles can be calcined.
  • the calcination is a process for heating the particles to have a more dense structure on the surface and inside thereof, and removing organic substances that fill the pores.
  • calcination is performed at 400 to 700 ° C for 3 hours To 8 hours, more specifically, from 500 ° C to 600 ° C for 4 hours to 5 hours, but the present invention is not limited thereto.
  • porous silica particles can be surface-modified.
  • the surface modification can be performed inside the surface and / or pores.
  • the surface of the particle and the inside of the pore may be surface-modified in the same manner or may be surface-modified differently.
  • the surface modification may allow the particles to be charged or have hydrophilic and / or hydrophobic properties.
  • the surface modification can be carried out, for example, by reacting a compound having substituents such as hydrophilic, hydrophobic, cationic, and anionic to be introduced with the particles, and the compound can be, for example, an alkoxysilane having a C1 to C10 alkoxy group But is not limited thereto.
  • the alkoxysilane is one having at least one of the above-mentioned alkoxy groups and may have, for example, 1 to 3 substituents, and may have a substituent to be introduced or a substituted substituent at a site where an alkoxy group is not bonded.
  • an alkoxysilane When the alkoxysilane is reacted with the porous silicon particles, an alkoxysilane can be bonded to the surface and / or pores of the porous silicon particles by forming a covalent bond between the silicon atom and the oxygen atom, and the alkoxysilane has a substituent
  • the substituent may be introduced into the surface and / or pores of the porous silicon particles.
  • the above reaction can be carried out by reacting the porous silica particles dispersed in a solvent with an alkoxysilane.
  • the solvent can be water and / or organic solvent
  • the organic solvent includes, for example, ethers (especially cyclic ethers) such as 1,4-dioxane; Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone,?
  • the charging to the positive charge can be carried out by reacting with an alkoxysilane having a basic group such as a nitrogen-containing group such as an amino group or an aminoalkyl group.
  • an alkoxysilane having a basic group such as a nitrogen-containing group such as an amino group or an aminoalkyl group.
  • Specific examples thereof include N- [3- (Trimethoxysilyl) propyl] ethylenediamine, N1- (3-Trimethoxysilylpropyl) diethylenetriamine, (3-Aminopropyl) trimethoxysilane, N- [3- propyl] silane, 3- (2-Aminoethylamino) propyldimethoxymethylsilane, and the like, but the present invention is not limited thereto.
  • the charging of the negative charge can be carried out, for example, by reacting with an alkoxysilane having an acidic group such as a carboxyl group, a sulfonic acid group or a thiol group.
  • an alkoxysilane having an acidic group such as a carboxyl group, a sulfonic acid group or a thiol group.
  • 3-Mercaptopropyl) trimethoxysilane can be used, but is not limited thereto.
  • the hydrophilic property may be a hydrophilic property such as a hydrophilic group such as a hydroxyl group, a carboxyl group, an amino group, a carbonyl group, a sulfhydryl group, a phosphate group, a thiol group, an ammonium group, an ester group, an imide group, a thioimide group, a keto group, A polyoxyethylene group, a polyoxyethylene group, a polyoxyethylene group, a polyoxyethylene group, a polyoxyethylene group, a polyoxyethylene group, a polyethylene glycol group and the like.
  • the hydrophobic property may be a hydrophobic substituent, for example, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, a substituted or unsubstituted A C2 to C30 heteroaryl group, a halogen group, an ester group of C1 to C30, a halogen-containing group, and the like.
  • a hydrophobic substituent for example, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, a substituted or unsubstituted A C2 to C30 heteroaryl group, a halogen group, an ester group of C1 to C30
  • trimethoxy (octadecyl) silane trimethoxy n-octylsilane, trimethoxy propyl silane
  • isobutyl trimethoxy silane trimethoxy (7-octen- silane, trimethoxy (2-phenylethyl) silane, vinyltrimethoxysilane, cyanomethyl, 3- (trimethoxysilyl) propyl] trithiocarbonate and (3-Bromopropyl) trimethoxysilane.
  • hydrophobic substituent in the pores for enhancing the bonding strength with the poorly soluble (hydrophobic) physiologically active substance through surface modification, and the hydrophobic substituent is present on the surface of the particle in terms of ease of use and formulation And a substituent for binding another substance other than the physiologically active substance may be present on the surface.
  • the surface modification may be performed in combination.
  • two or more surface modification may be performed on the outer surface or inside the pores.
  • a compound containing a carboxyl group may be bonded to an amide-introduced silica particle with an amide bond to change positively charged particles to have different surface characteristics, but the present invention is not limited thereto.
  • the reaction of the porous silica particles with the alkoxysilane can be carried out, for example, under heating.
  • the heating may be performed at a temperature of, for example, 80 to 180 DEG C, for example, 80 to 160 DEG C, 80 to 150 DEG C, 100 to 160 DEG C, 100 to 150 DEG C, 110 to 150 DEG C, But is not limited thereto.
  • the reaction of the porous silica particles with the alkoxysilane may be carried out, for example, for 4 to 20 hours, for example 4 to 18 hours, 4 to 16 hours, 6 to 18 hours, 6 to 16 hours , 8 hours to 18 hours, 8 hours to 16 hours, 8 hours to 14 hours, 10 hours to 14 hours, and the like.
  • the reaction temperature, time, and the amount of the compound used for surface modification can be selected depending on the desired degree of surface modification.
  • the reaction conditions vary depending on the hydrophilicity, hydrophobicity, and electric charge of the physiologically active substance,
  • the hydrophilic property, the hydrophobicity, and the electric charge level of the physiologically active substance can be controlled. For example, when a physiologically active substance has a strong negative charge at a neutral pH, the reaction temperature can be increased or the reaction time can be lengthened and the compound throughput can be increased in order to allow the porous silica particles to have a strong positive charge , But is not limited thereto.
  • the porous silica particles may be prepared by, for example, preparing pores of small pores, expanding pores, modifying the surface, or modifying pores inside.
  • the particle preparation and the pore expansion process of the small pores can be performed by the processes described above, and the cleaning and drying processes can be performed after the particle preparation of the small pores and after the pore expansion process.
  • the separation of the unreacted material may be preceded by washing before washing, for example, by separating the supernatant by centrifugation.
  • the centrifugation may be performed at, for example, 6,000 to 10,000 rpm, for example, 3 minutes to 60 minutes, specifically 3 minutes to 30 minutes, 3 minutes to 30 minutes, 5 minutes To 30 minutes, and the like, but the present invention is not limited thereto.
  • the cleaning after the particle preparation of the small pores may be carried out by any method / condition within the range exemplified above, but is not limited thereto.
  • the purging after the pore expansion can be performed under more relaxed conditions than in the previous examples. For example, washing may be performed within 3 times, but is not limited thereto.
  • the surface modification and the internal reforming of the pores can be performed by the processes described above, and the processes can be performed in the order of the surface modification and the internal pore modification, and the particle washing process is further performed between the two processes .
  • the inside of the pores are filled with the reaction liquid such as the particles used for the preparation of the particles and the pore expansion, Only the surface can be modified. The particles may then be washed to remove the reaction liquid inside the pores.
  • the washing of the particles between the surface modification and the pore interior modification process may be performed using water and / or an organic solvent. Specifically, since the materials soluble in each solvent are different, water and an organic solvent may be used once or several times, Water or an organic solvent can be washed once or several times. The number of times may be, for example, 2 or more, 10 or less, specifically 3 or more and 10 or less, 4 or more or 8 or less, 4 or more, 6 or less, and the like.
  • the washing may be carried out under centrifugation, for example, at 6,000 to 10,000 rpm, for example, for 3 to 60 minutes, specifically for 3 to 30 minutes, Min to 30 min, 5 min to 30 min, and the like, but the present invention is not limited thereto.
  • the washing may be performed by filtering the particles with a filter without centrifugation.
  • the filter may have pores smaller than the diameter of the porous silica particles.
  • the water and the organic solvent may be used once or several times at the time of the washing, and the water or the organic solvent may be washed once or several times.
  • the number of times may be, for example, 2 or more, 10 or less, specifically 3 or more and 10 or less, 4 or more or 8 or less, 4 or more, 6 or less, and the like.
  • the drying may be performed at, for example, 20 ° C to 100 ° C, but is not limited thereto, and may be performed in a vacuum state.
  • the physiologically active substance such as siRNA or dsRNA of the present invention can be carried on the surface and / or inside the pores of the porous silica particles.
  • the support may be carried out, for example, by mixing the porous silica particles in the solvent and the physiologically active substance.
  • the solvent can be water and / or organic solvent
  • the organic solvent includes, for example, ethers such as 1,4-dioxane (particularly, cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone, and cyclohexanone; Carbon-based aromatic compounds such as benzene, toluene and xylene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Etc. may be used.
  • PBS phosphate buffered saline solution
  • SBF Simulated Body Fluid
  • borate-buffered saline borate-buffered saline
  • Tris-buffered saline Tris-buffered saline, etc. may be used as the solvent.
  • the ratio of the porous silica particles to the physiologically active substance is not particularly limited and may be, for example, a weight ratio of 1: 0.05 to 0.8, such as 1: 0.05 to 0.7, 1: 0.05 to 0.6, 1: 0.8, 1: 0.1 to 0.6, 1: 0.2 to 0.8, 1: 0.2 to 0.6, and the like.
  • the physiologically active substance such as siRNA or dsRNA of the present invention carried on the porous silica particles can be gradually released over an extended period of time. Such slow release may be continuous or non-continuous, linear or non-linear, and may vary due to the characteristics of the porous silica particles and / or their interaction with the bioactive material.
  • the physiologically active substance supported on the porous silica particles is released while the porous silica particles are biodegraded.
  • the porous silica particles according to the present invention may be slowly decomposed to release the supported physiologically active substance slowly. This can be controlled, for example, by controlling the surface area, particle size, pore diameter, surface area and / or porosity of the porous silica particles, the degree of compactness of the surface, and the like.
  • the physiologically active substance carried on the porous silica particles can be released while diffusing from the porous silica particles, which is affected by the relationship between the porous silica particles, the physiologically active substance, and the environment for releasing the physiologically active substance , And it is possible to control the release of the biologically active substance by controlling this. For example, by strengthening or weakening the binding force of the porous silica particles with the physiologically active substance by surface modification.
  • the surface of the particle and / or the inside of the pore may have a hydrophobic substituent to increase the binding force between the porous silica particle and the physiologically active substance, Whereby the physiologically active substance can be released in a sustained manner.
  • the porous silica particles are surface-modified with an alkoxysilane having a hydrophobic substituent.
  • “poorly soluble” is a meant to include (for water) in that the insoluble (insoluble), substantially insoluble (practically insoluble) or very slightly soluble (only slightly soluble) This "Pharmaceutical Science,” 18 th Edition ( USP, Remington, published by Mack Publishing Company).
  • the water-insoluble physiologically active substance may have a water solubility of less than 10 g / L, specifically less than 5 g / L, more specifically less than 1 g / L at 25 ° C under 1 atm, but is not limited thereto.
  • the surface and / or the pores of the particles may have a hydrophilic substituent and the binding strength between the porous silica particles and the physiologically active substance may be increased, It can be released slowly.
  • the porous silica particles may be surface-modified with an alkoxysilane having a hydrophilic substituent.
  • the water-soluble physiologically active substance may have a water solubility of 10 g / L or more at 25 ° C and 1 atm, but is not limited thereto.
  • the surface and / or pore of the particle may be charged with opposite charge to increase the binding force between the porous silica particle and the physiologically active substance, Material may be released slowly.
  • the porous silica particles may be surface-modified with an alkoxysilane having an acidic group or a basic group.
  • the surface and / or pore interior of the particle may be negatively charged at a neutral pH, whereby the binding force between the porous silica particle and the physiologically active substance Is increased, so that the physiologically active substance can be released slowly.
  • the porous silica particles may be surface-modified with an alkoxysilane having an acidic group such as a carboxyl group (-COOH) or a sulfonic acid group (-SO 3 H).
  • the surface and / or pores of the particles may be positively charged, thereby increasing the binding force between the porous silica particles and the physiologically active substance, Material may be released slowly.
  • the porous silica particles may be surface-modified with an alkoxysilane having a basic group such as an amino group or other nitrogen-containing groups.
  • the physiologically active substance may be released for a period of, for example, 7 days to 1 year or more depending on the type of treatment required, the release environment, and the porous silica particles used.
  • the porous silica particles can be decomposed 100% as biodegradable, the supported physiologically active substance can be 100% released.
  • the pharmaceutical composition for preventing or treating liver cancer comprising siRNA or dsRNA of the present invention may further comprise a pharmaceutically acceptable carrier and may be formulated together with a carrier.
  • a pharmaceutically acceptable carrier refers to a carrier or diluent that does not irritate the organism and does not interfere with the biological activity and properties of the administered compound.
  • the pharmaceutical carrier which is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used.
  • diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • composition of the present invention can be applied to any formulation containing the siRNA or dsRNA of the present invention as an active ingredient, and can be manufactured into oral or parenteral formulations.
  • the pharmaceutical formulations of the present invention may be administered orally, rectally, nasal, topical (including under the ball and tongue), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous And intravenous), or forms suitable for administration by inhalation or insufflation.
  • composition of the present invention is administered in a pharmaceutically effective amount. Effective dose levels will depend on factors well known in the art and other medical disciplines including the type of disease, severity of the patient, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, Can be determined.
  • the composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.
  • the dosage of the composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, severity of disease and the like.
  • the amount of the drug accumulated in the body, and / or the degree of specific activity of the siRNA or dsRNA of the present invention to be used. May be calculated on the basis of the EC50 generally measured as effective in the in vivo animal model and in vitro, for example from 0.01 [mu] g to 1 g per kg of body weight and may be divided into daily, weekly, monthly, May be administered once or several times per unit period, or may be continuously administered for a long period using an infusion pump.
  • the number of repeated administrations is determined in consideration of the duration of the drug in the body, the drug concentration in the body, and the like.
  • the composition may be administered for recurrence, even after treatment according to the course of the disease treatment.
  • composition of the present invention may further contain one or more active ingredients which exhibit the same or similar functions with respect to the treatment of liver cancer, or a compound which maintains / increases the solubility and / or absorbency of the active ingredient. Also optionally, it may further comprise a chemotherapeutic agent, an anti-inflammatory agent, an antiviral agent and / or an immunomodulator.
  • compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal.
  • the formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.
  • Human liver cancer cell line (SNU-449) and murine Hepa-1c1c7 liver cancer cell line were obtained from Korean Cell Line Bank (Seoul, Korea). All cell lines were cultured in EMEM (American Type) supplemented with 10% fetal bovine serum (FBS, Lonza) and 100 units / mL penicillin-streptomycin (Invitrogen, Carlsbad, CA) at 37 ° C and 5% CO 2 in a humidified incubator Culture Collection, Manassas, Va.), RPMI-1640 or DMEM medium (Lonza, Walkersville, Md.).
  • EMEM American Type
  • FBS fetal bovine serum
  • penicillin-streptomycin Invitrogen, Carlsbad, CA
  • siRNA and dsRNA used in this experiment were synthesized by Lemonex (Seoul, Korea) and the gene ORF sequence (BANF1: NM_003860, PLOD3: NM_001084, SF3B4: NM_005850) in the pcDNA3.1 + / C- (K) Human BANF1, PLOD3, and SF3B4 expression plasmids were purchased from Genscript TM (Piscataway, NJ, USA). Transfection was performed using Lipofectamine RNAiMAX or Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's manual.
  • the cell line was supplied to a 12-well plate in 30% confluence. After transfusion or inhibition treatment, the cells were incubated with 0.5 mg / mL of (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) at 37 ° C for 1 hour every 24 hours .
  • the formazan crystal was dissolved in DMSO and the absorbance at 570 nm was read using a VICTOR3 TM multilabel plate reader (PerkinElmer, Boston, Mass.).
  • cell proliferation assay cell lines were fed to 24 well plates in 30% confluence. After transfection, cells were treated with 5-bromo-2'-deoxyuridine (BrdU) reagent for 2 h and fixed at room temperature for 30 min. Cells were incubated with anti-BrdU antibody for 1 hour at room temperature. Unbound antibody was removed by washing buffer. Horseradish peroxidase-conjugated secondary antibody was added to each well. The substrate solution was added and the reaction was stopped after 30 minutes in the stop solution. The final product was quantified at 490 nm by a VICTOR3 TM multilabel plate reader (PerkinElmer).
  • RhdU 5-bromo-2'-deoxyuridine
  • Transwell plates and cell culture inserts were used for in vitro cell motility and invasion analysis.
  • (BD Biosciences) was diluted to 0.3 mg / ml with coating buffer (0.01 M Tris, 0.7% NaCl, pH 8.0) for coating of the invasion assays and 100 ⁇ l of Macri Gel was applied to the upper compartment of the cell culture insert Lt; / RTI > After incubation at 37 ° C for 1 hour, the cell culture insert was ready for seeding.
  • the cells 5% FBS a serum present in chemoattractant- were properly seeded into the cell culture insert of the pre-culture medium (0.5 ⁇ 10 5 cells / well for the motility assay, 1 ⁇ 10 5 cells / well for the invasion assay).
  • the migrated or invaded cells were stained with a Diff-Quik staining kit (Sysmex, Japan). Cells were photographed at 200x magnification on an Axiovert 200 inverted microscope (Zeiss, Jena, Germany). Cells were enumerated in three randomized clocks.
  • Transfected cells were fed into wells of 6 well plates. At 100% confluence, a scratch was made on the same layer using a micropipette tip. Photographs of the same area of the wound were taken at 0 h and 24 h using an IX70 fluorescence inverted microscope (Olympus, Tokyo, Japan).
  • xenograft tumorigenicity evaluation 1 x 10 7 transduced cells were mixed in 0.2 ml PBS (pH 7.4) and 30% (v / v) Matrigel Matrix (BD Biosciences). Cell suspensions were subcutaneously injected into Balb / c-nude mice at 6 weeks of age. Rats were examined twice a week for confirmation of tumor formation at the injection site. Tumor volume was calculated as 0.5 x length (L) x width 2 (W 2 ). Each experimental group consisted of 10 rats, and tumor growth was quantified by caliper measurements in three orthogonal directions. Results were expressed as mean tumor volume and 95% confidence interval. The H-ras12V activated homotypic transgenic mice were provided by Dr.
  • Example 12- (1) -2) The porous silica nanoparticles of Example 12- (1) -2) -2, 80 ⁇ l of InViVojection TM RNAi-nano reagent, which is specific for BANF1, PLOD3 and SF3B4, No. DHMSN-vivo RNA; Lemonex Inc., Seoul, Korea) and prepared in 200 [mu] l PBS.
  • a mixture of siRNA or dsRNA and nanoparticles was injected into the H-ras transgenic HCC mouse model by weekly intravenous injection from week 9 to week 23. Ultrasonography was taken at 17, 19, and 21 weeks with an ultrasound machine (Affiniti 50, Philips, Seoul, Korea).
  • reaction solution was centrifuged at 8000 rpm for 10 minutes at 25 DEG C to remove the supernatant, centrifuged at 8000 rpm for 10 minutes at 25 DEG C, and washed five times with ethanol and distilled water alternately.
  • TMB trimethyl benzene
  • the reaction was carried out by starting at 25 ° C and heating at a rate of 10 ° C / min and then slowly cooling down at a rate of 1 to 10 ° C / min in an autoclave.
  • the cooled reaction solution was centrifuged at 8000 rpm for 10 minutes at 25 DEG C to remove supernatant, centrifuged at 8000 rpm for 10 minutes at 25 DEG C, and washed five times with ethanol and distilled water alternately.
  • the porous silica particles prepared in 2) were placed in a glass vial and heated at 550 ° C for 5 hours. After completion of the reaction, the particles were gradually cooled to room temperature to prepare particles.
  • Porous silica particles were prepared in the same manner as in item (1) of Example 11, except that the reaction conditions at the time of pore expansion were changed to 140 ° C and 72 hours.
  • Porous silica particles were prepared in the same manner as in item (1) of Example 11, except that 5-fold larger vessels were used and all the materials were used in a 5-fold capacity.
  • Porous silica particles were prepared in the same manner as in Example 11- (1) except that 920 ml of distilled water and 850 ml of methanol were used in the preparation of small pore particles.
  • Porous silica particles were prepared in the same manner as in Example 11- (1) except that 800 ml of distilled water, 1010 ml of methanol and 10.6 g of CTAB were used in the preparation of small pore particles.
  • Porous silica particles were prepared in the same manner as in Example 11- (1), except that 620 ml of distilled water, 1380 ml of methanol and 7.88 g of CTAB were used in the preparation of the small pore particles.
  • Porous silica particles were prepared in the same manner as in item (1) of Example 11 except that 2.5 mL of TMB was used at the time of pore expansion.
  • Porous silica particles were prepared in the same manner as in item (1) of Example 11 except that 4.5 mL of TMB was used at the time of pore expansion.
  • Porous silica particles were prepared in the same manner as in Example 11- (1) except that 11 mL of TMB was used at the time of pore expansion.
  • Porous silica particles were prepared in the same manner as in item (1) of Example 11 except that 12.5 mL of TMB was used at the time of pore expansion.
  • Example 11- (1) -2 The small pore particles were reacted with TMB in the same manner as in Example 11- (1) -2), cooled, and centrifuged to remove the supernatant. Thereafter, the mixture was centrifuged under the same conditions as in Example 11- (1) -2), washed three times with ethanol and distilled water alternately and then dried under the same conditions as in Example 11- (1) -2) Silica particles (pore diameter 10 to 15 nm, particle diameter 200 nm) were obtained.
  • the reaction solution in the previous step remains in the pores, and the inside of the pores are not modified.
  • the cooled reaction solution was centrifuged at 8000 rpm for 10 minutes to remove the supernatant, centrifuged at 8000 rpm for 10 minutes at 25 DEG C, and washed five times with ethanol and distilled water alternately.
  • Example 11- (4) The porous silica particles of Example 11- (4) were reacted with (3-Aminopropyl) triethoxysilane (APTES) to positively charge.
  • APTES (3-Aminopropyl) triethoxysilane
  • porous silica particles were dispersed in 10 mL of toluene in a 100 mL round bottom flask with a bath sonicator. Then, 1 mL of APTES was added, and the mixture was stirred at 400 rpm and stirred at 130 DEG C for 12 hours.
  • the mixture was slowly cooled to room temperature, centrifuged at 8000 rpm for 10 minutes to remove the supernatant, centrifuged at 8000 rpm for 10 minutes at 25 ° C, and washed 5 times with ethanol and distilled water.
  • Example 11- (1) The porous silica particles of Example 11- (1) were reacted with (3-aminopropyl) triethoxysilane (APTES) to positively charge, except that 0.4 ml of APTES was added and the reaction time was changed to 3 hours Was modified in the same manner as in Example 12- (1) -1).
  • APTES (3-aminopropyl) triethoxysilane
  • Example 11- (9) The porous silica particles of Example 11- (9) were reacted with (3-Aminopropyl) triethoxysilane (APTES) and positively charged.
  • APTES (3-Aminopropyl) triethoxysilane
  • Example 11- (10) were reacted with (3-Aminopropyl) triethoxysilane (APTES) to positively charge.
  • APTES (3-Aminopropyl) triethoxysilane
  • Example 11- (1) The porous silica particles of Example 11- (1) were reacted with trimethoxy (propyl) silane to introduce a propyl group into the surface and pores, except that 0.35 ml of trimethoxy (propyl) silane was added instead of APTES and reacted for 12 hours And the modification was carried out in the same manner as in Example 12- (1).
  • Example 11- (1) The porous silica particles of Example 11- (1) were reacted with trimethoxy-n-octylsilane to introduce a propyl group into the surface and pores, except that 0.5 ml of trimethoxy-n-octylsilane was added instead of APTES and reacted for 12 hours And the modification was carried out in the same manner as in Example 12- (1).
  • Example 11- (1) The porous silica particles of Example 11- (1) were reacted with succinic anhydride to negatively charge.
  • DMSO dimethyl sulfoxide
  • 80 mg of succinic anhydride was added instead of APTES
  • the reaction was carried out in the same manner as in Example 12- (1) -1) except that DMSO was used instead of distilled water at the time of washing.
  • Example 12- (3) -2 100 mg of the porous silica nanoparticles of Example 12- (3) -2) was dispersed in 1 mL of a 1 M aqueous sulfuric acid solution and 20 mL of 30% aqueous hydrogen peroxide and stirred at room temperature to induce an oxidation reaction to oxidize the thiol group with a sulfonic acid group. Thereafter, it was washed and dried in the same manner as in Example 12- (1) -1).
  • GFP green fluorescent protein
  • Example 2 Analysis of inhibitory rate of indicator gene expression of siRNA or dsRNA of the present invention
  • SiRNA, dsRNA validation test for inhibition of human BANF1, transcript variant 1, mRNA (Gene Bank number: NM_003860.3) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) 5 87.73 11 73.18 17 84.11 23 76.24 6 79.64 12 85.44 18 88.36 24 87.7 7 82.3 13 69.57 19 87.83 25 62.57 8 76.21 14 77.3 20 67.72 26 72.92 9 89.6 15 82.92 21 82.29 27 65.58 10 83.42 16 91.38 22 63.23 28 72.91
  • SiRNA, dsRNA validity test for inhibition of human BANF1, transcript variant 2, mRNA (Gene Bank number: NM_001143985.1) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) 29 92.55 37 81.64 45 83.22 53 84.31 30 91.49 38 68.4 46 78.16 54 64.9 31 86.44 39 79.72 47 73.48 55 74.72 32 77.1 40 91.6 48 68.3 33 73.82 41 87.37 49 85.27 34 76.6 42 53.77 50 88.74 35 88.33 43 86.39 51 92.32 36 82.53 44 68.63 52 74.8
  • SiRNA, dsRNA validity test for expression suppression of human human PLOD3 gene sequence (Gene Bank number: NM_001084.4) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) 56 87.62 73 76.8 90 72.7 107 74.5 57 78.13 74 68.27 91 83.69 108 86.25 58 92.72 75 77.44 92 85.3 109 83.7 59 83.49 76 86.26 93 76.62 110 74.13 60 86.8 77 84.3 94 82.11 111 76.29 61 64.29 78 81.52 95 83.46 112 73.52 62 73.33 79 79.35 96 71.25 113 82.86 63 85.83 80 76.63 97 72.73 114 73.52
  • SiRNA for the inhibition of the expression of the human SF3B4 gene sequence (human SF3B4 gene sequence (Gene Bank number: NM_005850.4), dsRNA validity test Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) Base sequence number Expression inhibition rate (%) 121 83.71 131 74.32 141 78.33 151 73.12 122 81.83 132 92.19 142 72.45 152 78.66 123 87.62 133 84.72 143 76.72 153 82.5 124 86.39 134 81.3 144 81.36 154 76.63 125 78.64 135 83.4 145 83.2 155 62.95 126 82.7 136 88.63 146 72.41 156 89.6 127 84.25 137 78.25 147 73.64 157 77.2 128 74.11 138 85.1 148
  • SiRNAs comprising the sense RNA having the sequence shown in Table 11 below and the antisense RNA consisting of the complementary sequence to the Hepa-1c1c7 and SNU-449 cell lines of Example 1 were subjected to the methods of Examples 1-2 or 1-8, respectively After in vitro transfection, the expression levels of the corresponding indicator factors of each siRNA were measured by Western blotting, and the results are shown in Fig.
  • SEQ ID NO: Sense RNA sequence The naming in Figure 1 Target gene SEQ ID NO: 311 5'-CCUCAGCGUUUCAAUCUUUU-3 ' Banf1 Mouse BANF1 gene SEQ ID NO: 312 5'-CGACUGCAGAAUCUCCUCUUU-3 ' Plod3 Mouse PLOD3 gene SEQ ID NO: 313 5'-CUGCUUUACGAUACUUUCAUU-3 ' Sf3b4 Mouse SF3B4 gene SEQ ID NO: 314 5'-CCUACGCCACCAAUUUCGU-3 ' Control - SEQ ID NO: 28 5'-AAGAAGCUGGAGGAAAGGGGUUU-3 ' BANF1 Human BANF1 gene SEQ ID NO: 119 5'-GCAUCUGGAGCUUUCUGUA UU-3 ' PLOD3 Human PLOD3 gene SEQ ID NO: 136 5'-GCAGUACCUCUGUAACCGU UU-3 ' SF3B4 Human SF3B4
  • SiRNAs comprising the sense RNA having the sequence shown in Table 12 below and the antisense RNA consisting of the complementary sequence of the SNU-449 cell line of Example 1-1 were in vitro transfected according to the method of Example 1-2, The migration and invasion responses of the corresponding surface factors of the siRNAs were analyzed according to the methods of Examples 1-5, and the scratch wound healing ability was analyzed according to the method of Examples 1-6, and the results are shown in FIG.
  • SEQ ID NO: Sense RNA sequence The naming in Figure 2 Target gene SEQ ID NO: 314 5'-CCUACGCCACCAAUUUCGU-3 ' Control - SEQ ID NO: 28 5'-AAGAAGCUGGAGGAAAGGGGUUU-3 ' BANF1 Human BANF1 gene SEQ ID NO: 119 5'-GCAUCUGGAGCUUUCUGUA UU-3 ' PLOD3 Human PLOD3 gene SEQ ID NO: 136 5'-GCAGUACCUCUGUAACCGU UU-3 ' SF3B4 Human SF3B4 gene
  • N-cadherin, Fibronectin, Snail, and Slug which are representative epithelial-mesenchymal transition (EMT) regulatory proteins related to the metastasis of liver cancer cells, can be inhibited by inhibiting the expression of each marker by the siRNA or dsRNA of the present invention.
  • SiRNAs consisting of the sense RNA having the sequence of Table 12 and the antisense RNA consisting of the complementary sequence were in vitro transfected in the SNU-449 cell line of Example 1 according to the method of Example 1-2 Then, the amount of expression of the corresponding indicator factors and the expression level of the EMT regulatory proteins of each siRNA were analyzed according to the method of Examples 1-9, and the results are shown in FIG. 3 (A).
  • SiRNAs consisting of the sense RNA having the sequence of Table 12 and the antisense RNA consisting of the complementary sequence were transfected in vitro in the SNU-449 cell line of Example 1 according to the method of Example 1-2, and transfected cells After injection into athymic nude mice, the size of the liver tumor and the survival rate of the mice were analyzed and the results are shown in FIG. 3 (B).
  • knockdown of the marker factors via the siRNA or dsRNA of the present invention can increase the overall tumor growth rate And the average tumor volume is decreased.
  • the tumor-free survival rate is significantly higher than that of the control group. More specifically, when 50 days have elapsed after subcutaneous transfection of the transfected cells, In the control group, tumors were found in 6 rats of 10 rats, but in the experimental group, tumors were found in 1 to 2 rats among 10 rats, indicating that the siRNA or dsRNA of the present invention can effectively inhibit the growth of liver tumors Able to know.
  • SiRNAs consisting of sense RNA having the sequence of Table 13 below and antisense RNA consisting of the complementary sequence were transfected in vivo according to the method of Examples 1-8 and the number of tumors 4 (A).
  • the level of inhibition of the expression of the respective indicator genes of the siRNAs carried on the porous nanoparticles was analyzed by the method of Example 1-9 and shown in FIG. 4 (B).
  • SEQ ID NO: Sense RNA sequence The naming in Figure 1 Target gene SEQ ID NO: 311 5'-CCUCAGCGUUUCAAUCUUUU-3 ' Banf1 Mouse BANF1 gene SEQ ID NO: 312 5'-CGACUGCAGAAUCUCCUCUUU-3 ' Plod3 Mouse PLOD3 gene SEQ ID NO: 313 5'-CUGCUUUACGAUACUUUCAUU-3 ' Sf3b4 Mouse SF3B4 gene
  • FIG. 5 is a photograph of the porous silica particles of 11- (1)
  • FIG. 6 is a photograph of the porous silica particles of 11- (2)
  • 7 is a photograph of small pore particles of 11- (1)
  • FIG. 8 is a comparative photograph of small pore particles of 11- (1) and 11- (3) have.
  • porous silica particles were almost completely decomposed after 360 hours of biodegradation.
  • a 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles in a cylindrical permeable membrane having pores having a diameter of 50 kDa,
  • a t is the absorbance of the porous silica particles measured after passage of time t from the measurement of A 0 ).
  • porous silica particle powder 5 mg was dissolved in 5 ml of SBF (pH 7.4). Thereafter, 5 ml of the porous silica particle solution was placed in the permeable membrane having the pore diameter of 50 kDa shown in Fig. 15 ml of SBF was added to the outer membrane, and SBF of the outer membrane was replaced every 12 hours.
  • the decomposition of the porous silica particles was carried out at 37 ° C with 60 rpm of horizontal stirring.
  • porous silica particles of the examples have significantly larger t than the control.
  • t which is the ratio of the absorbance of the positively charged particles to 1/2, was 24 or more.
  • the release solvent Prior to 24 hours, the release solvent was withdrawn at 0.5, 1, 2, 4, 8, 12, 24 hours, then every 24 hours thereafter, 0.5 ml of the release solvent was recovered for fluorescence measurement, SBF was added.
  • the time at which 50% of the siRNA was released was about 40 hours or more.
  • Balb / c nude male (5 weeks old) was purchased from Orient Bio Inc., and 3 million HeLa cells (cervical cancer cells) were dispersed in sterilized 1x PBS.
  • Xenograft tumors were subcutaneously injected into mice to grow 70 mm when the solidification of the 3 tumor size is confirmed, PBS, FITC- porous silica particles (example 12- (1) -2) -2 of porous silica particles), porous FITC- loaded with siRNA of example 1-13 (Silica particles of Example 12- (1) -2) -2) were injected into each mouse tumor, and the fluorescence intensity and distribution were measured immediately before, immediately after, and after 48 hours by FOBI fluorescence in vivo imaging system (Neo science, Korea).
  • the FITC label was prepared by dispersing 50 mg of silica particles in 1 mL of DMSO and adding 25 ⁇ g of FITC-NHS solution (2.5 mg / mL) (10 ⁇ l) The reaction mixture was reacted for 18 hours, and the reaction product was purified by centrifugation (8500 rpm, 10 minutes). The supernatant was discarded and the precipitated particles were collected and dispersed evenly in ethanol. This was repeated 3-4 times with ethanol- distilled water, And purified until no color was observed.
  • the control was administered alone in PBS, the siRNA of Example 1-13 alone, the siRNA alone of Example 1-13, the FITC-DDV of FITC alone, the complex
  • the siRNA transferred into the body to be loaded into the body has a longer period of maintaining the activity, After staying longer, we can confirm that the fluorescence is strong even after 48 hours.

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Abstract

The present invention provides siRNA or dsRNA, which can effectively inhibit the expression of three highly expressed markers in liver cancer, and a pharmaceutical composition comprising the same can obtain an excellent effect of preventing or treating liver cancer through RNAi.

Description

간암의 예방 또는 치료용 약학적 조성물Pharmaceutical composition for preventing or treating liver cancer
본 발명은 간암의 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating liver cancer.
간암(Hepatocellular carcinoma, HCC)은 전세계 암 관련 사망의 두 번째 주요 원인이다. HCC는 최근에 발생률이 증가하고 있는 소수의 암 중 하나이다.Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. HCC is one of the few cancers that have recently increased in incidence.
간암의 일차적 치료법은 외과적 절제술이며 초기 치료 단계에서 치유 치료(curative treatment)가 가능한 환자는 소수이다. 절제술(Resection)과 경피적 절제술(percutaneous ablation)은 5년 후 재발률이 70 %에 이르러 생존율과 밀접한 관련이 있다. The primary treatment for hepatocellular carcinoma is surgical resection, and few patients are eligible for curative treatment at the initial treatment stage. Resection and percutaneous ablation have a recurrence rate of 70% after 5 years and are closely related to the survival rate.
다른 암과 마찬가지로 HCC는 다발성 종양 진행 과정을 특징으로 가진다. 손상된 간 조직은 초기 단계에서 이형성 결절(dysplastic nodules, DNs)이라 불리는 작은 결절성 과증식 병변(small nodular hypercellular lesions)으로 진화한다. 이 전 암성 병변은 모호한 결절 형태의 작고 잘 분화된 간세포로 발전한 후 간질성 모양과 잦은 미세 혈관 침범을 특징으로 하는 진행성 간세포로 정의되는 조기 간암(eHCC)으로 진행된다. 다단계 간암 발생에 대한 현재의 지식에 근거하여, 고위험군 환자에 대해 밀접하게 추적 조사가 이루어졌으며 작은 크기를 가지며 원인이 밝혀지지 않은 한 병변의 수가 증가하는 것이 진단 영상을 통해 감지된다. 초음파 유도 바늘 생검은 이러한 병변에 대해 수행된다. 암으로 조직학적 진단을 받으면 치료를 받는다. 그러나 eHCC는 최소의 이형성을 나타내며 명확한 침습적 또는 파괴적인 성장이 결여되어 있다. 그러므로 간 병리학자 조차도 재생 결절, 전암 병변 및 조기 병변을 구별하는 것이 종종 어렵다. 이러한 이유로 초기 HCC의 조직학적 진단을 표준화하고 적절한 치료를 유도하는 객관적인 분자 마커의 발견이 열망되고 있다. 또한 간암종의 정확한 진단과 관련이 있는 바이오 마커의 발견은 간암종으로 진행될 가능성이 있는 전암 병변을 확인하고 외과적으로 절제 가능한 병변을 결정하는 데 도움이 될 수 있으므로 의사가 간암 환자의 수술 범위를 설계하는 데 도움이 될 수 있다. 전암 병소에서 HCC의 발생 가능성을 예측하는 추가 분자 마커의 확인은 외과적 절제술 후 재발 위험이 있는 환자를 확인하는 데 도움이 될 수 있다.Like other cancers, HCC is characterized by multiple tumor progression. The damaged liver tissue evolves in the early stages to small nodular hypercellular lesions called dysplastic nodules (DNs). This pre-cancerous lesion develops into small, well-differentiated hepatocytes with an ambiguous nodular pattern and then progresses to early hepatocarcinoma (eHCC), defined as progressive hepatocellular carcinoma characterized by an interstitial appearance and frequent microvascular invasion. Based on current knowledge of the occurrence of multilevel hepatocellular carcinoma, high-risk patients are closely followed up, and diagnostic images show that the number of lesions increases with the size of the lesion and the cause is unknown. An ultrasound-guided needle biopsy is performed on these lesions. When the cancer is diagnosed histologically, it is treated. However, eHCC exhibits minimal dysplasia and lacks clear invasive or destructive growth. Therefore, even in hepatopathologists, it is often difficult to distinguish recurrent nodules, precancerous lesions, and early lesions. For this reason, the discovery of objective molecular markers that standardize histologic diagnosis of early HCC and induce appropriate therapy is eagerly awaited. In addition, the detection of biomarkers related to the accurate diagnosis of hepatocellular carcinoma may help to identify precancerous lesions that may progress to hepatocellular carcinoma and to determine surgically resectable lesions. It can help you design. Identification of additional molecular markers that predict the likelihood of HCC in precancerous lesions can help identify patients at risk for recurrence following surgical resection.
본 연구에서는 간 병리학자가 정의한 단계적 간암 조직의 임상 병리학 적 및 유전자 발현 데이터를 통합하여 잠재적 원인 유전자를 식별하기 위한 유전자 선별 전략을 수립하고자 하였다. 이를 통해 초기 단계의 HCC 의 원인으로 추정되는 유전자 10개를 선별할 수 있었다.In this study, we tried to establish a gene selection strategy to identify potential causative genes by integrating clinicopathological and gene expression data of staged hepatocarcinoma tissues defined by hepatopathologist. This led to the selection of 10 genes that were thought to be the cause of early stage HCC.
임상적 및 실험적 연구 결과에 따르면 10 개의 추정 운전 유전자 중에서 procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 및 splicing factor 3b subunit 4에 대한 장벽이 전암 병소에서 HCC를 나타낼 수 있었으며 glyphican 3, glutamine synthetase 및 heatshock protein 70과 같은 현재 HCC 진단 마커 트리오에 비해 간암 환자의 대규모 지반에서 eHCC를 진단 할 수 있었다.Clinical and experimental studies have shown that barriers to procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 and splicing factor 3b subunit 4 among the 10 putative driving genes could indicate HCC in precancerous lesions, and glyphican 3, glutamine synthetase and heatshock eHCC could be diagnosed in a large scale of liver cancer patients compared to the current HCC diagnostic marker trio such as protein 70.
생체 외 실험과 생체 내 종양 형성 분석 결과를 통해 BANF1, PLOD3 및 SF3B4 유전자의 표적 파괴는 HCC 세포의 종양 및 전이 특성을 억제한다는 것을 확인할 수 있었다. SFB4의 과도한 과잉 반응은 p27과 HCC 세포에서 슬러그(Slug)를 증강시켜 악성 간 변환 및 증식에 기여하는 상피 간엽 전이(EMT)을 억제하여 세포주기 검사점을 방해하여 이어맞추기 복합체(spliceosome)의 활동과잉을 야기하고, KLF4 종양의 성장을 진압하는 선택적 스프라이싱 변종의 생산을 가속화 시킨다는 것을 알 수 있었다.In vivo experiments and in vivo tumor formation analysis showed that target destruction of BANF1, PLOD3 and SF3B4 genes inhibited the tumor and metastatic characteristics of HCC cells. Excessive overexpression of SFB4 enhances slugging in p27 and HCC cells, inhibiting the epithelial mesenchymal transition (EMT), which contributes to the transformation and proliferation of malignant cells, interfering with the cell cycle checkpoints and activating spliceosome activity And accelerates the production of selective sprime variants that suppress the growth of KLF4 tumors.
우리의 결과는 새로운 간암 진단 마커 BANF1, PLOD3 및 SF3B가 간 종양 형성에서 간세포의 조기 악성 형질 전환에 기여하고 간 악성 종양의 분자 치료법에 대한 유망한 표적임을 제안한다.Our results suggest that the new liver cancer diagnostic markers BANF1, PLOD3 and SF3B contribute to early malignant transformation of hepatocytes in liver tumor formation and are promising targets for molecular therapy of liver malignancy.
본 발명의 목적은 초기 간암 세포에서 고발현되는 특이 유전자를 knockdown 시키는 간암의 예방 또는 치료용 약학적 조성물을 제공하는 것에 있다.It is an object of the present invention to provide a pharmaceutical composition for preventing or treating liver cancer that knockdown a specific gene highly expressed in early liver cancer cells.
1. 서열번호 5 내지 157의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는1. an siRNA comprising a sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 5 to 157 and an antisense RNA consisting of the sequence complementary thereto; or
서열번호 158 내지 310의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA를 포함하는 간암 예방 또는 치료용 약학적 조성물.A dsRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 158 to 310;
2. 위 1에 있어서,2. In the above 1,
상기 조성물은 서열번호 5 내지 12, 14 내지 19, 21, 23, 24, 26, 28 내지 34, 35 내지 37, 39 내지 41, 43, 45 내지 47, 49 내지 53, 55 내지 60, 62 내지 73, 75 내지 81, 84 내지 87, 89 내지 98, 100 내지 102, 105 내지 116, 118 내지 128, 130 내지 154, 156 및 157 의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는Wherein said composition comprises at least one of SEQ ID NOS: 5-12, 14-19, 21, 23, 24, 26, 28-34, 35-37, 39-41, 43, 45-47, 49-53, 55-60, , At least one sequence selected from the group consisting of SEQ ID NOs: 75 to 81, 84 to 87, 89 to 98, 100 to 102, 105 to 116, 118 to 128, 130 to 154, 156 and 157, RTI ID = 0.0 > siRNA < / RTI > or
서열번호 158 내지 165, 167 내지 172, 174, 176, 177, 179, 181 내지 187, 188 내지 190, 192 내지 194, 196, 198 내지 200, 202 내지 206, 208 내지 213, 215 내지 226, 228 내지 234, 237 내지 240, 242 내지 251, 253 내지 255, 258 내지 269, 271 내지 281, 283 내지 307, 309 및 310의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA를 포함하는 간암 예방 또는 치료용 약학적 조성물.181 to 187, 188 to 190, 192 to 194, 196, 198 to 200, 202 to 206, 208 to 213, 215 to 226, 228 to 188, 187 to 179, 181 to 187, A prophylactic or therapeutic agent for liver cancer comprising at least one sequence selected from the group consisting of SEQ ID NOs: 234, 237 to 240, 242 to 251, 253 to 255, 258 to 269, 271 to 281, 283 to 307, 309 and 310 A pharmaceutical composition.
3. 위 1에 있어서,3. In above 1,
상기 조성물은 서열번호 5 내지 28의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는An siRNA consisting of a sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 5 to 28 and an antisense RNA comprising a sequence complementary thereto; or
서열번호 158 내지 181의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA를 포함하는 간암 예방 또는 치료용 약학적 조성물.A dsRNA comprising at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 158 to 181;
4. 위 1에 있어서,4. In above 1,
상기 조성물은 서열번호 29 내지 55의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는An siRNA consisting of a sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 29 to 55 and an antisense RNA comprising a sequence complementary thereto; or
서열번호 182 내지 208의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA를 포함하는 간암 예방 또는 치료용 약학적 조성물.A dsRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 182 to 208;
5. 위 1에 있어서,5. In above 1,
상기 조성물은 서열번호 56 내지 120의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는An siRNA consisting of a sense RNA consisting of at least one sequence selected from the group consisting of SEQ ID NOS: 56-120 and an antisense RNA consisting of a sequence complementary thereto; or
서열번호 209 내지 273의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA를 포함하는 간암 예방 또는 치료용 약학적 조성물.A dsRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 209 to 273.
6. 위 1에 있어서,6. In above 1,
상기 조성물은 서열번호 121 내지 157의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는The composition comprising siRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of SEQ ID NOS: 121 to 157 and antisense RNA having a sequence complementary thereto; or
서열번호 274 내지 310의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA를 포함하는 간암 예방 또는 치료용 약학적 조성물.26. A pharmaceutical composition for preventing or treating liver cancer, comprising a dsRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 274 to 310.
7. 위 1 내지 6 중 어느 한 항에 있어서,7. The compound according to any one of claims 1 to 6,
상기 siRNA 또는 dsRNA는 리포좀, 리포펙타민, 덴드리머, 마이셀, 다공성 실리카 입자, 아미노클레이, 금나노입자, 자성나노입자, 그래핀, 산화그래핀, 키토산, 덱스트란, 펙틴, 이산화망간 2차원시트, PVA, 젤라틴, 실리카, 글라스입자, 프로타민, 엑소좀, 폴리에틸렌이민, N-부틸 시아노아크릴레이트, 젤폼, 젤라틴, 에탄올, 나노크리스탈, 나노튜브, 탄소나노입자, 히알루론산, 산화철, 폴리락틱산, 폴리부틸시아노아크릴레이트, 알부민, 리피드입자, 폴리에틸렌글라이콜, 폴리-L-굴루로닉 알지네이트, 폴리글리콜릭-폴리액틱산, 폴리디옥사논, 폴리글리콜산-co-카프로락톤, 폴리프로필렌 및 하이드로겔로 이루어진 군에서 선택된 적어도 하나인 담지체에 담지된 것인 조성물.The siRNA or dsRNA may be selected from the group consisting of liposomes, lipofectamines, dendrimers, micelles, porous silica particles, amino clay, gold nanoparticles, magnetic nanoparticles, graphene, oxidized graphene, chitosan, dextran, pectin, , Gelatin, silica, glass particles, protamine, exosome, polyethyleneimine, N-butylcyanoacrylate, gel foam, gelatin, ethanol, nanocrystals, nanotubes, carbon nanoparticles, hyaluronic acid, iron oxide, polylactic acid, poly Polyglycolic acid, polydioxanone, polyglycolic acid-co-caprolactone, polypropylene and polyglycolic acid, and mixtures thereof. Wherein the carrier is supported on at least one support selected from the group consisting of hydrogels.
8. 위 7에 있어서,8. In above 7,
상기 담지체는 하기 수학식 1의 흡광도의 비가 1/2가 되는 t가 20 이상인 다공성 실리카 입자인 조성물:Wherein the support is porous silica particles having a t of 20 or more, wherein the ratio of absorbance of the following formula (1) is 1/2:
[수학식 1][Equation 1]
At/A0 A t / A 0
(식 중, A0는 상기 다공성 실리카 입자 1mg/ml 현탁액 5ml를 직경 50kDa의 기공을 갖는 원통형 투과막에 넣고 측정된 다공성 실리카 입자의 흡광도이고,Wherein A 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles in a cylindrical permeable membrane having pores having a diameter of 50 kDa,
상기 투과막 외부에는 상기 투과막과 접하며, 상기 현탁액과 동일한 용매 15ml가 위치하고, 상기 투과막 내외부는 37℃에서 60rpm 수평 교반되며,15 ml of the same solvent as that of the suspension was placed on the outside of the permeable membrane, and the inside and the outside of the permeable membrane were horizontally stirred at 60 rpm at 37 ° C,
상기 현탁액의 pH는 7.4이고, At는 A0의 측정시로부터 t시간 경과 후에 측정된 다공성 실리카 입자의 흡광도임).The pH of the suspension is 7.4 and A t is the absorbance of the porous silica particles measured after passage of time from the measurement of A 0 ).
9. 위 8에 있어서, 상기 t는 40 이상인 조성물.9. The composition of claim 8, wherein t is greater than or equal to 40.
10. 위 8에 있어서, 10. In above 8,
상기 siRNA는 서열번호 28, 119 및 136의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 것이고,Wherein the siRNA comprises at least one sequence selected from the group consisting of SEQ ID NOS: 28, 119, and 136, and an antisense RNA comprising a sequence complementary thereto,
상기 dsRNA는 서열번호 181, 272 및 289로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 것인 조성물.Wherein the dsRNA comprises at least one sequence selected from the group consisting of SEQ ID NOS: 181, 272, and 289.
11. 위 8에 있어서,11. In above 8,
상기 다공성 실리카 입자는 친수성 치환기 또는 소수성 치환기를 갖는 것인 조성물.Wherein the porous silica particle has a hydrophilic substituent or a hydrophobic substituent.
12. 위 8에 있어서,12. In above 8,
상기 다공성 실리카 입자는 알데하이드기, 케토기, 카바메이트기, 설페이트기, 설포네이트기, 아미노기, 아민기, 아미노알킬기, 실릴기, 카르복실기, 술폰산기, 티올기, 암모늄기, 설프히드릴기, 포스페이트기, 에스터기, 이미드기, 싸이오이미드기, 케토기, 에터기, 인덴기, 설포닐기, 메틸포스포네이트기, 폴리에틸렌글리콜기, 치환 또는 비치환된 C1 내지 C30의 알킬기, 치환 또는 비치환된 C3 내지 C30의 사이클로알킬기, 치환 또는 비치환된 C6 내지 C30의 아릴기 및 C1 내지 C30의 에스테르기로 이루어진 군에서 선택된 적어도 하나의 친수성 치환기를 갖는 것인 조성물.The porous silica particles may be in the form of particles of an aliphatic group having an aldehyde group, a keto group, a carbamate group, a sulfate group, a sulfonate group, an amino group, an amine group, an aminoalkyl group, a silyl group, a carboxyl group, a sulfonic acid group, a thiol group, , A substituted or unsubstituted C 1 to C 30 alkyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aryl group, A substituted or unsubstituted C 6 to C 30 aryl group, and a C 1 to C 30 ester group, in the presence of at least one hydrophilic substituent selected from the group consisting of a substituted or unsubstituted C 3 to C 30 cycloalkyl group, a substituted or unsubstituted C 6 to C 30 aryl group, and a C 1 to C 30 ester group.
