WO2022060208A1 - Antisense oligonucleotides - Google Patents
Antisense oligonucleotides Download PDFInfo
- Publication number
- WO2022060208A1 WO2022060208A1 PCT/KR2021/012989 KR2021012989W WO2022060208A1 WO 2022060208 A1 WO2022060208 A1 WO 2022060208A1 KR 2021012989 W KR2021012989 W KR 2021012989W WO 2022060208 A1 WO2022060208 A1 WO 2022060208A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- modified nucleotides
- oligonucleotide
- nucleotides
- aso
- modified
- Prior art date
Links
- 238000012230 antisense oligonucleotides Methods 0.000 title claims abstract description 105
- 239000000074 antisense oligonucleotide Substances 0.000 title claims abstract description 104
- 108020000948 Antisense Oligonucleotides Proteins 0.000 title abstract description 5
- 108091034117 Oligonucleotide Proteins 0.000 claims description 152
- 125000003729 nucleotide group Chemical group 0.000 claims description 86
- 239000002773 nucleotide Substances 0.000 claims description 36
- 102000011117 Transforming Growth Factor beta2 Human genes 0.000 claims description 33
- 101800000304 Transforming growth factor beta-2 Proteins 0.000 claims description 33
- 206010028980 Neoplasm Diseases 0.000 claims description 15
- 239000002777 nucleoside Substances 0.000 claims description 14
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 claims description 13
- 201000011510 cancer Diseases 0.000 claims description 10
- 201000010099 disease Diseases 0.000 claims description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 10
- 108020004707 nucleic acids Proteins 0.000 claims description 9
- 102000039446 nucleic acids Human genes 0.000 claims description 9
- 150000007523 nucleic acids Chemical class 0.000 claims description 9
- 150000003833 nucleoside derivatives Chemical class 0.000 claims description 9
- 125000003835 nucleoside group Chemical group 0.000 claims description 9
- 239000008194 pharmaceutical composition Substances 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 5
- 206010016654 Fibrosis Diseases 0.000 claims description 4
- 230000004761 fibrosis Effects 0.000 claims description 4
- 208000026278 immune system disease Diseases 0.000 claims description 4
- 229920000233 poly(alkylene oxides) Polymers 0.000 claims description 3
- 230000002018 overexpression Effects 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 description 64
- 108020004999 messenger RNA Proteins 0.000 description 50
- 239000012096 transfection reagent Substances 0.000 description 36
- 239000000203 mixture Substances 0.000 description 31
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 27
- 230000014509 gene expression Effects 0.000 description 20
- 239000001963 growth medium Substances 0.000 description 18
- 238000004445 quantitative analysis Methods 0.000 description 17
- 101500025624 Homo sapiens Transforming growth factor beta-2 Proteins 0.000 description 16
- 239000002299 complementary DNA Substances 0.000 description 14
- 239000002609 medium Substances 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 14
- 238000001890 transfection Methods 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 14
- 238000010805 cDNA synthesis kit Methods 0.000 description 12
- 230000008859 change Effects 0.000 description 12
- 239000012153 distilled water Substances 0.000 description 12
- 238000002123 RNA extraction Methods 0.000 description 11
- 239000003242 anti bacterial agent Substances 0.000 description 11
- 229940088710 antibiotic agent Drugs 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 10
- 201000009030 Carcinoma Diseases 0.000 description 9
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 9
- 238000010804 cDNA synthesis Methods 0.000 description 8
- 230000005764 inhibitory process Effects 0.000 description 8
- 230000028327 secretion Effects 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 101000700734 Homo sapiens Serine/arginine-rich splicing factor 9 Proteins 0.000 description 6
- 239000012098 Lipofectamine RNAiMAX Substances 0.000 description 6
- 239000012980 RPMI-1640 medium Substances 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 102000051805 human SRSF9 Human genes 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 238000010899 nucleation Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 238000003753 real-time PCR Methods 0.000 description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 5
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 5
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 5
- 201000005202 lung cancer Diseases 0.000 description 5
- 208000020816 lung neoplasm Diseases 0.000 description 5
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 5
- 201000001441 melanoma Diseases 0.000 description 5
- 201000002528 pancreatic cancer Diseases 0.000 description 5
- 208000008443 pancreatic carcinoma Diseases 0.000 description 5
- 238000011002 quantification Methods 0.000 description 5
- 206010006187 Breast cancer Diseases 0.000 description 4
- 208000026310 Breast neoplasm Diseases 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 201000005296 lung carcinoma Diseases 0.000 description 4
- 210000000496 pancreas Anatomy 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 238000008157 ELISA kit Methods 0.000 description 3
- 239000002543 antimycotic Substances 0.000 description 3
- 239000003184 complementary RNA Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 125000005642 phosphothioate group Chemical group 0.000 description 3
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 108020004394 Complementary RNA Proteins 0.000 description 2
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 2
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 201000005249 lung adenocarcinoma Diseases 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- -1 ribose pentose Chemical group 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000012421 spiking Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- ZIBGPFATKBEMQZ-UHFFFAOYSA-N triethylene glycol Chemical compound OCCOCCOCCO ZIBGPFATKBEMQZ-UHFFFAOYSA-N 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 108020005544 Antisense RNA Proteins 0.000 description 1
- 238000011729 BALB/c nude mouse Methods 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 208000009956 adenocarcinoma Diseases 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 201000010989 colorectal carcinoma Diseases 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000012207 quantitative assay Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000005760 tumorsuppression Effects 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 238000012447 xenograft mouse model Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
Definitions
- the present invention relates to an antisense oligonucleotide and a pharmaceutical composition comprising the same.
- tRNA fetches the corresponding amino acid according to the mRNA information from the ribosome, and the protein is produced through the process of linking it with a peptide bond.
- the complementary DNA or RNA binds to mRNA to form a double strand, thereby preventing protein production.
- RNA oligonucleotide capable of inhibiting its function by binding to an mRNA (sense RNA) that can be transcribed and translated into a protein is referred to as an antisense RNA or an antisense oligonucleotide.
- an antisense oligonucleotide of SEQ ID NO: 1 and the like are known as an antisense oligonucleotide targeting TGF- ⁇ , a tumor promoting factor.
- the antisense oligonucleotide itself needs to solve the problem of rapid degradation in vivo and also improve specificity so that it binds only to the mRNA of the target gene, so that it can be developed as a drug that can treat actual diseases.
- the present inventors have studied to develop an antisense oligonucleotide with improved ability to inhibit TGF- ⁇ protein expression.
- a novel antisense oligonucleotide in which the structure of some sugars among known antisense oligonucleotides is modified has excellent TGF- ⁇ inhibitory effect, and , it was confirmed that the stability was high.
- the present invention is to provide a novel antisense oligonucleotide and a pharmaceutical composition containing the same.
- the present invention relates to a novel antisense oligonucleotide comprising at least one modified nucleoside among the oligonucleotides of 5'-CGGCATGTCTATTTTGTA-3' of SEQ ID NO: 1.
- the present invention relates to an antisense oligonucleotide of SEQ ID NO: 1, wherein at least one nucleoside of the oligonucleotide has a modified nucleotide.
- nucleoside-modified nucleotide is a nucleoside is 2'-O-methoxyethyl (MOE) modified nucleotide, 2'-fluoro (F) modified nucleotide, 2'-O-methyl ( O-Me) modified nucleotides, locked nucleic acid (LNA) modified nucleotides, ethylene-bridged nucleic acid (ENA) modified nucleotides, restricted ethyl (cET: (R/S)-constrained ethyl ) modified nucleotides or polyalkylene oxide (eg, triethylene glycol (TEG)) modified nucleotides.
- MOE methoxyethyl
- F fluoro
- O-Me 2'-O-methyl
- LNA locked nucleic acid
- ENA ethylene-bridged nucleic acid
- cET restricted ethyl
- R/S restricted ethyl
- TAG triethylene glycol
- the 2' position of the ribose pentose of the nucleoside to be modified is 2'-O-methoxyethyl (MOE) form, 2'-fluoro form, or 2' -O-methyl form, or LNA or ENA form in which the oxygen at the 2' position of the ribose pentose of the modified nucleoside is connected to the carbon at the 4' position, or the oxygen at the 2' position and the 4' position of the LNA It may be in the form of cET in which a bridge connecting carbons is replaced with a methyl group.
- MOE 2'-O-methoxyethyl
- the oligonucleotides of the invention comprise one or more modified nucleosides at the 5'-end and/or the 3' end.
- each of the 5'-end or the 3'-end of the oligonucleotide of the present invention may include one or more modified nucleosides.
- the oligonucleotides of the present invention may include nucleotides having one or more modified nucleosides at the 5'-end or the 3'-end, respectively, ie, "modified nucleotides".
- the oligonucleotide according to the present invention is an oligonucleotide in which one or more nucleotides of the 5'-end or one or more nucleotides of the 3'-end of the oligonucleotide are modified nucleotides.
- the oligonucleotide according to the present invention is an oligonucleotide in which one or more nucleotides of the 5'-end and one or more nucleotides of the 3'-end of the oligonucleotide are modified nucleotides.
- the first to n-th nucleotides of the 5'-end of the oligonucleotide or the first to m-th nucleotides of the 3'-end of the oligonucleotide may be modified nucleotides.
- the first to n-th nucleotides of the 5'-end of the oligonucleotide and the first to m-th nucleotides of the 3'-end of the oligonucleotide may be modified nucleotides.
- n and m are each independently an integer of any one of 1 to 9, and preferably, n and m are each independently an integer of any one of 1 to 6.
- the oligonucleotide of the present invention may further include a modified nucleotide between the modified nucleosides present at the 5'-end or the 3'-end.
- the oligonucleotide may further include one or more modified nucleotides between the (n+1)-th nucleotide of the 5'-end and the (m+1)-th nucleotide of the 3'-end.
- Preferred examples of the oligonucleotide of the present invention may be any one of the antisense oligonucleotides of SEQ ID NO: 1 shown in Nos. 1 to 25 of the table below.
- modified nucleotides of the oligonucleotides shown in Nos. 1 to 21 of the Table are 2'-O-methoxyethyl (MOE) modified nucleotides
- the modified nucleotides of the oligonucleotides shown in Nos. 22 to 25 of the Table are locked nucleic acids ( LNA) is preferably an oligonucleotide that is a modified nucleotide.
- LNA locked nucleic acids
- the bond between nucleosides, whether or not the nucleosides are modified may be phosphodiester or phosphothioate.
- oligonucleotide should be understood as a concept that also includes pharmaceutically acceptable salts of oligonucleotides.
- the modification of the nucleoside as described above may be prepared through organic chemical synthesis methods known in the art to which the present invention pertains.
- the present invention also relates to a pharmaceutical composition comprising the modified antisense oligonucleotide as described above.
- the present invention contains the modified antisense oligonucleotide as described above, and diseases (eg, malignant tumors, benign tumors, immune diseases, fibrosis or ophthalmic diseases, etc.) related to overexpression of TGF- ⁇ 2 protein It relates to a pharmaceutical composition for preventing or treating.
- diseases eg, malignant tumors, benign tumors, immune diseases, fibrosis or ophthalmic diseases, etc.
- the oligonucleotide of the present invention was treated in a solid cancer cell line, it was confirmed that the effect of inhibiting the expression of TGF- ⁇ 2 protein was more excellent, the effect of killing cancer cells was excellent, and the stability in plasma was also improved.
- the oligonucleotide of the present invention can be usefully used as a pharmaceutical composition for treating diseases related to TGF- ⁇ expression, for example, malignant tumors, benign tumors, immune diseases, fibrosis, ophthalmic diseases, and the like.
- 1 is a graph confirming the change in TGF- ⁇ 2 mRNA expression by treating A549 with 20 nM of ASO.
- FIG. 2 is a graph confirming the change in TGF- ⁇ 2 mRNA expression by treating PANC-1 with ASO 20 nM.
- 3 is a graph confirming the change in TGF- ⁇ 2 mRNA expression by treating A549 with 5 nM of ASO.
- FIGS. 5 and 6 show changes in human TGF- ⁇ 2 mRNA level (level) when A549, PANC-1 is treated with ASO at 1 uM (Free uptake (meaning no transfection reagent)).
- TGF- ⁇ 2 mRNA expression levels for each cell line of 4 types of carcinoma (Breast cancer, Pancreatic cancer, Lung cancer, and Melanoma).
- Figure 11 shows changes in human TGF- ⁇ 2 mRNA level after ASO treatment in 4 types of carcinoma (Lung adenocarcinoma (A549), Pancreatic cancer (Panc-1), Breast cancer (MDA-MB-231), Melanoma (Hs294T)) .
- carcinoma Lung adenocarcinoma (A549), Pancreatic cancer (Panc-1), Breast cancer (MDA-MB-231), Melanoma (Hs294T)
- FIG. 12 is a diagram showing changes in body weight after administration of ASO to xenograft mice.
- FIG. 13 is a diagram showing tumor volume after administration of ASO to xenograft mice.
- FIG. 14 is a diagram showing the tumor suppression rate after administration of ASO to xenograft mice.
- the oligonucleotide of the present invention was treated in a solid cancer cell line, it was confirmed that the effect of inhibiting the expression of TGF- ⁇ 2 protein was more excellent, the effect of killing cancer cells was excellent, and the stability in plasma was also improved.