13. 위 8에 있어서,13. In above 8,
상기 다공성 실리카 입자는 외부 표면 또는 기공 내부가 중성의 pH에서 양전하 또는 음전하를 띠는 것인 조성물.Wherein the porous silica particles have a positive or negative charge at an external surface or pore interior at neutral pH.
14. 위 8에 있어서,14. The method of claim 8,
상기 다공성 실리카 입자는 외부 표면 및 기공 내부가 중성의 pH에서 양전하를 띠는 것인 조성물.Wherein the porous silica particles are positively charged at neutral pH at the outer surface and inside the pores.
15. 위 8에 있어서,15. The method of claim 8,
상기 다공성 실리카 입자는 평균 직경이 100 내지 400nm이고, 기공 직경이 4 내지 30nm인 것인 조성물.Wherein the porous silica particles have an average diameter of 100 to 400 nm and a pore diameter of 4 to 30 nm.
본 발명의 약학적 조성물은 초기 간암 세포에서 발현되는 유전자를 특이적으로 knockdown 시켜 간암의 발병을 막고, 간암세포의 전이와 증식을 억제함으로써 간암의 예방 및 치료의 효과를 제공한다.The pharmaceutical composition of the present invention provides a preventive and therapeutic effect of liver cancer by specifically knocking down the genes expressed in early liver cancer cells to prevent the development of liver cancer and inhibiting the metastasis and proliferation of liver cancer cells.
도 1은 실시예 1의 Hepa-1c1c7, SNU-449 세포주에 대하여 표 11의 서열을 갖는 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA들을 각각 실시예 1-2 또는 1-8의 방법에 따라 in vitro transfection 시킨 후, 각 siRNA들이 대응하는 지표인자들의 발현량을 Western blotting으로 측정한 결과를 나타낸 도이다.Fig. 1 shows siRNAs comprising the sense RNA having the sequence of Table 11 and the antisense RNA consisting of the complementary sequence to the Hepa-1c1c7 and SNU-449 cell strains of Example 1, , And then the amount of expression of corresponding indicator factors of each siRNA was measured by Western blotting.
도 2는 실시예 1-1의 SNU-449 세포주에 대하여 표 12의 서열을 갖는 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA들을 실시예 1-2의 방법에 따라 in vitro transfection 시킨 후, 각 siRNA들이 대응하는 지표인자들의 migration과 invasion 반응을 실시예 1-5의 방법에 따라 분석하였고, 실시예 1-6의 방법에 따라 scratch wound healing 능력을 분석한 결과를 나타낸 도이다.FIG. 2 shows the results of in vitro transfection of the SNU-449 cell line of Example 1-1 with siRNAs consisting of sense RNA having the sequence of Table 12 and antisense RNA of the complementary sequence according to the method of Example 1-2 , Migration and invasion responses of the corresponding surface factors of each siRNA were analyzed according to the methods of Examples 1-5, and the results of analysis of the scratch wound healing ability according to the method of Example 1-6 are shown.
도 3(A)는 실시예 1의 SNU-449 세포주에 표 12의 서열을 갖는 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA들을 실시예 1-2의 방법에 따라 in vitro transfection 시킨 후, 각 siRNA들이 대응하는 지표인자들의 발현량과 상기 EMT 조절 단백질들의 발현량을 실시예 1-9의 방법에 따라 분석한 결과를 나타낸 도이고, 도 3(B)는 상기 (A)의 transfection된 cell을 흉선 결실 쥐(athymic nude mice)의 피하에 주입한 후, 간 종양의 크기와 쥐의 생존률을 분석한 결과를 나타낸 도이다.FIG. 3 (A) shows the results of in vitro transfection of SNU-449 cell line of Example 1 with siRNAs consisting of sense RNA having the sequence of Table 12 and antisense RNA consisting of the complementary sequence according to the method of Example 1-2 FIG. 3 (B) is a graph showing the results of analysis of the expression level of the EMT regulatory proteins and the expression level of the corresponding indicator factors of the respective siRNAs according to the method of Example 1-9, In a subcutaneous tissue of athymic nude mice, and then analyzed the size of the liver tumor and the survival rate of the mouse.
도 4(A)는 표 13의 서열을 갖는 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA들을 실시예 1-8의 방법에 따라 in vivo transfection 시켰고, 그 과정과 초음파 이미지 및 시간의 경과에 따른 종양의 개수를 나타낸 도이고, 도 4(B)는 다공성 나노입자에 담지된 상기 siRNA들의 각 지표 유전자들의 발현 억제수준을 실시예 1-9의 방법으로 분석한 결과를 나타낸 도이다.4 (A) shows siRNAs consisting of sense RNA having the sequence of Table 13 and antisense RNA consisting of the complementary sequence thereof in vivo according to the method of Example 1-8, and the process, ultrasound image, FIG. 4 (B) is a graph showing the results of analysis of inhibitory levels of the expression levels of the respective indicator genes of the siRNAs carried on the porous nanoparticles by the method of Example 1-9. FIG.
도 5는 다공성 실리카 입자의 현미경 사진이다.5 is a photomicrograph of a porous silica particle.
도 6은 다공성 실리카 입자의 현미경 사진이다.6 is a photomicrograph of the porous silica particles.
도 7은 다공성 실리카 입자의 제조 공정 중의 소기공 입자의 현미경 사진이다.FIG. 7 is a microphotograph of a small pore particle during the production of porous silica particles.
도 8은 소기공 입자의 현미경 사진이다.8 is a micrograph of the small pore particle.
도 9는 다공성 실리카 입자의 생분해성을 확인할 수 있는 현미경 사진이다.9 is a photomicrograph showing the biodegradability of the porous silica particles.
도 10은 원통형 투과막을 구비한 튜브이다.10 is a tube having a cylindrical permeable membrane.
도 11은 다공성 실리카 입자의 시간 경과에 따른 흡광도 감소 결과를 나타낸 도이다.11 is a graph showing the results of absorbance reduction with time of the porous silica particles.
도 12는 다공성 실리카 입자의 시간 경과에 따른 입경별 흡광도 감소 결과를 나타낸 도이다.12 is a graph showing the results of absorbance reduction by particle size of porous silica particles over time.
도 13은 다공성 실리카 입자의 시간 경과에 따른 기공 직경별 흡광도 감소 결과를 나타낸 도이다.13 is a graph showing the results of absorbance reduction of pore diameters with time of porous silica particles.
도 14는 다공성 실리카 입자의 시간 경과에 따른 환경의 pH별 흡광도 감소 결과를 나타낸 도이다.14 is a graph showing the results of absorbance reduction of pH of the environment over time of the porous silica particles.
도 15는 다공성 실리카 입자의 시간 경과에 따른 흡광도 감소 결과를 나타낸 도이다.15 is a graph showing the results of absorbance reduction with time of the porous silica particles.
도 16은 다공성 실리카 입자의 생리활성물질 방출을 확인하는 튜브이다.16 is a tube for confirming release of physiologically active substance from porous silica particles.
도 17은 다공성 실리카 입자에 담지된 생리활성물질의 시간 경과에 따른 방출 정도를 나타낸 도이다.17 is a graph showing the release of the physiologically active substance carried on the porous silica particles with time.
도 18은 다공성 실리카 입자에 siRNA를 담지하여 생쥐 내에서 siRNA의 방출을 확인한 현미경 사진이다.18 is a photomicrograph showing the release of siRNA in mice by loading siRNA onto porous silica particles.
본 발명에서 사용되는 용어에 대한 정의는 이하와 같다.The terms used in the present invention are defined as follows.
"siRNA"는 RNA 방해 또는 유전자 사일런싱을 매개할 수 있는 핵산 분자를 의미한다.　siRNA는 표적 유전자의 발현을 억제할 수 있기 때문에 효율적인 유전자 녹다운 방법으로서 또는 유전자치료 방법으로 제공된다. siRNA　분자는 센스 가닥(표적 유전자의 mRNA 서열에 상응하는(corresponding) 서열)과 안티센스 가닥(표적 유전자의 mRNA 서열에 상보적인 서열)이 서로 반대쪽에 위치하여 이중쇄를 이루는 구조를 가질 수 있다. 또한, siRNA　분자는, 자기-상보성(self-complementary) 센스 및 안티센스 가닥을 가지는 단일쇄 구조를 가질 수 있다.　siRNA는 RNA끼리 짝을 이루는 이중사슬 RNA 부분이 완전히 쌍을 이루는 것에 한정되지 않고 미스매치(대응하는 염기가 상보적이지 않음), 벌지(일방의 사슬에 대응하는 염기가 없음) 등에 의하여 쌍을 이루지 않는 부분이 포함될 수 있다. siRNA　말단 구조는 표적 유전자의 발현을 RNAi(RNA interference) 효과에 의하여 억제할 수 있는 것이면 평활(blunt) 말단 혹은 점착(cohesive) 말단 모두 가능하다. 점착 말단 구조는 3'-말단 돌출 구조와 5'-말단 돌출 구조 모두 가능하다. 또한, siRNA　분자는 자기-상보성 센스 및 안티센스 가닥 사이에 짧은 뉴클레오타이드 서열(예컨대, 약 5-15 nt)이 삽입된 형태를 가질 수 있으며, 이 경우 뉴클레오타이드 서열의 발현에 의해 형성된　siRNA　분자는 분자내 혼성화에 의하여 헤어핀 구조를 형성하게 되며, 전체적으로는 스템-앤드-루프 구조를 형성하게 된다. 이 스템-앤드-루프 구조는 인 비트로(in vitro) 또는 인 비보(in vivo)에서 프로세싱되어 RNAi를 매개할 수 있는 활성의　siRNA　분자를 생성한다." siRNA " means a nucleic acid molecule capable of mediating RNA interference or gene silencing. Since siRNA can inhibit expression of a target gene, it is provided as an efficient gene knockdown method or as a gene therapy method. The siRNA molecule may have a structure in which the sense strand (the corresponding sequence corresponding to the mRNA sequence of the target gene) and the antisense strand (the sequence complementary to the mRNA sequence of the target gene) are located on opposite sides to form a double strand. In addition, siRNA molecules may have a single stranded structure with self-complementary sense and antisense strands. The siRNA is not limited to a complete pair of double-stranded RNA portions that are paired with each other, but is paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) May be included. The siRNA terminal structure is capable of blunt or cohesive termini as long as it can inhibit the expression of the target gene by RNA interference (RNAi) effect. The sticky end structure can be a 3'-end protruding structure and a 5'-end protruding structure. In addition, siRNA molecules may have a form in which a short nucleotide sequence (e.g., about 5-15 nt) is inserted between the self-complementary sense and antisense strands, in which case the siRNA molecule formed by the expression of the nucleotide sequence To form a hairpin structure, which in turn forms a stem-and-loop structure. This stem-and-loop structure is processed in vitro or in vivo to produce siRNA molecules that are capable of mediating RNAi.
"dsRNA"는 siRNA의 전구체 분자로서, 표적세포의 DICER 효소(Ribonuclease III)를 포함하는 RISC 복합체와 만나 siRNA로 절단되고, 이 과정에서 RNAi가 발생한다. dsRNA는 siRNA 보다 수 뉴클레오티드 만큼 긴 서열을 갖고, 센스 가닥(표적 유전자의 mRNA 서열에 상응하는(corresponding) 서열)과 안티센스 가닥(표적 유전자의 mRNA 서열에 상보적인 서열)이 서로 반대쪽에 위치하여 이중쇄를 이루는 구조를 가질 수 있다.The "dsRNA" is a siRNA precursor molecule that meets the RISC complex containing the DICER enzyme of the target cell (Ribonuclease III) and is cleaved into siRNA, which in turn generates RNAi. The dsRNA has a sequence that is several nucleotides longer than the siRNA, and the sense strand (the corresponding sequence corresponding to the mRNA sequence of the target gene) and the antisense strand (the sequence complementary to the mRNA sequence of the target gene) As shown in FIG.
"핵산"은 임의의 DNA 또는 RNA, 예를 들어, 조직 샘플에 존재하는 염색체, 미토콘드리아, 바이러스 및/또는 세균 핵산을 포함하는 의미이다. 이중가닥 핵산 분자의 하나 또는 두개 모두의 가닥을 포함하고, 무손상 핵산 분자의 임의의 단편 또는 일부를 포함한다.&Quot; Nucleic acid " is meant to include any DNA or RNA, such as chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in a tissue sample. Includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of the intact nucleic acid molecule.
"유전자"는 단백질 코딩 또는 전사시에 또는 다른 유전자 발현의 조절시에 기능적 역할을 갖는 임의의 핵산 서열 또는 그의 일부를 의미한다. 유전자는 기능적 단백질을 코딩하는 모든 핵산 또는 단백질을 코딩 또는 발현하는 핵산의 일부만으로 이루어질 수 있다. 핵산 서열은 엑손, 인트론, 개시 또는 종료 영역, 프로모터 서열, 다른 조절 서열 또는 유전자에 인접한 특유한 서열 내에 유전자 이상을 포함할 수 있다.&Quot; Gene " means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.
"유전자 발현"이란 용어는 일반적으로 생물학적 활성이 있는 폴리펩티드가 DNA 서열로부터 생성되고 세포에서 생물학적 활성을 나타내는 세포 과정을 의미한다. 그런 의미로, 유전자 발현은 전사 및 해독 과정을 포함할 뿐만 아니라, 유전자 또는 유전자 산물의 생물학적 활성에 영향을 끼칠 수 있는 전사후 및 해독후 과정을 포함한다. 상기 과정들은 RNA 합성, 가공 및 수송뿐만 아니라, 폴립펩티드 합성, 수송 및 폴리펩티드의 해독후 변형을 포함하지만, 이들에 국한되는 것은 아니다. 단백질 산물을 암호화하지 않는 유전자, 예컨대, siRNA 유전자의 경우에, "유전자 발현"이란 용어는 전구체 siRNA가 유전자로부터 생성되는 과정을 의미한다. 통상, 상기 과정은, 단백질 암호 유전자에 대해 RNA 폴리머라제 II에 의해 유도되는 전사와는 달리, siRNA 유전자의 전사 산물이 해독되어 단백질을 생성하지 않지만, 전사로 언급된다. 그럼에도 불구하고, siRNA 유전자로부터 성숙 siRNA의 생성은 그 용어가 본원에 사용되는 대로 "유전자 발현"이란 용어에 의해 포함된다The term " gene expression " generally refers to a cellular process in which a biologically active polypeptide is produced from a DNA sequence and exhibits biological activity in the cell. In this sense, gene expression includes post-transcriptional and post-transcriptional processes that not only involve transcription and translation processes, but can also affect the biological activity of the gene or gene product. Such procedures include, but are not limited to, RNA synthesis, processing and transport as well as polyp peptide synthesis, transport and post-translational modification of the polypeptide. In the case of a gene that does not encode a protein product, such as an siRNA gene, the term " gene expression " refers to a process in which a precursor siRNA is produced from a gene. Normally, this process is referred to as transcription, although the transcription product of the siRNA gene is not translated to produce a protein, unlike the transcription induced by RNA polymerase II on the protein coding gene. Nevertheless, the generation of mature siRNAs from siRNA genes is encompassed by the term " gene expression " as that term is used herein
"표적 유전자 (target gene)"란 용어는 본원에 개시되는 주제의 방법 및 조성물을 사용하여 조절하기 위해 표적으로 삼는 유전자를 의미한다. 그러므로, 표적 유전자는 그 발현 레벨이 mRNA 또는 폴리펩티드 레벨로 siRNA에 의해 하향 조절되는 핵산 서열을 포함한다. 유사하게, "표적 RNA" 또는 "표적 mRNA"란 용어는 siRNA가 결합하여 표적 유전자의 발현의 조절을 유도할 표적 유전자의 전사체를 의미한다.The term " target gene " refers to a gene that is targeted for modulation using methods and compositions of the subject matter disclosed herein. Thus, the target gene comprises a nucleic acid sequence whose expression level is down-regulated by the siRNA to an mRNA or polypeptide level. Similarly, the term " target RNA " or " target mRNA " refers to a transcript of a target gene that will bind siRNA and induce modulation of expression of the target gene.
"전사 (transcription)"란 용어는 유전자의 암호 서열에 존재하는 구조 정보의 RNA로서 발현을 유도하는 유전자와 RNA 폴리머라제의 상호작용을 포함하는 세포 과정을 의미한다.The term " transcription " refers to a cellular process involving the interaction of an RNA polymerase with a gene that induces expression as RNA of structural information present in the coding sequence of the gene.
"하향 조절(down-regulation)"이라는 표현은, 정상조직세포에 비하여, 활성화된 세포에서 세포 내 전사(gene transcription) 또는 번역(gene translation)에 의해서 특정 유전자의 mRNA로의 발현 또는 단백질로 발현량이 현저하게 감소된 것을 의미한다.The term " down-regulation " refers to the expression of a specific gene into mRNA or the expression level of a protein by an intracellular transcription or gene translation in an activated cell, .
"치료"는 이롭거나 바람직한 임상적 결과를 수득하기 위한 접근을 의미한다. 본 발명의 목적을 위해서, 이롭거나 바람직한 임상적 결과는 비제한적으로, 증상의 완화, 질병 범위의 감소, 질병 상태의 안정화 (즉, 악화되지 않음), 질병 진행의 지연 또는 속도의 감소, 질병 상태의 개선 또는 일시적 완화 및 경감 (부분적이거나 전체적으로), 검출가능하거나 또는 검출되지 않거나의 여부를 포함한다. 또한, "치료"는 치료를 받지 않았을 때 예상되는 생존율과 비교하여 생존율을 늘이는 것을 의미할 수도 있다. 치료는 치료학적 치료 및 예방적 또는 예방조치 방법 모두를 가리킨다. 상기 치료들은 예방되는 장애뿐만 아니라 이미 발생한 장애에 있어서 요구되는 치료를 포함한다.&Quot; Treatment " means an approach to obtaining beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of the disease state, (Either partially or totally), detectable or undetected, whether or not an improvement or temporary relief or reduction Also, " treatment " may mean increasing the survival rate compared to the expected survival rate when not receiving treatment. Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include treatments required for disorders that have already occurred as well as disorders to be prevented.
"예방"은 관련 질환의 발병을 억제 또는 지연시키는 모든 행위를 의미한다. 본원의 조성물은 초기 증상, 또는 나타나기 전에 투여할 경우 관련 질환을 예방할 수 있다는 것은 당업자에게 자명할 것이다.&Quot; Prevention " means any act that inhibits or delays the onset of a related disease. It will be apparent to those skilled in the art that the compositions herein may prevent early onset symptoms, or related disorders when administered prior to appearance.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 서열번호 5 내지 157의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는The present invention provides a siRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 5 to 157 and an antisense RNA comprising a sequence complementary thereto; or
서열번호 158 내지 310의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA를 포함하는 간암 예방 또는 치료용 약학적 조성물을 제공한다.A dsRNA comprising at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 158 to 310. The present invention also provides a pharmaceutical composition for preventing or treating liver cancer.
본 발명의 siRNA 또는 dsRNA는 인간을 포함한 동물, 예를 들어 원숭이(monkeys), 돼지(pigs), 말(horses), 소(cows), 양(sheeps), 개(dogs), 고양이(cats), 생쥐(mice), 토끼(rabbits) 등으로부터 유래할 수 있으며, 바람직하게는 인간 유래의 것일 수 있다. The siRNA or dsRNA of the present invention can be used in an animal including humans, such as monkeys, pigs, horses, cows, sheeps, dogs, cats, Mice, rabbits, and the like, and preferably may be derived from humans.
본 발명의 siRNA 또는 dsRNA는 이를 구성하는 핵산 분자의 작용성 등가물, 예를 들어, 본 발명의 siRNA 또는 dsRNA의 일부 염기서열이 결실(deletion), 치환(substitution) 또는 삽입(insertion)에 의해 변형되었지만, 본 발명의 siRNA 또는 dsRNA와 기능적으로 동일한 작용을 할 수 있는 변이체(variants)를 포함하는 개념이다.The siRNA or dsRNA of the present invention may be modified by deletion, substitution or insertion of a functional equivalent of the nucleic acid molecule constituting it, for example, a part of the base sequence of the siRNA or dsRNA of the present invention , And variants capable of functionally functioning with the siRNA or dsRNA of the present invention.
본 발명의 siRNA 또는 dsRNA는 표준 분자 생물학 기술, 예를 들어 화학적 합성 방법 또는 재조합 방법을 이용하여 분리 또는 제조하거나, 시판되는 것을 사용할 수 있다. 또한, 본 발명의 조성물은 본 발명의 siRNA 또는 dsRNA 자체뿐만 아니라, 세포 내에서 본 발명의 siRNA 또는 dsRNA의 발현율을 증가시킬 수 있는 기타의 물질, 예를 들어 화합물, 천연물, 신규 단백질 등을 포함할 수 있다.The siRNA or dsRNA of the present invention can be isolated or prepared using standard molecular biology techniques, such as chemical synthesis methods or recombinant methods, or commercially available. In addition, the composition of the present invention includes siRNA or dsRNA itself of the present invention, as well as other substances capable of increasing the expression rate of the siRNA or dsRNA of the present invention in a cell, for example, a compound, a natural product, .
한편, 본 발명의 siRNA 또는 dsRNA는 세포 내 발현을 위한 벡터에 포함되어 제공될 수 있다.Meanwhile, the siRNA or dsRNA of the present invention can be provided as a vector for intracellular expression.
본 발명의 siRNA 또는 dsRNA는 DNA 및 DEAE-덱스트란의 복합체, DNA 및 핵 단백질의 복합체, DNA 및 지질의 복합체 등의 다양한 형질전환 기술을 이용하여 세포 내로 도입시킬 수 있는데, 이를 위해 본 발명의 siRNA 또는 dsRNA는 세포 내로의 효율적인 도입을 가능하게 하는 전달체 내에 포함된 형태일 수 있다. 상기 전달체는 바람직하게는 벡터이며, 바이러스 벡터 및 비바이러스 벡터 모두 사용 가능하다. 바이러스 벡터(viral vector)로서 예를 들면, 렌티바이러스(lentivirus), 레트로바이러스(retrovirus), 아데노바이러스(adenovirus), 허피스바이러스(herpes virus) 및 아비폭스바이러스(avipox virus) 벡터 등을 사용할 수 있으며, 바람직하게는 렌티바이러스 벡터이지만, 이에 제한되는 것은 아니다. 렌티바이러스는 레트로바이러스의 일종으로 핵공(nucleopore)이나 완전한 핵막으로의 능동도입을 가능하게 하는 사전-통합 복합체(바이러스 "쉘(shell)")의 친핵성으로 인해 분열 세포 뿐만 아니라 미분열 세포도 감염시킬 수 있는 특징이 있다.The siRNA or dsRNA of the present invention can be introduced into cells using various transformation techniques such as a complex of DNA and DEAE-dextran, a complex of DNA and nuclear protein, a complex of DNA and lipid, etc. To this end, Or the dsRNA may be in a form that is contained within a carrier that enables efficient introduction into the cell. The carrier is preferably a vector, and both viral vectors and non-viral vectors are usable. As the viral vector, for example, lentivirus, retrovirus, adenovirus, herpes virus and avipox virus vector can be used, Preferably a lentiviral vector, but is not limited thereto. Lentiviruses are a type of retrovirus that is not only a mitotic cell but also a mitotic cell due to the nucleophilicity of a nucleopore or a pre-integrated complex (a virus "shell") that allows active incorporation into a complete nuclear membrane There is a feature that can be made.
또한, 본 발명의 siRNA 또는 dsRNA를 포함하는 벡터는 선별마커를 추가로 포함하는 것이 바람직하다. 상기 "선별마커(selection marker)"란 본 발명의 siRNA 또는 dsRNA가 도입된 세포의 선별을 용이하게 하기 위한 것이다. 상기 벡터에서 사용할 수 있는 선별마커로는 벡터의 도입 여부를 용이하게 검출 또는 측정할 수 있는 유전자라면, 특별히 한정되지 않으나, 대표적으로 약물 내성, 영양 요구성, 세포 독성제에 대한 내성 또는 표면 단백질의 발현과 같은 선택가능 표현형을 부여하는 마커들, 예를 들어 GFP(녹색 형광 단백질), 퓨로마이신(puromycin), 네오마이신(Neomycin: Neo), 하이그로마이신(hygromycin: Hyg), 히스티디놀 디하이드로게나제(histidinol dehydrogenase gene: hisD) 및 구아닌 포스포리보실트랜스퍼라제(guanine phosphosribosyltransferase: Gpt) 등이 있으며, 바람직하게는 GFP(녹색 형광 단백질) 및 퓨로마이신 마커를 사용할 수 있다.In addition, the vector comprising siRNA or dsRNA of the present invention preferably further comprises a selection marker. The " selection marker " is intended to facilitate screening of cells into which siRNA or dsRNA of the present invention has been introduced. The selectable marker used in the vector is not particularly limited as long as it is a gene capable of easily detecting or measuring the introduction of a vector. Typically, the selectable marker is selected from the group consisting of resistance to drugs, tolerance to cytotoxic agents, Such as GFP (green fluorescent protein), puromycin, neomycin (Neo), hygromycin (Hyg), histidine dehydrogenase Histidinol dehydrogenase gene hisD) and guanine phosphosribosyltransferase (Gpt). Preferably, GFP (green fluorescent protein) and puromycin marker can be used.
본 발명은 조성물은 하기 표 1의 서열번호 5 내지 28의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는 하기 표 1의 서열번호 158 내지 181의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA를 포함한다.The present invention relates to a composition comprising siRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 5 to 28 of Table 1 and antisense RNA comprising the sequence complementary thereto; Or at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 158 to 181 in Table 1 below.
하기 표 1의 서열번호 5 내지 28의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는 하기 표 1의 서열번호 158 내지 181의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA는 인간 BANF1 유전자의 variant 1 서열(서열번호 1)을 타겟하는 것으로, 인간 BANF1 유전자 variant 1의 발현을 RNAi를 통해 억제함으로써 간암의 예방 또는 치료 효과를 가진다.SiRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 5 to 28 of Table 1 and antisense RNA consisting of the complementary sequence thereof; Or at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 158 to 181 in Table 1 targets the variant 1 sequence (SEQ ID NO: 1) of the human BANF1 gene, and the expression of the human BANF1 gene variant 1 RNAi, thereby preventing or treating liver cancer.
Target sequence 5:5'- CAA GAA GCT GGA GGA AAG-3'(Position in gene sequence: 601)Target sequence 5: 5'-CAA GAA GCT GGA GGA AAG-3 '(Position in gene sequence: 601) GC content: 45.0%GC content: 45.0%
서열번호 5 Sense strand:5'-CAA GAA GCU GGA GGA AAG UU-3'SEQ ID NO: 5 Sense strand: 5'-CAA GAA GCU GGA GGA AAG UU-3 '
서열번호 326 Antisense strand:5'- CUU UCC UCC AGC UUC UUG UU-3'SEQ ID NO: 326 Antisense strand: 5'- CUU UCC UCC AGC UUC UUG UU-3 '
서열번호 158 dsRNA:5'- CAA GAA GCU GGA GGA AAG UU UCU AAA G-3'SEQ ID NO: 158 dsRNA: 5'-CAA GAA GCU GGA GGA AAG UU UCU AAA G-3 '
Target sequence 6:5'-GAA AGA TGA AGA CCT CTT CC-3'(Position in gene sequence: 667)Target sequence 6: 5'-GAA AGA TGA AGA CCT CTT-3 '(Position in gene sequence: 667) GC content: 40.9%GC content: 40.9%
서열번호 6 Sense strand:5'-GAA AGA UGA AGA CCU CUU CCU U-3'SEQ ID NO: 6 Sense strand: 5'-GAA AGA UGA AGA CCU CUU CCU U-3 '
서열번호 327 Antisense strand:5'-GGA AGA GGU CUU CAU CU UCU U-3'SEQ ID NO: 327 Antisense strand: 5'-GGA AGA GGU CUU CAU CU UCU U-3 '
서열번호 159 dsRNA:5'- GAA AGA UGA AGA CCU CUU CCU UUC UAA AG-3'SEQ ID NO: 159 dsRNA: 5'-GAA AGA UGA AGA CCU CUU CCU UUC UAA AG-3 '
Target sequence 7:5'-GGA ATG GCT GAA AGA CAC TT-3'(Position in gene sequence: 688)Target sequence 7: 5'-GGA ATG GCT GAA AGA CAC TT-3 '(Position in gene sequence: 688) GC content: 40.9%GC content: 40.9%
서열번호 7 Sense strand:5'-GGA AUG GCU GAA AGA CAC UUU U-3'SEQ ID NO: 7 Sense strand: 5'-GGA AUG GCU GAA AGA CAC UUU U-3 '
서열번호 328 Antisense strand:5'-AAG UGU CUU UCA GCC AUU CCU U-3'SEQ ID NO: 328 Antisense strand: 5'-AAG UGU CUU UCA GCC AUU CCUI-3 '
서열번호 160 dsRNA:5'- GGA AUG GCU GAA AGA CAC UUU UUC UAA AG-3'SEQ ID NO: 160 dsRNA: 5'-GGA AUG GCU GAA AGA CAC UUU UUC UAA AG-3 '
Target sequence 8:5'-CCA GTG TTC CCA GTT CCC-3'(Position in gene sequence: 1)Target sequence 8: 5'-CCA GTG TTC CCA GTT CCC-3 '(Position in gene sequence: 1) GC content: 55.0%GC content: 55.0%
서열번호 8 Sense strand:5'-CCA GUG UUC CCA GUU CCC UU-3'SEQ ID NO: 8 Sense strand: 5'-CCA GUG UUC CCA GUU CCC UU-3 '
서열번호 329 Antisense strand:5'-GGG AAC UGG GAA CAC UGG UU-3'SEQ ID NO: 329 Antisense strand: 5'-GGG AAC UGG GAA CAC UGG UU-3 '
서열번호 161 dsRNA:5'-CCA GUG UUC CCA GUU CCC UU UCU AAA G-3'SEQ ID NO: 161 dsRNA: 5'-CCA GUG UUC CCA GUU CCC UU UCU AAA G-3 '
Target sequence 9:5'-CCA GTC CAA CTG CGA GGA-3'(Position in gene sequence: 19)Target sequence 9: 5'-CCA GTC CAA CTG CGA GGA-3 '(Position in gene sequence: 19) GC content: 55.0%GC content: 55.0%
서열번호 9 Sense strand:5'- CCA GUC CAA CUG CGA GGA UU-3'SEQ ID NO: 9 Sense strand: 5'-CCA GUC CAA CUG CGA GGA UU-3 '
서열번호 330 Antisense strand:5'- UCCUCGCAGUUGGACUGG UU-3'SEQ ID NO: 330 Antisense strand: 5'- UCCUCGCAGUUGGACUGG UU-3 '
서열번호 162 dsRNA:5'- CCAGUCCAACUGCGAGGA UU UCU AAA G-3'SEQ ID NO: 162 dsRNA: 5'-CCAGUCCAACUGCGAGGA UU UCU AAA G-3 '
Target sequence 10:5'-CGA CGT GAG TCT GAG TCT-3'(Position in gene sequence: 41)Target sequence 10: 5'-CGA CGT GAG TCT GAG TCT-3 '(Position in gene sequence: 41) GC content: 50.0%GC content: 50.0%
서열번호 10 Sense strand:5'- CGA CGU GAG UCU GAG UCU UU-3'SEQ ID NO: 10 Sense strand: 5'-CGA CGU GAG UCU GAG UCU UU-3 '
서열번호 331 Antisense strand:5'- AGACUCAGACUCACGUCG UU-3'SEQ ID NO: 331 Antisense strand: 5'- AGACUCAGACACACGUCG UU-3 '
서열번호 163 dsRNA:5'- CGACGUGAGUCUGAGUCU UU UCU AAA G-3'SEQ ID NO: 163 dsRNA: 5'-CGACGUGAGUCUGAGUCUU UU UCU AAA G-3 '
Target sequence 11:5'-GTC CGT CTT CTA ACT CTT-3'(Position in gene sequence: 116)Target sequence 11: 5'-GTC CGT CTT CTA ACT CTT-3 '(Position in gene sequence: 116) GC content: 40.0%GC content: 40.0%
서열번호 11 Sense strand:5'- GUC CGU CUU CUA ACU CUU UU-3'SEQ ID NO: 11 Sense strand: 5'- GUC CGU CUU CUA ACU CUU UU-3 '
서열번호 332 Antisense strand:5'- AAGAGUUAGAAGACGGAC UU-3'SEQ ID NO: 332 Antisense strand: 5'- AAGAGUUAGAAGACGGAC UU-3 '
서열번호 164 dsRNA:5'- GUCCGUCUUCUAACUCUU UU UCU AAA G-3'SEQ ID NO: 164 dsRNA: 5'- GUCCGUCUUCUAACUCUU UU UCU AAA G-3 '
Target sequence 12:5'-CGT CAA GCC TAA GTC CTT-3'(Position in gene sequence: 149)Target sequence 12: 5'-CGT CAA GCC TAA GTC CTT-3 '(Position in gene sequence: 149) GC content: 45.0%GC content: 45.0%
서열번호 12 Sense strand:5'- CGUCAAGCCUAAGUCCUU UU-3'SEQ ID NO: 12 Sense strand: 5'- CGUCAAGCCUAAGUCCUU UU-3 '
서열번호 333 Antisense strand:5'- AAGGACUUAGGCUUGACG UU-3'SEQ ID NO: 333 Antisense strand: 5'- AAGGACUUAGGCUUGACG UU-3 '
서열번호 165 dsRNA:5'- CGUCAAGCCUAAGUCCUU UU UCU AAA G-3'SEQ ID NO: 165 dsRNA: 5'- CGUCAAGCCUAAGUCCUU UU UCU AAA G-3 '
Target sequence 13:5'-GCA GAG AAA GGA AGT CCT-3'(Position in gene sequence: 185)Target sequence 13: 5'-GCA GAG AAA GGA AGT CCT-3 '(Position in gene sequence: 185) GC content: 45.0%GC content: 45.0%
서열번호 13 Sense strand:5'- GCAGAGAAAGGAAGUCCU UU-3'SEQ ID NO: 13 Sense strand: 5'-GCAGAGAAAGGAAGUCCU UU-3 '
서열번호 334 Antisense strand:5'- AGGACUUCCUUUCUCUGC UU-3'SEQ ID NO: 334 Antisense strand: 5'- AGGACUUCCUUUCUCUGC UU-3 '
서열번호 166 dsRNA:5'- GCAGAGAAAGGAAGUCCU UU UCU AAA G-3'SEQ ID NO: 166 dsRNA: 5'-GCAGAGAAAGGAAGUCCU UU UCU AAA G-3 '
Target sequence 14:5'-CGA GAA GCG AGA CCT TAG-3'(Position in gene sequence: 234)Target sequence 14: 5'-CGA GAA GCG AGA CCT TAG-3 '(Position in gene sequence: 234) GC content: 50.0%GC content: 50.0%
서열번호 14 Sense strand:5'- CGAGAAGCGAGACCUUAG UU-3'SEQ ID NO: 14 Sense strand: 5'-CGAGAAGCGAGACCUUAG UU-3 '
서열번호 335 Antisense strand:5'- CUAAGGUCUCGCUUCUCG UU-3'SEQ ID NO: 335 Antisense strand: 5'- CUAAGGUCUCGCUUCUCG UU-3 '
서열번호 167 dsRNA:5'- CGAGAAGCGAGACCUUAG UU UCU AAA G-3'SEQ ID NO: 167 dsRNA: 5'-CGAGAAGCGAGACCUUAG UU UCU AAA G-3 '
Target sequence 15:5'-CCT CAA CTC TAT AGC TCT-3'(Position in gene sequence: 319)Target sequence 15: 5'-CCT CAA CTC TAT AGC TCT-3 '(Position in gene sequence: 319) GC content: 40.0%GC content: 40.0%
서열번호 15 Sense strand:5'- CCUCAACUCUAUAGCUCU UU-3'SEQ ID NO: 15 Sense strand: 5'- CCUCAACUCUAUAGCUCU UU-3 '
서열번호 336 Antisense strand:5'- AGAGCUAUAGAGUUGAGG UU-3'SEQ ID NO: 336 Antisense strand: 5'- AGAGCUAUAGAGUUGAGG UU-3 '
서열번호 168 dsRNA:5'- CCUCAACUCUAUAGCUCU UU UCU AAA G-3'SEQ ID NO: 168 dsRNA: 5'-CCUCAACUCUAUAGCUCU UU UCU AAA G-3 '
Target sequence 16:5'-CTA GTG GCT TGA GGT ATC-3'(Position in gene sequence: 423)Target sequence 16: 5'-CTA GTG GCT TGA GGT ATC-3 '(Position in gene sequence: 423) GC content: 45.0%GC content: 45.0%
서열번호 16 Sense strand:5'- CUAGUGGCUUGAGGUAUC UU-3'SEQ ID NO: 16 Sense strand: 5'- CUAGUGGCUUGAGGUAUC UU-3 '
서열번호 337 Antisense strand:5'- GAUACCUCAAGCCACUAG UU-3'SEQ ID NO: 337 Antisense strand: 5'- GAUACCUCAAGCCACUAG UU-3 '
서열번호 169 dsRNA:5'- CUAGUGGCUUGAGGUAUC UU UCU AAA G-3'SEQ ID NO: 169 dsRNA: 5'- CUAGUGGCUUGAGGUAUC UU UCU AAA G-3 '
Target sequence 17:5'-GGA TTA AGC CTG ATC AAG-3'(Position in gene sequence: 491)Target sequence 17: 5'-GGA TTA AGC CTG ATC AAG-3 '(Position in gene sequence: 491) GC content: 40.0%GC content: 40.0%
서열번호 17 Sense strand:5'- GGAUUAAGCCUGAUCAAG UU-3'SEQ ID NO: 17 Sense strand: 5'- GGAUUAAGCCUGAUCAAG UU-3 '
서열번호 338 Antisense strand:5'- CUUGAUCAGGCUUAAUCC UU-3'SEQ ID NO: 338 Antisense strand: 5'- CUUGAUCAGGCUUAAUCC UU-3 '
서열번호 170 dsRNA:5'- GGAUUAAGCCUGAUCAAG UU UCU AAA G-3'SEQ ID NO: 170 dsRNA: 5'-GGAUUAAGCCUGAUCAAG UU UCU AAA G-3 '
Target sequence 18:5'-GAC TGC TTC GGA TGC CTT-3'(Position in gene sequence: 734)Target sequence 18: 5'-GAC TGC TTC GGA TGC CTT-3 '(Position in gene sequence: 734) GC content: 50.0%GC content: 50.0%
서열번호 18 Sense strand:5'- GACUGCUUCGGAUGCCUU UU-3'SEQ ID NO: 18 Sense strand: 5'- GACUGCUUCGGAUGCCUU UU-3 '
서열번호 339 Antisense strand:5'- AAGGCAUCCGAAGCAGUC UU-3'SEQ ID NO: 339 Antisense strand: 5'- AAGGCAUCCGAAGCAGUC UU-3 '
서열번호 171 dsRNA:5'- GACUGCUUCGGAUGCCUU UU UCU AAA G-3'SEQ ID NO: 171 dsRNA: 5'- GACUGCUUCGGAUGCCUU UU UCU AAA G-3 '
Target sequence 19:5'-CCT TCT TGT GAT GCT CTC-3'(Position in gene sequence: 768)Target sequence 19: 5'-CCT TCT TGT GAT GCT CTC-3 '(Position in gene sequence: 768) GC content: 45.0%GC content: 45.0%
서열번호 19 Sense strand:5'- CCUUCUUGUGAUGCUCUC UU-3'SEQ ID NO: 19 Sense strand: 5'- CCUUCUUGUGAUGCUCUC UU-3 '
서열번호 340 Antisense strand:5'- GAGAGCAUCACAAGAAGG UU-3'SEQ ID NO: 340 Antisense strand: 5'-GAGAGCAUCACAAGAAGG UU-3 '
서열번호 172 dsRNA:5'- CCUUCUUGUGAUGCUCUC UU UCU AAA G-3'SEQ ID NO: 172 dsRNA: 5'-CCUUCUUGUGAUGUCUCUC UU UCU AAA G-3 '
Target sequence 20:5'-CCT CAT CCA GAG TTT GCA-3'(Position in gene sequence: 808) Target sequence 20: 5'-CCT CAT CCA GAG TTT GCA-3 '(Position in gene sequence: 808) GC content: 45.0%GC content: 45.0%
서열번호 20 Sense strand:5'- CCUCAUCCAGAGUUUGCA UU-3'SEQ ID NO: 20 Sense strand: 5'- CCUCAUCCAGAGUUUGCA UU-3 '
서열번호 341 Antisense strand:5'- UGCAAACUCUGGAUGAGG UU-3'SEQ ID NO: 341 Antisense strand: 5'- UGCAAACUCUGGAUGAGG UU-3 '
서열번호 173 dsRNA:5'- CCUCAUCCAGAGUUUGCA UU UCU AAA G-3'SEQ ID NO: 173 dsRNA: 5'- CCUCAUCCAGAGUUUGCA UU UCU AAA G-3 '
Target sequence 21:5'-CCT GTC CTC TAC GAA GGA-3'(Position in gene sequence: 845)Target sequence 21: 5'-CCT GTC CTC TAC GAA GGA-3 '(Position in gene sequence: 845) GC content: 50.0%GC content: 50.0%
서열번호 21 Sense strand:5'- CCUGUCCUCUACGAAGGA UU-3'SEQ ID NO: 21 Sense strand: 5'-CCUGUCCUCUACGAAGGA UU-3 '
서열번호 342 Antisense strand:5'- UCCUUCGUAGAGGACAGG UU-3'SEQ ID NO: 342 Antisense strand: 5'- UCCUUCGUAGAGGACAGG UU-3 '
서열번호 174 dsRNA:5'- CCUGUCCUCUACGAAGGA UU UCU AAA G-3'SEQ ID NO: 174 dsRNA: 5'- CCUGUCCUCUACGAAGGA UU UCU AAA G-3 '
Target sequence 22:5'-GAT TGC TAT TGT CGT ACT CA-3'(Position in gene sequence: 866)Target sequence 22: 5'-GAT TGC TAT TGT CGT ACT CA-3 '(Position in gene sequence: 866) GC content: 36.36%GC content: 36.36%
서열번호 22 Sense strand:5'- GAUUGCUAUUGUCGUACUCA UU-3'SEQ ID NO: 22 Sense strand: 5'- GAUUGCUAUUGUCGUACUCA UU-3 '
서열번호 343 Antisense strand:5'- UGAGUACGACAAUAGCAAUC UU-3'SEQ ID NO: 343 Antisense strand: 5'- UGAGUACGACAAUAGCAAUC UU-3 '
서열번호 175 dsRNA:5'- sense UU UCU AAA G-3'SEQ ID NO: 175 dsRNA: 5'-sense UU UCU AAA G-3 '
Target sequence 23:5'-GGA TTC TCG CTC TTG CAT-3'(Position in gene sequence: 947)Target sequence 23: 5'-GGA TTC TCG CTC TTG CAT-3 '(Position in gene sequence: 947) GC content: 45.0%GC content: 45.0%
서열번호 23 Sense strand:5'- GGAUUCUCGCUCUUGCAU UU-3'SEQ ID NO: 23 Sense strand: 5'- GGAUUCUCGCUCUUGCAU UU-3 '
서열번호 344 Antisense strand:5'- AUGCAAGAGCGAGAAUCC UU-3'SEQ ID NO: 344 Antisense strand: 5'- AUGCAAGAGCGAGAAUCC UU-3 '
서열번호 176 dsRNA:5'- GGAUUCUCGCUCUUGCAU UU UCU AAA G-3'SEQ ID NO: 176 dsRNA: 5'-GGAUUCUCGCUCUUGCAU UU UCU AAA G-3 '
Target sequence 24:5'-GGT GAC AGT TAC CAG CTT-3'(Position in gene sequence: 999)Target sequence 24: 5'-GGT GAC AGT TAC CAG CTT-3 '(Position in gene sequence: 999) GC content: 45.0%GC content: 45.0%
서열번호 24 Sense strand:5'- GGUGACAGUUACCAGCUU UU-3'SEQ ID NO: 24 Sense strand: 5'- GGUGACAGUUACCAGCUU UU-3 '
서열번호 345 Antisense strand:5'- AAGCUGGUAACUGUCACC UU-3'SEQ ID NO: 345 Antisense strand: 5'- AAGCUGGUAACUGUCACC UU-3 '
서열번호 177 dsRNA:5'- GGUGACAGUUACCAGCUU UU UCU AAA G-3'SEQ ID NO: 177 dsRNA: 5'-GGUGACAGUUACCAGCUU UU UCU AAA G-3 '
Target sequence 25:5'-CCT CAC TTT CAA TCC GTT-3'(Position in gene sequence: 1054)Target sequence 25: 5'-CCT CAC TTT CAA TCC GTT-3 '(Position in gene sequence: 1054) GC content: 40.0%GC content: 40.0%
서열번호 25 Sense strand:5'- CCUCACUUUCAAUCCGUU UU-3'SEQ ID NO: 25 Sense strand: 5'- CCUCACUUUCAAUCCGUU UU-3 '
서열번호 346 Antisense strand:5'- AACGGAUUGAAAGUGAGG UU-3'SEQ ID NO: 346 Antisense strand: 5'- AACGGAUUGAAAGUGAGG UU-3 '
서열번호 178 dsRNA:5'- CCUCACUUUCAAUCCGUU UU UCU AAA G-3'SEQ ID NO: 178 dsRNA: 5'- CCUCACUUUCAAUCCGUU UU UCU AAA G-3 '
Target sequence 26:5'-GCA GAA CAG TCA CTG TCC-3'(Position in gene sequence: 1096)Target sequence 26: 5'-GCA GAA CAG TCA CTG TCC-3 '(Position in gene sequence: 1096) GC content: 50.0%GC content: 50.0%
서열번호 26 Sense strand:5'- GCAGAACAGUCACUGUCC UU-3'SEQ ID NO: 26 Sense strand: 5'-GCAGAACAGUCACUGUCC UU-3 '
서열번호 347 Antisense strand:5'- GGACAGUGACUGUUCUGC UU-3'SEQ ID NO: 347 Antisense strand: 5'- GGACAGUGACUGUUCUGC UU-3 '
서열번호 179 dsRNA:5'- GCAGAACAGUCACUGUCC UU UCU AAA G-3'SEQ ID NO: 179 dsRNA: 5'-GCAGAACAGUCACUGUCC UU UCU AAA G-3 '
Target sequence 27:5'-GAT CAA TAA AGT CAG TGG CT-3'(Position in gene sequence: 1128)Target sequence 27: 5'-GAT CAA TAA AGT CAG TGG CT-3 '(Position in gene sequence: 1128) GC content: 36.36%GC content: 36.36%
서열번호 27 Sense strand:5'- GAUCAAUAAAGUCAGUGGCU UU-3'SEQ ID NO: 27 Sense strand: 5'- GAUCAAUAAAGUCAGUGGCU UU-3 '
서열번호 348 Antisense strand:5'- AGCCACUGACUUUAUUGAUC UU-3'SEQ ID NO: 348 Antisense strand: 5'- AGCCACUGACUUUAUUGAUC UU-3 '
서열번호 180 dsRNA:5'- GAUCAAUAAAGUCAGUGGCU UU UCU AAA G-3'SEQ ID NO: 180 dsRNA: 5'- GAUCAAUAAAGUCAGUGGCU UU UCU AAA G-3 '
Target sequence 28:5'-AAG AAG CTG GAG GAA AGG GGT -3'Target sequence 28: 5'-AAG AAG CTG GAG GAA AGG GGT -3 ' GC content: 47.83%GC content: 47.83%
서열번호 28 Sense strand:5'- AAGAAGCUGGAGGAAAGGGGU UU-3'SEQ ID NO: 28 Sense strand: 5'- AAGAAGCUGGAGGAAAGGGGU UU-3 '
서열번호 349 Antisense strand:5'- CCCCUUUCCUCCGCUUCUU UU-3'SEQ ID NO: 349 Antisense strand: 5'- CCCCUUUCCUCCGCUUCUU UU-3 '
서열번호 181 dsRNA:5'- AAGAAGCUGGAGGAAAGGGGU UU UCU AAA G-3'SEQ ID NO: 181 dsRNA: 5'- AAGAAGCUGGAGGAAAGGGGU UU UCU AAA G-3 '
본 발명은 조성물은 하기 표 2의 서열번호 29 내지 55의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는 하기 표 1의 서열번호 182 내지 208의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA를 포함한다.The present invention is a siRNA comprising a sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 29 to 55 of Table 2 and an antisense RNA comprising a sequence complementary thereto; Or at least one sequence selected from the group consisting of SEQ ID NOS: 182 to 208 in Table 1 below.
하기 표 2의 서열번호 29 내지 55의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는 하기 표 2의 서열번호 182 내지 208의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA는 인간 BANF1 유전자의 variant 2 서열(서열번호 2)을 타겟하는 것으로, 인간 BANF1 유전자 variant 2의 발현을 RNAi를 통해 억제함으로써 간암의 예방 또는 치료 효과를 가진다.An siRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 29 to 55 of Table 2 and antisense RNAs comprising the sequence complementary thereto; Or at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 182 to 208 shown in Table 2 below targets the variant 2 sequence (SEQ ID NO: 2) of the human BANF1 gene and the expression of the human BANF1 gene variant 2 RNAi, thereby preventing or treating liver cancer.
Target sequence 29:5'-ATG ACA ACC TCC CAA AAG CA-3'(Position in gene sequence: 452)Target sequence 29: 5'-ATG ACA ACC TCC CAA AAGCA-3 '(Position in gene sequence: 452) GC content: 40.9%GC content: 40.9%
서열번호 29 Sense strand:5'- AUGACAACCUCCCAAAAGCA UU-3'SEQ ID NO: 29 Sense strand: 5'- AUGACAACCUCCCAAAAGCA UU-3 '
서열번호 350 Antisense strand:5'- UGCUUUUGGGAGGUUGUCAU UU-3'SEQ ID NO: 350 Antisense strand: 5'- UGCUUUUGGGAGGUUGUCUCUU UU-3 '
서열번호 182 dsRNA:5'- AUGACAACCUCCCAAAAGCA UU UCU AAA G-3'SEQ ID NO: 182 dsRNA: 5'- AUGACAACCUCCCAAAAGCA UU UCU AAA G-3 '
Target sequence 30:5'-CCG AGA CTT CGT GGC AGA-3'(Position in gene sequence: 472)Target sequence 30: 5'-CCG AGA CTT CGT GGC AGA-3 '(Position in gene sequence: 472) GC content: 40.9%GC content: 40.9%
서열번호 30 Sense strand:5'- CCGAGACUUCGUGGCAGA UU-3'SEQ ID NO: 30 Sense strand: 5'- CCGAGACUUCGUGGCAGA UU-3 '
서열번호 351 Antisense strand:5'- UCUGCCACGAAGUCUCGG UU-3'SEQ ID NO: 351 Antisense strand: 5'-UCUGCCACGAAGUCUCGG UU-3 '
서열번호 183 dsRNA:5'- CCGAGACUUCGUGGCAGA UU UCU AAA G-3'SEQ ID NO: 183 dsRNA: 5'-CCGAGACUUCGUGGCAGA UU UCU AAA G-3 '
Target sequence 31:5'-AGC CTG GCT GGG ATT-3'(Position in gene sequence: 515)Target sequence 31: 5'-AGC CTG GCT GGG ATT-3 '(Position in gene sequence: 515) GC content: 52.94%GC content: 52.94%
서열번호 31 Sense strand:5'- AGCCUGGCUGGGAUU UU-3'SEQ ID NO: 31 Sense strand: 5'- AGCCUGGCUGGGAUU UU-3 '
서열번호 352 Antisense strand:5'- AAUCCCAGCCAGGCU UU-3'SEQ ID NO: 352 Antisense strand: 5'-AAUCCCAGCCAGGCU UU-3 '
서열번호 184 dsRNA:5'- AGCCUGGCUGGGAUU UU UCU AAA G-3'SEQ ID NO: 184 dsRNA: 5'- AGCCUGGCUGGGAUU UU UCU AAA G-3 '
Target sequence 32:5'-CAA GAA GCT GGA GGA AA-3'(Position in gene sequence: 544)Target sequence 32: 5'-CAA GAA GCT GGA GGA AA-3 '(Position in gene sequence: 544) GC content: 42.1%GC content: 42.1%
서열번호 32 Sense strand:5'- CAAGAAGCUGGAGGAAA UU-3'SEQ ID NO: 32 Sense strand: 5'-CAAGAAGCUGGAGGAAA UU-3 '
서열번호 353 Antisense strand:5'- UUUCCUCCAGCUUCUUG UU-3'SEQ ID NO: 353 Antisense strand: 5'- UUUCCUCCAGCUUCUUG UU-3 '
서열번호 185 dsRNA:5'- CAAGAAGCUGGAGGAAA UU UCU AAA G-3'SEQ ID NO: 185 dsRNA: 5'-CAAGAAGCUGGAGGAAA UU UCU AAA G-3 '
Target sequence 33:5'-CCA GTT TCT GGT GCT AAA GA-3'(Position in gene sequence: 592)Target sequence 33: 5'-CCA GTT TCT GGT GCT AAA GA-3 '(Position in gene sequence: 592) GC content: 40.9%GC content: 40.9%
서열번호 33 Sense strand:5'- CCAGUUUCUGGUGCUAAAGA UU-3'SEQ ID NO: 33 Sense strand: 5'- CCAGUUUCUGGUGCUAAAGA UU-3 '
서열번호 354 Antisense strand:5'- UCUUUAGCACCAGAAACUGG UU-3'SEQ ID NO: 354 Antisense strand: 5'- UCUUUAGCACCAGAAACUGG UU-3 '
서열번호 186 dsRNA:5'- CCAGUUUCUGGUGCUAAAGA UU UCU AAA G-3'SEQ ID NO: 186 dsRNA: 5'-CCAGUUUCUGGUGCUAAAGA UU UCU AAA G-3 '
Target sequence 34:5'-AAG ATG AAG ACC TCT TCC-3'(Position in gene sequence: 612)Target sequence 34: 5'-AAG ATG AAG ACC TCT TCC-3 '(Position in gene sequence: 612) GC content: 40.0%GC content: 40.0%
서열번호 34 Sense strand:5'- AAGAUGAAGACCUCUUCC UU-3'SEQ ID NO: 34 Sense strand: 5'- AAGAUGAAGACCUCUUCC UU-3 '
서열번호 355 Antisense strand:5' GGAAGAGGUCUUCAUCUU UU-3'SEQ ID NO: 355 Antisense strand: 5 'GGAAGAGGUCUUCAUCUU UU-3'
서열번호 187 dsRNA:5'- AAGAUGAAGACCUCUUCC UU UCU AAA G-3'SEQ ID NO: 187 dsRNA: 5'- AAGAUGAAGACCUCUUCC UU UCU AAA G-3 '
Target sequence 35:5'-GGA CTG CTT CGG ATG CCT T-3'(Position in gene sequence: 676)Target sequence 35: 5'-GGA CTG CTG CGG ATG CCT T-3 '(Position in gene sequence: 676) GC content: 52.38%GC content: 52.38%
서열번호 35 Sense strand:5'- GGACUGCUUCGGAUGCCUU UU-3'SEQ ID NO: 35 Sense strand: 5'- GGACUGCUUCGGAUGCCUU UU-3 '
서열번호 356 Antisense strand:5'- AAGGCAUCCGAAGCAGUCC UU-3'SEQ ID NO: 356 Antisense strand: 5'- AAGGCAUCCGAAGCAGUCC UU-3 '
서열번호 188 dsRNA:5'- GGACUGCUUCGGAUGCCUU UU UCU AAA G-3'SEQ ID NO: 188 dsRNA: 5'- GGACUGCUUCGGAUGCCUU UU UCU AAA G-3 '
Target sequence 36:5'-AGT GGT GCG ACG CCT TCT T-3'(Position in gene sequence: 698)Target sequence 36: 5'-AGT GGT GCG ACG CCT TCT T-3 '(Position in gene sequence: 698) GC content: 52.38%GC content: 52.38%
서열번호 36 Sense strand:5'- AGUGGUGCGACGCCUUCUU UU-3'SEQ ID NO: 36 Sense strand: 5'- AGUGGUGCGACGCCUUCUU UU-3 '
서열번호 357 Antisense strand:5'- AAGAAGGCGUCGCACCACU UU-3'SEQ ID NO: 357 Antisense strand: 5'- AAGAAGGCGUCGCACCACU UU-3 '
서열번호 189 dsRNA:5'- AGUGGUGCGACGCCUUCUU UU UU UCU AAA G-3'SEQ ID NO: 189 dsRNA: 5'-AGUGGUGCGACGCCUUCUU UU UU UCU AAA G-3 '
Target sequence 37:5'-CTC TCT GGG AAG CTC TCA AT-3'(Position in gene sequence: 724)Target sequence 37: 5'-CTC TCT GGG AAG CTC TCA AT-3 '(Position in gene sequence: 724) GC content: 52.38%GC content: 52.38%
서열번호 37 Sense strand:5'- AGUGGUGCGACGCCUUCUU UU-3'SEQ ID NO: 37 Sense strand: 5'- AGUGGUGCGACGCCUUCUU UU-3 '
서열번호 358 Antisense strand:5'- AAGAAGGCGUCGCACCACU UU-3'SEQ ID NO: 358 Antisense strand: 5'- AAGAAGGCGUCGCACCACU UU-3 '
서열번호 190 dsRNA:5'- AGUGGUGCGACGCCUUCUU UU UCU AAA G-3'SEQ ID NO: 190 dsRNA: 5'-AGUGGUGCGACGCCUUCUU UU UCU AAA G-3 '
Target sequence 38:5'-TTG CTA TTG TCG TAC TCA CC-3'(Position in gene sequence: 811)Target sequence 38: 5'-TTG CTA TTG TCG TAC TCA CC-3 '(Position in gene sequence: 811) GC content: 40.9%GC content: 40.9%
서열번호 38 Sense strand:5'- UUGCUAUUGUCGUACUCACC UU-3'SEQ ID NO: 38 Sense strand: 5'- UUGCUAUUGUCGUACUCACC UU-3 '
서열번호 359 Antisense strand:5'- GGUGAGUACGACAAUAGCAA UU-3'SEQ ID NO: 359 Antisense strand: 5'-GGUGAGUACGACAAUAGCAA UU-3 '
서열번호 191 dsRNA:5'- UUGCUAUUGUCGUACUCACC UU UCU AAA G-3'SEQ ID NO: 191 dsRNA: 5'- UUGCUAUUGUCGUACUCACC UU UCU AAA G-3 '
Target sequence 39:5'-GAT TCT CGC TCT TGC ATG-3'(Position in gene sequence: 891)Target sequence 39: 5'-GAT TCT CGC TCT TGC ATG-3 '(Position in gene sequence: 891) GC content: 45.0%GC content: 45.0%
서열번호 39 Sense strand:5'- GAUUCUCGCUCUUGCAUG UU-3'SEQ ID NO: 39 Sense strand: 5'- GAUUCUCGCUCUUGCAUG UU-3 '
서열번호 360 Antisense strand:5'- CAUGCAAGAGCGAGAAUC UU-3'SEQ ID NO: 360 Antisense strand: 5'- CAUGCAAGAGCGAGAAUC UU-3 '
서열번호 192 dsRNA:5'- GAUUCUCGCUCUUGCAUG UU UCU AAA G-3'SEQ ID NO: 192 dsRNA: 5'-GAUUCUCGCUCUUGCAUG UU UCU AAA G-3 '
Target sequence 40:5'-CAG TTC CCT GGT GAC AGT TA-3'(Position in gene sequence: 933)Target sequence 40: 5'-CAG TTC CCT GGT GAC AGT TA-3 '(Position in gene sequence: 933) GC content: 45.45%GC content: 45.45%
서열번호 40 Sense strand:5'- CAGUUCCCUGGUGACAGUUA UU-3'SEQ ID NO: 40 Sense strand: 5'- CAGUUCCCUGGUGACAGUUA UU-3 '
서열번호 361 Antisense strand:5'- UAACUGUCACCAGGGAACUG UU-3'SEQ ID NO: 361 Antisense strand: 5'-UAACUGUCACCAGGGAACUG UU-3 '
서열번호 193 dsRNA:5'- CAGUUCCCUGGUGACAGUUA UU UCU AAA G-3'SEQ ID NO: 193 dsRNA: 5'-CAGUUCCCUGGUGACAGUUA UU UCU AAA G-3 '
Target sequence 41:5'-CCA GCT TTC CTG AAT GGA-3'(Position in gene sequence: 953)Target sequence 41: 5'-CCA GCT TTC CTG AAT GGA-3 '(Position in gene sequence: 953) GC content: 45.0%GC content: 45.0%
서열번호 41 Sense strand:5'- CCAGCUUUCCUGAAUGGA UU-3'SEQ ID NO: 41 Sense strand: 5'- CCAGCUUUCCUGAAUGGA UU-3 '
서열번호 362 Antisense strand:5'- UCCAUUCAGGAAAGCUGG UU-3'SEQ ID NO: 362 Antisense strand: 5'- UCCAUUCAGGAAAGCUGG UU-3 '
서열번호 194 dsRNA:5'- CCAGCUUUCCUGAAUGGA UU UCU AAA G-3'SEQ ID NO: 194 dsRNA: 5'-CCAGCUUUCCUGAAUGGA UU UCU AAA G-3 '
Target sequence 42:5'-CTC ACT TTC AAT CCG TTT GA-3'(Position in gene sequence: 998) Target sequence 42: 5'-CTC ACT TTC AAT CCG TTT GA-3 '(Position in gene sequence: 998) GC content: 36.36%GC content: 36.36%
서열번호 42 Sense strand:5'- CUCACUUUCAAUCCGUUUGA UU-3'SEQ ID NO: 42 Sense strand: 5'- CUCACUUUCAAUCCGUUUGA UU-3 '
서열번호 363 Antisense strand:5'- UCAAACGGAUUGAAAGUGAG UU-3'SEQ ID NO: 363 Antisense strand: 5'-UCAAACGGAUUGAAAGUGAG UU-3 '
서열번호 195 dsRNA:5'- CUCACUUUCAAUCCGUUUGA UU UCU AAA G-3'SEQ ID NO: 195 dsRNA: 5'- CUCACUUUCAAUCCGUUUGA UU UCU AAA G-3 '
Target sequence 43:5'-CAG AAC AGT CAC TGT CCT TG-3'(Position in gene sequence: 1039)Target sequence 43: 5'-CAG AAC AGT CAC TGT CCT TG-3 '(Position in gene sequence: 1039) GC content: 45.45%GC content: 45.45%
서열번호 43 Sense strand:5'- CAGAACAGUCACUGUCCUUG UU-3'SEQ ID NO: 43 Sense strand: 5'- CAGAACAGUCACUGUCCUUG UU-3 '
서열번호 364 Antisense strand:5'- CAAGGACAGUGACUGUUCUG UU-3'SEQ ID NO: 364 Antisense strand: 5'- CAAGGACAGUGACUGUUCUG UU-3 '
서열번호 196 dsRNA:5'- CAGAACAGUCACUGUCCUUG UU UCU AAA G-3'SEQ ID NO: 196 dsRNA: 5'-CAGAACAGUCACUGUCCUUG UU UCU AAA G-3 '
Target sequence 44:5'-CAC CAG TCC AAC TGC GAG-3'(Position in gene sequence: 8)Target sequence 44: 5'-CAC CAG TCC AAC TGC GAG-3 '(Position in gene sequence: 8) GC content: 55.0%GC content: 55.0%
서열번호 44 Sense strand:5'- CACCAGUCCAACUGCGAG UU-3'SEQ ID NO: 44 Sense strand: 5'-CACCAGUCCAACUGCGAG UU-3 '
서열번호 365 Antisense strand:5'- CUCGCAGUUGGACUGGUG UU-3'SEQ ID NO: 365 Antisense strand: 5'- CUCGCAGUUGGACUGGUG UU-3 '
서열번호 197 dsRNA:5'- CACCAGUCCAACUGCGAG UU UCU AAA G-3'SEQ ID NO: 197 dsRNA: 5'-CACCAGUCCAACUGCGAG UU UCU AAA G-3 '
Target sequence 45:5'-TGC GAC GTG AGT CTG AGT CT-3'(Position in gene sequence: 39)Target sequence 45: 5'-TGC GAC GTG AGT CTG AGT CT-3 '(Position in gene sequence: 39) GC content: 50.0%GC content: 50.0%
서열번호 45 Sense strand:5'- UGCGACGUGAGUCUGAGUCU UU-3'SEQ ID NO: 45 Sense strand: 5'-UGCGACGUGAGUCUGAGUCU UU-3 '
서열번호 366 Antisense strand:5'- AGACUCAGACUCACGUCGCA UU-3'SEQ ID NO: 366 Antisense strand: 5'- AGACUCAGACACACGUCGCA UU-3 '
서열번호 198 dsRNA:5'- UGCGACGUGAGUCUGAGUCU UU UCU AAA G-3'SEQ ID NO: 198 dsRNA: 5'- UGCGACGUGAGUCUGAGUCUU UU UCU AAA G-3 '
Target sequence 46:5'-CCT CCG AAA ACC GTA CTT-3'(Position in gene sequence: 63)Target sequence 46: 5'-CCT CCG AAA ACC GTA CTT-3 '(Position in gene sequence: 63) GC content: 45.0%GC content: 45.0%
서열번호 46 Sense strand:5'- CCUCCGAAAACCGUACUU UU-3'SEQ ID NO: 46 Sense strand: 5'- CCUCCGAAAACCGUACUU UU-3 '
서열번호 367 Antisense strand:5'- AAGUACGGUUUUCGGAGG UU-3'SEQ ID NO: 367 Antisense strand: 5'- AAGUACGGUUUUCGGAGG UU-3 '
서열번호 199 dsRNA:5'- CCUCCGAAAACCGUACUU UU UCU AAA G-3'SEQ ID NO: 199 dsRNA: 5'- CCUCCGAAAACCGUACUU UU UCU AAA G-3 '
Target sequence 47:5'-CCT TGT CCG TCT TCT AAC TC-3'(Position in gene sequence: 113)Target sequence 47: 5'-CCT TGT CCG TCT TCT AAC TC-3 '(Position in gene sequence: 113) GC content: 45.45%GC content: 45.45%
서열번호 47 Sense strand:5'- CCUUGUCCGUCUUCUAACUC UU-3'SEQ ID NO: 47 Sense strand: 5'-CCUUGUCCGUCUUCUAACUC UU-3 '
서열번호 368 Antisense strand:5'- GAGUUAGAAGACGGACAAGG UU-3'SEQ ID NO: 368 Antisense strand: 5'- GAGUUAGAAGACGGACAAGG UU-3 '
서열번호 200 dsRNA:5'- CCUUGUCCGUCUUCUAACUC UU UCU AAA G-3'SEQ ID NO: 200 dsRNA: 5'-CCUUGUCCGUCUUCUAACUC UU UCU AAA G-3 '
Target sequence 48:5'-CCA GGT CCG TCA AGC CTA A-3'(Position in gene sequence: 142)Target sequence 48: 5'-CCA GGT CCG TCA AGC CTA A-3 '(Position in gene sequence: 142) GC content: 52.38%GC content: 52.38%
서열번호 48 Sense strand:5'- CCAGGUCCGUCAAGCCUAA UU-3'SEQ ID NO: 48 Sense strand: 5'-CCAGGUCCGUCAAGCCUAA UU-3 '
서열번호 369 Antisense strand:5'- UUAGGCUUGACGGACCUGG UU-3'SEQ ID NO: 369 Antisense strand: 5'- UUAGGCUUGACGGACCUGG UU-3 '
서열번호 201 dsRNA:5'- CCAGGUCCGUCAAGCCUAA UU UCU AAA G-3'SEQ ID NO: 201 dsRNA: 5'-CCAGGUCCGUCAAGCCUAA UU UCU AAA G-3 '
Target sequence 49:5'-GCA GCA GAG AAA GGA AGT-3'(Position in gene sequence: 182)Target sequence 49: 5'-GCA GCA GAG AAA GGA AGT-3 '(Position in gene sequence: 182) GC content: 45.0%GC content: 45.0%
서열번호 49 Sense strand:5'- GCAGCAGAGAAAGGAAGU UU-3'SEQ ID NO: 49 Sense strand: 5'-GCAGCAGAGAAAGGAAGU UU-3 '
서열번호 370 Antisense strand:5'- UACUUCCUUUCUCUGCUGC UU-3'SEQ ID NO: 370 Antisense strand: 5'- UACUUCCUUUCUCUGCUGC UU-3 '
서열번호 202 dsRNA:5'- GCAGCAGAGAAAGGAAGU UU UCU AAA G-3'SEQ ID NO: 202 dsRNA: 5'- GCAGCAGAGAAAGGAAGU UU UCU AAA G-3 '
Target sequence 50:5'-CCT ATC TCC CTC AGA ACT-3'(Position in gene sequence: 214)Target sequence 50: 5'-CCT ATC TCC CTC AGA ACT-3 '(Position in gene sequence: 214) GC content: 45.0%GC content: 45.0%
서열번호 50 Sense strand:5'- CCUAUCUCCCUCAGAACU UU-3'SEQ ID NO: 50 Sense strand: 5'-CCUAUCUCCCUCAGAACU UU-3 '
서열번호 371 Antisense strand:5'- AGUUCUGAGGGAGAUAGG UU-3'SEQ ID NO: 371 Antisense strand: 5'- AGUUCUGAGGGAGAUAGG UU-3 '
서열번호 203 dsRNA:5'- CCUAUCUCCCUCAGAACU UU UCU AAA G-3'SEQ ID NO: 203 dsRNA: 5'-CCUAUCUCCCUCAGAACU UU UCU AAA G-3 '
Target sequence 51:5'-GAG AAG CGA GAC CTT AGA AG-3'(Position in gene sequence: 236)Target sequence 51: 5'-GAG AAG CGA GAC CTT AGA-3 '(Position in gene sequence: 236) GC content: 45.45%GC content: 45.45%
서열번호 51 Sense strand:5'- GAGAAGCGAGACCUUAGAAG UU-3'SEQ ID NO: 51 Sense strand: 5'-GAGAAGCGAGACCUUAGAAG UU-3 '
서열번호 372 Antisense strand:5'- CUUCUAAGGUCUCGCUUCUC UU-3'SEQ ID NO: 372 Antisense strand: 5'- CUUCUAAGGUCUCGCUUCUC UU-3 '
서열번호 204 dsRNA:5'- GAGAAGCGAGACCUUAGAAG UU UCU AAA G-3'SEQ ID NO: 204 dsRNA: 5'-GAGAAGCGAGACCUUAGAAG UU UCU AAA G-3 '
Target sequence 52:5'-GCC TCA ACT CTA TAG CTC TA-3'(Position in gene sequence: 319)Target sequence 52: 5'-GCC TCA ACT CTA TAG CTC TA-3 '(Position in gene sequence: 319) GC content: 40.9%GC content: 40.9%
서열번호 52 Sense strand:5'- GCCUCAACUCUAUAGCUCUA UU-3'SEQ ID NO: 52 Sense strand: 5'- GCCUCAACUCUAUAGCUCUA UU-3 '
서열번호 373 Antisense strand:5'- UAGAGCUAUAGAGUUGAGGC UU-3'SEQ ID NO: 373 Antisense strand: 5'- UAGAGCUAUAGAGUUGAGGC UU-3 '
서열번호 205 dsRNA:5'- GCCUCAACUCUAUAGCUCUA UU UCU AAA G-3'SEQ ID NO: 205 dsRNA: 5'- GCCUCAACUCUAUAGCUCUA UU UCU AAA G-3 '
Target sequence 53:5'-CCA ACG TGG AAT GTT TCT-3'(Position in gene sequence: 354)Target sequence 53: 5'-CCA ACG TGG AAT GTT TCT-3 '(Position in gene sequence: 354) GC content: 40.0%GC content: 40.0%
서열번호 53 Sense strand:5'- CCAACGUGGAAUGUUUCU UU-3'SEQ ID NO: 53 Sense strand: 5'- CCAACGUGGAAUGUUUCU UU-3 '
서열번호 374 Antisense strand:5'- AGAAACAUUCCACGUUGG UU-3'SEQ ID NO: 374 Antisense strand: 5'- AGAAACAUUCCACGUUGG UU-3 '
서열번호 206 dsRNA:5'- CCAACGUGGAAUGUUUCU UU UCU AAA G-3'SEQ ID NO: 206 dsRNA: 5'- CCAACGUGGAAUGUUUCU UU UCU AAA G-3 '
Target sequence 54:5'-GAA GCG GAA GTG GAA GAA AGT T-3'(Position in gene sequence: 401)Target sequence 54: 5'-GAA GCG GAA GTG GAA GAA AGT T-3 '(Position in gene sequence: 401) GC content: 41.67%GC content: 41.67%
서열번호 54 Sense strand:5'- GAAGCGGAAGUGGAAGAAAGUU UU-3'SEQ ID NO: 54 Sense strand: 5'- GAAGCGGAAGUGGAAGAAAGUU UU-3 '
서열번호 375 Antisense strand:5'- AACUUUCUUCCACUUCCGCUUC UU-3'SEQ ID NO: 375 Antisense strand: 5'- AACUUUCUUCCACUUCCGCUUC UU-3 '
서열번호 207 dsRNA:5'- GAAGCGGAAGUGGAAGAAAGUU UU UCU AAA G-3'SEQ ID NO: 207 dsRNA: 5'- GAAGCGGAAGUGGAAGAAAGUU UU UCU AAA G-3 '
Target sequence 55:5'-CTA GTG GCT TGA GAT TAA GC-3'(Position in gene sequence: 423)Target sequence 55: 5'-CTA GTG GCT TGA GAT TAA GC-3 '(Position in gene sequence: 423) GC content: 40.9%GC content: 40.9%
서열번호 55 Sense strand:5'- CUAGUGGCUUGAGAUUAAGC UU-3'SEQ ID NO: 55 Sense strand: 5'- CUAGUGGCUUGAGAUUAAGC UU-3 '
서열번호 376 Antisense strand:5'- GCUUAAUCUCAAGCCACUAG UU-3'SEQ ID NO: 376 Antisense strand: 5'-GCUUAAUCUCAAGCCACUAG UU-3 '
서열번호 208 dsRNA:5'- CUAGUGGCUUGAGAUUAAGC UU UCU AAA G-3'SEQ ID NO: 208 dsRNA: 5'- CUAGUGGCUUGAGAUUAAGC UU UCU AAA G-3 '
본 발명은 조성물은 하기 표 3의 서열번호 56 내지 120의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는 하기 표 3의 서열번호 209 내지 273의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA를 포함한다.The present invention is a siRNA comprising a sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 56-120 of Table 3 and an antisense RNA consisting of the sequence complementary thereto; Or at least one sequence selected from the group consisting of SEQ ID NOS: 209 to 273 of Table 3 below.
하기 표 3의 서열번호 56 내지 120의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는 하기 표 3의 서열번호 209 내지 273의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA는 인간 PLOD3 유전자의 서열(서열번호 3)을 타겟하는 것으로, 인간 PLOD3 유전자의 발현을 RNAi를 통해 억제함으로써 간암의 예방 또는 치료 효과를 가진다.SiRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of SEQ ID NOS: 56-120 of Table 3 and antisense RNA consisting of the sequence complementary thereto; Or at least one sequence selected from the group consisting of SEQ ID NOS: 209 to 273 of Table 3, targets the sequence of the human PLOD3 gene (SEQ ID NO: 3) and inhibits the expression of the human PLOD3 gene through RNAi Thereby preventing or treating liver cancer.
Target sequence 56:5'-CCA GAG AAG CTG CTG GTG AT-3'(Position in gene sequence: 562)Target sequence 56: 5'-CCA GAG AAG CTG CTG GTG AT-3 '(Position in gene sequence: 562) GC content: 50.0%GC content: 50.0%
서열번호 56 Sense strand:5'- CCAGAGAAGCUGCUGGUGAU UU-3'SEQ ID NO: 56 Sense strand: 5'- CCAGAGAAGCUGCUGGUGAU UU-3 '
서열번호 377 Antisense strand:5'- AUCACCAGCAGCUUCUCUGG UU-3'SEQ ID NO: 377 Antisense strand: 5'- AUCACCAGCAGCUUCUCUGG UU-3 '
서열번호 209 dsRNA:5'- CCAGAGAAGCUGCUGGUGAU UU UCU AAA G-3'SEQ ID NO: 209 dsRNA: 5'- CCAGAGAAGCUGCUGGUGAU UU UCU AAA G-3 '
Target sequence 57:5'-CCA CAG CTG AAA CCG AGG-3'(Position in gene sequence: 590)Target sequence 57: 5'-CCA CAG CTG AAA CCG AGG-3 '(Position in gene sequence: 590) GC content: 55.0%GC content: 55.0%
서열번호 57 Sense strand:5'- CCACAGCUGAAACCGAGG UU-3'SEQ ID NO: 57 Sense strand: 5'- CCACAGCUGAAACCGAGG UU-3 '
서열번호 378 Antisense strand:5'- CCUCGGUUUCAGCUGUGG UU-3'SEQ ID NO: 378 Antisense strand: 5'- CCUCGGUUUCAGCUGUGG UU-3 '
서열번호 210 dsRNA:5'- CCACAGCUGAAACCGAGG UU UCU AAA G-3'SEQ ID NO: 210 dsRNA: 5'- CCACAGCUGAAACCGAGG UU UCU AAA G-3 '
Target sequence 58:5'-CTC TGC GGA GTT CTT C-3'(Position in gene sequence: 627)Target sequence 58: 5'-CTC TGC GGA GTT CTT C-3 '(Position in gene sequence: 627) GC content: 50.0%GC content: 50.0%
서열번호 58 Sense strand:5'- CUCUGCGGAGUUCUUC UU-3'SEQ ID NO: 58 Sense strand: 5'-CUCUGCGGAGUUCUUC UU-3 '
서열번호 379 Antisense strand:5'- GAAGAACUCCGCAGAG UU-3'SEQ ID NO: 379 Antisense strand: 5'- GAAGAACUCCGCAGAG UU-3 '
서열번호 211 dsRNA:5'- CUCUGCGGAGUUCUUC UU UCU AAA G-3'SEQ ID NO: 211 dsRNA: 5'-CUCUGCGGAGUUCUUC UU UCU AAA G-3 '
Target sequence 59:5'-AAC TAC ACT GTG CGG ACC-3'(Position in gene sequence: 643)Target sequence 59: 5'-AAC TAC ACT GTG CGG ACC-3 '(Position in gene sequence: 643) GC content: 50.0%GC content: 50.0%
서열번호 59 Sense strand:5'- AACUACACUGUGCGGACC UU-3'SEQ ID NO: 59 Sense strand: 5'- AACUACACUGUGCGGACC UU-3 '
서열번호 380 Antisense strand:5'- GGUCCGCACAGUGUAGUU UU-3'SEQ ID NO: 380 Antisense strand: 5'- GGUCCGCACAGUGUAGUU UU-3 '
서열번호 212 dsRNA:5'- AACUACACUGUGCGGACC UU UCU AAA G-3'SEQ ID NO: 212 dsRNA: 5'- AACUACACUGUGCGGACC UU UCU AAA G-3 '
Target sequence 60:5'-GTG ATG TGG CTC GAA CAG-3'(Position in gene sequence: 689)Target sequence 60: 5'-GTG ATG TGG CTC GAA CAG-3 '(Position in gene sequence: 689) GC content: 50.0%GC content: 50.0%
서열번호 60 Sense strand:5'- GUGAUGUGGCUCGAACAG UU-3'SEQ ID NO: 60 Sense strand: 5'-GUGAUGUGGCUCGAACAG UU-3 '
서열번호 381 Antisense strand:5'- CUGUUCGAGCCACAUCAC UU-3'SEQ ID NO: 381 Antisense strand: 5'- CUGUUCGAGCCACAUCAC UU-3 '
서열번호 213 dsRNA:5'- GUGAUGUGGCUCGAACAG UU UCU AAA G-3'SEQ ID NO: 213 dsRNA: 5'-GUGAUGUGGCUCGAACAG UU UCU AAA G-3 '
Target sequence 61:5'-GGT TAA AGA AGG AAA TGG AG-3'(Position in gene sequence: 731)Target sequence 61: 5'-GGT TAA AGA AGG AAA TGG AG-3 '(Position in gene sequence: 731) GC content: 36.36%GC content: 36.36%
서열번호 61 Sense strand:5'- GGUUAAAGAAGGAAAUGGAG UU-3'SEQ ID NO: 61 Sense strand: 5'- GGUUAAAGAAGGAAAUGGAG UU-3 '
서열번호 382 Antisense strand:5'- CUCCAUUUCCUUCUUUAACC UU-3'SEQ ID NO: 382 Antisense strand: 5'- CUCCAUUUCCUUCUUUAACC UU-3 '
서열번호 214 dsRNA:5'- GGUUAAAGAAGGAAAUGGAG UU UCU AAA G-3'SEQ ID NO: 214 dsRNA: 5'-GGUUAAAGAAGGAAAUGGAG UU UCU AAA G-3 '
Target sequence 62:5'-GGA GGA TAT GAT CAT CAT GT-3'(Position in gene sequence: 765)Target sequence 62: 5'-GGA GGA TAT GAT CAT CAT GT-3 '(Position in gene sequence: 765) GC content: 36.36%GC content: 36.36%
서열번호 62 Sense strand:5'- GGAGGAUAUGAUCAUCAUGU UU-3'SEQ ID NO: 62 Sense strand: 5'- GGAGGAUAUGAUCAUCAUGU UU-3 '
서열번호 383 Antisense strand:5'- ACAUGAUGAUCAUAUCCUCC UU-3'SEQ ID NO: 383 Antisense strand: 5'- ACAUGAUGAUCAUAUCCUCC UU-3 '
서열번호 215 dsRNA:5'- GGAGGAUAUGAUCAUCAUGU UU UCU AAA G-3'SEQ ID NO: 215 dsRNA: 5'- GGAGGAUAUGAUCAUCAUGU UU UCU AAA G-3 '
Target sequence 63:5'-GGA TAG CTA CGA CGT GAT-3'(Position in gene sequence: 789)Target sequence 63: 5'-GGA TAG CTA CGA CGT GAT-3 '(Position in gene sequence: 789) GC content: 45.0%GC content: 45.0%
서열번호 63 Sense strand:5'- GGAUAGCUACGACGUGAU UU-3'SEQ ID NO: 63 Sense strand: 5'- GGAUAGCUACGACGUGAU UU-3 '
서열번호 384 Antisense strand:5'- AUCACGUCGUAGCUAUCC UU-3'SEQ ID NO: 384 Antisense strand: 5'- AUCACGUCGUAGCUAUCC UU-3 '
서열번호 216 dsRNA:5'- GGAUAGCUACGACGUGAU UU UCU AAA G-3'SEQ ID NO: 216 dsRNA: 5'- GGAUAGCUACGACGUGAU UU UCU AAA G-3 '
Target sequence 64:5'-CAC AGA GCT GCT GAA GAA-3'(Position in gene sequence: 822)Target sequence 64: 5'-CAC AGA GCT GCT GAA GAA-3 '(Position in gene sequence: 822) GC content: 45.0%GC content: 45.0%
서열번호 64 Sense strand:5'- CACAGAGCUGCUGAAGAA UU-3'SEQ ID NO: 64 Sense strand: 5'-CACAGAGCUGCUGAAGAA UU-3 '
서열번호 385 Antisense strand:5'- UUCUUCAGCAGCUCUGUG UU-3'SEQ ID NO: 385 Antisense strand: 5'- UUCUUCAGCAGCUCUGUG UU-3 '
서열번호 217 dsRNA:5'- CACAGAGCUGCUGAAGAA UU UCU AAA G-3'SEQ ID NO: 217 dsRNA: 5'-CACAGAGCUGCUGAAGAA UU UCU AAA G-3 '
Target sequence 65:5'-TGC TCT TCT CTG CAG AGA-3'(Position in gene sequence: 863)Target sequence 65: 5'-TGC TCT TCT CTG CAG AGA-3 '(Position in gene sequence: 863) GC content: 45.0%GC content: 45.0%
서열번호 65 Sense strand:5'- UGCUCUUCUCUGCAGAGA UU-3'SEQ ID NO: 65 Sense strand: 5'-UGCUCUUCUCUGCAGAGA UU-3 '
서열번호 386 Antisense strand:5'- UCUCUGCAGAGAAGAGCA UU-3'SEQ ID NO: 386 Antisense strand: 5'-UCUCUGCAGAGAAGAGCA UU-3 '
서열번호 218 dsRNA:5'- UGCUCUUCUCUGCAGAGA UU UCU AAA G-3'SEQ ID NO: 218 dsRNA: 5'-UGCUCUUCUCUGCAGAGA UU UCU AAA G-3 '
Target sequence 66:5'-GCT TCC TCA ATT CTG GTG G-3'(Position in gene sequence: 941)Target sequence 66: 5'-GCT TCC TCA ATT CTG GTG G-3 '(Position in gene sequence: 941) GC content: 47.62%GC content: 47.62%
서열번호 66 Sense strand:5'- GCUUCCUCAAUUCUGGUGG UU-3'SEQ ID NO: 66 Sense strand: 5'-GCUUCCUCAAUUCUGGUGG UU-3 '
서열번호 387 Antisense strand:5'- CCACCAGAAUUGAGGAAGC UU-3'SEQ ID NO: 387 Antisense strand: 5'- CCACCAGAAUUGAGGAAGC UU-3 '
서열번호 219 dsRNA:5'- GCUUCCUCAAUUCUGGUGG UU UCU AAA G-3'SEQ ID NO: 219 dsRNA: 5'-GCUUCCUCAAUUCUGGUGG UU UCU AAA G-3 '
Target sequence 67:5'-ATT CAT CGG TTT TGC CAC CA-3'(Position in gene sequence: 950)Target sequence 67: 5'-ATT CAT CGG TTT TGC CACCA-3 '(Position in gene sequence: 950) GC content: 40.9%GC content: 40.9%
서열번호 67 Sense strand:5'- AUUCAUCGGUUUUGCCACCA UU-3'SEQ ID NO: 67 Sense strand: 5'- AUUCAUCGGUUUUGCCACCA UU-3 '
서열번호 388 Antisense strand:5'- UGGUGGCAAAACCGAUGAAU UU-3'SEQ ID NO: 388 Antisense strand: 5'- UGGUGGCAAAACCGAUGAAU UU-3 '
서열번호 220 dsRNA:5'- AUUCAUCGGUUUUGCCACCA UU UCU AAA G-3'SEQ ID NO: 220 dsRNA: 5'- AUUCAUCGGUUUUGCCACCA UU UCU AAA G-3 '
Target sequence 68:5'-AGT GGA AGT ACA AGG ATG A-3'(Position in gene sequence: 1001)Target sequence 68: 5'-AGT GGA AGT ACA AGG ATG A-3 '(Position in gene sequence: 1001) GC content: 38.1%GC content: 38.1%
서열번호 68 Sense strand:5'- AGUGGAAGUACAAGGAUGA UU-3'SEQ ID NO: 68 Sense strand: 5'- AGUGGAAGUACAAGGAUGA UU-3 '
서열번호 389 Antisense strand:5'- UCAUCCUUGUACUUCCACU UU-3'SEQ ID NO: 389 Antisense strand: 5'-UCAUCCUUGUACUUCCACU UU-3 '
서열번호 221 dsRNA:5'- AGUGGAAGUACAAGGAUGA UU UCU AAA G-3'SEQ ID NO: 221 dsRNA: 5'- AGUGGAAGUACAAGGAUGA UU UCU AAA G-3 '
Target sequence 69:5'-CAG CCT TAA TCT GGA TCA-3'(Position in gene sequence: 1080)Target sequence 69: 5'-CAG CCT TAA TCT GGA TCA-3 '(Position in gene sequence: 1080) GC content: 40.0%GC content: 40.0%
서열번호 69 Sense strand:5'- CAGCCUUAAUCUGGAUCA UU-3'SEQ ID NO: 69 Sense strand: 5'- CAGCCUUAAUCUGGAUCA UU-3 '
서열번호 390 Antisense strand:5'- UGAUCCAGAUUAAGGCUG UU-3'SEQ ID NO: 390 Antisense strand: 5'- UGAUCCAGAUUAAGGCUG UU-3 '
서열번호 222 dsRNA:5'- CAGCCUUAAUCUGGAUCA UU UCU AAA G-3'SEQ ID NO: 222 dsRNA: 5'-CAGCCUUAAUCUGGAUCA UU UCU AAA G-3 '
Target sequence 70:5'-GTC TCG GAT CTT TCA GAA CC-3'(Position in gene sequence: 1101)Target sequence 70: 5'-GTC TCG GAT CTT TCA GAA CC-3 '(Position in gene sequence: 1101) GC content: 45.45%GC content: 45.45%
서열번호 70 Sense strand:5'- GUCUCGGAUCUUUCAGAACC UU-3'SEQ ID NO: 70 Sense strand: 5'- GUCUCGGAUCUUUCAGAACC UU-3 '
서열번호 391 Antisense strand:5'- GGUUCUGAAAGAUCCGAGAC UU-3'SEQ ID NO: 391 Antisense strand: 5'- GGUUCUGAAAGAUCCGAGAC UU-3 '
서열번호 223 dsRNA:5'- GUCUCGGAUCUUUCAGAACC UU UCU AAA G-3'SEQ ID NO: 223 dsRNA: 5'- GUCUCGGAUCUUUCAGAACC UU UCU AAA G-3 '
Target sequence 71:5'-GGC TTT AGA TGA AGT GGT-3'(Position in gene sequence: 1128) Target sequence 71: 5'-GGC TTT AGA TGA AGT GGT-3 '(Position in gene sequence: 1128) GC content: 40.0%GC content: 40.0%
서열번호 71 Sense strand:5'- GGCUUUAGAUGAAGUGGU UU-3'SEQ ID NO: 71 Sense strand: 5'- GGCUUUAGAUGAAGUGGU UU-3 '
서열번호 392 Antisense strand:5'- ACCACUUCAUCUAAAGCC UU-3'SEQ ID NO: 392 Antisense strand: 5'- ACCACUUCAUCUAAAGCC UU-3 '
서열번호 224 dsRNA:5'- GGCUUUAGAUGAAGUGGU UU UCU AAA G-3'SEQ ID NO: 224 dsRNA: 5'- GGCUUUAGAUGAAGUGGU UU UCU AAA G-3 '
Target sequence 72:5'-GTT TGA TCG GAA CCG TGT-3'(Position in gene sequence: 1152)Target sequence 72: 5'-GTT TGA TCG GAA CCG TGT-3 '(Position in gene sequence: 1152) GC content: 45.0%GC content: 45.0%
서열번호 72 Sense strand:5'- GUUUGAUCGGAACCGUGU UU-3'SEQ ID NO: 72 Sense strand: 5'- GUUUGAUCGGAACCGUGU UU-3 '
서열번호 393 Antisense strand:5'- ACACGGUUCCGAUCAAAC UU-3'SEQ ID NO: 393 Antisense strand: 5'- ACACGGUUCCGAUCAAAC UU-3 '
서열번호 225 dsRNA:5'- GUUUGAUCGGAACCGUGU UU UCU AAA G-3'SEQ ID NO: 225 dsRNA: 5'- GUUUGAUCGGAACCGUGU UU UCU AAA G-3 '
Target sequence 73:5'-TTG TGG TCC ATG GAA ACG-3'(Position in gene sequence: 1205)Target sequence 73: 5'-TTG TGG TCC ATG GAA ACG-3 '(Position in gene sequence: 1205) GC content: 45.0%GC content: 45.0%
서열번호 73 Sense strand:5'- UUGUGGUCCAUGGAAACG UU-3'SEQ ID NO: 73 Sense strand: 5'-UUGUGGUCCAUGGAAACG UU-3 '
서열번호 394 Antisense strand:5'- CGUUUCCAUGGACCACAA UU-3'SEQ ID NO: 394 Antisense strand: 5'- CGUUUCCAUGGACCACAA UU-3 '
서열번호 226 dsRNA:5'- UUGUGGUCCAUGGAAACG UU UCU AAA G-3'SEQ ID NO: 226 dsRNA: 5'- UUGUGGUCCAUGGAAACG UU UCU AAA G-3 '
Target sequence 74:5'-CCA CTA AGC TGC AGC TCA A-3'(Position in gene sequence: 1223)Target sequence 74: 5'-CCA CTA AGC TGC AGC TCAA-3 '(Position in gene sequence: 1223) GC content: 47.62%GC content: 47.62%
서열번호 74 Sense strand:5'- CCACUAAGCUGCAGCUCAA UU-3'SEQ ID NO: 74 Sense strand: 5'- CCACUAAGCUGCAGCUCAA UU-3 '
서열번호 395 Antisense strand:5'- UUGAGCUGCAGCUUAGUGG UU-3'SEQ ID NO: 395 Antisense strand: 5'- UUGAGCUGCAGCUUAGUGG UU-3 '
서열번호 227 dsRNA:5'- CCACUAAGCUGCAGCUCAA UU UCU AAA G-3'SEQ ID NO: 227 dsRNA: 5'-CCACUAAGCUGCAGCUCAA UU UCU AAA G-3 '
Target sequence 75:5'-CCA ATG GCT GGA CTC CTG A-3'(Position in gene sequence: 1265)Target sequence 75: 5'-CCA ATG GCT GGA CTC CTGA-3 '(Position in gene sequence: 1265) GC content: 52.38%GC content: 52.38%
서열번호 75 Sense strand:5'- CCAAUGGCUGGACUCCUGA UU-3'SEQ ID NO: 75 Sense strand: 5'- CCAAUGGCUGGACUCCUGA UU-3 '
서열번호 396 Antisense strand:5'- TCAGGAGTCCAGCCATTGG UU-3'SEQ ID NO: 396 Antisense strand: 5'-TCAGGAGTCCAGCCATTGG UU-3 '
서열번호 228 dsRNA:5'- CCAAUGGCUGGACUCCUGA UU UCU AAA G-3'SEQ ID NO: 228 dsRNA: 5'-CCAAUGGCUGGACUCCUGA UU UCU AAA G-3 '
Target sequence 76:5'-GCT GTG GCT TCT GCA ACC A-3'(Position in gene sequence: 1289)Target sequence 76: 5'-GCT GTG GCT TCT GCA ACCA-3 '(Position in gene sequence: 1289) GC content: 52.38%GC content: 52.38%
서열번호 76 Sense strand:5'- GCUGUGGCUUCUGCAACCA UU-3'SEQ ID NO: 76 Sense strand: 5'- GCUGUGGCUUCUGCAACCA UU-3 '
서열번호 397 Antisense strand:5'- UGGUUGCAGAAGCCACAGC UU-3'SEQ ID NO: 397 Antisense strand: 5'- UGGUUGCAGAAGCCACAGC UU-3 '
서열번호 229 dsRNA:5'- GCUGUGGCUUCUGCAACCA UU UCU AAA G-3'SEQ ID NO: 229 dsRNA: 5'-GCUGUGGCUUCUGCAACCA UU UCU AAA G-3 '
Target sequence 77:5'-GTG TTT GTG GAA CAG CCT-3'(Position in gene sequence: 1360)Target sequence 77: 5'-GTG TTT GTG GAA CAG CCT-3 '(Position in gene sequence: 1360) GC content: 45.0%GC content: 45.0%
서열번호 77 Sense strand:5'- GUGUUUGUGGAACAGCCU UU-3'SEQ ID NO: 77 Sense strand: 5'- GUGUUUGUGGAACAGCCU UU-3 '
서열번호 398 Antisense strand:5'- AGGCUGUUCCACAAACAC UU-3'SEQ ID NO: 398 Antisense strand: 5'- AGGCUGUUCCACAAACAC UU-3 '
서열번호 230 dsRNA:5'- GUGUUUGUGGAACAGCCU UU UCU AAA G-3'SEQ ID NO: 230 dsRNA: 5'-GUGUUUGUGGAACAGCCU UU UCU AAA G-3 '
Target sequence 78:5'-GCT GCT ACT CCT GGA CTA T-3'(Position in gene sequence: 1407)Target sequence 78: 5'-GCT GCT ACT CCT GGA CTA T-3 '(Position in gene sequence: 1407) GC content: 47.62%GC content: 47.62%
서열번호 78 Sense strand:5'- GCUGCUACUCCUGGACUAU UU-3'SEQ ID NO: 78 Sense strand: 5'- GCUGCUACUCCUGGACUAU UU-3 '
서열번호 399 Antisense strand:5'- AUAGUCCAGGAGUAGCAGC UU-3'SEQ ID NO: 399 Antisense strand: 5'- AUAGUCCAGGAGUAGCAGC UU-3 '
서열번호 231 dsRNA:5'- GCUGCUACUCCUGGACUAU UU UCU AAA G-3'SEQ ID NO: 231 dsRNA: 5'-GCUGCUACUCCUGGACUAU UU UCU AAA G-3 '
Target sequence 79:5'-TTC CTG CAC AAC AAC GAG GT-3'(Position in gene sequence: 1447)Target sequence 79: 5'-TTC CTG CAC AAC AAC GAG GT-3 '(Position in gene sequence: 1447) GC content: 45.45%GC content: 45.45%
서열번호 79 Sense strand:5'- UUCCUGCACAACAACGAGGU UU-3'SEQ ID NO: 79 Sense strand: 5'- UUCCUGCACAACAACGAGGU UU-3 '
서열번호 400 Antisense strand:5'- ACCUCGUUGUUGUGCAGGAA UU-3'SEQ ID NO: 400 Antisense strand: 5'- ACCUCGUUGUUGUGCAGGAA UU-3 '
서열번호 232 dsRNA:5'- UUCCUGCACAACAACGAGGU UU UCU AAA G-3'SEQ ID NO: 232 dsRNA: 5'- UUCCUGCACAACAACGAGGU UU UCU AAA G-3 '
Target sequence 80:5'-CCA CAT CGC TGA CTC CTG-3'(Position in gene sequence: 1479)Target sequence 80: 5'-CCA CAT CGC TGA CTC CTG-3 '(Position in gene sequence: 1479) GC content: 55.0%GC content: 55.0%
서열번호 80 Sense strand:5'- CCACAUCGCUGACUCCUG UU-3'SEQ ID NO: 80 Sense strand: 5'- CCACAUCGCUGACUCCUG UU-3 '
서열번호 401 Antisense strand:5'- CAGGAGUCAGCGAUGUGG UU-3'SEQ ID NO: 401 Antisense strand: 5'-CAGGAGUCAGCGAUGUGG UU-3 '
서열번호 233 dsRNA:5'- CCACAUCGCUGACUCCUG UU UCU AAA G-3'SEQ ID NO: 233 dsRNA: 5'-CCACAUCGCUGACUCCUG UU UCU AAA G-3 '
Target sequence 81:5'-AGC TCC AGG ACC ACT TCT CA-3'(Position in gene sequence: 1502)Target sequence 81: 5'-AGC TCC AGG ACC ACT TCTCA-3 '(Position in gene sequence: 1502) GC content: 50.0%GC content: 50.0%
서열번호 81 Sense strand:5'- AGCUCCAGGACCACUUCUCA UU-3'SEQ ID NO: 81 Sense strand: 5'- AGCUCCAGGACCACUUCUCA UU-3 '
서열번호 402 Antisense strand:5'- TGAGAAGTGGTCCTGGAGCT UU-3'SEQ ID NO: 402 Antisense strand: 5'- TGAGAAGTGGTCCTGGAGCT UU-3 '
서열번호 234 dsRNA:5'- AGCUCCAGGACCACUUCUCA UU UCU AAA G-3'SEQ ID NO: 234 dsRNA: 5'-AGCUCCAGGACCACUUCUCA UU UCU AAA G-3 '
Target sequence 82:5'-ATG GCC ATG GAC CTG TGT-3'(Position in gene sequence: 1576)Target sequence 82: 5'-ATG GCC ATG GAC CTG TGT-3 '(Position in gene sequence: 1576) GC content: 50.0%GC content: 50.0%
서열번호 82 Sense strand:5'- AUGGCCAUGGACCUGUGU UU-3'SEQ ID NO: 82 Sense strand: 5'- AUGGCCAUGGACCUGUGU UU-3 '
서열번호 403 Antisense strand:5'- ACACAGGUCCAUGGCCAU UU-3'SEQ ID NO: 403 Antisense strand: 5'- ACACAGGUCCAUGGCCAU UU-3 '
서열번호 235 dsRNA:5'- AUGGCCAUGGACCUGUGU UU UCU AAA G-3'SEQ ID NO: 235 dsRNA: 5'- AUGGCCAUGGACCUGUGU UU UCU AAA G-3 '
Target sequence 83:5'-CGA GTG TGA GTT CTA CTT CA-3'(Position in gene sequence: 1605)Target sequence 83: 5'-CGA GTG TGA GTT CTA CTTCA-3 '(Position in gene sequence: 1605) GC content: 40.9%GC content: 40.9%
서열번호 83 Sense strand:5'- CGAGUGUGAGUUCUACUUCA UU-3'SEQ ID NO: 83 Sense strand: 5'- CGAGUGUGAGUUCUACUUCA UU-3 '
서열번호 404 Antisense strand:5'- UGAAGUAGAACUCACACUCG UU-3'SEQ ID NO: 404 Antisense strand: 5'- UGAAGUAGAACUCACACUCG UU-3 '
서열번호 236 dsRNA:5'- CGAGUGUGAGUUCUACUUCA UU UCU AAA G-3'SEQ ID NO: 236 dsRNA: 5'-CGAGUGUGAGUUCUACUUCA UU UCU AAA G-3 '
Target sequence 84:5'-GCT GTC CTC ACC AAC CTG-3'(Position in gene sequence: 1639)Target sequence 84: 5'-GCT GTC CTC ACC AAC CTG-3 '(Position in gene sequence: 1639) GC content: 55.0%GC content: 55.0%
서열번호 84 Sense strand:5'- GCUGUCCUCACCAACCUG UU-3'SEQ ID NO: 84 Sense strand: 5'- GCUGUCCUCACCAACCUG UU-3 '
서열번호 405 Antisense strand:5'- CAGGUUGGUGAGGACAGC UU-3'SEQ ID NO: 405 Antisense strand: 5'- CAGGUUGGUGAGGACAGC UU-3 '
서열번호 237 dsRNA:5'- GCUGUCCUCACCAACCUG UU UCU AAA G-3'SEQ ID NO: 237 dsRNA: 5'-GCUGUCCUCACCAACCUG UU UCU AAA G-3 '
Target sequence 85:5'-CTG CGT ATC CTC ATT GAG-3'(Position in gene sequence: 1663)Target sequence 85: 5'-CTG CGT ATC CTC ATT GAG-3 '(Position in gene sequence: 1663) GC content: 45.0%GC content: 45.0%
서열번호 85 Sense strand:5'- CUGCGUAUCCUCAUUGAG UU-3'SEQ ID NO: 85 Sense strand: 5'- CUGCGUAUCCUCAUUGAG UU-3 '
서열번호 406 Antisense strand:5'- CUCAAUGAGGAUACGCAG UU-3'SEQ ID NO: 406 Antisense strand: 5'- CUCAAUGAGGAUACGCAG UU-3 '
서열번호 238 dsRNA:5'- CUGCGUAUCCUCAUUGAG UU UCU AAA G-3'SEQ ID NO: 238 dsRNA: 5'-CUGCGUAUCCUCAUUGAG UU UCU AAA G-3 '