- Human lung cancer cell line A549 and human pancreatic cancer cell line PANC-1 were each treated with antisense oligonucleotide (ASO) 20 nM (w/transfection reagent), and changes in human TGF- ⁇ 2 mRNA level were confirmed.
- ASO antisense oligonucleotide
- A549 and PANC-1 cell lines was treated with ASO 1 ⁇ M (free uptake (meaning no transfection reagent)), and then human TGF- ⁇ 2 mRNA level changes were confirmed.
- RPMI 1640 (10% FBS, 1% Antibiotic-Antimycotic)
- DMEM 1% Antibiotic-Antimycotic
- McCoy's 5A 10% FBS, 1% Antibiotic-Antimycotic
- the mixture containing ASO and the mixture containing the transfection reagent were mixed 1:1 and reacted at room temperature for 5 minutes. This mixture was dispensed to the cell line in which the medium was replaced, and incubated in an incubator (37° C., CO 2 5%) for 24 hours or 48 hours depending on the purpose of the test.
- RNAiMAX total volume 6 well 200,000 to 300,000 7.5 ⁇ l 2 ml 96 wells 5,000 to 10,000 0.3 ⁇ l 100 ⁇ l
- An appropriate number of cells were seeded according to the characteristics of the cells and the plate size, and cultured for 24 hours.
- ASO was put in a medium containing 10% FBS according to the concentration.
- the mixture containing ASO was dispensed on the cell-seeded plate, it was cultured in an incubator (37° C., CO 2 5%) for 24 hours or 48 hours depending on the purpose of the test.
- cDNA was synthesized with 2 ⁇ g of the extracted RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis kit, Thermo Sceientific, K1622). Synthesis was carried out under synthetic conditions according to the manufacturer's instructions, and after PCR was completed, cDNA was diluted with distilled water (DW) to 20ng/ ⁇ l.
- a cDNA synthesis kit RevertAid First Strand cDNA Synthesis kit, Thermo Sceientific, K1622. Synthesis was carried out under synthetic conditions according to the manufacturer's instructions, and after PCR was completed, cDNA was diluted with distilled water (DW) to 20ng/ ⁇ l.
- PCR was performed by preparing a mixture under the composition and conditions as shown in Tables 4 to 6 below.
- the supernatant was recovered and centrifuged (13,000 rpm, 1 min).
- nucleotides in bold underlined text indicate modified nucleotides.
- A001 is the known antisense oligonucleotide of SEQ ID NO: 1, and A001-01M to A001-021M and A001-01L, A001-04L, A001-06L and A001-10L below are modified according to the present invention Examples of antisense oligonucleotides comprising nucleotides.
- the modified nucleotides of the oligonucleotides indicated by A001-01M to A001-021M in the table above are 2'-O-methoxyethyl (MOE) modified nucleotides, and A001-01L, A001-04L, A001-06L and
- the modified nucleotides of the oligonucleotides designated A001-10L are oligonucleotides that are locked nucleic acid (LNA) modified nucleotides.
- LNA locked nucleic acid
- the bond between nucleosides in the oligonucleotides of the table above is a phosphothioate bond, whether or not the nucleoside is modified.
- the change in the mRNA level of TGF- ⁇ 2 was compared for each candidate, and the efficacy of the ASO of the present invention was compared with A001 did
- the culture medium for cell line A549 (Lung carcinoma) was RPMI1640+10%FBS+1% Antibiotics, and the culture medium for cell line PANC-1 (Pancreas Epithelioid carcinoma) was DMEM+10%FBS+1% Antibiotics.
- Transfection Change the medium 24 hours after cell seeding (after removing the existing culture, add 1.8 ml of media containing no antibiotics and 10% FBS), 20 times the final concentration of ASO to be tested (400 nM) It was put in plain media without addition of anything, and the transfection reagent (Lipofectamine RNAiMAX) was also put in plain media without addition to a concentration of 7.5 ul/100 ul. The mixture containing ASO and the mixture containing the transfection reagent were mixed 1:1 and reacted at room temperature for 5 minutes (at this time, the concentration of ASO becomes 200 nM).
- RNA extraction and quantification After removing all the culture medium 24 hours after transfection, RNA was extracted using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, RNA was quantified by absorptiometry using a micro-volume plate (Take 3, Biotek) and a multi plate reader (Synergy H1, Biotek).
- cDNA was synthesized with 2 ug of the extracted RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622). Synthesis conditions were carried out according to the manufacturer's instruction, and after PCR was completed, cDNA was diluted with distilled water (DW) to 20 ng/ul.
- a cDNA synthesis kit RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622. Synthesis conditions were carried out according to the manufacturer's instruction, and after PCR was completed, cDNA was diluted with distilled water (DW) to 20 ng/ul.
- Figure 2 shows the inhibition ratio of human TGF- ⁇ 2 mRNA when PANC-1 is treated with ASO 20nM (with transfection reagent).
- Example 5 Based on the experimental results of Example 5, ASO with relatively good efficacy was selected, and cell lines (A549, PANC-1) were treated at a concentration of 5 nM ASO in the presence of a transfection reagent, and then the mRNA level of TGF- ⁇ 2 was Changes were compared by ASO to compare the efficacy of ASO to the known A001 ASO.
- the culture medium RPMI1640+10%FBS+1% Antibiotics was used, and for the cell line PANC-1 (Pancreas Epithelioid carcinoma), the culture medium DMEM+10%FBS+1% Antibiotics was used.
- Cells were seeded at 2x10 5 cells / 2 ml per well in a 6 well plate.
- RNA extraction and quantification After removing all the culture medium 24 hours after transfection, RNA was extracted using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, RNA was quantified by absorptiometry using a micro-volume plate (Take 3, Biotek) and a multi-plate reader (Synergy H1, Biotek).
- cDNA was synthesized with 2 ug of the extracted RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622). Synthesis conditions were carried out according to the manufacturer's instruction, and after PCR was completed, cDNA was diluted with distilled water (DW) to 20 ng/ul.
- a cDNA synthesis kit RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622. Synthesis conditions were carried out according to the manufacturer's instruction, and after PCR was completed, cDNA was diluted with distilled water (DW) to 20 ng/ul.
- 3 is a graph confirming the change in TGF- ⁇ 2 mRNA expression by treating A549 with 5 nM of ASO.
- the culture medium RPMI1640+10%FBS+1% Antibiotics was used, and for the cell line Panc-1 (Pancreas Epithelioid carcinoma), the culture medium DMEM+10%FBS+1% Antibiotics was used.
- RNA extraction and quantification After removing all the culture medium 24 hours after transfection, RNA was extracted using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, RNA was quantified by absorptiometry using a micro-volume plate (Take 3, Biotek) and a multi-plate reader (Synergy H1, Biotek).
- cDNA was synthesized with 2 ug of the extracted RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622). Synthesis conditions were carried out according to the manufacturer's instruction, and after PCR was completed, cDNA was diluted with distilled water (DW) to 20 ng/ul.
- a cDNA synthesis kit RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622. Synthesis conditions were carried out according to the manufacturer's instruction, and after PCR was completed, cDNA was diluted with distilled water (DW) to 20 ng/ul.
- the medium was replaced (after removing the existing culture medium, 1.8 ml of a medium containing 10% FBS without antibiotics was added).
- the ASO to be tested was serially diluted with plain media without any addition so as to be 20 times the concentration to be treated (50-1600 nM).
- the transfection reagent (Lipofectamine RNAiMAX) was also put in plain media to which nothing was added to a concentration of 7.5 ul/100 ul.
- the mixture containing ASO and the mixture containing the transfection reagent were mixed 1:1 and reacted at room temperature for 5 minutes (at this time, the concentration of ASO becomes 10 times the concentration to be treated).
- RNA extraction and quantification After collecting the culture medium 48 hours after transfection, RNA was extracted using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, RNA was quantified by absorptiometry using a micro-volume plate (Take 3, Biotek) and a multi-plate reader (Synergy H1, Biotek).
- cDNA was synthesized with 2ug of the extracted RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622). Synthesis conditions were carried out according to the manufacturer's instruction, and after PCR was completed, cDNA was diluted with distilled water (DW) to 20 ng/ul.
- a cDNA synthesis kit RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622. Synthesis conditions were carried out according to the manufacturer's instruction, and after PCR was completed, cDNA was diluted with distilled water (DW) to 20 ng/ul.
- ASO spiked plasma was sampled and the amount of remaining ASO was analyzed by HPLC.
- ASO containing nucleotides modified according to the present invention was maintained at 50% or more until 24 hours.
- the amount of ASO detected even after 24 hours was about 80% or more.
- the nucleotide according to the present invention has improved stability.
- the transfection reagent (Lipofectamine RNAiMAX) was also put in plain media to which nothing was added to a concentration of 7.8 ul/100 ul.
- RNA extraction and quantification After collecting the culture medium 48 hours after transfection, RNA was extracted using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, RNA was quantified by absorptiometry using a micro-volume plate (Take 3, Biotek) and a multi-plate reader (Synergy H1, Biotek).
- cDNA was synthesized with 2ug of the extracted RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622). Synthesis conditions were performed according to the manufacturer’s instructions. After completion of PCR, cDNA was diluted with distilled water (DW) to 20 ng/ul.
- DW distilled water
- TGF- ⁇ 2 protein expression quantitative assay (ELISA):
- TGF- ⁇ 2 mRNA expression levels for each cell line of 4 types of carcinoma (Breast cancer, Pancreatic cancer, Lung cancer, and Melanoma).
- Figure 11 shows changes in human TGF- ⁇ 2 mRNA level after ASO treatment in 4 types of carcinoma (Lung adenocarcinoma (A549), Pancreatic cancer (Panc-1), Breast cancer (MDA-MB-231), Melanoma (Hs294T)) .
- carcinoma Lung adenocarcinoma (A549), Pancreatic cancer (Panc-1), Breast cancer (MDA-MB-231), Melanoma (Hs294T)
- the human TGF-b2 mRNA level was significantly reduced compared to the control group for 4 cell lines.
- mice/group Balb/c nude mouse, male (5 weeks old), 6 mice/group
- mice were acclimatized for one week after wearing, and tumor cells (A549) were transplanted 13 days before group separation (day 0).
- Groups were separated 13 days after tumor cell transplantation.
- the tumor size was about 80-100 mm 3 .
- Treatment A total of 5 times (day 0, 2, 4, 7, 9) was administered at a dose of 15 mg/kg, 10 ml/kg by subcutaneous injection (s.c.) or intraperitoneal injection (i.p.).
- the experimental schedule is as follows:
- the oligonucleotide of the present invention can be usefully used as a pharmaceutical composition for treating diseases related to TGF- ⁇ expression, for example, malignant tumors, benign tumors, immune diseases, fibrosis, ophthalmic diseases, and the like.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Public Health (AREA)
- Biotechnology (AREA)
- Animal Behavior & Ethology (AREA)
- Biophysics (AREA)
- General Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plant Pathology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Antisense oligonucleotides are disclosed.
Description
본 발명은 안티센스 올리고뉴클레오타이드 및 이를 포함하는 의약 조성물에 관한 것이다.The present invention relates to an antisense oligonucleotide and a pharmaceutical composition comprising the same.
세포 내의 DNA 염기 서열 정보는 mRNA로 전사(transcription)된 후, 리보솜에서 mRNA의 정보에 따라 tRNA가 대응되는 아미노산을 가져오고, 이를 펩타이드 결합으로 연결시키는 과정을 통하여 단백질이 생산된다.After DNA nucleotide sequence information in the cell is transcribed into mRNA, tRNA fetches the corresponding amino acid according to the mRNA information from the ribosome, and the protein is produced through the process of linking it with a peptide bond.
이러한 유전자 발현 과정 중에, 만일 mRNA와 상보적인 염기서열을 가진 한가닥 DNA 또는 RNA가 존재하게 된다면, 이 상보적 DNA 또는 RNA는 mRNA와 결합하여 이중사슬을 형성하게 되어, 단백질 생산을 막을 수 있다.During the gene expression process, if single-stranded DNA or RNA having a base sequence complementary to mRNA is present, the complementary DNA or RNA binds to mRNA to form a double strand, thereby preventing protein production.
상기와 같이 전사되어 단백질로 번역될 수 있는 mRNA(센스 RNA)와 이중사슬로 결합하여 그 기능을 저해시킬 수 있는 상보적 관계의 DNA 또는 RNA의 올리고뉴클레오타이드를 안티센스 RNA 또는 안티센스 올리고뉴클레오타이드라 칭한다.As described above, a complementary DNA or RNA oligonucleotide capable of inhibiting its function by binding to an mRNA (sense RNA) that can be transcribed and translated into a protein is referred to as an antisense RNA or an antisense oligonucleotide.
현재, 종양촉진 인자인 TGF-β를 타겟으로 하는 안티센스 올리고뉴클레오타이드로서 서열번호 1의 안티센스 올리고뉴클레오타이드 등이 공지되어 있다.Currently, an antisense oligonucleotide of SEQ ID NO: 1 and the like are known as an antisense oligonucleotide targeting TGF-β, a tumor promoting factor.
안티센스 뉴클레오타이드를 사용하여, 표적 유전자(예를 들어, 바이러스와 같은 병원체 또는 발암 유전자)의 기능을 저해시켜 질병을 치료하려는 연구가 진행되고 있다.Research is underway to treat diseases by inhibiting the function of a target gene (eg, a pathogen such as a virus or an oncogene) using antisense nucleotides.