Target sequence 86:5'-GAG AAC AGG AAG GTG ATC-3'(Position in gene sequence: 1681)Target sequence 86: 5'-GAG AAC AGG AAG GTG ATC-3 '(Position in gene sequence: 1681) GC content: 45.0%GC content: 45.0%
서열번호 86 Sense strand:5'- GAGAACAGGAAGGUGAUC UU-3'SEQ ID NO: 86 Sense strand: 5'-GAGAACAGGAAGGUGAUC UU-3 '
서열번호 407 Antisense strand:5'- GAUCACCUUCCUGUUCUC UU-3'SEQ ID NO: 407 Antisense strand: 5'- GAUCACCUUCCUGUUCUC UU-3 '
서열번호 239 dsRNA:5'- GAGAACAGGAAGGUGAUC UU UCU AAA G-3'SEQ ID NO: 239 dsRNA: 5'-GAGAACAGGAAGGUGAUC UU UCU AAA G-3 '
Target sequence 87:5'-CAA GCT GTG GTC CAA CTT-3'(Position in gene sequence: 1722)Target sequence 87: 5'-CAA GCT GTG GTC CAA CTT-3 '(Position in gene sequence: 1722) GC content: 45.0%GC content: 45.0%
서열번호 87 Sense strand:5'- CAAGCUGUGGUCCAACUU UU-3'SEQ ID NO: 87 Sense strand: 5'- CAAGCUGUGGUCCAACUU UU-3 '
서열번호 408 Antisense strand:5'- AAGUUGGACCACAGCUUG UU-3'SEQ ID NO: 408 Antisense strand: 5'- AAGUUGGACCACAGCUUG UU-3 '
서열번호 240 dsRNA:5'- CAAGCUGUGGUCCAACUU UU UCU AAA G-3'SEQ ID NO: 240 dsRNA: 5'-CAAGCUGUGGUCCAACUU UU UCU AAA G-3 '
Target sequence 88:5'-GAG GAC TAC GTG GAG CTG-3'(Position in gene sequence: 1780)Target sequence 88: 5'-GAG GAC TAC GTG GAG CTG-3 '(Position in gene sequence: 1780) GC content: 55.0%GC content: 55.0%
서열번호 88 Sense strand:5'- GAGGACUACGUGGAGCUG UU-3'SEQ ID NO: 88 Sense strand: 5'-GAGGACUACGUGGAGCUG UU-3 '
서열번호 409 Antisense strand:5'- CAGCUCCACGUAGUCCUC UU-3'SEQ ID NO: 409 Antisense strand: 5'-CAGCUCCACGUAGUCUCUC UU-3 '
서열번호 241 dsRNA:5'- GAGGACUACGUGGAGCUG UU UCU AAA G-3'SEQ ID NO: 241 dsRNA: 5'-GAGGACUACGUGGAGCUG UU UCU AAA G-3 '
Target sequence 89:5'-GTG TGT GGA ATG TAC CAT AC-3'(Position in gene sequence: 1817)Target sequence 89: 5'-GTG TGT GGA ATG TAC CAT AC-3 '(Position in gene sequence: 1817) GC content: 40.9%GC content: 40.9%
서열번호 89 Sense strand:5'- GUGUGUGGAAUGUACCAUAC UU-3'SEQ ID NO: 89 Sense strand: 5'- GUGUGUGGAAUGUACCAUAC UU-3 '
서열번호 410 Antisense strand:5'- GUAUGGUACAUUCCACACAC UU-3'SEQ ID NO: 410 Antisense strand: 5'- GUAUGGUACAUUCCACACAC UU-3 '
서열번호 242 dsRNA:5'- GUGUGUGGAAUGUACCAUAC UU UCU AAA G-3'SEQ ID NO: 242 dsRNA: 5'-GUGUGUGGAAUGUACCAUAC UU UCU AAA G-3 '
Target sequence 90:5'-AGA GGG ATG TGT TCT CGG G-3'(Position in gene sequence: 1888)Target sequence 90: 5'-AGA GGG ATG TGT TCT CGG G-3 '(Position in gene sequence: 1888) GC content: 52.38%GC content: 52.38%
서열번호 90 Sense strand:5'- AGAGGGAUGUGUUCUCGGG UU-3'SEQ ID NO: 90 Sense strand: 5'- AGAGGGAUGUGUUCUCGGG UU-3 '
서열번호 411 Antisense strand:5'- CCCGAGAACACAUCCCUCU UU-3'SEQ ID NO: 411 Antisense strand: 5'- CCCGAGAACACAUCCCUCU UU-3 '
서열번호 243 dsRNA:5'- AGAGGGAUGUGUUCUCGGG UU UCU AAA G-3'SEQ ID NO: 243 dsRNA: 5'-AGAGGGAUGUGUUCUCGGG UU UCU AAA G-3 '
Target sequence 91:5'-CCT TCT GTA AGA GCT TTC GA-3'(Position in gene sequence: 1931)Target sequence 91: 5'-CCT TCT GTA AGA GCT TTC GA-3 '(Position in gene sequence: 1931) GC content: 40.9%GC content: 40.9%
서열번호 91 Sense strand:5'- CCUUCUGUAAGAGCUUUCGA UU-3'SEQ ID NO: 91 Sense strand: 5'- CCUUCUGUAAGAGCUUUCGA UU-3 '
서열번호 412 Antisense strand:5'- UCGAAAGCUCUUACAGAAGG UU-3'SEQ ID NO: 412 Antisense strand: 5'-UCGAAAGCUCUUACAGAAGG UU-3 '
서열번호 244 dsRNA:5'- CCUUCUGUAAGAGCUUUCGA UU UCU AAA G-3'SEQ ID NO: 244 dsRNA: 5'-CCUUCUGUAAGAGCUUUCGA UU UCU AAA G-3 '
Target sequence 92:5'-ACA AGG GCA TCT TCC TCC AT-3'(Position in gene sequence: 1952)Target sequence 92: 5'-ACA AGG GCA TCT TCC TCC AT-3 '(Position in gene sequence: 1952) GC content: 45.45%GC content: 45.45%
서열번호 92 Sense strand:5'- ACAAGGGCAUCUUCCUCCAU UU-3'SEQ ID NO: 92 Sense strand: 5'- ACAAGGGCAUCUUCCUCCAU UU-3 '
서열번호 413 Antisense strand:5'- AUGGAGGAAGAUGCCCUUGU UU-3'SEQ ID NO: 413 Antisense strand: 5'- AUGGAGGAAGAUGCCCUUGU UU-3 '
서열번호 245 dsRNA:5'- ACAAGGGCAUCUUCCUCCAU UU UCU AAA G-3'SEQ ID NO: 245 dsRNA: 5'- ACAAGGGCAUCUUCCUCCAU UU UCU AAA G-3 '
Target sequence 93:5'-CTG AGC AAT CAG CAT GAA-3'(Position in gene sequence: 1972)Target sequence 93: 5'-CTG AGC AAT CAG CAT GAA-3 '(Position in gene sequence: 1972) GC content: 40.0%GC content: 40.0%
서열번호 93 Sense strand:5'- CUGAGCAAUCAGCAUGAA UU-3'SEQ ID NO: 93 Sense strand: 5'- CUGAGCAAUCAGCAUGAA UU-3 '
서열번호 414 Antisense strand:5'- UUCAUGCUGAUUGCUCAG UU-3'SEQ ID NO: 414 Antisense strand: 5'- UUCAUGCUGAUUGCUCAG UU-3 '
서열번호 246 dsRNA:5'- CUGAGCAAUCAGCAUGAAUU UCU AAA G-3'SEQ ID NO: 246 dsRNA: 5'-CUGAGCAAUCAGCAUGAAUU UCU AAA G-3 '
Target sequence 94:5'-CCA CTT CCA GAT ACG ACA -3'(Position in gene sequence: 2006)Target sequence 94: 5'-CCA CTT CCA GAT ACG ACA -3 '(Position in gene sequence: 2006) GC content: 45.0%GC content: 45.0%
서열번호 94 Sense strand:5'- CCACUUCCAGAUACGACA UU-3'SEQ ID NO: 94 Sense strand: 5'- CCACUUCCAGAUACGACA UU-3 '
서열번호 415 Antisense strand:5'- UGUCGUAUCUGGAAGUGG UU-3'SEQ ID NO: 415 Antisense strand: 5'- UGUCGUAUCUGGAAGUGG UU-3 '
서열번호 247 dsRNA:5'- CCACUUCCAGAUACGACA UU UCU AAA G-3'SEQ ID NO: 247 dsRNA: 5'- CCACUUCCAGAUACGACA UU UCU AAA G-3 '
Target sequence 95:5'-ACC TCT GGC AGA TCT TCG A-3'(Position in gene sequence: 2042)Target sequence 95: 5'-ACC TCT GGC AGA TCT TCG A-3 '(Position in gene sequence: 2042) GC content: 47.62%GC content: 47.62%
서열번호 95 Sense strand:5'- ACCUCUGGCAGAUCUUCGA UU-3'SEQ ID NO: 95 Sense strand: 5'-ACCUCUGGCAGAUCUUCGA UU-3 '
서열번호 416 Antisense strand:5'- UCGAAGAUCUGCCAGAGGU UU-3'SEQ ID NO: 416 Antisense strand: 5'-UCGAAGAUCUGCCAGAGGU UU-3 '
서열번호 248 dsRNA:5'- ACCUCUGGCAGAUCUUCGA UU UCU AAA G-3'SEQ ID NO: 248 dsRNA: 5'-ACCUCUGGCAGAUCUUCGA UU UCU AAA G-3 '
Target sequence 96:5'-CGT CGA CTG GAA GGA GCA-3'(Position in gene sequence: 2067)Target sequence 96: 5'-CGT CGA CTG GAA GGA GCA-3 '(Position in gene sequence: 2067) GC content: 55.0%GC content: 55.0%
서열번호 96 Sense strand:5'- CGUCGACUGGAAGGAGCA UU-3'SEQ ID NO: 96 Sense strand: 5'- CGUCGACUGGAAGGAGCA UU-3 '
서열번호 417 Antisense strand:5'- UGCUCCUUCCAGUCGACG UU-3'SEQ ID NO: 417 Antisense strand: 5'- UGCUCCUUCCAGUCGACG UU-3 '
서열번호 249 dsRNA:5'- CGUCGACUGGAAGGAGCA UU UCU AAA G-3'SEQ ID NO: 249 dsRNA: 5'-CGUCGACUGGAAGGAGCA UU UCU AAA G-3 '
Target sequence 97:5'-GTA CAT CCA CGA GAA CTA-3'(Position in gene sequence: 2085)Target sequence 97: 5'-GTA CAT CCA CGA GAA CTA-3 '(Position in gene sequence: 2085) GC content: 40.0%GC content: 40.0%
서열번호 97 Sense strand:5'- GUACAUCCACGAGAACUA UU-3'SEQ ID NO: 97 Sense strand: 5'- GUACAUCCACGAGAACUA UU-3 '
서열번호 418 Antisense strand:5'- UAGUUCUCGUGGAUGUAC UU-3'SEQ ID NO: 418 Antisense strand: 5'-UAGUUCUCGUGGAUGUAC UU-3 '
서열번호 250 dsRNA:5'- GUACAUCCACGAGAACUA UU UCU AAA G-3'SEQ ID NO: 250 dsRNA: 5'-GUACAUCCACGAGAACUA UU UCU AAA G-3 '
Target sequence 985'-AAG GAA TCG TGG AGC AGC CA-3'(Position in gene sequence: 2123)Target sequence 985'-AAG GAA TCG TGG AGC AGCCA-3 '(Position in gene sequence: 2123) GC content: 50.0%GC content: 50.0%
서열번호 98 Sense strand:5'- AAGGAAUCGUGGAGCAGCCA UU-3'SEQ ID NO: 98 Sense strand: 5'- AAGGAAUCGUGGAGCAGCCA UU-3 '
서열번호 419 Antisense strand:5'- UGGCUGCUCCACGAUUCCUU UU-3'SEQ ID NO: 419 Antisense strand: 5'- UGGCUGCUCCACGAUUCCUU UU-3 '
서열번호 251 dsRNA:5'- AAGGAAUCGUGGAGCAGCCA UU UCU AAA G-3'SEQ ID NO: 251 dsRNA: 5'- AAGGAAUCGUGGAGCAGCCA UU UCU AAA G-3 '
Target sequence 995'-CTG CTG TCA GAA CAA ATG-3'(Position in gene sequence: 2167)Target sequence 995'-CTG CTG TCA GAA CAA ATG-3 '(Position in gene sequence: 2167) GC content: 40.0%GC content: 40.0%
서열번호 99 Sense strand:5'- CUGCUGUCAGAACAAAUG UU-3'SEQ ID NO: 99 Sense strand: 5'- CUGCUGUCAGAACAAAUG UU-3 '
서열번호 420 Antisense strand:5'- CAUUUGUUCUGACAGCAG UU-3'SEQ ID NO: 420 Antisense strand: 5'-CAUUUGUUCUGACAGCAG UU-3 '
서열번호 252 dsRNA:5'- CUGCUGUCAGAACAAAUG UU UCU AAA G-3'SEQ ID NO: 252 dsRNA: 5'-CUGCUGUCAGAACAAAUG UU UCU AAA G-3 '
Target sequence 100:5'-TGT GAT GAG CTG GTG GCA GA-3'(Position in gene sequence: 2185)Target sequence 100: 5'-TGT GAT GAG CTG GTG GCA GA-3 '(Position in gene sequence: 2185) GC content: 50.0%GC content: 50.0%
서열번호 100 Sense strand:5'- UGUGAUGAGCUGGUGGCAGA UU-3'SEQ ID NO: 100 Sense strand: 5'- UGUGAUGAGCUGGUGGCAGA UU-3 '
서열번호 421 Antisense strand:5'- UCUGCCACCAGCUCAUCACA UU-3'SEQ ID NO: 421 Antisense strand: 5'- UCUGCCACCAGCUCAUCACA UU-3 '
서열번호 253 dsRNA:5'- UGUGAUGAGCUGGUGGCAGA UU UCU AAA G-3'SEQ ID NO: 253 dsRNA: 5'-UGUGAUGAGCUGGUGGCAGA UU UCU AAA G-3 '
Target sequence 101:5'-GCA TGA GGA TTC AAG GCT-3'(Position in gene sequence: 2238)Target sequence 101: 5'-GCA TGA GGA TTC AAG GCT-3 '(Position in gene sequence: 2238) GC content: 45.0%GC content: 45.0%
서열번호 101 Sense strand:5'- GCAUGAGGAUUCAAGGCU UU-3'SEQ ID NO: 101 Sense strand: 5'-GCAUGAGGAUUCAAGGCU UU-3 '
서열번호 422 Antisense strand:5'- AGCCUUGAAUCCUCAUGC UU-3'SEQ ID NO: 422 Antisense strand: 5'- AGCCUUGAAUCCUCAUGC UU-3 '
서열번호 254 dsRNA:5'- GCAUGAGGAUUCAAGGCU UU UCU AAA G-3'SEQ ID NO: 254 dsRNA: 5'-GCAUGAGGAUUCAAGGCU UU UCU AAA G-3 '
Target sequence 102:5'-CTG GAG GCT ACG AGA ATG T-3'(Position in gene sequence: 2256)Target sequence 102: 5'-CTG GAG GCT ACG AGA ATG T-3 '(Position in gene sequence: 2256) GC content: 47.62%GC content: 47.62%
서열번호 102 Sense strand:5'- CUGGAGGCUACGAGAAUGU UU-3'SEQ ID NO: 102 Sense strand: 5'- CUGGAGGCUACGAGAAUGU UU-3 '
서열번호 423 Antisense strand:5'- ACAUUCUCGUAGCCUCCAG UU-3'SEQ ID NO: 423 Antisense strand: 5'- ACAUUCUCGUAGCCUCCAG UU-3 '
서열번호 255 dsRNA:5'- CUGGAGGCUACGAGAAUGU UU UCU AAA G-3'SEQ ID NO: 255 dsRNA: 5'- CUGGAGGCUACGAGAAUGU UU UCU AAA G-3 '
Target sequence 103:5'-TGG ACA TCC ACA TGA AGC-3'(Position in gene sequence: 2285)Target sequence 103: 5'-TGG ACA TCC ACA TGA AGC-3 '(Position in gene sequence: 2285) GC content: 45.0%GC content: 45.0%
서열번호 103 Sense strand:5'- UGGACAUCCACAUGAAGC UU-3'SEQ ID NO: 103 Sense strand: 5'- UGGACAUCCACAUGAAGC UU-3 '
서열번호 424 Antisense strand:5'- GCUUCAUGUGGAUGUCCA UU-3'SEQ ID NO: 424 Antisense strand: 5'-GCUUCAUGUGGAUGUCCA UU-3 '
서열번호 256 dsRNA:5'- UGGACAUCCACAUGAAGC UU UCU AAA G-3'SEQ ID NO: 256 dsRNA: 5'- UGGACAUCCACAUGAAGC UU UCU AAA G-3 '
Target sequence 104:5'-TAC GAG GAC CAG TGG CTG CA-3'(Position in gene sequence: 2311)Target sequence 104: 5'-TAC GAG GAC CAG TGG CTGCA-3 '(Position in gene sequence: 2311) GC content: 54.54%GC content: 54.54%
서열번호 104 Sense strand:5'- UACGAGGACCAGUGGCUGCA UU-3'SEQ ID NO: 104 Sense strand: 5'-UACGAGGACCAGUGGCUGCA UU-3 '
서열번호 425 Antisense strand:5'- TGCAGCCACTGGTCCTCGTA UU-3'SEQ ID NO: 425 Antisense strand: 5'- TGCAGCCACTGGTCCTCGTA UU-3 '
서열번호 257 dsRNA:5'- UACGAGGACCAGUGGCUGCA UU UCU AAA G-3'SEQ ID NO: 257 dsRNA: 5'- UACGAGGACCAGUGGCUGCA UU UCU AAA G-3 '
Target sequence 105:5'-CAT GAC CGA GAG CCT GTT T-3'(Position in gene sequence: 2355)Target sequence 105: 5'-CAT GAC CGA GAG CCT GTT T-3 '(Position in gene sequence: 2355) GC content: 47.62%GC content: 47.62%
서열번호 105 Sense strand:5'- CAUGACCGAGAGCCUGUUU UU-3'SEQ ID NO: 105 Sense strand: 5'-CAUGACCGAGAGCCUGUUU UU-3 '
서열번호 426 Antisense strand:5'- AAACAGGCUCUCGGUCAUG UU-3'SEQ ID NO: 426 Antisense strand: 5'- AAACAGGCUCUCGGUCAUG UU-3 '
서열번호 258 dsRNA:5'- CAUGACCGAGAGCCUGUUU UU UCU AAA G-3'SEQ ID NO: 258 dsRNA: 5'-CAUGACCGAGAGCCUGUUU UU UCU AAA G-3 '
Target sequence 106:5'-GTG ATG AAC TTT GTG GTT CG-3'(Position in gene sequence: 2401)Target sequence 106: 5'-GTG ATG AAC TTT GTG GTT CG-3 '(Position in gene sequence: 2401) GC content: 40.9%GC content: 40.9%
서열번호 106 Sense strand:5'- GUGAUGAACUUUGUGGUUCG UU-3'SEQ ID NO: 106 Sense strand: 5'- GUGAUGAACUUUGUGGUUCG UU-3 '
서열번호 427 Antisense strand:5'- CGAACCACAAAGUUCAUCAC UU-3'SEQ ID NO: 427 Antisense strand: 5'- CGAACCACAAAGUUCAUCAC UU-3 '
서열번호 259 dsRNA:5'- GUGAUGAACUUUGUGGUUCG UU UCU AAA G-3'SEQ ID NO: 259 dsRNA: 5'- GUGAUGAACUUUGUGGUUCG UU UCU AAA G-3 '
Target sequence 107:5'-AGA CGA GCA GCC GTC TCT-3'(Position in gene sequence: 2429)Target sequence 107: 5'-AGA CGA GCA GCC GTC TCT-3 '(Position in gene sequence: 2429) GC content: 55.0%GC content: 55.0%
서열번호 107 Sense strand:5'- AGACGAGCAGCCGUCUCU UU-3'SEQ ID NO: 107 Sense strand: 5'- AGACGAGCAGCCGUCUCU UU-3 '
서열번호 428 Antisense strand:5'- AGAGACGGCUGCUCGUCU UU-3'SEQ ID NO: 428 Antisense strand: 5'- AGAGACGGCUGCUCGUCU UU-3 '
서열번호 260 dsRNA:5'- AGACGAGCAGCCGUCUCU UU UCU AAA G-3'SEQ ID NO: 260 dsRNA: 5'-AGACGAGCAGCCGUCUCU UU UCU AAA G-3 '
Target sequence 108:5'-GAC TCA TCC ACC TTC ACC CT-3'(Position in gene sequence: 2461)Target sequence 108: 5'-GAC TCA TCC ACC TTC ACC CT-3 '(Position in gene sequence: 2461) GC content: 50.0%GC content: 50.0%
서열번호 108 Sense strand:5'- GACUCAUCCACCUUCACCCU UU-3'SEQ ID NO: 108 Sense strand: 5'- GACUCAUCCACCUUCACCCU UU-3 '
서열번호 429 Antisense strand:5'- AGGGUGAAGGUGGAUGAGUC UU-3'SEQ ID NO: 429 Antisense strand: 5'- AGGGUGAAGGUGGAUGAGUC UU-3 '
서열번호 261 dsRNA:5'- GACUCAUCCACCUUCACCCUUU UCU AAA G-3'SEQ ID NO: 261 dsRNA: 5'- GACUCAUCCACCUUCACCCUUU UCU AAA G-3 '
Target sequence 109:5'-TTC CTG CGC TAC GAC TGT GT-3'(Position in gene sequence: 2533)Target sequence 109: 5'-TTC CTG CGC TAC GAC TGT GT-3 '(Position in gene sequence: 2533) GC content: 50.0%GC content: 50.0%
서열번호 109 Sense strand:5'- UUCCUGCGCUACGACUGUGU UU-3'SEQ ID NO: 109 Sense strand: 5'- UUCCUGCGCUACGACUGUGU UU-3 '
서열번호 430 Antisense strand:5'- ACACAGUCGUAGCGCAGGAA UU-3'SEQ ID NO: 430 Antisense strand: 5'- ACACAGUCGUAGCGCAGGAA UU-3 '
서열번호 262 dsRNA:5'- UUCCUGCGCUACGACUGUGU UU UCU AAA G-3'SEQ ID NO: 262 dsRNA: 5'- UUCCUGCGCUACGACUGUGU UU UCU AAA G-3 '
Target sequence 110:5'-CAC ACG CTA CAT CAT GGT GT-3'(Position in gene sequence: 2637)Target sequence 110: 5'-CAC ACG CTA CAT CAT GGT GT-3 '(Position in gene sequence: 2637) GC content: 45.45%GC content: 45.45%
서열번호 110 Sense strand:5'- CACACGCUACAUCAUGGUGU UU-3'SEQ ID NO: 110 Sense strand: 5'- CACACGCUACAUCAUGGUGU UU-3 '
서열번호 431 Antisense strand:5'- ACACCAUGAUGUAGCGUGUG UU-3'SEQ ID NO: 431 Antisense strand: 5'- ACACCAUGAUGUAGCGUGUG UU-3 '
서열번호 263 dsRNA:5'- CACACGCUACAUCAUGGUGU UU UCU AAA G-3'SEQ ID NO: 263 dsRNA: 5'- CACACGCUACAUCAUGGUGU UU UCU AAA G-3 '
Target sequence 111:5'-TGC CAT TGT GCC TTT TTA GG-3'(Position in gene sequence: 2701)Target sequence 111: 5'-TGC CAT TGT GCC TTT TTA GG-3 '(Position in gene sequence: 2701) GC content: 40.9%GC content: 40.9%
서열번호 111 Sense strand:5'- UGCCAUUGUGCCUUUUUAGG UU-3'SEQ ID NO: 111 Sense strand: 5'- UGCCAUUGUGCCUUUUUAGG UU-3 '
서열번호 432 Antisense strand:5'- CCUAAAAAGGCACAAUGGCA UU-3'SEQ ID NO: 432 Antisense strand: 5'-CCUAAAAAGGCACAAUGGCA UU-3 '
서열번호 264 dsRNA:5'- UGCCAUUGUGCCUUUUUAGG UU UCU AAA G-3'SEQ ID NO: 264 dsRNA: 5'- UGCCAUUGUGCCUUUUUAGG UU UCU AAA G-3 '
Target sequence112:5'-CAC TTC CTG AGT TCA TGT TC-3'(Position in gene sequence: 2769)Target sequence 112: 5'-CAC TTC CTG AGT TCA TGT TC-3 '(Position in gene sequence: 2769) GC content: 40.9%GC content: 40.9%
서열번호 112 Sense strand:5'- CACUUCCUGAGUUCAUGUUC UU-3'SEQ ID NO: 112 Sense strand: 5'- CACUUCCUGAGUUCAUGUUC UU-3 '
서열번호 433 Antisense strand:5'- GAACAUGAACUCAGGAAGUG UU-3'SEQ ID NO: 433 Antisense strand: 5'- GAACAUGAACUCAGGAAGUG UU-3 '
서열번호 265 dsRNA:5'- CACUUCCUGAGUUCAUGUUC UU UCU AAA G-3'SEQ ID NO: 265 dsRNA: 5'-CACUUCCUGAGUUCAUGUUC UU UCU AAA G-3 '
Target sequence 113:5'-CCT GAA CTG AAT ATG TCA CC-3'(Position in gene sequence: 2796)Target sequence 113: 5'-CCT GAA CTG AAT ATG TCA CC-3 '(Position in gene sequence: 2796) GC content: 40.9%GC content: 40.9%
서열번호 113 Sense strand:5'- CCUGAACUGAAUAUGUCACC UU-3'SEQ ID NO: 113 Sense strand: 5'- CCUGAACUGAAUAUGUCACC UU-3 '
서열번호 434 Antisense strand:5'- GGUGACAUAUUCAGUUCAGG UU-3'SEQ ID NO: 434 Antisense strand: 5'- GGUGACAUAUUCAGUUCAGG UU-3 '
서열번호 266 dsRNA:5'- CCUGAACUGAAUAUGUCACC UU UCU AAA G-3'SEQ ID NO: 266 dsRNA: 5'-CCUGAACUGAAUAUGUCACC UU UCU AAA G-3 '
Target sequence 114:5'-CGC AGT CTC ACT CTG AAT AAA-3'(Position in gene sequence: 2937)Target sequence 114: 5'-CGC AGT CTC ACT CTG AAT AAA-3 '(Position in gene sequence: 2937) GC content: 40.9%GC content: 40.9%
서열번호 114 Sense strand:5'-CGCAGUCUCACUCUGAAUAAA UU-3'SEQ ID NO: 114 Sense strand: 5'-CGCAGUCUCACUCUGAAUAAA UU-3 '
서열번호 435 Antisense strand:5'- UUUAUUCAGAGUGAGACUGCG UU-3'SEQ ID NO: 435 Antisense strand: 5'- UUUAUUCAGAGUGAGACUGCG UU-3 '
서열번호 267 dsRNA:5'- CGCAGUCUCACUCUGAAUAAAUU UCU AAA G-3'SEQ ID NO: 267 dsRNA: 5'-CGCAGUCUCACUCUGAAUAAAUU UCU AAA G-3 '
Target sequence 115:5'-GGA CAG TTT GTA AGT CTT G-3'(Position in gene sequence: 2958)Target sequence 115: 5'-GGA CAG TTT GTA AGT CTT G-3 '(Position in gene sequence: 2958) GC content: 38.06%GC content: 38.06%
서열번호 115 Sense strand:5'- GGACAGUUUGUAAGUCUUG UU-3'SEQ ID NO: 115 Sense strand: 5'- GGACAGUUUGUAAGUCUUG UU-3 '
서열번호 436 Antisense strand:5'- CAAGACUUACAAACUGUCC UU-3'SEQ ID NO: 436 Antisense strand: 5'- CAAGACUUACAAACUGUCC UU-3 '
서열번호 268 dsRNA:5'- GGACAGUUUGUAAGUCUUG UU UCU AAA G-3'SEQ ID NO: 268 dsRNA: 5'- GGACAGUUUGUAAGUCUUG UU UCU AAA G-3 '
Target sequence 116:5'-TCA CTT CCC CTG TCC AGG TT-3'(Position in gene sequence: 121)Target sequence 116: 5'-TCA CTT CCC CTG TCC AGG TT-3 '(Position in gene sequence: 121) GC content: 50.0%GC content: 50.0%
서열번호 116 Sense strand:5'- UCACUUCCCCUGUCCAGGUU UU-3'SEQ ID NO: 116 Sense strand: 5'- UCACUUCCCCUGUCCAGGUU UU-3 '
서열번호 437 Antisense strand:5'- AACCUGGACAGGGGAAGUGA UU-3'SEQ ID NO: 437 Antisense strand: 5'- AACCUGGACAGGGGAAGUGA UU-3 '
서열번호 269 dsRNA:5'- UCACUUCCCCUGUCCAGGUU UU UCU AAA G-3'SEQ ID NO: 269 dsRNA: 5'-UCACUUCCCCUGUCCAGGUU UU UCU AAA G-3 '
Target sequence 117:5'-TCA GCT TCC ACA TGT GTC AA-3'(Position in gene sequence: 141)Target sequence 117: 5'-TCA GCT TCC ACA TGT GTC AA-3 '(Position in gene sequence: 141) GC content: 40.9%GC content: 40.9%
서열번호 117 Sense strand:5'-UCAGCUUCCACAUGUGUCAA UU-3'SEQ ID NO: 117 Sense strand: 5'-UCAGCUUCCACAUGUGUCUCA UU-3 '
서열번호 438 Antisense strand:5'-UUGACACAUGUGGAAGCUGA UU-3'SEQ ID NO: 438 Antisense strand: 5'-UUGACACAUGUGGAAGCUGA UU-3 '
서열번호 270 dsRNA:5'- UCAGCUUCCACAUGUGUCAA UU UCU AAA G-3'SEQ ID NO: 270 dsRNA: 5'- UCAGCUUCCACAUGUGUCUCA UU UCU AAA G-3 '
Target sequence 118:5'-GAC AAT CCT CGC CTT GTC T-3'(Position in gene sequence: 241)Target sequence 118: 5'-GAC AAT CCT CGC CTT GTC T-3 '(Position in gene sequence: 241) GC content: 47.62%GC content: 47.62%
서열번호 118 Sense strand:5'- GACAAUCCUCGCCUUGUCU UU-3'SEQ ID NO: 118 Sense strand: 5'- GACAAUCCUCGCCUUGUCU UU-3 '
서열번호 439 Antisense strand:5'- AGACAAGGCGAGGAUUGUC UU-3'SEQ ID NO: 439 Antisense strand: 5'- AGACAAGGCGAGGAUUGUC UU-3 '
서열번호 271 dsRNA:5'- GACAAUCCUCGCCUUGUCU UU UCU AAA G-3'SEQ ID NO: 271 dsRNA: 5'- GACAAUCCUCGCCUUGUCU UU UCU AAA G-3 '
Target sequence 119:5'-CAT CTG GAG CTT TCT GTA GC-3'(Position in gene sequence: 270)Target sequence 119: 5'-CAT CTG GAG CTT TCT GTA GC-3 '(Position in gene sequence: 270) GC content: 45.45%GC content: 45.45%
서열번호 119 Sense strand:5'- GCAUCUGGAGCUUUCUGUA UU-3'SEQ ID NO: 119 Sense strand: 5'- GCAUCUGGAGCUUUCUGUA UU-3 '
서열번호 440 Antisense strand:5'- UACAGAAAGCUCCAGAUGC UU-3'SEQ ID NO: 440 Antisense strand: 5'-UACAGAAAGCUCCAGAUGC UU-3 '
서열번호 272 dsRNA:5'- GCAUCUGGAGCUUUCUGUA UU UCU AAA G-3'SEQ ID NO: 272 dsRNA: 5'-GCAUCUGGAGCUUUCUGUA UU UCU AAA G-3 '
Target sequence 120:5'-GAG ATC CCA GGA TCC TGG-3'(Position in gene sequence: 414)Target sequence 120: 5'-GAG ATC CCA GGA TCC TGG-3 '(Position in gene sequence: 414) GC content: 55.0%GC content: 55.0%
서열번호 120 Sense strand:5'- GAGAUCCCAGGAUCCUGG UU-3'SEQ ID NO: 120 Sense strand: 5'-GAGAUCCCAGGAUCUCUGG UU-3 '
서열번호 441 Antisense strand:5'- CCAGGAUCCUGGGAUCUC UU-3'SEQ ID NO: 441 Antisense strand: 5'-CCAGGAUCCUGGGAUCUC UU-3 '
서열번호 273 dsRNA:5'- GAGAUCCCAGGAUCCUGG UU UCU AAA G-3'SEQ ID NO: 273 dsRNA: 5'-GAGAUCCCAGGAUCUCUGG UU UCU AAA G-3 '
본 발명은 조성물은 하기 표 4의 서열번호 121 내지 157의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는 하기 표 4의 서열번호 274 내지 310의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA를 포함한다.The present invention is a siRNA comprising a sense RNA consisting of at least one sequence selected from the group consisting of SEQ ID NOs: 121 to 157 shown in Table 4 below and an antisense RNA comprising a sequence complementary thereto; Or at least one sequence selected from the group consisting of SEQ ID NOS: 274 to 310 shown in Table 4 below.
하기 표 4의 서열번호 121 내지 157의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는 하기 표 4의 서열번호 274 내지 310의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA는 인간 SF3B4 유전자의 서열(서열번호 4)을 타겟하는 것으로, 인간 SF3B4 유전자의 발현을 RNAi를 통해 억제함으로써 간암의 예방 또는 치료 효과를 가진다.An siRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of SEQ ID NOs: 121 to 157 of Table 4 and antisense RNA consisting of the sequence complementary thereto; Or at least one sequence selected from the group consisting of SEQ ID NOS: 274 to 310 shown in Table 4 below, targets the sequence of the human SF3B4 gene (SEQ ID NO: 4) and inhibits the expression of the human SF3B4 gene through RNAi Thereby preventing or treating liver cancer.
Target sequence 121:5'-AAT CAG GAT GCC ACT GTG TA-3'(Position in gene sequence: 521)Target sequence 121: 5'-AAT CAG GAT GCC ACT GTG TA-3 '(Position in gene sequence: 521) GC content: 40.9%GC content: 40.9%
서열번호 121 Sense strand:5'- AAUCAGGAUGCCACUGUGUA UU-3'SEQ ID NO: 121 Sense strand: 5'- AAUCAGGAUGCCACUGUGUA UU-3 '
서열번호 442 Antisense strand:5'- UACACAGUGGCAUCCUGAUU UU-3'SEQ ID NO: 442 Antisense strand: 5'- UACACAGUGGCAUCCUGAUU UU-3 '
서열번호 274 dsRNA:5'- AAUCAGGAUGCCACUGUGUA UU UCU AAA G-3'SEQ ID NO: 274 dsRNA: 5'- AAUCAGGAUGCCACUGUGUA UU UCU AAA G-3 '
Target sequence 122:5'-CTG GAT GAG AAG GTT AGT GA-3'(Position in gene sequence: 551)Target sequence 122: 5'-CTG GAT GAG AAG GTT AGT GA-3 '(Position in gene sequence: 551) GC content: 40.9%GC content: 40.9%
서열번호 122 Sense strand:5'- CUGGAUGAGAAGGUUAGUGA UU-3'SEQ ID NO: 122 Sense strand: 5'- CUGGAUGAGAAGGUUAGUGA UU-3 '
서열번호 443 Antisense strand:5'- UCACUAACCUUCUCAUCCAG UU-3'SEQ ID NO: 443 Antisense strand: 5'- UCACUAACCUUCUCAUCCAG UU-3 '
서열번호 275 dsRNA:5'- CUGGAUGAGAAGGUUAGUGA UU UCU AAA G-3'SEQ ID NO: 275 dsRNA: 5'- CUGGAUGAGAAGGUUAGUGA UU UCU AAA G-3 '
Target sequence 123:5'-TGT GGG AAC TGT TTC TCC AG-3'(Position in gene sequence: 579)Target sequence 123: 5'-TGT GGG AAC TGT TTC TCC AG-3 '(Position in gene sequence: 579) GC content: 45.45%GC content: 45.45%
서열번호 123 Sense strand:5'- UGUGGGAACUGUUUCUCCAG UU-3'SEQ ID NO: 123 Sense strand: 5'- UGUGGGAACUGUUUCUCCAG UU-3 '
서열번호 444 Antisense strand:5'- CUGGAGAAACAGUUCCCACA UU-3'SEQ ID NO: 444 Antisense strand: 5'- CUGGAGAAACAGUUCCCACA UU-3 '
서열번호 276 dsRNA:5'- UGUGGGAACUGUUUCUCCAG UU UCU AAA G-3'SEQ ID NO: 276 dsRNA: 5'-UGUGGGAACUGUUUCUCCAG UU UCU AAA G-3 '
Target sequence 124:5'-CTG GAC CAG TAG TCA ACA-3'(Position in gene sequence: 599)Target sequence 124: 5'-CTG GAC CAG TAG TCA ACA-3 '(Position in gene sequence: 599) GC content: 45.0%GC content: 45.0%
서열번호 124 Sense strand:5'- CUGGACCAGUAGUCAACA UU-3'SEQ ID NO: 124 Sense strand: 5'- CUGGACCAGUAGUCAACA UU-3 '
서열번호 445 Antisense strand:5'- UGUUGACUACUGGUCCAG UU-3'SEQ ID NO: 445 Antisense strand: 5'- UGUUGACUACUGGUCCAG UU-3 '
서열번호 277 dsRNA:5'- CUGGACCAGUAGUCAACA UU UCU AAA G-3'SEQ ID NO: 277 dsRNA: 5'- CUGGACCAGUAGUCAACA UU UCU AAA G-3 '
Target sequence 125:5'-CCA AAG GAT AGA GTC ACT G-3'(Position in gene sequence: 626)Target sequence 125: 5'-CCA AAG GAT AGA GTC ACT G-3 '(Position in gene sequence: 626) GC content: 42.86%GC content: 42.86%
서열번호 125 Sense strand:5'- CCAAAGGAUAGAGUCACUG UU-3'SEQ ID NO: 125 Sense strand: 5'-CCAAAGGAUAGAGUCUCUG UU-3 '
서열번호 446 Antisense strand:5'- CAGUGACUCUAUCCUUUGG UU-3'SEQ ID NO: 446 Antisense strand: 5'-CAGUGACUCUAUCCUUUGG UU-3 '
서열번호 278 dsRNA:5'- CCAAAGGAUAGAGUCACUG UU UCU AAA G-3'SEQ ID NO: 278 dsRNA: 5'-CCAAAGGAUAGAGUCACUG UU UCU AAA G-3 '
Target sequence 126:5'-CAG CAC CAA GGC TAT GGC TTT-3'(Position in gene sequence: 647)Target sequence 126: 5'-CAG CAC CAA GGC TAT GGC TTT-3 '(Position in gene sequence: 647) GC content: 47.83%GC content: 47.83%
서열번호 126 Sense strand:5'- CAGCACCAAGGCUAUGGCUUU UU-3'SEQ ID NO: 126 Sense strand: 5'- CAGCACCAAGGCUAUGGCUUU UU-3 '
서열번호 447 Antisense strand:5'- AAAGCCAUAGCCUUGGUGCUG UU-3'SEQ ID NO: 447 Antisense strand: 5'- AAAGCCAUAGCCUUGGUGCUG UU-3 '
서열번호 279 dsRNA:5'- CAGCACCAAGGCUAUGGCUUU UU UCU AAA G-3'SEQ ID NO: 279 dsRNA: 5'-CAGCACCAAGGCUAUGGCUUU UU UCU AAA G-3 '
Target sequence 127:5'-GTG GAA TTC TTG AGT GAG GA-3'(Position in gene sequence: 668)Target sequence 127: 5'-GTG GAA TTC TTG AGT GAG GA-3 '(Position in gene sequence: 668) GC content: 40.9%GC content: 40.9%
서열번호 127 Sense strand:5'- GUGGAAUUCUUGAGUGAGGA UU-3'SEQ ID NO: 127 Sense strand: 5'- GUGGAAUUCUUGAGUGAGGA UU-3 '
서열번호 448 Antisense strand:5'- UCCUCACUCAAGAAUUCCAC UU-3'SEQ ID NO: 448 Antisense strand: 5'- UCCUCACUCAAGAAUUCCAC UU-3 '
서열번호 280 dsRNA:5'- GUGGAAUUCUUGAGUGAGGA UU UCU AAA G-3'SEQ ID NO: 280 dsRNA: 5'- GUGGAAUUCUUGAGUGAGGA UU UCU AAA G-3 '
Target sequence 128:5'-GCT GAC TAT GCC ATT AAG AT-3'(Position in gene sequence: 692)Target sequence 128: 5'-GCT GAC TAT GCC ATT AAG AT-3 '(Position in gene sequence: 692) GC content: 36.36%GC content: 36.36%
서열번호 128 Sense strand:5'- GCUGACUAUGCCAUUAAGAU UU-3'SEQ ID NO: 128 Sense strand: 5'- GCUGACUAUGCCAUUAAGAU UU-3 '
서열번호 449 Antisense strand:5'- AUCUUAAUGGCAUAGUCAGC UU-3'SEQ ID NO: 449 Antisense strand: 5'- AUCUUAAUGGCAUAGUCAGC UU-3 '
서열번호 281 dsRNA:5'- GCUGACUAUGCCAUUAAGAU UU UCU AAA G-3'SEQ ID NO: 281 dsRNA: 5'- GCUGACUAUGCCAUUAAGAU UU UCU AAA G-3 '
Target sequence 129:5'-ACA TGA TCA AAC TCT ATG G-3'(Position in gene sequence: 717)Target sequence 129: 5'-ACA TGA TCA AAC TCT ATG G-3 '(Position in gene sequence: 717) GC content: 33.33%GC content: 33.33%
서열번호 129 Sense strand:5'- ACAUGAUCAAACUCUAUGG UU-3'SEQ ID NO: 129 Sense strand: 5'- ACAUGAUCAAACUCUAUGG UU-3 '
서열번호 450 Antisense strand:5'- CCAUAGAGUUUGAUCAUGU UU-3'SEQ ID NO: 450 Antisense strand: 5'- CCAUAGAGUUUGAUCAUGU UU-3 '
서열번호 282 dsRNA:5'- ACAUGAUCAAACUCUAUGG UU UCU AAA G-3'SEQ ID NO: 282 dsRNA: 5'- ACAUGAUCAAACUCUAUGG UU UCU AAA G-3 '
Target sequence 130:5'-GGT GAA CAA AGC ATC AGC-3'(Position in gene sequence: 748)Target sequence 130: 5'-GGT GAA CAA AGC ATC AGC-3 '(Position in gene sequence: 748) GC content: 45.0%GC content: 45.0%
서열번호 130 Sense strand:5'- GGUGAACAAAGCAUCAGC UU-3'SEQ ID NO: 130 Sense strand: 5'- GGUGAACAAAGCAUCAGC UU-3 '
서열번호 451 Antisense strand:5'- GCUGAUGCUUUGUUCACC UU-3'SEQ ID NO: 451 Antisense strand: 5'-GCUGAUGCUUUGUUCACC UU-3 '
서열번호 283 dsRNA:5'- GGUGAACAAAGCAUCAGC UU UCU AAA G-3'SEQ ID NO: 283 dsRNA: 5'-GGUGAACAAAGCAUCAGC UU UCU AAA G-3 '
Target sequence 131:5'-CCT GAG ATT GAT GAG AAG-3'(Position in gene sequence: 818)Target sequence 131: 5'-CCT GAG ATT GAT GAG AAG-3 '(Position in gene sequence: 818) GC content: 40.0%GC content: 40.0%
서열번호 131 Sense strand:5'- CCUGAGAUUGAUGAGAAG UU-3'SEQ ID NO: 131 Sense strand: 5'- CCUGAGAUUGAUGAGAAG UU-3 '
서열번호 452 Antisense strand:5'- CUUCUCAUCAAUCUCAGG UU-3'SEQ ID NO: 452 Antisense strand: 5'-CUUCUCAUCAAUCUCAGG UU-3 '
서열번호 284 dsRNA:5'- CCUGAGAUUGAUGAGAAG UU UCU AAA G-3'SEQ ID NO: 284 dsRNA: 5'-CCUGAGAUUGAUGAGAAG UU UCU AAA G-3 '
Target sequence 132:5'-GGT CAT CTT ACA AAC CC-3'(Position in gene sequence: 865)Target sequence 132: 5'-GGT CAT CTT ACA AAC CC-3 '(Position in gene sequence: 865) GC content: 42.1%GC content: 42.1%
서열번호 132 Sense strand:5'- GGUCAUCUUACAAACCC UU-3'SEQ ID NO: 132 Sense strand: 5'- GGUCAUCUUACAAACCC UU-3 '
서열번호 453 Antisense strand:5'- GGGUUUGUAAGAUGACC UU-3'SEQ ID NO: 453 Antisense strand: 5'- GGGUUUGUAAGAUGACC UU-3 '
서열번호 285 dsRNA:5'- GGUCAUCUUACAAACCC UU UCU AAA G-3'SEQ ID NO: 285 dsRNA: 5'-GGUCAUCUUACAAACCC UU UCU AAA G-3 '
Target sequence 133:5'-CCT GAC ACA GGC AAC TCC-3'(Position in gene sequence: 899)Target sequence 133: 5'-CCT GAC ACA GGC AAC TCC-3 '(Position in gene sequence: 899) GC content: 55.0%GC content: 55.0%
서열번호 133 Sense strand:5'- CCUGACACAGGCAACUCC UU-3'SEQ ID NO: 133 Sense strand: 5'- CCUGACACAGGCAACUCC UU-3 '
서열번호 454 Antisense strand:5'- GGAGUUGCCUGUGUCAGG UU-3'SEQ ID NO: 454 Antisense strand: 5'-GGAGUUGCCUGUGUCAGAG UU-3 '
서열번호 286 dsRNA:5'- CCUGACACAGGCAACUCC UU UCU AAA G-3'SEQ ID NO: 286 dsRNA: 5'- CCUGACACAGGCAACUCC UU UCU AAA G-3 '
Target sequence 134:5'-GCT TCA TTT GAT GCT TCG GA-3'(Position in gene sequence: 941)Target sequence 134: 5'-GCT TCA TTT GAT GCT TCG GA-3 '(Position in gene sequence: 941) GC content: 40.9%GC content: 40.9%
서열번호 134 Sense strand:5'- GCUUCAUUUGAUGCUUCGGA UU-3'SEQ ID NO: 134 Sense strand: 5'-GCUUCAUUUGAUGCUUCGGA UU-3 '
서열번호 455 Antisense strand:5'- UCCGAAGCAUCAAAUGAAGC UU-3'SEQ ID NO: 455 Antisense strand: 5'-UCCGAAGCAUCAAAUGAAGC UU-3 '
서열번호 287 dsRNA:5'- GCUUCAUUUGAUGCUUCGGA UU UCU AAA G-3'SEQ ID NO: 287 dsRNA: 5'-GCUUCAUUUGAUGCUUCGGA UU UCU AAA G-3 '
Target sequence 135:5'-TGC AGC AAT TGA AGC CAT GA-3'(Position in gene sequence: 961)Target sequence 135: 5'-TGC AGC AAT TGA AGC CAT GA-3 '(Position in gene sequence: 961) GC content: 40.9%GC content: 40.9%
서열번호 135 Sense strand:5'- UGCAGCAAUUGAAGCCAUGA UU-3'SEQ ID NO: 135 Sense strand: 5'- UGCAGCAAUUGAAGCCAUGA UU-3 '
서열번호 456 Antisense strand:5'- UCAUGGCUUCAAUUGCUGCA UU-3'SEQ ID NO: 456 Antisense strand: 5'-UCAUGGCUUCAAUUGCUGCA UU-3 '
서열번호 288 dsRNA:5'- UGCAGCAAUUGAAGCCAUGA UU UCU AAA G-3'SEQ ID NO: 288 dsRNA: 5'- UGCAGCAAUUGAAGCCAUGA UU UCU AAA G-3 '
Target sequence 136:5'-GCA GTA CCT CTG TAA CCG T-3'(Position in gene sequence: 985) Target sequence 136: 5'-GCA GTA CCT CTG TAA CCG T-3 '(Position in gene sequence: 985) GC content: 47.62%GC content: 47.62%
서열번호 136 Sense strand:5'- GCAGUACCUCUGUAACCGU UU-3'SEQ ID NO: 136 Sense strand: 5'-GCAGUACCUCUGUAACCGU UU-3 '
서열번호 457 Antisense strand:5'- ACGGUUACAGAGGUACUGC UU-3'SEQ ID NO: 457 Antisense strand: 5'- ACGGUUACAGAGGUACUGC UU-3 '
서열번호 289 dsRNA:5'- GCAGUACCUCUGUAACCGU UU UCU AAA G-3'SEQ ID NO: 289 dsRNA: 5'- GCAGUACCUCUGUAACCGU UU UCU AAA G-3 '
Target sequence 137:5'-CAC CGT ATC TTA TGC CTT CA-3'(Position in gene sequence: 1009)Target sequence 137: 5'-CAC CGT ATC TTA TGC CTT CA-3 '(Position in gene sequence: 1009) GC content: 40.9%GC content: 40.9%
서열번호 137 Sense strand:5'- CACCGUAUCUUAUGCCUUCA UU-3'SEQ ID NO: 137 Sense strand: 5'- CACCGUAUCUUAUGCCUUCA UU-3 '
서열번호 458 Antisense strand:5'- UGAAGGCAUAAGAUACGGUG UU-3'SEQ ID NO: 458 Antisense strand: 5'- UGAAGGCAUAAGAUACGGUG UU-3 '
서열번호 290 dsRNA:5'- CACCGUAUCUUAUGCCUUCA UU UCU AAA G-3'SEQ ID NO: 290 dsRNA: 5'- CACCGUAUCUUAUGCCUUCA UU UCU AAA G-3 '
Target sequence 138:5'-GAA CGA CTT CTG GCA GCT CA-3'(Position in gene sequence: 1067)Target sequence 138: 5'-GAA CGA CTT CTG GCA GCT CA-3 '(Position in gene sequence: 1067) GC content: 50.0%GC content: 50.0%
서열번호 138 Sense strand:5'- GAACGACUUCUGGCAGCUCA UU-3'SEQ ID NO: 138 Sense strand: 5'- GAACGACUUCUGGCAGCUCA UU-3 '
서열번호 459 Antisense strand:5'- UGAGCUGCCAGAAGUCGUUC UU-3'SEQ ID NO: 459 Antisense strand: 5'-UGAGCUGCCAGAAGUCGUUC UU-3 '
서열번호 291 dsRNA:5'- GAACGACUUCUGGCAGCUCA UU UCU AAA G-3'SEQ ID NO: 291 dsRNA: 5'- GAACGACUUCUGGCAGCUCA UU UCU AAA G-3 '
Target sequence 139:5'-CCT CAT CAG CTG TTT GCA GA-3'(Position in gene sequence: 1112)Target sequence 139: 5'-CCT CAT CAG CTG TTT GCA GA-3 '(Position in gene sequence: 1112) GC content: 45.45%GC content: 45.45%
서열번호 139 Sense strand:5'- CCUCAUCAGCUGUUUGCAGA UU-3'SEQ ID NO: 139 Sense strand: 5'- CCUCAUCAGCUGUUUGCAGA UU-3 '
서열번호 460 Antisense strand:5'- UCUGCAAACAGCUGAUGAGG UU-3'SEQ ID NO: 460 Antisense strand: 5'-UCUGCAAACAGCUGAUGAGG UU-3 '
서열번호 292 dsRNA:5'- CCUCAUCAGCUGUUUGCAGA UU UCU AAA G-3'SEQ ID NO: 292 dsRNA: 5'-CCUCAUCAGCUGUUUGCAGA UU UCU AAA G-3 '
Target sequence 140:5'-TGG TCA TGG ACA CTC ACA TC-3'(Position in gene sequence: 1351)Target sequence 140: 5'-TGG TCA TGG ACA CTC ACA TC-3 '(Position in gene sequence: 1351) GC content: 45.45%GC content: 45.45%
서열번호 140 Sense strand:5'- UGGUCAUGGACACUCACAUC UU-3'SEQ ID NO: 140 Sense strand: 5'- UGGUCAUGGACACUCACAUC UU-3 '
서열번호 461 Antisense strand:5'- GAUGUGAGUGUCCAUGACCA UU-3'SEQ ID NO: 461 Antisense strand: 5'- GAUGUGAGUGUCCAUGACCA UU-3 '
서열번호 293 dsRNA:5'- UGGUCAUGGACACUCACAUC UU UCU AAA G-3'SEQ ID NO: 293 dsRNA: 5'- UGGUCAUGGACACUCACAUC UU UCU AAA G-3 '
Target sequence 141:5'-GAT GTC TCA GAT GCA GCT-3'(Position in gene sequence: 1408)Target sequence 141: 5'-GAT GTC TCA GAT GCA GCT-3 '(Position in gene sequence: 1408) GC content: 45.0%GC content: 45.0%
서열번호 141 Sense strand:5'- GAUGUCUCAGAUGCAGCU UU-3'SEQ ID NO: 141 Sense strand: 5'- GAUGUCUCAGAUGCAGCU UU-3 '
서열번호 462 Antisense strand:5'- AGCUGCAUCUGAGACAUC UU-3'SEQ ID NO: 462 Antisense strand: 5'- AGCUGCAUCUGAGACAUC UU-3 '
서열번호 294 dsRNA:5'- GAUGUCUCAGAUGCAGCU UU UCU AAA G-3'SEQ ID NO: 294 dsRNA: 5'-GAUGUCUCAGAUGCAGCU UU UCU AAA G-3 '
Target sequence 142:5'-CCT CAT GGC TTA GGA CAT-3'(Position in gene sequence: 1439)Target sequence 142: 5'-CCT CAT GGC TTA GGA CAT-3 '(Position in gene sequence: 1439) GC content: 45.0%GC content: 45.0%
서열번호 142 Sense strand:5'- CCUCAUGGCUUAGGACAU UU-3'SEQ ID NO: 142 Sense strand: 5'- CCUCAUGGCUUAGGACAU UU-3 '
서열번호 463 Antisense strand:5'- AUGUCCUAAGCCAUGAGG UU-3'SEQ ID NO: 463 Antisense strand: 5'- AUGUCCUAAGCCAUGAGG UU-3 '
서열번호 295 dsRNA:5'- CCUCAUGGCUUAGGACAU UU UCU AAA G-3'SEQ ID NO: 295 dsRNA: 5'- CCUCAUGGCUUAGGACAU UU UCU AAA G-3 '
Target sequence 143:5'-TCA CAT TTT CCT TCC TCC TG-3'(Position in gene sequence: 1771)Target sequence 143: 5'-TCA CAT TTT CCT TCC TCC TG-3 '(Position in gene sequence: 1771) GC content: 40.9%GC content: 40.9%
서열번호 143 Sense strand:5'- UCACAUUUUCCUUCCUCCUG UU-3'SEQ ID NO: 143 Sense strand: 5'-UCACAUUUUCCUUCCUCCUG UU-3 '
서열번호 464 Antisense strand:5'- CAGGAGGAAGGAAAAUGUGA UU-3'SEQ ID NO: 464 Antisense strand: 5'- CAGGAGGAAGGAAAAUGUGA UU-3 '
서열번호 296 dsRNA:5'- UCACAUUUUCCUUCCUCCUG UU UCU AAA G-3'SEQ ID NO: 296 dsRNA: 5'-UCACAUUUUCCUUCCUCCUG UU UCU AAA G-3 '
Target sequence 144:5'-CCT TGG ACC AAT CAG AGA TG-3'(Position in gene sequence: 1818)Target sequence 144: 5'-CCT TGG ACC AAT CAG AGA TG-3 '(Position in gene sequence: 1818) GC content: 45.45%GC content: 45.45%
서열번호 144 Sense strand:5'- CCUUGGACCAAUCAGAGAUG UU-3'SEQ ID NO: 144 Sense strand: 5'-CCUUGGACCAAUCAGAGAUG UU-3 '
서열번호 465 Antisense strand:5'- CAUCUCUGAUUGGUCCAAGG UU-3'SEQ ID NO: 465 Antisense strand: 5'- CAUCUCUGAUUGGUCCAAGG UU-3 '
서열번호 297 dsRNA:5'- CCUUGGACCAAUCAGAGAUG UU UCU AAA G-3'SEQ ID NO: 297 dsRNA: 5'-CCUUGGACCAAUCAGAGAUG UU UCU AAA G-3 '
Target sequence 145:5'-GGC AAA GGT ACT AAT CCC TT-3'(Position in gene sequence: 1852)Target sequence 145: 5'-GGC AAA GGT ACT AAT CCC TT-3 '(Position in gene sequence: 1852) GC content: 40.9%GC content: 40.9%
서열번호 145 Sense strand:5'- GGCAAAGGUACUAAUCCCUU UU-3'SEQ ID NO: 145 Sense strand: 5'- GGCAAAGGUACUAAUCCCUU UU-3 '
서열번호 466 Antisense strand:5'- AAGGGAUUAGUACCUUUGCC UU-3'SEQ ID NO: 466 Antisense strand: 5'- AAGGGAUUAGUACCUUUGCC UU-3 '
서열번호 298 dsRNA:5'- GGCAAAGGUACUAAUCCCUU UU UCU AAA G-3'SEQ ID NO: 298 dsRNA: 5'-GGCAAAGGUACUAAUCCCUU UU UCU AAA G-3 '
Target sequence 146:5'-TTC CAC AGG AGG TAT TTC-3'(Position in gene sequence: 1911)Target sequence 146: 5'-TTC CAC AGG AGG TAT-3 '(Position in gene sequence: 1911) GC content: 40.0%GC content: 40.0%
서열번호 146 Sense strand:5'- UUCCACAGGAGGUAUUUC UU-3'SEQ ID NO: 146 Sense strand: 5'- UUCCACAGGAGGUAUUUC UU-3 '
서열번호 467 Antisense strand:5'- GAAAUACCUCCUGUGGAA UU-3'SEQ ID NO: 467 Antisense strand: 5'- GAAAUACCUCCUGUGGAA UU-3 '
서열번호 299 dsRNA:5'- UUCCACAGGAGGUAUUUC UU UCU AAA G-3'SEQ ID NO: 299 dsRNA: 5'- UUCCACAGGAGGUAUUUC UU UCU AAA G-3 '
Target sequence 147:5'-GGT CCT GAG TAT TTT GCA-3'(Position in gene sequence: 1940)Target sequence 147: 5'-GGT CCT GAG TAT TTT GCA-3 '(Position in gene sequence: 1940) GC content: 40.0%GC content: 40.0%
서열번호 147 Sense strand:5'- GGUCCUGAGUAUUUUGCA UU-3'SEQ ID NO: 147 Sense strand: 5'- GGUCCUGAGUAUUUUGCA UU-3 '
서열번호 468 Antisense strand:5'- UGCAAAAUACUCAGGACC UU-3'SEQ ID NO: 468 Antisense strand: 5'- UGCAAAAUACUCAGGACC UU-3 '
서열번호 300 dsRNA:5'- GGUCCUGAGUAUUUUGCA UU UCU AAA G-3'SEQ ID NO: 300 dsRNA: 5'- GGUCCUGAGUAUUUUGCA UU UCU AAA G-3 '
Target sequence 148:5'-CCA AAT CTG CAA GAA GGC T-3'(Position in gene sequence: 18)Target sequence 148: 5'-CCA AAT CTG CAA GAA GGC T-3 '(Position in gene sequence: 18) GC content: 42.86%GC content: 42.86%
서열번호 148 Sense strand:5'- CCAAAUCUGCAAGAAGGCU UU-3'SEQ ID NO: 148 Sense strand: 5'-CCAAAUCUGCAAGAAGGCU UU-3 '
서열번호 469 Antisense strand:5'- AGCCUUCUUGCAGAUUUGG UU-3'SEQ ID NO: 469 Antisense strand: 5'- AGCCUUCUUGCAGAUUUGG UU-3 '
서열번호 301 dsRNA:5'- CCAAAUCUGCAAGAAGGCU UU UCU AAA G-3'SEQ ID NO: 301 dsRNA: 5'-CCAAAUCUGCAAGAAGGCU UU UCU AAA G-3 '
Target sequence 149:5'-GGA ACT CTT CAG CAC ATC CTT-3'(Position in gene sequence: 95)Target sequence 149: 5'-GGA ACT CTT CAG CAC ATC CTT-3 '(Position in gene sequence: 95) GC content: 43.48%GC content: 43.48%
서열번호 149 Sense strand:5'- GGAACUCUUCAGCACAUCCUU UU-3'SEQ ID NO: 149 Sense strand: 5'- GGAACUCUUCAGCACAUCCUU UU-3 '
서열번호 470 Antisense strand:5'- AAGGAUGUGCUGAAGAGUUCC UU-3'SEQ ID NO: 470 Antisense strand: 5'- AAGGAUGUGCUGAAGAGUUCC UU-3 '
서열번호 302 dsRNA:5'- GGAACUCUUCAGCACAUCCUU UU UCU AAA G-3'SEQ ID NO: 302 dsRNA: 5'-GGAACUCUUCAGCACAUCCUU UU UCU AAA G-3 '
Target sequence 150:5'-CTC TGG ACA ACA GAA GAA GA-3'(Position in gene sequence: 116)Target sequence 150: 5'-CTC TGG ACA ACA GAA GAA-3 '(Position in gene sequence: 116) GC content: 40.9%GC content: 40.9%
서열번호 150 Sense strand:5'- CUCUGGACAACAGAAGAAGA UU-3'SEQ ID NO: 150 Sense strand: 5'- CUCUGGACAACAGAAGAAGA UU-3 '
서열번호 471 Antisense strand:5'- UCUUCUUCUGUUGUCCAGAG UU-3'SEQ ID NO: 471 Antisense strand: 5'-UCUUCUUCUGUUGUCCAGAG UU-3 '
서열번호 303 dsRNA:5'- CUCUGGACAACAGAAGAAGA UU UCU AAA G-3'SEQ ID NO: 303 dsRNA: 5'- CUCUGGACAACAGAAGAAGA UU UCU AAA G-3 '
Target sequence 151:5'-TGA GAG CAG TGT GAT TCT-3'(Position in gene sequence: 201)Target sequence 151: 5'-TGA GAG CAG TGT GAT TCT-3 '(Position in gene sequence: 201) GC content: 40.0%GC content: 40.0%
서열번호 151 Sense strand:5'- UGAGAGCAGUGUGAUUCU UU-3'SEQ ID NO: 151 Sense strand: 5'- UGAGAGCAGUGUGAUUCU UU-3 '
서열번호 472 Antisense strand:5'- AGAAUCACACUGCUCUCA UU-3'SEQ ID NO: 472 Antisense strand: 5'-AGAAUCACACUGCUCUCA UU-3 '
서열번호 304 dsRNA:5'- UGAGAGCAGUGUGAUUCU UU UCU AAA G-3'SEQ ID NO: 304 dsRNA: 5'- UGAGAGCAGUGUGAUUCU UU UCU AAA G-3 '
Target sequence 152:5'-CAA GTC TAG CAG TGC AT-3'(Position in gene sequence: 221)Target sequence 152: 5'-CAA GTC TAG CAG TGC AT-3 '(Position in gene sequence: 221) GC content: 42.1%GC content: 42.1%
서열번호 152 Sense strand:5'- CAAGUCUAGCAGUGCAU UU-3'SEQ ID NO: 152 Sense strand: 5'-CAAGUCUAGCAGUGCAU UU-3 '
서열번호 473 Antisense strand:5'- AUGCACUGCUAGACUUG UU-3'SEQ ID NO: 473 Antisense strand: 5'- AUGCACUGCUAGACUUG UU-3 '
서열번호 305 dsRNA:5'- CAAGUCUAGCAGUGCAU UU UCU AAA G-3'SEQ ID NO: 305 dsRNA: 5'-CAAGUCUAGCAGUGCAU UU UCU AAA G-3 '
Target sequence 153:5'-CTC GCT AAG ACA ACT AGC A-3'(Position in gene sequence: 270)Target sequence 153: 5'-CTC GCT AAG ACA ACT AGCA-3 '(Position in gene sequence: 270) GC content: 42.86%GC content: 42.86%
서열번호 153 Sense strand:5'-CUCGCUAAGACAACUAGCA UU-3'SEQ ID NO: 153 Sense strand: 5'-CUCGCUAAGACAACUAGCA UU-3 '
서열번호 474 Antisense strand:5'- UGCUAGUUGUCUUAGCGAGA UU-3'SEQ ID NO: 474 Antisense strand: 5'- UGCUAGUUGUCUUAGCGAGA UU-3 '
서열번호 306 dsRNA:5'- CUCGCUAAGACAACUAGCA UU UCU AAA G-3'SEQ ID NO: 306 dsRNA: 5'- CUCGCUAAGACAACUAGCA UU UCU AAA G-3 '
Target sequence 154:5'-CAG GTT AAG TTT CGG AGG CT-3'(Position in gene sequence: 331)Target sequence 154: 5'-CAG GTT AAG TTT CGG AGG CT-3 '(Position in gene sequence: 331) GC content: 45.45%GC content: 45.45%
서열번호 154 Sense strand:5'- CAGGUUAAGUUUCGGAGGCU UU-3'SEQ ID NO: 154 Sense strand: 5'- CAGGUUAAGUUUCGGAGGCU UU-3 '
서열번호 475 Antisense strand:5'- AGCCUCCGAAACUUAACCUG UU-3'SEQ ID NO: 475 Antisense strand: 5'- AGCCUCCGAAACUUAACCUG UU-3 '
서열번호 307 dsRNA:5'- CAGGUUAAGUUUCGGAGGCU UU UCU AAA G-3'SEQ ID NO: 307 dsRNA: 5'-CAGGUUAAGUUUCGGAGGCU UU UCU AAA G-3 '
Target sequence 155:5'-GCT TCC AGG CAC CTC CTC TT-3'(Position in gene sequence: 369)Target sequence 155: 5'-GCT TCC AGG CAC CTC CTC TT-3 '(Position in gene sequence: 369) GC content: 54.54%GC content: 54.54%
서열번호 155 Sense strand:5'- GCUUCCAGGCACCUCCUCUU UU-3'SEQ ID NO: 155 Sense strand: 5'-GCUUCCAGGCACCUCCUCUU UU-3 '
서열번호 476 Antisense strand:5'- AAGAGGAGGUGCCUGGAAGC UU-3'SEQ ID NO: 476 Antisense strand: 5'- AAGAGGAGGUGCCUGGAAGC UU-3 '
서열번호 308 dsRNA:5'- GCUUCCAGGCACCUCCUCUU UU UCU AAA G-3'SEQ ID NO: 308 dsRNA: 5'-GCUUCCAGGCACCUCCUCUU UU UCU AAA G-3 '
Target sequence 156:5'-GAA GTG GAA GTC GTG CTG AG-3'(Position in gene sequence: 427)Target sequence 156: 5'-GAA GTG GAA GTC GTG CTG AG-3 '(Position in gene sequence: 427) GC content: 50.0%GC content: 50.0%
서열번호 156 Sense strand:5'- GAAGUGGAAGUCGUGCUGAG UU-3'SEQ ID NO: 156 Sense strand: 5'- GAAGUGGAAGUCGUGCUGAG UU-3 '
서열번호 477 Antisense strand:5'- CUCAGCACGACUUCCACUUC UU-3'SEQ ID NO: 477 Antisense strand: 5'- CUCAGCACGACUUCCACUUC UU-3 '
서열번호 309 dsRNA:5'- GAAGUGGAAGUCGUGCUGAG UU UCU AAA G-3'SEQ ID NO: 309 dsRNA: 5'-GAAGUGGAAGUCGUGCUGAG UU UCU AAA G-3 '
Target sequence 157:5'-GAT CTC TTT CGC CAT GGC TG-3'(Position in gene sequence: 481)Target sequence 157: 5'-GAT CTC TTT CGC CAT GGC TG-3 '(Position in gene sequence: 481) GC content: 50.0%GC content: 50.0%
서열번호 157 Sense strand:5'- GAUCUCUUUCGCCAUGGCUG UU-3'SEQ ID NO: 157 Sense strand: 5'- GAUCUCUUUCGCCAUGGCUG UU-3 '
서열번호 478 Antisense strand:5'- CAGCCAUGGCGAAAGAGAUC UU-3'SEQ ID NO: 478 Antisense strand: 5'- CAGCCAUGGCGAAAGAGAUC UU-3 '
서열번호 310 dsRNA:5'- GAUCUCUUUCGCCAUGGCUG UU UCU AAA G-3'SEQ ID NO: 310 dsRNA: 5'- GAUCUCUUUCGCCAUGGCUG UU UCU AAA G-3 '
본 발명의 siRNA 또는 dsRNA는 담지체에 담지된 것일 수 있고, 담지체의 종류에 있어 RNA분자를 담지할 수 있는 것으로서 당업계에 공지된 것이라면 특별한 제한은 없으나, 예를 들면, 리포좀, 리포펙타민, 덴드리머, 마이셀, 다공성 실리카 입자, 아미노클레이, 금나노입자, 자성나노입자, 그래핀, 산화그래핀, 키토산, 덱스트란, 펙틴, 이산화망간 2차원시트, PVA, 젤라틴, 실리카, 글라스입자, 프로타민, 엑소좀, 폴리에틸렌이민, N-부틸 시아노아크릴레이트, 젤폼, 젤라틴, 에탄올, 나노크리스탈, 나노튜브, 탄소나노입자, 히알루론산, 산화철, 폴리락틱산, 폴리부틸시아노아크릴레이트, 알부민, 리피드입자, 폴리에틸렌글라이콜, 폴리-L-굴루로닉 알지네이트, 폴리글리콜릭-폴리액틱산, 폴리디옥사논, 폴리글리콜산-co-카프로락톤, 폴리프로필렌 및 하이드로겔로 이루어진 군에서 선택된 적어도 하나일 수 있으나, 바람직하게는 높은 RNA 담지율, 서방성, 생분해성 등의 장점을 갖는 다공성 실리카 입자일 수 있다.The siRNA or dsRNA of the present invention may be one that is carried on a carrier and is capable of carrying RNA molecules in the kind of the carrier. The siRNA or dsRNA is not particularly limited as long as it is known in the art. For example, liposome, lipofectamine , Dendrimers, micelles, porous silica particles, amino clays, gold nanoparticles, magnetic nanoparticles, graphene, oxidized graphene, chitosan, dextran, pectin, manganese dioxide two-dimensional sheet, PVA, gelatin, silica, But are not limited to, polyvinyl pyrrolidone, polyvinyl pyrrolidone, polyvinyl pyrrolidone, polyvinyl pyrrolidone, polyvinyl pyrrolidone, polyvinyl pyrrolidone, polyvinyl pyrrolidone, polyvinyl pyrrolidone, polyvinyl pyrrolidone, , Polyethylene glycol, poly-L-guluronic alginate, polyglycolic-polyacrylic acid, polydioxanone, polyglycolic acid-co-caprolactone, polypropylene and hydrogel But it may preferably be a porous silica particle having advantages such as high RNA retention, sustained release, biodegradability and the like.
본 발명의 siRNA 또는 dsRNA는 다공성 실리카 입자에 담지된 것일 수 있고, 상기 입자는 실리카(SiO2) 소재의 입자이며, 나노 사이즈의 입경을 갖는다.The siRNA or dsRNA of the present invention may be carried on a porous silica particle, and the particle is a particle of silica (SiO 2 ) and has a nano-sized particle size.
상기 다공성 실리카 입자는 다공성 입자로서, 나노사이즈의 기공을 갖아 그 표면 및/또는 기공 내부에 본 발명의 siRNA 또는 dsRNA와 같은 생리활성물질을 담지할 수 있다.The porous silica particles are porous particles having nano-sized pores and can carry physiologically active substances such as siRNA or dsRNA of the present invention on the surface and / or pores thereof.
상기 다공성 실리카 입자는 생분해성 입자로서, 생리활성물질을 담지하여 체내에 투여되었을 때 체내에서 생분해되면서 생리활성물질을 방출할 수 있다. 즉, 다공성 실리카 입자가 생분해되면서 생리활성물질이 방출되는데, 본 발명에 따른 다공성 실리카 입자는 체내에서 서서히 분해되어 담지된 생리활성물질이 서방성을 갖도록 할 수 있다. 예를 들면, 하기 수학식 1의 흡광도의 비가 1/2이 되는 t가 20 이상이다:The porous silica particles are biodegradable particles, and when physiologically active substances are supported on the body, they can be biodegraded in the body and release physiologically active substances. That is, biodegradation of the porous silica particles results in the release of the physiologically active substance. The porous silica particles according to the present invention may be slowly decomposed in the body, so that the supported physiologically active substance may have sustained release properties. For example, t at which the ratio of the absorbance of the following formula (1) is 1/2 is 20 or more:
[수학식 1][Equation 1]
At/A0 A t / A 0
(식 중, A0는 상기 다공성 실리카 입자 1mg/ml 현탁액 5ml를 직경 50kDa의 기공을 갖는 원통형 투과막에 넣고 측정된 다공성 실리카 입자의 흡광도이고,Wherein A 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles in a cylindrical permeable membrane having pores having a diameter of 50 kDa,
상기 투과막 외부에는 상기 투과막과 접하며, 상기 현탁액과 동일한 용매 15ml가 위치하고, 상기 투과막 내외부는 37℃에서 60rpm 수평 교반되며,15 ml of the same solvent as that of the suspension was placed on the outside of the permeable membrane, and the inside and the outside of the permeable membrane were horizontally stirred at 60 rpm at 37 ° C,
상기 현탁액의 pH는 7.4이고,The pH of the suspension was 7.4,
At는 상기 A0의 측정시로부터 t시간 경과 후에 측정된 다공성 실리카 입자의 흡광도임).A t is the absorbance of the porous silica particles measured after passage of time t from the measurement of A 0 ).
상기 수학식 1은 다공성 실리카 입자가 체내와 유사한 환경에서 어느 정도의 속도로 분해되는지를 의미하는 것이다.Equation (1) means that the porous silica particles are decomposed at a certain rate in an environment similar to the inside of the body.
상기 수학식 1에서의 흡광도 A0, At는 예를 들면 원통형 투과막에 다공성 실리카 입자 및 현탁액을 넣고, 투과막 외부에도 동일한 현탁액을 넣고 측정된 것일 수 있다.The absorbances A 0 and A t in Equation (1) may be measured by, for example, placing the porous silica particles and suspension in a cylindrical permeable membrane and putting the same suspension in the outside of the permeable membrane.
상기 다공성 실리카 입자는 생분해성으로서, 현탁액 내에서 서서히 분해될 수 있고, 직경 50kDa는 약 5nm에 해당하는 것으로서 생분해된 다공성 실리카 입자는 직경 50kDa의 투과막을 통과할 수 있고, 원통형 투과막은 60rpm 수평 교반 하에 있으므로 현탁액이 고루 섞일 수 있으며 분해된 다공성 실리카 입자는 투과막 외부로 나올 수 있다.The porous silica particles are biodegradable and can be slowly decomposed in the suspension. The porous silica particles having a diameter of 50 kDa corresponding to about 5 nm can pass through the permeable membrane having a diameter of 50 kDa and the cylindrical permeable membrane can be permeated under a condition of 60 rpm Therefore, the suspension may be uniformly mixed, and the decomposed porous silica particles may come out of the permeable membrane.
상기 수학식 1에서의 흡광도는 예를 들어 투과막 외부의 현탁액이 새로운 현탁액으로 교체되는 환경 하에 측정된 것일 수 있다. 현탁액은 지속적으로 교체되는 것일 수 있고, 일정 기간마다 교체되는 것일 수 있으며, 상기 일정 기간은 정기 또는 비정기적인 기간일 수 있다. 예를 들어 1시간 내지 1주일의 범위 내에서, 1시간 간격, 2시간 간격, 3시간 간격, 6시간 간격, 12시간 간격, 24시간 간격, 2일 간격, 3일 간격, 4일 간격, 7일 간격 등으로 교체될 수 있으나 이에 제한되는 것은 아니다The absorbance in Equation (1) may be measured under an environment in which, for example, the suspension outside the permeable membrane is replaced with a new suspension. The suspension may be constantly being replaced, replaced at regular intervals, and the period may be a periodic or irregular period. For example, in the range of 1 hour to 1 week, an interval of 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 7 Day interval, etc., but is not limited thereto
상기 흡광도의 비가 1/2가 된다는 것은 t시간 후에 흡광도가 초기 흡광도의 절반이 된다는 것인 바, 이는 다공성 실리카 입자의 대략 절반이 분해되었다는 의미이다.When the ratio of the absorbance is 1/2, the absorbance becomes half of the initial absorbance after t time, meaning that about half of the porous silica particles are decomposed.
상기 현탁액은 완충용액일 수 있고, 구체적인 예를 들면, PBS(phosphate buffered saline) 및 SBF(simulated body fluid)로 이루어진 군에서 선택된 1종 이상일 수 있으며, 보다 구체적으로는 PBS일 수 있다.The suspension may be a buffer solution, and may be at least one selected from the group consisting of, for example, PBS (phosphate buffered saline) and SBF (simulated body fluid), and more specifically, PBS.
상기 수학식 1의 흡광도의 비가 1/2이 되는 t가 20 이상으로, 예를 들면 t는 20 내지 120일 수 있고, 예를 들어 상기 범위 내에서 20 내지 96, 20 내지 72, 30 내지 70, 40 내지 70, 50 내지 65 등일 수 있으나, 이에 제한되는 것은 아니다.T may be 20 or more, for example, t may be 20 to 120, for example, 20 to 96, 20 to 72, 30 to 70, 40 to 70, 50 to 65, and the like.
상기 다공성 실리카 입자는 상기 수학식 1의 흡광도의 비가 1/5가 되는 t가 예를 들면 70 내지 140일 수 있고, 예를 들어 상기 범위 내에서 80 내지 140, 80 내지 120, 80 내지 110, 70 내지 140, 70 내지 120, 70 내지 110 등일 수 있으나, 이에 제한되는 것은 아니다.The porous silica particles may have a ratio t of 1/5 of the absorbance of the formula (1), for example, 70 to 140, and may be, for example, in the range of 80 to 140, 80 to 120, 80 to 110, To 140, 70 to 120, 70 to 110, and the like.
상기 다공성 실리카 입자는 상기 수학식 1의 흡광도의 비가 1/20가 되는 t가 예를 들면 130 내지 220일 수 있고, 예를 들어 상기 범위 내에서 130 내지 200, 140 내지 200, 140 내지 180, 150 내지 180 등일 수 있으나, 이에 제한되는 것은 아니다.For example, the porous silica particles may have a ratio t of 1/20 of the absorbance of the formula (1), for example, 130 to 220, and for example, within the range of 130 to 200, 140 to 200, 140 to 180, 150 To 180, and the like.
상기 다공성 실리카 입자는 측정되는 흡광도가 0.01 이하가 되는 t가 예를 들면 250 이상, 예를 들어, 300 이상, 350 이상, 400 이상, 500 이상, 1000 이상 등일 수 있으며, 그 상한은 2000일 수 있으나, 이에 제한되는 것은 아니다.The porous silica particles may have a t of, for example, not less than 250, such as not less than 300, not less than 350, not less than 400, not less than 500, not less than 1000, and the like, , But is not limited thereto.
상기 다공성 실리카 입자에서 상기 수학식 1의 흡광도의 비와 t는 높은 양의 상관 관계를 갖는 것으로서, 예를 들면 피어슨 상관 계수가 0.8 이상일 수 있고, 예를 들어, 0.9 이상, 0.95 이상일 수 있다.In the porous silica particle, the ratio of the absorbance of Equation 1 to t has a high positive correlation. For example, the Pearson correlation coefficient may be 0.8 or more, for example, 0.9 or more and 0.95 or more.
상기 수학식 1의 t는 다공성 실리카 입자가 체내와 유사한 환경에서 어느 정도의 속도로 분해되는지를 의미하는 것으로서, 이는 예를 들면 다공성 실리카 입자의 표면적, 입경, 기공 직경, 표면 및/또는 기공 내부의 치환기, 표면의 치밀함 정도 등을 조절함으로써 조절될 수 있다.T represents the degree of decomposition of the porous silica particles at a certain rate in an environment similar to that of the body. This means that the surface area, the particle size, the pore diameter, the surface of the porous silica particles, and / The substituent, the degree of compactness of the surface, and the like.
예를 들면, 입자의 표면적을 증가시켜 t를 감소시키거나, 표면적을 감소시켜 t를 증가시킬 수 있다. 표면적은 입자의 직경, 기공의 직경을 조절함으로써 조절될 수 있다. 또한, 표면 및/또는 기공 내부에 치환기를 위치시켜 다공성 실리카 입자가 환경(용매 등)에 직접 노출되는 것을 줄여 t를 증가시킬 수 있다. 또한, 다공성 실리카 입자에 생리활성물질을 담지시키고 생리활성물질과 다공성 실리카 입자 간의 친화도를 증가시켜, 다공성 실리카 실리카 입자가 환경에 직접 노출되는 것을 줄여 t를 증가시킬 수 있다. 또한, 입자의 제조시에 표면을 보다 치밀하게 제조하여 t를 증가시킬 수도 있다. 상기에는 수학식 1의 t를 조절할 수 있는 다양한 예시를 서술하였으나, 이에 제한되는 것은 아니다.For example, the surface area of a particle can be increased to decrease t, or the surface area can be decreased to increase t. The surface area can be controlled by controlling the diameter of the particles and the diameter of the pores. It is also possible to increase the t by placing substituents in the surface and / or pores to reduce the direct exposure of the porous silica particles to the environment (solvent, etc.). Also, it is possible to increase the affinity between the physiologically active substance and the porous silica particles by supporting the physiologically active substance on the porous silica particles, and to reduce the direct exposure of the porous silica silica particles to the environment, thereby increasing t. It is also possible to increase the t by fabricating the surface more densely during the production of the particles. While various examples have been described above for adjusting t in Equation 1, the present invention is not limited thereto.
상기 다공성 실리카 입자는 예를 들면 구형 입자일 수 있으나, 이에 제한되는 것은 아니다.The porous silica particles may be, for example, spherical particles, but are not limited thereto.
상기 다공성 실리카 입자는 평균 직경이 예를 들면 100nm 내지 1000nm일 수 있고, 예를 들어 상기 범위 내에서 예를 들면 100nm 내지 800nm, 100nm 내지 500nm, 100nm 내지 400nm, 100nm 내지 300nm, 100nm 내지 200nm일 수 있으나, 이에 제한되는 것은 아니다.The average diameter of the porous silica particles may be, for example, 100 nm to 1000 nm, and may be within the above range, for example, 100 nm to 800 nm, 100 nm to 500 nm, 100 nm to 400 nm, 100 nm to 300 nm, , But is not limited thereto.
상기 다공성 실리카 입자는 평균 기공 직경이 예를 들면 1nm 내지 100nm일 수 있고, 예를 들어 상기 범위 내에서 예를 들면 4nm 내지 100nm, 4nm 내지 50nm, 4nm 내지 30nm, 10nm 내지 30nm일 수 있으나, 이에 제한되는 것은 아니다. 상기와 같은 큰 직경을 가져 다량의 생리활성물질을 담지할 수 있고, 크기가 큰 생리활성물질의 담지도 가능하다.The porous silica particles may have an average pore diameter of, for example, 1 nm to 100 nm, and may be within the above range, for example, 4 nm to 100 nm, 4 nm to 50 nm, 4 nm to 30 nm, 10 nm to 30 nm, It is not. It is possible to carry a large amount of the physiologically active substance and to carry the physiologically active substance having a large size.
상기 다공성 실리카 입자는 BET 표면적이 예를 들면 200m2/g 내지 700m2/g일 수 있다. 예를 들어 상기 범위 내에서 200m2/g 내지 700m2/g, 200m2/g 내지 650m2/g, 250m2/g 내지 650m2/g, 300m2/g 내지 700m2/g, 300m2/g 내지 650m2/g, 300m2/g 내지 600m2/g, 300m2/g 내지 550m2/g, 300m2/g 내지 500m2/g, 300m2/g 내지 450m2/g 등일 수 있으나, 이에 제한되는 것은 아니다.The porous silica particles may have a BET surface area of, for example, 200 m 2 / g to 700 m 2 / g. For example, within the range of 200m 2 / g to 700m 2 / g, 200m 2 / g to 650m 2 / g, 250m 2 / g to 650m 2 / g, 300m 2 / g to 700m 2 / g, 300m 2 / g to 650 m 2 / g, 300 m 2 / g to 600 m 2 / g, 300 m 2 / g to 550 m 2 / g, 300 m 2 / g to 500 m 2 / g, 300 m 2 / g to 450 m 2 / But is not limited thereto.
상기 다공성 실리카 입자는 g당 부피가 예를 들면 0.7ml 내지 2.2ml일 수 있다. 예를 들어 상기 범위 내에서 0.7ml 내지 2.0ml, 0.8ml 내지 2.2ml, 0,8 ml 내지 2.0ml, 0.9 ml 내지 2.0ml, 1.0 ml 내지 2.0ml 등일 수 있으나, 이에 제한되는 것은 아니다. g당 부피가 과도하게 작아지면 분해 속도가 너무 빨라질 수 있고, 과도하게 큰 입자는 제조가 어렵거나, 온전한 형상을 가질 수 없을 수 있다.The porous silica particles may have a volume per gram of, for example, 0.7 ml to 2.2 ml. For example, it may be within the range of 0.7 ml to 2.0 ml, 0.8 ml to 2.2 ml, 0.8 ml to 2.0 ml, 0.9 ml to 2.0 ml, 1.0 ml to 2.0 ml, and the like. If the volume per gram is too small, the rate of decomposition may become too high, and excessively large particles may be difficult to manufacture or may not have a perfect shape.
상기 다공성 실리카 입자는 외부 표면 및/또는 기공 내부에 친수성 치환기 및/또는 소수성 치환기가 존재할 수 있다. 예를 들면 표면 및 기공 내부 모두 친수성 치환기만 존재하거나, 소수성 치환기만 존재할 수도 있고, 표면 또는 기공 내부에만 친수성 치환기가 존재하거나, 소수성 치환기가 존재할 수도 있고, 표면에는 친수성 치환기, 기공 내부에는 소수성 치환기가 존재할 수도 있고, 그 반대의 경우도 가능하다.The porous silica particles may have a hydrophilic substituent and / or a hydrophobic substituent on the outer surface and / or inside the pore. For example, only hydrophilic substituents may be present on both the surface and the pores, only hydrophobic substituents may be present, hydrophilic substituents may be present only on the surface or pores, hydrophobic substituents may be present, hydrophilic substituents may be present on the surface, Or vice versa, and vice versa.
상기 다공성 실리카 입자에 담지된 생리활성물질의 방출은 주로 입자의 분해에 의해 수행되는 것인 바, 상기 치환기의 조절로 생리활성물질 방출 환경에 대한 다공성 실리카 입자의 상호 작용이 조절되어 입자 자체의 분해 속도가 조절되어 생리활성물질 방출 속도가 조절될 수 있고, 또한, 생리활성물질은 입자로부터 확산되어 방출될 수도 있는데, 상기 치환기의 조절로 생리활성물질의 입자에 대한 결합력이 조절되어 생리활성물질 방출이 조절될 수 있다.The release of the physiologically active substance carried on the porous silica particles is mainly performed by the decomposition of the particles, and the interaction of the porous silica particles with respect to the release environment of the physiologically active substance is controlled by the control of the substituent, The rate of release of the physiologically active substance can be controlled by adjusting the rate of the physiologically active substance, and the physiologically active substance can be diffused and released from the particles. Can be adjusted.
또한, 난용성(소수성) 생리활성물질과의 결합력 증진을 위해 기공 내부에는 소수성 치환기가 존재하고, 사용, 제형화의 용이성 등의 측면에서 입자의 표면은 친수성 치환기가 존재하도록 하는 등의 처리도 가능하다.In order to improve the bonding strength with the poorly soluble (hydrophobic) physiologically active substance, a hydrophobic substituent is present inside the pore, and a hydrophilic substituent may be present on the surface of the particle in view of ease of use and formulation Do.
친수성 치환기는 예를 들면 알데하이드기, 케토기, 카바메이트기, 설페이트기, 설포네이트기, 아미노기, 아민기, 아미노알킬기, 실릴기, 카르복실기, 술폰산기, 티올기, 암모늄기, 설프히드릴기, 포스페이트기, 에스터기, 이미드기, 싸이오이미드기, 케토기, 에터기, 인덴기, 설포닐기, 메틸포스포네이트기, 폴리에틸렌글리콜기, 치환 또는 비치환된 C1 내지 C30의 알킬기, 치환 또는 비치환된 C3 내지 C30의 사이클로알킬기, 치환 또는 비치환된 C6 내지 C30의 아릴기 및 C1 내지 C30의 에스테르기 등을 들 수 있고, 소수성 치환기는 예를 들면 치환 또는 비치환된 C1 내지 C30의 알킬기, 치환 또는 비치환된 C3 내지 C30의 사이클로알킬기, 치환 또는 비치환된 C6 내지 C30의 아릴기, 치환 또는 비치환된 C2 내지 C30의 헤테로아릴기, 할로겐기, C1 내지 C30의 에스테르기, 및 할로겐 함유기 등을 들 수 있다.The hydrophilic substituent may be, for example, an aldehyde group, a keto group, a carbamate group, a sulfate group, a sulfonate group, an amino group, an amine group, an aminoalkyl group, a silyl group, a carboxyl group, a sulfonic acid group, a thiol group, A substituted or unsubstituted C 1 to C 30 alkyl group, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C1 to C30 alkyl group, An unsubstituted C 3 to C 30 cycloalkyl group, a substituted or unsubstituted C 6 to C 30 aryl group, and a C 1 to C 30 ester group, and the hydrophobic substituent includes, for example, a substituted or unsubstituted A substituted or unsubstituted C6 to C30 aryl group, a substituted or unsubstituted C2 to C30 heteroaryl group, a halogen group, a C1 to C30 alkyl group, a substituted or unsubstituted C3 to C30 aryl group, Esthetic And the like group, and a halogen-containing group.
상기 '치환 또는 비치환'에 있어서, '치환'되는 작용기는 알데하이드기, 케토기, 카바메이트기, 설페이트기, 설포네이트기, 아미노기, 아민기, 아미노알킬기, 실릴기, 카르복실기, 술폰산기, 티올기, 암모늄기, 설프히드릴기, 포스페이트기, 에스터기, 이미드기, 싸이오이미드기, 케토기, 에터기, 인덴기, 설포닐기, 메틸포스포네이트기 및 폴리에틸렌글리콜기로 이루어진 군에서 선택된 적어도 하나일 수 있으나 이에 제한되지 않는다.The 'substituted' functional group in the 'substituted or unsubstituted' may be an aldehyde group, a keto group, a carbamate group, a sulfate group, a sulfonate group, an amino group, an amine group, an aminoalkyl group, a silyl group, a carboxyl group, At least one member selected from the group consisting of an alkyl group, an alkoxy group, an alkoxy group, an ammonium group, a sulfhydryl group, a phosphate group, an ester group, an imide group, a thioimide group, a keto group, an ether group, an indene group, a sulfonyl group, a methylphosphonate group and a polyethylene glycol group But is not limited thereto.
또한, 상기 다공성 실리카 입자는 외부 표면 및/또는 기공 내부가 양전하 및/또는 음전하로 대전된 것일 수 있다. 예를 들면 표면 및 기공 내부 모두 양전하로 대전되거나, 음전하로 대전될 수 있고, 표면 또는 기공 내부만 양전하로 대전되거나, 음전하로 대전될 수 있고, 표면은 양전하, 기공 내부는 음전하로 대전될 수 있고, 그 반대의 경우도 가능하다.Further, the porous silica particles may be those in which the outer surface and / or the inside of the pores are positively charged and / or negatively charged. For example, both the surface and the pores can be positively charged, negatively charged, only positively charged on the surface or pore, negatively charged, surface can be positively charged, negatively charged inside the pore , And vice versa.
상기 대전은 예를 들면 양이온성 치환기 또는 음이온성 치환기가 존재함으로써 된 것일 수 있다.The charging may be performed, for example, by the presence of a cationic substituent or an anionic substituent.
양이온성 치환기는 예를 들면 염기성기로서 아미노기, 그 외 질소함유기 등일 수 있고, 음이온성 치환기는 예를 들면 산성기로서 카르복시기(-COOH), 술폰산기(-SO3H), 티올기(-SH) 등일 수 있으나, 이에 제한되는 것은 아니다.The cationic substituent may be, for example, an amino group or other nitrogen-containing group as a basic group, and the anionic substituent may be, for example, a carboxyl group (-COOH), a sulfonic acid group (-SO 3 H) SH), and the like.
마찬가지로 상기 대전에 의해 상기 치환기의 조절로 생리활성물질 방출 환경에 대한 다공성 실리카 입자의 상호 작용이 조절되어 입자 자체의 분해 속도가 조절되어 생리활성물질 방출 속도가 조절될 수 있고, 또한, 생리활성물질은 입자로부터 확산되어 방출될 수도 있는데, 상기 치환기의 조절로 생리활성물질의 입자에 대한 결합력이 조절되어 생리활성물질 방출이 조절될 수 있다.Similarly, by the above-mentioned charging, the interaction of the porous silica particles with respect to the release environment of the physiologically active substance is controlled by controlling the substituent, so that the rate of decomposition of the particles themselves can be controlled to control the release rate of the physiologically active substance, May be diffused and released from the particles. By controlling the substituent, the binding force of the physiologically active substance to the particles can be controlled and the release of the physiologically active substance can be controlled.
또한, 상기 다공성 실리카 입자는 그 표면 및/또는 기공 내부에 상기 외에 생리활성물질의 담지, 생리활성물질의 표적 세포로의 이동, 그 외 기타 목적을 위한 물질의 담지 또는 그외 추가 치환기의 결합 등을 위한 치환기가 존재할 수 있으며, 이에 결합된 항체, 리간드, 세포투과성 펩타이드 또는 압타머 등을 더 포함할 수 있다.In addition, the porous silica particles may be coated on the surface and / or inside of the pores by carrying a physiologically active substance, transferring a physiologically active substance to a target cell, carrying a substance for other purposes, And may further include an antibody, a ligand, a cell permeable peptide, an umbilical cord or the like coupled thereto.
전술한 표면 및/또는 기공 내부의 치환기, 전하, 결합물질 등은 예를 들면 표면 개질에 의해 부가될 수 있다.Substituents, charges, bonding substances, etc. in the surface and / or pores described above can be added, for example, by surface modification.
상기 표면 개질은 예를 들면 도입하고자 하는 치환기를 갖는 화합물을 입자와 반응시켜 수행할 수 있고, 상기 화합물은 예를 들면 C1 내지 C10 알콕시기를 갖는 알콕시실란일 수 있으나, 이에 제한되는 것은 아니다. 상기 알콕시실란은 상기 알콕시기를 1개 이상 갖는 것으로서, 예를 들면 1 내지 3개 가질 수 있고, 알콕시기가 결합되지 않은 부위에 도입하고자 하는 치환기가 있거나 이로 치환된 치환기가 있을 수 있다.The surface modification can be performed, for example, by reacting a compound having a substituent to be introduced with a particle, and the compound may be, for example, an alkoxysilane having a C1 to C10 alkoxy group, but is not limited thereto. The alkoxysilane is one having at least one of the above-mentioned alkoxy groups and may have, for example, 1 to 3 substituents, and may have a substituent to be introduced or a substituted substituent at a site where an alkoxy group is not bonded.
상기 다공성 실리카 입자는 예를 들면 소기공의 입자 제조 및 기공 확장 공정을 거쳐 제조된 것일 수 있고, 필요에 따라 하소(calcination) 공정, 표면 개질 공정 등을 더 거쳐 제조된 것일 수 있다. 하소 및 표면 개질 공정을 모두 거친 경우는 하소 이후에 표면 개질 된 것일 수 있다.The porous silica particles may be prepared by, for example, preparing fine pore particles and a pore expansion process, and may be manufactured by further performing a calcination process, a surface modification process or the like, if necessary. When both the calcination and the surface modification process are performed, the surface modification may be performed after the calcination.
상기 소기공의 입자는 예를 들면 평균 기공 직경이 1nm 내지 5nm인 입자일 수 있다.The particles of the small pores may be, for example, particles having an average pore diameter of 1 nm to 5 nm.
상기 소기공의 입자는 용매에 계면활성제와 실리카 전구물질을 넣고 교반 및 균질화시켜 얻어질 수 있다.The small pore particles can be obtained by adding a surfactant and a silica precursor to a solvent and stirring and homogenizing the same.
상기 용매는 물 및/또는 유기용매일 수 있고, 유기용매는 예를 들면 1,4-디옥산 등의 에테르류(특히 고리형상 에테르류); 클로로포름, 염화메틸렌, 4염화탄소, 1,2-디클로로에탄, 디클로로에틸렌, 트리클로로에틸렌, 퍼클로로에틸렌, 디클로로프로판, 염화아밀, 1,2-디브로모에탄 등의 할로겐화 탄화수소류; 아세톤, 메틸이소부틸케톤, γ-부티로락톤, 1,3-디메틸-이미다졸리디논, 메틸에틸케톤, 시클로헥사논, 시클로펜타논, 4-하이드록시-4-메틸-2-펜타논 등의 케톤류; 벤젠, 톨루엔, 크실렌, 테트라메틸벤젠 등의 탄소계 방향족류 ; N,N-디메틸포름아미드, N,N-디부틸포름아미드, N,N-디메틸아세트아미드, N-메틸피롤리돈 등의 알킬아미드류; 메탄올, 에탄올, 프로판올, 부탄올 등의 알코올류; 에틸렌글리콜모노에틸에테르, 에틸렌글리콜모노메틸에테르, 에틸렌글리콜모노부틸에테르, 디에틸렌글리콜모노에틸에테르, 디에틸렌글리콜모노메틸에테르, 디에틸렌글리콜모노부틸에테르, 프로필렌글리콜모노메틸에테르, 프로필렌글리콜모노에틸에테르, 디프로필렌글리콜디에틸에테르, 트리에틸렌글리콜모노에틸에테르 등의 글리콜에테르류(셀로솔브); 그외 디메틸아세트아미드(DMAc), N,N-디에틸아세트아미드, 디메틸포름아미드(DMF), 디에틸포름아미드(DEF), N,N-디메틸아세트아미드(DMAc), N-메틸피롤리돈(NMP), N-에틸피롤리돈(NEP), 1,3-디메틸-2-이미다졸리디논, N,N-디메틸메톡시아세트아미드, 디메틸술폭사이드, 피리딘, 디메틸술폰, 헥사메틸포스포아미드, 테트라메틸우레아, N-메틸카르로락탐, 테트라히드로퓨란, m-디옥산, P-디옥산, 1,2-디메톡시에탄 등을 사용할 수 있고, 구체적으로는 알코올, 보다 구체적으로 메탄올을 사용할 수 있으나, 이에 제한되는 것은 아니다.The solvent can be water and / or organic solvent, and the organic solvent includes, for example, ethers (especially cyclic ethers) such as 1,4-dioxane; Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone,? -Butyrolactone, 1,3-dimethyl-imidazolidinone, methyl ethyl ketone, cyclohexanone, cyclopentanone, 4-hydroxy- Of ketones; Carbon-based aromatic compounds such as benzene, toluene, xylene, and tetramethylbenzene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether , Glycol ethers (cellosolve) such as dipropylene glycol diethyl ether and triethylene glycol monoethyl ether; Other solvents such as dimethylacetamide (DMAc), N, N-diethylacetamide, dimethylformamide (DMF), diethylformamide (DEF), N, N-dimethylacetamide (DMAc) NMP), N-ethylpyrrolidone (NEP), 1,3-dimethyl-2-imidazolidinone, N, N-dimethylmethoxyacetamide, dimethylsulfoxide, pyridine, dimethylsulfone, hexamethylphosphoamide , Tetramethylurea, N-methylcarrolactam, tetrahydrofuran, m-dioxane, P-dioxane, 1,2-dimethoxyethane and the like can be used, and specifically, alcohols, more specifically, methanol But is not limited thereto.
상기 용매에 있어, 물과 유기 용매의 혼합 용매 사용시 그 비율은 예를 들면 물과 유기용매를 1: 0.7 내지 1.5의 부피비, 예를 들어, 1: 0.8 내지 1.3의 부피비로 사용할 수 있으나, 이에 제한되는 것은 아니다.In the solvent, when the mixed solvent of water and the organic solvent is used, for example, water and the organic solvent may be used in a volume ratio of 1: 0.7 to 1.5, for example, 1: 0.8 to 1.3 by volume, It is not.