그러나, 안티센스 올리고뉴클레오타이드 자체가 생체 내에서 신속하게 분해되는 문제를 해결하고, 또한 표적 유전자의 mRNA에만 결합하도록 특이성을 향상시켜야 실제 질병을 치료할 수 있는 의약품으로서 개발될 수 있을 것이다.However, the antisense oligonucleotide itself needs to solve the problem of rapid degradation in vivo and also improve specificity so that it binds only to the mRNA of the target gene, so that it can be developed as a drug that can treat actual diseases.
본 발명자들은 TGF-β 단백질 발현의 억제 능력이 향상된 안티센스 올리고뉴클레오타이드를 개발하기 위하여 연구한 결과, 공지의 안티센스 올리고뉴클레오타이드 중 일부 당의 구조를 변형시킨 신규의 안티센스 올리고뉴클레오타이드가 TGF-β 억제 효과가 우수하고, 안정성이 높다는 점을 확인하였다.The present inventors have studied to develop an antisense oligonucleotide with improved ability to inhibit TGF-β protein expression. As a result, a novel antisense oligonucleotide in which the structure of some sugars among known antisense oligonucleotides is modified has excellent TGF-β inhibitory effect, and , it was confirmed that the stability was high.
따라서, 본 발명은 신규의 안티센스 올리고뉴클레오타이드 및 이를 함유하는 의약 조성물을 제공하기 위한 것이다.Accordingly, the present invention is to provide a novel antisense oligonucleotide and a pharmaceutical composition containing the same.
본 발명은 서열번호 1의 5'-CGGCATGTCTATTTTGTA-3'의 올리고뉴클레오타이드 중 적어도 하나의 변형된 뉴클레오사이드를 포함하는 신규의 안티센스 올리고뉴클레오타이드에 관한 것이다.The present invention relates to a novel antisense oligonucleotide comprising at least one modified nucleoside among the oligonucleotides of 5'-CGGCATGTCTATTTTGTA-3' of SEQ ID NO: 1.
구체적으로, 본 발명은 서열번호 1의 안티센스 올리고뉴클레오타이드로서, 상기 올리고뉴클레오타이드의 하나 이상의 뉴클레오사이드가 변형된 뉴클레오타이드를 갖는 안티센스 올리고뉴클레오타이드에 관한 것이다.Specifically, the present invention relates to an antisense oligonucleotide of SEQ ID NO: 1, wherein at least one nucleoside of the oligonucleotide has a modified nucleotide.
여기에서, 뉴클레오사이드가 변형된 뉴클레오타이드는, 뉴클레오사이드가 2'-O-메톡시에틸(MOE) 변형된 뉴클레오타이드, 2'-플루오로(F) 변형된 뉴클레오타이드, 2'-O-메틸(O-Me) 변형된 뉴클레오타이드, 잠긴핵산(LNA: locked nucleic acid) 변형된 뉴클레오타이드, 에틸렌 가교 핵산(ENA: ethylene-bridged nucleic acid) 변형된 뉴클레오타이드, 제한된 에틸(cET: (R/S)-constrained ethyl) 변형된 뉴클레오타이드 또는 폴리알킬렌 옥사이드(예를 들어, 트리에틸렌 글리콜(TEG)) 변형된 뉴클레오타이드일 수 있다. (이하에서는, 특별한 언급이 없는 한, 위와 같이 뉴클레오사이드가 변형된 뉴클레오타이드를 "변형된 뉴클레오타이드"라 칭하기로 한다)Here, the nucleoside-modified nucleotide is a nucleoside is 2'-O-methoxyethyl (MOE) modified nucleotide, 2'-fluoro (F) modified nucleotide, 2'-O-methyl ( O-Me) modified nucleotides, locked nucleic acid (LNA) modified nucleotides, ethylene-bridged nucleic acid (ENA) modified nucleotides, restricted ethyl (cET: (R/S)-constrained ethyl ) modified nucleotides or polyalkylene oxide (eg, triethylene glycol (TEG)) modified nucleotides. (Hereinafter, unless otherwise specified, nucleotides modified with nucleosides as above will be referred to as "modified nucleotides")
예를 들어, 본 발명에 따른 안티센스 올리고뉴클레오타이드는, 변형되는 뉴클레오사이드의 리보스 5탄당의 2' 위치가 2'-O-메톡시에틸(MOE) 형태, 2'-플루오로 형태, 또는 2'-O-메틸 형태이거나, 변형되는 뉴클레오사이드의 리보스 5탄당의 2' 위치의 산소가 4' 위치의 탄소와 연결되는 LNA 또는 ENA 형태이거나, 상기 LNA의 2' 위치의 산소 및 4' 위치의 탄소를 연결하는 브릿지가 메틸기로 치한된 cET 형태일 수 있다.For example, in the antisense oligonucleotide according to the present invention, the 2' position of the ribose pentose of the nucleoside to be modified is 2'-O-methoxyethyl (MOE) form, 2'-fluoro form, or 2' -O-methyl form, or LNA or ENA form in which the oxygen at the 2' position of the ribose pentose of the modified nucleoside is connected to the carbon at the 4' position, or the oxygen at the 2' position and the 4' position of the LNA It may be in the form of cET in which a bridge connecting carbons is replaced with a methyl group.
상기 예시한 변형 뉴클레오사이드의 구조는 다음과 같이 나타낼 수 있다:The structure of the modified nucleoside exemplified above can be represented as follows:
본 발명의 올리고뉴클레오타이드는 5'- 단부 및/또는 3' 단부에 하나 이상의 변형된 뉴클레오사이드를 포함한다. 예를 들어, 본 발명의 올리고뉴클레오타이드의 5'-단부 또는 3'-단부에 각각 1개 또는 그 이상의 변형된 뉴클레오사이드를 포함할 수 있다. 따라서, 본 발명의 올리고뉴클레오타이드의 5'-단부 또는 3'-단부에 각각 1개 또는 그 이상의 변형된 뉴클레오사이드를 갖는 뉴클레오타이드, 즉 "변형된 뉴클레오타이드"를 포함할 수 있다.The oligonucleotides of the invention comprise one or more modified nucleosides at the 5'-end and/or the 3' end. For example, each of the 5'-end or the 3'-end of the oligonucleotide of the present invention may include one or more modified nucleosides. Accordingly, the oligonucleotides of the present invention may include nucleotides having one or more modified nucleosides at the 5'-end or the 3'-end, respectively, ie, "modified nucleotides".
구체적으로, 본 발명에 따른 올리고뉴클레오타이드는 상기 올리고뉴클레오타이드의 5'-단부의 하나 이상의 뉴클레오타이드 또는 3'-단부의 하나 이상의 뉴클레오타이드가 변형된 뉴클레오타이드인 올리고뉴클레오타이드이다.Specifically, the oligonucleotide according to the present invention is an oligonucleotide in which one or more nucleotides of the 5'-end or one or more nucleotides of the 3'-end of the oligonucleotide are modified nucleotides.
또한, 본 발명에 따른 올리고뉴클레오타이드는 상기 올리고뉴클레오타이드의 5'-단부의 하나 이상의 뉴클레오타이드 및 3'-단부의 하나 이상의 뉴클레오타이드가 변형된 뉴클레오타이드인 올리고뉴클레오타이드이다.In addition, the oligonucleotide according to the present invention is an oligonucleotide in which one or more nucleotides of the 5'-end and one or more nucleotides of the 3'-end of the oligonucleotide are modified nucleotides.
본 발명에서, 상기 올리고뉴클레오타이드의 5'-단부의 첫번째 내지 n번째 뉴클레오타이드 또는 상기 올리고뉴클레오타이드의 3'-단부의 첫번째 내지 m번째 뉴클레오타이드가 변형된 뉴클레오타이드일 수 있다 In the present invention, the first to n-th nucleotides of the 5'-end of the oligonucleotide or the first to m-th nucleotides of the 3'-end of the oligonucleotide may be modified nucleotides.
또한, 본 발명에서, 상기 올리고뉴클레오타이드의 5'-단부의 첫번째 내지 n번째 뉴클레오타이드 및 상기 올리고뉴클레오타이드의 3'-단부의 첫번째 내지 m번째 뉴클레오타이드가 변형된 뉴클레오타이드일 수 있다Also, in the present invention, the first to n-th nucleotides of the 5'-end of the oligonucleotide and the first to m-th nucleotides of the 3'-end of the oligonucleotide may be modified nucleotides.
여기에서 n 및 m은 각각 독립적으로 1 내지 9 중 어느 하나의 정수이며, 바람직하게는 n 및 m은 각각 독립적으로 1 내지 6 중 어느 하나의 정수이다.Here, n and m are each independently an integer of any one of 1 to 9, and preferably, n and m are each independently an integer of any one of 1 to 6.
또한, 본 발명의 올리고뉴클레오타이드는 상기 5'-단부 또는 3'-단부에 존재하는 변형된 뉴클레오사이드 사이에 변형된 뉴클레오타이드를 더 포함할 수 있다.In addition, the oligonucleotide of the present invention may further include a modified nucleotide between the modified nucleosides present at the 5'-end or the 3'-end.
즉, 상기 올리고뉴클레오타이드의 5'-단부의 (n+1)번째 뉴클레오타이드 및 3'-단부의 (m+1)번째 뉴클레오타이드 사이에 하나 이상의 변형된 뉴클레오타이드를 더 포함할 수 있다.That is, the oligonucleotide may further include one or more modified nucleotides between the (n+1)-th nucleotide of the 5'-end and the (m+1)-th nucleotide of the 3'-end.
본 발명의 올리고뉴클레오타이드의 바람직한 예는 하기 표의 1번 내지 25번으로 나타낸 서열번호 1의 안티센스 올리고뉴클레오타이드 중 어느 하나일 수 있다.Preferred examples of the oligonucleotide of the present invention may be any one of the antisense oligonucleotides of SEQ ID NO: 1 shown in Nos. 1 to 25 of the table below.
하기 표 1에서 밑줄로 표시된 뉴클레오타이드가 변형된 뉴클레오타이드를 나타낸다.Nucleotides underlined in Table 1 below represent modified nucleotides.
상기 표의 1번 내지 21번으로 나타낸 올리고뉴클레오타이드의 변형된 뉴클레오타이드는 2'-O-메톡시에틸(MOE) 변형된 뉴클레오타이드이고, 표의 22번 내지 25번으로 나타낸 올리고뉴클레오타이드의 변형된 뉴클레오타이드는 잠긴핵산(LNA) 변형된 뉴클레오타이드인 올리고뉴클레오타이드인 것이 바람직하다.The modified nucleotides of the oligonucleotides shown in Nos. 1 to 21 of the Table are 2'-O-methoxyethyl (MOE) modified nucleotides, and the modified nucleotides of the oligonucleotides shown in Nos. 22 to 25 of the Table are locked nucleic acids ( LNA) is preferably an oligonucleotide that is a modified nucleotide.
본 발명의 올리고뉴클레오타이드에서, 뉴클레오사이드 사이의 결합은, 당해 뉴클레오사이드가 변형된 것인지 여부와 상관없이, 포스포디에스테르이거나 또는 포스포티오에이트일 수 있다.In the oligonucleotide of the present invention, the bond between nucleosides, whether or not the nucleosides are modified, may be phosphodiester or phosphothioate.
본 발명의 올리고뉴클레오타이드는 양이온과 함께 염을 형성할 수 있다. 따라서, 본 명세서에서 "올리고뉴클레오타이드"는 올리고뉴클레오타이드의 약학적으로 허용가능한 염도 포함하는 개념으로 이해되어야 한다.The oligonucleotides of the present invention can form salts with cations. Therefore, in the present specification, "oligonucleotide" should be understood as a concept that also includes pharmaceutically acceptable salts of oligonucleotides.
본 발명에서 상기한 바와 같은 뉴클레오사이드의 변형은 본 발명이 속하는 기술분야에서 공지된 유기화학적 합성 방법을 통하여 제조될 수 있다.In the present invention, the modification of the nucleoside as described above may be prepared through organic chemical synthesis methods known in the art to which the present invention pertains.
본 발명은 또한 상기와 같이 변형된 안티센스 올리고뉴클레오타이드를 포함하는 의약조성물에 관한 것이다.The present invention also relates to a pharmaceutical composition comprising the modified antisense oligonucleotide as described above.
예를 들어, 본 발명은 상기와 같이 변형된 안티센스 올리고뉴클레오타이드를 포함하며, TGF-β2 단백질의 과발현과 관련있는 질병(예를 들어, 악성 종양, 양성 종양, 면역질환, 섬유증 또는 안과질환 등)을 예방 또는 치료하기 위한 의약 조성물에 관한 것이다.For example, the present invention contains the modified antisense oligonucleotide as described above, and diseases (eg, malignant tumors, benign tumors, immune diseases, fibrosis or ophthalmic diseases, etc.) related to overexpression of TGF-β2 protein It relates to a pharmaceutical composition for preventing or treating.
본 발명의 올리고뉴클레오타이드를 고형암 세포주에 처리하였을 때, TGF-β2 단백질의 발현 억제 효과가 더 우수하게 나타나고, 암세포의 사멸 효과도 우수하였으며, 혈장에서의 안정성도 개선됨을 확인하였다.When the oligonucleotide of the present invention was treated in a solid cancer cell line, it was confirmed that the effect of inhibiting the expression of TGF-β2 protein was more excellent, the effect of killing cancer cells was excellent, and the stability in plasma was also improved.