상기 계면활성제는 예를 들면 CTAB(cetyltrimethylammonium bromide), TMABr(hexadecyltrimethylammonium bromide), TMPrCl(hexadecyltrimethylpyridinium chloride), TMACl(tetramethylammonium chloride) 등일 수 있고, 구체적으로는 CTAB를 사용할 수 있다.The surfactant may be, for example, CTAB (cetyltrimethylammonium bromide), hexadecyltrimethylammonium bromide (TMABr), hexadecyltrimethylpyridinium chloride (TMPrCl), tetramethylammonium chloride (TMACl), and the like.
상기 계면활성제는 예를 들면 용매 1리터당 1g 내지 10g, 예를 들어 상기 범위 내에서 1g 내지 8g, 2g 내지 8g, 3g 내지 8g 등의 양으로 첨가될 수 있으나, 이에 제한되는 것은 아니다.The surfactant may be added in an amount of, for example, 1 g to 10 g per liter of solvent, for example, in the range of 1 g to 8 g, 2 g to 8 g, 3 g to 8 g, and the like.
상기 실리카 전구 물질은 용매에 계면활성제를 첨가하여 교반한 후에 첨가될 수 있다. 실리카 전구물질은 예를 들면 TMOS(Tetramethyl orthosilicate)일 수 있으나, 이에 제한되는 것은 아니다.The silica precursor may be added after stirring and adding a surfactant to the solvent. The silica precursor may be, for example, TMOS (Tetramethyl orthosilicate), but is not limited thereto.
상기 교반은 예를 들면 10분 내지 30분간 수행될 수 있으나, 이에 제한되는 것은 아니다.The stirring may be carried out, for example, for 10 minutes to 30 minutes, but is not limited thereto.
상기 실리카 전구물질은 예를 들면 용매 1리터당 0.5ml 내지 5ml, 예를 들어 상기 범위 내에서 0.5ml 내지 4ml, 0.5ml 내지 3ml, 0.5ml 내지 2ml, 1ml 내지 2ml 등으로 첨가될 수 있으나, 이에 제한되는 것은 아니고, 필요에 따라 촉매로서 수산화나트륨을 더 사용할 수 있으며, 이는 용매에 계면활성제를 첨가한 후 실리카 전구물질의 첨가 전에 교반하면서 첨가될 수 있다.The silica precursor may be added, for example, in an amount of 0.5 ml to 5 ml per liter of solvent, for example, 0.5 ml to 4 ml, 0.5 ml to 3 ml, 0.5 ml to 2 ml, 1 ml to 2 ml, But may be further added with sodium hydroxide as a catalyst as needed, which may be added with stirring before addition of the surfactant to the solvent, followed by addition of the silica precursor.
상기 수산화나트륨은 예를 들면 1M 수산화나트륨 수용액 기준으로 용매 1리터당 0.5ml 내지 8ml, 예를 들어 상기 범위 내에서 0.5ml 내지 5ml, 0.5ml 내지 4ml, 1ml 내지 4ml, 1ml 내지 3ml 2ml 내지 3ml 등일 수 있으나, 이에 제한되는 것은 아니다.The sodium hydroxide may be, for example, 0.5 ml to 8 ml per liter of solvent, for example 1 ml of sodium hydroxide solution, 0.5 ml to 5 ml, 0.5 ml to 4 ml, 1 ml to 4 ml, 1 ml to 3 ml, But is not limited thereto.
상기 실리카 전구 물질의 첨가 후에 용액을 교반하며 반응시킬 수 있다. 교반은 예를 들면 2시간 내지 15시간 할 수 있고, 예를 들어 상기 범위 내에서 3시간 내지 15시간, 4시간 내지 15시간, 4시간 내지 13시간, 5시간 내지 12시간, 6 시간 내지 12시간, 6시간 내지 10시간 등일 수 있으나, 이에 제한되는 것은 아니다. 교반 시간(반응 시간)이 너무 짧은 경우에는 결정핵 생성(nucleation)이 부족할 수 있다.After the addition of the silica precursor, the solution can be reacted with stirring. The stirring may be carried out for example for 2 hours to 15 hours, for example, within the above range for 3 hours to 15 hours, 4 hours to 15 hours, 4 hours to 13 hours, 5 hours to 12 hours, 6 hours to 12 hours , 6 hours to 10 hours, and the like, but is not limited thereto. If the stirring time (reaction time) is too short, nucleation may be insufficient.
상기 교반 이후에는 용액을 숙성(aging)시킬 수 있다. 숙성은 예를 들면 8시간 내지 24시간 할 수 있고, 예를 들어 상기 범위 내에서 8시간 내지 20시간, 8시간 내지 18시간, 8시간 내지 16시간, 8시간 내지 14시간, 10시간 내지 16시간, 10시간 내지 14시간 등일 수 있으나, 이에 제한되는 것은 아니다.After agitation, the solution may be aged. The aging can be carried out, for example, for 8 hours to 24 hours, for example within the above range, 8 to 20 hours, 8 to 18 hours, 8 to 16 hours, 8 to 14 hours, 10 to 16 hours , 10 hours to 14 hours, and the like, but is not limited thereto.
이후, 반응산물을 세척 및 건조시켜 다공성 실리카 입자를 얻을 수 있고, 필요에 따라 세척 전에 미반응 물질의 분리가 선행될 수 있으며, 이는 예를 들면 원심분리로 상등액을 분리하여 수행될 수 있다.Thereafter, the reaction product may be washed and dried to obtain porous silica particles, and if necessary, separation of the unreacted material may be preceded by washing, for example, by separating the supernatant liquid by centrifugation.
상기 원심분리는 예를 들면 6,000 내지 10,000rpm으로 수행될 수 있으며, 그 시간은 예를 들면 3분 내지 60분, 예를 들어 상기 범위 내에서 3분 내지 30분, 3분 내지 30분, 5분 내지 30분 등으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The centrifugation can be performed at, for example, 6,000 to 10,000 rpm, for example, for 3 minutes to 60 minutes, for example, 3 minutes to 30 minutes, 3 minutes to 30 minutes, 5 minutes To 30 minutes, and the like, but the present invention is not limited thereto.
상기 세척은 물 및/또는 유기용매로 할 수 있고, 구체적으로는 용매별로 녹일 수 있는 물질이 상이하므로 물과 유기용매를 1회 또는 수회 번갈아 사용할 수 있으며, 물 또는 유기용매 단독으로도 1회 또는 수회 세척할 수 있다. 상기 수회는 예를 들면 2회 이상, 10회 이하, 예를 들어, 3회 이상 10회 이하, 4회 이상 8회 이하, 4회 이상 6회 이하 등일 수 있다.The washing may be performed using water and / or an organic solvent. Specifically, since the materials soluble in each solvent are different, water and an organic solvent may be used once or several times alternately. Alternatively, water or an organic solvent may be used once or several times It can be washed several times. The number of times may be, for example, 2 or more, 10 or less, for example, 3 or more and 10 or less, 4 or more or 8 or less, 4 or more or 6 or less, and the like.
상기 유기용매는 예를 들면 1,4-디옥산 등의 에테르류(특히 고리형상 에테르류); 클로로포름, 염화메틸렌, 4염화탄소, 1,2-디클로로에탄, 디클로로에틸렌, 트리클로로에틸렌, 퍼클로로에틸렌, 디클로로프로판, 염화아밀, 1,2-디브로모에탄 등의 할로겐화 탄화수소류; 아세톤, 메틸이소부틸케톤, γ-부티로락톤, 1,3-디메틸-이미다졸리디논, 메틸에틸케톤, 시클로헥사논, 시클로펜타논, 4-하이드록시-4-메틸-2-펜타논 등의 케톤류; 벤젠, 톨루엔, 크실렌, 테트라메틸벤젠 등의 탄소계 방향족류 ; N,N-디메틸포름아미드, N,N-디부틸포름아미드, N,N-디메틸아세트아미드, N-메틸피롤리돈 등의 알킬아미드류; 메탄올, 에탄올, 프로판올, 부탄올 등의 알코올류; 에틸렌글리콜모노에틸에테르, 에틸렌글리콜모노메틸에테르, 에틸렌글리콜모노부틸에테르, 디에틸렌글리콜모노에틸에테르, 디에틸렌글리콜모노메틸에테르, 디에틸렌글리콜모노부틸에테르, 프로필렌글리콜모노메틸에테르, 프로필렌글리콜모노에틸에테르, 디프로필렌글리콜디에틸에테르, 트리에틸렌글리콜모노에틸에테르 등의 글리콜에테르류(셀로솔브); 그외 디메틸아세트아미드(DMAc), N,N-디에틸아세트아미드, 디메틸포름아미드(DMF), 디에틸포름아미드(DEF), N,N-디메틸아세트아미드(DMAc), N-메틸피롤리돈(NMP), N-에틸피롤리돈(NEP), 1,3-디메틸-2-이미다졸리디논, N,N-디메틸메톡시아세트아미드, 디메틸술폭사이드, 피리딘, 디메틸술폰, 헥사메틸포스포아미드, 테트라메틸우레아, N-메틸카르로락탐, 테트라히드로퓨란, m-디옥산, P-디옥산, 1,2-디메톡시에탄 등을 사용할 수 있고, 구체적으로는 알코올, 보다 구체적으로 에탄올을 사용할 수 있으나, 이에 제한되는 것은 아니다.The organic solvent includes, for example, ethers such as 1,4-dioxane (particularly, cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone,? -Butyrolactone, 1,3-dimethyl-imidazolidinone, methyl ethyl ketone, cyclohexanone, cyclopentanone, 4-hydroxy- Of ketones; Carbon-based aromatic compounds such as benzene, toluene, xylene, and tetramethylbenzene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether , Glycol ethers (cellosolve) such as dipropylene glycol diethyl ether and triethylene glycol monoethyl ether; Other solvents such as dimethylacetamide (DMAc), N, N-diethylacetamide, dimethylformamide (DMF), diethylformamide (DEF), N, N-dimethylacetamide (DMAc) NMP), N-ethylpyrrolidone (NEP), 1,3-dimethyl-2-imidazolidinone, N, N-dimethylmethoxyacetamide, dimethylsulfoxide, pyridine, dimethylsulfone, hexamethylphosphoamide , Tetramethylurea, N-methylcarrolactam, tetrahydrofuran, m-dioxane, P-dioxane, 1,2-dimethoxyethane and the like can be used. Specifically, alcohols, more specifically, ethanol But is not limited thereto.
상기 세척은 원심분리 하에 수행될 수 있으며, 예를 들면 6,000 내지 10,000rpm으로 수행될 수 있으며, 그 시간은 예를 들면 3분 내지 60분, 예를 들어 상기 범위 내에서 3분 내지 30분, 3분 내지 30분, 5분 내지 30분 등으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The washing may be carried out under centrifugation, for example, at a speed of 6,000 to 10,000 rpm, for example, for 3 minutes to 60 minutes, for example 3 minutes to 30 minutes, Min to 30 min, 5 min to 30 min, and the like, but the present invention is not limited thereto.
상기 세척은 원심분리를 하지 않고, 필터로 입자를 걸러내어 수행될 수도 있다. 필터는 다공성 실리카 입자의 직경 이하의 기공을 가지는 것일 수 있다. 반응액을 그러한 필터로 걸러내면 입자만이 필터 위에 남고, 그 필터 위에 물 및/또는 유기용매를 부어 세척할 수 있다.The washing may be performed by filtering the particles with a filter without centrifugation. The filter may have pores smaller than the diameter of the porous silica particles. When the reaction solution is filtered with such a filter, only the particles remain on the filter, and water and / or an organic solvent can be poured onto the filter to wash.
상기 세척 시에 물과 유기용매를 1회 또는 수회 번갈아 사용할 수 있으며, 물 또는 유기용매 단독으로도 1회 또는 수회 세척할 수 있다. 상기 수회는 예를 들면 2회 이상, 10회 이하, 예를 들어, 3회 이상 10회 이하, 4회 이상 8회 이하, 4회 이상 6회 이하 등일 수 있다.The water and the organic solvent may be used once or several times at the time of the washing, and the water or the organic solvent may be washed once or several times. The number of times may be, for example, 2 or more, 10 or less, for example, 3 or more and 10 or less, 4 or more or 8 or less, 4 or more or 6 or less, and the like.
상기 건조는 예를 들면 20℃ 내지 100℃로 수행될 수 있으나, 이에 제한되는 것은 아니고, 진공 상태에서 수행될 수도 있다.The drying may be performed at, for example, 20 ° C to 100 ° C, but is not limited thereto, and may be performed in a vacuum state.
이후 상기 얻어진 다공성 실리카 입자의 기공을 확장하는데, 이는 기공 팽창제를 사용하여 수행될 수 있다.Then, the pores of the obtained porous silica particles are expanded, which can be carried out using a pore-expanding agent.
상기 기공 팽창제는 예를 들면 트리메틸벤젠, 트리에틸벤젠, 트리프로필벤젠, 트리부틸벤젠, 트리펜틸벤젠, 트리헥실벤젠, 톨루엔, 벤젠 등을 사용할 수 있고, 구체적으로, 트리메틸벤젠을 사용할 수 있으나, 이에 제한되는 것은 아니다.As the pore-expanding agent, for example, trimethylbenzene, triethylbenzene, tripropylbenzene, tributylbenzene, tripentylbenzene, trihexylbenzene, toluene, benzene and the like can be used. Specifically, trimethylbenzene can be used. But is not limited to.
또한, 상기 기공 팽창제는 예를 들면 N,N-디메틸헥사데실아민(N,N-dimethylhexadecylamine,DMHA)를 사용할 수 있으나, 이에 제한되는 것은 아니다.The pore-expanding agent may be, for example, N, N-dimethylhexadecylamine (DMHA), but is not limited thereto.
상기 기공 확장은 예를 들면 용매 중의 다공성 실리카 입자를 기공 팽창제와 혼합하고, 가열하여 반응시켜 수행될 수 있다.The pore expansion can be performed, for example, by mixing the porous silica particles in a solvent with a pore-expanding agent, and heating and reacting.
상기 용매는 예를 들면 물 및/또는 유기용매일 수 있고, 유기용매는 예를 들면 1,4-디옥산 등의 에테르류(특히 고리형상 에테르류); 클로로포름, 염화메틸렌, 4염화탄소, 1,2-디클로로에탄, 디클로로에틸렌, 트리클로로에틸렌, 퍼클로로에틸렌, 디클로로프로판, 염화아밀, 1,2-디브로모에탄 등의 할로겐화 탄화수소류; 아세톤, 메틸이소부틸케톤, 시클로헥산온 등의 케톤류; 벤젠, 톨루엔, 크실렌 등의 탄소계 방향족류 ; N,N-디메틸포름아미드, N,N-디부틸포름아미드, N,N-디메틸아세트아미드, N-메틸피롤리돈 등의 알킬아미드류; 메탄올, 에탄올, 프로판올, 부탄올 등의 알코올류; 등을 사용할 수 있고, 구체적으로는 알코올, 보다 구체적으로 에탄올을 사용할 수 있으나, 이에 제한되는 것은 아니다.The solvent can be, for example, water and / or organic solvents, and the organic solvent can be, for example, ethers (especially cyclic ethers) such as 1,4-dioxane; Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone, and cyclohexanone; Carbon-based aromatic compounds such as benzene, toluene and xylene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Etc., and specifically ethanol, more specifically ethanol, may be used, but the present invention is not limited thereto.
상기 다공성 실리카 입자는 예를 들면 용매 1리터당 10g 내지 200g, 예를 들어 상기 범위 내에서 10g 내지 150g, 10g 내지 100g, 30g 내지 100g, 40g 내지 100g, 50g 내지 100g, 50g 내지 80g, 60g 내지 80g 등의 비율로 첨가될 수 있으나, 이에 제한되는 것은 아니다.The porous silica particles may be present in an amount of from 10 g to 200 g per liter of solvent, for example from 10 g to 150 g, 10 g to 100 g, 30 g to 100 g, 40 g to 100 g, 50 g to 100 g, 50 g to 80 g, But the present invention is not limited thereto.
상기 다공성 실리카 입자는 용매 중에 고르게 분산되어 있는 것일 수 있고, 예를 들면 용매에 다공성 실리카 입자를 첨가하고 초음파 분산시킨 것일 수 있다. 혼합용매를 사용하는 경우에는 제1 용매에 다공성 실리카 입자를 분산시킨 후에 제2 용매를 첨가한 것일 수 있다.The porous silica particles may be uniformly dispersed in a solvent, for example, the porous silica particles may be added to a solvent and ultrasonically dispersed. When a mixed solvent is used, the second solvent may be added after dispersing the porous silica particles in the first solvent.
상기 기공 팽창제는 예를 들면 용매 100부피부에 대하여 10 내지 200부피부, 상기 범위 내에서, 10 내지 150부피부, 10 내지 100부피부, 10 내지 80부피부, 30 내지 80부피부, 30 내지 70부피부 등의 비율로 첨가될 수 있으나, 이에 제한되는 것은 아니다.The pore-expanding agent may be, for example, 10 to 200 parts of skin to 100 parts of skin, 10 to 150 parts of skin, 10 to 100 parts of skin, 10 to 80 parts of skin, 30 to 80 parts of skin, 70 parts skin, and the like, but the present invention is not limited thereto.
상기 반응은 예를 들면 120℃ 내지 180℃로 수행될 수 있다. 예를 들어 상기 범위 내에서 120℃ 내지 170℃, 120℃ 내지 160℃, 120℃ 내지 150℃, 130℃ 내지 180℃, 130℃ 내지 170℃, 130℃ 내지 160℃, 130℃ 내지 150℃ 등으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The reaction can be carried out, for example, at 120 ° C to 180 ° C. For example, within the above-mentioned range, it is possible to use a temperature of 120 to 170 ° C, 120 to 160 ° C, 120 to 150 ° C, 130 to 180 ° C, 130 to 170 ° C, 130 to 160 ° C, 130 to 150 ° C, But is not limited thereto.
상기 반응은 예를 들면 24시간 내지 96시간 수행 수행될 수 있다. 예를 들어 상기 범위 내에서 30시간 내지 96시간, 30시간 내지 96시간, 30시간 내지 80시간, 30시간 내지 72시간, 24시간 내지 80시간, 24시간 내지 72시간, 36시간 내지 96시간, 36시간 내지 80시간, 36시간 내지 72시간, 36시간 내지 66시간, 36시간 내지 60시간, 48시간 내지 96시간, 48시간 내지 88시간, 48시간 내지 80시간, 48시간 내지 72시간 등일 수 있으나, 이에 제한되는 것은 아니다.The reaction can be carried out, for example, for 24 hours to 96 hours. For example, within the above range from 30 hours to 96 hours, 30 hours to 96 hours, 30 hours to 80 hours, 30 hours to 72 hours, 24 hours to 80 hours, 24 hours to 72 hours, 36 hours to 96 hours, 36 48 hours to 48 hours, 48 hours to 80 hours, 48 hours to 72 hours, and the like, for example, from 1 hour to 80 hours, 36 hours to 72 hours, 36 hours to 66 hours, 36 hours to 60 hours, But is not limited thereto.
상기 예시한 범위 내에서 시간 및 온도를 조절하여 반응이 과다하지 않으면서 충분히 수행될 수 있도록 할 수 있다. 예를 들면 반응 온도가 낮아지면 반응 시간을 늘리거나, 반응 온도가 낮아지면 반응 시간을 짧게하는 등에 의할 수 있다. 반응이 충분하지 않으면 기공의 확장이 충분하지 못할 수 있고, 반응이 과다하게 진행되면 기공의 과다 확장에 의해 입자가 붕괴될 수 있다.It is possible to control the time and the temperature within the above-mentioned range so that the reaction can be performed sufficiently without excess. For example, the reaction time may be increased when the reaction temperature is lowered, or the reaction time may be shortened when the reaction temperature is lowered. If the reaction is insufficient, the expansion of the pores may not be sufficient, and if the reaction proceeds excessively, the particles may collapse due to over-expansion of the pores.
상기 반응은 예를 들면 단계적으로 승온시켜 수행될 수 있다. 구체적으로, 상온에서 상기 온도까지 0.5℃/분 내지 15℃/분의 속도로 단계적으로 승온시켜 수행될 수 있으며, 예를 들어 상기 범위 내에서 1℃/분 내지 15℃/분, 3℃/분 내지 15℃/분, 3℃/분 내지 12℃/분, 3℃/분 내지 10℃/분 등일 수 있으나, 이에 제한되는 것은 아니다.The reaction can be carried out, for example, by raising the temperature stepwise. Specifically, it can be performed by raising the temperature from room temperature to the temperature stepwise at a rate of 0.5 ° C / min to 15 ° C / min, for example, within a range of 1 ° C / min to 15 ° C / Min to 15 ° C / min, 3 ° C / min to 12 ° C / min, 3 ° C / min to 10 ° C / min, and the like.
상기 반응 이후에는 반응액을 서서히 냉각시킬 수 있으며, 예를 들면 단계적으로 감온하여 냉각시킬 수 있다. 구체적으로 상기 온도에서 상온까지 0.5℃/분 내지 20℃/분의 속도로 단계적으로 감온시켜 수행될 수 있으며, 예를 들어 상기 범위 내에서 1℃/분 내지 20℃/분, 3℃/분 내지 20℃/분, 3℃/분 내지 12℃/분, 3℃/분 내지 10℃/분 등일 수 있으나, 이에 제한되는 것은 아니다.After the reaction, the reaction solution may be gradually cooled, for example, it may be cooled stepwise. Specifically, it may be performed by gradually warming the temperature to room temperature at a rate of 0.5 ° C / minute to 20 ° C / minute. For example, the temperature may be 1 ° C / minute to 20 ° C / 20 ° C / min, 3 ° C / min to 12 ° C / min, 3 ° C / min to 10 ° C / min, and the like.
상기 냉각 이후에 반응 산물을 세척 및 건조시켜 기공이 확장된 다공성 실리카 입자를 얻을 수 있고, 필요에 따라 세척 전에 미반응 물질의 분리가 선행될 수 있으며, 이는 예를 들면 원심분리로 상등액을 분리하여 수행될 수 있다.After the cooling, the reaction product may be washed and dried to obtain pore-expanded porous silica particles, and if necessary, separation of the unreacted material may be preceded by separation of the supernatant by, for example, centrifugation .
상기 원심분리는 예를 들면 6,000 내지 10,000rpm으로 수행될 수 있으며, 그 시간은 예를 들면 3분 내지 60분, 예를 들어 상기 범위 내에서 3분 내지 30분, 3분 내지 30분, 5분 내지 30분 등으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The centrifugation can be performed at, for example, 6,000 to 10,000 rpm, for example, for 3 minutes to 60 minutes, for example, 3 minutes to 30 minutes, 3 minutes to 30 minutes, 5 minutes To 30 minutes, and the like, but the present invention is not limited thereto.
상기 세척은 물 및/또는 유기용매로 할 수 있고, 구체적으로는 용매별로 녹일 수 있는 물질이 상이하므로 물과 유기용매를 1회 또는 수회 번갈아 사용할 수 있으며, 물 또는 유기용매 단독으로도 1회 또는 수회 세척할 수 있다. 상기 수회는 예를 들면 2회 이상, 10회 이하, 예를 들어, 3회, 4회, 5회, 6회, 7회, 8회 등일 수 있다.The washing may be performed using water and / or an organic solvent. Specifically, since the materials soluble in each solvent are different, water and an organic solvent may be used once or several times alternately. Alternatively, water or an organic solvent may be used once or several times It can be washed several times. The number of times may be, for example, two times or more and ten times or less, for example, three times, four times, five times, six times, seven times, eight times, and the like.
상기 유기용매는 예를 들면 1,4-디옥산 등의 에테르류(특히 고리형상 에테르류); 클로로포름, 염화메틸렌, 4염화탄소, 1,2-디클로로에탄, 디클로로에틸렌, 트리클로로에틸렌, 퍼클로로에틸렌, 디클로로프로판, 염화아밀, 1,2-디브로모에탄 등의 할로겐화 탄화수소류; 아세톤, 메틸이소부틸케톤, 시클로헥산온 등의 케톤류 ; 벤젠, 톨루엔, 크실렌 등의 탄소계 방향족류 ; N,N-디메틸포름아미드, N,N-디부틸포름아미드, N,N-디메틸아세트아미드, N-메틸피롤리돈 등의 알킬아미드류; 메탄올, 에탄올, 프로판올, 부탄올 등의 알코올류; 등을 사용할 수 있고, 구체적으로는 알코올, 보다 구체적으로 에탄올을 사용할 수 있으나, 이에 제한되는 것은 아니다.The organic solvent includes, for example, ethers such as 1,4-dioxane (particularly, cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone, and cyclohexanone; Carbon-based aromatic compounds such as benzene, toluene and xylene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Etc., and specifically ethanol, more specifically ethanol, may be used, but the present invention is not limited thereto.
상기 세척은 원심분리 하에 수행될 수 있으며, 예를 들면 6,000 내지 10,000rpm으로 수행될 수 있으며, 그 시간은 예를 들면 3분 내지 60분, 예를 들어 상기 범위 내에서 3분 내지 30분, 3분 내지 30분, 5분 내지 30분 등으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The washing may be carried out under centrifugation, for example, at a speed of 6,000 to 10,000 rpm, for example, for 3 minutes to 60 minutes, for example 3 minutes to 30 minutes, Min to 30 min, 5 min to 30 min, and the like, but the present invention is not limited thereto.
상기 세척은 원심분리를 하지 않고, 필터로 입자를 걸러내어 수행될 수도 있다. 필터는 다공성 실리카 입자의 직경 이하의 기공을 가지는 것일 수 있다. 반응액을 그러한 필터로 걸러내면 입자만이 필터 위에 남고, 그 필터 위에 물 및/또는 유기용매를 부어 세척할 수 있다.The washing may be performed by filtering the particles with a filter without centrifugation. The filter may have pores smaller than the diameter of the porous silica particles. When the reaction solution is filtered with such a filter, only the particles remain on the filter, and water and / or an organic solvent can be poured onto the filter to wash.
상기 세척 시에 물과 유기용매를 1회 또는 수회 번갈아 사용할 수 있으며, 물 또는 유기용매 단독으로도 1회 또는 수회 세척할 수 있다. 상기 수회는 예를 들면 2회 이상, 10회 이하, 예를 들어, 3회 이상 10회 이하, 4회 이상 8회 이하, 4회 이상 6회 이하 등일 수 있다.The water and the organic solvent may be used once or several times at the time of the washing, and the water or the organic solvent may be washed once or several times. The number of times may be, for example, 2 or more, 10 or less, for example, 3 or more and 10 or less, 4 or more or 8 or less, 4 or more or 6 or less, and the like.
상기 건조는 예를 들면 20℃ 내지 100℃로 수행될 수 있으나, 이에 제한되는 것은 아니고, 진공 상태에서 수행될 수도 있다.The drying may be performed at, for example, 20 ° C to 100 ° C, but is not limited thereto, and may be performed in a vacuum state.
이후, 얻어진 입자는 하소될 수 있는데, 하소는 입자를 가열하여 그 표면 및 내부를 좀 더 치밀한 구조를 갖게 하고, 기공을 채우는 유기물들을 제거하는 공정으로, 예를 들면 400℃ 내지 700℃에서 3시간 내지 8시간, 구체적으로 500℃ 내지 600℃에서 4시간 내지 5시간 수행될 수 있으나, 이에 제한되는 것은 아니다.Then, the obtained particles can be calcined. The calcination is a process for heating the particles to have a more dense structure on the surface and inside thereof, and removing organic substances that fill the pores. For example, calcination is performed at 400 to 700 ° C for 3 hours To 8 hours, more specifically, from 500 ° C to 600 ° C for 4 hours to 5 hours, but the present invention is not limited thereto.
이후, 얻어진 다공성 실리카 입자는 표면개질 될 수 있다.Then, the obtained porous silica particles can be surface-modified.
상기 표면 개질은 표면 및/또는 기공 내부에 수행될 수 있다. 입자 표면과 기공 내부는 동일하게 표면개질될 수도 있고, 서로 다르게 표면개질될 수도 있다.The surface modification can be performed inside the surface and / or pores. The surface of the particle and the inside of the pore may be surface-modified in the same manner or may be surface-modified differently.
상기 표면 개질을 통해 입자가 대전되도록 하거나, 친수성 및/또는 소수성 성질을 갖도록 할 수 있다. 표면 개질은 예를 들면 도입하고자 하는 친수성, 소수성, 양이온성, 음이온성 등의 치환기를 갖는 화합물을 입자와 반응시켜 수행할 수 있고, 상기 화합물은 예를 들면 C1 내지 C10 알콕시기를 갖는 알콕시실란일 수 있으나, 이에 제한되는 것은 아니다. 상기 알콕시실란은 상기 알콕시기를 1개 이상 갖는 것으로서, 예를 들면 1 내지 3개 가질 수 있고, 알콕시기가 결합되지 않은 부위에 도입하고자 하는 치환기가 있거나 이로 치환된 치환기가 있을 수 있다.The surface modification may allow the particles to be charged or have hydrophilic and / or hydrophobic properties. The surface modification can be carried out, for example, by reacting a compound having substituents such as hydrophilic, hydrophobic, cationic, and anionic to be introduced with the particles, and the compound can be, for example, an alkoxysilane having a C1 to C10 alkoxy group But is not limited thereto. The alkoxysilane is one having at least one of the above-mentioned alkoxy groups and may have, for example, 1 to 3 substituents, and may have a substituent to be introduced or a substituted substituent at a site where an alkoxy group is not bonded.
상기 알콕시실란을 다공성 실리콘 입자와 반응시키면 실리콘 원자와 산소 원자간 공유 결합이 형성되어 알콕시실란이 다공성 실리콘 입자의 표면 및/또는 기공 내부와 결합될 수 있고, 상기 알콕시실란은 도입하고자 하는 치환기를 가지고 있는 바, 해당 치환기가 다공성 실리콘 입자의 표면 및/또는 기공 내부에 도입될 수 있다.When the alkoxysilane is reacted with the porous silicon particles, an alkoxysilane can be bonded to the surface and / or pores of the porous silicon particles by forming a covalent bond between the silicon atom and the oxygen atom, and the alkoxysilane has a substituent The substituent may be introduced into the surface and / or pores of the porous silicon particles.
상기 반응은 용매에 분산시킨 다공성 실리카 입자를 알콕시실란과 반응시켜 수행할 수 있다.The above reaction can be carried out by reacting the porous silica particles dispersed in a solvent with an alkoxysilane.
상기 용매는 물 및/또는 유기용매일 수 있고, 유기용매는 예를 들면 1,4-디옥산 등의 에테르류(특히 고리형상 에테르류); 클로로포름, 염화메틸렌, 4염화탄소, 1,2-디클로로에탄, 디클로로에틸렌, 트리클로로에틸렌, 퍼클로로에틸렌, 디클로로프로판, 염화아밀, 1,2-디브로모에탄 등의 할로겐화 탄화수소류; 아세톤, 메틸이소부틸케톤, γ-부티로락톤, 1,3-디메틸-이미다졸리디논, 메틸에틸케톤, 시클로헥사논, 시클로펜타논, 4-하이드록시-4-메틸-2-펜타논 등의 케톤류; 벤젠, 톨루엔, 크실렌, 테트라메틸벤젠 등의 탄소계 방향족류 ; N,N-디메틸포름아미드, N,N-디부틸포름아미드, N,N-디메틸아세트아미드, N-메틸피롤리돈 등의 알킬아미드류; 메탄올, 에탄올, 프로판올, 부탄올 등의 알코올류; 에틸렌글리콜모노에틸에테르, 에틸렌글리콜모노메틸에테르, 에틸렌글리콜모노부틸에테르, 디에틸렌글리콜모노에틸에테르, 디에틸렌글리콜모노메틸에테르, 디에틸렌글리콜모노부틸에테르, 프로필렌글리콜모노메틸에테르, 프로필렌글리콜모노에틸에테르, 디프로필렌글리콜디에틸에테르, 트리에틸렌글리콜모노에틸에테르 등의 글리콜에테르류(셀로솔브); 그외 디메틸아세트아미드(DMAc), N,N-디에틸아세트아미드, 디메틸포름아미드(DMF), 디에틸포름아미드(DEF), N,N-디메틸아세트아미드(DMAc), N-메틸피롤리돈(NMP), N-에틸피롤리돈(NEP), 1,3-디메틸-2-이미다졸리디논, N,N-디메틸메톡시아세트아미드, 디메틸술폭사이드, 피리딘, 디메틸술폰, 헥사메틸포스포아미드, 테트라메틸우레아, N-메틸카르로락탐, 테트라히드로퓨란, m-디옥산, P-디옥산, 1,2-디메톡시에탄 등을 사용할 수 있고, 구체적으로는 톨루엔을 사용할 수 있으나, 이에 제한되는 것은 아니다.The solvent can be water and / or organic solvent, and the organic solvent includes, for example, ethers (especially cyclic ethers) such as 1,4-dioxane; Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Acetone, methyl isobutyl ketone,? -Butyrolactone, 1,3-dimethyl-imidazolidinone, methyl ethyl ketone, cyclohexanone, cyclopentanone, 4-hydroxy- Of ketones; Carbon-based aromatic compounds such as benzene, toluene, xylene, and tetramethylbenzene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether , Glycol ethers (cellosolve) such as dipropylene glycol diethyl ether and triethylene glycol monoethyl ether; Other solvents such as dimethylacetamide (DMAc), N, N-diethylacetamide, dimethylformamide (DMF), diethylformamide (DEF), N, N-dimethylacetamide (DMAc) NMP), N-ethylpyrrolidone (NEP), 1,3-dimethyl-2-imidazolidinone, N, N-dimethylmethoxyacetamide, dimethylsulfoxide, pyridine, dimethylsulfone, hexamethylphosphoamide , Tetramethylurea, N-methylcarrolactam, tetrahydrofuran, m-dioxane, p-dioxane, 1,2-dimethoxyethane and the like can be used. Specifically, toluene can be used. It is not.
상기 양전하로의 대전은 예를 들면 아미노기, 아미노알킬기 등 질소함유기 등의 염기성기를 갖는 알콕시실란과 반응시켜 수행할 수 있다. 구체적으로는 N-[3-(Trimethoxysilyl)propyl]ethylenediamine, N1-(3-Trimethoxysilylpropyl)diethylenetriamine, (3-Aminopropyl)trimethoxysilane, N-[3-(Trimethoxysilyl)propyl]aniline, Trimethoxy[3-(methylamino)propyl]silane, 3-(2-Aminoethylamino)propyldimethoxymethylsilane 등을 사용할 수 있으나, 이에 제한되는 것은 아니다.The charging to the positive charge can be carried out by reacting with an alkoxysilane having a basic group such as a nitrogen-containing group such as an amino group or an aminoalkyl group. Specific examples thereof include N- [3- (Trimethoxysilyl) propyl] ethylenediamine, N1- (3-Trimethoxysilylpropyl) diethylenetriamine, (3-Aminopropyl) trimethoxysilane, N- [3- propyl] silane, 3- (2-Aminoethylamino) propyldimethoxymethylsilane, and the like, but the present invention is not limited thereto.
상기 음전하로의 대전은 예를 들면 카르복시기, 술폰산기, 티올기 등의 산성기를 갖는 알콕시실란과 반응시켜 수행할 수 있다. 구체적으로는 (3-Mercaptopropyl) trimethoxysilane 등을 사용할 수 있으나, 이에 제한되는 것은 아니다.The charging of the negative charge can be carried out, for example, by reacting with an alkoxysilane having an acidic group such as a carboxyl group, a sulfonic acid group or a thiol group. Specifically, (3-Mercaptopropyl) trimethoxysilane can be used, but is not limited thereto.
상기 친수성 성질은 친수성기, 예를 들면 히드록시기, 카르복시기, 아미노기, 카르보닐기, 설프히드릴기, 포스페이트기, 티올기, 암모늄기, 에스터기, 이미드기, 티오이미드기, 케토기, 에터기, 인덴기, 설포닐기, 폴리에틸렌글리콜기 등을 갖는 알콕시실란과 반응시켜 갖도록 할 수 있다. 구체적으로는, N-[3-(Trimethoxysilyl)propyl]ethylenediamine, N1-(3-Trimethoxysilylpropyl)diethylenetriamine, (3-Aminopropyl)trimethoxysilane, (3-Mercaptopropyl) trimethoxysilane, Trimethoxy[3-(methylamino)propyl]silane, 3-(2-Aminoethylamino)propyldimethoxymethylsilane 등을 사용할 수 있으나, 이에 제한되는 것은 아니다.The hydrophilic property may be a hydrophilic property such as a hydrophilic group such as a hydroxyl group, a carboxyl group, an amino group, a carbonyl group, a sulfhydryl group, a phosphate group, a thiol group, an ammonium group, an ester group, an imide group, a thioimide group, a keto group, A polyoxyethylene group, a polyoxyethylene group, a polyoxyethylene group, a polyoxyethylene group, a polyoxyethylene group, a polyethylene glycol group and the like. Specific examples thereof include N- [3- (Trimethoxysilyl) propyl] ethylenediamine, N1- (3-Trimethoxysilylpropyl) diethylenetriamine, (3-Aminopropyl) trimethoxysilane, (3-Mercaptopropyl) trimethoxysilane, Trimethoxy [3- 3- (2-aminoethylamino) propyldimethoxymethylsilane, and the like, but the present invention is not limited thereto.
상기 소수성 성질은 소수성 치환기, 예를 들면 치환 또는 비치환된 C1 내지 C30의 알킬기, 치환 또는 비치환된 C3 내지 C30의 사이클로알킬기, 치환 또는 비치환된 C6 내지 C30의 아릴기, 치환 또는 비치환된 C2 내지 C30의 헤테로아릴기, 할로겐기, C1 내지 C30의 에스테르기, 할로겐 함유기 등을 갖는 알콕시실란과 반응시켜 갖도록 할 수 있다. 구체적으로는, Trimethoxy(octadecyl)silane, Trimethoxy-n-octylsilane, Trimethoxy(propyl)silane, Isobutyl(trimethoxy)silane, Trimethoxy(7-octen-1-yl)silane, Trimethoxy(3,3,3-trifluoropropyl)silane, Trimethoxy(2-phenylethyl)silane, Vinyltrimethoxysilane, Cyanomethyl, 3-(trimethoxysilyl)propyl] trithiocarbonate, (3-Bromopropyl)trimethoxysilane 등을 사용할 수 있으나, 이에 제한되는 것은 아니다.The hydrophobic property may be a hydrophobic substituent, for example, a substituted or unsubstituted C1 to C30 alkyl group, a substituted or unsubstituted C3 to C30 cycloalkyl group, a substituted or unsubstituted C6 to C30 aryl group, a substituted or unsubstituted A C2 to C30 heteroaryl group, a halogen group, an ester group of C1 to C30, a halogen-containing group, and the like. Specific examples thereof include trimethoxy (octadecyl) silane, trimethoxy n-octylsilane, trimethoxy propyl silane, isobutyl trimethoxy silane, trimethoxy (7-octen- silane, trimethoxy (2-phenylethyl) silane, vinyltrimethoxysilane, cyanomethyl, 3- (trimethoxysilyl) propyl] trithiocarbonate and (3-Bromopropyl) trimethoxysilane.
그 외에 표면 개질을 통해 난용성(소수성) 생리활성물질과의 결합력 증진을 위해 기공 내부에는 소수성 치환기가 존재하고, 사용, 제형화의 용이성 등의 측면에서 입자의 표면은 친수성 치환기가 존재하도록 하는 등의 처리도 가능하고, 표면에 생리활성물질 외에 다른 물질을 결합시키기 위한 치환기가 존재할 수도 있다.In addition, there is a hydrophobic substituent in the pores for enhancing the bonding strength with the poorly soluble (hydrophobic) physiologically active substance through surface modification, and the hydrophobic substituent is present on the surface of the particle in terms of ease of use and formulation And a substituent for binding another substance other than the physiologically active substance may be present on the surface.
또한, 상기 표면 개질은 복합적으로 수행될 수도 있다. 예를 들어, 외부 표면 또는 기공 내부에 2회 이상의 표면 개질이 수행될 수도 있다. 구체적인 예를 들자면, 아미노기가 도입된 실리카 입자에 카르복실기를 포함하는 화합물을 아미드 결합으로 결합시켜 양전하로 대전된 입자를 다른 표면특성을 가지게 변화시킬 수 있으나, 이에 제한되는 것은 아니다.In addition, the surface modification may be performed in combination. For example, two or more surface modification may be performed on the outer surface or inside the pores. As a specific example, a compound containing a carboxyl group may be bonded to an amide-introduced silica particle with an amide bond to change positively charged particles to have different surface characteristics, but the present invention is not limited thereto.
상기 다공성 실리카 입자의 알콕시실란과의 반응은 예를 들면 가열 하에 수행될 수 있다.The reaction of the porous silica particles with the alkoxysilane can be carried out, for example, under heating.
상기 가열은 예를 들면 80℃ 내지 180℃, 예를 들어 상기 범위 내에서 80℃ 내지 160℃, 80℃ 내지 150℃, 100℃ 내지 160℃, 100℃ 내지 150℃, 110℃ 내지 150℃ 등으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The heating may be performed at a temperature of, for example, 80 to 180 DEG C, for example, 80 to 160 DEG C, 80 to 150 DEG C, 100 to 160 DEG C, 100 to 150 DEG C, 110 to 150 DEG C, But is not limited thereto.
상기 다공성 실리카 입자의 알콕시실란과의 반응은 예를 들면 4시간 내지 20시간, 예를 들어 상기 범위 내에서 4시간 내지 18시간, 4시간 내지 16시간, 6시간 내지 18시간, 6시간 내지 16시간, 8시간 내지 18시간, 8시간 내지 16시간, 8시간 내지 14시간, 10시간 내지 14시간 등으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The reaction of the porous silica particles with the alkoxysilane may be carried out, for example, for 4 to 20 hours, for example 4 to 18 hours, 4 to 16 hours, 6 to 18 hours, 6 to 16 hours , 8 hours to 18 hours, 8 hours to 16 hours, 8 hours to 14 hours, 10 hours to 14 hours, and the like.
상기 반응 온도, 시간, 그리고 표면개질에 사용되는 화합물의 양 등은 표면개질하고자 하는 정도에 따라 선택될 수 있는 것으로서, 생리활성물질의 친수성, 소수성, 전하 정도에 따라 반응 조건을 달리하여 다공성 실리카 입자의 친수성, 소수성, 전하 정도를 조절함으로써, 생리활성물질 방출 속도를 조절할 수 있다. 예를 들면, 생리활성물질이 중성의 pH에서 강한 음전하를 띠는 경우에는 다공성 실리카 입자가 강한 양전하를 띠도록 하기 위해, 반응 온도를 높이거나 반응 시간을 길게 할 수 있으며, 화합물 처리량을 늘릴 수 있으나, 이에 제한되는 것은 아니다.The reaction temperature, time, and the amount of the compound used for surface modification can be selected depending on the desired degree of surface modification. The reaction conditions vary depending on the hydrophilicity, hydrophobicity, and electric charge of the physiologically active substance, The hydrophilic property, the hydrophobicity, and the electric charge level of the physiologically active substance can be controlled. For example, when a physiologically active substance has a strong negative charge at a neutral pH, the reaction temperature can be increased or the reaction time can be lengthened and the compound throughput can be increased in order to allow the porous silica particles to have a strong positive charge , But is not limited thereto.
또한, 상기 다공성 실리카 입자는 예를 들면 소기공의 입자 제조, 기공 확장, 표면 개질, 기공 내부 개질 공정을 거쳐 제조된 것일 수도 있다.The porous silica particles may be prepared by, for example, preparing pores of small pores, expanding pores, modifying the surface, or modifying pores inside.
소기공의 입자 제조 및 기공 확장 공정은 전술한 바의 공정에 의할 수 있으며, 소기공의 입자 제조 이후, 그리고 기공 확장 공정 이후에 세척 및 건조 공정을 수행할 수 있다.The particle preparation and the pore expansion process of the small pores can be performed by the processes described above, and the cleaning and drying processes can be performed after the particle preparation of the small pores and after the pore expansion process.
필요에 따라 세척 전에 미반응 물질의 분리가 선행될 수 있고, 이는 예를 들면 원심분리로 상등액을 분리하여 수행될 수 있다.If necessary, the separation of the unreacted material may be preceded by washing before washing, for example, by separating the supernatant by centrifugation.
상기 원심분리는 예를 들면 6,000 내지 10,000rpm으로 수행될 수 있으며, 그 시간은 예를 들면 3분 내지 60분, 구체적으로, 상기 범위 내에서 3분 내지 30분, 3분 내지 30분, 5분 내지 30분 등으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The centrifugation may be performed at, for example, 6,000 to 10,000 rpm, for example, 3 minutes to 60 minutes, specifically 3 minutes to 30 minutes, 3 minutes to 30 minutes, 5 minutes To 30 minutes, and the like, but the present invention is not limited thereto.
상기 소기공의 입자 제조 이후의 세척은 앞서 예시한 범위 내의 방법/조건으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The cleaning after the particle preparation of the small pores may be carried out by any method / condition within the range exemplified above, but is not limited thereto.
상기 기공 확장 이후의 세척은 앞서 예시보다는 보다 완화된 조건으로 수행할 수 있다. 예를 들면, 세척을 3회 이내 수행할 수 있으나, 이에 제한되는 것은 아니다.The purging after the pore expansion can be performed under more relaxed conditions than in the previous examples. For example, washing may be performed within 3 times, but is not limited thereto.
상기 표면 개질과 기공 내부 개질은 각각 전술한 바의 공정에 의할 수 있으며, 표면 개질과 기공 내부 개질의 순서로 공정이 수행될 수 있고, 상기 두 공정 사이에 입자의 세척 공정이 추가로 수행될 수 있다.The surface modification and the internal reforming of the pores can be performed by the processes described above, and the processes can be performed in the order of the surface modification and the internal pore modification, and the particle washing process is further performed between the two processes .
상기 소기공의 입자 제조 및 기공 확장 이후에 세척을 보다 완화된 조건으로 수행하는 경우 기공 내부에 입자 제조, 기공 확장에 사용된 계면활성제 등의 반응액이 채워져 있어 표면 개질시에 기공 내부는 개질되지 않고 표면만 개질될 수 있다. 그러고 나서 입자를 세척하면 기공 내부의 반응액이 제거될 수 있다.When the preparation of the small pores and the cleaning after the pore expansion are carried out in a more relaxed condition, the inside of the pores are filled with the reaction liquid such as the particles used for the preparation of the particles and the pore expansion, Only the surface can be modified. The particles may then be washed to remove the reaction liquid inside the pores.
상기 표면 개질과 기공 내부 개질 공정 사이의 입자 세척은 물 및/또는 유기용매로 할 수 있고, 구체적으로는 용매별로 녹일 수 있는 물질이 상이하므로 물과 유기용매를 1회 또는 수회 번갈아 사용할 수 있으며, 물 또는 유기용매 단독으로도 1회 또는 수회 세척할 수 있다. 상기 수회는 예를 들면 2회 이상, 10회 이하, 구체적으로, 3회 이상 10회 이하, 4회 이상 8회 이하, 4회 이상 6회 이하 등일 수 있다.The washing of the particles between the surface modification and the pore interior modification process may be performed using water and / or an organic solvent. Specifically, since the materials soluble in each solvent are different, water and an organic solvent may be used once or several times, Water or an organic solvent can be washed once or several times. The number of times may be, for example, 2 or more, 10 or less, specifically 3 or more and 10 or less, 4 or more or 8 or less, 4 or more, 6 or less, and the like.
상기 세척은 원심분리 하에 수행될 수 있으며, 예를 들면 6,000 내지 10,000rpm으로 수행될 수 있으며, 그 시간은 예를 들면 3분 내지 60분, 구체적으로, 상기 범위 내에서 3분 내지 30분, 3분 내지 30분, 5분 내지 30분 등으로 수행될 수 있으나, 이에 제한되는 것은 아니다.The washing may be carried out under centrifugation, for example, at 6,000 to 10,000 rpm, for example, for 3 to 60 minutes, specifically for 3 to 30 minutes, Min to 30 min, 5 min to 30 min, and the like, but the present invention is not limited thereto.
상기 세척은 원심분리를 하지 않고, 필터로 입자를 걸러내어 수행될 수도 있다. 필터는 다공성 실리카 입자의 직경 이하의 기공을 가지는 것일 수 있다. 반응액을 그러한 필터로 걸러내면 입자만이 필터 위에 남고, 그 필터 위에 물 및/또는 유기용매를 부어 세척할 수 있다.The washing may be performed by filtering the particles with a filter without centrifugation. The filter may have pores smaller than the diameter of the porous silica particles. When the reaction solution is filtered with such a filter, only the particles remain on the filter, and water and / or an organic solvent can be poured onto the filter to wash.
상기 세척 시에 물과 유기용매를 1회 또는 수회 번갈아 사용할 수 있으며, 물 또는 유기용매 단독으로도 1회 또는 수회 세척할 수 있다. 상기 수회는 예를 들면 2회 이상, 10회 이하, 구체적으로, 3회 이상 10회 이하, 4회 이상 8회 이하, 4회 이상 6회 이하 등일 수 있다.The water and the organic solvent may be used once or several times at the time of the washing, and the water or the organic solvent may be washed once or several times. The number of times may be, for example, 2 or more, 10 or less, specifically 3 or more and 10 or less, 4 or more or 8 or less, 4 or more, 6 or less, and the like.
상기 건조는 예를 들면 20℃ 내지 100℃로 수행될 수 있으나, 이에 제한되는 것은 아니고, 진공 상태에서 수행될 수도 있다.The drying may be performed at, for example, 20 ° C to 100 ° C, but is not limited thereto, and may be performed in a vacuum state.
본 발명의 siRNA 또는 dsRNA와 같은 생리활성물질은 다공성 실리카 입자의 표면 및/또는 기공 내부에 담지될 수 있다.The physiologically active substance such as siRNA or dsRNA of the present invention can be carried on the surface and / or inside the pores of the porous silica particles.
상기 담지는 예를 들면 용매 중의 다공성 실리카 입자와 생리활성물질을 혼합하여 수행될 수 있다.The support may be carried out, for example, by mixing the porous silica particles in the solvent and the physiologically active substance.
상기 용매는 물 및/또는 유기용매일 수 있으며, 유기용매는 예를 들면 1,4-디옥산 등의 에테르류(특히 고리형상 에테르류); 클로로포름, 염화메틸렌, 4염화탄소, 1,2-디클로로에탄, 디클로로에틸렌, 트리클로로에틸렌, 퍼클로로에틸렌, 디클로로프로판, 염화아밀, 1,2-디브로모에탄 등의 할로겐화 탄화수소류; 아세톤, 메틸이소부틸케톤, 시클로헥산온 등의 케톤류; 벤젠, 톨루엔, 크실렌 등의 탄소계 방향족류; N,N-디메틸포름아미드, N,N-디부틸포름아미드, N,N-디메틸아세트아미드, N-메틸피롤리돈 등의 알킬아미드류; 메탄올, 에탄올, 프로판올, 부탄올 등의 알코올류; 등을 사용할 수 있다.The solvent can be water and / or organic solvent, and the organic solvent includes, for example, ethers such as 1,4-dioxane (particularly, cyclic ethers); Halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, dichloroethylene, trichlorethylene, perchlorethylene, dichloropropane, amyl chloride and 1,2-dibromoethane; Ketones such as acetone, methyl isobutyl ketone, and cyclohexanone; Carbon-based aromatic compounds such as benzene, toluene and xylene; Alkyl amides such as N, N-dimethylformamide, N, N-dibutylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; Alcohols such as methanol, ethanol, propanol and butanol; Etc. may be used.