따라서, 본 발명의 올리고뉴클레오타이드는 TGF-β의 발현과 관련있는 질병, 예를 들어, 악성 종양, 양성 종양, 면역질환, 섬유증, 안과질환 등을 치료하기 위한 의약 조성물로서 유용하게 사용될 수 있다.Therefore, the oligonucleotide of the present invention can be usefully used as a pharmaceutical composition for treating diseases related to TGF-β expression, for example, malignant tumors, benign tumors, immune diseases, fibrosis, ophthalmic diseases, and the like.
도 1은 A549를 ASO 20nM 로 처리하여 TGF-β2 mRNA 발현 변화를 확인한 그래프이다.1 is a graph confirming the change in TGF-β2 mRNA expression by treating A549 with 20 nM of ASO.
도 2는 PANC-1을 ASO 20nM 로 처리하여 TGF-β2 mRNA 발현 변화를 확인한 그래프이다.2 is a graph confirming the change in TGF-β2 mRNA expression by treating PANC-1 with ASO 20 nM.
도 3은 A549를 ASO 5nM 로 처리하여 TGF-β2 mRNA 발현 변화를 확인한 그래프이다.3 is a graph confirming the change in TGF-β2 mRNA expression by treating A549 with 5 nM of ASO.
도 4는 PANC-1을 ASO 5nM 로 처리하여 TGF-β2 mRNA 발현 변화를 확인한 그래프이다.4 is a graph confirming the change in TGF-β2 mRNA expression by treating PANC-1 with ASO 5nM.
도 5 및 도 6은 A549, PANC-1에 ASO를 1uM (Free uptake(형질주입 시약이 없는 것을 의미함)) 처리 시 Human TGF-β2 mRNA 레벨(level) 변화를 나타낸 것이다.5 and 6 show changes in human TGF-β2 mRNA level (level) when A549, PANC-1 is treated with ASO at 1 uM (Free uptake (meaning no transfection reagent)).
도 7은 A549를 ASO로 농도 별로 처리 후, human TGF-β2 mRNA 레벨과 protein 분비 레벨 변화를 나타낸 것이다.7 shows changes in human TGF-β2 mRNA level and protein secretion level after A549 was treated with ASO for each concentration.
도 8은 뉴클레오타이드를 변형하기 전의 뉴클레오타이드 백본 차이에 따른 안정성 개선 효과 여부를 확인한 실험 결과이다.8 is an experimental result confirming whether the stability improvement effect according to the difference in the nucleotide backbone before nucleotide modification.
도 9는 뉴클레오타이드를 변형한 후의 혈장 내 안정성 개선 효과를 확인한 실험 결과이다.9 is an experimental result confirming the effect of improving plasma stability after nucleotide modification.
도 10은 4종 암종 (Breast cancer, Pancreatic cancer, Lung cancer, Melanoma) 세포주별 TGF-β2 mRNA 발현 레벨을 비교한 것이다.10 is a comparison of TGF-β2 mRNA expression levels for each cell line of 4 types of carcinoma (Breast cancer, Pancreatic cancer, Lung cancer, and Melanoma).
도 11은 4종 암종 (Lung adenocarcinoma (A549), Pancreatic cancer (Panc-1), Breast cancer (MDA-MB-231), Melanoma (Hs294T))에 ASO 처리 후 human TGF-β2 mRNA 레벨 변화를 나타낸 것이다.Figure 11 shows changes in human TGF-β2 mRNA level after ASO treatment in 4 types of carcinoma (Lung adenocarcinoma (A549), Pancreatic cancer (Panc-1), Breast cancer (MDA-MB-231), Melanoma (Hs294T)) .
도 12는 제노그라프트 마우스에 ASO를 투여한 후, 체중 변화를 나타낸 도면이다.12 is a diagram showing changes in body weight after administration of ASO to xenograft mice.
도 13은 제노그라프트 마우스에 ASO를 투여한 후, 종양 부피를 나타낸 도면이다.13 is a diagram showing tumor volume after administration of ASO to xenograft mice.
도 14는 제노그라프트 마우스에 ASO를 투여한 후, 종양 억제 비율을 나타낸 도면이다.14 is a diagram showing the tumor suppression rate after administration of ASO to xenograft mice.
본 발명의 올리고뉴클레오타이드를 고형암 세포주에 처리하였을 때, TGF-β2 단백질의 발현 억제 효과가 더 우수하게 나타나고, 암세포의 사멸 효과도 우수하였으며, 혈장에서의 안정성도 개선됨을 확인하였다.When the oligonucleotide of the present invention was treated in a solid cancer cell line, it was confirmed that the effect of inhibiting the expression of TGF-β2 protein was more excellent, the effect of killing cancer cells was excellent, and the stability in plasma was also improved.
이하에서는, 본 발명을 실시예에 의거하여 상세하게 설명하고자 한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐이며, 본 발명의 사상이나 범위가 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail based on examples. However, the following examples are only for illustrating the present invention, and the spirit or scope of the present invention is not limited thereto.
0. 실험 개요0. Experiment Overview
가. 인간 폐암 세포주인 A549 및 인간 췌장암 세포주인 PANC-1 각각을 안티센스 올리고뉴클레오타이드(ASO) 20 nM (w/형질주입 시약)로 처리한 후, human TGF-β2 mRNA 레벨 변화를 확인하였다.go. Human lung cancer cell line A549 and human pancreatic cancer cell line PANC-1 were each treated with antisense oligonucleotide (ASO) 20 nM (w/transfection reagent), and changes in human TGF-β2 mRNA level were confirmed.
나. A549 및 PANC-1 세포주 각각을 ASO 5 nM (w/형질주입 시약)로 처리한 후, human TGF-β2 mRNA 레벨 변화를 확인하였다.me. After each of A549 and PANC-1 cell lines were treated with ASO 5 nM (w/transfection reagent), changes in human TGF-β2 mRNA levels were confirmed.
다. A549 및 PANC-1 세포주 각각을 ASO 1 μM (free uptake(형질주입 시약이 없는 것을 의미함))로 처리한 후, human TGF-β2 mRNA 레벨 변화를 확인하였다.all. Each of A549 and PANC-1 cell lines was treated with ASO 1 μM (free uptake (meaning no transfection reagent)), and then human TGF-β2 mRNA level changes were confirmed.
라. 형질주입 시약이 있는 조건에서 ASO를 농도별로 처리한 후, human TGF-β2 mRNA 및 단백질 분비(protein secretion)의 용량의존적 억제 효과를 확인하였다.La. After treatment with each concentration of ASO in the presence of a transfection reagent, the dose-dependent inhibitory effect of human TGF-β2 mRNA and protein secretion was confirmed.
마. ASO를 인간의 혈장 시료에 스파이킹(spiking)한 후, 안정성을 측정하였다.mind. After spiking ASO into human plasma samples, stability was measured.
바. 4종의 암세포주를 ASO 40 nM (w/형질주입 시약)로 처리한 후, mRNA 레벨 및 분비물 레벨의 변화를 확인하였다.bar. After 4 cancer cell lines were treated with ASO 40 nM (w/transfection reagent), changes in mRNA levels and secretion levels were confirmed.
사. 폐암 A549 제토그라프트(xenograft) 마우스 모델에서 A001-01M의 항암 효과를 평가하였다.buy. The anticancer effect of A001-01M was evaluated in the lung cancer A549 xenograft mouse model.
1. 실험 방법 (공통사항)1. Experimental method (common)
가. 세포 배양go. cell culture
하기 표 2와 같이 세포 타입에 따라, RPMI 1640 (10% FBS, 1% Antibiotic-Antimycotic), DMEM (10% FBS, 1% Antibiotic-Antimycotic) 또는 McCoy’s 5A (10% FBS, 1% Antibiotic-Antimycotic) 등의 배지를 사용하였으며, 세포는 37℃, CO2 5.0% 환경에서 배양하였다.As shown in Table 2 below, depending on the cell type, RPMI 1640 (10% FBS, 1% Antibiotic-Antimycotic), DMEM (10% FBS, 1% Antibiotic-Antimycotic) or McCoy's 5A (10% FBS, 1% Antibiotic-Antimycotic) The medium was used, and the cells were cultured at 37° C., CO 2 5.0% environment.
| 조직group | 세포주cell line | 진단명Diagnosis | 배양배지culture medium |
| 폐lung | A549A549 | 암종 (carcinoma)Carcinoma | RMPI 1640RMPI 1640 |
| 췌장pancreas | Panc-1Panc-1 | 상피 암종 (Epithelioid carcinoma)Epithelioid carcinoma | DMEDDMED |
| 직장rectal | HCT116HCT116 | 결직장암 (Colorectal carcinoma)Colorectal carcinoma | McCoy's 5AMcCoy's 5A |
| 유방breast | MDA-MB-231MDA-MB-231 | 선암 (Adenocarcinoma)Adenocarcinoma | RPMI 1640RPMI 1640 |
| 피부skin | Hs294THs294T | 흑색종 (Melanoma)Melanoma | DMEMDMEM |
나. 형질주입 시약 (Lipofectamine RNAiMAX)을 사용한 형질주입me. Transfection using transfection reagent (Lipofectamine RNAiMAX)
세포의 특성 및 플레이트 크기에 따라 적절한 수의 세포를 시딩하고, 24시간 배양한 후에 배지를 교체하였다. ASO를 농도에 맞추어 FBS가 포함되어 있지 않은 배지에 넣고, 형질주입 시약도 FBS가 포함되어 있지 않은 배지에 넣었다.An appropriate number of cells were seeded according to the characteristics of the cells and the plate size, and the medium was replaced after culturing for 24 hours. ASO was added to the FBS-free medium according to the concentration, and the transfection reagent was also added to the FBS-free medium.
ASO가 들어간 혼합물과 형질주입 시약이 들어간 혼합물을 1:1로 섞어 5분간 상온에서 반응시켰다. 배지를 교체한 세포주에 이 혼합물을 분주하고, 시험목적에 따라 24시간 또는 48시간 동안 인큐베이터(37℃, CO2 5%)에서 배양하였다.The mixture containing ASO and the mixture containing the transfection reagent were mixed 1:1 and reacted at room temperature for 5 minutes. This mixture was dispensed to the cell line in which the medium was replaced, and incubated in an incubator (37° C., CO 2 5%) for 24 hours or 48 hours depending on the purpose of the test.
세포 시딩 및 형질주입 시약은 하기 표 3과 같다:Cell seeding and transfection reagents are shown in Table 3:
| 플레이트plate | 세포 수number of cells | 리포펙타민 RNAiMAXLipofectamine RNAiMAX | 전체 부피total volume |
| 6 웰6 well | 200,000 ~ 300,000200,000 to 300,000 |
7.5㎕7.5 |
2 ㎖2 ml |
| 96 웰96 wells | 5,000 ~ 10,0005,000 to 10,000 |
0.3㎕0.3 |
100 ㎕100 μl |
다. 형질주입 시약없이 형질주입all. Transfection without transfection reagent
세포의 특성 및 플레이트 크기에 따라 적절한 수의 세포를 시딩하고, 24시간 배양하였다.An appropriate number of cells were seeded according to the characteristics of the cells and the plate size, and cultured for 24 hours.
ASO를 농도에 맞추어 10% FBS가 포함된 배지에 넣었다.ASO was put in a medium containing 10% FBS according to the concentration.
ASO가 들어간 혼합물을 세포가 시딩된 플레이트에 분주한 후, 시험목적에 따라 24시간 또는 48시간 동안 인큐베이터(37℃, CO2 5%)에서 배양하였다.After the mixture containing ASO was dispensed on the cell-seeded plate, it was cultured in an incubator (37° C., CO 2 5%) for 24 hours or 48 hours depending on the purpose of the test.
2. 2.
TGFTGF
--
β2β2
mRNAmRNA
정량 분석 quantitative analysis
가. RNA 추출 및 정량go. RNA extraction and quantitation
형질주입 후 24시간 또는 48시간 경과하였을 때에, 배양액을 모두 제거하고, RNA 정제(preparation) 키트(Rneasy Plus Mini kit, Qiagen, Cat# 74136)를 이용하여 RNA를 추출하고, 이후 마이크로-부피 플레이트(Take 3, Biotek)와 멀티 플레이트 리더(Reader)(Synergy H1, Biotek)를 이용하여 흡광측정법으로 RNA를 정량하였다.24 hours or 48 hours after transfection, all of the culture medium was removed, RNA was extracted using an RNA preparation kit (Rneasy Plus Mini kit, Qiagen, Cat# 74136), and then micro-volume plates ( RNA was quantified by absorptiometry using Take 3, Biotek) and a multi-plate reader (Synergy H1, Biotek).
나. cDNA 합성me. cDNA synthesis
추출한 RNA 2㎍으로 cDNA 합성 키트(RevertAid First Strand cDNA Synthesis kit, Thermo Sceientific, K1622)를 이용하여 cDNA를 합성하였다. 합성은 제조사의 지시사항에 따른 합성 조건 하에서 진행하였고, PCR 완료 후에 cDNA를 20ng/㎕가 되도록 증류수(DW)로 희석하였다.cDNA was synthesized with 2㎍ of the extracted RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis kit, Thermo Sceientific, K1622). Synthesis was carried out under synthetic conditions according to the manufacturer's instructions, and after PCR was completed, cDNA was diluted with distilled water (DW) to 20ng/μl.
TGF-β2 mRNA 정량 분석을 위하여 real-time PCR을 진행하였다. 레퍼런스 유전자로는 인간 SRSF9 가 사용되었다.Real-time PCR was performed for quantitative analysis of TGF-β2 mRNA. Human SRSF9 was used as the reference gene.
하기 표 4 내지 6과 같은 조성, 조건으로 혼합물을 제조하여 PCR을 진행하였다.PCR was performed by preparing a mixture under the composition and conditions as shown in Tables 4 to 6 below.
[표 4] PCR 혼합물 조성[Table 4] PCR mixture composition
[표 5] 프라이머 정보[Table 5] Primer information
[표 6] PCR 조건[Table 6] PCR conditions
3. TGF-β2 단백질 발현 정량 분석 (ELISA)3. TGF-β2 Protein Expression Quantitative Analysis (ELISA)
적절한 수의 세포를 시딩하고, 24시간 후에 ASO를 형질주입하였다(형질주입 시약은 없는 조건).An appropriate number of cells were seeded and transfected with ASO 24 hours later (without transfection reagent).
형질주입하고 48시간 경과 시에, 상층액을 회수하고, 이를 원심분리하였다 (13,000rpm, 1분).At 48 hours after transfection, the supernatant was recovered and centrifuged (13,000 rpm, 1 min).
상층액만 회수하여 TGF-β2 ELISA 키트(R&D)를 사용하여 제조사의 지시에 따라 정량 분석을 시행하였다.Only the supernatant was recovered and quantitative analysis was performed using the TGF-β2 ELISA kit (R&D) according to the manufacturer's instructions.
4. 4.
ASOASO
물질의 제조 manufacture of substances
하기 표의 ASO 물질을 통상적인 방법으로(예를 들어, 국제공개번호 WO 03/085110호 등에 기재된 방법을 참조하여) 제조하였다:The ASO materials in the table below were prepared in a conventional manner (see, for example, the method described in International Publication No. WO 03/085110, et al.):
상기 표 7에서 밑줄로 표시된 굵은 글씨체의 뉴클레오타이드가 변형된 뉴클레오타이드를 나타낸다.In Table 7, nucleotides in bold underlined text indicate modified nucleotides.
상기 표 7에서 A001은 공지의 서열번호 1의 안티센스 올리고뉴클레오타이드이며, 그 아래의 A001-01M 내지 A001-021M 및 A001-01L, A001-04L, A001-06L과 A001-10L은 본 발명에 따라 변형된 뉴클레오타이드를 포함하는 안티센스 올리고뉴클레오타이드의 예이다.In Table 7, A001 is the known antisense oligonucleotide of SEQ ID NO: 1, and A001-01M to A001-021M and A001-01L, A001-04L, A001-06L and A001-10L below are modified according to the present invention Examples of antisense oligonucleotides comprising nucleotides.
구체적으로, 상기 표의 A001-01M 내지 A001-021M 으로 나타낸 올리고뉴클레오타이드의 변형된 뉴클레오타이드는 2'-O-메톡시에틸(MOE) 변형된 뉴클레오타이드이고, 표의 A001-01L, A001-04L, A001-06L과 A001-10L 로 나타낸 올리고뉴클레오타이드의 변형된 뉴클레오타이드는 잠긴핵산(LNA) 변형된 뉴클레오타이드인 올리고뉴클레오타이드이다. 상기 표의 올리고뉴클레오타이드에서 뉴클레오사이드 사이의 결합은, 당해 뉴클레오사이드가 변형된 것인지 여부와 상관없이, 포스포티오에이트 결합이다.Specifically, the modified nucleotides of the oligonucleotides indicated by A001-01M to A001-021M in the table above are 2'-O-methoxyethyl (MOE) modified nucleotides, and A001-01L, A001-04L, A001-06L and The modified nucleotides of the oligonucleotides designated A001-10L are oligonucleotides that are locked nucleic acid (LNA) modified nucleotides. The bond between nucleosides in the oligonucleotides of the table above is a phosphothioate bond, whether or not the nucleoside is modified.
실험예Experimental example
5. A549 및 5. A549 and
PANCPANC
-1 세포주에 대한 -1 for cell lines
ASOASO
20nM20nM
처리 결과 processing result
형질주입 시약이 있는 조건에서 ASO를 20nM 농도로 세포주 (A549, PANC-1)에 처리한 후, TGF-β2의 mRNA 레벨의 변화를 후보물질 별로 비교하고, 본 발명의 ASO의 효능을 A001과 비교하였다.After treating the cell line (A549, PANC-1) with ASO at a concentration of 20 nM in the presence of a transfection reagent, the change in the mRNA level of TGF-β2 was compared for each candidate, and the efficacy of the ASO of the present invention was compared with A001 did
세포주 A549 (Lung carcinoma)의 배양배지는 RPMI1640+10%FBS+1% Antibiotics 이고, 세포주 PANC-1 (Pancreas Epithelioid carcinoma)의 배양배지는 DMEM+10%FBS+1% Antibiotics 이었다.The culture medium for cell line A549 (Lung carcinoma) was RPMI1640+10%FBS+1% Antibiotics, and the culture medium for cell line PANC-1 (Pancreas Epithelioid carcinoma) was DMEM+10%FBS+1% Antibiotics.
6 well plate 에 well 당 2x105 cells / 2 ml 의 세포를 시딩하였다.2x10 5 cells / 2 ml of cells per well were seeded in a 6 well plate.
형질주입: 세포 시딩 24시간 후 배지를 교체하고(기존 배양액 제거 후, antibiotics를 포함하지 않고, 10% FBS가 포함된 media를 1.8 ml 넣어 줌), 시험할 ASO를 최종 농도의 20배 (400nM)가 되도록 아무것도 첨가되지 않은 plain media에 넣고, 형질주입 시약 (Lipofectamine RNAiMAX)도 7.5 ul/100 ul 농도가 되도록 아무것도 첨가되지 않은 plain media에 넣었다. ASO가 들어간 혼합물과 형질주입 시약이 들어간 혼합물을 1:1로 섞어 5분간 상온에서 반응시켰다(이 때 ASO의 농도는 200 nM이 됨). 반응시킨 ASO 및 형질주입 시약 혼합물의 최종 농도가 20nM이 되도록, 배지를 교체한 세포주에 200 ul씩 분주한 후 24시간 동안 배양하였다 (CO2 incubator (37℃, CO2 5.0%))Transfection: Change the medium 24 hours after cell seeding (after removing the existing culture, add 1.8 ml of media containing no antibiotics and 10% FBS), 20 times the final concentration of ASO to be tested (400 nM) It was put in plain media without addition of anything, and the transfection reagent (Lipofectamine RNAiMAX) was also put in plain media without addition to a concentration of 7.5 ul/100 ul. The mixture containing ASO and the mixture containing the transfection reagent were mixed 1:1 and reacted at room temperature for 5 minutes (at this time, the concentration of ASO becomes 200 nM). 200 ul each was dispensed to the cell line in which the medium was replaced so that the final concentration of the reacted ASO and transfection reagent mixture was 20 nM, and then cultured for 24 hours (CO 2 incubator (37° C., CO 2 5.0%))
TGF-β2 mRNA 정량 분석:TGF-β2 mRNA quantitative analysis:
- RNA 추출 및 정량 : 형질주입 24시간 후에 배양액을 모두 제거한 후, RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136)를 이용하여 RNA를 추출하였다. RNA 추출 후 micro-volume plate (Take 3, Biotek)와 multi plate reader (Synergy H1, Biotek)를 이용하여 흡광측정법으로 RNA 정량하였다.- RNA extraction and quantification: After removing all the culture medium 24 hours after transfection, RNA was extracted using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, RNA was quantified by absorptiometry using a micro-volume plate (Take 3, Biotek) and a multi plate reader (Synergy H1, Biotek).
- cDNA synthesis : 추출한 RNA 2 ug으로 cDNA 합성 kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622)를 이용하여 cDNA 합성하였다. 합성 조건은 manufacturer’s instruction에 따라 진행하고, PCR 완료 후 cDNA를 20 ng/ul가 되도록 증류수(DW)로 희석하였다.- cDNA synthesis: cDNA was synthesized with 2 ug of the extracted RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622). Synthesis conditions were carried out according to the manufacturer's instruction, and after PCR was completed, cDNA was diluted with distilled water (DW) to 20 ng/ul.
- TGF-β2 mRNA 정량 분석을 위한 real-time PCR 진행 : Reference gene으로 human SRSF9 을 사용하여, 상기 표 4 내지 6과 동일한 조성 및 조건으로 PCR 혼합물을 제조하여, PCR을 진행하였다.- Real-time PCR for quantitative analysis of TGF-β2 mRNA: Using human SRSF9 as a reference gene, a PCR mixture was prepared under the same composition and conditions as in Tables 4 to 6, and PCR was performed.
- Human TGF-β2의 mRNA 발현 억제율(inhibition rate, %)을 비처리 세포( untreated cell: 세포주에 ASO없이 형질주입 시약만 처리)에 대한 상대적인 비율로 계산하여 결과를 확인하였다.- Human TGF-β2 mRNA expression inhibition rate (inhibition rate, %) was calculated as a relative ratio to untreated cells (cell line treated with transfection reagent only without ASO) to confirm the results.
도 1은 A549를 ASO 20nM (형질주입 시약과 함께) 처리 시 Human TGF-β2 mRNA 레벨 변화를 확인한 것이다.1 shows the change in human TGF-β2 mRNA level when A549 is treated with ASO 20nM (with transfection reagent).
도 2는 PANC-1을 ASO 20nM (형질주입 시약과 함께) 처리 시 human TGF-β2 mRNA 억제 비율을 나타낸 것이다.Figure 2 shows the inhibition ratio of human TGF-β2 mRNA when PANC-1 is treated with ASO 20nM (with transfection reagent).
실험 결과는 하기 표 8과 같다:The experimental results are shown in Table 8 below:
실험예Experimental example
6. A549, 6. A549,
PANCPANC
-1 세포주에 -1 in cell line
ASO를ASO
5 nM (w/ 형질주입 시약) 처리 후 human Human after treatment with 5 nM (w/ transfection reagent)
TGFTGF
--
β2β2
mRNAmRNA
레벨 변화 확인 Check the level change
실시예 5의 실험 결과를 토대로 하여 효능이 상대적으로 좋은 ASO를 선별하고, 형질주입 시약이 있는 조건에서 ASO 5nM 농도로 세포주(A549, PANC-1)를 처리한 후, TGF-β2의 mRNA 레벨의 변화를 ASO 별로 비교히여, ASO의 효능을 공지의 A001 ASO와 비교하였다.Based on the experimental results of Example 5, ASO with relatively good efficacy was selected, and cell lines (A549, PANC-1) were treated at a concentration of 5 nM ASO in the presence of a transfection reagent, and then the mRNA level of TGF-β2 was Changes were compared by ASO to compare the efficacy of ASO to the known A001 ASO.
세포주 A549 (Lung carcinoma)는 배양배지 RPMI1640+10%FBS+1% Antibiotics를 사용하고, 세포주 PANC-1(Pancreas Epithelioid carcinoma)는 배양배지 DMEM+10%FBS+1% Antibiotics를 사용하였다.For the cell line A549 (Lung carcinoma), the culture medium RPMI1640+10%FBS+1% Antibiotics was used, and for the cell line PANC-1 (Pancreas Epithelioid carcinoma), the culture medium DMEM+10%FBS+1% Antibiotics was used.
6 well plate 에 well 당 2x105 cells / 2 ml 로 세포를 시딩하였다.Cells were seeded at 2x10 5 cells / 2 ml per well in a 6 well plate.
형질주입:Transfection:
- 세포 시딩 24시간 후 배지를 교체하고(기존 배양액 제거 후, antibiotics를 포함하지 않고, 10% FBS가 포함된 배지를 1.8 ml 넣어 줌), 시험할 ASO를 최종 농도의 20배(100nM)가 되도록 아무것도 첨가되지 않은 plain media에 넣고, 형질주입 시약(Lipofectamine RNAiMAX)도 7.5 ul/100 ul 농도가 되도록 아무것도 첨가되지 않은 plain media에 넣었다.- Replace the medium 24 hours after cell seeding (after removing the existing culture medium, add 1.8 ml of medium containing 10% FBS without antibiotics), and add 20 times (100 nM) of the final concentration of ASO to be tested It was put in plain media without addition of anything, and the transfection reagent (Lipofectamine RNAiMAX) was also put in plain media without adding anything to a concentration of 7.5 ul/100 ul.
- ASO가 들어간 혼합물과 형질주입 시약이 들어간 혼합물을 1:1로 섞어 5분간 상온에서 반응시켰다(이 때 ASO의 농도는 50 nM이 됨).- The mixture containing ASO and the mixture containing the transfection reagent were mixed 1:1 and reacted at room temperature for 5 minutes (ASO concentration becomes 50 nM).