또한, 상기 용매로 PBS(phosphate buffered saline solution), SBF(Simulated Body Fluid), Borate-buffered saline, Tris-buffered saline 등을 사용할 수도 있다. In addition, PBS (phosphate buffered saline solution), SBF (Simulated Body Fluid), borate-buffered saline, Tris-buffered saline, etc. may be used as the solvent.
상기 다공성 실리카 입자와 생리활성물질의 비율은 특별히 한정되지 않으며, 예를 들면 중량비가 1: 0.05 내지 0.8, 예를 들어 상기 범위 내에서 1: 0.05 내지 0.7, 1:0.05 내지 0.6, 1: 0.1 내지 0.8, 1: 0.1 내지 0.6, 1: 0.2 내지 0.8, 1: 0.2 내지 0.6 등일 수 있다.The ratio of the porous silica particles to the physiologically active substance is not particularly limited and may be, for example, a weight ratio of 1: 0.05 to 0.8, such as 1: 0.05 to 0.7, 1: 0.05 to 0.6, 1: 0.8, 1: 0.1 to 0.6, 1: 0.2 to 0.8, 1: 0.2 to 0.6, and the like.
상기 다공성 실리카 입자에 담지된 본 발명 siRNA 또는 dsRNA와 같은 생리활성물질은 연장된 시간에 걸쳐 점진적으로 방출될 수 있다. 이와 같이 느린 방출은 연속성 또는 비연속성, 선형 또는 비선형일 수 있으며, 다공성 실리카 입자의 특징 및/또는 그와 생리활성물질과의 상호작용에 기인하여 달라질 수 있다.The physiologically active substance such as siRNA or dsRNA of the present invention carried on the porous silica particles can be gradually released over an extended period of time. Such slow release may be continuous or non-continuous, linear or non-linear, and may vary due to the characteristics of the porous silica particles and / or their interaction with the bioactive material.
상기 다공성 실리카 입자에 담지된 생리활성물질은 다공성 실리카 입자가 생분해되면서 방출되는데, 본 발명에 따른 다공성 실리카 입자는 서서히 분해되어 담지된 생리활성물질이 서방적으로 방출되도록 할 수 있다. 이는 예를 들면 다공성 실리카 입자의 표면적, 입경, 기공 직경, 표면 및/또는 기공 내부의 치환기, 표면의 치밀함 정도 등을 조절함으로써 조절될 수 있으나, 이에 제한되는 것은 아니다.The physiologically active substance supported on the porous silica particles is released while the porous silica particles are biodegraded. The porous silica particles according to the present invention may be slowly decomposed to release the supported physiologically active substance slowly. This can be controlled, for example, by controlling the surface area, particle size, pore diameter, surface area and / or porosity of the porous silica particles, the degree of compactness of the surface, and the like.
또한, 상기 다공성 실리카 입자에 담지된 생리활성물질은 다공성 실리카 입자로부터 이탈되어 확산되면서도 방출될 수 있고, 이는 다공성 실리카 입자와 생리활성물질, 생리활성물질 방출 환경과의 관계에 영향을 받는 것인 바, 이를 조절하여 생리활성물질 방출을 조절할 수 있다. 예를 들면 표면개질에 의해 다공성 실리카 입자의 생리활성물질과의 결합력을 강화 또는 약화시킴으로써 조절할 수 있다.In addition, the physiologically active substance carried on the porous silica particles can be released while diffusing from the porous silica particles, which is affected by the relationship between the porous silica particles, the physiologically active substance, and the environment for releasing the physiologically active substance , And it is possible to control the release of the biologically active substance by controlling this. For example, by strengthening or weakening the binding force of the porous silica particles with the physiologically active substance by surface modification.
보다 구체적인 예를 들자면, 담지된 생리활성물질이 난용성(소수성)인 경우에는 입자의 표면 및/또는 기공 내부가 소수성 치환기를 가져 다공성 실리카 입자와 생리활성물질과의 결합력이 증가된 것일 수 있고, 이에 의해 생리활성물질이 서방적으로 방출될 수 있다. 이는 예를 들면 다공성 실리카 입자가 소수성 치환기를 갖는 알콕시실란으로 표면개질된 것일 수 있다.More specifically, in the case where the supported physiologically active substance is poorly soluble (hydrophobic), the surface of the particle and / or the inside of the pore may have a hydrophobic substituent to increase the binding force between the porous silica particle and the physiologically active substance, Whereby the physiologically active substance can be released in a sustained manner. This may be, for example, that the porous silica particles are surface-modified with an alkoxysilane having a hydrophobic substituent.
본 명세서에서 "난용성"은 (물에 대해) 불용성(insoluble), 실질적으로 불용성(practically insoluble) 또는 극히 약간의 가용성(only slightly soluble)인 것을 포함하는 의미로서 이는 "Pharmaceutical Science" 18th Edition(U.S.P., Remington, Mack Publishing Company 발행)에 정의되어 있는 용어이다.In this specification, "poorly soluble" is a meant to include (for water) in that the insoluble (insoluble), substantially insoluble (practically insoluble) or very slightly soluble (only slightly soluble) This "Pharmaceutical Science," 18 th Edition ( USP, Remington, published by Mack Publishing Company).
상기 난용성 생리활성물질은 예를 들면 1기압, 25℃에서 수용해도가 10g/L 미만, 구체적으로 5g/L 미만, 보다 구체적으로 1g/L 미만일 수 있으나, 이에 제한되는 것은 아니다.The water-insoluble physiologically active substance may have a water solubility of less than 10 g / L, specifically less than 5 g / L, more specifically less than 1 g / L at 25 ° C under 1 atm, but is not limited thereto.
상기 담지된 생리활성물질이 수용성(친수성)인 경우에는 입자의 표면 및/또는 기공 내부가 친수성 치환기를 가져 다공성 실리카 입자와 생리활성물질과의 결합력이 증가된 것일 수 있고, 이에 의해 생리활성물질이 서방적으로 방출될 수 있다. 이는 예를 들면 다공성 실리카 입자가 친수성 치환기를 갖는 알콕시실란으로 표면개질된 것일 수 있다.When the supported physiologically active substance is water-soluble (hydrophilic), the surface and / or the pores of the particles may have a hydrophilic substituent and the binding strength between the porous silica particles and the physiologically active substance may be increased, It can be released slowly. For example, the porous silica particles may be surface-modified with an alkoxysilane having a hydrophilic substituent.
상기 수용성 생리활성물질은 예를 들면 1기압, 25℃에서 수용해도가 10g/L 이상일 수 있으나, 이에 제한되는 것은 아니다.For example, the water-soluble physiologically active substance may have a water solubility of 10 g / L or more at 25 ° C and 1 atm, but is not limited thereto.
상기 담지된 생리활성물질이 전하를 띠는 경우에는 입자의 표면 및/또는 기공 내부가 그와 반대 전하로 대전되어 다공성 실리카 입자와 생리활성물질과의 결합력이 증가된 것일 수 있고, 이에 의해 생리활성물질이 서방적으로 방출될 수 있다. 이는 예를 들면 다공성 실리카 입자가 산성기 또는 염기성기를 갖는 알콕시실란으로 표면개질된 것일 수 있다.When the supported physiologically active substance has an electric charge, the surface and / or pore of the particle may be charged with opposite charge to increase the binding force between the porous silica particle and the physiologically active substance, Material may be released slowly. For example, the porous silica particles may be surface-modified with an alkoxysilane having an acidic group or a basic group.
구체적으로, 상기 생리활성물질이 중성의 pH에서 양전하를 띠는 것이라면 입자의 표면 및/또는 기공 내부가 중성의 pH에서 음전하로 대전되는 것일 수 있고, 이에 의해 다공성 실리카 입자와 생리활성물질과의 결합력이 증가되어 생리활성물질이 서방적으로 방출될 수 있다. 이는 예를 들면 다공성 실리카 입자가 카르복시기(-COOH), 술폰산기(-SO3H) 등의 산성기를 갖는 알콕시실란으로 표면개질된 것일 수 있다.Specifically, if the physiologically active substance is positively charged at a neutral pH, the surface and / or pore interior of the particle may be negatively charged at a neutral pH, whereby the binding force between the porous silica particle and the physiologically active substance Is increased, so that the physiologically active substance can be released slowly. For example, the porous silica particles may be surface-modified with an alkoxysilane having an acidic group such as a carboxyl group (-COOH) or a sulfonic acid group (-SO 3 H).
또한, 상기 생리활성물질이 중성의 pH에서 음전하를 띠는 것이라면 입자의 표면 및/또는 기공 내부가 양전하로 대전되는 것일 수 있고, 이에 의해 다공성 실리카 입자와 생리활성물질과의 결합력이 증가되어 생리활성물질이 서방적으로 방출될 수 있다. 이는 예를 들면 다공성 실리카 입자가 아미노기, 그 외 질소함유기 등의 염기성기를 갖는 알콕시실란으로 표면개질된 것일 수 있다.In addition, if the physiologically active substance has a negative charge at neutral pH, the surface and / or pores of the particles may be positively charged, thereby increasing the binding force between the porous silica particles and the physiologically active substance, Material may be released slowly. For example, the porous silica particles may be surface-modified with an alkoxysilane having a basic group such as an amino group or other nitrogen-containing groups.
상기 생리활성물질은 필요한 치료 유형, 방출 환경, 사용되는 다공성 실리카 입자에 의존하여 예를 들면 7일 내지 1년 또는 그 이상의 기간 동안 방출될 수 있다. The physiologically active substance may be released for a period of, for example, 7 days to 1 year or more depending on the type of treatment required, the release environment, and the porous silica particles used.
또한, 상기 다공성 실리카 입자는 생분해성으로서 100% 분해될 수 있으므로, 이에 담지된 생리활성물질은 100% 방출될 수 있다.In addition, since the porous silica particles can be decomposed 100% as biodegradable, the supported physiologically active substance can be 100% released.
본 발명의 siRNA 또는 dsRNA를 포함하는 간암 예방 또는 치료용 약학적 조성물은 약학적으로 허용가능한 담체를 추가로 포함할 수 있으며, 담체와 함께 제제화될 수 있다. 본 발명에서 용어, "약학적으로 허용가능한 담체"란 생물체를 자극하지 않고 투여 화합물의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 말한다. 액상 용액으로 제제화되는 조성물에 있어서 허용되는 약제학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로오스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다.The pharmaceutical composition for preventing or treating liver cancer comprising siRNA or dsRNA of the present invention may further comprise a pharmaceutically acceptable carrier and may be formulated together with a carrier. As used herein, the term " pharmaceutically acceptable carrier " refers to a carrier or diluent that does not irritate the organism and does not interfere with the biological activity and properties of the administered compound. Examples of the pharmaceutical carrier which is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
본 발명의 조성물은 본 발명의 siRNA 또는 dsRNA를 유효성분으로 포함하는 어떠한 제형으로도 적용가능하며, 경구용 또는 비경구용 제형으로 제조할 수 있다. 본 발명의 약학적 제형은 구강(oral), 직장(rectal), 비강(nasal), 국소(topical; 볼 및 혀 밑을 포함), 피하, 질(vaginal) 또는 비경구(parenteral; 근육내, 피하 및 정맥내를 포함) 투여에 적당한 것 또는 흡입(inhalation) 또는 주입(insufflation)에 의한 투여에 적당한 형태를 포함한다.The composition of the present invention can be applied to any formulation containing the siRNA or dsRNA of the present invention as an active ingredient, and can be manufactured into oral or parenteral formulations. The pharmaceutical formulations of the present invention may be administered orally, rectally, nasal, topical (including under the ball and tongue), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous And intravenous), or forms suitable for administration by inhalation or insufflation.
본 발명의 조성물은 약학적으로 유효한 양으로 투여한다. 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The composition of the present invention is administered in a pharmaceutically effective amount. Effective dose levels will depend on factors well known in the art and other medical disciplines including the type of disease, severity of the patient, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, Can be determined. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.
본 발명의 조성물의 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설률 및 질환의 중증도 등에 따라 그 범위가 매우 다양하며, 적정한 투여량은 예를 들면 환자의 체내에 축적된 약물의 양 및/또는 사용되는 본 발명의 siRNA 또는 dsRNA의 구체적 효능정도에 따라 달라질 수 있다. 일반적으로 인비보 동물모델 및 인비트로에서 효과적인 것으로 측정된 EC50을 기초로 계산될 수 있으며, 예를 들면 체중 1kg당 0.01 μg 내지 1 g 일 수 있으며, 일별, 주별, 월별 또는 연별의 단위 기간으로, 단위 기간 당 일회 내지 수회 나누어 투여될 수 있으며, 또는 인퓨전 펌프를 이용하여 장기간 연속적으로 투여될 수 있다. 반복투여 횟수는 약물이 체내 머무는 시간, 체내 약물 농도 등을 고려하여 결정된다. 질환 치료 경과에 따라 치료가 된 후라도, 재발을 위해 조성물이 투여될 수 있다.The dosage of the composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, severity of disease and the like. The amount of the drug accumulated in the body, and / or the degree of specific activity of the siRNA or dsRNA of the present invention to be used. May be calculated on the basis of the EC50 generally measured as effective in the in vivo animal model and in vitro, for example from 0.01 [mu] g to 1 g per kg of body weight and may be divided into daily, weekly, monthly, May be administered once or several times per unit period, or may be continuously administered for a long period using an infusion pump. The number of repeated administrations is determined in consideration of the duration of the drug in the body, the drug concentration in the body, and the like. The composition may be administered for recurrence, even after treatment according to the course of the disease treatment.
본 발명의 조성물은 간암의 치료와 관련하여 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 또는 유효성분의 용해성 및/또는 흡수성을 유지/증가시키는 화합물을 추가로 함유할 수 있다. 또한 선택적으로, 화학치료제, 항염증제, 항바이러스제 및/또는 면역조절제 등을 추가로 포함할 수 있다.The composition of the present invention may further contain one or more active ingredients which exhibit the same or similar functions with respect to the treatment of liver cancer, or a compound which maintains / increases the solubility and / or absorbency of the active ingredient. Also optionally, it may further comprise a chemotherapeutic agent, an anti-inflammatory agent, an antiviral agent and / or an immunomodulator.
또한, 본 발명의 조성물은 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 사용하여 제형화될 수 있다. 제형은 분말, 과립, 정제, 에멀젼, 시럽, 에어로졸, 연질 또는 경질 젤라틴 캅셀, 멸균 주사용액, 멸균 분말의 형태일 수 있다.In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.
이하, 본 발명을 구체적으로 설명하기 위해 실시예를 들어 상세하게 설명하기로 한다.BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to examples.
실시예 1. 실험재료 및 방법Example 1. Experimental Materials and Methods
1. 세포배양1. Cell culture
인간 간암 세포주(SNU-449) 및 쥐 Hepa-1c1c7 간암 세포주는 한국세포주은행(서울, 대한민국)으로부터 얻었다. 모든 세포주는 가습 인큐베이터 내 37℃, 5% CO2 조건에서, 10% fetal bovine serum (FBS, Lonza) 및 100 단위/mL의 페니실린-스트렙토마이신(Invitrogen, Carlsbad, CA)를 함유한 EMEM (American Type Culture Collection, Manassas, VA), RPMI-1640 또는 DMEM 배지(Lonza, Walkersville, MD)에서 배양되었다. Human liver cancer cell line (SNU-449) and murine Hepa-1c1c7 liver cancer cell line were obtained from Korean Cell Line Bank (Seoul, Korea). All cell lines were cultured in EMEM (American Type) supplemented with 10% fetal bovine serum (FBS, Lonza) and 100 units / mL penicillin-streptomycin (Invitrogen, Carlsbad, CA) at 37 ° C and 5% CO 2 in a humidified incubator Culture Collection, Manassas, Va.), RPMI-1640 or DMEM medium (Lonza, Walkersville, Md.).
2. siRNA 및 dsRNA의 합성 및 Transfection 2. Synthesis and Transfection of siRNA and dsRNA
본 실험에 사용된 siRNA 및 dsRNA는 Lemonex(서울, 대한민국)에 의해 합성되었고, pcDNA3.1+/C-(K)-DYK 플라스미드 내 유전자 ORF 서열(BANF1: NM_003860, PLOD3: NM_001084, SF3B4: NM_005850)을 서브클로닝하는 인간의 BANF1, PLOD3, SF3B4 발현 플라스미드는 GenscriptTM (Piscataway, NJ, USA)에서 구입하였다. 형질주입은 제조사의 메뉴얼에 따라 Lipofectamine RNAiMAX 또는 Lipofectamine 2000 시약(Invitrogen)을 이용하여 수행되었다. The siRNA and dsRNA used in this experiment were synthesized by Lemonex (Seoul, Korea) and the gene ORF sequence (BANF1: NM_003860, PLOD3: NM_001084, SF3B4: NM_005850) in the pcDNA3.1 + / C- (K) Human BANF1, PLOD3, and SF3B4 expression plasmids were purchased from Genscript TM (Piscataway, NJ, USA). Transfection was performed using Lipofectamine RNAiMAX or Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's manual.
3. RNA, DNA extraction, RT-PCR and qRT-PCR3. RNA, DNA extraction, RT-PCR and qRT-PCR
빙결 조직 및 세포로부터 총 RNA가 트리졸 시약(invitrogen)을 이용하여 분리되었다. 1μg의 총 RNA가 Tetro cDNA 합성 키트(Bioline, London, UK)를 이용하여 제조사의 메뉴얼에 따라 cDNA로 역전사되었다. RT-PCR 반응이 nTaq DNA 폴리머라제(Enzynomics, 대전, 대한민국)로 수행되었고, 검출은 Gel Doc XR 이미지 시스템(Bio-Rad, Hercules, CA) 내의 에티듐 브로마이드를 이용하였다. qRT-PCR은 SensiFAST SYBR No-ROX Kit (Bioline)로 수행되었고 iQTM-5 (Bio-Rad)에 의해 실시간 모니터되었다. 3회 반복 실험으로부터의 평균 Ct(threshold cycle)가 계산을 위해 사용되었다. 정상화된 유전자 발현이 상대 정량화 방법을 이용하여 결정되었다. 결과는 3회 반복 실험의 평균값으로 표현되었다. 조직 및 세포로부터의 유전체 DNA는 메뉴얼에 따라 DNAzol 시약(Invitrogen)을 이용하여 분리되었다. 복제수 변이 분석(copy number variation analysis)을 위해, SF3B4 유전체 DNA 영역이 20쌍(비종양 및 종양) HCC 조직으로부터 엑손-1부터 인트론-1까지의 프라이머 세트로 Genbank accession No.NC_000001.11의 유전체 서열에 따라 증폭되었다. qRT-PCR이 전술한 바에 따라 수행되었고 glyceraldehyde-3-phosphate dehydrogenase가 endogenous loading control로 사용되었다. RT-PCR 및 qRT-PCR에 사용된 프라이머 서열은 표 5에 나타내었다.Total RNA was isolated from free tissue and cells using invitrogen. 1 μg of total RNA was reverse transcribed with cDNA using Tetro cDNA synthesis kit (Bioline, London, UK) according to the manufacturer's manual. RT-PCR reactions were performed with nTaq DNA polymerase (Enzynomics, Taejon, Korea) and detection was performed using ethidium bromide in a Gel Doc XR imaging system (Bio-Rad, Hercules, Calif.). qRT-PCR was performed with SensiFAST SYBR No-ROX Kit (Bioline) and was monitored in real time by iQ TM -5 (Bio-Rad). The mean Ct (threshold cycle) from three replicate experiments was used for the calculation. Normalized gene expression was determined using a relative quantification method. The results were expressed as mean values of three replicate experiments. Tissue and genomic DNA from cells were isolated using DNAzol reagent (Invitrogen) according to the manual. For copy number variation analysis, the SF3B4 genomic DNA region was amplified from 20 pairs (non-tumor and tumor) HCC tissues with a primer set from exon-1 to intron-1 and the genome of Genbank accession No. NC_000001.11 Lt; / RTI > qRT-PCR was performed as described above and glyceraldehyde-3-phosphate dehydrogenase was used as an endogenous loading control. The primer sequences used for RT-PCR and qRT-PCR are shown in Table 5.
GeneGene Accession No.Accession No. PrimerPrimer Nucleotide sequenceNucleotide sequence 서열번호SEQ ID NO:
BANF1BANF1 NM_003860NM_003860 ForwardForward 5'-GAACCGTTACGGGAACTGAA-3’5'-GAACCGTTACGGGAACTGAA-3 ' 서열번호 316SEQ ID NO: 316
ReverseReverse 5'-CCCGGAAGAGGTCTTCATCT-3’5'-CCCGGAAGAGGTCTTCATCT-3 ' 서열번호 317SEQ ID NO: 317
PLOD3PLOD3 NM_001084NM_001084 ForwardForward 5'-CAGCTCCAGGACCACTTCTC-3’5'-CAGCTCCAGGACCACTTCTC-3 ' 서열번호 318SEQ ID NO: 318
ReverseReverse 5'-GAGCGGGCGTAGTACTCATC-3’5'-GAGCGGGCGTAGTACTCATC-3 ' 서열번호 319SEQ ID NO: 319
SF3B4SF3B4 NM_005850NM_005850 ForwardForward 5'-CTCAGATGCAGCTTGCACAC-3’5'-CTCAGATGCAGCTTGCACAC-3 ' 서열번호 320SEQ ID NO: 320
ReverseReverse 5'-GGAGGGCCAGTGTATCCAT-3’5'-GGAGGGCCAGTGTATCCAT-3 ' 서열번호 321SEQ ID NO: 321
GAPDHGAPDH NM_002046NM_002046 ForwardForward 5'-ACCAGGTGGTCTCCTCTGAC-3'5'-ACCAGGTGGTCTCCTCTGAC-3 ' 서열번호 322SEQ ID NO: 322
ReverseReverse 5'-TGCTGTAGCCAAATTCGTTG-3'5'-TGCTGTAGCCAAATTCGTTG-3 ' 서열번호 323SEQ ID NO: 323
4. Cell Growth and Proliferation assay4. Cell Growth and Proliferation Assay
세포 성장 분석을 위해 세포주는 30% confluence의 12웰 플레이트에 공급되었다. 형질 주입 또는 저해 치료 이후에, 세포는 매 24시간마다 37℃에서 1시간동안 0.5mg/mL의 (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)와 함께 배양되었다. formazan crystal을 DMSO에 녹이고, VICTOR3™ multilabel plate reader (PerkinElmer, Boston, MA)를 이용하여 570nm에서의 흡광도를 읽었다. For cell growth analysis, the cell line was supplied to a 12-well plate in 30% confluence. After transfusion or inhibition treatment, the cells were incubated with 0.5 mg / mL of (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) at 37 ° C for 1 hour every 24 hours . The formazan crystal was dissolved in DMSO and the absorbance at 570 nm was read using a VICTOR3 ™ multilabel plate reader (PerkinElmer, Boston, Mass.).
세포 증식 분석을 위해 세포주는 24웰 플레이트에 30% confluence에 공급되었다. 형질 주입 이후에, 세포는 5-bromo-2'-deoxyuridine (BrdU) 시약으로 2시간 처리되고 상온에서 30분간 고정되었다. 세포는 상온에서 1시간 동안 항-BrdU 항체와 함께 배양되었다. 결합하지 않은 항체는 워싱 버퍼(washing buffer)에 의해 제거되었다. Horseradish peroxidase-conjugated 2차 항체가 각 웰에 첨가되었다. 기질 용액이 첨가되고 반응은 스탑 용액으로 30분 이후에 멈춰졌다. 최종 산물은 VICTOR3™ multilabel plate reader (PerkinElmer)에 의해 490nm에서 정량화되었다.For cell proliferation assay, cell lines were fed to 24 well plates in 30% confluence. After transfection, cells were treated with 5-bromo-2'-deoxyuridine (BrdU) reagent for 2 h and fixed at room temperature for 30 min. Cells were incubated with anti-BrdU antibody for 1 hour at room temperature. Unbound antibody was removed by washing buffer. Horseradish peroxidase-conjugated secondary antibody was added to each well. The substrate solution was added and the reaction was stopped after 30 minutes in the stop solution. The final product was quantified at 490 nm by a VICTOR3 ™ multilabel plate reader (PerkinElmer).
5. Cell motility and invasion assay5. Cell motility and invasion assay
시험관 내 세포 운동성 및 침윤 분석을 위해 Transwell plates and cell culture inserts (BD Biosciences)를 사용했다. 침입 분석의 코팅을 위해 코팅 버퍼(0.01 M 트리스, 0.7% NaCl, pH 8.0)로 매크리젤(BD Biosciences)을 0.3 mg/ml 농도로 희석하고 100 μl의 매크리젤을 세포 배양 인서트의 상부 구획에 코팅하였다. 37℃에서 1 시간 동안 배양한 후, 세포 배양 인서트를 시딩할 준비가 완료되었다. si-SF3B4 형질 주입 이후에, 세포는 5% FBS가 화학유인물질로 존재하는 세럼-프리 배지의 세포 배양 인서트 내로 적절하게 시드되었다(0.5×105 cells/well for the motility assay, 1×105 cells/well for the invasion assay). 37℃에서 6시간(migration assay) 또는 12시간(invasion assay) 배양 후에, 이동 또는 침입된 세포들은 Diff-Quik staining kit(Sysmex, Japan)를 이용하여 염색되었다. 세포를 200배 배율에서 Axiovert 200 도립 현미경(inverted microscope) (Zeiss, Jena, Germany)으로 사진찍었다. 세포는 3개의 무작위 시계에서 열거되었다.Transwell plates and cell culture inserts (BD Biosciences) were used for in vitro cell motility and invasion analysis. (BD Biosciences) was diluted to 0.3 mg / ml with coating buffer (0.01 M Tris, 0.7% NaCl, pH 8.0) for coating of the invasion assays and 100 μl of Macri Gel was applied to the upper compartment of the cell culture insert Lt; / RTI > After incubation at 37 ° C for 1 hour, the cell culture insert was ready for seeding. After si-SF3B4 transfected injection, the cells 5% FBS a serum present in chemoattractant-were properly seeded into the cell culture insert of the pre-culture medium (0.5 × 10 5 cells / well for the motility assay, 1 × 10 5 cells / well for the invasion assay). After 6 h (migration assay) or 12 h (invasion assay) at 37 ° C, the migrated or invaded cells were stained with a Diff-Quik staining kit (Sysmex, Japan). Cells were photographed at 200x magnification on an Axiovert 200 inverted microscope (Zeiss, Jena, Germany). Cells were enumerated in three randomized clocks.
6. Wound healing assay6. Wound healing assay
형질주입 세포는 6웰 플레이트의 웰 내에 공급되었다. 100% confluence에서, micropipette tip을 이용하여 스크래치가 동일한 층 상에 만들어졌다. 상처의 동일 영역의 사진이 IX70 fluorescence inverted microscope (올림푸스, 도쿄, 일본)를 이용하여 0시간 및 24시간 후에 찍어졌다.Transfected cells were fed into wells of 6 well plates. At 100% confluence, a scratch was made on the same layer using a micropipette tip. Photographs of the same area of the wound were taken at 0 h and 24 h using an IX70 fluorescence inverted microscope (Olympus, Tokyo, Japan).
7. 쥐 간암 모델7. Rat Liver Cancer Model
이종이식(xenograft) 종양형성 평가를 위해, 1×107 개의 형질주입된 세포가 0.2ml PBS(pH 7.4) 및 30% (v/v) 매트리겔 매트릭스(BD Biosciences)에 혼합되었다. 세포 현탁액은 6주령의 Balb/c-nude mice에 피하 주사되었다. 쥐는 주사 부위에서의 종양 형성 확인을 위해 주당 2회 검사되었다. 종양 부피는 0.5 × 길이(L) × 폭2(W2)로 계산되었다. 각 실험 그룹은 10마리의 쥐로 이루어지고, 종양 성장은 세 직각 방향으로 캘리퍼스를 이용하여 측정함으로써 정량화되었다. 결과는 평균 종양 부피 및 95% 신뢰구간으로 표현되었다. H-ras12V 활성화 동형유전 형질전환쥐는 유대열 박사 (Laboratory of Human Genomics, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea)로부터 제공되었다. 형질전환쥐는 H-ras12V 활성화되었다. 수컷쥐는 자연히 15주령부터 HCC가 발달하기 시작했다. 외과적으로 35주령의 5마리 쥐로부터 비종양 부위 및 HCC mass를 얻고, 병리학적 스코어링에 의해 3쌍의 HCC 조직을 선별했다. 디에틸니트로스아민(Diethylnitrosamine (DEN))이 HCC 도입을 위해 사용되었다.For xenograft tumorigenicity evaluation, 1 x 10 7 transduced cells were mixed in 0.2 ml PBS (pH 7.4) and 30% (v / v) Matrigel Matrix (BD Biosciences). Cell suspensions were subcutaneously injected into Balb / c-nude mice at 6 weeks of age. Rats were examined twice a week for confirmation of tumor formation at the injection site. Tumor volume was calculated as 0.5 x length (L) x width 2 (W 2 ). Each experimental group consisted of 10 rats, and tumor growth was quantified by caliper measurements in three orthogonal directions. Results were expressed as mean tumor volume and 95% confidence interval. The H-ras12V activated homotypic transgenic mice were provided by Dr. Jae-Yeol Lee (Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea). Transgenic mice were H-ras12V activated. Male rats naturally began to develop HCC at 15 weeks of age. Surgically, non-tumor sites and HCC mass were obtained from five rats at 35 weeks of age and three pairs of HCC tissues were selected by pathological scoring. Diethylnitrosamine (DEN) was used for HCC incorporation.
8. 다공성 실리카 입자(Mesoporous nanoparticle) Transfection8. Porous silica particles (Mesoporous nanoparticle) Transfection
BANF1, PLOD3, SF3B4에 특이적인 siRNA가 3nmol의 80 ㎕ InViVojectionTM RNAi-nano reagent (실시예 12-(1)-2)-②의 다공성 실리카 나노입자, Cat. No. DHMSN-vivoRNA; Lemonex Inc., Seoul, Korea)에 담지되었고, 200 ㎕ PBS에 준비되었다. siRNA 또는 dsRNA와 나노입자의 혼합물은 9주부터 23주까지 매주 꼬리 정맥 주사에 의해 H-ras transgenic HCC 마우스 모델에 주사되었다. 초음파 기계(Affiniti 50, Philips, Seoul, Korea)로 17, 19, 21 주에 초음파검사 사진을 찍었다.The porous silica nanoparticles of Example 12- (1) -2) -2, 80 μl of InViVojection ™ RNAi-nano reagent, which is specific for BANF1, PLOD3 and SF3B4, No. DHMSN-vivo RNA; Lemonex Inc., Seoul, Korea) and prepared in 200 [mu] l PBS. A mixture of siRNA or dsRNA and nanoparticles was injected into the H-ras transgenic HCC mouse model by weekly intravenous injection from week 9 to week 23. Ultrasonography was taken at 17, 19, and 21 weeks with an ultrasound machine (Affiniti 50, Philips, Seoul, Korea).
9. Western blotting analysis9. Western blotting analysis
세포들은 단백질 추출 완충액(50 mM HEPES, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 mM Na2P2O7, 1 mM Na3VO4, 100 x Halt protease inhibitor cocktail)에서 용해시켰다. 동일한 양의 단백질을 함유하는 용해물을 SDS-PAGE로 분리하고 폴리비닐리덴 불화물 (PVDF) 막 (Bio-Rad) 상으로 옮겼다. 블롯을 5 % 탈지유로 블로킹하고 각 항체(표 6)와 함께 배양 하였다.Cells were washed twice with protein extraction buffer (50 mM HEPES, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 mM Na 2 P 2 O 7 , 1 mM Na 3 VO 4 , 100 x Halt protease inhibitor cocktail. Lysates containing the same amount of protein were separated by SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The blots were blocked with 5% skim milk and incubated with each antibody (Table 6).
ProteinProtein ManufacturerManufacturer Catalog No.Catalog No. DilutionDilution
BANF1BANF1 Santa CruzSanta Cruz sc-33787sc-33787 1 : 2001: 200
PLOD3PLOD3 ProteintechProteintech 11027-1-AP11027-1-AP 1 : 10001: 1000
SF3B4SF3B4 AbcamAbcam ab157117ab157117 1 : 10001: 1000
E-cadherinE-cadherin BD BiosciencesBD Biosciences 610404610404 1 : 10001: 1000
N-cadherinN-cadherin BD BiosciencesBD Biosciences 610920610920 1 : 10001: 1000
FibronectinFibronectin Santa CruzSanta Cruz sc-9068sc-9068 1 : 10001: 1000
SnailSnail AbcamAbcam ab78105ab78105 1 : 10001: 1000
SlugSlug Cell SignalingCell Signaling #9585# 9585 1 : 5001: 500
GAPDHGAPDH Santa CruzSanta Cruz sc-32233sc-32233 1 : 10001: 1000
10. 통계적 분석10. Statistical analysis
생존 곡선은 Kaplan-Meier product limit method를 이용하여 그려졌고, 생존 곡선 간의 유의성 차이는 log-rank test를 이용하여 결정되었다. 모든 실험은 3회 이상 수행되었고, 모든 샘플은 3회 분석되었다. 결과는 평균 ± 표준편차 또는 평균의 표준 오차로 나타내었다. 실험 그룹간의 차이의 통계적 유의성은 GraphpadTM 7.0 software를 이용하여 student’s t-tests로 평가되었다. 통계적 유의성은 p<0.05에서 결정되었다. chi-square test(2-sided)는 파라미터간의 연관을 결정하였다.Survival curves were plotted using the Kaplan-Meier product limit method and the difference in survival curves was determined using the log-rank test. All experiments were performed more than 3 times, and all samples were analyzed 3 times. Results are expressed as mean ± standard deviation or standard error of mean. Statistical significance of the differences between the experimental groups was assessed using Student's t-tests using Graphpad TM 7.0 software. Statistical significance was determined at p <0.05. The chi-square test (2-sided) determined the association between the parameters.
11. 다공성 실리카 입자(Mesoporous nanoparticle)의 제조11. Preparation of porous silica particles (mesoporous nanoparticle)
(1) 입자 1의 제조(1) Preparation of Particle 1
1) 소기공 입자의 제조1) Preparation of small pore particles
2 L 둥근바닥플라스크에 증류수 (DW) 960 mL 과 MeOH 810 mL을 넣었다. 상기 플라스크에 CTAB 7.88 g을 넣은 후 교반하면서 1M NaOH 4.52 mL를 빠르게 넣었다. 10분 동안 교반시켜 균일한 혼합액을 넣은 후 TMOS 2.6 mL를 넣었다. 6시간 동안 교반하여 균일하게 혼합한 후, 24시간 동안 숙성시켰다.A 2 L round bottom flask was charged with 960 mL of distilled water (DW) and 810 mL of MeOH. 7.88 g of CTAB was added to the flask and 4.52 mL of 1 M NaOH was rapidly added with stirring. The mixture was stirred for 10 minutes to give a homogeneous mixed solution, and 2.6 mL of TMOS was added. Stirred for 6 hours, homogeneously mixed, and aged for 24 hours.
이후 상기 반응액을 25℃에서 10분간 8000rpm에서 원심분리하여 상등액을 제거하고, 25℃에서 10분간 8000rpm에서 원심분리하며 에탄올 및 증류수로 번갈아가며 5회 세척하였다.Then, the reaction solution was centrifuged at 8000 rpm for 10 minutes at 25 DEG C to remove the supernatant, centrifuged at 8000 rpm for 10 minutes at 25 DEG C, and washed five times with ethanol and distilled water alternately.
이후 70℃ 오븐에서 건조시켜 1.5g의 분말형의 소기공 다공성 실리카 입자(기공 평균 직경 2nm, 입경 200nm)를 얻었다.Thereafter, the resultant was dried in an oven at 70 ° C to obtain 1.5 g of powdery pore-size porous silica particles (average pore diameter 2 nm, particle diameter 200 nm).
2) 기공 확장2) Expansion of porosity
1.5g의 소기공 다공성 실리카 입자 분말을 에탄올 10ml에 첨가하여1.5 g of small pore porous silica particle powder was added to 10 ml of ethanol
초음파 분산시키고, 물 10ml, TMB (trimethyl benzene) 10ml를 첨가하여 초음파 분산시켰다.10 ml of water and 10 ml of trimethyl benzene (TMB) were added thereto, followed by ultrasonic dispersion.
이 후 상기 분산액을 오토클레이브에 넣고 160℃, 48시간 반응시켰다.Thereafter, the dispersion was placed in an autoclave and reacted at 160 DEG C for 48 hours.
반응은 25℃에서 시작하여 10℃/분의 속도로 승온시켜 수행하였고, 이 후 오토클레이브 내에서 1~10℃/분의 속도로 서서히 냉각시켰다.The reaction was carried out by starting at 25 ° C and heating at a rate of 10 ° C / min and then slowly cooling down at a rate of 1 to 10 ° C / min in an autoclave.
냉각된 반응액을 25℃에서 10분간 8000rpm에서 원심분리하여 상등액을 제거하고, 25℃에서 10분간 8000rpm에서 원심분리하며 에탄올 및 증류수로 번갈아가며 5회 세척하였다.The cooled reaction solution was centrifuged at 8000 rpm for 10 minutes at 25 DEG C to remove supernatant, centrifuged at 8000 rpm for 10 minutes at 25 DEG C, and washed five times with ethanol and distilled water alternately.
이 후 70℃ 오븐에서 건조시켜 분말형의 다공성 실리카 입자(기공 직경 10~15nm, 입경 200nm)를 얻었다.Thereafter, the resultant was dried in an oven at 70 DEG C to obtain powdery porous silica particles (pore diameter: 10 to 15 nm, particle size: 200 nm).
3) 하소3) Calcination
상기 2)에서 제조된 다공성 실리카 입자를 유리 vial에 담아 550℃에서 5시간 동안 가열하고, 반응 종료 후 상온으로 서서히 식혀 입자를 제조하였다.The porous silica particles prepared in 2) were placed in a glass vial and heated at 550 ° C for 5 hours. After completion of the reaction, the particles were gradually cooled to room temperature to prepare particles.
(2) 입자 2의 제조(2) Production of Particle 2
기공 확장시의 반응 조건을 140℃, 72시간으로 변경한 것을 제외하고는 실시예 11-(1)과 동일한 방법으로 다공성 실리카 입자를 제조하였다.Porous silica particles were prepared in the same manner as in item (1) of Example 11, except that the reaction conditions at the time of pore expansion were changed to 140 ° C and 72 hours.
(3) 입자 3의 제조 (10L 스케일)(3) Preparation of Particle 3 (10 L scale)
5배 큰 용기를 사용하고, 각 물질을 모두 5배 용량으로 사용한 것을 제외하고는 실시예 11-(1)과 동일한 방법으로 다공성 실리카 입자를 제조하였다.Porous silica particles were prepared in the same manner as in item (1) of Example 11, except that 5-fold larger vessels were used and all the materials were used in a 5-fold capacity.
(4) 입자 4의 제조 (입경 300nm)(4) Production of Particle 4 (particle diameter 300 nm)
소기공 입자의 제조시에 증류수 920ml, 메탄올 850ml를 사용한 것을 제외하고는 실시예 11-(1)과 동일한 방법으로 다공성 실리카 입자를 제조하였다.Porous silica particles were prepared in the same manner as in Example 11- (1) except that 920 ml of distilled water and 850 ml of methanol were used in the preparation of small pore particles.
(5) 입자 5의 제조 (입경 500nm)(5) Preparation of Particle 5 (particle diameter 500 nm)
소기공 입자의 제조시에 증류수 800ml, 메탄올 1010ml, CTAB 10.6g을 사용한 것을 제외하고는 실시예 11-(1)과 동일한 방법으로 다공성 실리카 입자를 제조하였다.Porous silica particles were prepared in the same manner as in Example 11- (1) except that 800 ml of distilled water, 1010 ml of methanol and 10.6 g of CTAB were used in the preparation of small pore particles.
(6) 입자 6의 제조 (입경 1000nm)(6) Production of Particle 6 (particle diameter 1000 nm)
소기공 입자의 제조시에 증류수 620ml, 메탄올 1380ml, CTAB 7.88g을 사용한 것을 제외하고는 실시예 11-(1)과 동일한 방법으로 다공성 실리카 입자를 제조하였다.Porous silica particles were prepared in the same manner as in Example 11- (1), except that 620 ml of distilled water, 1380 ml of methanol and 7.88 g of CTAB were used in the preparation of the small pore particles.
(7) 입자 7의 제조 (기공 직경 4nm)(7) Preparation of particle 7 (pore diameter: 4 nm)
기공 확장시에 TMB를 2.5mL를 사용한 것을 제외하고는 실시예 11-(1)과 동일한 방법으로 다공성 실리카 입자를 제조하였다.Porous silica particles were prepared in the same manner as in item (1) of Example 11 except that 2.5 mL of TMB was used at the time of pore expansion.
(8) 입자 8의 제조 (기공 직경 7nm)(8) Production of Particle 8 (pore diameter 7 nm)
기공 확장시에 TMB를 4.5mL를 사용한 것을 제외하고는 실시예 11-(1)과 동일한 방법으로 다공성 실리카 입자를 제조하였다.Porous silica particles were prepared in the same manner as in item (1) of Example 11 except that 4.5 mL of TMB was used at the time of pore expansion.
(9) 입자 9의 제조 (기공 직경 17nm)(9) Production of Particle 9 (pore diameter: 17 nm)
기공 확장시에 TMB를 11mL를 사용한 것을 제외하고는 실시예 11-(1)과 동일한 방법으로 다공성 실리카 입자를 제조하였다.Porous silica particles were prepared in the same manner as in Example 11- (1) except that 11 mL of TMB was used at the time of pore expansion.
(10) 입자 10의 제조 (기공 직경 23nm)(10) Preparation of particle 10 (pore diameter: 23 nm)
기공 확장시에 TMB를 12.5mL를 사용한 것을 제외하고는 실시예 11-(1)과 동일한 방법으로 다공성 실리카 입자를 제조하였다.Porous silica particles were prepared in the same manner as in item (1) of Example 11 except that 12.5 mL of TMB was used at the time of pore expansion.
(11) 입자 11의 제조 (이중개질)(11) Production of particle 11 (dual modification)
1) 소기공 입자의 제조1) Preparation of small pore particles
실시예 11-(1)과 동일한 방법으로 소기공 입자를 제조하였다.Small pore particles were prepared in the same manner as in Example 11- (1).
2) 기공 확장2) Expansion of porosity
실시예 11-(1)-2)와 동일한 방법으로 소기공 입자를 TMB와 반응시키고 냉각시키고 원심분리하여 상등액을 제거하였다. 이후 실시예 11-(1)-2)와 동일 조건으로 원심분리하며 에탄올 및 증류수로 번갈아가며 3회 세척하고, 이후 실시예 11-(1)-2)와 동일 조건으로 건조하여 분말형의 다공성 실리카 입자(기공 직경 10~15nm, 입경 200nm)를 얻었다.The small pore particles were reacted with TMB in the same manner as in Example 11- (1) -2), cooled, and centrifuged to remove the supernatant. Thereafter, the mixture was centrifuged under the same conditions as in Example 11- (1) -2), washed three times with ethanol and distilled water alternately and then dried under the same conditions as in Example 11- (1) -2) Silica particles (pore diameter 10 to 15 nm, particle diameter 200 nm) were obtained.
3) 표면 개질3) Surface modification
기공이 확장된 다공성 실리카 입자 0.8g 내지 1g을 50mL의 톨루엔에 분산시킨 후, (3-aminopropyl)triethoxysilane를 5mL 넣어주어 120℃로 환류한 채로 12시간 가열하였다. 해당 과정은 상기 서술된 세척과정 및 건조과정을 거친 뒤 1mL의 트레에틸렌글리콜(PEG3, 2-[2-(2-methoxyethoxy)ethoxy]acetic acid)와 100mg의 EDC(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) 및 200mg의 N-Hydroxysuccinimide(NHS)를 30mL의 PBS에 분산시켜서 상온에서 교반한 채로 12시간 동안 반응을 보낸다. 이후 생성물은 상기의 세척 및 건조과정을 거친다.0.8 g to 1 g of porous silica particles having expanded pores were dispersed in 50 mL of toluene, and then 5 mL of (3-aminopropyl) triethoxysilane was added thereto, followed by heating at 120 ° C for 12 hours. After the washing and drying processes described above, 1 mL of PEG3, 2- [2- (2-methoxyethoxy) ethoxy] acetic acid and 100 mg of EDC (1-Ethyl-3- -dimethylaminopropyl) carbodiimide) and 200 mg of N-Hydroxysuccinimide (NHS) were dispersed in 30 mL of PBS, and the mixture was stirred at room temperature for 12 hours. The product is then subjected to the above-described washing and drying processes.
기공 내부에 이전 단계의 반응액이 남아 있어, 기공 내부는 개질 되지 않는다.The reaction solution in the previous step remains in the pores, and the inside of the pores are not modified.
4) 기공 내부 세척4) Internal pore cleaning
표면개질된 입자 분말 800mg을 2M HCl/에탄올 40ml에 녹이고, 12시간 강하게 교반 하에 환류시켰다.800 mg of the surface-modified particle powder was dissolved in 40 ml of 2M HCl / ethanol and refluxed with vigorous stirring for 12 hours.
이후 냉각된 반응액을 10분간 8000rpm에서 원심분리하여 상등액을 제거하고, 25℃에서 10분간 8000rpm에서 원심분리하며 에탄올 및 증류수로 번갈아가며 5회 세척하였다.Then, the cooled reaction solution was centrifuged at 8000 rpm for 10 minutes to remove the supernatant, centrifuged at 8000 rpm for 10 minutes at 25 DEG C, and washed five times with ethanol and distilled water alternately.
이후 70℃ 오븐에서 건조시켜 분말형의 다공성 실리카 입자를 얻었다.Thereafter, the resultant was dried in an oven at 70 DEG C to obtain powdery porous silica particles.
5) 기공 내부 개질5) Internal reforming of porosity
① 후술하는 실시예 2. (2) 1)의 방법과 동일한 방법으로 기공 내부에 프로필기를 도입하였다.(1) A propyl group was introduced into the pores in the same manner as in Example 2 (2) 1) described later.
② 후술하는 실시예 2. (2) 2)의 방법과 동일한 방법으로 기공 내부에 옥틸기를 도입하였다.(2) An octyl group was introduced into the pores in the same manner as in Example 2 (2) 2).
12. 다공성 실리카 입자의 표면 개질12. Surface modification of porous silica particles
(1) 양전하로의 대전(1) Competition with positive charge
1) 입경 300nm의 입자1) Particles having a particle size of 300 nm
실시예 11-(4)의 다공성 실리카 입자를 (3-Aminopropyl)triethoxysilane (APTES)와 반응시켜 양전하로 대전시켰다.The porous silica particles of Example 11- (4) were reacted with (3-Aminopropyl) triethoxysilane (APTES) to positively charge.
구체적으로, 100 mL 둥근 바닥 플라스크에 100 mg의 다공성 실리카 입자를 10 mL의 톨루엔에 bath sonicator로 분산시켰다. 이후 1 mL의 APTES를 첨가하고 400 rpm으로 교반하며 130℃에서 교반하며 12시간 동안 반응시켰다.Specifically, 100 mg of porous silica particles were dispersed in 10 mL of toluene in a 100 mL round bottom flask with a bath sonicator. Then, 1 mL of APTES was added, and the mixture was stirred at 400 rpm and stirred at 130 DEG C for 12 hours.
반응 후에 상온까지 서서히 식히고, 10분간 8000rpm에서 원심분리하여 상등액을 제거하고, 25℃에서 10분간 8000rpm에서 원심분리하며 에탄올 및 증류수로 번갈아가며 5회 세척하였다.After the reaction, the mixture was slowly cooled to room temperature, centrifuged at 8000 rpm for 10 minutes to remove the supernatant, centrifuged at 8000 rpm for 10 minutes at 25 ° C, and washed 5 times with ethanol and distilled water.
이후 70℃ 오븐에서 건조시켜 표면 및 기공 내부에 아미노기를 갖는 분말형의 다공성 실리카 입자를 얻었다.Thereafter, the resultant was dried in an oven at 70 DEG C to obtain powdery porous silica particles having an amino group on the surface and pores.
2) 입경 200nm의 입자2) Particles having a particle diameter of 200 nm
① 실시예 11-(1)의 다공성 실리카 입자를 (3-Aminopropyl)triethoxysilane (APTES)와 반응시켜 양전하로 대전시켰으며, APTES를 0.4ml 첨가하고, 반응 시간을 3시간으로 한 것을 제외하고는 실시예 12-(1)-1)의 방법과 동일하게 개질하였다.(1) The porous silica particles of Example 11- (1) were reacted with (3-aminopropyl) triethoxysilane (APTES) to positively charge, except that 0.4 ml of APTES was added and the reaction time was changed to 3 hours Was modified in the same manner as in Example 12- (1) -1).
② 실시예 11-(9)의 다공성 실리카 입자를 (3-Aminopropyl)triethoxysilane (APTES)와 반응시켜 양전하로 대전시켰으며, 그 외 방법은 실시예 12-(1)-1)의 방법과 동일하게 개질하였다.(2) The porous silica particles of Example 11- (9) were reacted with (3-Aminopropyl) triethoxysilane (APTES) and positively charged. The other method was the same as that of Example 12- Lt; / RTI &gt;
③ 실시예 11-(10)의 다공성 실리카 입자를 (3-Aminopropyl)triethoxysilane (APTES)와 반응시켜 양전하로 대전시켰으며, 그 외 방법은 실시예 12-(1)-1)의 방법과 동일하게 개질하였다.(3) The porous silica particles of Example 11- (10) were reacted with (3-Aminopropyl) triethoxysilane (APTES) to positively charge. The other method was the same as that of Example 12- Lt; / RTI &gt;
(2) 소수성기의 도입(2) introduction of a hydrophobic group
1) 프로필기1) propyl group
실시예 11-(1)의 다공성 실리카 입자를 Trimethoxy(propyl)silane와 반응시켜 표면 및 기공 내부에 프로필기를 도입하였으며, APTES 대신에 Trimethoxy(propyl)silane를 0.35ml 첨가하고, 12시간 반응시킨 것을 제외하고는 실시예 12-(1)과 동일한 방법으로 개질을 수행하였다.The porous silica particles of Example 11- (1) were reacted with trimethoxy (propyl) silane to introduce a propyl group into the surface and pores, except that 0.35 ml of trimethoxy (propyl) silane was added instead of APTES and reacted for 12 hours And the modification was carried out in the same manner as in Example 12- (1).
2) 옥틸기2) Octyl group
실시예 11-(1)의 다공성 실리카 입자를 Trimethoxy-n-octylsilane와 반응시켜 표면 및 기공 내부에 프로필기를 도입하였으며, APTES 대신에 Trimethoxy-n-octylsilane를 0.5ml 첨가하고, 12시간 반응시킨 것을 제외하고는 실시예 12-(1)과 동일한 방법으로 개질을 수행하였다.The porous silica particles of Example 11- (1) were reacted with trimethoxy-n-octylsilane to introduce a propyl group into the surface and pores, except that 0.5 ml of trimethoxy-n-octylsilane was added instead of APTES and reacted for 12 hours And the modification was carried out in the same manner as in Example 12- (1).