- 반응시킨 ASO 및 형질주입 시약 혼합물의 최종 농도가 5 nM이 되도록, 배지를 교체한 세포주에 200 ul씩 분주한 후 24시간 동안 배양하였다(CO2 incubator (37℃, CO2 5.0%))- In order that the final concentration of the reacted ASO and transfection reagent mixture is 5 nM, 200 ul of each of the medium-exchanged cell lines was dispensed and cultured for 24 hours (CO 2 incubator (37° C., CO 2 5.0%))
TGF-β2 mRNA 정량 분석:TGF-β2 mRNA quantitative analysis:
- RNA 추출 및 정량 : 형질주입 24시간 후에 배양액을 모두 제거한 후, RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136)를 이용하여 RNA를 추출하였다. RNA 추출 후 micro-volume plate (Take 3, Biotek)와 multi plate reader (Synergy H1, Biotek)를 이용하여 흡광측정법으로 RNA를 정량하였다.- RNA extraction and quantification: After removing all the culture medium 24 hours after transfection, RNA was extracted using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, RNA was quantified by absorptiometry using a micro-volume plate (Take 3, Biotek) and a multi-plate reader (Synergy H1, Biotek).
- cDNA synthesis : 추출한 RNA 2 ug으로 cDNA 합성 kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622)를 이용하여 cDNA를 합성하였다. 합성 조건은 manufacturer’s instruction에 따라 진행하고, PCR 완료 후 cDNA를 20 ng/ul가 되도록 증류수(DW)로 희석하였다.- cDNA synthesis: cDNA was synthesized with 2 ug of the extracted RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622). Synthesis conditions were carried out according to the manufacturer's instruction, and after PCR was completed, cDNA was diluted with distilled water (DW) to 20 ng/ul.
- TGF-β2 mRNA 정량 분석을 위한 real-time PCR 진행 : Reference gene으로 human SRSF9 을 사용하였다. 상기 표 4 내지 6과 동일한 조성 및 조건으로 PCR 혼합물을 제조하여, PCR을 진행하였다.- Real-time PCR for quantitative analysis of TGF-β2 mRNA: Human SRSF9 was used as a reference gene. A PCR mixture was prepared under the same composition and conditions as in Tables 4 to 6, and PCR was performed.
- Human TGF-β2의 mRNA 발현 억제율 (inhibition rate, %)을 untreated cell (세포주에 ASO없이 형질주입 시약만 처리)에 대한 상대적인 비율로 계산하여 결과를 확인하였다.- Human TGF-β2 mRNA expression inhibition rate (inhibition rate, %) was calculated as a relative ratio to untreated cells (cell line treated with transfection reagent only without ASO) to confirm the results.
도 3은 A549를 ASO 5nM 로 처리하여 TGF-β2 mRNA 발현 변화를 확인한 그래프이다.3 is a graph confirming the change in TGF-β2 mRNA expression by treating A549 with 5 nM of ASO.
도 4는 PANC-1을 ASO 5nM 로 처리하여 TGF-β2 mRNA 발현 변화를 확인한 그래프이다.4 is a graph confirming the change in TGF-β2 mRNA expression by treating PANC-1 with ASO 5nM.
실험 결과는 하기 표 9와 같다:The experimental results are shown in Table 9 below:
실험예Experimental example
7. A549, 7. A549,
PANCPANC
-1 세포주에 -1 in cell line
ASO를ASO
1 One
uMuM
(free uptake) 처리 후 human TGF-β2 (free uptake) human TGF-β2 after treatment
mRNAmRNA
레벨 변화 확인 Check the level change
실험예 5 및 6의 실험 결과를 토대로 효능이 상대적으로 좋은 ASO를 선별하고, 형질주입 시약이 없는 조건(free uptake)에서 ASO 1 uM 농도로 세포주(A549, PANC-1)를 처리한 후, TGF-β2의 mRNA 레벨의 변화를 후보물질 별로 비교하였다.Based on the experimental results of Experimental Examples 5 and 6, ASO with relatively good efficacy was selected, and cell lines (A549, PANC-1) were treated with ASO 1 uM concentration in free uptake without transfection reagent, and then TGF Changes in the mRNA level of -β2 were compared for each candidate substance.
실험방법:Experimental method:
세포주 A549 (Lung carcinoma)는 배양배지 RPMI1640+10%FBS+1% Antibiotics를 사용하고, 세포주 Panc-1 (Pancreas Epithelioid carcinoma)는 배양배지 DMEM+10%FBS+1% Antibiotics를 사용하였다.For the cell line A549 (Lung carcinoma), the culture medium RPMI1640+10%FBS+1% Antibiotics was used, and for the cell line Panc-1 (Pancreas Epithelioid carcinoma), the culture medium DMEM+10%FBS+1% Antibiotics was used.
2x105 cells / 1.8 ml / well in 6-well plate의 세포를 시딩하였다.Cells of 2x10 5 cells / 1.8 ml / well in 6-well plate were seeded.
형질주입:Transfection:
- 세포 시딩 후 24시간 배양하였다.- Cells were cultured for 24 hours after seeding.
- ASO를 최종 처리할 농도의 10배 (10 uM) 농도가 되도록 10% FBS가 포함된 배지에 넣었다.- ASO was placed in a medium containing 10% FBS so that the concentration was 10 times (10 uM) of the concentration to be treated.
- ASO가 들어간 혼합물을 세포가 시딩된 plate에 최종 농도가 1 uM이 되도록 200 ul씩 분주한 후, 24시간 동안 배양하였다(CO2 incubator (37℃, CO2 5.0%)).- 200 ul of the mixture containing ASO was dispensed to the cell-seeded plate at a final concentration of 1 uM, and incubated for 24 hours (CO 2 incubator (37°C, CO 2 5.0%)).
TGF-β2 mRNA 정량 분석:TGF-β2 mRNA quantitative analysis:
- RNA 추출 및 정량 : 형질주입 24시간 후에 배양액을 모두 제거한 후, RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136)를 이용하여 RNA를 추출하였다. RNA 추출 후 micro-volume plate (Take 3, Biotek)와 multi plate reader (Synergy H1, Biotek)를 이용하여 흡광측정법으로 RNA를 정량하였다.- RNA extraction and quantification: After removing all the culture medium 24 hours after transfection, RNA was extracted using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, RNA was quantified by absorptiometry using a micro-volume plate (Take 3, Biotek) and a multi-plate reader (Synergy H1, Biotek).
- cDNA synthesis : 추출한 RNA 2 ug으로 cDNA 합성 kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622)를 이용하여 cDNA를 합성하였다. 합성 조건은 manufacturer’s instruction에 따라 진행하고, PCR 완료 후 cDNA를 20 ng/ul가 되도록 증류수(DW)로 희석하였다.- cDNA synthesis: cDNA was synthesized with 2 ug of the extracted RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622). Synthesis conditions were carried out according to the manufacturer's instruction, and after PCR was completed, cDNA was diluted with distilled water (DW) to 20 ng/ul.
- TGF-β2 mRNA 정량 분석을 위한 real-time PCR 진행 : Reference gene으로 human SRSF9 을 사용하고, 상기 표 4 내지 6과 동일한 조성 및 조건으로 PCR 혼합물을 제조하여, PCR을 진행하였다. - Real-time PCR for quantitative analysis of TGF-β2 mRNA: Human SRSF9 was used as a reference gene, and a PCR mixture was prepared under the same composition and conditions as in Tables 4 to 6, and PCR was performed.
- Human TGF-β2의 mRNA 발현 억제율 (inhibition rate, %)을 비처리된 세포에 대한 상대적인 비율로 계산하여 결과를 확인하고, 하기 표 10 및 도 5와 도 6에 나타내었다.- Human TGF-β2 mRNA expression inhibition rate (inhibition rate, %) was calculated as a relative ratio to untreated cells to confirm the results, and are shown in Table 10 and FIGS. 5 and 6 below.
실험예Experimental example
8. 형질주입 시약이 있는 조건에서 8. In the presence of transfection reagent
ASO를ASO
농도별로 처리 후, human TGF-β2 RNA 및 protein 분비의 용량의존적 억제 확인 After treatment by concentration, dose-dependent inhibition of human TGF-β2 RNA and protein secretion was confirmed.
형질주입 시약이 있는 조건에서 ASO를 농도별 (5-160 nM)로 세포주 (A549)에 처리한 후, TGF-β2의 mRNA 레벨과 분비 레벨의 변화를 확인하였다.After the cell line (A549) was treated with ASO at each concentration (5-160 nM) in the presence of the transfection reagent, changes in the mRNA level and secretion level of TGF-β2 were confirmed.
실험방법:Experimental method:
세포주 A549 (Lung carcinoma)는 배양배지 RPMI1640+10%FBS+1% Antibiotics를 사용하였다.For cell line A549 (Lung carcinoma), culture medium RPMI1640+10%FBS+1% Antibiotics was used.
2x105 cells / 2 ml / well in 6-well plate로 세포를 시딩하였다.Cells were seeded with 2x10 5 cells / 2 ml / well in 6-well plate.
형질주입:Transfection:
- 세포 시딩 24시간 후. 배지를 교체하였다(기존 배양액 제거 후, 1.8 ml의 antibiotics를 포함하지 않고, 10% FBS가 포함된 배지를 넣어 줌). 시험할 ASO를 처리할 농도의 20배가 되도록 (50-1600nM) 아무것도 첨가되지 않은 plain media로 연속 희석하였다(serial dilution).- 24 hours after cell seeding. The medium was replaced (after removing the existing culture medium, 1.8 ml of a medium containing 10% FBS without antibiotics was added). The ASO to be tested was serially diluted with plain media without any addition so as to be 20 times the concentration to be treated (50-1600 nM).
- 형질주입 시약 (Lipofectamine RNAiMAX)도 7.5 ul/100 ul 농도가 되도록 아무것도 첨가되지 않은 plain media에 넣었다.- The transfection reagent (Lipofectamine RNAiMAX) was also put in plain media to which nothing was added to a concentration of 7.5 ul/100 ul.
- ASO가 들어간 혼합물과 형질주입 시약이 들어간 혼합물을 1:1로 섞어 5분간 상온에서 반응시켰다(이 때 ASO의 농도는 처리할 농도의 10배가 됨).- The mixture containing ASO and the mixture containing the transfection reagent were mixed 1:1 and reacted at room temperature for 5 minutes (at this time, the concentration of ASO becomes 10 times the concentration to be treated).
- 반응시킨 ASO 및 형질주입 시약 혼합물의 최종 농도가 되도록, 배지를 교체한 세포주에 200 ul씩 분주한 후 48시간 동안 배양하였다. (CO2 incubator (37℃, CO2 5.0%))- In order to obtain the final concentration of the reacted ASO and transfection reagent mixture, 200 ul of each of the medium-exchanged cell lines was dispensed and cultured for 48 hours. (CO 2 incubator (37℃, CO 2 5.0%))
TGF-β2 mRNA 정량 분석:TGF-β2 mRNA quantitative analysis:
- RNA 추출 및 정량 : 형질주입 48시간 후에 배양액을 따로 모아 둔 후, RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136)를 이용하여 RNA를 추출하였다. RNA 추출 후 micro-volume plate (Take 3, Biotek)와 multi plate reader (Synergy H1, Biotek)를 이용하여 흡광측정법으로 RNA를 정량하였다.- RNA extraction and quantification: After collecting the culture medium 48 hours after transfection, RNA was extracted using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, RNA was quantified by absorptiometry using a micro-volume plate (Take 3, Biotek) and a multi-plate reader (Synergy H1, Biotek).
- cDNA synthesis : 추출한 RNA 2ug으로 cDNA 합성 kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622)를 이용하여 cDNA를 합성하였다. 합성 조건은 manufacturer’s instruction에 따라 진행하고, PCR 완료 후 cDNA를 20 ng/ul가 되도록 증류수(DW)로 희석하였다.- cDNA synthesis: cDNA was synthesized with 2ug of the extracted RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622). Synthesis conditions were carried out according to the manufacturer's instruction, and after PCR was completed, cDNA was diluted with distilled water (DW) to 20 ng/ul.
- TGF-β2 mRNA 정량 분석을 위한 real-time PCR 진행 : Reference gene으로 human SRSF9 을 사용하였다. 상기 표 4 내지 6과 동일한 조성 및 조건으로 PCR 혼합물을 제조하여, PCR을 진행하였다.- Real-time PCR for quantitative analysis of TGF-β2 mRNA: Human SRSF9 was used as a reference gene. A PCR mixture was prepared under the same composition and conditions as in Tables 4 to 6, and PCR was performed.
TGF-β2 단백질 발현 정량 분석 (ELISA)Quantitative analysis of TGF-β2 protein expression (ELISA)
- 따로 모아둔 배양약을 원심 분리하여 상층액만 회수하여 TGF-β2 ELISA kit (R&D)로 manufacturer’s instruction에 따라 정량 분석을 진행하였다.- Separately collected culture medium was centrifuged to recover only the supernatant, and quantitative analysis was performed with the TGF-β2 ELISA kit (R&D) according to the manufacturer’s instructions.
실험 결과를 표 11 및 도 7에 나타내었다.The experimental results are shown in Table 11 and FIG. 7 .
A001-01M에 의하여 human TGF-β2 mRNA 레벨이 감소하면서 단백질 분비 레벨이 감소함을 확인하였다.It was confirmed that the protein secretion level decreased while the human TGF-β2 mRNA level was decreased by A001-01M.
실험예Experimental example
9. 9.
ASO를ASO
인간 혈장에 in human plasma
스파이킹한spiked
후 안정성 측정 Post-stability measurement
실험방법: Experimental method:
인간의 혈장에 ASO의 최종 농도가 5 uM이 되도록 스파이킹(spiking)하고, 37℃ 수조에서 인큐베이션하였다. 시간별(0, 1, 2, 4, 18시간)로 ASO 스파이킹한 후혈장을 샘플링하여 잔존하는 ASO의 양을 HPLC로 분석하였다. 0시간에 검출된 ASO의 양을 기준으로 남아있는 ASO 양의 비율을 계산하였다.It was spiked so that the final concentration of ASO in human plasma was 5 uM, and incubated in a water bath at 37°C. After spiking with ASO by time (0, 1, 2, 4, 18 hours), plasma was sampled and the amount of remaining ASO was analyzed by HPLC. Based on the amount of ASO detected at time 0, the ratio of the remaining amount of ASO was calculated.
실험 결과를 도 8에 도시하였다. 포스포디에스테르 뉴클레오타이드인 경우, 4시간 이후에는 ASO가 검출되지 아니하였다. 포스포티오에이트 뉴클레오타이드인 경우, 18시간까지도 약 50%가 남아있는 것으로 검출되었다. 따라서, 포스포티오에이트가 백본인 경우에, 혈장 중에서의 안정성이 향상된다는 것을 확인할 수 있었다.The experimental results are shown in FIG. 8 . In the case of phosphodiester nucleotides, ASO was not detected after 4 hours. In the case of phosphothioate nucleotides, it was detected that about 50% remained until 18 hours. Therefore, it could be confirmed that when phosphothioate is the backbone, stability in plasma is improved.
실험방법:Experimental method:
인간의 혈장에 ASO의 최종 농도가 5uM이 되도록 스파이킹하여, 37℃ 수조에서 인큐베이션하였다. 시간별로(0, 4, 24시간) ASO 스파이킹한 플라스마를 샘플링하여 남아있는 ASO의 양을 HPLC로 분석하였다.It was spiked so that the final concentration of ASO in human plasma was 5 uM, and incubated in a 37 degreeC water bath. Hourly (0, 4, 24 hours) ASO spiked plasma was sampled and the amount of remaining ASO was analyzed by HPLC.
0시간에 검출된 ASO의 양을 기준으로 남아있는 ASO 양의 비율을 계산하였다.Based on the amount of ASO detected at time 0, the ratio of the remaining amount of ASO was calculated.
실험 결과Experiment result
실험 결과를 도 9에 도시하였다.The experimental results are shown in FIG. 9 .
본 발명에 따라 변형된 뉴클레오타이드를 포함하는 ASO는 24시간까지 50% 이상을 유지하였다.ASO containing nucleotides modified according to the present invention was maintained at 50% or more until 24 hours.
특히, A001-04M를 제외한 다른 대부분의 ASO들은 24시간 후에도 검출되는 ASO의 양이 약 80% 이상이었다.In particular, in most ASOs except for A001-04M, the amount of ASO detected even after 24 hours was about 80% or more.
따라서, 본 발명에 따른 뉴클레오타이드는 안정성이 개선됨을 알 수 있다.Therefore, it can be seen that the nucleotide according to the present invention has improved stability.
실험예Experimental example
10. 4종 10. 4 types
암세포주에in cancer cell lines
ASO를ASO
40 nM (w/ 형질주입 시약) 처리 후 After treatment with 40 nM (w/ transfection reagent)
mRNAmRNA
레벨, 분비 레벨 변화 확인 Check the level, secretion level change
실험내용 : 형질주입 시약이 있는 조건에서 ASO를 40 nM 농도로 하기 표 12의 4종 세포주 (A549, Panc-1, MDA-MB-231, Hs294T)에 처리한 후, TGF-β2의 mRNA 레벨 및 단백질 분비 레벨의 변화를 확인하였다.Experimental details: After treatment with ASO at a concentration of 40 nM in the presence of transfection reagent to the four cell lines (A549, Panc-1, MDA-MB-231, Hs294T) shown in Table 12 below, the mRNA level of TGF-β2 and Changes in the protein secretion level were confirmed.
실험방법:Experimental method:
- 2x105 cells / 2 ml / well in 6-well plate로 세포를 시딩하였다.- Cells were seeded with 2x10 5 cells / 2 ml / well in 6-well plate.
형질주입:Transfection:
- 세포 시딩 24시간 후 배지를 교체하고(기존 배양액 제거 후, 1.8 ml의 antibiotics를 포함하지 않고, 10% FBS가 포함된 media를 넣어 줌), 시험할 ASO를 처리할 농도의 20배가 되도록 (800 nM) 아무것도 첨가되지 않은 plain media로 연속 희석하였다.- Replace the medium 24 hours after cell seeding (after removing the existing culture, 1.8 ml of media containing 10% FBS without antibiotics), and 20 times the concentration to be treated with ASO to be tested (800 nM) was serially diluted with plain media to which nothing was added.
- 형질주입 시약 (Lipofectamine RNAiMAX)도 7.8 ul/100 ul 농도가 되도록 아무것도 첨가되지 않은 plain media에 넣었다.- The transfection reagent (Lipofectamine RNAiMAX) was also put in plain media to which nothing was added to a concentration of 7.8 ul/100 ul.
- ASO가 들어간 혼합물 및 형질주입 시약이 들어간 혼합물을 1:1로 섞어 5분간 상온에서 반응시켰다.(이 때 ASO의 농도는 처리할 농도의 10배가 됨)- The mixture containing ASO and the mixture containing the transfection reagent were mixed 1:1 and reacted at room temperature for 5 minutes. (ASO concentration is 10 times the concentration to be treated)
- 반응시킨 ASO 및 형질주입 시약 혼합물의 최종 농도가 되도록, 배지를 교체한 세포주에 200 ul씩 분주한 후 48시간 동안 배양하였다. (CO2 incubator (37℃, CO2 5.0%))- In order to obtain the final concentration of the reacted ASO and transfection reagent mixture, 200 ul of each of the medium-exchanged cell lines was dispensed and cultured for 48 hours. (CO 2 incubator (37℃, CO 2 5.0%))
TGF-β2 mRNA 정량 분석:TGF-β2 mRNA quantitative analysis:
- RNA 추출 및 정량 : 형질주입 48시간 후에 배양액을 따로 모아 둔 후, RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136)를 이용하여 RNA를 추출하였다. RNA 추출 후, micro-volume plate (Take 3, Biotek)와 multi plate reader (Synergy H1, Biotek)를 이용하여 흡광측정법으로 RNA를 정량하였다.- RNA extraction and quantification: After collecting the culture medium 48 hours after transfection, RNA was extracted using an RNA preparation kit (Rneasy Plus Mini Kit, Qiagen, Cat# 74136). After RNA extraction, RNA was quantified by absorptiometry using a micro-volume plate (Take 3, Biotek) and a multi-plate reader (Synergy H1, Biotek).
- cDNA synthesis : 추출한 RNA 2ug으로 cDNA 합성 kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622)를 이용하여 cDNA를 합성하였다. 합성 조건은 manufacturer’s instruction에 따라 진행하였다. PCR 완료 후 cDNA를 20 ng/ul가 되도록 증류수(DW)로 희석하였다.- cDNA synthesis: cDNA was synthesized with 2ug of the extracted RNA using a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific, K1622). Synthesis conditions were performed according to the manufacturer’s instructions. After completion of PCR, cDNA was diluted with distilled water (DW) to 20 ng/ul.
- TGF-β2 mRNA 정량 분석을 위한 real-time PCR 진행 : Reference gene으로 human SRSF9 을 사용하였다. 상기 표 4 내지 6과 동일한 조성 및 조건으로 PCR 혼합물을 제조하여, PCR을 진행하였다.- Real-time PCR for quantitative analysis of TGF-β2 mRNA: Human SRSF9 was used as a reference gene. A PCR mixture was prepared under the same composition and conditions as in Tables 4 to 6, and PCR was performed.
TGF-β2 단백질 발현 정량 분석 (ELISA):TGF-β2 protein expression quantitative assay (ELISA):
- 따로 모아둔 배양약을 원심 분리하여 상층액만 회수하여 TGF-β2 ELISA kit (R&D)로 manufacturer’s instruction에 따라 정량 분석을 진행하였다.- Separately collected culture medium was centrifuged to recover only the supernatant, and quantitative analysis was performed with the TGF-β2 ELISA kit (R&D) according to the manufacturer’s instructions.
도 10은 4종 암종 (Breast cancer, Pancreatic cancer, Lung cancer, Melanoma) 세포주별 TGF-β2 mRNA 발현 레벨을 비교한 것이다.10 is a comparison of TGF-β2 mRNA expression levels for each cell line of 4 types of carcinoma (Breast cancer, Pancreatic cancer, Lung cancer, and Melanoma).
도 11은 4종 암종 (Lung adenocarcinoma (A549), Pancreatic cancer (Panc-1), Breast cancer (MDA-MB-231), Melanoma (Hs294T))에 ASO 처리 후 human TGF-β2 mRNA 레벨 변화를 나타낸 것이다.Figure 11 shows changes in human TGF-β2 mRNA level after ASO treatment in 4 types of carcinoma (Lung adenocarcinoma (A549), Pancreatic cancer (Panc-1), Breast cancer (MDA-MB-231), Melanoma (Hs294T)) .
실험 결과는 하기 표 13과 같다:The experimental results are shown in Table 13 below:
실험 결과, 세포주를 4개에 대하여 Human TGF-b2 mRNA 레벨이 대조군에 비하여 크게 감소되었다.As a result of the experiment, the human TGF-b2 mRNA level was significantly reduced compared to the control group for 4 cell lines.
실험예Experimental example
11. Lung cancer (A549) 11. Lung cancer (A549)
제노그라프트xenograft
마우스 모델에서 A001-01M의 항암 효능 평가 Evaluation of anticancer efficacy of A001-01M in mouse model
A549 xenograft model:A549 xenograft model:
Mouse : Balb/c nude mouse, 수컷 (5 주령), 6마리/군Mouse : Balb/c nude mouse, male (5 weeks old), 6 mice/group
마우스 입고 후, 일주일 간 순화시키고, 군을 분리(day 0)하기 13일 전에 종양 세포(A549)를 이식하였다.Mice were acclimatized for one week after wearing, and tumor cells (A549) were transplanted 13 days before group separation (day 0).
종양 세포 이식 후 13일째에 군을 분리하였다. 종양 크기는 약 80-100mm3 이었다.Groups were separated 13 days after tumor cell transplantation. The tumor size was about 80-100 mm 3 .
처리: 총 5회 (day 0, 2, 4, 7, 9) 피하 주사(s.c.) 또는 복강내 주사(i.p.)로 15 mg/kg, 10 ml/kg 용량으로 투여하였다.Treatment: A total of 5 times ( day 0, 2, 4, 7, 9) was administered at a dose of 15 mg/kg, 10 ml/kg by subcutaneous injection (s.c.) or intraperitoneal injection (i.p.).
모니터링 : 주2회, 체중 및 종양 부피를 측정하였다.Monitoring: Twice a week, body weight and tumor volume were measured.
실험 스케줄은 다음과 같다:The experimental schedule is as follows:
군 정보는 하기 표 14와 같다:Group information is shown in Table 14 below:
체중 측정 결과는 하기 표 15 및 도 12와 같다:The weight measurement results are shown in Table 15 and FIG. 12 below:
종양 부피 측정 결과는 하기 표 16 및 도 13과 같다:The tumor volume measurement results are shown in Table 16 and Figure 13 below:
실험 결과, 본 발명에 따른 ASO(A001-01M) 투여 시 종양 성장이 공지의 ASO(A001)에 비하여 크게 억제됨을 확인할 수 있었다(도 14 참조).As a result of the experiment, it was confirmed that when ASO (A001-01M) according to the present invention was administered, tumor growth was significantly inhibited compared to the known ASO (A001) (see FIG. 14 ).
본 발명의 올리고뉴클레오타이드는 TGF-β의 발현과 관련있는 질병, 예를 들어, 악성 종양, 양성 종양, 면역질환, 섬유증, 안과질환 등을 치료하기 위한 의약 조성물로서 유용하게 사용될 수 있다.The oligonucleotide of the present invention can be usefully used as a pharmaceutical composition for treating diseases related to TGF-β expression, for example, malignant tumors, benign tumors, immune diseases, fibrosis, ophthalmic diseases, and the like.
<110> Autotelic Bio Inc.<110> Autotelic Bio Inc.