(3) 음전하로의 대전(3) Battle of negative charge
1) 카르복실기1) carboxyl group
실시예 11-(1)의 다공성 실리카 입자를 succinic anhydride와 반응시켜 음전하로 대전시켰으며, 톨루엔 대신에 DMSO(dimethyl sulfoxide)를 사용하고, APTES 대신에 80 mg의 succinic anhydride를 첨가하여 24시간 동안 상온에서 교반하며 반응시키고, 세척 시에 증류수 대신에 DMSO를 사용한 것을 제외하고는 실시예 12-(1)-1)의 방법과 동일하게 개질하였다.The porous silica particles of Example 11- (1) were reacted with succinic anhydride to negatively charge. DMSO (dimethyl sulfoxide) was used instead of toluene, 80 mg of succinic anhydride was added instead of APTES, And the reaction was carried out in the same manner as in Example 12- (1) -1) except that DMSO was used instead of distilled water at the time of washing.
2) 티올기2) thiol group
APTES 대신에 MPTES 1.1 mL를 사용한 것을 제외하고는 실시예 12-(1)-1)의 방법과 동일하게 개질하였다.(1) -1) except that 1.1 mL of MPTES was used instead of APTES.
3) 술폰산기3) Sulfonic acid group
실시예 12-(3)-2)의 다공성 실리카 나노입자 100mg를 1M 황산수용액을 1 mL와 30% 과산화수소수 20 mL에 분산하여 상온에서 교반하여 산화반응을 유도하여 티올기를 술폰산기로 산화시켰다. 이후 실시예 12-(1)-1)의 방법과 동일하게 세척 및 건조시켰다.100 mg of the porous silica nanoparticles of Example 12- (3) -2) was dispersed in 1 mL of a 1 M aqueous sulfuric acid solution and 20 mL of 30% aqueous hydrogen peroxide and stirred at room temperature to induce an oxidation reaction to oxidize the thiol group with a sulfonic acid group. Thereafter, it was washed and dried in the same manner as in Example 12- (1) -1).
13. 다공성 실리카 입자에의 siRNA 또는 dsRNA의 담지13. Carrying of siRNA or dsRNA onto porous silica particles
녹색형광단백질(Green Fluorescence Protein, GFP)을 표적으로 하는 21 base pair duplex siRNA를 ㈜바이오니아에 합성의뢰하여 구입하였다(서열: sense; 5'-GGCUACGUCCAGGAGCGCACC-3'(서열번호 324), antisense; 5'- UGCGCUCCUGGACGUAGCCUU-3'(서열번호 325)).5'-GGCUACGUCCAGGAGCGCACC-3 '(SEQ ID NO: 324), antisense; 5' -GGCUACGACCAGGAGCGCACC-3 '(SEQ ID NO: 324) was purchased by synthesizing 21 base pair duplex siRNA targeting green fluorescent protein (GFP) - UGCGCUCCUGGACGUAGCCUU-3 '(SEQ ID NO: 325)).
실시예 12-(1)-2)-②의 다공성 실리카 입자 10μg과 50pmol의 상기 siRNA을 1xPBS 조건에서 섞은 후 상온에서 30분간 두고 적재가 되도록 하였다.10 占 퐂 of the porous silica particles of Example 12- (1) -2) -② and 50 pmol of the siRNA were mixed in 1xPBS conditions and allowed to stand for 30 minutes at room temperature.
실시예 2. 본 발명 siRNA 또는 dsRNA의 지표 유전자 발현 억제율 분석Example 2. Analysis of inhibitory rate of indicator gene expression of siRNA or dsRNA of the present invention
상기 실시예 1의 1 내지 3에 따른 실험방법에 따라, 본 발명 siRNA 및 dsRNA 들의 지표 유전자(BANF1 variant 1, BANF1 variant 2, PLOD3, SF3B4) 발현 억제율을 분석하였고, 그 결과를 하기 표 7 내지 10에 나타내었다. (BANF1 variant 1, BANF1 variant 2, PLOD3, and SF3B4) expression of the siRNA and dsRNA of the present invention were analyzed according to the experimental method according to Example 1, and the results are shown in Tables 7 to 10 Respectively.
하기 표 7 내지 10을 참조하면, 본 발명의 모든 siRNA 및 dsRNA는 상기 지표 유전자들의 발현을 우수한 비율로 억제하였음을 확인할 수 있다.Referring to Tables 7 to 10, it can be confirmed that all the siRNAs and dsRNAs of the present invention suppressed the expression of the indicator genes at a good ratio.
인간 BANF1, transcript variant 1, mRNA (Gene Bank number: NM_003860.3)의 발현 억제에 대한 siRNA, dsRNA 유효성 검정SiRNA, dsRNA validation test for inhibition of human BANF1, transcript variant 1, mRNA (Gene Bank number: NM_003860.3)
염기서열번호Base sequence number 발현억제율(%)Expression inhibition rate (%) 염기서열번호Base sequence number 발현억제율(%)Expression inhibition rate (%) 염기서열번호Base sequence number 발현억제율(%)Expression inhibition rate (%) 염기서열번호Base sequence number 발현억제율(%)Expression inhibition rate (%)
55 87.7387.73 1111 73.1873.18 1717 84.1184.11 2323 76.2476.24
66 79.6479.64 1212 85.4485.44 1818 88.3688.36 2424 87.787.7
77 82.382.3 1313 69.5769.57 1919 87.8387.83 2525 62.5762.57
88 76.2176.21 1414 77.377.3 2020 67.7267.72 2626 72.9272.92
99 89.689.6 1515 82.9282.92 2121 82.2982.29 2727 65.5865.58
1010 83.4283.42 1616 91.3891.38 2222 63.2363.23 2828 72.9172.91
인간 BANF1, transcript variant 2, mRNA (Gene Bank number: NM_001143985.1)의 발현 억제에 대한 siRNA, dsRNA 유효성 검정SiRNA, dsRNA validity test for inhibition of human BANF1, transcript variant 2, mRNA (Gene Bank number: NM_001143985.1)
염기서열번호Base sequence number 발현억제율(%)Expression inhibition rate (%) 염기서열번호Base sequence number 발현억제율(%)Expression inhibition rate (%) 염기서열번호Base sequence number 발현억제율(%)Expression inhibition rate (%) 염기서열번호Base sequence number 발현억제율(%)Expression inhibition rate (%)
2929 92.5592.55 3737 81.6481.64 4545 83.2283.22 5353 84.3184.31
3030 91.4991.49 3838 68.468.4 4646 78.1678.16 5454 64.964.9
3131 86.4486.44 3939 79.7279.72 4747 73.4873.48 5555 74.7274.72
3232 77.177.1 4040 91.691.6 4848 68.368.3
3333 73.8273.82 4141 87.3787.37 4949 85.2785.27
3434 76.676.6 4242 53.7753.77 5050 88.7488.74
3535 88.3388.33 4343 86.3986.39 5151 92.3292.32
3636 82.5382.53 4444 68.6368.63 5252 74.874.8
인간 인간 PLOD3 유전자 서열(Gene Bank number: NM_001084.4)의 발현 억제에 대한siRNA, dsRNA 유효성 검정SiRNA, dsRNA validity test for expression suppression of human human PLOD3 gene sequence (Gene Bank number: NM_001084.4)
염기서열번호Base sequence number 발현억제율(%)Expression inhibition rate (%) 염기서열번호Base sequence number 발현억제율(%)Expression inhibition rate (%) 염기서열번호Base sequence number 발현억제율(%)Expression inhibition rate (%) 염기서열번호Base sequence number 발현억제율(%)Expression inhibition rate (%)
5656 87.6287.62 7373 76.876.8 9090 72.772.7 107107 74.574.5
5757 78.1378.13 7474 68.2768.27 9191 83.6983.69 108108 86.2586.25
5858 92.7292.72 7575 77.4477.44 9292 85.385.3 109109 83.783.7
5959 83.4983.49 7676 86.2686.26 9393 76.6276.62 110110 74.1374.13
6060 86.886.8 7777 84.384.3 9494 82.1182.11 111111 76.2976.29
6161 64.2964.29 7878 81.5281.52 9595 83.4683.46 112112 73.5273.52
6262 73.3373.33 7979 79.3579.35 9696 71.2571.25 113113 82.8682.86
6363 85.8385.83 8080 76.6376.63 9797 72.7372.73 114114 73.5273.52
6464 82.6882.68 8181 85.3285.32 9898 87.687.6 115115 73.7173.71
6565 76.9276.92 8282 62.7262.72 9999 69.9169.91 116116 82.5582.55
6666 91.6491.64 8383 64.364.3 100100 81.3881.38 117117 69.7269.72
6767 85.7785.77 8484 77.1377.13 101101 78.2778.27 118118 77.877.8
6868 79.179.1 8585 83.7883.78 102102 74.874.8 119119 86.4986.49
6969 82.482.4 8686 86.7186.71 103103 69.7669.76 120120 71.371.3
7070 84.6384.63 8787 82.3382.33 104104 62.4562.45
7171 89.2689.26 8888 68.4668.46 105105 87.2287.22
7272 76.476.4 8989 74.8874.88 106106 82.782.7
인간 SF3B4 유전자 서열(인간 SF3B4 유전자 서열(Gene Bank number: NM_005850.4)의 발현 억제에 대한 siRNA, dsRNA 유효성 검정SiRNA for the inhibition of the expression of the human SF3B4 gene sequence (human SF3B4 gene sequence (Gene Bank number: NM_005850.4), dsRNA validity test
염기서열번호Base sequence number 발현억제율(%)Expression inhibition rate (%) 염기서열번호Base sequence number 발현억제율(%)Expression inhibition rate (%) 염기서열번호Base sequence number 발현억제율(%)Expression inhibition rate (%) 염기서열번호Base sequence number 발현억제율(%)Expression inhibition rate (%)
121121 83.7183.71 131131 74.3274.32 141141 78.3378.33 151151 73.1273.12
122122 81.8381.83 132132 92.1992.19 142142 72.4572.45 152152 78.6678.66
123123 87.6287.62 133133 84.7284.72 143143 76.7276.72 153153 82.582.5
124124 86.3986.39 134134 81.381.3 144144 81.3681.36 154154 76.6376.63
125125 78.6478.64 135135 83.483.4 145145 83.283.2 155155 62.9562.95
126126 82.782.7 136136 88.6388.63 146146 72.4172.41 156156 89.689.6
127127 84.2584.25 137137 78.2578.25 147147 73.6473.64 157157 77.277.2
128128 74.1174.11 138138 85.185.1 148148 86.7786.77
129129 63.3663.36 139139 81.6881.68 149149 84.884.8
130130 76.2576.25 140140 83.5783.57 150150 82.5682.56
실시예Example 3. 다공성 실리카 입자의 RNA 전달의 우수성 확인 3. Confirm the superiority of RNA delivery of porous silica particles
실시예 1의 Hepa-1c1c7, SNU-449 세포주에 대하여 하기 표 11의 서열을 갖는 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA들을 각각 실시예 1-2 또는 1-8의 방법에 따라 in vitro transfection 시킨 후, 각 siRNA들이 대응하는 지표인자들의 발현량을 Western blotting으로 측정하였고, 그 결과를 도 1에 나타내었다.SiRNAs comprising the sense RNA having the sequence shown in Table 11 below and the antisense RNA consisting of the complementary sequence to the Hepa-1c1c7 and SNU-449 cell lines of Example 1 were subjected to the methods of Examples 1-2 or 1-8, respectively After in vitro transfection, the expression levels of the corresponding indicator factors of each siRNA were measured by Western blotting, and the results are shown in Fig.
도 1을 참조하면, 표 11의 siRNA들을 Transfection 시켰을 때 각 지표인자들을 효과적으로 억제하였고, 특히 다공성 실리카 입자에 담지하여 Transfection 시킨 경우 발현량의 억제 수준이 더 높음을 확인할 수 있다.Referring to FIG. 1, when the siRNAs shown in Table 11 were transfected, the respective surface factors were effectively inhibited. Especially, when the siRNAs were transfected with the porous silica particles, the level of inhibition of expression was higher.
서열번호SEQ ID NO: Sense RNA 서열Sense RNA sequence 도 1에서의 명명The naming in Figure 1 타겟유전자Target gene
서열번호 311SEQ ID NO: 311 5'-CCUCAGCGUUUCAAUCUUUUU-3’5'-CCUCAGCGUUUCAAUCUUUUU-3 ' Banf1Banf1 Mouse BANF1 geneMouse BANF1 gene
서열번호 312SEQ ID NO: 312 5'-CGACUGCAGAAUCUCCUCUUU-3’5'-CGACUGCAGAAUCUCCUCUUU-3 ' Plod3Plod3 Mouse PLOD3 geneMouse PLOD3 gene
서열번호 313SEQ ID NO: 313 5'-CUGCUUUACGAUACUUUCAUU-3’5'-CUGCUUUACGAUACUUUCAUU-3 ' Sf3b4Sf3b4 Mouse SF3B4 geneMouse SF3B4 gene
서열번호 314SEQ ID NO: 314 5'-CCUACGCCACCAAUUUCGU-3'5'-CCUACGCCACCAAUUUCGU-3 ' ControlControl --
서열번호 28SEQ ID NO: 28 5'-AAGAAGCUGGAGGAAAGGGGUUU-3'5'-AAGAAGCUGGAGGAAAGGGGUUU-3 ' BANF1BANF1 Human BANF1 geneHuman BANF1 gene
서열번호 119SEQ ID NO: 119 5'-GCAUCUGGAGCUUUCUGUA UU-3'5'-GCAUCUGGAGCUUUCUGUA UU-3 ' PLOD3PLOD3 Human PLOD3 geneHuman PLOD3 gene
서열번호 136SEQ ID NO: 136 5'-GCAGUACCUCUGUAACCGU UU-3'5'-GCAGUACCUCUGUAACCGU UU-3 ' SF3B4SF3B4 Human SF3B4 geneHuman SF3B4 gene
실시예Example 4. 본 발명  4. Invention siRNAsiRNA 또는  or dsRNA의dsRNA 간암세포의 전이능력  Metastatic potential of liver cancer cells 억제능Inhibition 확인 Confirm
1. Cell motility and invasion assay 및 Wound healing assay1. Cell motility and invasion assay and Wound healing assay
실시예 1-1의 SNU-449 세포주에 대하여 하기 표 12의 서열을 갖는 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA들을 실시예 1-2의 방법에 따라 in vitro transfection 시킨 후, 각 siRNA들이 대응하는 지표인자들의 migration과 invasion 반응을 실시예 1-5의 방법에 따라 분석하였고, 실시예 1-6의 방법에 따라 scratch wound healing 능력을 분석하였으며, 그 결과를 도 2에 나타내었다.SiRNAs comprising the sense RNA having the sequence shown in Table 12 below and the antisense RNA consisting of the complementary sequence of the SNU-449 cell line of Example 1-1 were in vitro transfected according to the method of Example 1-2, The migration and invasion responses of the corresponding surface factors of the siRNAs were analyzed according to the methods of Examples 1-5, and the scratch wound healing ability was analyzed according to the method of Examples 1-6, and the results are shown in FIG.
서열번호SEQ ID NO: Sense RNA 서열Sense RNA sequence 도 2에서의 명명The naming in Figure 2 타겟유전자Target gene
서열번호 314SEQ ID NO: 314 5'-CCUACGCCACCAAUUUCGU-3'5'-CCUACGCCACCAAUUUCGU-3 ' ControlControl --
서열번호 28SEQ ID NO: 28 5'-AAGAAGCUGGAGGAAAGGGGUUU-3'5'-AAGAAGCUGGAGGAAAGGGGUUU-3 ' BANF1BANF1 Human BANF1 geneHuman BANF1 gene
서열번호 119SEQ ID NO: 119 5'-GCAUCUGGAGCUUUCUGUA UU-3'5'-GCAUCUGGAGCUUUCUGUA UU-3 ' PLOD3PLOD3 Human PLOD3 geneHuman PLOD3 gene
서열번호 136SEQ ID NO: 136 5'-GCAGUACCUCUGUAACCGU UU-3'5'-GCAGUACCUCUGUAACCGU UU-3 ' SF3B4SF3B4 Human SF3B4 geneHuman SF3B4 gene
도 2(A)을 참조하면, 상기 표 12의 siRNA들을 transfection 시켜 각 지표인자들을 knockdown 시킨 cell들은 대조군에 비해 현저히 migration과 invasion 반응이 저하됨을 확인할 수 있고, 도 2(B)를 참조하면, 대조군에 비해 현저히 wound-healing 능력이 저하됨을 확인할 수 있다. 이 결과들은 본 발명의 siRNA 또는 dsRNA가 간암 세포들의 전이능력을 억제하고, 악성으로 진행되는 것을 저하시킬 수 있음을 보여주는 것이다.2 (A), cells transfected with the siRNAs shown in Table 12 and knockdown of the respective surface factors were observed to exhibit significantly lower migration and invasion responses than the control group. Referring to FIG. 2 (B) And the wound-healing ability is remarkably lower than that of the wound-healing ability. These results show that the siRNA or dsRNA of the present invention can inhibit the metastatic ability of hepatocellular carcinoma cells and decrease the malignant progression.
2. 2. EMTEMT 조절 단백질들의  Regulatory proteins 억제능Inhibition 확인 Confirm
간암 세포들의 전이와 관련한 대표적인 EMT(epithelial-mesenchymal transition) 조절 단백질들인 N-cadherin, Fibronectin, Snail 및 Slug의 발현을 본 발명 siRNA 또는 dsRNA가 각 지표인자들의 발현을 억제함으로서 간암의 전이를 억제할 수 있는지를 확인하기 위하여, 실시예 1의 SNU-449 세포주에 상기 표 12의 서열을 갖는 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA들을 실시예 1-2의 방법에 따라 in vitro transfection 시킨 후, 각 siRNA들이 대응하는 지표인자들의 발현량과 상기 EMT 조절 단백질들의 발현량을 실시예 1-9의 방법에 따라 분석하였고, 그 결과를 도 3(A)에 나타내었다.Expression of N-cadherin, Fibronectin, Snail, and Slug, which are representative epithelial-mesenchymal transition (EMT) regulatory proteins related to the metastasis of liver cancer cells, can be inhibited by inhibiting the expression of each marker by the siRNA or dsRNA of the present invention. , SiRNAs consisting of the sense RNA having the sequence of Table 12 and the antisense RNA consisting of the complementary sequence were in vitro transfected in the SNU-449 cell line of Example 1 according to the method of Example 1-2 Then, the amount of expression of the corresponding indicator factors and the expression level of the EMT regulatory proteins of each siRNA were analyzed according to the method of Examples 1-9, and the results are shown in FIG. 3 (A).
도 3(A)를 참조하면, 상기 표 12의 siRNA들을 transfection 시켜 각 지표인자들을 knockdown 시킨 cell들은 대조군에 비해 유의미하게 각 지표인자들의 발현량은 물론, EMT(epithelial-mesenchymal transition) 조절 단백질들인 N-cadherin, Fibronectin, Snail 및 Slug의 발현량도 억제됨을 확인할 수 있다. 이 결과는 본 발명의 siRNA 또는 dsRNA가 각 대응하는 지표인자들의 발현을 선택적으로 억제하여 간암 세포들의 잠재적 전이능력을 억제할 수 있음을 보여주는 것이다.3 (A), cells transfected with the siRNAs shown in Table 12 and knockdown of the respective surface factors were significantly higher than those of the control group, as well as the expression amounts of the respective surface factors, as well as the epithelial-mesenchymal transition (EMT) -cadherin, Fibronectin, Snail, and Slug. This result shows that the siRNA or dsRNA of the present invention can inhibit the potential transfer ability of liver cancer cells by selectively inhibiting the expression of each corresponding indicator factor.
실시예 5. 본 발명 siRNA 또는 dsRNA의 종양 성장 억제능 확인Example 5. Confirmation of tumor growth inhibition ability of siRNA or dsRNA of the present invention
실시예 1의 SNU-449 세포주에 상기 표 12의 서열을 갖는 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA들을 실시예 1-2의 방법에 따라 in vitro transfection 시킨 후, transfection 된 cell을 흉선 결실 쥐(athymic nude mice)의 피하에 주입한 후, 간 종양의 크기와 쥐의 생존률을 분석하여 그 결과를 도 3(B)에 도시하였다.SiRNAs consisting of the sense RNA having the sequence of Table 12 and the antisense RNA consisting of the complementary sequence were transfected in vitro in the SNU-449 cell line of Example 1 according to the method of Example 1-2, and transfected cells After injection into athymic nude mice, the size of the liver tumor and the survival rate of the mice were analyzed and the results are shown in FIG. 3 (B).
도 3(B)의 왼쪽 이미지를 참조하면, 대부분의 실험군은 대조군에 비하여 간 종양의 크기가 현저하게 작음을 확인할 수 있는데, 이로써 본 발명 siRNA 또는 dsRNA를 통한 지표인자들의 knockdown이 전체적인 종양의 성장률을 저하시키고, 평균적인 종양 부피를 감소시킴을 알 수 있다.Referring to the left image of FIG. 3 (B), it can be seen that most of the experimental groups have a significantly smaller hepatic tumor size than the control group. Thus, knockdown of the marker factors via the siRNA or dsRNA of the present invention can increase the overall tumor growth rate And the average tumor volume is decreased.
도 3(B)의 오른쪽 이미지를 참조하면 실험군의 생존률(tumor-free survival rate)이 대조군의 생존률보다 현저히 높음을 확인할 수 있는데, 보다 구체적으로 상기 transfection 된 cell 피하주입 후 50일이 경과하였을 때, 대조군은 10개의 쥐 중 6개의 쥐에서 종양이 발견되었으나, 실험군은 10개의 쥐 중 1 내지 2개의 쥐에서 종양이 발견되었고, 이로써 본 발명 siRNA 또는 dsRNA이 간 종양의 성장을 효과적으로 억제할 수 있음을 알 수 있다.Referring to the right image of FIG. 3 (B), it can be seen that the tumor-free survival rate is significantly higher than that of the control group. More specifically, when 50 days have elapsed after subcutaneous transfection of the transfected cells, In the control group, tumors were found in 6 rats of 10 rats, but in the experimental group, tumors were found in 1 to 2 rats among 10 rats, indicating that the siRNA or dsRNA of the present invention can effectively inhibit the growth of liver tumors Able to know.
실시예 6. 본 발명 siRNA 또는 dsRNA의 간암 예방 효능 확인Example 6. Confirmation of liver cancer prevention efficacy of siRNA or dsRNA of the present invention
하기 표 13의 서열을 갖는 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA들을 실시예 1-8의 방법에 따라 in vivo transfection 시켰고, 그 과정과 초음파 이미지 및 시간의 경과에 따른 종양의 개수를 도 4(A)에 나타내었고, 다공성 나노입자에 담지된 상기 siRNA들의 각 지표 유전자들의 발현 억제수준을 실시예 1-9의 방법으로 분석하여 도 4(B)에 나타내었다.SiRNAs consisting of sense RNA having the sequence of Table 13 below and antisense RNA consisting of the complementary sequence were transfected in vivo according to the method of Examples 1-8 and the number of tumors 4 (A). The level of inhibition of the expression of the respective indicator genes of the siRNAs carried on the porous nanoparticles was analyzed by the method of Example 1-9 and shown in FIG. 4 (B).
도 4(A)를 참조하면, 다공성 나노입자만 주입한 대조군의 경우, 주입 후 17주가 경과하며 3 내지 4개의 쥐에서 큰 복수개의 간 종양이 발견된 것에 비해, 상기 siRNA를 담지한 다공성 나노입자를 주입한 실험군의 경우, 주입 후 19주가 경과하며 2 내지 4개의 쥐에서 상대적으로 작은 간 종양만이 발견되었음을 확인할 수 있고, 도 4(B)를 참조하면, 대조군에 비해 실험군의 경우 지표 유전자들의 발현량을 in vivo 상에서 현저히 감소시킴을 Western blotting 결과로 확인할 수 있다. 이 결과는 in vivo 상으로도 본 발명 siRNA 또는 dsRNA가 각 지표인자들의 발현을 효과적으로 억제하여 간 종양 생성 억제 및 간암의 예방에 우수한 효능을 발휘함을 알 수 있다.4 (A), in the case of the control group injected with only the porous nanoparticles, 17 weeks after the injection, a large number of hepatic tumors were found in 3 to 4 mice, whereas the number of the porous nanoparticles , It was confirmed that only a relatively small hepatic tumor was found in 2 to 4 rats and 19 weeks after the injection, and as shown in Fig. 4 (B), in the case of the experimental group, Western blotting can significantly confirm the reduction in expression level in vivo. These results indicate that the siRNA or dsRNA of the present invention effectively inhibits the expression of the respective marker factors in vivo, and exerts excellent effects in inhibiting hepatoma formation and prevention of liver cancer.
서열번호SEQ ID NO: Sense RNA 서열Sense RNA sequence 도 1에서의 명명The naming in Figure 1 타겟유전자Target gene
서열번호 311SEQ ID NO: 311 5'-CCUCAGCGUUUCAAUCUUUUU-3’5'-CCUCAGCGUUUCAAUCUUUUU-3 ' Banf1Banf1 Mouse BANF1 geneMouse BANF1 gene
서열번호 312SEQ ID NO: 312 5'-CGACUGCAGAAUCUCCUCUUU-3’5'-CGACUGCAGAAUCUCCUCUUU-3 ' Plod3Plod3 Mouse PLOD3 geneMouse PLOD3 gene
서열번호 313SEQ ID NO: 313 5'-CUGCUUUACGAUACUUUCAUU-3’5'-CUGCUUUACGAUACUUUCAUU-3 ' Sf3b4Sf3b4 Mouse SF3B4 geneMouse SF3B4 gene
실시예Example 7. 다공성 실리카 입자의 형성 및 기공 확장의 확인 7. Formation of Porous Silica Particles and Confirmation of Pore Expansion
실시예 11-(1) 내지 (3)의 입자의 소기공 입자, 제조된 다공성 실리카 입자를 현미경으로 관찰하여, 소기공 입자가 균일하게 생성되었는지, 기공이 충분히 확장되어 다공성 실리카 입자가 균일하게 형성되었는지를 확인하였다(도 5 내지 8).Small pores of the particles of Example 11- (1) - (3) and the prepared porous silica particles were observed with a microscope to confirm whether the small pore particles were uniformly formed and the pores were sufficiently expanded to form the porous silica particles uniformly (Figs. 5 to 8).
도 5는 11-(1)의 다공성 실리카 입자의 사진, 도 6는 11-(2)의 다공성 실리카 입자의 사진으로 기공이 충분히 확장된 구형의 다공성 실리카 입자가 고르게 생성된 것을 확인할 수 있고, 도 7은 11-(1)의 소기공 입자의 사진이고, 도 8은 11-(1)과 11-(3)의 소기공 입자의 비교 사진으로, 구형의 소기공 입자가 고르게 생성된 것을 확인할 수 있다.FIG. 5 is a photograph of the porous silica particles of 11- (1), FIG. 6 is a photograph of the porous silica particles of 11- (2) 7 is a photograph of small pore particles of 11- (1), and FIG. 8 is a comparative photograph of small pore particles of 11- (1) and 11- (3) have.
실시예 8. 다공성 실리카 입자의 생분해성 확인Example 8. Confirmation of biodegradability of porous silica particles
실시예 11-(1)의 다공성 실리카 입자의 생분해성 확인을 위해 37℃, SBF(pH 7.4)에서의 생분해 정도를 0시간, 120시간, 360시간에 현미경으로 관찰하였고, 이는 도 9에 나타내었다.In order to confirm the biodegradability of the porous silica particles of Example 11- (1), the degree of biodegradation at 37 ° C and SBF (pH 7.4) was observed with a microscope at 0 hours, 120 hours, and 360 hours, .
도 9를 참조하면 다공성 실리카 입자가 생분해되어 360시간 경과 후에는 거의 다 분해된 것을 확인할 수 있다.Referring to FIG. 9, it can be confirmed that the porous silica particles were almost completely decomposed after 360 hours of biodegradation.
실시예 9. 다공성 실리카 입자의 흡광도비 측정Example 9. Measurement of absorbance ratio of porous silica particles
1. 측정 방법1. Measurement method
시간별 하기 수학식 1에 따른 흡광도비를 측정하였다:The absorbance ratio according to the following formula (1) was measured:
[수학식 1][Equation 1]
At/A0 A t / A 0
(식 중, A0는 상기 다공성 실리카 입자 1mg/ml 현탁액 5ml를 직경 50kDa의 기공을 갖는 원통형 투과막에 넣고 측정된 다공성 실리카 입자의 흡광도이고,Wherein A 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles in a cylindrical permeable membrane having pores having a diameter of 50 kDa,
상기 투과막 외부에는 상기 투과막과 접하며, 상기 현탁액과 동일한 용매 15ml가 위치하고, 상기 투과막 내외부는 37℃에서 60rpm 수평 교반되며,15 ml of the same solvent as that of the suspension was placed on the outside of the permeable membrane, and the inside and the outside of the permeable membrane were horizontally stirred at 60 rpm at 37 ° C,
At는 상기 A0의 측정시로부터 t시간 경과 후에 측정된 다공성 실리카 입자의 흡광도임).A t is the absorbance of the porous silica particles measured after passage of time t from the measurement of A 0 ).
구체적으로, 다공성 실리카 입자 분말 5mg을 SBF (pH 7.4) 5ml에 녹였다. 이 후 5ml의 다공성 실리카 입자 용액을 도 10에 도시된 직경 50 kDa의 기공을 갖는 투과막에 넣었다. 외부막에 15ml의 SBF를 첨가하고, 외부막의 SBF는 12시간마다 교체하였다. 다공성 실리카 입자의 분해는 37℃에서 60rpm 수평 교반하며 수행되었다.Specifically, 5 mg of the porous silica particle powder was dissolved in 5 ml of SBF (pH 7.4). Thereafter, 5 ml of the porous silica particle solution was placed in the permeable membrane having the pore diameter of 50 kDa shown in Fig. 15 ml of SBF was added to the outer membrane, and SBF of the outer membrane was replaced every 12 hours. The decomposition of the porous silica particles was carried out at 37 ° C with 60 rpm of horizontal stirring.
이 후 UV-vis spectroscopy에 의해 흡광도를 측정하였고, λ = 640 nm에서 분석되었다.The absorbance was then measured by UV-vis spectroscopy and analyzed at λ = 640 nm.
2. 흡광도 비 측정 결과2. Absorbance ratio measurement results
실시예 11-(1)의 다공성 실리카 입자의 흡광도비를 상기 방법에 따라 측정하였고, 그 결과는 도 11에 나타내었다.The absorbance ratio of the porous silica particles of Example 11- (1) was measured according to the above method, and the results are shown in Fig.
도 11을 참조하면 흡광도비가 1/2가 되는 t가 약 58시간으로 굉장히 천천히 분해되는 것을 확인할 수 있다.Referring to FIG. 11, it can be seen that t at which the absorbance ratio becomes 1/2 is decomposed very slowly to about 58 hours.
3. 입경별 측정 결과3. Measurement results by particle size
실시예 11-(1),(5),(6)의 다공성 실리카 입자의 흡광도를 상기 수학식 1에 따라 측정하였고, 그 결과는 도 12에 나타내었다(현탁액과 용매로는 SBF를 사용).The absorbance of the porous silica particles of Examples 11- (1), (5) and (6) was measured according to Equation 1 above and the results are shown in FIG. 12 (using SBF as a suspension and a solvent).
도 12를 참조하면, 입경의 증가에 따라 t가 감소함을 알 수 있다.Referring to FIG. 12, it can be seen that t decreases with increasing grain size.
4. 기공 평균 직경별 측정 결과4. Measurement results of pore diameter average diameter
실시예 11-(1),(9)의 다공성 실리카 입자, 그리고 컨트롤로서 실시예 11-(1)의 소기공 다공성 실리카 입자의 흡광도를 상기 수학식 1에 따라 측정하였고, 그 결과는 도 13에 나타내었다(현탁액과 용매로는 SBF를 사용).The absorbance of the porous silica particles of Examples 11- (1), (9) and the small pore porous silica particles of Example 11- (1) as a control was measured according to the above formula (1) (SBF is used as suspension and solvent).
도 13을 참조하면, 실시예의 다공성 실리카 입자는 컨트롤에 비해 t가 상당히 큰 것을 확인할 수 있다.Referring to FIG. 13, it can be seen that the porous silica particles of the examples have significantly larger t than the control.
5. pH별 측정 결과5. Measurement results by pH
실시예 11-(4)의 다공성 실리카 입자의 pH별 흡광도를 측정하였다. 흡광도는 SBF에서, 그리고 pH 2, 5, 및 7.4의 Tris에서 측정하였고, 그 결과는 도 14에 나타내었다.The absorbance of the porous silica particles of Example 11- (4) was measured by pH. Absorbance was measured in SBF and in Tris at pH 2, 5, and 7.4, and the results are shown in FIG.
도 14를 참조하면, pH 별 t의 차이는 있으나, 모두 흡광도의 비가 1/2이 되는 t가 24 이상이었다.Referring to FIG. 14, there was a difference in t by pH, but all of the t values at which the ratio of the absorbance was 1/2 were 24 or more.
6. 대전6. Daejeon
실시예 12-(1)-1)의 다공성 실리카 입자의 흡광도를 측정하였고, 그 결과는 도 15에 나타내었다(현탁액과 용매로는 Tris(pH 7.4)를 사용).The absorbance of the porous silica particles of Example 12- (1) -1) was measured, and the results are shown in Fig. 15 (using Tris (pH 7.4) as a suspension and a solvent).
도 15를 참조하면, 양전하로 대전된 입자도 흡광도의 비가 1/2이 되는 t가 24 이상이었다.Referring to Fig. 15, t, which is the ratio of the absorbance of the positively charged particles to 1/2, was 24 or more.
실시예 10. 다공성 실리카 입자에 담지된 siRNA 또는 dsRNA의 방출Example 10. Release of siRNA or dsRNA carried on porous silica particles
실시예 1-13의 siRNA를 로딩한 다공성 실리카 입자 10μl를 SBF(pH 7.4, 37℃)에 재부유시키고, 기공 직경 20kDa의 투과막(도 16의 튜브)에 넣었다. 이 후, 투과 튜브를 1.5ml의 SBF에 담갔다. siRNA의 방출은 37℃에서 60rpm 수평 교반하며 수행되었다.10 mu l of the porous silica particles loaded with the siRNA of Examples 1-13 were resuspended in SBF (pH 7.4, 37 DEG C) and placed in a permeation membrane (the tube of Fig. 16) having a pore diameter of 20 kDa. The permeate tube was then immersed in 1.5 ml of SBF. The release of siRNA was performed at 37 ° C with 60 rpm horizontal stirring.
24시간 이전에는 0.5, 1, 2, 4, 8, 12, 24시간 경과한 시간에 방출 용매를 회수하고, 그 이후는 24시간 간격으로, 0.5ml의 방출 용매를 형광 측정을 위해 회수하고 등량의 SBF를 첨가하였다.Prior to 24 hours, the release solvent was withdrawn at 0.5, 1, 2, 4, 8, 12, 24 hours, then every 24 hours thereafter, 0.5 ml of the release solvent was recovered for fluorescence measurement, SBF was added.
상기 siRNA의 형광 강도는 670 nm 파장(λex=647 nm)에서 측정하여 siRNA의 방출 정도를 측정하였고, 그 결과는 도 17에 나타내었다.The fluorescence intensity of the siRNA was measured at a wavelength of 670 nm (? Ex = 647 nm) to measure the degree of siRNA release, and the results are shown in FIG.
도 17을 참조하면 siRNA가 50% 방출된 시간이 약 40시간 이상인 것을 확인할 수 있다.Referring to FIG. 17, it can be seen that the time at which 50% of the siRNA was released was about 40 hours or more.
실시예Example 11. 다공성 실리카 입자에  11. To porous silica particles 담지된Supported siRNAsiRNA 또는  or dsRNA의dsRNA 표적  Target 전달여부Delivery 확인 Confirm
동물수준에서의 siRNA 전달 연구에 적합한 수준의 전달체 역할이 가능함을 검증하고자, 마우스(쥐)를 대상으로 생리활성물질 방출에 따른 종양 억제 정도를 확인하였다.In order to verify that the delivery of siRNAs at an animal level is suitable for the study of siRNA delivery, the degree of tumor suppression due to the release of biologically active substances in mice (rats) was confirmed.
Balb/c nude 수컷 (5주령)을 ㈜오리엔트바이오에서 구입하여 멸균된 1x PBS에 300만개의 HeLa 세포(자궁경부암세포)를 분산시켜 마우스에 피하주사 이종이식(Xenograft) 종양을 성장시키고, 70 mm3 크기의 고형화된 종양이 확인되었을 때, PBS, FITC-다공성 실리카 입자(실시예 12-(1)-2)-②의 다공성 실리카 입자), 실시예 1-13의 siRNA를 적재한 FITC-다공성 실리카 입자(실시예 12-(1)-2)-②의 다공성 실리카 입자)를 각각 마우스 종양 내 주사 투여하고 투여 직전, 투여 직후, 그리고 48시간 이후에 대해 형광 세기 및 분포를 FOBI Fluorescence in vivo imaging system (Neo science, Korea) 기기를 통해 관찰하였다.Balb / c nude male (5 weeks old) was purchased from Orient Bio Inc., and 3 million HeLa cells (cervical cancer cells) were dispersed in sterilized 1x PBS. Xenograft tumors were subcutaneously injected into mice to grow 70 mm when the solidification of the 3 tumor size is confirmed, PBS, FITC- porous silica particles (example 12- (1) -2) -② of porous silica particles), porous FITC- loaded with siRNA of example 1-13 (Silica particles of Example 12- (1) -2) -②) were injected into each mouse tumor, and the fluorescence intensity and distribution were measured immediately before, immediately after, and after 48 hours by FOBI fluorescence in vivo imaging system (Neo science, Korea).
상기 FITC 표지는 실리카 입자 50mg을 1 mL DMSO (dimethyl sulfoxide)에 분산시키고 FITC-NHS(N-hydroxycuccinimide) 용액 (2.5 mg/mL) 25μg(10μl)을 넣고 알루미늄 호일로 빛을 차단한 상태로 상온에서 18시간 반응시키고, 반응물을 원심분리(8500 rpm, 10 분)로 정제하여 상층액은 버리고 가라앉은 입자를 모아 에탄올에 고르게 분산시키고 이를 에탄올-증류수로 교차하여 3-4번 반복하여 상층액에 FITC 색이 보이지 않을때까지 정제하여 수행하였다.The FITC label was prepared by dispersing 50 mg of silica particles in 1 mL of DMSO and adding 25 μg of FITC-NHS solution (2.5 mg / mL) (10 μl) The reaction mixture was reacted for 18 hours, and the reaction product was purified by centrifugation (8500 rpm, 10 minutes). The supernatant was discarded and the precipitated particles were collected and dispersed evenly in ethanol. This was repeated 3-4 times with ethanol- distilled water, And purified until no color was observed.
상기 실험결과를 나타낸 도 18을 참조하면, control은 PBS 단독 투여, 실시예 1-13의 siRNA는 실시예 1-13의 siRNA 단독 투여, FITC-DDV가 FITC로 표기한 다공성 실리카 입자 단독 투여, complex가 실시예 1-13의 siRNA가 로딩되고 FITC 표지된 다공성 실리카 입자의 투여를 나타내는 것으로서, 이를 참조하면, 입자에 적재하여 체내로 전달된 siRNA는 활성을 유지하는 기간이 더 길고, 주입된 부위에서 더 오래 머물어서, 48시간이 지나도 강한 형광을 나타내는 것을 확인할 수 있다.18 showing the results of the above experiments, the control was administered alone in PBS, the siRNA of Example 1-13 alone, the siRNA alone of Example 1-13, the FITC-DDV of FITC alone, the complex Refers to the administration of FITC-labeled porous silica particles loaded with the siRNA of Examples 1-13. When referring to this, the siRNA transferred into the body to be loaded into the body has a longer period of maintaining the activity, After staying longer, we can confirm that the fluorescence is strong even after 48 hours.

Claims (15)

  1. 서열번호 5 내지 157의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는An siRNA consisting of a sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 5 to 157 and an antisense RNA consisting of the sequence complementary thereto; or
    서열번호 158 내지 310의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA를 포함하는 간암 예방 또는 치료용 약학적 조성물.A dsRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 158 to 310;
  2. 청구항 1에 있어서,The method according to claim 1,
    상기 조성물은 서열번호 5 내지 12, 14 내지 19, 21, 23, 24, 26, 28 내지 34, 35 내지 37, 39 내지 41, 43, 45 내지 47, 49 내지 53, 55 내지 60, 62 내지 73, 75 내지 81, 84 내지 87, 89 내지 98, 100 내지 102, 105 내지 116, 118 내지 128, 130 내지 154, 156 및 157 의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는Wherein said composition comprises at least one of SEQ ID NOS: 5-12, 14-19, 21, 23, 24, 26, 28-34, 35-37, 39-41, 43, 45-47, 49-53, 55-60, , At least one sequence selected from the group consisting of SEQ ID NOs: 75 to 81, 84 to 87, 89 to 98, 100 to 102, 105 to 116, 118 to 128, 130 to 154, 156 and 157, RTI ID = 0.0 &gt; siRNA &lt; / RTI &gt; or
    서열번호 158 내지 165, 167 내지 172, 174, 176, 177, 179, 181 내지 187, 188 내지 190, 192 내지 194, 196, 198 내지 200, 202 내지 206, 208 내지 213, 215 내지 226, 228 내지 234, 237 내지 240, 242 내지 251, 253 내지 255, 258 내지 269, 271 내지 281, 283 내지 307, 309 및 310의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA를 포함하는 간암 예방 또는 치료용 약학적 조성물.181 to 187, 188 to 190, 192 to 194, 196, 198 to 200, 202 to 206, 208 to 213, 215 to 226, 228 to 188, 187 to 179, 181 to 187, A prophylactic or therapeutic agent for liver cancer comprising at least one sequence selected from the group consisting of SEQ ID NOs: 234, 237 to 240, 242 to 251, 253 to 255, 258 to 269, 271 to 281, 283 to 307, 309 and 310 A pharmaceutical composition.
  3. 청구항 1에 있어서,The method according to claim 1,
    상기 조성물은 서열번호 5 내지 28의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는An siRNA consisting of a sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 5 to 28 and an antisense RNA comprising a sequence complementary thereto; or
    서열번호 158 내지 181의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA를 포함하는 간암 예방 또는 치료용 약학적 조성물.A dsRNA comprising at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 158 to 181;
  4. 청구항 1에 있어서,The method according to claim 1,
    상기 조성물은 서열번호 29 내지 55의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는An siRNA consisting of a sense RNA consisting of at least one sequence selected from the group consisting of the sequences of SEQ ID NOS: 29 to 55 and an antisense RNA comprising a sequence complementary thereto; or
    서열번호 182 내지 208의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA를 포함하는 간암 예방 또는 치료용 약학적 조성물.A dsRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 182 to 208;
  5. 청구항 1에 있어서,The method according to claim 1,
    상기 조성물은 서열번호 56 내지 120의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는An siRNA consisting of a sense RNA consisting of at least one sequence selected from the group consisting of SEQ ID NOS: 56-120 and an antisense RNA consisting of a sequence complementary thereto; or
    서열번호 209 내지 273의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA를 포함하는 간암 예방 또는 치료용 약학적 조성물.A dsRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 209 to 273.
  6. 청구항 1에 있어서,The method according to claim 1,
    상기 조성물은 서열번호 121 내지 157의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 siRNA; 또는The composition comprising siRNA consisting of sense RNA consisting of at least one sequence selected from the group consisting of SEQ ID NOS: 121 to 157 and antisense RNA having a sequence complementary thereto; or
    서열번호 274 내지 310의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 dsRNA를 포함하는 간암 예방 또는 치료용 약학적 조성물.26. A pharmaceutical composition for preventing or treating liver cancer, comprising a dsRNA comprising at least one sequence selected from the group consisting of SEQ ID NOS: 274 to 310.
  7. 청구항 1 내지 6 중 어느 한 항에 있어서,7. The method according to any one of claims 1 to 6,
    상기 siRNA 또는 dsRNA는 리포좀, 리포펙타민, 덴드리머, 마이셀, 다공성 실리카 입자, 아미노클레이, 금나노입자, 자성나노입자, 그래핀, 산화그래핀, 키토산, 덱스트란, 펙틴, 이산화망간 2차원시트, PVA, 젤라틴, 실리카, 글라스입자, 프로타민, 엑소좀, 폴리에틸렌이민, N-부틸 시아노아크릴레이트, 젤폼, 젤라틴, 에탄올, 나노크리스탈, 나노튜브, 탄소나노입자, 히알루론산, 산화철, 폴리락틱산, 폴리부틸시아노아크릴레이트, 알부민, 리피드입자, 폴리에틸렌글라이콜, 폴리-L-굴루로닉 알지네이트, 폴리글리콜릭-폴리액틱산, 폴리디옥사논, 폴리글리콜산-co-카프로락톤, 폴리프로필렌 및 하이드로겔로 이루어진 군에서 선택된 적어도 하나인 담지체에 담지된 것인 조성물.The siRNA or dsRNA may be selected from the group consisting of liposomes, lipofectamines, dendrimers, micelles, porous silica particles, amino clay, gold nanoparticles, magnetic nanoparticles, graphene, oxidized graphene, chitosan, dextran, pectin, , Gelatin, silica, glass particles, protamine, exosome, polyethyleneimine, N-butylcyanoacrylate, gel foam, gelatin, ethanol, nanocrystals, nanotubes, carbon nanoparticles, hyaluronic acid, iron oxide, polylactic acid, poly Polyglycolic acid, polydioxanone, polyglycolic acid-co-caprolactone, polypropylene and polyglycolic acid, and mixtures thereof. Wherein the carrier is supported on at least one support selected from the group consisting of hydrogels.
  8. 청구항 7에 있어서,The method of claim 7,
    상기 담지체는 하기 수학식 1의 흡광도의 비가 1/2가 되는 t가 20 이상인 다공성 실리카 입자인 조성물:Wherein the support is porous silica particles having a t of 20 or more, wherein the ratio of absorbance of the following formula (1) is 1/2:
    [수학식 1][Equation 1]
    At/A0 A t / A 0
    (식 중, A0는 상기 다공성 실리카 입자 1mg/ml 현탁액 5ml를 직경 50kDa의 기공을 갖는 원통형 투과막에 넣고 측정된 다공성 실리카 입자의 흡광도이고,Wherein A 0 is the absorbance of the porous silica particles measured by placing 5 ml of the 1 mg / ml suspension of the porous silica particles in a cylindrical permeable membrane having pores having a diameter of 50 kDa,
    상기 투과막 외부에는 상기 투과막과 접하며, 상기 현탁액과 동일한 용매 15ml가 위치하고, 상기 투과막 내외부는 37℃에서 60rpm 수평 교반되며,15 ml of the same solvent as that of the suspension was placed on the outside of the permeable membrane, and the inside and the outside of the permeable membrane were horizontally stirred at 60 rpm at 37 ° C,
    상기 현탁액의 pH는 7.4이고, At는 A0의 측정시로부터 t시간 경과 후에 측정된 다공성 실리카 입자의 흡광도임).The pH of the suspension is 7.4 and A t is the absorbance of the porous silica particles measured after passage of time from the measurement of A 0 ).
  9. 청구항 8에 있어서, 상기 t는 40 이상인 조성물.The composition of claim 8, wherein t is greater than or equal to 40.
  10. 청구항 8에 있어서, The method of claim 8,
    상기 siRNA는 서열번호 28, 119 및 136의 서열로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 센스 RNA 및 이에 상보적인 서열로 이루어진 안티센스 RNA로 이루어진 것이고,Wherein the siRNA comprises at least one sequence selected from the group consisting of SEQ ID NOS: 28, 119, and 136, and an antisense RNA comprising a sequence complementary thereto,
    상기 dsRNA는 서열번호 181, 272 및 289로 이루어진 군에서 선택된 적어도 하나의 서열로 이루어진 것인 조성물.Wherein the dsRNA comprises at least one sequence selected from the group consisting of SEQ ID NOS: 181, 272, and 289.
  11. 청구항 8에 있어서,The method of claim 8,
    상기 다공성 실리카 입자는 친수성 치환기 또는 소수성 치환기를 갖는 것인 조성물.Wherein the porous silica particle has a hydrophilic substituent or a hydrophobic substituent.
  12. 청구항 8에 있어서,The method of claim 8,
    상기 다공성 실리카 입자는 알데하이드기, 케토기, 카바메이트기, 설페이트기, 설포네이트기, 아미노기, 아민기, 아미노알킬기, 실릴기, 카르복실기, 술폰산기, 티올기, 암모늄기, 설프히드릴기, 포스페이트기, 에스터기, 이미드기, 싸이오이미드기, 케토기, 에터기, 인덴기, 설포닐기, 메틸포스포네이트기, 폴리에틸렌글리콜기, 치환 또는 비치환된 C1 내지 C30의 알킬기, 치환 또는 비치환된 C3 내지 C30의 사이클로알킬기, 치환 또는 비치환된 C6 내지 C30의 아릴기 및 C1 내지 C30의 에스테르기로 이루어진 군에서 선택된 적어도 하나의 친수성 치환기를 갖는 것인 조성물.The porous silica particles may be in the form of particles of an aliphatic group having an aldehyde group, a keto group, a carbamate group, a sulfate group, a sulfonate group, an amino group, an amine group, an aminoalkyl group, a silyl group, a carboxyl group, a sulfonic acid group, a thiol group, , A substituted or unsubstituted C 1 to C 30 alkyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aryl group, A substituted or unsubstituted C 6 to C 30 aryl group, and a C 1 to C 30 ester group, in the presence of at least one hydrophilic substituent selected from the group consisting of a substituted or unsubstituted C 3 to C 30 cycloalkyl group, a substituted or unsubstituted C 6 to C 30 aryl group, and a C 1 to C 30 ester group.
  13. 청구항 8에 있어서,The method of claim 8,
    상기 다공성 실리카 입자는 외부 표면 또는 기공 내부가 중성의 pH에서 양전하 또는 음전하를 띠는 것인 조성물.Wherein the porous silica particles have a positive or negative charge at an external surface or pore interior at neutral pH.
  14. 청구항 8에 있어서,The method of claim 8,
    상기 다공성 실리카 입자는 외부 표면 및 기공 내부가 중성의 pH에서 양전하를 띠는 것인 조성물.Wherein the porous silica particles are positively charged at neutral pH at the outer surface and inside the pores.
  15. 청구항 8에 있어서,The method of claim 8,
    상기 다공성 실리카 입자는 평균 직경이 100 내지 400nm이고, 기공 직경이 4 내지 30nm인 것인 조성물.Wherein the porous silica particles have an average diameter of 100 to 400 nm and a pore diameter of 4 to 30 nm.
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JP2022520467A (en) * 2019-02-22 2022-03-30 レモネックス インコーポレイテッド Pharmaceutical composition for immunoreactivity or prevention or treatment of cancer
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