<120> Antisense Oligonucleotide<120> Antisense Oligonucleotide
<130> KR20P090211<130> KR20P090211
<150> KR 10-2020-0121849<150> KR 10-2020-0121849
<151> 2020-09-21<151> 2020-09-21
<160> 1<160> 1
<170> KoPatentIn 3.0<170> KoPatentIn 3.0
<210> 1<210> 1
<211> 18<211> 18
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> Antisense Oligonucleotide<223> Antisense Oligonucleotide
<400> 1<400> 1
cggcatgtct attttgta 18 cggcatgtct attttgta 18
Claims (11)
- 서열번호 1의 안티센스 올리고뉴클레오타이드로서,As the antisense oligonucleotide of SEQ ID NO: 1,상기 올리고뉴클레오타이드의 하나 이상의 뉴클레오타이드는 뉴클레오사이드가 변형된 뉴클레오타이드이며,At least one nucleotide of the oligonucleotide is a nucleotide modified with a nucleoside,여기에서, 변형된 뉴클레오타이드는 2'-O-메톡시에틸(MOE) 변형된 뉴클레오타이드, 2'-플루오로(F) 변형된 뉴클레오타이드, 2'-O-메틸(O-Me) 변형된 뉴클레오타이드, 잠긴핵산(LNA: locked nucleic acid) 변형된 뉴클레오타이드, 에틸렌 가교 핵산(ENA: ethylene-bridged nucleic acid) 변형된 뉴클레오타이드, 제한된 에틸(cET: (R/S)-constrained ethyl) 변형된 뉴클레오타이드 및 폴리알킬렌 옥사이드 변형된 뉴클레오타이드로 이루어진 군으로부터 선택된 것인 올리고뉴클레오타이드.wherein the modified nucleotides are 2'-O-methoxyethyl (MOE) modified nucleotides, 2'-fluoro (F) modified nucleotides, 2'-O-methyl (O-Me) modified nucleotides, locked Nucleic acid (LNA) modified nucleotides, ethylene-bridged nucleic acid (ENA) modified nucleotides, cET: (R/S)-constrained ethyl modified nucleotides and polyalkylene oxides An oligonucleotide selected from the group consisting of modified nucleotides.
- 제1항에 있어서, 상기 올리고뉴클레오타이드의 5'-단부의 하나 이상의 뉴클레오타이드 또는 3'-단부의 하나 이상의 뉴클레오타이드가 변형된 뉴클레오타이드인 올리고뉴클레오타이드.The oligonucleotide according to claim 1, wherein one or more nucleotides of the 5'-end or one or more nucleotides of the 3'-end of the oligonucleotide are modified nucleotides.
- 제1항에 있어서, 상기 올리고뉴클레오타이드의 5'-단부의 하나 이상의 뉴클레오타이드 및 3'-단부의 하나 이상의 뉴클레오타이드가 변형된 뉴클레오타이드인 올리고뉴클레오타이드.The oligonucleotide according to claim 1, wherein at least one nucleotide of the 5'-end and at least one nucleotide of the 3'-end of the oligonucleotide is a modified nucleotide.
- 제2항에 있어서, 상기 올리고뉴클레오타이드의 5'-단부의 첫번째 내지 n번째 뉴클레오타이드 또는 상기 올리고뉴클레오타이드의 3'-단부의 첫번째 내지 m번째 뉴클레오타이드가 상기 변형된 뉴클레오타이드인 올리고뉴클레오타이드 (여기에서 n 및 m은 각각 독립적으로 1 내지 9 중 어느 하나의 정수이다).3. The oligonucleotide according to claim 2, wherein the first to n nucleotides of the 5'-end of the oligonucleotide or the first to m nucleotides of the 3'-end of the oligonucleotide are the modified nucleotides, wherein n and m are each independently an integer of any one of 1 to 9).
- 제3항에 있어서, 상기 올리고뉴클레오타이드의 5'-단부의 첫번째 내지 n번째 뉴클레오타이드 및 상기 올리고뉴클레오타이드의 3'-단부의 첫번째 내지 m번째 뉴클레오타이드가 상기 변형된 뉴클레오타이드인 올리고뉴클레오타이드.4. The oligonucleotide according to claim 3, wherein the first to nth nucleotides of the 5'-end of the oligonucleotide and the first to mth nucleotides of the 3'-end of the oligonucleotide are the modified nucleotides.
- 제5항에 있어서, 상기 올리고뉴클레오타이드의 5'-단부의 (n+1)번째 뉴클레오타이드 및 3'-단부의 (m+1)번째 뉴클레오타이드 사이에 하나 이상의 변형된 뉴클레오타이드를 더 포함하는 올리고뉴클레오타이드.6. The oligonucleotide of claim 5, further comprising one or more modified nucleotides between the (n+1)-th nucleotide of the 5'-end and the (m+1)-th nucleotide of the 3'-end of the oligonucleotide.
- 하기 표 1의 1번 내지 25번으로 나타낸 서열번호 1의 안티센스 올리고뉴클레오타이드로서,As the antisense oligonucleotide of SEQ ID NO: 1 shown in Nos. 1 to 25 of Table 1 below,하기 표 1에서 밑줄로 표시된 뉴클레오타이드가 변형된 뉴클레오타이드이며,Nucleotides underlined in Table 1 are modified nucleotides,여기에서, 변형된 뉴클레오타이드는 2'-O-메톡시에틸(MOE) 변형된 뉴클레오타이드, 2'-플루오로(F) 변형된 뉴클레오타이드, 2'-O-메틸(O-Me) 변형된 뉴클레오타이드, 잠긴핵산(LNA: locked nucleic acid) 변형된 뉴클레오타이드, 에틸렌 가교 핵산(ENA: ethylene-bridged nucleic acid) 변형된 뉴클레오타이드, 제한된 에틸(cET: (R/S)-constrained ethyl) 변형된 뉴클레오타이드 및 폴리알킬렌 옥사이드 변형된 뉴클레오타이드로 이루어진 군으로부터 선택된 것인 올리고뉴클레오타이드:wherein the modified nucleotides are 2'-O-methoxyethyl (MOE) modified nucleotides, 2'-fluoro (F) modified nucleotides, 2'-O-methyl (O-Me) modified nucleotides, locked Nucleic acid (LNA) modified nucleotides, ethylene-bridged nucleic acid (ENA) modified nucleotides, cET: (R/S)-constrained ethyl modified nucleotides and polyalkylene oxides An oligonucleotide selected from the group consisting of modified nucleotides:[표 1][Table 1]
- 제7항에 있어서,8. The method of claim 7,표 1의 1번 내지 21번으로 나타낸 올리고뉴클레오타이드의 변형된 뉴클레오타이드는 2'-O-메톡시에틸(MOE) 변형된 뉴클레오타이드이고,The modified nucleotides of the oligonucleotides shown in Nos. 1 to 21 of Table 1 are 2'-O-methoxyethyl (MOE) modified nucleotides,표 1의 22번 내지 25번으로 나타낸 올리고뉴클레오타이드의 변형된 뉴클레오타이드는 잠긴핵산(LNA) 변형된 뉴클레오타이드인 올리고뉴클레오타이드.The modified nucleotides of the oligonucleotides indicated by Nos. 22 to 25 of Table 1 are locked nucleic acid (LNA) modified nucleotides.
- 제1항 내지 제8항 중 어느 한 항에 있어서, 이웃한 뉴클레오사이드 사이의 결합은 포스포디에스테르 결합 또는 포스포티오에이트 결합인 올리고뉴클레오타이드.The oligonucleotide according to any one of claims 1 to 8, wherein the bond between neighboring nucleosides is a phosphodiester bond or a phosphothioate bond.
- 제9항에 따른 올리고뉴클레오타이드를 포함하며, TGF-β2 단백질의 과발현과 관련있는 질병을 예방 또는 치료하기 위한 의약 조성물.A pharmaceutical composition comprising the oligonucleotide according to claim 9, for preventing or treating a disease related to overexpression of TGF-β2 protein.
- 제10항에 있어서, 악성 종양, 양성 종양, 면역질환, 섬유증 또는 안과질환을 예방 또는 치료하기 위한 의약 조성물.The pharmaceutical composition according to claim 10, for preventing or treating a malignant tumor, a benign tumor, an immune disease, fibrosis, or an ophthalmic disease.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2020-0121849 | 2020-09-21 | ||
| KR20200121849 | 2020-09-21 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2022060208A1 true WO2022060208A1 (en) | 2022-03-24 |
Family
ID=80777223
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2021/012989 WO2022060208A1 (en) | 2020-09-21 | 2021-09-23 | Antisense oligonucleotides |
Country Status (2)
| Country | Link |
|---|---|
| KR (1) | KR20220039639A (en) |
| WO (1) | WO2022060208A1 (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010512773A (en) * | 2006-12-22 | 2010-04-30 | アンティセンス ファルマ ゲゼルシャフト ミット ベシュレンクテル ハフツング | Oligonucleotide, protein, and / or peptide-polymer conjugate |
| US20100160208A1 (en) * | 2003-12-19 | 2010-06-24 | Antisense Pharma Gmbh | Pharmaceutical composition |
| KR20180103817A (en) * | 2016-02-09 | 2018-09-19 | 오토텔릭 엘엘씨 | Compositions and methods for treating cancer |
| KR102108599B1 (en) * | 2013-03-27 | 2020-05-08 | 이사르나 테라퓨틱스 게엠베하 | Modified tgf-beta oligonucleotides |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3555952B2 (en) | 1993-04-30 | 2004-08-18 | バイオグノスティック・ゲゼルシャフト・フュア・バイオモレキュラーレ・ダイアグノスティック・ミット・ベシュレンクテル・ハフツング | Antisense-oligonucleotides for treating the immunosuppressive effect of transforming growth factor-β (TGF-β) |
-
2021
- 2021-09-23 KR KR1020210126028A patent/KR20220039639A/en unknown
- 2021-09-23 WO PCT/KR2021/012989 patent/WO2022060208A1/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100160208A1 (en) * | 2003-12-19 | 2010-06-24 | Antisense Pharma Gmbh | Pharmaceutical composition |
| JP2010512773A (en) * | 2006-12-22 | 2010-04-30 | アンティセンス ファルマ ゲゼルシャフト ミット ベシュレンクテル ハフツング | Oligonucleotide, protein, and / or peptide-polymer conjugate |
| KR102108599B1 (en) * | 2013-03-27 | 2020-05-08 | 이사르나 테라퓨틱스 게엠베하 | Modified tgf-beta oligonucleotides |
| KR20180103817A (en) * | 2016-02-09 | 2018-09-19 | 오토텔릭 엘엘씨 | Compositions and methods for treating cancer |
Non-Patent Citations (1)
| Title |
|---|
| PETER HAU , PIOTR JACHIMCZAK ,ULRICH BOGDAHN: "Treatment of malignant gliomas with TGF-β2 antisense oligonucleotides. ", EXPERT REVIEW OF ANTICANCER THERAPY, vol. 9, no. 11, 1 January 2009 (2009-01-01), GB , pages 1663 - 1674, XP009535063, ISSN: 1473-7140, DOI: 10.1586/era.09.138 * |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20220039639A (en) | 2022-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2018030608A1 (en) | Nanoliposome carrier composition containing hybrid of cas9 protein and guide rna | |
| WO2019135639A1 (en) | Novel polypeptide and method for producing imp using same | |
| WO2012074277A2 (en) | Novel hybrid promoter and recombinant vector comprising the same | |
| WO2019050071A1 (en) | Composition for preventing or treating liver fibrosis, containing exosome or exosome-derived ribonucleic acid | |
| WO2015156649A1 (en) | Peptide having fibrosis inhibitory activity and composition containing same | |
| WO2017022962A1 (en) | Composition for preventing or treating immune disease, comprising ripk inhibitor as active ingredient | |
| WO2016140492A1 (en) | Novel dna-rna hybrid regular tetrahedron structure or rna tetrahedron structure | |
| WO2018008903A1 (en) | Adipocyte-targeting non-viral gene delivery complex comprising dual plasmid vector | |
| WO2021010723A1 (en) | Melanophilin antisense oligonucleotides | |
| WO2022060208A1 (en) | Antisense oligonucleotides | |
| WO2023075043A1 (en) | Antisense oligonucleotides | |
| WO2018026212A2 (en) | Method for producing fibrosis disease model, and use of fibrosis disease model | |
| WO2019031804A9 (en) | E. coli and corynebacterium glutamicum shuttle vector for regulating expression of target gene | |
| WO2022145949A2 (en) | Composition comprising tead4 inhibitor for preventing or treating colorectal cancer | |
| WO2018056583A1 (en) | Composition for skin whitening comprising sh3bp4-inhibiting material, and method for screening sh3bp4-inhibiting material | |
| EP3837274A1 (en) | Acetyl-coa carboxylase2 antisense oligonucleotides | |
| WO2016178519A1 (en) | Composition for inhibiting muscle aging or treating muscle aging related diseases | |
| WO2021033973A1 (en) | Composition for inhibiting myeloid-derived suppressor cell comprising mitf inhibitor as active ingredient | |
| WO2009145573A2 (en) | Recombinant escherichia coli for producing recombinant human variation interferon-beta proteins wherein methionine at the amino end is removed, and preparation method thereof | |
| WO2024177416A1 (en) | Pharmaceutical composition for treating egfr targeted therapy-resistant cancer | |
| WO2020050602A1 (en) | Screening system for human-transformed cartilage cell line-based cartilage disease treatment agents | |
| WO2024071935A1 (en) | Composition for treating cancer containing histone deacetylase inhibitor, and anticancer adjuvant for cancer immunotherapy | |
| WO2015126033A1 (en) | Composition for controlling stem cell pluripotency, containing lin28a methylation inhibitor, and method for screening for lin28a methylation inhibitor | |
| WO2017164486A1 (en) | Composition for inhibiting resistance to anticancer agents, containing tesk1 inhibitor, and method for screening for tesk1 inhibitor | |
| WO2024058589A1 (en) | Chimeric antigen receptor cell prepared using genetic scissor knock-in, and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21869830 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 21869830 Country of ref document: EP Kind code of ref document: A1 |